Parte 2 Bio

132 Chapter 6: How Cells Read the Genome: From DNA to Protein
TABLE 6–4 Summary of rounds of selection for a ribozyme with RNA ligation
activity (Problem 6–98).
Ligation conditions
Error-prone Ligation rate
Round PCR MgCl2 (mM) Time (hours) (per hour)
0 0.000003
1 No 60 16 <0.000004
2 No 60 16 0.0008
3 No 60 16 0.0094
4 No 60 16 0.027
5 Yes 60 0.50 0.16
6 Yes 60 0.17 0.40
7 Yes 60 0.02 0.86
8 No 4 0.12 3.2
9 No 2.5 0.17 4.5
10 No 2.5 0.17 8.0
same, although 11 of the 15 have very similar sequences. Your advisor is

excited by your results and has asked you to give the next departmental
seminar. You know from your conversations with other students that you
will need to prepare careful explanations for the questions listed below.
A. Why did you use error-prone PCR, which can introduce mutations, in
some of the rounds of amplification?
B. Why did you reduce the time and Mg2+ concentration—both changes
increase the difficulty of ligation—in successive rounds of selection?
C. How much of an improvement in ligation rate have you found in your 10
rounds of selection and amplification?
D. Why is there still such diversity among the RNA molecules after 10 rounds
of selection and amplification?
MCAT STYLE
Passage 1 (Questions 6–99 to 6–102)
Congenital heart defects are present in 1–2% of newborns and cause a significant
number of stillbirths. They are also a major cause of heart disease in adults. Many
congenital heart defects are due to mutations in genes for transcription factors
that control expression of genes that are required for normal heart development.
For example, over 30 different mutations have been found in the gene for tran-
scription factor Tbx5 in patients with congenital heart defects. To carry out its
functions, Tbx5 must bind to another transcription factor called Nkx2-5, which
also plays a role in heart development and is mutated in many individuals with
congenital heart defects. Imagine that you can isolate developing cardiac cells
with homozygous Tbx5 mutations and study the molecular defects caused by the
mutations.
6–99 In one mutant cell line, you find that the normal, mature Tbx5 mRNA
is present at its usual levels, but that full-length Tbx5 protein cannot be
detected. What kind of mutation most likely accounts for this phenotype?
A. A mutation that blocks binding of Tbx5 to Nkx2-5
B. A mutation that causes a splicing defect
C. A mutation that disrupts the normal folding of Tbx5
D. A mutation that introduces a premature stop codon
MCAT STYLE 133
6–100 In another mutant cell line, you find that levels of the mature, properly
spliced Tbx5 mRNA are greatly reduced. What kind of mutation could
account for this phenotype?
I. A short deletion in the coding region that preserves the reading frame
II. A short deletion in the coding region that disrupts the reading frame
III. A mutation in the promotor region of the Tbx5 gene
A. I
B. I and II
C. I and III
D. II and III
6–101 In another mutant cell line, you find that full-length Tbx5 mRNA and pro-
tein are present at normal levels, yet individuals carrying this mutation
have severe defects in heart development. What kind of mutation could
cause this phenotype?
A. A mutation that blocks association of the Tbx5 mRNA with the ribosome
B. A mutation that blocks export of the Tbx5 mRNA from the nucleus
C. A mutation that causes a splicing defect
D. A mutation that disrupts binding of Tbx5 with Nkx2-5
6–102 Which one of the following is a plausible mechanism by which Tbx5
could control initiation of transcription?
A. Activation of transcription elongation factors
B. Recruitment of RNA polymerase II to promoters
C. Recruitment of splicing factors to RNA polymerase II
D. Regulation of 5 cap formation
A key discovery in cell biology came from studying autoimmune diseases. Sera
from patients with autoimmune diseases were found to contain antibodies that
bind to cellular proteins. To characterize these proteins further, the antibodies
were used to purify the proteins they recognized. In one case, it was found that the
antibodies bound to small nuclear ribonucleoprotein complexes or snRNPs (pro-
nounced “snurps”). The RNAs within each complex were snRNAs (small nuclear
RNAs). The functions of these snRNPs were initially mysterious, but it was eventu-
ally found that they are the key functional components of the spliceosome.
6–103 Which one of the following would have provided key evidence that
snRNPs are involved in the splicing of pre-mRNA?
A. Association of snRNPs with ribosomes
B. Base-pairing between snRNAs and pre-mRNA splice sites
C. Introns within snRNAs
D. Lariat structures within snRNAs
6–104 Autoimmune antibodies from patients were used to determine the local-
ization of snRNPs in the cell. Which of the following best describes where
snRNP particles would be found within the cell?
A. In the cytoplasm, near ribosomes
B. In the cytoplasm, near the nucleus
C. In the nucleus, near RNA polymerase II
D. In the nucleus, near RNA polymerase III
6–105 Which one of the following proteins might be expected to be found in a
snRNP?
A. A DNA helicase
B. An ATPase
C. An enhancer protein
D. A site-specific ribonuclease
Gene Regulation by the Dorsal Gradient
in the Drosophila Embryo.
This stunning image shows cross
sections through a Drosophila embryo at
about the 14th nuclear division cycle after
fertilization. The image on the left shows
the gradient of Dorsal protein as revealed
by a fluorescent antibody and, on the
right, the resulting pattern of expression
of a number of genes detected by in situ
hybridization with fluorescent nucleic acid
probes. At least 50 genes are controlled
by Dorsal, some activated, others
repressed.
Key to gene names: dpp,
decapentaplegic; ind, intermediate
neuroblasts defective; sog, short
gastrulation; sna, snail; vnd, ventral
neuroblasts defective.
Chapter 7 135
CHAPTER
Control of Gene Expression 7

AN OVERVIEW OF GENE CONTROL IN THIS CHAPTER
TERMS TO LEARN AN OVERVIEW OF GENE CONTROL

mRNA degradation control RNA transport control
protein activity control transcriptional control CONTROL OF TRANSCRIPTION BY
RNA localization control translational control SEQUENCE-SPECIFIC
RNA processing control DNA-BINDING PROTEINS
DEFINITIONS TRANSCRIPTION REGULATORS

SWITCH GENES ON AND OFF
Match each definition below with its term from the list above.
MOLECULAR GENETIC
7–1 Regulates which RNAs are exported from the nucleus.
MECHANISMS THAT CREATE AND
7–2 Regulates when and how often a given gene sequence is made into RNA. MAINTAIN SPECIALIZED CELL
7–3 Regulates which mRNA molecules are selectively destabilized in the TYPES
cytoplasm.
MECHANISMS THAT REINFORCE
7–4 Regulates which mRNAs are selected to be used for protein synthesis by CELL MEMORY IN PLANTS AND
ribosomes. ANIMALS
7–5 Regulates the splicing and modification of RNA transcripts.
POST-TRANSCRIPTIONAL
TRUE/FALSE CONTROLS
Decide whether each of these statements is true or false, and then explain why. REGULATION OF GENE
7–6 When the nucleus of a fully differentiated carrot cell is injected into a EXPRESSION BY NONCODING
frog egg whose nucleus has been removed, the injected donor nucleus is RNAs
capable of programming the recipient egg to produce a normal carrot.
7–7 The differences in the patterns of proteins produced in different spe-
cialized cell types are accurately reflected in the patterns of expressed
mRNAs.
larger
THOUGHT PROBLEMS
7–8 A small portion of a two-dimensional display of proteins from human
brain is shown in Figure 7–1. These proteins were separated on the basis
of size in one dimension and electrical charge (isoelectric point) in the
other. Not all protein spots on such displays are products of different
smaller
genes; some represent modified forms of a protein that migrate to dif-

ferent positions. Pick out a couple of sets of spots that could represent acidic basic
proteins that differ by the number of phosphates they carry. Explain the Figure 7–1 Two-dimensional separation of
basis for your selection. proteins from the human brain (Problem
7–8). The proteins were displayed using two-
7–9 In principle, a eukaryotic cell can regulate gene expression at any step in dimensional gel electrophoresis. Only a small
the pathway from DNA to the active protein (Figure 7–2). portion of the protein spectrum is shown.
136 Chapter 7: Control of Gene Expression
NUCLEUS CYTOSOL Figure 7–2 Six steps at which the

pathway for eukaryotic gene expression
modified can be controlled (Problem 7–9).
protein
primary RNA
DNA mRNA mRNA protein
transcripts
amino
acids
nucleotides
A. Place the types of control listed below at appropriate places on the dia-
gram in Figure 7–2.
1. mRNA degradation control
2. protein activity control
3. RNA processing control
4. RNA transport and localization control
5. transcriptional control
6. translational control
B. Which of the types of control listed above are unlikely to be used in bac-
teria?
DATA HANDLING
7–10 In the original cloning of sheep from somatic cells, the success rate was
very low. For example, only one lamb (Dolly) was born from 277 zygotes
Figure
that were reconstructed using nuclei7-02
derived from breast cells and enu-
cleated, unfertilized eggs. Other experiments using nuclei from embry-
Problem
onic or fetal lamb cells had a higher7-10
success rate, albeit still a low one.
Given the rarity of successful events, it was critical to eliminate inadvert-
ent mating—of either the oocyte donor or the surrogate mother—as the
source of newborn lambs. To determine whether the cloned animals
were derived from the donor nuclei, the researchers analyzed DNA
microsatellites (short, repetitive DNA sequences) at four loci in surrogate
mothers and donor cells (Figure 7–3). These loci were chosen because
many different lengths are present in sheep populations.
A. Do the results in Figure 7–3 argue that the lambs were derived from the
transplanted nuclei, or from an inadvertent mating? Explain your answer.
B. What would the results have looked like for the alternative you did not
choose in question A?
7–11 One of the rare examples of developmentally programmed genome
rearrangements in mammals occurs during the generation of antibody
diversity in the immune system. In B cells, which produce antibodies,
the variable (V) and constant (C) segments of immunoglobulin genes
are brought close together by deletion of a long segment of DNA that lies
Figure 7–3 Microsatellite analysis of
seven surrogate mothers, the three
ORIGIN OF DONOR CELL NUCLEI different nuclear donor cell types, and
embryo fetus breast the seven lambs that were born (Problem
7–10). Four polymorphic loci were used in
SURROGATE
bs
bs
the analysis. The surrogate mothers are

m
m
lls
lls
lls
locus MOTHERS
la
la
la
ce
ce
ce
arranged left to right in the same order

1 as the lambs they gave birth to. Nuclear
donor cells were derived from embryo,
fetus, or breast. At each of the four
2
polymorphic loci, flanking polymerase
chain reaction (PCR) primers were used
3
to amplify DNA that included a particular
microsatellite. Microsatellites with
4 different numbers of repeats give rise to
different length PCR products.
CONTROL OF TRANSCRIPTION BY SEQUENCE-SPECIFIC DNA-BINDING PROTEINS 137
between them. Digestion of unrearranged germ-line DNA with a restric- GERM LINE B CELLS
tion nuclease that cuts in DNA flanking the V and C segments generates PROBE C V C V
two bands on a Southern blot when hybridized to radioactive probes
loading
specific for the V and the C segments (Figure 7–4). Would you expect the slots
V- and C-segment probes to hybridize to the same or different DNA frag-
ments after digestion of B cell DNA with the same restriction nuclease?
Sketch a possible pattern of hybridization to B cell DNA that is consistent
with your expectations. Explain the basis for your pattern of hybridiza-
tion. (Without a lot more information you cannot predict the exact pat-
tern, so focus on the general features of the pattern.)
MEDICAL LINKS
Figure 7–4 Southern blot of DNAs from
7–12 Comparisons of the patterns of mRNA levels across different human cell the germ line and B cells (Problem
types show that the level of expression of almost every active gene is dif- 7–11). Germ-line DNA and B cell DNA
were digested with the same restriction
ferent. The patterns of mRNA abundance are so characteristic of cell type nuclease. Only the hybridization to
that they can be used to determine the tissue of origin of cancer cells, even germ-line DNA is shown.
though the cells may have metastasized to different parts of the body. By
definition, however, cancer cells are different from their noncancerous
precursor cells. How do you suppose then that patterns of mRNA expres-
sion might be used to determine the tissue source of a human cancer?
Figure 7-4
CONTROL OF TRANSCRIPTION BY SEQUENCE-
Problem 7-12
SPECIFIC DNA-BINDING PROTEINS
TERMS TO LEARN
cis-regulatory sequence transcription regulator
DEFINITIONS
7–13 A protein that binds to a specific DNA sequence and influences the rate
at which a gene is transcribed.
7–14 A short segment of DNA, 5–10 nucleotide pairs in length, in the neigh-
borhood of the promoter, that serves as a binding site for a protein that
modulates gene expression.
TRUE/FALSE
Decide whether each of these statements is true or false, and then explain why.
7–15 Because the individual contacts are weak, the interactions between regu-
latory proteins and DNA are among the weakest in biology.
7–16 In terms of the way it interacts with DNA, the helix–loop–helix motif is
more closely related to the leucine zipper motif than it is to the helix–
turn–helix motif.
THOUGHT PROBLEMS
7–17 Figure 7–5 shows a short stretch of a DNA helix displayed as a space-
filling model. Indicate the major and minor grooves and provide a length
scale. Is it possible to tell the polarity of each of the strands in this figure?
7–18 Explain how DNA-binding proteins can make sequence-specific con-
tacts to a double-stranded DNA molecule without breaking the hydro- Figure 7–5 A space-filling model of a DNA
gen bonds that hold the bases together. Indicate how, by making such duplex (Problem 7–17).
MAJOR GROOVE MAJOR GROOVE
H H
H H
H N H O H C O H N H
N H N
H N H N N H N H N N
N N N N
O H N O H
cytosine H guanine thymine adenine
MINOR GROOVE MINOR GROOVE
contacts, a protein can distinguish a C-G from a T-A base pair. Use Figure 7–6 C-G and T-A base pairs
Figure 7–6 to indicate what sorts of noncovalent bonds (hydrogen bonds, (Problem 7–18).
electrostatic attractions, or hydrophobic interactions) could be used to
discriminate between C-G and T-A. (You do not need to specify any par-
ticular amino acid on the protein.)
7–19 What are the two fundamental components of a genetic switch?
7–20 The nucleus of a eukaryotic cell is much larger than a bacterium, and it
contains much more DNA. As a consequence, a transcription regulator
in a eukaryotic cell must be able to select its specific binding site from
among many more unrelated sequences than does a transcription regu-
lator in a bacterium. Does this present any special problems for eukary-
otic gene regulation?
Consider the following situation. Assume that the eukaryotic nucleus
and the bacterial cell each have a single copy of the same DNA binding
site. In addition, assume that the nucleus is 500 times the volume of the
bacterium, and has 500 times as much DNA. If the concentration of the
transcription regulator that binds the site were the same in the nucleus
and in the bacterium, would the regulator occupy its binding site equally
as well in the eukaryotic nucleus as it does
Figurein the bacterium? Explain
7-6
your answer.
7–21 One type of zinc finger motif consists Problem
of an helix 7-22
and a sheet held
together by a zinc ion (Figure 7–7). When this motif binds to DNA, the
helix is positioned in the major groove, where it makes specific contacts
with the bases. This type of zinc finger is often found in a cluster with
additional zinc fingers, an arrangement that allows strong and specific
DNA–protein interactions to be built up through a repeating basic struc-
tural unit. Why do you suppose this motif is thought to enjoy a particular C
advantage over other DNA-binding motifs when the strength and speci- H 24
ficity of the DNA–protein interaction needs to be adjusted during evolu- C7
tion?
Zn
H 20
7–22 Many transcription regulators form dimers of identical or slightly differ- C4
ent subunits on the DNA. Suggest two advantages of dimerization.
7–23 Genetic analyses in bacteria in the 1950s provided the first evidence
for the existence of transcription regulators. The lambda repressor, one
such regulator, is encoded by the bacterial virus, bacteriophage lambda. N
The lambda repressor binds as a dimer to critical sites on the bacterio-
phage lambda genome to keep the lytic genes turned off, which allows
the bacteriophage lambda genome to be maintained as a silent resident
in the bacterial genome. Each molecule of the repressor consists of an Figure 7–7 Zinc finger DNA-binding motif
(Problem 7–21). The zinc ion interacts with
N-terminal DNA-binding domain and a C-terminal dimerization domain Cys (C) and His (H) side chains so that
(Figure 7–8). Upon induction (for example, by irradiation with ultravio- the helix is held tightly to one end of the
let light), the genes for lytic growth are expressed, bacteriophage lambda sheet.
Figure 7–8 Domains of the lambda

C C C C C C cleavage
site
repressor and the binding of repressor
+ dimers to DNA (Problem 7–23).
N N N N N N
repressor monomers repressor dimer DNA-binding site
progeny are produced, and the bacterial cell lyses to release the viral
progeny. Induction is initiated by cleavage of the lambda repressor at a
site between the DNA-binding domain and the dimerization domain. In
the absence of bound repressor, RNA polymerase initiates transcription
of the lytic genes, triggering lytic growth. Given that the number (concen-
tration) of DNA-binding domains is unchanged by cleavage of the repres-
sor, why do you suppose its cleavage results in its removal from the DNA?
7–24 The differentiation of muscle cells from the somites of the developing
embryo is controlled by myogenin, a member of the MyoD family of
helix–loop–helix transcription regulators. Myogenin functions as a het-
erodimer with another member of the MyoD family of helix–loop–helix
proteins (Figure 7–9A). The activity of myogenin must be carefully con-
trolled lest it trigger premature expression of the muscle program of
Figure
cell differentiation. The myogenin gene7-8
is turned on in advance of the
time when it is needed, but myogenin is prevented from functioning
by phosphorylation of its DNA-binding
Problem 7-28 domain and by its tight bind-
ing to Id, a helix–loop–helix protein that lacks a DNA-binding domain
(Figure 7–9B). Explain how phosphorylation of the DNA-binding domain
and dimerization with Id might act to keep myogenin nonfunctional.
CALCULATIONS
7–25 One method for determining the DNA sites bound by a transcription
regulator is to mix random sequences of DNA with the regulator, sepa-
rate the bound sequences from the starting mixture, amplify the bound
sequences by PCR, and then repeat this binding-release-amplification
cycle until a consensus sequence emerges. But is it reasonable to expect
that all possible consensus-site binding sequences will actually be pre-
sent in the starting sample of oligonucleotides?
(A) MyoD–HLH HETERODIMER (B) PHOSPHO–MyoD/Id

BOUND TO DNA HETERODIMER
HLH MyoD Id MyoD
Figure 7–9 The transcription regulator myogenin (Problem 7–24). (A) Myogenin as
part of a heterodimer bound to DNA. HLH stands for helix–loop–helix. (B) An inactive
form of phosphorylated myogenin bound to Id.
TABLE 7–1 Nucleotide sequences of selected and amplified DNAs

(Problem 7–25).
GAATTCGCCTCGAGCACATCATTGCCCATATATGGCACGACAGGATCC
GAATTCGCCTCTTCTAATGCCCATATATGGACTTGCTCGACAGGATCC
GGATCCTGTCGGTCCTTTATGCCCATATATGGTCATTGAGGCGAATTC
GAATTCGCCTCATGCCCATATATGGCAATAGGTGTTTCGACAGGATCC
GAATTCGCCTCTATGCCCATATAAGGCGCCACTACCCCGACAGGATCC
GAATTCGCCTCGTTCCCAGTATGCCCATATATGGACACGACAGGATCC
GGATCCTGTCGACACCATGCCCATATTTGGTATGCTCGAGGCGAATTC
GAATTCGCCTCATTTATGAACATGCCCTTATAAGGACCGACAGGATCC
GAATTCGCCTCTAATACTGCAATGCCCAAATAAGGAGCGACAGGATCC
GAATTCGCCTCATGCCCAAATATGGTCATCACCTACACGACAGGATCC
Underlined sequences correspond to PCR primer sites. The sequences have been
ordered so that the binding sites are all oriented in the same direction (to make it
easier to pick out the consensus sequence by eye).
Consider the following specific example. You wish to test all possi-
ble consensus sequences that are 14 nucleotides long. You synthesize a
population of oligonucleotides that carry a central 26-nucleotide-long
random sequence, flanked on either side by 25-nucleotide-long defined
sequences to serve as primer sites for PCR amplification. You convert the
single-stranded oligonucleotides to double-stranded ones, using one of
the PCR primers. You begin the first cycle of binding with a 0.4 ng sample
of the synthesized population of double-stranded oligonucleotides.
A. How many double-stranded oligonucleotides, 76-nucleotide pairs long,
are present in the 0.4 ng sample with which you started the experiment?
(The average mass of a nucleotide pair is 660 daltons.)
B. Assuming that the starting population of oligonucleotides was truly ran-
dom in the 26 central nucleotides, would you expect to find all possible
14-base-pair-long sequences represented in the starting sample?
C. The sequences of 10 individual clones resulting from this procedure are
shown in Table 7–1. What is the consensus sequence to which the tran-
scription regulator binds?
DATA HANDLING
7–26 When Jacob and Wollman tried to check the genetic linkage between
the Gal gene and an integrated bacteriophage lambda genome (termed
a lambda prophage), they discovered a surprising phenomenon they recipient
referred to as “erotic induction” (which was later called zygotic induction lambda– lambda+
for publication). In a bacterial mating used to determine genetic linkage,
lambda+ lambda–
no no
a portion of the chromosome is transferred via a narrow tube from the lysis lysis
donor
donor bacterium to the recipient. Jacob and Wollman found that if the
donated chromosome carried a lambda prophage, but the recipient cell lysis +
no
lambda
did not, lambda growth was induced in the recipient cell, which then phage
lysis
lysed, producing lambda phage. If the recipient cell carried the lambda
prophage, however, no lysis was observed. A summary of results from all
their matings is shown in Figure 7–10. Figure 7–10 Results of matings between
bacteria with and without lambda
A. Are these results consistent with the notion that a transcription regulator prophages (Problem 7–26). Lambda–
encoded by the prophage normally represses the bacteriophage’s lytic indicates the absence of a prophage;
genes. Why or why not? lambda+ indicates its presence.
B. Suppose that the prophage prevented lytic growth by expressing a tran- (A) EXPERIMENTAL SET-UP
scription regulator that turned on a gene for an anti-lysis protein. Would bulk flow of
RNA polymerase
the results of the matings have been the same or different? Explain your molecules
answer.
7–27 Transcription regulators often find their specific sites much faster than aligned
DNA
would be anticipated by simple three-dimensional diffusion. The Lac molecules
repressor, for example, associates with the Lac operator—its DNA bind-
ing site—more than 100 times faster than expected for three-dimensional
diffusion. Clearly, the repressor must find the operator by mechanisms
that reduce the dimensionality or volume of the search in order to hasten (B) SINGLE RNA POLYMERASE MOLECULES
target acquisition.
Several techniques have been used to investigate this problem. One
of the most elegant used fluorescent RNA polymerase molecules that
could be followed individually. An array of DNA molecules was aligned
in parallel by an electrophoretic technique and anchored to a glass slide.
Fluorescent RNA polymerase molecules were then allowed to flow across
them at an oblique angle (Figure 7–11A). Traces of individual RNA poly-
merases showed that about half flowed in the same direction as the bulk
and about half deviated from the bulk flow in a characteristic manner
Figure 7–11 Interactions of individual
(Figure 7–11B). If the RNA polymerase molecules were first incubated RNA polymerase molecules with DNA
with short DNA fragments containing a strong promoter, all the traces (Problem 7–27). (A) Experimental set-up.
followed the bulk flow. DNA molecules are aligned and anchored
A. Offer an explanation for why some RNA polymerase molecules deviated to a glass slide at their ends, and highly
from the bulk flow as shown in Figure 7–11B. Why did incubation with fluorescent RNA polymerase molecules
are allowed to flow across them.
short DNA fragments containing a strong promoter eliminate traces that (B) Traces of two individual RNA
deviated from the bulk flow? polymerase molecules. The one on the
B. Do these results suggest an explanation for how transcription regulators left has traveled with the bulk flow, and
manage to find their sites faster than expected by diffusion? the one on the right has deviated from it.
C. Based on your explanation, would you expect a transcription regulator The scale bar is 10 μm.
to find its target site faster in a population of short DNA molecules or in
a population of long DNA molecules? Assume that the concentration of Figure 7-11
DNA is the same in both populations and that both populations have the
same number of target sites.
Problem 7-32
7–28 The binding of a transcription regulator to a DNA sequence can cause
the DNA to bend to make appropriate contacts with groups on the sur-
face of the protein. Such protein-induced DNA bending can be readily
detected by the way the protein–DNA complexes migrate through poly-
acrylamide gels. The rate of migration of bent DNA depends on the aver-
age distance between its ends as it gyrates in solution: the more bent the
DNA, the closer together the ends are on average, and the more slowly
it migrates. If there are two sites of bending, the end-to-end distance
depends on whether the bends are in the same (cis) or opposite (trans)
directions (Figure 7–12A).
You have shown that the catabolite activator protein (CAP) causes
DNA to bend by more than 90° when it binds to its regulatory site. You
wish to know the details of the bent structure. Specifically, is the DNA
at the center of the CAP-binding site bent so that the minor groove of
the DNA helix is on the inside, or is it bent so that the major groove is
on the inside? To answer this, you prepare two kinds of constructs, as
shown in Figure 7–12B. In one, you place two CAP-binding sequences
on either side of a central site into which you insert a series of DNA seg-
ments that vary from 10 to 20 nucleotide pairs in length. In the other, you
flank the insertion site with one CAP-binding sequence and one (A5N5)4
sequence, which is known to bend with the major groove on the inside.
You measure the migration of the CAP-bound constructs relative to the
corresponding CAP-bound DNA with no insert. You then plot the relative
migration versus the number of nucleotide pairs between the centers of
bending (Figure 7–12C and D).
(A) DNA BENDING BY CAP (B) TWO BENDY CONSTRUCTS Figure 7–12 Bending of DNA by CAP
binding (Problem 7–28). (A) The cis and
CAP insert CAP trans configurations of a pair of bends.
cis (B) Two constructs used to investigate
DNA bending by CAP binding.
centers of bending (C) Relative migration as a function of
the number of nucleotides between the
centers of bending in the CAP–CAP
trans (A5N5)4 insert CAP construct. (D) Relative migration as a
function of the number of nucleotides
between the centers of bending in the
centers of bending (A5N5)4–CAP construct.
(C) CAP–CAP (D) (A5N5)4–CAP

1.2 1.2
relative migration
1.1 1.1
1.0 1.0
0.9 0.9
0.8 0.8
77 79 81 83 85 87 89 93 95 97 99 101 103 105
nucleotides between nucleotides between
centers of bending centers of bending
A. Assuming that there are 10.6 nucleotides per turn of the DNA helix, esti-
mate the number of turns that separate the centers of bending of the
two CAP-binding sites at the point of minimum relative migration. How
many helical turns separate the centers of bending at the point of maxi-
mum relative migration?
B. Is the relationship betweenFigure 11-04
the relative migration and the separation of
the centers of bending of the CAP sites what you would expect, assuming
that the cis configurationProblem 11-37 than the trans configuration
migrates slower
(Figure 7–12C)? Explain your answer.
C. How many helical turns separate the centers of bending at the point of
minimum migration of the construct with one CAP site and one (A5N5)4
site (Figure 7–12D)?
D. Which groove of the helix faces the inside of the bend at the center of
bending of the CAP site?
7–29 The Fos and Jun oncogenes encode proteins that form a heterodimeric
transcription regulator. Leucine zipper domains in each protein mediate
their dimerization through coiled-coil interactions. Dimerization juxta-
poses the DNA-binding domains of each protein, positioning them for
interaction with regulatory sites in DNA. The dynamics of Fos–Jun inter-
action in the presence and absence of AP-1 DNA (the sequence to which
the Fos–Jun heterodimer binds) were investigated by fluorescence reso-
nance energy transfer (FRET), which is well suited for the rapid measure-
ments that are necessary in such studies.
Fos was tagged with fluorescein (Fos–F) and Jun was tagged with
rhodamine (Jun–R), as shown in Figure 7–13A. Fluorescein absorbs
light at 490 nm and emits light at 530 nm, whereas rhodamine absorbs
light at 530 nm and emits light at 603 nm. When Fos–F and Jun–R are
brought into close proximity through heterodimerization, light energy
absorbed by fluorescein at 490 nm is efficiently transferred to rhoda-
mine through nonradiative energy transfer and emitted by rhodamine at
603 nm. Dimerization thus decreases fluorescence by fluorescein at
530 nm and increases fluorescence by rhodamine at 603 nm, as shown in
Figure 7–13B. In the presence of AP-1 DNA, FRET is even more efficient
(A) rhodamine fluorescein (B) EMISSION SPECTRA (C) EXCHANGE KINETICS
fluorescein + AP-1 DNA

530 nm 1.0
relative fluorescence at 603 nm

1.0 Fos–F
+ Fos
relative fluorescence
0.8
Jun–R Fos–F 0.8
Fos–F 0.6
0.6
+ Jun–R
rhodamine
AP-1 DNA 0.4 0.4
603 nm
Fos–F + Jun–R − AP-1 DNA
+ AP-1 DNA 0.2
0.2
0 0
500 540 580 620 660 0 200 400 600 800 1000
wavelength (nm) time (seconds)
Figure 7–13 Dynamics of Fos–Jun heterodimerization in the presence and absence of AP-1 DNA (Problem 7–29).
(A) Arrangement of fluorophores in Fos–F and Jun–R. In the dimer, the excited fluorescein on Fos transfers energy to the
rhodamine on Jun. (B) Emission spectra of Fos–F alone, Fos–F with Jun–R, and Fos–F with Jun–R and AP-1 DNA. The
emission spectra were recorded between 500 and 700 nm after excitation at 490 nm. (C) Analysis of the exchange of
Fos–F and Jun–R in the presence and absence of AP-1 DNA. Rhodamine fluorescence at 603 nm was followed over time
after excitation of fluorescein at 490 nm. A 10-fold excess of unlabeled Fos was added at the time indicated by the arrow.
(Figure 7–13B), indicating that binding to the DNA brings the two fluoro-
phores into even closer proximity.
FRET was also used to measure the ability of Fos–Jun dimers to
exchange subunits with monomers in solution in the presence and
absence of AP-1 DNA (Figure 7–13C). Fos–F and Jun–R were preincu-
bated in the absence of DNA to allow free heterodimers to form, or in
its presence to allow DNA-bound heterodimers to form. A 10-fold excess
of Fos (without fluorescein) was then added to both solutions, and rho-
damine fluorescence at 603 nm was followed after excitation at 490 nm
(Figure 7–13C).
A. Do free heterodimers exchange subunits with added unlabeled Fos?
Do heterodimers bound to DNA exchange subunits? How can you tell?
Figure 7-13
Explain any significant differences in the behavior of free and DNA-
bound heterodimers.
Problem
B. In most cells, there are many distinct leucine 7-34
zipper transcription regula-
tors, several of which can interact to form a variety of heterodimers. If the
results in Figure 7–13C were typical of the leucine zipper proteins, what
would they imply about the leucine zipper heterodimers in cells?
7–30 The binding of a protein to DNA can protect it from chemical cleavage,
which is the basis for the technique known as DNA footprinting. You
have used DNA footprinting to determine the DNA binding site of a
transcription regulator after labeling one strand. To check your results,
you repeat the experiment after labeling the other strand of the duplex.
You find that the footprints are slightly offset from one another relative
to the sequence of the DNA (Figure 7–14). If the protein binds to the
same duplex in both cases, how can the footprints on the two strands be Figure 7–14 DNA footprints (Problem
different? 7–30). The sequences of the DNA from
the top and bottom strands around the
footprint are shown. The 5 ends of the
top top or bottom strands were labeled with
strand no protein 32P. Chemical cleavage was used to
5ʹ-CTGTGTGTATGCTGGGAAGGACTT introduce DNA breaks, which are fairly
top
strand + protein randomly distributed, as shown by the
roughly equal intensities of the individual
bottom bands. Each band corresponds to a
strand + protein fragment of DNA that differs from those
GACACACATACGACCCTTCCTGAA– 5ʹ on either side by one nucleotide.
TRANSCRIPTION REGULATORS AS GENE SWITCHES

TERMS TO LEARN
cis-regulatory sequence gene control region
gene promoter
DEFINITIONS
7–31 Nucleotide sequence in DNA to which RNA polymerase binds to begin
transcription.
7–32 The whole expanse of DNA involved in regulating and initiating tran-
scription of a gene.
7–33 The segment of DNA that is transcribed into RNA.
TRUE/FALSE
7–34 Many transcription regulators in eukaryotes can act even when they are
bound to DNA thousands of nucleotide pairs away from the promoter
they influence.
7–35 The close-packed arrangement of bacterial genes and genetic switches
may have developed from more extended forms of switches in response
to the evolutionary pressure to maintain a small genome.
THOUGHT PROBLEMS
7–36 The genes encoding the enzymes for arginine biosynthesis are located
at several positions around the genome of E. coli. The ArgR transcription
regulator coordinates their expression. The activity of ArgR is modulated
by arginine. Upon binding arginine, ArgR dramatically changes its affin-
ity for the cis-regulatory sequences in the promoters of the genes for the
arginine biosynthetic enzymes. Given that ArgR is a transcription repres-
sor, would you expect that ArgR would bind more tightly, or less tightly, to
the regulatory sequences when arginine is abundant? If ArgR functioned
instead as a transcription activator, would you expect the binding of argi-
nine to increase, or to decrease, its affinity for its regulatory sequences?
Explain your answers.
7–37 Bacterial cells can take up the amino acid tryptophan from their sur-
roundings, or, if the external supply is insufficient, they can synthesize
tryptophan from small molecules in the cell. The tryptophan repres-
sor inhibits transcription of the genes in the tryptophan operon, which
encodes the tryptophan biosynthetic enzymes. Upon binding trypto-
phan, the tryptophan repressor binds to a site in the promoter of the
operon.
A. Why is tryptophan-dependent binding to the operon a useful property
for the tryptophan repressor?
B. What would you expect to happen to the regulation of the tryptophan
biosynthetic enzymes in cells that express a mutant form of the trypto-
phan repressor that (i) cannot bind to DNA or (ii) binds to DNA even
when no tryptophan is bound to it?
C. What would happen in scenarios (i) and (ii) if the cell produced normal
tryptophan repressor from a second, unmutated copy of the gene?
7–38 In Figure 7–15, the bacterial activator protein CAP and the Lac repres-
sor have been placed in the four possible combinations on their binding
TRANSCRIPTION REGULATORS AS GENE SWITCHES 145
GLUCOSE LACTOSE OPERON ACTIVITY Figure 7–15 Arrangement of binding sites

and the four possible combinations of
transcription regulators on the promoter
for the Lac operon (Problem 7–38).
Lac repressor
CAP Lac repressor
CAP RNA polymerase
LacZ
sites in the promoter for the Lac operon. Each combination of transcrip-
tion regulators corresponds to the expected binding in a particular mix-
ture of glucose and lactose. For each of the four combinations, indicate
on the left-hand side of the figure which sugars are present and, on the
right-hand side, whether the operon is expected to be turned on or off.
7–39 Imagine that you have created a fusion between the Trp operon,
which encodes the enzymes for tryptophan biosynthesis, and the Lac
operon, which encodes the enzymes necessary for lactose utiliza-
tion (Figure 7–16). Under which set of conditions (A–F below) will
-galactosidase be expressed in the strain that carries the fused operon?
A. Only when lactose and glucose are both absent.
B. Only when lactose and glucose are both present.
C. Only when lactose is absent and glucose is present.
D. Only when lactose is present and glucose is absent.
E. Only when tryptophan is absent.
F. Only when tryptophan is present.
7–40 When cis-regulatory sequences were initially found to influence activ-
Figure 7-62
ity at distant promoters, two principal models were invoked to explain
this action at a distance. In the “DNA looping” model, direct interactions
Answerregulators
between transcription 7-50 bound at cis-regulatory sequences and
the distant promoters were proposed to stimulate RNA polymerase. In
the “scanning” model, RNA polymerase (or a transcription regulator)
was proposed to bind at the regulatory sequence and then slide along the
DNA until it reached the promoter. These two models were distinguished
using a cis-regulatory sequence on one piece of DNA and the β-globin
gene with its promoter on a separate piece of DNA (Figure 7–17). The
β-globin gene was not expressed from the mixture of pieces. However, Figure 7–16 Separated (normal) and fused
when the two segments of DNA were joined via a protein linker, the Trp and Lac operons (Problem 7–39).
β-globin gene was expressed.
Trp Lac
regulatory Trp operon regulatory Lac operon
region region
E D C B A z y a
encodes
β-galactosidase
FUSION
Trp
regulatory Trp–Lac fusion operon
region
E D C z y a
encodes
β-galactosidase
biotin Figure 7–17 Stimulation of β-globin gene

expression by a cis-regulatory sequence
linked via a protein bridge (Problem
cis-regulatory β-globin
sequence 7–40). Each DNA molecule carries biotin
+ avidin
attached to one end, as shown. In the
presence of the protein avidin, the
two molecules are linked together and
transcription occurs, as shown by the
arrow above the β-globin gene.
cis-regulatory β-globin
sequence
How does this experiment distinguish between the DNA looping

model and the scanning model? Explain your answer.
7–41 Some transcription regulators bind to DNA and cause the double helix to
bend at a sharp angle. Such “bending proteins” can affect the initiation
of transcription without directly contacting any other protein. Can you
devise a plausible explanation for how such proteins might work to mod-
ulate transcription? Draw a diagram that illustrates your explanation.
7–42 The yeast Gal4 transcription activator comprises two domains: a DNA-
binding domain and an activation domain. The DNA-binding domain
allows Gal4 to bind to appropriate DNA sequences located near genes
that are required for metabolism of the sugar galactose. The activation
domain binds to components of the transcriptional machinery (includ-
ing RNA polymerase), attracting them to the promoter, so the regulated
genes can be turned on. In the absence of Gal4, the galactose genes can-
not be turned on. When Gal4 is expressed normally, the genes can be
maximally activated. When Gal4 is massively overexpressed, however,
the galactose genes are turned off. Why do you suppose that too much
Gal4 squelches expression of the galactose genes?
Figure 7-19
7–43 How are histone modification enzymes and chromatin remodeling com-
plexes recruited to unmodified chromatin, and how are they thought to
Problem 7-52
aid in the activation of transcription from previously silent genes?
7–44 How is it that protein–protein interactions that are too weak to cause pro-
teins to assemble in solution can nevertheless allow the same proteins to
assemble into complexes on DNA?
7–45 Consider the following argument: “If the expression of every gene
depends on a set of transcription regulators, then the expression of
these transcription regulators must also depend on the expression of
other transcription regulators, and their expression must depend on the
expression of still other transcription regulators, and so on. Cells would
therefore need an infinite number of genes, most of which would code
for transcription regulators.” How does the cell get by without having to
achieve the impossible?
DATA HANDLING
7–46 E. coli proliferates faster on the monosaccharide glucose than it does on
the disaccharide lactose for two reasons: (1) lactose is taken up more
slowly than glucose and (2) lactose must be hydrolyzed to glucose and
galactose (by -galactosidase) before it can be further metabolized.
When E. coli is grown on a medium containing a mixture of glucose
and lactose, it proliferates with complex kinetics (Figure 7–18, squares).
The bacteria proliferate faster at the beginning than at the end, and
there is a lag between these two phases when they virtually stop divid-
ing. Assays of the concentrations of the two sugars in the medium show
TRANSCRIPTION REGULATORS AS GENE SWITCHES 147
Figure 7–18 Proliferation of E. coli

β-galactosidase on a mixture of glucose and lactose
(Problem 7–46).
108 10
β−galactosidase (arbitrary units)

glucose concentration (mM)
glucose
bacteria per mL
bacteria
107 5
106 0
0 100 200
time (minutes)
that glucose falls to very low levels after a few cell doublings (Figure 7–18,
circles), but lactose remains high until near the end of the experimental
time course (not shown). Although the concentration of lactose is high
throughout most of the experiment, -galactosidase, which is regulated
as part of the Lac operon, is not induced until more than 100 minutes
have passed (Figure 7–18, triangles).
A. Explain the kinetics of bacterial proliferation during the experiment.
Account for the rapid initial rate, the slower final rate, and the delay in
the middle of the experiment.
B. Explain why the Lac operon is not induced by lactose during the rapid
initial phase of bacterial proliferation.
7–47 Transcription of the bacterial gene encoding the enzyme glutamine
Figure
synthetase is regulated by the 7-21of nitrogen in the cell. The key
availability
transcription regulator is the NtrC protein, which stimulates transcrip-
tion when it is phosphorylated. Transcription
Problem 7-60 of the gene for glutamine
synthetase can be achieved in vitro by adding RNA polymerase, a special
sigma factor, and phosphorylated NtrC. Although NtrC binds regardless
of its phosphorylation state and RNA polymerase binds strongly to the (A) NtrC- glutamine synthetase
promoter in the absence of NtrC, transcription is absolutely dependent binding sites gene
on phosphorylated NtrC.
Activation of transcription by NtrC was characterized using three dif-
ferent templates: the normal gene with five regulatory sequences, a gene promoter
with all of the NtrC-binding sites deleted, and a gene with three NtrC-
binding sites at its 3 end (Figure 7–19). For half-maximal rates of tran- (B) glutamine synthetase
gene
scription, the normal gene (Figure 7–19A) required 5 nM NtrC, the gene
with 3 NtrC-binding sites (Figure 7–19C) required 10 nM NtrC, and the
gene without NtrC-binding sites (Figure 7–19B) required 50 nM NtrC.
promoter
A. If RNA polymerase can bind to the promoter of the glutamine synthetase
gene in the absence of NtrC, why do you suppose NtrC is needed to acti-
(C) glutamine synthetase NtrC-
vate transcription? gene binding sites
B. If NtrC can bind to its binding sites regardless of its state of phospho-
rylation, why do you suppose phosphorylation is necessary for transcrip-
tion? promoter
C. If NtrC can activate transcription even when its binding sites are absent,
what role do the binding sites play?
Figure 7–19 Three templates for studying
7–48 Coactivators do not bind to DNA, but instead serve as bridging mol- the role of NtrC in transcription of the
ecules that link transcription activators to general transcription factors glutamine synthetase gene (Problem
at the promoter. The SAGA complex in yeast is a large multiprotein com- 7–47). (A) The normal gene with intact
upstream regulatory sequences. (B) A
plex that is required for transcription of many genes. SAGA contains a gene with all of the NtrC-binding sites
variety of proteins, including a histone acetyl transferase and a subset of deleted. (C) A gene with three NtrC-
TATA-binding protein associated factors (TAFs). If SAGA functions as a binding sites at the 3 end of the gene.
(A) MAP OF THE Gal1-Gal10 LOCUS Figure 7–20 Analysis of SAGA

association with the Gal promoter
Gal10 Gal1 (Problem 7–48). (A) Organization of
UAS
the Gal10 and Gal1 genes. A common
UAS serves both promoters, which
A B C D E F G are indicated by green boxes. The
positions of the PCR fragments that
(B) PCR PRODUCTS FROM CHROMATIN IMMUNOPRECIPITATES were used for quantification are shown
by A–G below. (B) Results of chromatin
Spt3 Spt20
immunoprecipitation using antibodies
6
against Spt3 or Spt20. The strains
8 were grown in medium containing
raffinose (Raf) or galactose (Gal),
4 6 their chromatin was fragmented and
Gal/Raf
Gal/Raf
immunoprecipitated, and the DNA was
4 amplified by quantitative PCR. The PCR
2 products from strains grown on raffinose
2 were loaded onto the gels and run for a
few minutes before the PCR products
0 0 from strains grown on galactose, so that
IN A B C D E F G IN A B C D E F G
the products could be compared within
Gal the same lanes. Finally, the amounts
Raf of PCR products were measured,
normalized to the input chromatin before
immunoprecipitation (IN), and the ratio
(Gal/Raf) was calculated. About 50- to
100-fold less input chromatin (IN) was
loaded per lane than immunoprecipitated
coactivator, it should be physically present at the promoters it regulates chromatin, reflecting the inefficiency of
chromatin immunoprecipitation.
under appropriate conditions.
To test this idea, you focus on the regulation of the Gal1 and Gal10
genes by the Gal4 activator, which binds to cis-regulatory sequences
(termed UAS) adjacent to their promoters (Figure 7–20A, green boxes).
When cells are grown on galactose, the Gal1 and Gal10 genes are tran-
scribed; when the cells are grown on the sugar raffinose, the genes are
not transcribed. Figure 7-27
To determine whether the SAGA complex associates with Gal4 on the
chromosome, you use the technique
Problem 7-66 of chromatin immunoprecipita-
tion. You shear the chromatin to small pieces and precipitate those that
are bound to the SAGA complex, using antibodies against two compo-
nents of the complex—Spt3 and Spt20. You strip the precipitated chro-
matin of protein and analyze the DNA by quantitative PCR to measure
the amounts of specific DNA segments in the precipitates. The ratio of
precipitated DNA sequences from galactose-grown cells to those from
raffinose-grown cells (Gal/Raf ) is shown for several segments around the
galactose promoter in Figure 7–20B.
A. Which of the tested fragments would you have expected to be enriched if
SAGA behaved as a coactivator? Explain your answer.
B. Does SAGA meet the criteria for a coactivator? Is it physically present at
the promoter under appropriate conditions?
7–49 Genes expressed at the yeast mating type (Mat) locus on chromosome
III determine the mating type of a haploid yeast cell. Identical mating-
type genes also exist at two other loci—Hml and Hmr—on the same chro-
mosome (Figure 7–21A). At these two silent loci, the mating-type genes
are not expressed, even though all the signals for expression are present.
Each silent mating-type locus is bracketed by two short DNA sequences,
designated E and I, which bind a variety of proteins and serve to establish
and maintain repression of genes within each locus.
To investigate the function of these elements, you insert the Ura3
gene at different locations between E and I and outside the Hml locus,
as shown in Figure 7–21B. You then test the growth of these strains in
complete medium, in the absence of uracil (–uracil), and in the presence
of 5-fluoroorotic acid (+FOA) (Figure 7–21B). These growth conditions
MOLECULAR GENETIC MECHANISMS THAT CREATE AND MAINTAIN SPECIALIZED CELL TYPES 149
(A) YEAST SILENT MATING-TYPE LOCI
α2 α1 centromere a2 a1
E Hml I E Hmr I
3 kb
(B) INSERTION OF Ura3 GENES NEAR Hml

complete medium – uracil + FOA
A B C D E F
0 E Hml I 0
1 Ura3 1
2 Ura3 2
Ura3
3 3
4 Ura3 4
5 5
Ura3
6 Ura3 6
7 7
Ura3
8 Ura3 8
Ura3
9 9
10 Ura3 10
test for the activity of the Ura3 gene: in complete medium, expression of Figure 7–21 Effects of E and I elements
the Ura3 protein is irrelevant; in the absence of uracil, expression of Ura3 at Hml on the expression of the Ura3
gene inserted nearby (Problem 7–49).
is required for growth; and in the presence of FOA, expression of Ura3 is (A) Arrangement of E and I elements
lethal to the cell. around Hml and Hmr. The mating-type
Do these control elements specifically repress the mating-type genes, genes are shown as 1, 2, a1, and a2.
or do they act like insulators that control the expression of any gene (B) Locations of the Ura3 gene inserted
around the Hml locus. Orientations
placed between them?
of the Ura3 gene are indicated by the
direction of lettering. Growth phenotypes
of each strain are indicated in the panel
MOLECULAR GENETIC MECHANISMS THAT CREATE
Figure 7-31 on the right. Strain 0 is the parental
AND MAINTAIN SPECIALIZED CELL TYPES strain with no Ura3 gene; strains 1 to
10 have the Ura3 gene inserted as
TERMS TO LEARN Problem 7-68 indicated. Serial dilutions of each strain
were spotted on agar plates (with the
cell memory induced pluripotent stem (iPS) cell highest concentration on the left). The
agar contained complete medium,
complete medium minus uracil (–uracil),
DEFINITIONS or complete medium plus FOA (+FOA).
White areas indicate growth.
7–50 Cell derived from a fibroblast, by artificial expression of specific tran-
scription regulators, that looks and behaves like an embryonic stem cell.
7–51 The property that allows a proliferating cell to maintain its identity
through subsequent cell divisions.
TRUE/FALSE
7–52 In most organisms, extracellular molecules that bind to receptors on
the cell surface communicate the positional cues that allow cells in the
embryo to differentiate into appropriate cell types.
7–53 The fibroblasts that are converted to muscle cells by expression of the
transcription regulator MyoD have probably already accumulated a
number of transcription regulators that can cooperate with MyoD to
switch on muscle-specific genes.
7–54 Once cells have differentiated to their final specialized forms, they never (A) PROPHAGE STATE
again alter expression of their genes.
THOUGHT PROBLEMS cI gene Cro gene
7–55 Bacteriophage lambda can replicate as a prophage or lytically. In the NO Cro GENE
prophage state, the viral DNA is integrated into the bacterial chromo- TRANSCRIPTION
some and is copied once per cell division. In the lytic state, the viral DNA (B) LYTIC STATE
is released from the chromosome and replicates many times. This viral
DNA then produces viral coat proteins that enclose the replicated viral
genomes to form many new virus particles, which are released when the
cI gene Cro gene
bacterial cell bursts.
These two states are controlled by the transcription regulators cI
NO cI GENE
and Cro, which are encoded by the virus. In the prophage state, cI is TRANSCRIPTION
expressed; in the lytic state, Cro is expressed. In addition to regulating
the expression of other genes, cI is a repressor of transcription of the
Figure 7–22 Regulation of bacteriophage
gene that encodes Cro, and Cro is a repressor of the gene that encodes cI
lambda replication by cI and Cro (Problem
(Figure 7–22). 7–55). (A) The prophage state. (B) The
When bacteria containing a lambda prophage are briefly irradiated lytic state.
with ultraviolet (UV) light, cI protein is degraded.
A. What will happen next?
B. Will the change in question A be reversed when the UV light is switched
off?
C. How is this mechanism beneficial to the virus?
7–56 Imagine the two situations shown in Figure 7–23. In cell 1, a transient
signal induces the synthesis of protein A, which is a transcription activa-
tor that turns on many genes including its own. In cell 2, a transient sig-
nal induces the synthesis of protein R, which is a transcription repressor
that turns off many genes including its own. In which, if either, of these
Figure 7-32
situations will the descendants of the original cell “remember” that the
progenitor cell had experienced the transient signal? Explain your rea-
soning. Problem 7-78
7–57 Figure 7–24 shows a simple scheme by which three transcription regu-
lators might be used during development to create eight different cell
types. How many cell types could you create, using the same rules, with
four different transcription regulators? MyoD is a transcription regula-
tor that can trigger the entire program of muscle differentiation when
expressed in fibroblasts. How might you accomodate this observation
into the scheme shown in Figure 7–24?
DATA HANDLING
7–58 The protein encoded by the Even-skipped (Eve) gene of Drosophila is a
transcription regulator required for proper segmentation in the middle
(A) CELL 1
A A
transient
OFF signal A
A A A
transcription activator turns on transcription activator protein
of activator mRNA turns on its own
transcription
(B) CELL 2
transient R R Figure 7–23 Gene regulatory circuits and
OFF signal R cell memory (Problem 7–56). (A) Induction
R R R of synthesis of transcription activator
transcription repressor turns on transcription repressor protein A by a transient signal. (B) Induction of
of repressor mRNA turns off its own synthesis of transcription repressor R by
transcription a transient signal.
Figure 7–24 An illustration of

combinatorial gene control for
development (Problem 7–57). In this
simple, idealized scheme, a “decision”
INDUCE to make a new transcription regulator
TRANSCRIPTION (numbered symbols) is made after
REGULATOR 1 each cell division. In this scheme, the
daughter cell on the right is induced to
make the new transcription regulator.
LEFT 1 RIGHT Each transcription regulator is assumed
to continue to be expressed after it
INDUCE is induced, thereby allowing different
TRANSCRIPTION combinations of regulatory proteins to be
REGULATOR 2 built up. In this example, eight different
cell types have been created.
1
2 1
2
INDUCE
TRANSCRIPTION
REGULATOR 3
2 1 1 1
3 2 1 3
3 3 2 2
A B C D E F G H
of the body. It first appears about 2 hours after fertilization at a uniform

level in all the embryonic nuclei. Not long after that, it forms a pattern
of seven stripes across the embryo. Each stripe is under the control of a
separate module in the Eve promoter, which provides binding sites for
both repressors and activators of Eve transcription. In the case of stripe
2, there are multiple, overlapping binding sites for activators and repres-
sors (Figure 7–25). Two transcription activators, Hunchback (Hb) and
Bicoid (Bcd), and two transcription repressors, Giant (Gt) and Krüp-
pel (Kr), are required toFigure
give the 7-34
normal pattern. The binding sites for
these proteins have been mapped onto the 670-nucleotide segment
shown in Figure 7–25; deletion this upstream segment abolishes Eve
Problemof 7-81
expression in stripe 2. The patterns of expression of Hunchback, Bicoid,
Giant, and Krüppel in the embryo are shown in Figure 7–26. It seems that
Eve expression in stripe 2 occurs only in the region of the embryo that
expresses both transcription activators, but neither transcription repres-
sor: a simple enough rule.
To check if this rule is correct, you construct a β-galactosidase reporter
gene driven by a 5 kb upstream segment from the Eve promoter. (This
segment also includes the controlling elements for stripes 3 and 7.) In
addition to the normal upstream element, you make three mutant ver-
sions in which several of the binding sites in the Eve stripe 2 control seg-
ment have been deleted.
Construct 1. Deletion of all the Krüppel-binding sites
Construct 2. Deletion of all the Giant-binding sites
Construct 3. Deletion of two Bicoid-binding sites (indicated by in
Figure 7–25)
Eve stripe 2
control
element
β-galactosidase gene
Figure 7–25 Binding sites for transcription

repressors and activators of Eve stripe 2
REPRESSORS (Problem 7–58). The Eve stripe 2 control
Kr Kr Gt Gt Kr Gt Kr Kr Kr
segment, with additional DNA upstream
of the Eve promoter, was fused to the
Bcd Bcd Bcd Hb Bcd Hb Hb β-galactosidase gene, whose product can
ACTIVATORS Δ Δ be readily visualized in the embryo.
Eve
stripe 2
expression levels
Hunchback (Hb)
Giant Krüppel
(Gt) (Kr)
Bicoid (Bcd)
anterior posterior
pole pole
Figure 7–26 Expression of transcription repressors and

activators of Eve stripe 2 in the Drosophila embryo
(Problem 7–58).
You make flies containing these novel genetic constructs integrated

into their chromosomes and determine the patterns of -galactosidase
expression in their embryos, which are shown in Figure 7–27.
A. Match the mutant embryos to the mutant constructs.
B. You began these experiments to test the simple rule that Eve expres-
sion in stripe 2 occurs in the embryo where the two transcription activa-
tors are present and the two transcription repressors are absent. Do the
results with the mutant embryos confirm this rule? Explain your answer.
C. Offer a plausible explanation for why there is no expression of
-galactosidase at the anterior pole of mutant embryo D in Figure 7–27.
D. In the Eve stripe 2 control segment, the binding sites for the two tran-
scription activators do not overlap, nor do the binding sites for the two
transcription repressors; however, it is often the case that binding sites
for activators overlap binding sites for repressors. What does this overlap
suggest about the mode of genetic control of Eve in stripe 2, and what
might be the consequences for stripe morphology?
7–59 Hormone receptors for glucocorticoids, upon hormone binding, become
Figure 7-29
transcription regulators that activate a specific set of responsive genes.
The DNA- and hormone-binding sites occupy distinct regions of the
Problem receptor.
C-terminal half of the glucocorticoid 7-67 Hormone binding could stripe numbers
generate a functional DNA-binding protein either by altering receptor 2 3 7
(A) NORMAL
conformation to create a DNA-binding domain or by altering the confor-
mation to uncover a preexisting DNA-binding domain.
These possibilities were investigated by comparing the activities of
a series of C-terminal deletions (Figure 7–28). Segments of the cDNA
that encode N-terminal portions of the receptor were expressed in cells, (B) MUTANT
and their ability to activate a chloramphenicol acetyl transferase (Cat)
gene—linked to a glucocorticoid-responsive cis-regulatory sequence—
was measured. As shown in Figure 7–28, the full-length receptor (with its
C-terminus at position 0) displayed CAT activity only in the presence of (C) MUTANT
the glucocorticoid dexamethasone. Most of the mutant receptors failed
to activate CAT expression in the presence or absence of dexametha-
sone. In contrast, the four mutant receptors lacking 190, 200, 239, and 270
C-terminal amino acids activated CAT expression in the presence and (D) MUTANT
absence of dexamethasone.
How do these experiments distinguish between the proposed models
for hormone-dependent conversion of the normal receptor to a DNA-
binding form? Does hormone binding create a DNA-binding site or does anterior posterior
it uncover a preexisting one?
Figure 7–27 Embryonic expression of
7–60 One of the key transcription regulators produced by the yeast mating-type -galactosidase from constructs with
locus is a repressor protein known as 2. In haploid cells of the mating normal or mutated Eve stripe 2 control
type, 2 is essential for turning off a set of genes that are specific for the elements (Problem 7–58).
position of C-terminus in mutant receptors Figure 7–28 Effect of C-terminal deletions

on the activity of the glucocorticoid
receptor (Problem 7–59). The schematic
277
0
9
0
0
0
3
1
33
28
23
20
19
18
12
10
27
0
glucocorticoid receptor diagram at the top illustrates the
position of the DNA-binding site (DNA)
N-terminus DNA dexamethasone
and the glucocorticoid-binding site
(dexamethasone) in the receptor, as
well as the positions of the C-terminal
CAT ASSAY RESULTS
deletions. The lower diagram shows
3-acetylchloramphenicol the results of a standard CAT
1-acetylchloramphenicol assay. The lowest spot is unreacted
chloramphenicol; the upper spots
chloramphenicol show the attachment of acetyl groups
to one or the other of two positions
dexamethasone – + – + – + – + – + – + – + – + – + – + – +
331 287 270 239 200 190 180 123 101 27 0 on chloramphenicol. The presence (+)
or absence (–) of dexamethasone is
missing C-terminal amino acids
indicated below appropriate lanes.
a mating type. In a/ diploid cells, the 2 repressor collaborates with the

product of the a1 gene to turn off a set of haploid-specific genes in addi-
tion to the a mating-type-specific genes. Two distinct but related types of
conserved DNA sequences are found upstream of these two sets of regu-
lated genes: one in front of the a-specific genes and the other in front
of the haploid-specific genes. Given the relatedness of these upstream
sequences, it is most likely that 2 binds to both; however, its binding
properties must be modified in some way by the a1 protein before it can
recognize the haploid-specific sequence. You wish to understand the
nature of this modification. Does a1 catalyze covalent modification of 2,
or does it modify 2 by binding to it stoichiometrically?
To study these questions, you perform three types of experiment. In
the first, you measure the binding of a1 and 2, alone and together, to the
Figure
two kinds of upstream regulatory DNA 7-25
sites. As shown in Figure 7–29,
a1 alone does not bind radiolabeled DNA fragments that contain either
regulatory site (Figure 7–29,Problem 7-64 2 binds to a-specific frag-
lane 2), whereas
ments, but not to haploid-specific fragments (lane 3). The mixture of a1
and 2 binds to a-specific and haploid-specific fragments (lane 4).
In the second set of experiments, you add a vast excess of unlabeled
DNA containing the a-specific sequence, along with the mixture of
a1 and 2 proteins. Under these conditions, the haploid-specific frag-
ment is still bound (Figure 7–29, lane 5). Similarly, if you add an excess
of unlabeled haploid-specific DNA, the a-specific fragment is still bound
(lane 6).
In the third set of experiments, you vary the ratio of a1 relative to
2. When 2 is in excess, binding to the haploid-specific fragment is
decreased (Figure 7–29, lane 7); when a1 is in excess, binding to the
a-specific fragment is decreased (lane 8).
Figure 7–29 Binding of transcription

α2 − − + + + + + + regulators to fragments of DNA
containing the a-specific or haploid-
a1 − + − + + + low high
specific cis-regulatory sequences
unlabeled a-specific DNA − − − − + − − − (Problem 7–60). Various combinations
unlabeled haploid-specific DNA − − − − − + − − of regulators were incubated with a
mixture of a-specific and haploid-specific
radioactive DNA fragments (shown in
lane 1). At the end of the incubation, the
haploid-specific samples were precipitated with antibody
against the proteins, and the DNA
a-specific
fragments in the precipitate were run on
the gel. The gel was then placed against
x-ray film to expose the positions of the
1 2 3 4 5 6 7 8 radioactive DNA fragments.
A. In the presence of a1, is 2 present in two forms with different binding

specificities, or in one form that can bind to both regulatory sequences?
How do your experiments distinguish between these alternatives?
B. An 2 repressor with a small deletion in its DNA-binding domain does
not bind to DNA fragments containing the haploid-specific sequence.
When this mutant protein is expressed in a diploid cell along with nor-
mal 2 and a1 proteins, the haploid-specific genes are turned on. (These
genes are normally off in a diploid.) In the light of this result and your
other experiments, do you consider it more likely that a1 catalyzes a cova-
lent modification of 2, or that a1 modifies 2 by binding to it to form an
a1– 2 complex?
MECHANISMS THAT REINFORCE CELL MEMORY

IN PLANTS AND ANIMALS
TERMS TO LEARN
CG island genomic imprinting
DNA methylase monoallelic gene expression
DNA methylation X-inactivation
epigenetic inheritance X-inactivation center (XIC)
DEFINITIONS
7–61 Transcriptional silencing of gene expression on one of the two X chromo-
somes in the somatic cells of female mammals.
7–62 Addition of a –CH3 group to cytosines in CG sequences in vertebrate
DNA.
7–63 Heritable difference in the phenotype of a cell or an organism that does
not result from changes in the nucleotide sequence of the DNA.
7–64 Expression from only one of the two copies of a gene in the diploid
genome.
7–65 Situation in which one copy of a gene is either expressed or not expressed
in the embryo, depending on which parent it is inherited from.
7–66 Long region of DNA with a much greater than average density of CG
sequences, which usually remain unmethylated.
TRUE/FALSE
7–67 Because CG sequences in one strand are paired with GC sequences in
the other strand, two different methyl transferases are required to place
methyl groups on the two strands.
7–68 CG islands are thought to have arisen during evolution because they
were associated with portions of the genome that remained unmethyl-
ated in the germ line.
7–69 In most differentiated tissues, daughter cells retain a memory of gene
expression patterns that were present in the parent cell through mech-
anisms that do not involve changes in the sequence of their genomic
DNA.
MECHANISMS THAT REINFORCE CELL MEMORY IN PLANTS AND ANIMALS 155
(A) Figure 7–30 Pedigrees reflecting maternal

and paternal imprinting (Problem 7–71).
In one pedigree, the gene is paternally
imprinted; in the other, it is maternally
imprinted. In generations 3 and 4, only
one of the two parents in the indicated
matings is shown; the other parent
is a normal individual from outside
this pedigree. Affected individuals are
represented by red circles for females
and red squares for males. Dotted
(B)
yellow symbols indicate individuals that
carry the deletion but do not display the
phenotype.
THOUGHT PROBLEMS
7–70 Maintenance methyl transferase, de novo DNA methyl transferases, and
demethylating enzymes play crucial roles in the changes in methylation
patterns during development. Starting with the unfertilized egg, describe
in a general way how these enzymes bring about the observed changes in
genomic DNA methylation.
7–71 Examine the two pedigrees shown in Figure 7–30. One results from
deletion of a maternallyFigure
imprinted7-35
autosomal gene. The other pedigree
results from deletion of a paternally imprinted autosomal gene. In both
Problem(red
pedigrees, affected individuals 7-83
symbols) are heterozygous for the
deletion. These individuals are affected because one copy of the chromo-
some carries an imprinted, inactive gene, while the other carries a dele-
tion of the gene. Dotted yellow symbols indicate individuals that carry
the deleted locus, but do not display the mutant phenotype. Which pedi-
gree is based on paternal imprinting and which on maternal imprinting?
Explain your answer.
7–72 Imprinting occurs only in mammals, and why it should exist at all is a
mystery. One idea is that it represents an evolutionary end point in a tug
of war between the sexes. In most mammalian species, a female can mate
with multiple males, generating multiple embryos with different fathers.
If one father could cause more rapid growth of his embryo, it would pros-
per at the expense of the other embryos. While this would be good for the
father’s genes (in an evolutionary sense), it would drain the resources of
the mother, potentially putting her life at risk (not good for her genes).
Thus, it is in the mother’s interest to counter these paternal effects with
maternal changes that limit the growth of the embryo.
Based on this scenario, decide whether the Igf 2 gene, whose prod-
uct—insulin-like growth factor-2—is required for prenatal growth, is
more likely to be imprinted in the male or in the female.
DATA HANDLING
7–73 You are studying the role of DNA methylation in the control of gene
expression, using the human -globin gene as a test system (Figure
7–31). Globin mRNA can be detected when this gene is integrated into
the genome of mouse fibroblasts, even though it is expressed at much
lower levels than in red blood cells. If the gene is methylated at all 27 of
its CG sites before integration, its expression is blocked completely. You
are using this system to decide whether a single critical methylation site
is sufficient to determine globin expression.
exons 1 & 2 exon 3

transcription
unmethylated methylated
A 0
B 0
C 100
D 0
E 0
F 10
transcript
11 12 13 14 15 16
CG sequences around
promoter
Figure 7–31 Effects of methylation on transcription of the -globin gene (Problem

7–73). The methylated segments of the gene are shown in blue; unmethylated
segments are shown in red. The six CG sites around the promoter are shown in
more detail below the constructs. The level of expression of -globin RNA from
each construct is shown on the right as a percentage of the expression from a
fully unmethylated construct.
You create several different -globin constructs that are unmethylated

in specific regions of the gene. These constructs are illustrated in Figure
7–31, with the methylated regions shown in blue. The arrangement of six
Figure
methylation sites around the7-40
promoter is shown below the constructs.
Sites 11, 12, and 13 are unmethylated in construct E; sites 14, 15, and 16
are unmethylated Problem
in construct D; and all six sites are unmethylated in
7-91
construct F. You integrate these constructs in mouse fibroblasts, grow cell GENOTYPE
lines containing individual constructs, verify their methylation patterns,
XX X
X Y
XX
XX
Y
XO
and measure -globin RNA synthesis relative to cell lines containing the
XX
XY
XX
XX
a
X
i
fully unmethylated construct (Figure 7–31).
cDNA
Does the -globin transcription associated with the cell lines contain-
ing the constructs (Figure 7–31) indicate that a single critical site of meth- C
ylation determines whether the gene is expressed? How can you tell?
7–74 In female mammals, one X chromosome in each cell is chosen at random
A
for inactivation early in development. X-inactivation, which involves
more than 1000 genes in humans, is crucial for equalizing expression
of X-chromosome genes in males and females. A critical clue to the B
mechanism of X-inactivation came from the isolation of a large num-
ber of cDNAs for genes on the human X chromosome. Their expression
patterns were characterized in cells from normal males and females, in normal abnormal hybrid
cells from individuals with abnormal numbers of X chromosomes, and in cells cells cells
rodent:human hybrid cell lines that retained either one inactive human
X chromosome (Xi) or one active human X chromosome (Xa). Among all Figure 7–32 Northern analysis of gene
expression from cells with different
these cDNAs, there were three patterns of expression, as illustrated by numbers and types of X chromosomes
cDNAs A, B, and C in Figure 7–32. (Problem 7–74). RNA from cells was run
A. For each pattern of expression, decide whether the gene is expressed out on gels, blotted onto nitrocellulose,
from the active X, the inactive X, or both. Which pattern do you expect to probed with a mixture of radioactive
be the most common? Which pattern is the most surprising? cDNAs A, B, and C, and visualized by
autoradiography. The positions of the
B. From the results with cells from abnormal individuals, formulate a rule RNA bands that correspond to genes A,
as to how many chromosomes are inactivated, and how many remain B, and C were determined in a separate
active during X-inactivation. experiment.
Figure 7-42
POST-TRANSCRIPTIONAL CONTROLS 157
POST-TRANSCRIPTIONAL CONTROLS
TERMS TO LEARN
alternative RNA splicing regulated nuclear transport
internal ribosome entry site (IRES) RNA editing
post-transcriptional control
DEFINITIONS
7–75 Production of a functional RNA by insertion or alteration of individual
nucleotides in an RNA transcript after it has been synthesized.
7–76 A way to generate different proteins from the same gene by combining
different segments of the initial RNA transcript to make distinct mRNAs.
7–77 General term for a regulatory event that occurs after RNA polymerase has
bound to the gene’s promoter and begun RNA synthesis.
7–78 A sequence in the interior of an mRNA that folds into structures that bind
translation initiation proteins.
TRUE/FALSE
7–79 If the site of transcript cleavage and poly-A addition for a particular RNA
in one cell is downstream of the site used for cleavage and poly-A addi-
tion for the same RNA in a second cell, the protein produced from the
longer polyadenylated RNA will necessarily contain additional amino
acids at its C-terminus.
7–80 In one extreme case, a single gene in Drosophila—the Dscam gene—has
the potential to produce more than 38,000 different proteins by alterna-
tive splicing; thus, the complexity of this one gene rivals the complexity
of the whole human genome.
THOUGHT PROBLEMS
7–81 RNA polymerase II commonly terminates transcription of the HIV (the
human AIDS virus) genome a few hundred nucleotides after it begins,
unless helped along by a virus-encoded protein called Tat, which binds
to a specific hairpin structure in the nascent viral RNA. Tat then recruits
a collection of proteins, including the protein kinase Cdk9, which phos-
phorylates RNA polymerase, enhancing its ability to continue transcrip-
tion. Flavopiridol is the most potent inhibitor of Cdk9 yet discovered; it
blocks Cdk9-mediated phosphorylation. Would you expect flavopiridol
to interfere with HIV transcription? Why or why not?
7–82 Several distinct mechanisms for mRNA localization have been discov-
ered. They all require specific sequences in the mRNA itself, usually in
the 3 untranslated region (UTR). Briefly outline three mechanisms by
which cellular mRNAs might become localized in the cell.
7–83 Regulation of ferritin translation is controlled by the interaction between
a hairpin structure in the mRNA, termed an iron-response element
(IRE), and the iron-response protein (IRP) that binds to it. When the IRE
is bound by the IRP, translation is inhibited. The location of the IRE in the
mRNA is critical for its function. To work properly, it must be positioned
near the 5 end of the mRNA. If it is moved more than 60 nucleotides
downstream of the cap structure, the IRE no longer inhibits translation.
Why do you suppose there is such a critical position-dependence for reg-

ulation of translation by IRE and IRP?
7–84 Although essential for the cell, iron is also potentially toxic. It is main-
tained at optimal levels in mammalian cells by the actions of three pro-
teins. Transferrin binds to extracellular iron ions and delivers them to
cells; the transferrin receptor binds iron-loaded transferrin and brings it
into the cell; and ferritin binds iron (up to 4500 atoms in the internal cav-
ity in each complex) to provide an intracellular storage site.
The regulation of the transferrin receptor, like that of ferritin (see
Problem 7–83), is accomplished by IREs and IRPs, and, in both cases,
iron binding to IRPs prevents their binding to IREs. Nevertheless, the
mechanism of transferrin-receptor regulation is quite different from that
of ferritin. The transferrin receptor mRNA contains five IREs that are all
located in the 3 untranslated region of the mRNA (instead of at the 5 end,
as in ferritin mRNA). In addition, binding of IRPs to these IREs increases
the translation of transferrin receptor (as opposed to a decrease for fer-
ritin).
A. Does this opposite regulation of ferritin and transferrin receptor in
response to iron levels make biological sense? Consider the conse-
quences of high and low iron levels.
B. In the presence of iron, the transferrin receptor mRNA is rapidly degraded;
in the absence of iron, it is stable. Can you suggest a mechanism for how
iron levels might be linked to the stability of transferrin receptor mRNA?
7–85 Vg1 mRNA encodes a member of the TGF family of growth factors,
which is important for mesoderm induction. In Xenopus, Vg1 mRNA
is localized to the vegetal pole of the oocyte. Translation of Vg1 mRNA
does not occur until a late stage, after localization is complete. Analysis
of the 3 untranslated region identified two elements: a localization ele-
ment and a UA-rich translation control element. Translational regulation
was shown to be independent of the poly-A tail. By contrast, translational
repression was abolished when an internal ribosome entry site (IRES)
sequence was inserted into the mRNA upstream of the coding sequence.
How do you suppose translation of Vg1 mRNA is controlled?
DATA HANDLING
7–86 A repeated hexanucleotide element, TGCATG, has been shown to regu-
late the tissue-specific splicing of the fibronectin alternative exon EIIIB.
You wonder if it might also regulate alternative splicing in the calcitonin/
CGRP gene (Figure 7–33). In thyroid cells, an mRNA containing exons
1, 2, 3, and 4 encodes calcitonin. In neuronal cells, an mRNA containing
exons 1, 2, 3, 5, and 6 encodes calcitonin gene-related peptide (CGRP).
When you examine the calcitonin/CGRP gene you find five copies
of a related repeat, GCATG, within 500 nucleotides of the exon-4 splice
site (Figure 7–33). To analyze their potential role in alternative splicing,
you make several calcitonin/CGRP constructs in which the repeats have
been altered singly or in combination. You transfect the constructs into
HeLa cells, which normally give the calcitonin splicing pattern, and into
F9 cells, which normally give the CGRP splicing pattern. You find that
no single mutation alters the pattern seen with wild type (not shown);
however, various combinations of the mutations have dramatic effects
(Figure 7–33).
A. Why do you suppose the most dramatic changes were seen in HeLa cells
rather than in F9 cells?
B. Is there a particular combination of GCATG repeats that is critical for
proper calcitonin mRNA production in HeLa cells? If so, what is it?
7–87 In humans, two closely related forms of apolipoprotein B (ApoB) are
found in blood as constituents of the plasma lipoproteins. ApoB48,
POST-TRANSCRIPTIONAL CONTROLS 159
1 kb HeLa F9 Figure 7–33 Effects of mutations in

cells cells GCATG repeats on alternative splicing
of calcitonin/CGRP pre-mRNA (Problem
calcitonin CGRP 7–86). In HeLa cells, exon 3 is spliced
poly A poly A to exon 4 to make calcitonin mRNA; in
1 2 3 4 5 6
F9 cells, exon 3 is spliced to exon 5 to
HeLa F9
make CGRP mRNA. The positions of
the GCATG repeats around exon 4 are
GCATG repeat 1 2 3 4 5 CALC CGRP CALC CGRP
indicated. In the various constructs, the
wild type + + + + + ++++ − − ++++ presence of a GCATG repeat is indicated
construct A − − − − − − ++++ − ++++ by a plus, and its absence by a minus.
construct B + − − − + ++++ − − ++++ Production of calcitonin- (CALC) and
CGRP-specific spliced RNA is indicated
construct C + − − − − − ++++ − ++++
relative to wild type as follows: ++++ =
construct D − − − − + + +++ − ++++
80–100%, +++ = 60–80%, ++ = 40–60%,
construct E − − − + + − ++++ − ++++ + = 20–40%, and – = 0–20%.
construct F + + + − − ++++ − ++ ++
which has a molecular mass of 48 kilodaltons, is synthesized by the intes-

tine and is a key component of chylomicrons, the large lipoprotein par-
ticles responsible for delivery of dietary triglycerides to adipose tissue
for storage. ApoB100, which has a molecular mass of 100 kilodaltons, is
synthesized in the liver for formation of the much smaller, very-low-den-
sity lipoprotein particles used in the distribution of triglycerides to meet
energy needs. A classic set of studies defined the surprising relationship
between these two proteins.
Sequences of cloned cDNA copies of the mRNAs from these two tis-
sues revealed a single difference: cDNAs from intestinal cells had a T, as
part of a stop codon, at a point where the cDNAs from liver cells had a C,
as part of a glutamine codon (Figure 7–34). To verify the differences in the
mRNAs and to search for corresponding differences in the genome, RNA
Figure 7-47
and DNA were isolated from intestinal and liver cells and then subjected
to PCR amplification, using oligonucleotides that flanked the region of
Problem
interest. The amplified 7-112from the four samples were tested
DNA segments
for the presence of the T or C by hybridization to oligonucleotides con-
taining either the liver cDNA sequence (oligo-Q) or the intestinal cDNA
sequence (oligo-STOP). The results are shown in Table 7–2.
Are the two forms of ApoB produced by transcriptional control from
two different genes, by a processing control of the RNA transcript from
a single gene, or by differential cleavage of the protein product from a
single gene? Explain your reasoning.
7–88 The level of β-tubulin gene expression is established in cells by an unu-
sual regulatory pathway, in which the intracellular concentration of free
tubulin dimers (composed of one -tubulin and one -tubulin subunit)
regulates the rate of new -tubulin synthesis. The initial evidence for
such an autoregulatory pathway came from studies with drugs that cause
assembly or disassembly of all cellular tubulin. For example, treatment liver M I Q F D
of cells with colchicine, which causes microtubule depolymerization into cDNA ATGATACAATTTGAT
ApoB mRNA
TABLE 7–2 Hybridization of specific oligonucleotides to the amplified
segments from liver and intestine RNA and DNA (Problem 7–87).
intestine ATGATATAATTTGAT
RNA DNA cDNA M I *
Intestine Liver Intestine Liver Figure 7–34 Location of the sequence

difference in cDNA clones from ApoB
Oligo-Q + – + + RNA isolated from liver and intestine
(Problem 7–87). The encoded amino acid
Oligo-STOP – + – – sequences, in the one-letter code, are
Hybridization is indicated by +; absence of hybridization is indicated by –. shown aligned with the cDNA sequences.
The asterisk indicates a stop codon.
Figure 7–35 Effects of mutations on the regulation of -tubulin mRNA

stability (Problem 7–88). The wild-type sequence for the first 12 M R E I regulation
nucleotides of the coding portion of the gene is shown at the top, and the --ATGAGGGAAATC-- +
first four amino acids beginning with methionine (M) are indicated above -----T---------- -
the codons. The nucleotide changes in the 12 mutants are shown below; -----C---------- +
only the altered nucleotides are indicated. Regulation of mRNA stability
------C--------- -
is shown on the right: + indicates wild-type response to changes in
intracellular tubulin concentration and – indicates no response to changes. -------A-------- +
Vertical lines mark the position of the first nucleotide in each codon. --------C------- -
---------G------ -
----------G----- +
tubulin dimers, represses -tubulin synthesis 10-fold. Autoregulation of -----TA--------- -
-tubulin synthesis by tubulin dimers is accomplished at the level of -----C-C-------- +
-tubulin mRNA stability. The first 12 nucleotides of the coding portion -----G--A------- -
of the mRNA were found to contain the site responsible for this autoregu- -----G---T------ -
latory control. -----C----G----- +
Since the critical segment of the mRNA involves a coding region, it
is not clear whether the regulation of mRNA stability results from the
interaction of tubulin dimers with the RNA or with the nascent protein.
Either interaction might plausibly trigger a nuclease that would destroy
the mRNA.
These two possibilities were tested by mutagenizing the regulatory
region on a cloned version of the gene. The mutant genes were then
expressed in cells, and the stability of their mRNAs was assayed in the
presence of excess free tubulin dimers. The results from a dozen mutants
that affect a short region of the mRNA are shown in Figure 7–35.
Does the regulation of -tubulin mRNA stability result from an inter-
action with the RNA or from an interaction with the encoded protein?
Explain your reasoning.
7–89 In nematodes, the choice between spermatogenesis and oogenesis in the
hermaphrodite germ line depends on translational regulation of the Tra2
and Fem3 genes, as shown in Figure 7–36. When they are expressed, Tra2
promotes oogenesis and Fem3 promotes spermatogenesis. Translation
of each gene’s mRNA is regulated by the binding of proteins to elements
within their 3 untranslated regions. In each case, the protein-bound
mRNA, although stable, is not efficiently translated and has a short poly-
A tail. In each case, the mRNA in its unbound form is translationally
active and has a long poly-A tail. How do you suppose that the lengths of Figure 7-49
the poly-A tails might affect the efficiency of translation of these mRNAs?
7–90 To investigate the molecular mechanism by which the Tra2 regulatory Problem 7-114
elements (TGEs) control translation, you inject Tra2 mRNAs into Xeno-
pus oocytes and follow their fate. You have shown that an oocyte protein
binds to the TGEs and inhibits translation of injected Tra2 mRNA. Now
you wish to determine how mRNAs with bound proteins come to have
short poly-A tails. To investigate this question, you prepare two kinds
(A) Tra2 mRNA (B) Fem3 mRNA

GLD1 GLD1 long poly A
short
Tra2 TGE TGE Fem3 PME
poly A
translation OFF sperm translation ON sperm
long poly A FBF1 NOS

short
Tra2 TGE TGE Fem3 PME
poly A
translation ON eggs translation OFF eggs
Figure 7–36 Translational control of the choice between spermatogenesis and oogenesis (Problem
7–89). (A) Tra2 mRNA. (B) Fem3 mRNA. Control elements within the 3 untranslated regions of the
two genes are shown, along with the specific proteins that bind to those elements.
REGULATION OF GENE EXPRESSION BY NONCODING RNAs 161
mRNA with TGEs mRNA without TGEs Figure 7–37 Injection of radiolabeled
number mRNAs with and without TGEs into
1 4
24
8
6
102
2
8
6
102
of cells Xenopus one-cell embryos (Problem
16
32
64
12
25
51
16
32
64
12
25
51
1
2
4
8
2
4
8
7–90). The lengths of the poly-A tails are
indicated on the left. Cell number refers
to the number of cells in the embryos
when they were harvested.
65
of poly A
length
of radiolabeled Tra2 mRNA: one with the normal pair of TGE elements,
and one with those elements deleted (see Figure 7–36A). Each RNA has
a tail of 65 A nucleotides. You inject these mRNAs into one-cell Xenopus
embryos and then re-isolate the mRNAs at various cell stages thereafter.
(A) LINEAR mRNA
The re-isolated radiolabeled RNA was analyzed by gel electrophoresis
and autoradiography (Figure 7–37). UAA IRES AUG
A. Do the TGEs alter the overall stability of the mRNAs; that is, are the
mRNAs destroyed more rapidly in the presence or absence of the TGEs? TRANSLATE
How can you tell?
20 kd protein
B. Do the TGEs influence the length of the poly-A tails? How can you tell?
7–91 You are skeptical that IRESs really allow direct binding of the eukaryotic (B) CIRCULAR mRNA
translation machinery to the interior of an mRNA. As a critical test of
IRES
this notion, you prepare a set of linear and circular RNA molecules, with
and without IRESs (Figure 7–38A and B). You translate these various
RNAs in rabbit reticulocyte lysates and display the translation products UAA AUG
by sodium dodecyl sulfate (SDS) gel Figure 7-53 (Figure 7–38C). Do
electrophoresis
these results support or refute the idea that IRESs allow ribosomes to ini-
Problem 7-117
tiate translation of mRNAs in a cap-independent fashion? Explain your TRANSLATE
answer.
23 kd protein
REGULATION OF GENE EXPRESSION BY

(C) TRANSLATION ASSAY
NONCODING RNAs linear + − − + −
TERMS TO LEARN circular − + − − +
IRES + + − − −
CRISPR piRNA (piwi-interacting RNA)
kd
crRNAs RNA interference (RNAi)
long noncoding RNA (lncRNA) small interfering RNA (siRNA)
microRNA (miRNA)
DEFINITIONS 23
20
7–92 Natural defense mechanism in many organisms that is directed against
foreign RNA molecules, especially those that occur in double-stranded
form. Figure 7–38 Analysis of effects of IRESs
on translation (Problem 7–91). (A) Linear
7–93 A class of short noncoding RNAs that regulate gene expression; roughly mRNA that contains an IRES. The
one-third of human genes are thought to be regulated in this way. structure of the mRNA without the IRES
was the same. (B) Circular mRNA that
7–94 Small RNAs that transcriptionally silence intact transposon genes and contains an IRES. The structureFigure
of the 7-55
destroy any mRNA produced by them. mRNA without the IRES was the same.
Circular RNAs were prepared by ligating
7–95 A defense mechanism in bacteria that allows them to destroy viral invad- Problem 7-119
the ends together; they were purified
ers they have seen before. from the linear starting molecules by gel
electrophoresis. (C) Display of translation
7–96 An RNA longer than 200 nucleotides that does not encode a protein. products from various species of mRNA.
mouse Figure 7–39 Conservation of piRNA

chromosome 17 clusters among mammalian species
(Problem 7–101). The figure is a
schematic depiction of the distribution of
rat piRNAs cloned from a conserved region
chromosome 20
of the mouse, rat, and human genomes.
Segments that are transcribed from the
human top strand of the DNA are shown in blue;
chromosome 6 segments transcribed from the bottom
strand are shown in red. The three
genomes are aligned according to this
pattern of transcription.
TRUE/FALSE
7–97 Because siRNAs are so widespread among species, they are believed to
be the most ancient form of RNA interference, with miRNAs being a later
refinement.
7–98 Although the functions of lncRNAs are still mysterious, it is now clear that
they do not function as scaffolds for binding groups of proteins. (A)
polysome distribution
absorbance 254 nm
7–99 piRNAs and crRNAs serve analogous functions; they defend against for- wild type
Let7
eign invaders.
46
2
THOUGHT PROBLEMS
7–100 List and briefly discuss three features that make miRNAs especially use-
ful regulators of gene expression. (B)
mRNA distribution
20
7–101 piRNAs have several general characteristics: they are roughly 29–30 Daf12
nucleotides in length, they have a strong bias for U in the first nucleotide, mRNA level (percent of total) 10
they bind to the Piwi class of Argonaute proteins, they are found mostly
in germ cells, and they are clustered in conserved regions of the mouse, 0
rat, and human genomes (Figure 7–39). These characteristics suggest
Figure 7-201 20
Act1
an important function, but the role of not a single piRNA has yet been
defined. Why do you suppose that many biologists are convinced that 10
Problem
piRNAs serve a critical function7-219
in organisms?
0
2 4 6 8 10 12
7–102 If you insert a β-galactosidase gene lacking its own transcription con- fraction number
trol region into a cluster of piRNA genes in Drosophila, you find that
-galactosidase expression from a normal copy elsewhere in the genome Figure 7–40 Polysome distributions
is strongly inhibited in the fly’s germ cells. If the inactive β-galactosidase
of mRNAs in larval extracts with or
without Let7 miRNA (Problem 7–103).
gene is inserted outside the piRNA gene cluster, the normal gene is prop-
erly expressed. What do you suppose is the basis for this observation? (A) Polysome distribution. mRNAs with
attached ribosomes (polyribosomes)
How would you test your hypothesis? were isolated from wild-type and
Let7 mutant
Figurelarvae7-202
and separated by
DATA HANDLING centrifugation through a sucrose gradient.
The distributions of all mRNAs in normal
7–103 miRNAs were discovered in nematode worms, but it is still unclear Problem
(wild type; blue) and7-221
Let7 mutant larvae
(red) are shown in the top panel. The
whether miRNAs reduce protein expression by causing rapid mRNA deg- monoribosomes are shown to the left of
radation or by interfering with translation. The Let7 miRNA, for example, the dashed line and the polyribosomes
recognizes sites in the 3 end of the Daf12 mRNA, reducing Daf12 protein are shown to the right, with a few
synthesis. To investigate how Let7 miRNA controls protein synthesis from individual peaks numbered to indicate
how many ribosomes are present in each
Daf12 mRNA, you make extracts of wild-type larvae at a stage when Let7 polysome peak. (B) mRNA distribution.
miRNA levels are highest, and also make extracts of mutant larvae that do The polysome distribution was divided
Adapted from Figure 1 of Ding, X.C. and Grosshans,
not make Let7 miRNA at the same stage. Analysis of the polyribosomes EMBO into
Journal (2009)
12 fractions, 28:213-222
as indicated by the
(or polysomes) in normal and mutant larvae revealed no significant gray lines. The RNA was extracted from
difference, nor was there any difference in the distribution of a control each fraction and the amounts of Daf12
and Act1 mRNAs in each fraction were
mRNA from the Act1 gene (Figure 7–40A). By contrast, the distribution quantified. The distributions of Daf12
of Daf12 mRNA changed significantly in the presence of Let7 miRNA mRNA and Act1 mRNA in normal and
(Figure 7–40B). Let7 mutant strains are shown below.
REGULATION OF GENE EXPRESSION BY NONCODING RNAs 163
B A B A B A B A B A B A Figure 7–41 Analysis of expression

mutant ES cells of X-chromosome-specific alleles in
undifferentiated undifferentiated and differentiated ES
cells (Problem 7–104). Analysis of the
A and B alleles from individual cells is
B A B A B A B A B A B A
normal ES cells
indicated by the brackets.
differentiated
B A B A B A B A B A B A
mutant ES cells
differentiated
A. Does Let7 miRNA cause degradation of Daf12 mRNA? How can you tell?
B. How does Let7 miRNA alter the distribution of Daf12 mRNA? Is Daf12
mRNA in smaller or larger polysomes in the presence of Let7 miRNA?
C. Propose a mechanism by which Let7 miRNA reduces synthesis of Daf12
protein.
7–104 To determine whether the Xist gene, which encodes the Xist lncRNA, is
required for X-inactivation in mice, scientists deleted the Xist gene on
one X chromosome. Using female embryonic stem (ES) cells in which
genes on the two X chromosomes could be distinguished due to poly-
morphisms, they followed X-inactivation during differentiation of ES
cells in culture. ES cells normally maintain both X chromosomes in the
active state; however, when they are induced to differentiate, they ran-
domly inactivate one.
The scientists considered three hypotheses. (1) ES cells mutant for
one Xist gene would fail to register the presence of two X chromosomes
and thus fail to undergo X-inactivation. (2) The Xist knockout would pre-
vent X-inactivation of the targeted X chromosome, thus predisposing the
normal X chromosome to preferential X-inactivation. (3) The mutation
Figure 7-43
would have no effect on X-inactivation at all.
Using allele-specific (X-chromosome-specific) oligonucleotide
probes, they were able to Problem
determine 7-94
which allele of an X-chromosome
gene was expressed in individual cells. As shown in Figure 7–41, they
examined mutant ES cells that were undifferentiated, and mutant and
nonmutant ES cells that had undergone differentiation. Only a few cells
were examined in Figure 7–41, but analysis of many more confirmed the
patterns shown. The A allele marks the X chromosome whose Xist gene is
intact in the mutant ES cells; the B allele marks the X chromosome from
which the Xist gene was deleted.
Which of the three hypotheses do these results support? Explain your
reasoning.
7–105 Using strand-specific probes of transcription in the X-inactivation center,
a second gene was found to be transcribed in the opposite direction to
Xist and was named Tsix to indicate its antisense orientation. The Tsix
transcript, like the Xist transcript, has no significant open reading frame
and is thought to be a functional lncRNA. As shown in Figure 7–42, the
Tsix transcript extends all the way across the Xist gene. To determine
whether Tsix plays a role in counting X chromosomes, in choosing
which one to inactivate, or in silencing the inactive X, female embryonic
stem (ES) cells were generated in which the promoter for Tsix had been
deleted from one X chromosome. When these ES cells were allowed to
Tsix
Xist promoter Figure 7–42 Arrangement of Xist and
Tsix transcripts in the X-inactivation
center (Problem 7–105). Boxes indicate
Tsix the exons of Xist; exons for Tsix are not
0 10 20 30 40 50 kb shown. The promoter deletion in the Tsix
mutant is indicated.
differentiate into females, it was found that the X chromosome with the
Tsix deletion was always inactivated.
A. Is Tsix important for the counting, choice, or silencing of X chromo-
somes? Explain your answer.
B. Before X-inactivation, Tsix is expressed from both alleles, as is Xist. At the
onset of X-inactivation, Tsix expression becomes confined to the future
active X, whereas Xist expression is restricted to the future inactive X. Can
you suggest some possible ways that Tsix might regulate Xist?
MCAT STYLE
Embryonic stem cells differentiate into diverse cell types once they receive the
appropriate signals. Until then they are kept in an undifferentiated state by the
action of three transcription regulators called Oct4, Sox2, and Nanog. An import-
ant target of Oct4 and Sox2 is the FGF4 gene. Analysis of the mechanism by which
Oct4 and Sox2 promote transcription of FGF4 provided early clues to how they
work. By analyzing deletions of the DNA regions around the FGF4 gene, scien-
tists identified a DNA element that was required for the ability of Oct4 and Sox2
to promote transcription of FGF4. The element was located beyond the coding
sequence at the 3 end of the gene. Analysis of the DNA element revealed that it
contained binding sites for Oct4 and Sox2 that were separated by three nucleo-
tides. When the element was mutated to increase the spacing between the bind-
ing sites by 3 or more nucleotides, Oct4 and Sox2 could no longer stimulate tran-
scription of FGF4.
The level of expression of Oct4 has strong effects on stem cell differentiation.
When expressed at normal levels, Oct4 helps maintain the undifferentiated state.
Overexpression of Oct4 by as little as 1.5-fold, however, causes stem cells to dif-
ferentiate into mesoderm.
7–106 What term best describes the DNA element that is required for Oct4 and
Sox2 to promote FGF4 transcription?
A. Enhancer
B. Mediator
C. Promoter
D. TATA box
7–107 Which hypothesis best explains the observation that increasing the spac-
ing between the Oct4 and Sox2 binding sites prevented stimulation of
FGF4 transcription?
A. Oct4 and Sox2 block the spread of repressive chromatin into the 3’ end of
the FGF4 gene.
B. Oct4 and Sox2 must be properly positioned to interact with the RNA pol-
ymerase II complex.
C. Precise positioning of the binding sites promotes cooperative binding of
Oct4 and Sox2.
D. The position of Oct4 and Sox2 influences the spacing of nucleosomes
over the FGF4 gene.
7–108 Which one of the following actions best explains how a minor change in
Oct4 levels could lead to a big change in the differentiation state of cells?
A. Chromatin modification
B. Cooperative binding
C. Dimerization
D. Negative feedback

Treatment of mouse fibroblast cells with 5-azacytidine causes some of the cells
to differentiate into muscle cells. A search for proteins that are responsible for
MCAT STYLE 165
this differentiation identified a transcription regulator called MyoD. Transfection (A)

of cells with a vector that expresses MyoD from an inducible promoter caused
efficient differentiation of fibroblast cells into muscle cells when expression of MyoD early genes late genes
MyoD was turned on. Moreover, when expression of MyoD from the vector was
later turned off, the cells remained differentiated as muscle cells. MyoD controls
the transcription of numerous genes. Some of these genes are turned on early in (B)
the differentiation process, whereas others are turned on late. Interestingly, MyoD
binds to the promoters of the late genes immediately after MyoD expression is
MyoD early genes late genes
first turned on, yet expression of these genes does not occur until later.
7–109 Which of the following possible effects of 5-azacytidine would best
(C)
account for its ability to cause fibroblasts to differentiate into muscle
cells?
A. Activation of Mediator complex MyoD early genes late genes
B. Inhibition of a DNA demethylase
C. Inhibition of a specific riboswitch
D. Inhibition of histone modification (D)
7–110 Which one of the following mechanisms best explains how cells remain
MyoD early genes late genes
differentiated as muscle cells, even after the original pulse of MyoD
expression has been turned off?
A. Cooperative binding
B. Negative feedback Figure 7–43 Four potential MyoD
C. Positive feedback regulatory circuits (Problem 7–111).
D. Synergistic transcriptional activation
7–111 Which one of the regulatory circuits in Figure 7–43 best explains all of
the observations regarding the role of MyoD in induction of muscle cell
differentiation? MCAT 7.306
A 1.5 mL Eppendorf Tube.
An Eppendorf tube containing
10 microliters of solution. Most molecular
biology reactions are carried out in these
tiny plastic tubes, which were invented
in 1961 at the Eppendorf hospital
laboratories in the suburbs of Hamburg,
Germany, as part of a system for handling
very small volumes of clinical samples.
These tubes are inert, robust, and
inexpensive.
Chapter 8 167
CHAPTER
Analyzing Cells, Molecules,

and Systems 8
ISOLATING CELLS AND GROWING THEM IN CULTURE IN THIS CHAPTER
TERMS TO LEARN
ISOLATING CELLS AND
hybridoma monoclonal antibody GROWING THEM IN CULTURE
DEFINITIONS PURIFYING PROTEINS
Match each definition below with its term from the list above. ANALYZING PROTEINS
8–1 Antibody secreted by a hybridoma cell line. ANALYZING AND MANIPULATING
8–2 Cell line used in the production of monoclonal antibodies; obtained by DNA
fusing antibody-secreting B cells with cells of a lymphocyte tumor.
STUDYING GENE EXPRESSION
TRUE/FALSE AND FUNCTION
Decide whether each of these statements is true or false, and then explain why. MATHEMATICAL ANALYSIS
OF CELL FUNCTIONS
8–3 Laser-capture microdissection permits isolation of individual cells from
a sample of tissue.
8–4 Because a monoclonal antibody recognizes a specific antigenic site
(epitope), it binds only to the specific protein against which it was made.
THOUGHT PROBLEMS
8–5 A common step in the isolation of cells from a sample of animal tissue is
to treat it with trypsin, collagenase, and EDTA. Why is such a treatment
necessary, and what does each component accomplish? Why doesn’t
this treatment kill the cells?
8–6 Isolation of cells from tissues, fluorescence-activated cell sorting, and
laser-capture microdissection are just a few of the ways for generating
homogeneous cell populations. Why do you suppose it is important to
have a homogeneous cell population for many experiments?
8–7 Distinguish among the terms “primary culture,” “secondary culture,” and
“cell line.”
8–8 Consider the following two statements. “The most important advantage
of the hybridoma technique is that monoclonal antibodies can be made
against molecules that constitute only a minor component of a complex
mixture.” “The most important advantage of the hybridoma technique
is that antibodies that may be present as only minor components in
conventional antiserum can be produced in quantity in pure form as
monoclonal antibodies.” Are these two statements equivalent? Why or
why not?
168 Chapter 8: Analyzing Cells, Molecules, and Systems
8–9 Do you suppose it would be possible to raise an antibody against another 1 2 3 4 5 6 7 8 9 10

antibody? Explain your answer.
CALCULATIONS
p3
8–10 You want to isolate rare cells that are present in a population at a fre-
quency of 1 in 105 cells, and you need 10 of those cells to do an experi-
ment. If your fluorescence-activated cell sorter can sort cells at the rate
of 1000 per second, how long would it take to collect enough rare cells for
p2
your experiment?
p1
DATA HANDLING
q1
8–11 Panels of human–rodent cell hybrids that retain one or a few human
chromosomes, parts of human chromosomes, or radiation-induced frag-
ments of human chromosomes have proven enormously useful in map- q2
ping genes to defined locations. Now that the human genome has been
sequenced, it is a trivial matter to know a gene’s location if you have a
bit of sequence from the gene. Nevertheless, there are many instances
in which such panels of cells are still invaluable; for example, when you q3
know a phenotype, but not the identity of the gene responsible for it, as
in the following case. q4
You wish to map the location of the receptor for feline leukemia virus
type C (FeLV-C), which infects human cells but not rodent cells. Using
a panel of hybrid cells carrying whole chromosomes, you have shown
that FeLV-C infects only those hybrids carrying human chromosome 1. Figure 8–1 Mapping the gene for the
FeLV-C receptor using human–rodent
Using a second panel carrying portions of chromosome 1, you show that hybrid cell lines that carry portions of
FeLV-C infects several of the hybrid cell lines. The segments of chromo- human chromosome 1 (Problem 8–11).
some 1 that are present in the infectable hybrid cell lines are shown in The hybrid cell lines that could be
Figure 8–1. Where on chromosome 1 is the gene for the FeLV-C receptor infected by FeLV-C are shown, with
located? the portions of human chromosome 1
retained in the individual hybrid cell
lines indicated as blue lines. The
p and q designations refer to a standard
PURIFYING PROTEINS convention (the Paris nomenclature) for
describing chromosome positions. Short-
TERMS TO LEARN arm locations are labeled p (petit) and
long arms q (queue). Each chromosome
column chromatography high-performance liquid Problems p8.01/8.01
arm is divided into regions labeled
fusion protein chromatography (HPLC) p1, p2, p3, q1, q2, q3, etc., counting
purified cell-free system outward from the centromere. Regions
are delimited by specific landmarks,
DEFINITIONS which are distinct morphological
features, including the centromere and
certain prominent bands. Regions are
divided into bands labeled p11 (one-
8–12 General term for purification technique in which a mixture of proteins is one, not eleven), p12, etc. Sub-bands
are designated p11.1, p11.2, etc., and
passed through a cylinder containing a porous solid matrix. sub-sub bands are designated p11.11,
p11.12, etc. In all cases, the numbers
8–13 Type of chromatography that uses columns packed with special chro- increase from the centromere toward
matography resins composed of tiny spheres that attain a high degree of the telomere.
resolution, even at very fast flow rates.
8–14 Artificial product generated by linking the coding sequences for two dif-
ferent proteins, or protein segments, and expressing the hybrid gene in
cells.
TRUE/FALSE
8–15 It is possible to pellet hemoglobin by centrifugation at sufficiently high
speed.
PURIFYING PROTEINS 169
Figure 8–2 Backbone models of

tropomyosin and hemoglobin
(Problem 8–19).
hemoglobin tropomyosin
8–16 If the beads used in gel-filtration chromatography had pores of a uni-

form size, proteins would either be excluded from the pores or included
in them, but would not be further fractionated.
THOUGHT PROBLEMS
8–17 Describe how you would use preparative centrifugation to purify mito-
chondria fromProblems
a cell homogenate.
p8.02/8.02
8–18 Distinguish between velocity sedimentation and equilibrium sedimen-
tation. For what general purpose is each technique used? Which do you
suppose might be best suited for separating two proteins of different
size?
8–19 Tropomyosin, at 93 kd, sediments at 2.6S, whereas the 65-kd protein,
hemoglobin, sediments at 4.3S. (The sedimentation coefficient S is a lin-
ear measure of the rate of sedimentation: both increase or decrease in
parallel.) These proteins are drawn to scale in Figure 8–2. How is it that
the bigger protein sediments more slowly than the smaller one? Can you
think of an analogy from everyday experience that might help you with
this problem?
8–20 Distinguish among ion-exchange chromatography, hydrophobic chro-
matography, gel-filtration chromatography, and affinity chromatography
in terms of the column material and the basis for separation of a mixture
of proteins.
CALCULATIONS
8–21 The purification of a protein usually requires multiple steps and often
involves several types of column chromatography. A key component of
any purification is an assay for the desired protein. The assay can be a
band on a gel, a structure in the electron microscope, the ability to bind
to another molecule, or enzyme activity. The purification of an enzyme
is particularly instructive because the assay allows one to quantify the
extent of purification at each step. Consider the purification of the
enzyme shown in Table 8–1. The total volume, total protein, and total
enzyme activity are shown at each step.
A. For each step in the purification procedure, calculate the specific activity
of the enzyme (units of activity per mg of protein). How can you tell that
purification has occurred at each step?
B. Which of the purification steps was most effective? Which was least effec-
tive?
C. If you were to carry the purification through additional steps, how would
the specific activity change? How could you tell from specific activity
measurements that the enzyme was pure? How might you check on that
conclusion?
TABLE 8–1 Purification of an enzyme (Problem 8–21).

Procedure Total volume (mL) Total protein (mg) Total activity (units) Specific activity
(units/mg)
1. Crude extract 2000 15,000 150,000
2. Ammonium sulfate precipitation 320 4000 140,000
3. Ion-exchange chromatography 100 550 125,000
4. Gel-filtration chromatography 85 120 105,000
5. Affinity chromatography 8 5 75,000
D. If the enzyme is pure at the end of the purification scheme in Table 8–1,
what proportion of the protein in the starting cell does it represent?
DATA HANDLING
8–22 In the classic paper that demonstrated the semiconservative replication
of DNA, Meselson and Stahl began by showing that DNA itself will form
a band when subjected to equilibrium sedimentation. They mixed ran-
domly fragmented E. coli DNA with a solution of CsCl so that the final
solution had a density of 1.71 g/mL. As shown in Figure 8–3, with increas-
ing length of centrifugation at 70,000 times gravity, the DNA, which was
initially dispersed throughout the centrifuge tube, became concentrated
over time into a discrete band in the middle.
A. Describe what is happening with time and explain why the DNA forms a
discrete band.
B. What is the buoyant density of the DNA? (The density of the solution at
which DNA “floats” at equilibrium defines the “buoyant density” of the
DNA.) hours centrifugal field
C. Even if the DNA were centrifuged for twice as long—or even longer—the 0
width of the band remains about what is shown at the bottom of Figure
8–3. Why doesn’t the band become even more compressed? Suggest 2.1
some possible reasons to explain the thickness of the DNA band at equi- 4.3
librium.
6.4
8–23 The result of gel-filtration chromatography of six roughly spherical pro- 8.5
teins is shown in Figure 8–4. The identities of the proteins, their molecu-
10.7
lar masses, and their elution volumes are indicated in Table 8–2. (The
elution volume identifies when each protein came off the column.) 12.8
14.9
17.1
TABLE 8–2 Proteins separated by gel-filtration chromatography 19.2

(Problem 8–23).
21.3
Protein Molecular Molecular Elution volume
23.5
mass (kd) mass (log) (mL)
36.5
Ribonuclease A 13 4.11 250
43.5
Chymotrypsinogen 25 4.40 228
Ovalbumin 43 4.63 199 Figure 8–3 Ultraviolet (UV) absorption
photographs showing successive
Bovine serum albumin 67 4.83 176 stages in the banding of E. coli DNA
(Problem 8–22). DNA, which absorbs
Aldolase 158 5.20 146
UV light, shows up as dark regions in
Catalase 232 5.37 123 the photographs. The bottom of the
centrifuge tube is on the right.
Problems p8.03/8.03
PURIFYING PROTEINS 171
Figure 8–4 Elution profile for

0.4 proteins fractionated by gel-filtration
199 228 chromatography (Problem 8–23). The
absorbance at 280 nm
176 250 absorbance at 280 nm is a measure of
0.3 146
protein concentration. Each of the peaks
123
is identified by its elution volume.
0.2
0.1
0
0 50 100 150 200 250 300
elution volume (mL)
A. Why do the smaller proteins come off the column later than the larger
proteins?
B. Plot molecular mass versus elution volume. Now plot the log of the
molecular mass versus the elution volume. Which plot gives a straight
line? What do you suppose is the basis for that result?
8–24 In preliminary studies, you’ve determined that your partially purified
protein is stable (retains activity) between pH 5.0 and pH 7.5. On either
side of that pH range, the protein is no longer active. Your advisor now
wants you to do a quick experiment to determine conditions for ion-
exchange chromatography. He has left instructions for you. First, you’re
supposed to mix a bit of the crude preparation with a small amount of the
ion-exchange resin DEAE-Sepharose™ in a series of buffer solutions that
have a pH between 5.0 and 7.5.
Problems Next, you are to pellet the resin and assay
p8.04/8.04
the supernatant for the presence of your protein. Finally, he tells you to
use this information to pick the proper pH to do the ion-exchange chro-
matography. You have completed the first two steps and have obtained
the results shown in Figure 8–5. But you are a little uncertain as to how to
use the information to pick the pH for the chromatography.
A. At which end of the pH range is the charge on your protein more positive
and at which end is it more negative? [Over this pH range, the positively
charged amine groups on the DEAE-Sepharose beads (Figure 8–5A) are
unaffected.]
B. For the chromatography, should you pick a pH at which the protein binds
to the beads (pH 6.5 to 7.5) or a pH where it does not bind (pH 5.0 to 6.0)?
Explain your choice.
C. Should you pick a pH close to the boundary (that is, pH 6.0 or 6.5) or far
away from the boundary (that is, pH 5.0 or pH 7.5)? Explain your reason-
ing.
D. How will you carry out ion-exchange chromatography of your protein?
What are the various steps you will use to accomplish the separation of
your protein from others via ion-exchange chromatography?
Figure 8–5 Preliminary test to

(A) STRUCTURE OF DEAE-SEPHAROSE (B) TEST FOR CONDITIONS determine conditions for ion-exchange
chromatography (Problem 8–24).
protein in supernatant after mixing (A) Structure of the charged amine
+ + + − − − groups attached to Sepharose beads.
(B) Results of mixing your protein with
C2H5 DEAE-Sepharose beads. Samples of the
O CH2 CH2 N H protein protein were mixed with DEAE-Sepharose
solution beads in buffers at a range of pH values,
C2H5 and then the mixtures were centrifuged
DEAE- to pellet the beads. The presence of the
Sepharose protein in the supernatant is indicated by
pH 5.0 5.5 6.0 6.5 7.0 7.5 a +; its absence is indicated by a –.
ANALYZING PROTEINS
TERMS TO LEARN
chemical biology two-dimensional gel electrophoresis
nuclear magnetic resonance Western blotting (immunoblotting)
(NMR) spectroscopy x-ray crystallography
SDS polyacrylamide-gel
electrophoresis (SDS-PAGE)
DEFINITIONS
8–25 Analysis of the release of electromagnetic radiation by atomic nuclei in a
magnetic field, due to flipping of the orientation of their magnetic dipole
moments.
8–26 Technique for protein separation in which the protein mixture is run first
in one direction and then in a direction at right angles to the first.
8–27 Technique in which a protein mixture is separated by running it through
a gel containing a detergent that binds to and unfolds the proteins.
8–28 The main technique that has been used to discover the three-dimen-
sional structure of molecules, including proteins, at atomic resolution.
8–29 Technique by which proteins are separated by electrophoresis, immobi-
lized on a paper sheet, and then analyzed, usually by means of a labeled
antibody.
TRUE/FALSE
Decide whether the statement is true or false, and then explain why.
8–30 Given the inexorable march of technology, it seems inevitable that the
sensitivity of detection of molecules will ultimately be pushed beyond
the yoctomole level (10–24 mole).
THOUGHT PROBLEMS
8–31 How is it that smaller molecules move through a gel-filtration column
more slowly than larger molecules, whereas in SDS polyacrylamide-gel
electrophoresis (SDS-PAGE) the opposite is true: larger molecules move
more slowly than small molecules?
8–32 You are set to run your first SDS polyacrylamide-gel electrophoresis. You
have boiled your samples of protein in SDS in the presence of mercap-
toethanol and loaded them into the wells of a polyacrylamide gel. You
are now ready to attach the electrodes. Uh oh, does the positive electrode
(the anode) go at the top of the gel, where you loaded your proteins, or at
the bottom of the gel?
8–33 You hate the smell of mercaptoethanol. Since disulfide bonds in intra-
cellular proteins are very rare (see Problem 3–37), you have convinced
yourself that it is not necessary to treat a cytoplasmic homogenate with
mercaptoethanol prior to SDS-PAGE. You heat a sample of your homoge-
nate in SDS and subject it to electrophoresis. Much to your surprise, your
gel looks horrible; it is an ugly smear! You show your result to a fellow
student with a background in chemistry, and she suggests that you treat
your sample with N-ethylmaleimide (NEM), which reacts with free sulf-
hydryls. You run another sample of your homogenate after treating it
with NEM and SDS. Now the gel looks perfect!
ANALYZING PROTEINS 173
If the vast majority of intracellular proteins don’t have disulfide

bonds—and they don’t—why didn’t your original scheme work? And
how does treatment with NEM correct the problem?
8–34 For separation of proteins by two-dimensional polyacrylamide-gel elec-
trophoresis, what are the two types of electrophoresis that are used in
each dimension? Do you suppose it makes any difference which electro-
phoretic method is applied first? Why or why not?
8–35 Discuss the following statement: “With the ever-expanding databases
of protein sequences and structures, it will soon be possible to input an
amino acid sequence of an unknown protein and, by analogy to known
proteins, determine its structure and function. Thus, it will not be long
before biochemists are put out of work.”
8–36 Hybridoma technology allows one to generate monoclonal antibodies
to virtually any protein. Why is it, then, that genetically tagging proteins
with epitopes is such a commonly used technique, especially since an
epitope tag has the potential to interfere with the function of the protein?
8–37 Specific activity refers to the amount of radioactivity per unit amount
of substance, most commonly in biology expressed on a molar basis,
for example, as Ci/mmol. [One curie (Ci), which is the standard unit of
radioactive decay, corresponds to 2.22 × 1012 disintegrations per minute
(dpm).] If you examine Table 8–3, you will see that there seems to be an
inverse relationship between maximum specific activity and half-life. Do
you suppose this is just a coincidence or is there an underlying reason?
Explain your answer.
8–38 You just developed an autoradiograph after a two-week exposure. You
had incubated your protein with a cell-cycle kinase in the presence of
32P-ATP in hopes of demonstrating that it was indeed a substrate for the
kinase. You see the hint of a band on the gel at the right position, but it is
just too faint to be convincing. You show your result to your advisor and
tell him that you’ve put the blot against a fresh sheet of film, which you
plan to expose for a longer period of time. He gives you a sideways look
and tells you to do the experiment over again and use more radioactivity.
What’s wrong with your plan to reexpose the blot for a longer time?
CALCULATIONS
8–39 How many copies of a protein need to be present in a cell in order for it
to be visible as a band on an SDS gel? Assume that you can load 100 μg
of cell extract onto a gel and that you can detect 10 ng in a single band
by silver staining. The concentration of protein in cells is about 200 mg/
mL, and a typical mammalian cell has a volume of about 1000 μm3 and
a typical bacterium a volume of about 1 μm3. Given these parameters,
TABLE 8–3 Radioactive isotopes and some of their properties (Problem

8–37).
Radioactive Emission Half-life Maximum specific
isotope activity (Ci/mmol)
14C 5730 years 0.062
particle
3H 12.3 years 29
particle
35S 87.4 days 1490
particle
32P 14.3 days 9120
particle
calculate the number of copies of a 120-kd protein that would need to be

present in a mammalian cell and in a bacterium in order to give a detect-
able band on a gel. You might try an order-of-magnitude guess before
you make the calculations.
8–40 How many molecules of your labeled protein are required for detection
by autoradiography? You added 1 μL containing 10 μCi of -32P-ATP (a
negligible amount of ATP) to 9 μL of cell extract that had an ATP con-
centration of 1 mM. You incubated the mixture to allow transfer of phos-
phate to proteins in the extract. You then subjected 1 μL of the mixture to
SDS-PAGE, dried the gel, and placed it against a sheet of x-ray film. After
an overnight exposure you saw a barely detectable band in the location of
your protein. You know from previous experience that a protein labeled
at 1 count per minute per band (1 cpm equals 1 disintegration per min-
ute, dpm, for 32P) will form such a band after an overnight exposure. How
many molecules of labeled protein are in the band, if you assume 1 phos-
phate per molecule? (Some useful conversion factors for radioactivity are
shown in Table 3 on page 964.)
8–41 You want to know the sensitivity for detection of immunoblotting (West-
ern blotting), using an enzyme-linked second antibody to detect the
antibody directed against your protein (Figure 8–6A). You are using the
mouse monoclonal antibody 4G10, which is specific for phosphotyros-
ine residues, to detect phosphorylated proteins. You first phosphorylate
the myelin basic protein in vitro using a tyrosine protein kinase that adds
one phosphate per molecule. You then prepare a dilution series of the
phosphorylated protein and subject the samples to SDS-PAGE. Next, the
protein is transferred (blotted) onto a nitrocellulose filter, incubated with
the 4G10 antibody, and washed to remove unbound antibody. The blot
is then incubated with a second goat anti-mouse antibody that carries
horseradish peroxidase (HRP) conjugated to it, and any excess unbound
antibody is again washed away. You place the blot in a thin plastic bag,
add reagents that chemiluminesce when they react with HRP (Figure
8–6A), and place the bag against a sheet of x-ray film. When the film is
developed, you see the picture shown in Figure 8–6B.
A. Given the amounts of phosphorylated myelin basic protein indicated in
each lane in Figure 8–6B, calculate the detection limit of this method in
terms of molecules of protein per band.
B. Assuming that you were using monoclonal antibodies to detect proteins,
would you expect that the detection limit would depend on the molecu-
lar mass of the protein? Why or why not?
(A) SCHEMATIC DIAGRAM (B) IMMUNOBLOT

myelin basic protein (fmol)
light 40 20 10 5 2.5 1.2 0.6
NH2 O NH2 O Figure 8–6 Sensitivity of detection

of immunoblotting (Problem 8–41).
NH O−
(A) Schematic diagram of the experiment.
NH H2O2 O− MBP stands for myelin basic protein.
In the presence of hydrogen peroxide,
O HRP O
luminol horseradish peroxidase (HRP) converts
luminol to a chemiluminescent molecule
second antibody that emits light, which is detected by
exposure of an x-ray film. (B) Exposed
MBP
first antibody film of an immunoblot. The number of
femtomoles of myelin basic protein in
nitrocellulose membrane each band is indicated.
ANALYZING PROTEINS 175
DATA HANDLING kd
8–42 Figure 8–7 shows an autoradiograph of an SDS-PAGE separation of 116

radiolabeled proteins in a cell-free extract of sea urchin eggs. Alongside 97
are shown a set of radiolabeled marker proteins of defined molecular
67
mass. Two bands that contain known proteins—the small subunit of rib-
onucleotide reductase and cyclin B—are indicated. 56 cyclin B
A. Do the standard set of proteins migrate at a rate that is inversely propor-
ribonucleotide
tional to their molecular masses? That is to say, would you expect a pro- reductase
40
tein of 35 kd, for example, to migrate twice as far down the gel as a protein
of 70 kd? Do you suppose a plot of log molecular mass versus migration 35
would give a more linear relationship?
B. How would you use the standard set of proteins to estimate the molecu-
lar masses of ribonucleotide reductase and cyclin B? What would you
estimate the molecular masses of these two proteins to be? Figure 8–7 Autoradiograph of
C. The sequences of the genes for these two proteins give molecular masses radiolabeled proteins separated by
of 44 kd for ribonucleotide reductase and 46 kd for cyclin B. Can you offer SDS-PAGE (Problem 8–42). A set of
some possible reasons why the SDS-PAGE estimate for the molecular radiolabeled marker proteins with known
mass of cyclin B is so far off? molecular masses is shown in the left-
hand lane, along with their molecular
8–43 You have isolated the proteins from two adjacent spots after two-dimen- masses in kilodaltons. Radiolabeled
Problems p8.08/8.07
proteins from a sea urchin egg extract
sional polyacrylamide-gel electrophoresis and digested them with are shown in the right-hand lane. Arrows
trypsin. When the masses of the peptides were measured by MALDI-TOF mark the bands that correspond to cyclin
mass spectrometry, the peptides from the two proteins were found to B and the small subunit of ribonucleotide
be identical except for one (Figure 8–8). For this peptide, the mass-to- reductase.
charge (m/z) values differed by 80, a value that does not correspond to a
difference in amino acid sequence. (For example, glutamic acid instead
of valine at one position would give an m/z difference of around 30.) Can
you suggest a possible difference between the two peptides that might
account for the observed m/z difference?
8–44 You have raised four different monoclonal antibodies to Xenopus Orc1,
which is a component of the DNA replication origin recognition com-
plex (ORC) found in eukaryotes. You want to use the antibodies to immu-
TK 423
nopurify other members of ORC. To decide which of your monoclonal
15
37
47
b
1
A
antibodies—TK1, TK15, TK37, or TK47—is best suited for this purpose,
TK
TK
TK
m
you covalently attach them to beads, incubate them with a Xenopus egg
extract, spin the beads down and wash them carefully, and then solu-
bilize the bound proteins with SDS. You use SDS-PAGE to separate the kd
solubilized proteins and stain them, as shown in Figure 8–9. 116
A. From these results, which bands do you think arise from proteins that are 97
present in ORC?
B. Why do you suppose the various monoclonal antibodies give such differ- 66
ent results?
C. Which antibody do you think is the best one to use in future studies of
this kind? Why? 45
D. How might you determine which band on this gel is Orc1?
abundance
29
3706
20
18
abundance
3786
Figure 8–9 Immunoaffinity purification
of Xenopus ORC (Problem 8–44). The
monoclonal antibody mAb423 is specific
m/z (mass-to-charge ratio) for an antigen not found in Xenopus
extracts and thus serves as a control. The
Figure 8–8 Masses of peptides measured by MALDI-TOF positions of marker proteins are shown
mass spectrometry (Problem 8–43). Only the numbered at the left with their masses indicated in
peaks differ between the two protein samples. kilodaltons.
Problems p8.10/8.09
Problems p8.09/8.08
LexA DNA-binding VP16 activation domain Figure 8–10 Activation of transcription by

domain VP16 a hybrid transcription regulator (Problem
transcription 8–45).
LacZ gene LacZ gene

LexA-binding site
8–45 The yeast two-hybrid system relies on the cell’s own mechanisms to reveal
protein–protein interactions. This method takes advantage of the modu-
lar nature of many transcription regulators, which have one domain that
binds to DNA and another domain that activates transcription. Domains
can be interchanged by recombinant DNA methods, allowing hybrid
transcription regulators to be constructed. Thus, the DNA-binding
domain of the E. coli LexA repressor can be combined with the power-
ful VP16 activation domain from herpesvirus to activate transcription of
genes downstream of a LexA DNA binding site (Figure 8–10).
If the two domains of the transcription regulator can be brought into
proximity by protein–protein interactions, they will activate transcrip-
tion. This is the key feature of the two-hybrid system. Thus, if one mem-
ber of an interacting pair of proteins is fused to the DNA-binding domain
of LexA (to form the “bait”) and the other is fused to the VP16 activa-
tion domain (to form the “prey”), transcription will be activated when
the two hybrid proteins interact inside a yeast cell. It is possible to design
powerful screens for protein–protein interactions, if the gene whose tran-
scription is turned on is essential for growth or can give rise to a colored
product.
To check out the ability of the system to find proteins with which Ras
interacts, hybrid genes were constructed that contained the LexA DNA-
binding domain, one fused to Ras (LexA–Ras) and the other fused to
nuclear lamin (LexA–lamin). A second pair of constructs contained the
VP16 activation domain alone (VP16) or fused
Problems to the adenylyl cyclase
p8.11/8.10
gene (VP16–CYR). Adenylyl cyclase is known to interact with Ras and
serves as a positive control; nuclear lamins do not interact with Ras and
serve as a negative control. These plasmid constructs were introduced
into a strain of yeast containing copies of the His3 gene and the LacZ
gene, both with LexA-binding sites positioned immediately upstream.
Individual transformed colonies were tested for the ability to grow on
a plate lacking histidine, which requires expression of the His3 gene. In
addition, they were tested for ability to form blue colonies (as compared
to the normal white colonies) when grown in the presence of an appro-
priate substrate (XGAL) for -galactosidase. The set-up for the experi-
ment is outlined in Table 8–4.
TABLE 8–4 Experiments to test the two-hybrid system (Problem 8–45).

Plasmid constructs
Bait Prey Growth on plates lacking histidine Color on plates with XGAL
lexA–Ras
LexA–lamin
VP16
VP16–CYR
LexA–Ras VP16
LexA–Ras VP16–CYR
LexA–lamin VP16
LexA–lamin VP16–CYR
ANALYZING AND MANIPULATING DNA 177
A. Fill in Table 8–4 with your expectations. Use a plus sign to indicate growth
on plates lacking histidine and a minus sign to indicate no growth. Write
“blue” or “white” to indicate the color of colonies grown in the presence
of XGAL.
B. For any combinations of bait and prey in the table that you expect to
confer growth in the absence of histidine and to form blue colonies with
XGAL, sketch the structure of the active transcription regulator on the
LacZ gene.
C. If you want two proteins to be expressed in a single polypeptide chain,
what must you be careful to do when you fuse the two genes together?
ANALYZING AND MANIPULATING DNA

TERMS TO LEARN
bacterial artificial chromosome (BAC) genome annotation
cDNA clone genomic library
cDNA library hybridization
deep RNA sequencing (RNA-seq) open reading frame (ORF)
dideoxy sequencing (Sanger plasmid vector
sequencing) polymerase chain reaction (PCR)
DNA cloning recombinant DNA technology
DNA library restriction nuclease
DEFINITIONS
8–46 Small, circular DNA molecule that replicates independently of the
genome and can be used for DNA cloning.
8–47 A collection of clones that contain a variety of DNA segments from the
genome of an organism.
8–48 The process of marking out all the genes in a genome and ascribing a
biological function to each.
8–49 One of a large number of enzymes that can cleave a DNA molecule at any
site where a specific short sequence of nucleotides occurs.
8–50 A DNA clone of a DNA copy of an mRNA molecule.
8–51 Technique for generating multiple copies of specific regions of DNA by
the use of sequence-specific primers and multiple cycles of DNA synthe-
sis.
8–52 The sequencing of the entire repertoire of RNA from a cell or tissue.
8–53 Prokaryotic cloning vector that can accommodate large pieces of DNA
up to 1 million base pairs.
8–54 The process whereby two complementary nucleic acid strands form a
double helix.
TRUE/FALSE
8–55 Bacteria that make a specific restriction nuclease for defense against
viruses have evolved in such a way that their own genome does not con-
tain the recognition sequence for that nuclease.
8–56 Pulsed-field gel electrophoresis uses a strong electric field to separate

very long DNA molecules, stretching them out so that they travel end-
first through the gel at a rate that depends on their length.
8–57 By far the most important advantage of cDNA clones over genomic clones
is that they can contain the complete coding sequence of a gene.
8–58 If each cycle of PCR doubles the amount of DNA synthesized in the previ-
ous cycle, then 10 cycles will give a 103-fold amplification, 20 cycles will
give a 106-fold amplification, and 30 cycles will give a 109-fold amplifica-
tion.
THOUGHT PROBLEMS
8–59 Figure 8–11 shows a picture of DNA fragments that have been separated
by gel electrophoresis and then stained by ethidium bromide, a mole- Figure 8–11 DNA fragments separated
cule that fluoresces intensely under long-wavelength UV light when it is by gel electrophoresis and stained
bound to DNA. Such gels are a standard way of detecting the products with ethidium bromide (Problem 8–59).
of cleavage by restriction nucleases. For the DNA fragment shown in Each of the orange bands on the gel
represents a site where fragments of DNA
Figure 8–12, decide whether it will be cut by the restriction nucleases have migrated during electrophoresis.
EcoRI (5 -GAATTC), AluI (5 -AGCT), and PstI (5 -CTGCAG). For those Ethidium intercalates between base pairs
that cut the DNA, how many products will be produced? in double-stranded DNA. Removal of
ethidium from the aqueous environment
and fixing its orientation in the nonpolar
5′-AAGAATTGCGGAATTCGAGCTTAAGGGCCGCGCCGAAGCTTTAAA-3′ Problems
environment p8.14/8.11
of DNA enhance its
3′-TTCTTAACGCCTTAAGCTCGAATTCCCGGCGCGGCTTCGAAATTT-5′ fluorescence dramatically. When
irradiated with long-wavelength UV light,
Figure 8–12 A segment of double-stranded DNA (Problem 8–59). it fluoresces a bright orange.
8–60 The restriction nucleases BamHI and PstI cut their recognition sequences
as shown in Figure 8–13.
A. Indicate the 5 and 3 ends of the cut DNA molecules.
B. How would the ends be modified if you incubated the cut molecules with
DNA polymerase in the Problems
presence ofp8.15/8.12
all four dNTPs?
C. After the reaction in part B, could you still join the BamHI ends together
by incubation with T4 DNA ligase? Could you still join the PstI ends
together? (T4 DNA ligase will join blunt ends together as well as cohesive
ends.)
D. Will joining of the ends in part C regenerate the BamHI site? Will it regen-
erate the PstI site?
8–61 The restriction nuclease EcoRI recognizes the sequence 5 -GAATTC
and cleaves between the G and A to leave 5 protruding single strands
(like BamHI, see Figure 8–13A). PstI, on the other hand, recognizes the
sequence 5 -CTGCAG and cleaves between the A and G to leave 3 pro-
truding single strands (see Figure 8–13B). These two recognition sites are
displayed on the helical representations of DNA in Figure 8–14.
A. For each restriction site, indicate the position of cleavage on each strand
of the DNA.
(A) BamHI CLEAVAGE (B) PstI CLEAVAGE
5′ 3′ 5′ 3′
-- G G A T C C -- -- C T G C A G --
-- C C T A G G -- -- G A C G T C --
3′ 5′ 3′ 5′
Figure 8–13 Restriction nuclease
BamHI PstI cleavage of DNA (Problem 8–60).
(A) BamHI cleavage. (B) PstI cleavage.
-- G G A T C C -- -- C T G C A G -- Only the nucleotides that form the
-- C C T A G G -- -- G A C G T C -- recognition sites are shown.
B. From the positions of the cleavage sites, decide for each restriction 3′
nuclease whether you expect it to approach the recognition site from the
EcoRI
major-groove side or from the minor-groove side.
8–62 Which, if any, of the restriction nucleases listed in Table 8–5 will 5′
definitely cleave a segment of cDNA that encodes the peptide KIGPACF?

(See Tables 7 and 8, page 966, for the genetic code.) G
A
8–63 You wish to make a restriction map of a 3.0-kb BamHI restriction frag- A
minor
ment. You digest three samples of the fragment with EcoRI, HpaII, and groove T
a mixture of EcoRI and HpaII. You then separate the fragments by gel T
electrophoresis and visualize the DNA bands by staining with ethidium C
bromide (Figure 8–15). From these results, draw a restriction map that
shows the relative positions of the EcoRI and HpaII recognition sites and
the distances in kilobases (kb) between them. 5′
8–64 If you add DNA to wells at the top of a gel, should you place the posi-
tive electrode (anode) at the top or at the bottom of the gel? Explain your 3′
choice.
8–65 You want to clone a DNA fragment that has KpnI ends into a vector that 3′
has BamHI ends. The problem is that BamHI and KpnI ends are not
PstI
compatible: BamHI leaves a 5 overhang and KpnI leaves a 3 overhang
(Figure 8–16). A friend suggests that you try to link them with an oligo- 5′
nucleotide “splint” as shown in Figure 8–16. It is not immediately clear
to you that such a scheme will work because ligation requires an adja- C
cent 5 phosphate and 3 hydroxyl. Although molecules that are cleaved T
with restriction nucleases have appropriate ends, oligonucleotides are G
typically synthesized with hydroxyl groups at both ends. Also, although major
C groove
the junction shown in Figure 8–16 is BamHI–KpnI, the other junction is A
KpnI–BamHI, and you are skeptical that the same oligonucleotide could G
splint both junctions.
A. Draw a picture of the KpnI–BamHI junction and the oligonucleotide
splint that would be needed. Is this oligonucleotide the same or different 5′
from the one shown in Figure 8–16?
B. Draw a picture of the molecule after treatment with DNA ligase. Indicate
which if any of the nicks will be ligated. 3′
C. Will your friend’s scheme work?
Figure 8–14 Restriction sites on helical
8–66 How would a DNA sequencing reaction be affected if the ratio of dideox- DNA (Problem 8–61).
ynucleoside triphosphates (ddNTPs) to deoxynucleoside triphosphates
(dNTPs) were increased? What would the consequences be if the ratio
were decreased?
8–67 DNA sequencing of your own two -globin genes (one from each of your Problems p8.17/8.14
two copies of chromosome 11) reveals a mutation in one of the genes.
EcoRI
+
EcoRI HpaII HpaII
TABLE 8–5 A set of restriction

nucleases and their recognition
sequences (Problem 8–62).
1.7 kb
Restriction Recognition 1.6 kb
nuclease sequence 1.4 kb
1.2 kb
0.9 kb 0.9 kb
AluI AGCT
Sau96I GGNCC 0.5 kb Figure 8–15 Sizes of DNA bands
0.4 kb 0.4 kb produced by digestion of a 3.0-kb
HindIII AAGCTT fragment by EcoRI, HpaII, and a mixture
of the two (Problem 8–63). Sizes of the
N stands for any nucleotide.
fragments are shown in kilobases.
BamHI-cut vector DNA DNA to be amplified
5′ -GACCTGTGGAAGC CATACGGGATTGA-3′
3′ 3′ -CTGGACACCTTCG GTATGCCCTAACT-5′
5′
G GATCC
CCTAG G
5′ 3′ primers
5′ 3′ (1) 5′ -GACCTGTGGAAGC-3′ (5) 5′ -CATACGGGATTGA-3′
C GGTAC
CATGG C (2) 5′ -CTGGACACCTTCG-3′ (6) 5′ -GTATGCCCTAACT-3′
3′ 5′
(3) 5′ -CGAAGGTGTCCAG-3′ (7) 5′ -TGTTAGGGCATAC-3′
KpnI-cut fragment
(4) 5′ -GCTTCCACAGGTC-3′ (8) 5′ -TCAATCCCGTATG-3′
oligonucleotide “splint”
Figure 8–16 Scheme to use Figure 8–17 DNA to be amplified and potential PCR primers
5′ 3′ an oligonucleotide “splint” to (Problem 8–68).
GATCGTAC
link incompatible restriction
3′ 5′
G C ends (Problem 8–65).
CCTAG CATGG
5′ 3′
BamHI KpnI Problems p8.21/8.17
Given this information alone, how much should you worry about being a
carrier of an inherited disease that could be passed on to your children?
What other information would you like to have to assess your risk?
8–68Problems
You wantp8.19/8.16
to amplify the DNA between the two stretches of sequence
shown in Figure 8–17. Of the listed primers, choose the pair that will
allow you to amplify the DNA by PCR.
8–69 In the very first round of PCR using genomic DNA, the DNA primers
prime synthesis that terminates only when the cycle ends (or when a
random end of DNA is encountered). Yet, by the end of 20 to 30 cycles—
a typical amplification—the only visible product is defined precisely by
the ends of the DNA primers (Figure 8–18). In what cycle is a double-
stranded fragment of the correct size first generated?
8–70 You want to express a rare human protein in bacteria so you can make
large quantities of it. To aid in its purification, you decide to add a stretch
of six histidines to the N-terminus or the C-terminus of the protein. Such
histidine-tagged proteins bind tightly to Ni2+ columns but can be readily
eluted with a solution of EDTA or imidazole. This procedure allows an
enormous purification in one step.
The nucleotide sequence that encodes your protein is shown in
Figure 8–19. Design a pair of PCR primers, each with 18 nucleotides of
homology to the gene, that will amplify the coding sequence and add
an initiation codon followed by six histidine codons to the N-terminus.
Design a pair of primers that will add six histidine codons followed by a
stop codon to the C-terminus.
FIRST CYCLE OF PCR
5′
3′
5′
3′
20 CYCLES
Figure 8–18 Products of PCR after 1 and

20 cycles (Problem 8–69).
N-terminus H H H H
5′ GGT CGT ATG GCT ACT CGT CGC GCT GCT
M A T R R A A C C C C
+ H
H N N H H N N
C C
CTT GCT GCA AGT CTC TCT TAG AAG TGT 3′

H H
L A A S L S *
C-terminus
Figure 8–19 Nucleotide sequence around the N- and C-termini of the Figure 8–20 Structure of imidazole (Problem 8–71).
protein you want to modify (Problem 8–70). The encoded amino acid
sequence is indicated below each codon using the one-letter code.
The asterisk (*) indicates the stop codon. Only the top strand of the
double-stranded DNA is shown.
G A T C
8–71 You have now cloned in an expression
Problems vector both versions of the his-
p8.23/8.19
tidine-tagged protein you created in Problem 8–70. Neither construct
expresses particularly strongly in bacteria, but the product is soluble. You
pass the crude extract over a Ni2+ affinity column, which binds histidine-
tagged proteins specifically. After washing the column extensively, you 100
elute your protein from the column using a solution containing imida-
zole (Figure 8–20), which releases your protein.
When you subject the eluted protein to electrophoresis and stain the Problems p8.24/8.20
gel for protein, you are pleased to find bands in the eluate that are not
present when control bacteria are treated similarly. But you are puz-
zled to see that the construct tagged at the N-terminus gives a ladder of
shorter proteins below the full-length protein, whereas the C-terminally
tagged construct yields exclusively the full-length protein. The amount of
full-length protein is about the same for each construct.
A. Why does a solution of imidazole release a histidine-tagged protein from
the Ni2+ column?
B. Offer an explanation for the difference in the products generated by the
two constructs.
8–72 An example of a dideoxy sequencing gel is shown in Figure 8–21. Try
reading it. As read from the bottom of the gel to the top, the sequence cor-
responds to the mRNA for a protein. Can you find the open reading frame
in this sequence? What protein does it code for? 50
CALCULATIONS
8–73 The restriction nuclease Sau3A recognizes the sequence 5 -GATC and
cleaves on the 5 side (to the left) of the G. (Since the top and bottom
strands of most restriction sites read the same in the 5 -to-3 direction,
only one strand of the site need be shown.) The single-stranded ends
produced by Sau3A cleavage are identical to those produced by BamHI
cleavage (see Figure 8–13), allowing the two types of ends to be joined
together by incubation with DNA ligase. (You may find it helpful to draw
out the product of this ligation to convince yourself that it is true.)
A. What fraction of BamHI sites (5 -GGATCC) can be cut with Sau3A? What
fraction of Sau3A sites can be cut with BamHI?
B. If two BamHI ends are ligated together, the resulting site can be cleaved
again by BamHI. The same is true for two Sau3A ends. Suppose you ligate
a Sau3A end to a BamHI end. Can the hybrid site be cut with Sau3A? Can
it be cut with BamHI?
C. What do you suppose is the average size of DNA fragments produced by
digestion of chromosomal DNA with Sau3A? What’s the average size with
BamHI?
Figure 8–21 A dideoxy sequencing gel of a cloned segment of DNA
(Problem 8–72). The lanes are labeled G, A, T, and C to indicate which
ddNTP was included in the reaction. 1
protein sequence Figure 8–22 A degenerate oligonucleotide probe for the Factor VIII EcoRI BamHI
gene based on a stretch of amino acids from the protein (Problem
M Q K F N 8–75). Because more than one DNA triplet can encode each amino 4 kb
A A T T acid, a number of different nucleotide sequences are possible
ATGCA AA TT AA for each amino acid sequence. During synthesis, a mixture of insert
G G C C
nucleotides is included at each ambiguous position (for example,
degenerate oligonucleotide A plus G at position 6). As a result, the mixture of synthesized
oligonucleotides contains all possible sequences that might encode vector
the specific amino acid segment. Although only one of these
sequences in the genomic DNA will actually code for the protein, it
is impossible to tell in advance which one it is. The mixture of the Figure 8–23 Recombinant plasmid
possible sequences—called a degenerate oligonucleotide probe—is containing a cloned DNA segment
used to search a genomic library for the gene. (Problem 8–76).
8–74 To prepare a genomic library, it is necessary to fragment the genome so

that it can be cloned in a vector. A common method is to use a restriction
nuclease.
A. How many different DNA fragments would you expect to obtain if you
cleaved human genomic DNA with Sau3A (5 -GATC)? (Recall that there
are 3.2 × 109 base pairs in the haploid human genome.) How many would
you expect to get with EcoRI (5 -GAATTC)?
B. Human genomic libraries are often made from fragments obtained by
cleaving human DNA with Sau3A in such a way that the DNA is only par-
tially digested; that is, so that not all the Sau3A sites have been cleaved.
What is a possible reason for doing this?
8–75 A degenerate set of oligonucleotide probes for the Factor VIII gene for
Problems p8.27/8.22
blood clotting is shown in Figure 8–22. Each of these probes is only 15
nucleotides long. On average, how many exact matches to any single
15-nucleotide sequence would you expect to find in the human genome Problems p8.31/8.23
(3.2 × 109 base pairs)? How many matches to the collection of sequences
in the degenerate oligonucleotide probe would you expect to find? How
might you determine that a match corresponds to the Factor VIII gene?
DATA HANDLING
rs
ke
8–76 You have cloned a 4-kb segment of a gene into a plasmid vector (Figure
ar
m
8–23) and now wish to prepare a restriction map of the gene in prepara- kb
tion for other DNA manipulations. Your advisor left instructions on how
to do it, but she is now on vacation, so you are on your own. You follow 4.8
4.3
her instructions, as outlined below. 3.7
1. Cut the plasmid with EcoRI.
2. Add a radioactive label to the EcoRI ends. 2.3
3. Cut the labeled DNA with BamHI. 1.9
4. Purify the insert away from the vector. 1.4
5. Digest the labeled insert briefly with a restriction nuclease so that on 1.3
average each labeled molecule is cut about one time.
6. Repeat step 5 for several different restriction nucleases. 0.7
7. Run the partially digested samples side by side on an agarose gel.
8. Place the gel against x-ray film so that fragments with a radioactive
end can expose the film to produce an autoradiograph.
9. Draw the restriction map.
Your biggest problem thus far has been step 5; however, by decreasing 0.2
the amounts of nuclease and lowering the temperature, you were able to
find conditions for partial digestion. You have now completed step 8, and
your autoradiograph is shown in Figure 8–24.
Figure 8–24 Autoradiograph showing the
Unfortunately, your advisor was not explicit about how to construct a electrophoretic separation of the labeled
map from the data in the autoradiograph. She is due back tomorrow. Will fragments after partial digestion with the
you figure it out in time? three restriction nucleases represented by
the symbols (Problem 8–76). Numbers at
8–77 The DNA of certain animal viruses can integrate into a cell’s DNA the left indicate the sizes of a set of marker
as shown schematically in Figure 8–25. You want to know the fragments in kilobases.
Problems p8.32/8.24
Figure 8–25 Integration of viral DNA into (A) MAP OF VIRAL DNA
viral DNA
cell DNA (Problem 8–77).
EcoRI
cell DNA HpaII e

a
d
BglI
b
c
integrated BglI HpaII
viral DNA
structure of the viral genome as it exists in the integrated state. You digest (B) RESTRICTION DIGESTS
samples of viral DNA and DNA from cells that contain the integrated
EcoRI HpaII BglI
virus with restriction nucleases that cut the viral DNA at known sites virus cell virus cell virus cell
(Figure 8–26A). Subsequently, you separate the fragments by electro-
phoresis on agarose gels and visualize the bands that contain viral DNA
by hybridization with a viral DNA probe. You obtain the patterns shown
in Figure 8–26B.
From this information, decide in which of the five segments of the viral
genome (labeled a to e in Figure 8–26A) the integration event occurred.
MEDICAL LINKS
8–78 Many mutations that cause human genetic diseases involve the substitu-
tion of one nucleotide for another, as is the case for sickle-cell anemia Figure 8–26 Viral and cell DNA digested
(Figure 8–27A). An assay based on ligation of oligonucleotides provides with various restriction nucleases and
a rapid way to detect such specific single-nucleotide differences. This hybridized to a viral DNA probe (Problem
8–77). (A) Restriction sites on the viral
assay uses pairs of oligonucleotides: for each pair, one oligonucleotide genome. The DNA segments defined by
is labeled with biotin and the other with a radioactive (or fluorescent) these sites are indicated by the letters a
tag. In the assay shown in Figure 8–27B for the detection of the muta- to e. (B) Restriction digests of viral DNA
Problems
tion responsiblep8.34/8.25
for sickle-cell anemia, two pairs of oligonucleotides are and cellular DNA. Agarose gels separate
DNA fragments on the basis of size—the
hybridized to DNA from an individual and incubated in the presence of
smaller the fragment, the farther it moves
DNA ligase. Biotinylated oligonucleotides are then bound to streptavidin toward the bottom of the gel.
on a solid support and any associated radioactivity is visualized by auto-
radiography, as shown in Figure 8–27C.
A. Do you expect the A and S oligonucleotides to hybridize to both A and Problems p8.35/8.26
S DNA?
B. How does this assay distinguish between A and S DNA?
(A) β-GLOBIN SEQUENCE

normal (β A) sequence
A
ATGGTGCACCTGACTCCTG GGAGAAGGTCTGCCGTTACTG
T
sickle-cell mutant (β S) sequence
(B) OLIGONUCLEOTIDES
β A oligo
1 biotin-ATGGTGCACCTGACTCCTGA Figure 8–27 Oligonucleotide-ligation
radioactive oligo
32P-GGAGAAGGTCTGCCGTTACTG 3
assay (Problem 8–78). (A) Sequence of
β S oligo the -globin gene around the site of the
2 biotin-ATGGTGCACCTGACTCCTGT sickle-cell ( S) mutation. The normal A
sequence carries an A at the central
position; the sickle-cell mutant S
(C) ASSAY oligos oligos sequence has a T instead. (B) Specific
1 + 3 2 + 3 oligonucleotides for ligation assay.
(C) Assays to detect the single-nucleotide
difference between the A and S
β Aβ A homozygote
sequences. After hybridization to patient
β Aβ S heterozygote DNA, biotinylated oligonucleotides were
collected in a spot on a sheet of filter
β Sβ S homozygote paper and exposed to x-ray film to detect
radioactivity, which turns the film black.
(A) DMD GENE a b c d e f g h i sites amplified Figure 8–28 Multiplex PCR analysis
by PCR of six DMD patients (Problem 8–79).
(A) The DMD gene with the nine sites
amplified by PCR indicated by arrows.
The sizes of the PCR products are so
0 0.5 1.0 1.5 2.0 (Mb)
small on this scale that their location is
simply indicated. (B) Agarose gel display
(B) MULTIPLEX PCR ANALYSIS of amplified PCR products. “Normal”
DMD patients
indicates a normal male. The lane marked
normal A B C D E F 0 “0” shows a negative control with no
g
h
added DNA.
e
d
i
b
c
f
a
8–79 Duchenne’s muscular dystrophy (DMD) is among the most common

human genetic diseases, affecting approximately 1 in 3500 male births.
One-third of all new cases arise via new mutations. The DMD gene, which
is located on the X chromosome, is greater than 2 million base pairs in
length and contains at least 70 exons. Large deletions account for about
60% of all cases of the disease, and they tend to be concentrated around
two regions of the Problems
gene. p8.36/8.28
The very large size of the DMD gene complicates the analysis of muta-
tions. One rapid approach, which can detect about 80% of all deletions,
is termed multiplex PCR. It uses multiple pairs of PCR primers to amplify
nine different segments of the gene in the two most common regions for
deletions (Figure 8–28A). By arranging the PCR primers so that each pair
gives a different size product, it is possible to amplify and analyze all nine
segments in one PCR reaction. An example of multiplex PCR analysis of
six unrelated DMD males is shown in Figure 8–28B.
A. Describe the extent of the deletions, if any, in each of the six DMD
patients.
B. What additional control might you suggest to confirm your analysis of
patient F?
STUDYING GENE EXPRESSION AND FUNCTION

TERMS TO LEARN
allele green fluorescent protein (GFP)
chromatin immunoprecipitation haplotype block
complementation test phenotype
conditional mutation polymorphism
DNA microarray quantitative RT-PCR
epistasis analysis reverse genetics
genetic screen single-nucleotide polymorphism (SNP)
genetics transgene
genotype transgenic organism
DEFINITIONS
8–80 A search through a large collection of mutants for a mutant with a par-
ticular phenotype.
8–81 One of a number of common sequence variants that coexist in the popu-
lation.
STUDYING GENE EXPRESSION AND FUNCTION 185
8–82 One of a set of alternative forms of a gene.

8–83 The observable character of a cell or an organism.
8–84 Comparing the phenotypes of different combinations of mutations to
determine the order in which the genes act.
8–85 Ancestral chromosome segment that has been inherited with little
genetic rearrangement across generations.
8–86 Animal or plant that has been permanently engineered by gene deletion,
gene insertion, or gene replacement.
8–87 The genetic constitution of an individual cell or organism.
TRUE/FALSE
8–88 In an organism whose genome has been sequenced, identifying the
mutant gene responsible for an interesting phenotype is as easy for
mutations induced by chemical mutagenesis as it is for those generated
by insertional mutagenesis.
8–89 Loss-of-function mutations are usually recessive.
8–90 If two mutations have a synthetic phenotype, it usually means that the
mutations are in genes whose products operate in the same pathway.
THOUGHT PROBLEMS
8–91 Distinguish between the following genetic terms:
A. Locus and allele
B. Homozygous and heterozygous
C. Genotype and phenotype
D. Dominant and recessive
8–92 Explain the difference between a gain-of-function mutation and a dom-
inant-negative mutation. Why are both these types of mutation usually
dominant?
8–93 Discuss the following statement: “We would have no idea today of the
importance of insulin as a regulatory hormone if its absence were not
associated with the human disease diabetes. It is the dramatic conse-
quences of its absence that focused early efforts on the identification of
insulin and the study of its normal role in physiology.”
8–94 What are single-nucleotide polymorphisms (SNPs), and how can they be
used to locate a mutant gene?
8–95 Fatty acid synthase in mammalian cells is encoded by a single gene. This
remarkable protein carries out seven distinct biochemical reactions. The
mammalian fatty acid synthase gene is homologous to seven different
E. coli genes, each of which encodes one of the functions of the mamma-
lian protein. Do you think it is likely that the proteins in E. coli function
together as a complex? Why or why not?
8–96 How does reverse genetics differ from standard genetics?
8–97 The cells in an individual animal contain nearly identical genomes. In
an experiment, a tissue composed of multiple cell types is fixed and sub-
jected to in situ hybridization with a DNA probe to a particular gene. To
your surprise, the hybridization signal is much stronger in some cells
than in others. Explain this result.
(A) Figure 8–29 Site-directed mutagenesis

L R D P Q G G V I (Problem 8–98). (A) Sequence of DNA and
5′ -CTTAGAGACCCGCAGGGCGGCGTCATC- 3′ the encoded protein. (B) Conversion of
normal gene into a mutant gene.
(B)
your gene
CAG
GCC
mutator
oligo
DNA POLYMERASE,
DNA LIGASE
CAG
G C
C
INTRODUCE DNA INTO CELLS

AND CLONE RESULTING VIRUSES
CAG C GG
GTC GCC
wild-type gene mutant gene (Q → R)
8–98 From previous work, you suspect that the glutamine (Q) in the protein
segment in Figure 8–29A plays an important role at the active site. Your
advisor wants you to alter the protein in three ways: change the glu-
tamine to arginine (R) change the glutamine to glycine (G), and delete
the glutamine from the protein. You plan to accomplish these mutational
alterations on a version of your gene that is cloned into M13 viral DNA.
You want to hybridize an appropriate oligonucleotide to the M13 viral
DNA, so that when DNA polymerase extends the oligonucleotide around
the single-stranded M13 circle, it will complete a strand that encodes the
complement of theProblems p8.37/8.29
desired mutant protein. Design three 20-nucleotide-
long oligonucleotides that could be hybridized to the cloned gene on sin-
gle-stranded M13 viral DNA as the first step in effecting the mutational
changes (Figure 8–29B).
8–99 You have just gotten back the results from an RNA-seq analysis of mRNA
from liver. You had anticipated counting the number of reads of each
mRNA to determine the relative abundance of different mRNAs. But you
are puzzled because many of the mRNAs have given you results like those
shown in Figure 8–30. How is it that different parts of an mRNA can be
represented at different levels?
Figure 8–30 RNA-seq reads for a liver

reads mRNA (Problem 8–99). The exon structure
of the mRNA is indicated, with protein-
coding segments indicated in light blue
and untranslated regions in dark blue.
mRNA The numbers of sequencing reads are
indicated by the heights of the vertical
exons 1 2 3 4 5 lines above the mRNA.
brick-red white
DATA HANDLING Figure 8–31 Drosophila with different

color eyes (Problem 8–100). Wild-type
8–100 Early genetic studies in Drosophila laid the foundation for our current flies with brick-red eyes are shown on the
left and white-eyed flies are shown on the
understanding of genes. Drosophila geneticists were able to generate right. Flies with eye colors between red
mutant flies with a variety of easily observable phenotypic changes. and white are shown in between.
Alterations from the fly’s normal brick-red eye color have a venerable
history because the very first mutant found by Thomas Hunt Morgan
was a white-eyed fly (Figure 8–31). Since that time, a large number of
mutant flies with intermediate eye colors haveProblems p8.39/8.31
been isolated and given
names that challenge your color sense: garnet, ruby, vermilion, cherry,
coral, apricot, buff, and carnation. The mutations responsible for these
eye-color phenotypes are recessive. To determine whether the mutations
affected the same or different genes, flies homozygous for each muta-
tion were bred to one another in pairs and the eye colors of their progeny
were noted. In Table 8–6, brick-red wild-type eyes are shown as (+) and
other colors are indicated as (–).
A. How is it that flies with two different eye colors—ruby and white, for
example—give rise to progeny that all have brick-red eyes?
B. Which mutations affect different genes and which mutations are alleles
of the same gene?
C. How can alleles of the same gene give different eye colors? That is to say,
why don’t all the mutations in the same gene give the same phenotype?
8–101 You have designed and constructed a DNA microarray that carries 20,000
allele-specific oligonucleotides (ASOs). These ASOs correspond to the
wild-type and mutant alleles associated with 1000 human diseases. You
have designed the microarray so the ASO that hybridizes to the mutant
allele is located right below the ASO that hybridizes to the same site in
the wild-type sequence. This arrangement is illustrated in Figure 8–32 for
ASOs that are specific for the sickle-cell allele ( S) and the corresponding
TABLE 8–6 Complementation analysis of Drosophila eye-color mutations (Problem 8–100).

MUTATION white garnet ruby vermilion cherry coral apricot buff carnation
White – + + + – – – – +
Garnet – + + + + + + +
Ruby – + + + + + +
Vermilion – + + + + +
Cherry – – – – +
Coral – – – +
Apricot – – +
Buff – +
Carnation –
Brick-red eyes are indicated as (+). Other colors are indicated as (–).
(A) β-GLOBIN ALLELES (B) DNA MICROARRAY Figure 8–32 DNA microarray for detection
of disease alleles (Problem 8–101).
ASOβA (A) The -globin alleles. The wild-type
βA -globin gene ( A) and the sickle-cell
ASOβS allele ( S) are shown. The position of the
βS sickle-cell mutation is shown by a vertical
line. The ASOs are shown as short red
sickle-cell lines arranged above the sites in the
mutation ASOβA gene to which they hybridize. Because
ASOβS
the ASOs are located at corresponding
positions, each is specific for its allele:
the wild-type ASO will not hybridize to
the sickle-cell allele, nor will the sickle-
site in the wild-type allele ( A). ASO S hybridizes to the sickle-cell muta- cell ASO hybridize to the wild-type allele.
tion, and ASO A hybridizes to the corresponding position in the wild-type (B) DNA microarray. A tiny section of
the microarray is enlarged to illustrate the
allele. As a test of your microarray, you carry out hybridizations of DNA locations of the wild-type and sickle-cell
isolated from individuals who are homozygous for the wild-type allele, ASOs.
homozygous for the sickle-cell allele, or heterozygous for the wild-type
and sickle-cell alleles. For each DNA sample, draw the expected patterns
of hybridization to theProblems
globin ASOsp8.40/8.32
on your microarray.
8–102 Now that news of your disease-specific DNA microarray has gotten
around, you are being inundated with requests to analyze various sam-
ples. Just today you received requests from four physicians for help in
the prenatal diagnosis of the same disease. Each of the pregnant mothers
has a family history of this disease. You included on your array the five
alleles known to cause this disease (Figure 8–33). Each of these alleles
is recessive. You agree to help. You prepare samples of fetal DNA gotten
by amniocentesis and hybridize them to your microarrays. Your data
are shown in Figure 8–33C. Assuming that the five disease alleles shown
in Figure 8–33A are the only ones in the human population, decide for
each sample of DNA whether the individual will have the disease or not.
Explain your reasoning.
8–103 You’ve heard about this cool technique for targeted recombination into
embryonic stem (ES) cells that allows you to create a null allele or a con-
ditional allele more or less at the same time. As your friend explained it
to you, you first carry out standard gene targeting into ES cells by homol-
ogous recombination, as shown in Figure 8–34. The Neo gene, which
codes for resistance to the antibiotic neomycin, allows selection for ES
cells that have incorporated the vector. These cells can then be screened
by Southern blotting for those that have undergone a targeted event.
The really cool part is to flank the Neo gene and an adjacent exon or two
(A) DISEASE ALLELES (B) DNA MICROARRAY
1 2 3 4 5
wild type Figure 8–33 DNA microarray analysis
m1 of alleles present in prenatal samples
mutant 1 (Problem 8–102). (A) Wild-type and
m2 disease alleles. Vertical lines indicate the
mutant 2 sites of the mutations in the disease-
m3
causing alleles. The ASOs specific for
mutant 3 the disease mutations are shown as
m4 1 2 3 4 5 wild-type ASOs
red lines and labeled m1, m2, etc. The
mutant 4 m1 m2 m3 m4 m5 mutant ASOs
m5 corresponding ASOs that hybridize to the
mutant 5
wild-type gene at sites that correspond
to the position of the mutations are
labeled 1, 2, etc. (B) DNA microarray. The
arrangement of wild-type and mutant
(C) HYBRIDIZATION DATA ASOs is indicated. (C) Hybridization data.
JC BF HK TW Samples of fetal DNA were hybridized
to DNA arrays. Dark spots indicate sites
where hybridization occurred. Letters
identify the patients.
lox lox lox Figure 8–34 Targeted modification of a

2 3 Neo 4 gene in mouse ES cells (Problem 8–103).
Exons are shown as boxes; the promoter
+ is identified by the horizontal arrow. The
targeting vector is fully homologous to
1 2 3 4 5 the gene, except for the presence of the
three lox sites and the Neo gene, all of
HOMOLOGOUS which are in introns. Cre recombinase,
RECOMBINATION which can be introduced by transfection
of a Cre-expression vector, catalyzes
a site-specific recombination event
lox lox lox between a pair of lox sites in about
1 2 3 Neo 4 5 20% of transfected cells.
Cre RECOMBINASE
with lox sites. This technique is commonly referred to as “floxing.” Once

the modified ES cells have been identified, they can be exposed to the
Cre recombinase, which promotes site-specific recombination between
pairs of lox sites. One advantage is that it allows you to get rid of the Neo
gene and any bacterial DNA segments, which can sometimes influence
the phenotype. Problems p8.43/8.34
A. What possible products might you get from expression of Cre in modified
ES cells that carry three lox sites, as indicated in Figure 8–34?
B. Which product(s) would be a null allele?
C. Which product(s) would have a pair of lox sites but be an otherwise nor-
mal allele?
D. If you had one mouse that expressed Cre under the control of a tissue-
specific promoter, can you use the allele in part C (after you’ve put it into
the germ line of a mouse) as a conditional allele; that is, one whose defect
is expressed only in a particular tissue?
8–104 CRISPR/Cas9–guide RNA complexes hold enormous promise as aids for
genome engineering in plants and animals. The CRISPR system is almost
too good to be true. You want to test just how specific the Cas9–guide
RNA complexes are; that is, whether they really recognize individual sites
in the genome, which is the basis for their touted actions. You realize
that you can test their specificity using the “DNA curtain” assay you have
developed. You make the DNA curtain by tethering single molecules of
bacteriophage lambda DNA (about 50,000 nucleotides) at one end, and
then stretching them in the same direction by flowing buffer across the
slide. You incubate the DNA curtain with a highly fluorescent version of
Cas9—either loaded with the guide RNA or free—and visualize the dis-
tribution of the Cas9 by sensitive fluorescence microscopy, as shown in
Figure 8–35.
A. Phage lambda DNA has a single site that perfectly matches the guide
RNA. Does your experiment support the existence of a single site that is
recognized by the Cas9–guide RNA complex? Explain your answer.
(A)
RNA-loaded Cas9
λ2
5 μm
Figure 8–35 DNA curtain assay for target
binding by Cas9–guide RNA (Problem
(B) 8–104). (A) DNA curtain incubated with
RNA-free Cas9 Cas9–guide RNA that matches one site in
the lambda genome. Pink spots indicate
the sites where Cas9–guide RNA is
5 μm located. (B) DNA curtains incubated with
Cas9 in the absence of guide RNA.
B. One of the main concerns for using the CRISPR system is that the Cas9–
guide RNA will bind to other sites that are related to the intended target
(so-called off-target effects). Is there any evidence for off-target binding
in your experiment? Do you think the results would be any different if you
had a DNA curtain made from human chromosome 1 (about 250,000,000
nucleotides)?
MATHEMATICAL ANALYSIS OF CELL FUNCTIONS

TERMS TO LEARN
robustness
stochastic
DEFINITIONS
8–105 The ability of biological regulatory systems to function normally in the
face of frequent and sometimes extreme variations in external conditions
or the concentrations or activities of key components.
8–106 Describes random variations in protein content of individual cells result-
ing in variations in cell phenotypes.
TRUE/FALSE
8–107 To judge the biological importance of an interaction between protein A
and protein B, we need to know quantitative details about their concen-
trations, affinities, and kinetic behaviors.
8–108 The association constant, Ka, is equal to 1 minus the dissociation con-
stant, Kd; that is, Ka = 1 – Kd.
8–109 The rate of change in the concentration of any molecular species X is
given by the balance between its rate of appearance and its rate of disap-
pearance.
8–110 After a sudden increase in transcription, a protein with a slow rate of deg-
radation will reach a new steady-state level more quickly than a protein
with a rapid rate of degradation.
8–111 The Lac operon, which is turned on by a transcription activator and
turned off by a transcription repressor, is a classic example of an AND
NOT logic gate.
THOUGHT PROBLEMS
8–112 Consider the two network motifs illustrated in Figure 8–36. Both contain
only negative regulation by repressors, and yet one is a negative feedback
(A) (B)
ACTIVATING ACTIVATING
INPUT INPUT
GENE X GENE Y GENE X GENE Y GENE Z

Figure 8–36 Two network motifs
X Y X Y Z composed of transcriptional repressors
(Problem 8–112). (A) A two-gene network.
(B) A three-gene network.
MATHEMATICAL ANALYSIS OF CELL FUNCTIONS 191
(A) (B) (C) (D)

ACTIVATING ACTIVATING ACTIVATING ACTIVATING
INPUT INPUT INPUT INPUT
GENE X GENE X GENE X GENE X
X X X X
GENE Y GENE Y GENE Y GENE Y
Y Y Y Y
GENE Z GENE Z GENE Z GENE Z
Z Z Z Z
loop and the other is a positive feedback loop. Which is the negative and Figure 8–37 Network motifs composed
which is the positive feedback loop? Explain how networks made up of of transcription activators and repressors
(Problem 8–113).
negative regulatory elements can differ in their behavior.
8–113 Examine the network motifs in Figure 8–37. Decide which ones are nega-
tive feedback loops and which are positive. Explain your reasoning.
8–114 Imagine that a random perturbation positions a bistable system
precisely at the boundary between two stable states (at the orange dot in
Figure 8–38). How would the system respond?
8–115 For the network motif shown in Figure 8–39A, decide whether the pulse
of protein X output (Figure 8–39B) requires that the equilibrium constant
for the binding of transcription activator A (KA) must be much greater or
much less than the equilibrium constant for binding of the transcription
repressor R (KR). Explain your reasoning.
(A) SUDDEN (B)

ACTIVATING
1 INPUT
concentration of Y
2
rate of protein synthesis
A A
inactive
33
Figure 8-403
A
Problem 8-310 sudden protein X
activating
fast gene A R slow gene input
activation repression
concentration of X
GENE X
time
Figure 8–38 Perturbations of a bistable
system (Problem 8–114). As shown by the X
green lines, after perturbation 1 the system
returns to its original stable state (green dot
at left), and after perturbation 2, the system
moves to the other stable state (green dot at Figure 8–39 Network motif that generates a pulse of protein X (Problem
right). Perturbation 3 moves the system to 8–115). (A) Regulation of gene X by a transcription activator (green circle) and
the precise boundary between the two stable a transcription repressor (red circle). (B) Pulse of protein X in response to an
states (orange dot). activating input.
CALCULATIONS
8–116 Consider the situation in which a transcription regulator (R) binds to the
promoter (pX) for gene X. If the concentration of a transcription regula-
tor is 100 times 1/K, where K is the equilibrium constant for the binding
of the regulator to the promoter, what percentage of the promoters in a
population of cells will be occupied? What percentage of promoters will
be bound if [R] is equal to 1/K? If [R] is 100-fold less than 1/K? (The equa-
tion for bound fraction is [R:pX]/[pXT ] = K[R]/(1 + K[R]), where [R:pX] is the
complex of the regulator with the promoter and [pXT ] is the total concen-
tration of promoters.)
8–117 What is the molar concentration of a promoter sequence that is present
at one copy per E. coli? Assume E. coli is a rod that is 0.8 μm in diameter
and 3 μm in length. (The equation for the volume of a cylinder is r2h.)
8–118 If the concentration of a transcription repressor is 100 molecules per
E. coli, and it has a single binding site in the bacterial genome, what must
the equilibrium constant be for the binding site to be 99% occupied?
8–119 The Lac repressor regulates the expression of a set of genes for lactose
metabolism, which are adjacent to its binding site on the bacterial chro-
mosome. In the absence of lactose in the medium, the binding of the
repressor turns the genes off. When lactose is added, an inducer is gener-
ated that binds to the repressor and prevents it from binding to its DNA
target, thereby turning on gene expression.
Inside E. coli there are about 10 molecules of Lac repressor (10–8 M)
and 1 binding site (10–9 M) on the bacterial genome. The equilibrium
constant, K, for binding of the repressor to its binding site is 1013 M–1. In
the presence of lactose, when an inducer of gene expression binds to the
repressor, K for repressor binding to its DNA binding sites decreases to
1010 M–1.
A. In a population of bacteria growing in the absence of lactose, what frac-
tion of the binding sites would you expect to be bound by repressor?
B. In bacteria growing in the presence of lactose, what fraction of binding
sites would you expect to be bound by the repressor?
C. Given the information in this problem, would you expect the inducer to
turn on gene expression? Why or why not?
D. The Lac repressor binds nonspecifically to any sequence of DNA with a K
of about 106 M–1, which is a very low affinity. Can you suggest in a qualita-
tive way how such low-affinity, nonspecific binding might alter the cal-
culations in parts A and B and your conclusion in part C?
8–120 Equilibrium dialysis provides a simple method for determining the equi-
librium constant for binding of a ligand (L) by a protein (Pr). The protein
is confined inside a dialysis sac, formed by an artificial membrane with
pores too tiny for the protein to enter, but which the much smaller ligand
can freely permeate. The ligand, usually radiolabeled, is added to the
solution surrounding the dialysis sac; after equilibrium has been estab-
lished, the concentration of the ligand is measured in both compart-
ments. The concentration in the external compartment is the concentra-
tion of free (unbound) ligand and the concentration in the dialysis sac is
the sum of the bound (Pr–L) plus free ligand. By measuring these values
after various initial ligand concentrations, the value of the equilibrium
constant can be determined. By convention, the equilibrium is usually
considered for the dissociation reaction (Pr–L Pr + L), rather than the
association reaction (Pr + L Pr–L), and thus the equilibrium constant
in this case is referred to as the dissociation constant, Kd.
It is useful to look at the transformation of the standard equilibrium
relationship into the form commonly used to analyze the data. At equi-
librium,
MATHEMATICAL ANALYSIS OF CELL FUNCTIONS 193
[Pr][L]
Kd =
[Pr–L] 1.0
Given that the total protein concentration, [Pr]TOT, is the sum of the con-
centration of bound protein [Pr–L] and free protein [Pr], we can substi-
bound/free
tute [Pr]TOT – [Pr–L] for [Pr] and rearrange to give 0.5
[Pr]TOT[L] A B
[Pr–L] =
Kd + [L]
This is an equation for a rectangular hyperbola. It can be rearranged to
0
give a linear form, as was first done by George Scatchard in 1947 (hence, 2 4 6
graphs of such data are commonly known as Scatchard plots): IPTG bound (× 10–7 M)
[Pr–L] – [Pr–L] [Pr]TOT Figure 8–40 Scatchard plot of equilibrium

= + dialysis data for the binding of IPTG to
[L] Kd Kd the Lac repressor (Problem 8–120).
At constant protein concentration and a variety of ligand concentra-
tions, a plot of bound over free ligand ([Pr–L]/[L]) against bound ligand
([Pr–L]) gives a line with slope equal to –1/Kd and an x-intercept equal to
[Pr]TOT, which is the total concentration of binding sites.
In the early 1960s, when the nature of the genetically identified
repressors of bacterial gene expression had not been defined, Walter
Gilbert and Benno Müller-Hill used equilibrium dialysis to measure the
binding of an inducer of gene expression (IPTG) to the Lac repressor
protein. They used radiolabeled IPTG and two partially purified prepa-
rations of Lac repressor protein: one from wild-type cells and the other
from mutant cells in which induction of the lactose operon occurred at
lower concentrations of IPTG. The mutant cells were assumed to carry a
Lac repressor that bound IPTG more tightly. Their data are shown as a
Scatchard plot in Figure 8–40.
A. What are the Kd values for the two lines shown in the figure?
B. Which line corresponds to the wild-type Lac repressor and which to the
mutant (tighter IPTG-binding) repressor?
DATA HANDLING
8–121 Detailed analysis of the regulatory region of the Lac operon has revealed
Figure 3-101
surprising complexity. Instead of a single binding site for the Lac repres-
sor, as might be expected, there are three sites termed operators: O1, O2,
and O3, arrayed along the DNA as shown in Figure 8–41. To probe the Problem 3-26
functions of these three sites, you make a series of constructs in which
various combinations of operator sites are present. You examine their
92 bp 401 bp 2-mer 4-mer
1 O3 O1 O2 110 6700
2 O3 O1 90 3900
3 O1 O2 80 1400
4 O1 60 140
Figure 8–41 Repression of
-galactosidase by promoter regions that
5 O3 O2 1 5 contain different combinations of Lac
repressor binding sites (Problem 8–121).
6 O3 1 2 The base-pair (bp) separation of the
three operator sites is shown. Numbers
7 O2 1 1
at right refer to the level of repression,
with higher numbers indicating more
effective repression by dimeric (2-mer) or
8 1 1 tetrameric (4-mer) repressors.
ability to repress expression of -galactosidase, using either tetrameric

(wild type) or dimeric (mutant) forms of the Lac repressor. The dimeric
form of the repressor can bind to a single operator (with the same affinity
as the tetramer) with each monomer binding to half the site. The tetramer,
the form normally expressed in cells, can bind to two sites simultane-
ously. When you measure repression of -galactosidase expression, you
find the results shown in Figure 8–41, with higher numbers indicating
more effective repression.
A. Which single operator site is the most important for repression? How can
you tell?
B. Do combinations of operator sites (Figure 8–41, constructs 1, 2, 3, and 5)
substantially increase repression by the dimeric repressor? Do combina-
tions of operator sites substantially increase repression by the tetrameric
repressor? If the two repressors behave differently, offer an explanation
for the difference.
C. The wild-type repressor binds O3 very weakly when it is by itself on a seg-
ment of DNA. However, if O1 is included on the same segment of DNA,
the repressor binds O3 quite well. How can that be?
MCAT STYLE
Cancer is caused by aberrant versions of our own genes. Chronic myelogenous
leukemia, for example, is caused by a hybrid chromosome—the Philadelphia
chromosome—that is formed by fusion of broken segments of chromosomes 9
and 22. By chance, this fusion links the Bcr gene from chromosome 22 to the Abl
gene from chromosome 9. The Bcr-Abl fusion gene produces a chimeric protein
that combines the protein kinase activity of Abl with an activating segment of Bcr
to produce a hyperactive Bcr-Abl fusion protein that drives cells of the immune
system to proliferate excessively.
Drugs that specifically inhibit such hyperactive kinases are revolutionizing
cancer treatment. The first example of such a drug—imatinib—was discovered by
searching for compounds that bind and inhibit the Bcr-Abl protein kinase. Ima-
tinib caused rapid disappearance of cancer cells with minimal side effects. Typi-
cally, however, after a few months or years, the cancer returns, this time in a form
that is resistant to the drug. The appearance of drug-resistant cancers is a major
challenge for many inhibitor-based therapies.
8–122 Which of the following techniques would serve best as the basis for a
rapid, highly sensitive blood test to detect circulating cancer cells that
carry the Bcr-Abl gene?
A. DNA sequencing
B. Flow cytometry
C. PCR analysis
D. Western blotting
8–123 You hypothesize that drug resistance is caused by mutations in the fusion
gene that block binding of imatinib to the Bcr-Abl protein. What would
be your first step in testing this hypothesis?
A. Amplify the Bcr-Abl gene by PCR and sequence it to look for mutations in
the gene.
B. Chemically modify imatinib to search for new drugs that will kill the
resistant cells.
C. Determine whether the resistant cancer cells carry the Philadelphia
chromosome.
D. Purify Bcr-Abl protein from resistant cancer cells and test whether it
binds imatinib.
8–124 To test whether imatinib inhibits the activity of the Bcr-Abl kinases from
imatinib-resistant cancer cells from patients, you will need a lot of the
MCAT STYLE 195
Bcr-Abl protein. What would be the most rapid and direct way to obtain a
large amount of the protein?
A. Clone the Bcr-Abl gene from a genomic DNA library into an expression
vector and purify the protein after expression in bacteria.
B. Grow large amounts of drug-resistant cancer cells in culture and isolate
the protein after SDS polyacrylamide-gel electrophoresis.
C. Grow large amounts of drug-resistant cancer cells in culture and purify
the Bcr-Abl protein by column chromatography.
D. Use PCR to amplify the Bcr-Abl gene from cDNA, clone it into an expres-
sion vector, express the protein in bacteria, and then purify it.
8–125 As an alternative hypothesis, you consider that imatinib resistance arises
from mutations outside the coding region of the Bcr-Abl gene that cause
the gene to be expressed at abnormally high levels, thereby reducing the
effectiveness of imatinib. Which one of the following techniques would
you use to most rapidly test this hypothesis?
A. cDNA library analysis
B. In situ hybridization
C. Quantitative RT-PCR
D. Western blotting
8–126 If you find that there are no mutations in the Bcr-Abl coding region and
the gene is not overexpressed, what would you do next to define the basis
for imatinib resistance?
A. Look for mutations in regions neighboring the Bcr-Abl gene.
B. Sequence the genomic DNA from imatinib-resistant cells.
C. Use genetic analysis to identify the genes responsible for resistance.
D. Use genome-wide association studies to identify resistance genes.

Highly regulated proteolysis of cytoplasmic proteins plays an important role in
many cellular events. Biochemical analysis uncovered the molecular machinery
responsible for regulated proteolysis. Early studies established that damaged pro-
teins added to cell lysates were rapidly destroyed in an ATP-dependent manner.
Subsequently, it was shown that a small protein called ubiquitin was covalently
attached to proteins before they were destroyed. Column chromatography was
used to purify the proteins that attach ubiquitin to other proteins. These studies
led to the discovery of a biochemical pathway in which ubiquitin is first activated
via formation of a high-energy thioester bond with a protein called E1. E1 then
passes the ubiquitin to another protein called E2. Finally, ubiquitin is covalently
attached to the target protein. A multiprotein complex called E3 binds to both E2
and the target protein, which ensures that the ubiquitin is attached to the correct
target.
Components of the ubiquitin machinery were independently identified in
genetic screens for genes that regulate the cell cycle. These screens were based on
the hypothesis that mutations in genes necessary for specific steps in the cell cycle
should cause cells to arrest before completion of those steps. Thus, a mutation in a
gene required for chromosome segregation should cause cells to arrest in mitosis
before chromosomes have separated. Mutagenized yeast cells were screened for
mutations that cause cells to arrest at specific points in the cell cycle, which led
to a collection of cell-division control (Cdc) mutants. Three of these mutants—
Cdc16, Cdc23, and Cdc27—arrested in mitosis before chromosome segregation.
Subsequent work found that the protein products of these genes were required
for proteolytic destruction of specific proteins, which triggers chromosome seg-
regation.
8–127 As a first step toward identification of proteins that play a role in ATP-
dependent proteolytic destruction, cell extracts were passed over an
ion-exchange column. The ion-exchange column was then washed with
buffer and eluted with high salt. This resulted in two fractions: proteins
that bound to the column (bound fraction), and proteins that flowed
through the column (unbound fraction). No proteolytic activity could be

detected in either of these fractions, even though the starting extract had
robust activity. What should you do next?
A. Carry out additional purification steps and test for activity.
B. Combine bound and unbound fractions and test for activity.
C. Improve the sensitivity of the assay used to detect proteolysis.
D. Use a gel-filtration column instead of an ion-exchange column.
E. Give up and go home.
8–128 Analysis of the unbound fraction identified the small protein ubiquitin as
a key factor. The investigators then sought to discover additional proteins
in the cell lysates that work with ubiquitin in the proteolytic pathway.
Which one of the following techniques do you suppose they applied?
A. A BLAST search
B. Affinity chromatography
C. Gel-filtration chromatography
D. SDS gel electrophoresis
8–129 To isolate yeast mutants that cannot traverse the cell-division cycle, what
kind of mutation did the investigators need to obtain?
A. Conditional mutation
B. Gain-of-function mutation
C. Loss-of-function mutation
D. Null mutation
8–130 The Cdc16, Cdc23, and Cdc27 mutants displayed identical phenotypes,
which suggested that the encoded proteins work together to execute
common functions. Investigators hypothesized that they exist in a mul-
tiprotein complex. What technique would you use to test this hypothesis
most rapidly?
A. Co-immunoprecipitation
B. Ion-exchange chromatography
C. Nuclear magnetic resonance
D. X-ray diffraction
8–131 Cdc16, Cdc23, and Cdc27 were found to be components of the anaphase-
promoting complex (APC). The APC is an E3 complex that targets specific
proteins for destruction during mitosis. Investigators hypothesized that
the APC triggers chromosome segregation by destroying a protein called
securin, which inhibits chromosome segregation. To test this hypothesis,
they arrested yeast cells in G1 phase to synchronize the cell population.
By releasing the cells from the G1 arrest, they could study a population
of cells going through the cell cycle in synchrony. Starting with the syn-
chronized cells, which one of the following techniques would be most
useful for testing whether securin is proteolytically destroyed during the
cell cycle?
A. Hybridization
B. Immunoprecipitation
C. Mass spectrometry
D. Western blotting
Chapter 9 197
CHAPTER
Visualizing Cells 9
LOOKING AT CELLS IN THE LIGHT MICROSCOPE IN THIS CHAPTER
TERMS TO LEARN LOOKING AT CELLS IN THE

bright-field microscope green fluorescent protein (GFP) LIGHT MICROSCOPE
cell doctrine image processing
confocal microscope ion-sensitive indicator LOOKING AT CELLS AND
dark-field microscope light microscope MOLECULES IN THE ELECTRON
differential-interference-contrast limit of resolution MICROSCOPE
microscope microelectrode
fluorescence microscope phase-contrast microscope
fluorescence recovery after photoactivation
photobleaching (FRAP) superresolution
fluorescence resonance energy
transfer (FRET)
DEFINITIONS
9–1 Fluorescent protein (from a jellyfish) that is widely used as a marker for
monitoring the movements of proteins in living cells.
9–2 The minimal separation between two objects at which they appear dis-
tinct.
9–3 The normal light microscope in which the image is obtained by simple
transmission of light through the object being viewed.
9–4 Computer treatment of images gained from microscopy that reveals
information not immediately visible to the eye.
9–5 Similar to a light microscope but the illuminating light is passed through
one set of filters before the specimen, to select those wavelengths that
excite the dye, and through another set of filters before it reaches the eye,
to select only those wavelengths emitted when the dye fluoresces.
9–6 Type of light microscope that produces a clear image of a given plane
within a solid object. It uses a laser beam as a pinpoint source of illumi-
nation and scans across the plane to produce a two-dimensional optical
section.
9–7 Technique for monitoring the closeness of two fluorescently labeled
molecules (and thus their interaction) in cells.
9–8 Piece of fine glass tubing, pulled to an even finer tip, that is used to inject
electric current into cells.
198 Chapter 9: Visualizing Cells
TRUE/FALSE G
Decide whether each of these statements is true or false, and then explain why. F
9–9 Because the DNA double helix is only 2 nm wide—well below the limit of
resolution of the light microscope—it is impossible to see chromosomes E
in living cells without special stains.

9–10 Monoclonal antibodies are superior to the mixture of antibodies in
standard antisera raised against the same protein because they are abso-
lutely specific for that protein.
9–11 Caged molecules can be introduced into a cell and then activated by a
strong pulse of laser light at the precise time and cellular location chosen
by the experimenter.
9–12 Superresolution techniques allow fluorescently tagged molecules to be D
imaged to accuracies an order of magnitude below the classic diffraction
C
limit to resolution.
B
THOUGHT PROBLEMS
9–13 Examine the diagram of the light microscope in Figure 9–1. Identify and
label the eyepiece, the condenser, the light source, the objective, and the
A
specimen. At what two points in the light path is the image of the speci-
men magnified?
9–14 Why is it important to keep dust, fingerprints, and other smudges off the Figure 9–1 Schematic diagram of a light
lenses of a light microscope? microscope (Problem 9–13).
9–15 When light enters a medium with a different optical density, it bends in
a direction that depends on the refractive indices of the two media. Air
and glass, for example, have refractive indices of 1.00 and 1.51, respec-
tively. When light enters glass—the medium with the higher refractive Problems p9.01/9.01
index—it bends toward a line drawn normal to the surface (Figure 9–2A).
Conversely, when light exits glass into air, it bends away from the normal
line (Figure 9–2B). Using these principles, draw the paths of two parallel
light rays that pass through the hemispherical glass lens shown in Figure
9–2C. Will the two rays converge or diverge? Would the result be any dif-
ferent if the glass lens were flipped so that light entered the flat surface?
9–16 The diagrams in Figure 9–3 show the paths of light rays passing through
a specimen with a dry lens and with an oil-immersion lens. Offer an
(A) normal (B) normal
air
glass
glass
air DRY LENS OIL-IMMERSION LENS
(C)
glass objective
air lens
air oil
cover slip
Figure 9–2 Refraction of light at air–glass
slide
interfaces (Problem 9–15). (A) A ray of light
passing from air to glass. (B) A ray of light
passing from glass to air. (C) Two parallel rays Figure 9–3 Paths of light rays through dry and oil-immersion lenses (Problem
of light entering a glass lens. 9–16). The red circle at the origin of the light rays is the specimen.
Problems p9.02/9.02
LOOKING AT CELLS IN THE LIGHT MICROSCOPE 199
Figure 9–4 Diagram of the human eye

iris (Problem 9–17).
vitreous
humor retina
cornea
lens
aqueous
humor
explanation for why oil-immersion lenses should give better resolution.

Air, glass, and oil have refractive indices of 1.00, 1.51, and 1.51, respec-
tively. Problems p9.04/9.04
9–17 Figure 9–4 shows a diagram of the human eye. The refractive indices of
the components in the light path are: cornea 1.38, aqueous humor 1.33,
crystalline lens 1.41, and vitreous humor 1.38. Where does the main
refraction—the main focusing—occur? What role do you suppose the
lens plays?
9–18 Why do humans see so poorly under water? And why do goggles help?
9–19 Reading through a beaker filled with clear glass balls is impossible
(Figure 9–5). Do you suppose it would help to fill the beaker with water?
With immersion oil? Explain your reasoning.
Figure 9–5 Viewing an eye chart through
9–20 Examine the four photomicrographs of the same cell in Figure 9–6. a beaker filled with clear glass balls
Match each image to the technique listed below. (Problem 9–19).
1. Bright-field microscopy
2. Dark-field microscopy Problems p9.05/9.05
3. Nomarski differential-interference-contrast microscopy
4. Phase-contrast microscopy
9–21 Explain the difference between resolution and magnification.
9–22 Many fluorescent dyes that stain DNA require excitation by ultraviolet
light. Hoechst 33342, for example, binds to DNA and absorbs light at 352
nm and emits at 461 nm. If you want to use this membrane-permeant dye
in living cells, do you have to worry about ultraviolet light damage to the Figure 9–6 Four photomicrographs of the
DNA, which absorbs light maximally at around 260 nm? Why or why not? same cell (Problem 9–20).
(A) (B)
(C) (D)
50 µm
(A)
(B) +
CH3NH N +
NH
1.5
absorbance or emission
barrier +
barrier N NH
filter H
filter N
H
1.0
OCH2CH3
light
source 0.5
beam-splitter
absorbance emission
0
280 320 360 400 440 480 520 560 600
wavelength (nm)
Figure 9–7 Fluorescence microscopy (Problem 9–24). (A) The light path through a fluorescence microscope. (B) Absorption
and fluorescence emission spectra of Hoechst 33342 bound to DNA. The structure of Hoechst 33342 is shown above the
spectra.
9–23 Why is it, do you suppose, that a fluorescent molecule, having absorbed
a single photon of light at one wavelength, always emits a photon at a
longer wavelength?
9–24 A fluorescence microscope, which is shown schematically in Figure
9–7A, uses two filters and a beam-splitting (dichroic) mirror to excite the
sample and capture the emitted fluorescent light. Imagine that you wish
to view the fluorescence of a sample that has been stained with Hoechst
33342, a common stain for DNA. The absorption and fluorescence emis-
sion spectra for Hoechst 33342 are shown in Figure 9–7B.
A. Which of the following commercially available barrier filters would you
select to place between the light source and theProblems p9.07/9.07
sample? Which one
would you place between the sample and the eyepiece?
1. A filter that passes wavelengths between 300 nm and 380 nm.
2. A filter that passes all wavelengths above 420 nm.
B. How would you design your beam-splitting mirror? Which wavelengths
would be reflected and which would be transmitted?
9–25 Antibodies that bind to specific proteins are important tools for defin-
ing the locations of molecules in cells. The sensitivity of the primary
antibody—the antibody that reacts with the target molecule—is often
enhanced by using labeled secondary antibodies that bind to it. What
are the advantages and disadvantages of using secondary antibodies that
carry fluorescent tags versus those that carry bound enzymes?
9–26 The green fluorescent protein (GFP) was isolated as a cDNA from a spe-
cies of jellyfish that glows green. When the cDNA for GFP was introduced
into bacteria, the colonies they formed glowed pale green under ultra-
violet light. In these early studies, the following pertinent observations
provided important insights into how GFP becomes fluorescent.
1. When bacteria are grown anaerobically, they express normal amounts of
GFP but it is not fluorescent.
2. The denatured GFP found in insoluble protein aggregates (inclusion
bodies) in bacteria is not fluorescent.
3. The rate of appearance of fluorescence follows first-order kinetics, with a
time constant that is independent of the concentration of GFP.
Figure 9–8 A rainbow of colors produced

by modified GFPs (Problem 9–27).
4. Random mutations introduced into the cDNA coding for GFP produced
some proteins with appreciably brighter fluorescence and some with dif-
ferent colors.
Comment on what each of these observations says about GFP fluores-
cence. Problems p9.08/9.08
9–27 Figure 9–8 shows a series of modified GFPs that emit light in a range of
colors. How do you suppose the exact same chromophore can fluoresce
at so many different wavelengths?
CALCULATIONS
9–28 The resolving power of a light microscope depends on the width of the
cone of light that illuminates the specimen, the wavelength of the light
used, and the refractive index of the medium separating the specimen
from the objective and condenser lenses, according to the formula
resolution = 0.61
n sin
where equals the wavelength of light used, n is the refractive index, and
is half the angular width of the cone of rays collected by the objective
lens. Assuming an angular width of 120° ( = 60°), calculate the various
resolutions you would expect if the sample were illuminated with violet
light ( = 0.4 μm) or red light ( = 0.7 μm) in a refractive medium of air
(n = 1.00) or oil (n = 1.51). Which of these conditions would give you the
best resolution?
9–29 At a certain critical angle, which depends on the refractive indices of
the two media, light from the optically denser medium will be bent suf-
ficiently that it cannot escape and will be reflected back into the denser
medium. The formula that describes refraction is
ni sin i = nt sin t
where ni and nt are the refractive indices of the incident and transmit-
ting media, respectively, and i and t are the incident and transmitted
angles, respectively, as shown in Figure 9–9A. For a glass–air interface,
calculate the incident angle for a light ray that is bent so that it is parallel
to the surface (Figure 9–9B). The refractive indices for air and glass are
1.00 and 1.51, respectively.
(A) (B)
θt
θt
nt air Figure 9–9 Refraction at the interface
between media with different refractive
ni glass
indices (Problem 9–29). (A) The
relationship between incident and
θi
θi transmitted angles for materials with
different refractive indices such that
ni > nt. (B) The incident angle for a
glass–air interface such that the
transmitted (bent) ray is parallel to
the interface.
TABLE 9–1 Excitation properties of a few common biological

fluorophores (Problem 9–30).
Fluorophore Opea absorption Tpeb absorption Emission
maximum (nm) maximum (nm) maximum (nm)
DAPI 358 685 461
ER-Tracker 374 728 575
Mito-Tracker 579 1133 599
Alexa Fluor 488 491 985 515
FITC-IgG 490 947 525
aOne-photon excitation; bTwo-photon excitation.
DATA HANDLING
9–30 Fluorescent molecules can be excited by a single high-energy photon or
by multiple lower-energy photons. A list of commonly used biological
fluorophores is given in Table 9–1. Why do you suppose that the absorp-
tion maximum for two-photon excitation is about twice that for one-pho-
ton excitation?
9–31 You wish to attach fluorescent tags to two different proteins so that you
can follow them independently. The excitation and emission spectra for
cyan, green, and yellow fluorescent proteins (CFP, GFP, and YFP) are
shown in Figure 9–10. Can you pick any pair of these proteins? Or are
some pairs better than others? Explain your answer.
9–32 Consider a fluorescent detector designed to report the cellular location
of active protein tyrosine kinases. A blue (cyan) fluorescent protein (CFP)
and a yellow fluorescent protein (YFP) were fused to either end of a hybrid
EXCITATION
CFP GFP YFP
percent of maximum
EMISSION
CFP GFP YFP
percent of maximum
Figure 9–10 Excitation and emission

400 450 500 550 600 spectra for CFP, GFP, and YFP
wavelength (nm) (Problem 9–31).
(A) REPORTER (B) FRET Figure 9–11 Fluorescent reporter

434 nm protein designed to detect tyrosine
+ phosphatase
476 nm 1.3 phosphorylation (Problem 9–32).
CF Abl + ATP (A) Domain structure of reporter protein.
P Four domains are indicated: CFP, YFP,
1.2 tyrosine kinase substrate peptide, and
YFP/CFP
substrate a phosphotyrosine-binding domain.
peptide (B) FRET assay. YFP/CFP is normalized
1.1 to 1.0 at time zero. The reporter was
omit Abl or ATP
incubated in the presence (or absence)
YFP of Abl and ATP for the indicated times.
phosphotyrosine- 1.0
binding protein
Arrow indicates time of addition of a
0 5 10 15 20 25 30 tyrosine phosphatase.
time (hours)
protein domain. The hybrid protein segment consisted of a substrate

peptide recognized by the Abl protein tyrosine kinase and a phosphoty-
rosine-binding domain (Figure 9–11A). Stimulation of the CFP domain
does not cause emission by the YFP domain when the domains are sepa-
rated. When the CFP and YFP domains are brought close together, fluo-
rescence resonance energy transfer (FRET) allows excitation of CFP to
Problems p9.11/9.11
stimulate emission by YFP. FRET shows up experimentally as an increase
in the ratio of emission at 526 nm versus 476 nm (YFP/CFP) when CFP is
excited by 434 nm light.
Incubation of the reporter protein with Abl protein tyrosine kinase
in the presence of ATP gave an increase in YFP/CFP emission (Figure
9–11B). In the absence of ATP or the Abl protein, no FRET occurred.
FRET was also eliminated by addition of a tyrosine phosphatase (Figure
9–11B). Describe as best you can how the reporter protein detects active
Abl protein tyrosine kinase.
9–33 Cells activate Abl protein tyrosine kinase in response to platelet-derived
growth factor (PDGF). PDGF binds to the PDGF receptor, which activates
Src, which then activates Abl. It is unclear where in the cell Abl is active,
but one of the consequences of PDGF stimulation is the appearance of
membrane ruffles. To investigate this question, the reporter construct
described in Problem 9–32 was transfected into cells, which were then
stimulated by addition of PDGF. Using fluorescence microscopy, YFP
emission in response to CFP excitation (FRET) was followed in different
parts of the cell, with the results shown in Figure 9–12. What can you
infer about the cellular distribution of active Abl protein tyrosine kinase
in response to PDGF?
9–34 You are using a cameleon indicator to measure intracellular concentra-
tions of Ca2+. The indicator is composed of a central calmodulin domain,
which converts from an extended form to a much more compact form
upon calcium binding, and two flanking fluorescent proteins, with CFP
2.3
ruffles
2.2
2.1 cytoplasm
YFP/CFP
2.0
Figure 9–12 Time course of FRET in
various parts of the cell after addition
1.9
nucleus of PDGF (Problem 9–33). FRET was
measured as the increase in the ratio of
1.8
emission at 526 nm to that at 476 nm
0 10 20 30 40 50 60 (YFP/CFP) when CFP was excited by
time after adding PDGF (minutes) 434 nm light.
Time (milliseconds) Figure 9–13 TIRF microscopy of cells

0 300 600 900 1200 1500 expressing clathrin–GFP (Problem 9–35).
Each frame in this time course represents
300 msec. The yellow arrow indicates a
dot that disappears during the 1.5 sec
observation period. Green dots appear
white in this image.
attached at one end and YFP attached at the other. You have expressed
this indicator in cells and now wish to measure intracellular changes in
Ca2+ concentration in response to the biological process you are study-
ing. The instructions say to excite the cameleon at 440 nm and to measure
emission at 535 nm. How do you suppose the cameleon indicator works?
9–35 The formation of clathrin-coated vesicles is initiated by the assembly of a
basketlike framework of clathrin on the underside of the cell membrane,
which distorts the membrane into a shallow pit. These clathrin-coated
pits ultimately pinch off to form clathrin-coated vesicles. You want to
understand how pits are converted into vesicles, and have engineered a
Problems
cell line that expresses clathrin–GFP. p9.201/9.13
To focus on events at the membrane
surface, you use total internal reflection fluorescence (TIRF) microscopy
to examine the cell membrane and a thin slice of adjacent cytosol. You
are excited to find that the samples show many green dots under the
microscope and that some dots disappear over time (Figure 9–13).
A. Why do you suppose some dots remain unchanged, while others disap-
pear?
B. You are concerned that the green dots may not be functional assemblies,
but some kind of artifact. A colleague suggests that you test whether the
green dots really represent clathrin-coated structures by incubating the
cells with transferrin that is tagged with a red fluorophore. Transferrin,
which carries iron ions, binds to its receptors on the cell surface and is
taken into the cell in clathrin-coated vesicles. What might you expect to
see if you carried out this experiment?
LOOKING AT CELLS AND MOLECULES IN THE

ELECTRON MICROSCOPE
TERMS TO LEARN
cryoelectron microscopy negative staining
electron microscope (EM) scanning electron microscope (SEM)
electron-microscope tomography single-particle reconstruction
immunogold electron microscopy
DEFINITIONS
9–36 A contrast-enhancing technique for the electron microscope in which
a heavy-metal salt is used to create a reverse, or negative, image of the
object.
9–37 Type of microscope that uses a beam of electrons to create an image.
9–38 Electron microscopy technique in which the objects to be viewed, such
as macromolecules and viruses, are rapidly frozen.
9–39 Type of electron microscope that produces an image of the surface of an
object.
LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE 205
9–40 Electron microscopy technique in which cellular structures or molecules

of interest are labeled with antibodies tagged with electron-dense gold
particles, which show up as black spots on the image.
TRUE/FALSE
Decide whether this statement is true or false, and then explain why.
9–41 Transmission electron microscopy (TEM) and scanning electron micros-
copy (SEM) can both be used to examine a structure in the interior of a
thin section; TEM provides a projection view, while SEM captures elec-
trons scattered from the structure and gives a more three-dimensional
view.
THOUGHT PROBLEMS
9–42 A major challenge to electron microscopists from the beginning was to
convince others that what they observed in micrographs truly reflected
structures that were originally present in the living cell. Outline a current
approach to this problem.
9–43 The technique of negative staining uses heavy metals such as uranium to
provide contrast. If the heavy metals don’t actually bind to defined bio-
logical structures (which they don’t), how is it that they can help to make
such structures visible?
9–44 Heavy metals can be used to highlight the surface features of a sample.
In the technique called metal shadowing, a heavy metal such as plati-
num is evaporated at a shallow angle onto the specimen so as to deposit
a thin film. The thickness of the film depends on the surface features of
the sample and on their orientation relative to the source of platinum. By
stabilizing the metal film and eliminating the original specimen, it is pos-
sible to image the metal replica of the sample by transmission electron
microscopy, as shown in the micrographs in Figure 9–14.
It is sometimes difficult to tell bumps from pits in such micrographs
just by looking at the pattern of shadows, as illustrated in Figure 9–14 for
a set of shaded circles. In Figure 9–14A, the circles appear to be bumps;
however, when the picture is simply turned upside down (Figure 9–14B),
the circles seem to be pits. This is a classic illusion. The same illusion is
present in metal shadowing, as shown in Figure 9–14C and D. In one
micrograph, the membrane appears to be covered in bumps, while in the
same micrograph, turned upside down, the membrane looks heavily pit-
ted. Is it possible for an electron microscopist to be sure that one view is
correct, or is it all arbitrary? Explain your reasoning.
(A) (B) (C) (D)
Figure 9–14 Bumps and pits (Problem 9–44). (A) Shaded circles that look like bumps. (B) Shaded circles that look like
pits. (C) An electron micrograph oriented so that it appears to be covered with bumps. (D) The same electron micrograph
oriented so that it appears to be covered with pits.
Problems p9.13/9.14
9–45 Nuclear pore complexes, which are large assemblages of more than 30
different proteins, mediate the exchange of macromolecules between
the nucleus and cytoplasm. You have gently isolated nuclei from Dic-
tyostelium discoideum and frozen them in vitreous ice for examination
by cryoelectron tomography. You obtain a tomogram of a nucleus and
combine the images from 267 nuclear pore complexes. You expect that
individual nuclei will have been arrested in different states of transport
because the isolated nuclei were shown to be competent for transport.
Assuming that parts of the structure flex and move during the transport
process, how do you suppose that averaging structures in different states
will affect your final picture? Can you think of a way to improve the qual-
ity of the image you would get from this data set?
CALCULATIONS
9–46 The practical resolving power of modern electron microscopes is around
0.1 nm. The major reason for this constraint is the small numerical aper-
ture (n sin ), which is limited by (half the angular width of rays col-
lected at the objective lens). Assuming that the wavelength ( ) of the
electron is 0.004 nm and that the refractive index (n) is 1.0, calculate the
value for . How does that value compare with a of 60°, which is typical
for light microscopes?
resolution = 0.61
n sin
DATA HANDLING
9–47 You are studying two proteins that you think may be components of gap
junctions, which are structures in membranes that allow adjacent cells to
exchange small molecules. When cells are very rapidly frozen and then
cracked with a knife blade, the cells often fracture along membrane sur-
faces. When the freeze-fractured cells are viewed in the electron micro-
scope, gap junctions show up clearly as specialized areas densely pop-
ulated with membrane particles. To decide whether your proteins are
components of gap junctions, you have prepared antibodies against each
of them. To one antibody you’ve attached 15 nm gold particles; to the
other, 10 nm gold particles. You prepare freeze-fractured cells for elec-
tron microscopy and then incubate them with your gold-tagged antibod-
ies, with the results shown in Figure 9–15. Do these results indicate that
both proteins are part of gap junctions? Why or why not?
Figure 9–15 A freeze-fracture electron

micrograph of a gap junction (Problem
9–47). The central densely packed area
outlined in white is the gap junction. The
two leaflets of the plasma membrane are
indicated as EF (for external face) and
PF (for protoplasmic face). Gold particles
200 nm show up as black dots.
LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE 207
9–48 Aquaporin water channels, which are known to be located in the plasma
membrane, play a major role in water metabolism and osmoregulation
in many cells, including nerve cells of the brain and spinal cord. To deter-
mine their structural organization in the membrane, you have prepared a
highly specific antibody against the protein AQP4, which forms the water
channels in the astrocytes of the brain. You prepare a freeze-fractured
sample from the brain, incubate it with gold-tagged antibodies against
AQP4, and examine it by electron microscopy (Figure 9–16).
A. Are the gold particles (black dots) consistently associated with any par-
ticular structure?
B. Are there any examples of black dots that are not associated with these
structures? Are there any examples of structures that do not have black
dots? How do your answers to these questions affect your confidence
that the structure you’ve identified is the aquaporin water channel?
MCAT STYLE
Passage (Questions 9–49 to 9–51)
Many key advances in cell biology were dependent upon technical advances in
microscopy. This was particularly true in the case of the cytoskeleton, the dynamic
network of cytoplasmic fibers that is responsible for cell motility and chromosome
movement during mitosis. The two key components of the cytoskeleton—actin
filaments and microtubules—assemble by polymerization of individual subunits. 100 nm
Actin filaments and microtubules were first seen by electron microscopy, which Figure 9–16 Freeze-fracture micrograph
showed that they have diameters of 7 nm and 25 nm, respectively, and can form of an astrocyte membrane labeled with
very long polymers. Later development of light microscopy techniques for imag- gold-tagged antibodies against AQP4
ing individual microtubules and actin filaments under natural conditions revolu- (Problem 9–48).
tionized the field.
Problems p9.15/9.16
9–49 Imagine that you are developing techniques to visualize individual actin
filaments by light microscopy for the first time. Which of the following
approaches would you choose?
I. Assemble actin filaments from fluorescently tagged subunits and image
them with a fluorescence microscope.
II. Visualize actin filaments by using differential-interference-contrast
microscopy.
III. Combine standard light microscopic techniques with digital image pro-
cessing to enhance resolution of actin filaments.
A. I
B. II
C. III
D. I, II, and III
9–50 What would be the apparent diameter of actin filaments imaged using
the approach or approaches you selected in the previous question?
A. 7 nm
B. 50 nm
C. 100 nm
D. 200 nm
9–51 Microscopy has also played a key role in answering mechanistic ques-
tions about how enzymes work. Consider, for example, ATP synthase, the
remarkable multisubunit enzyme responsible for ATP synthesis in the
inner mitochondrial membrane. The structure of ATP synthase revealed
a central stalk surrounded by other subunits that form a ring 10 nm in
diameter. It was hypothesized that rotation of the stalk relative to the ring
subunits—powered by proton flow across the inner membrane—gener-
ated sufficient mechanical energy to force the conformational changes
needed to drive ATP synthesis. You would like to test this hypothesis by
directly visualizing rotation of the stalk relative to the ring. Which one of
the following ideas do you think would be the best approach for detect-
ing rotation of the stalk?
A. Attach a 2 μm-long actin filament assembled from fluorescent subunits
to the stalk and view its rotation by fluorescence microscopy.
B. Attach green fluorescent protein (GFP) to the proteins in the ring and use
fluorescence microscopy to observe rotation of the ring around the stalk.
C. Attach GFP to the stalk and RFP (red fluorescent protein) to the ring and
observe their relative rotation by fluorescence microscopy.
D. Attach multiple fluorescently labeled antibodies to the stalk and then
observe rotation of the stalk by fluorescence microscopy.
Chapter 10 209
CHAPTER
Membrane Structure 10
THE LIPID BILAYER IN THIS CHAPTER
TERMS TO LEARN THE LIPID BILAYER

amphiphilic hydrophobic liposome MEMBRANE PROTEINS
cholesterol lipid bilayer phosphoglyceride
ganglioside lipid droplet phospholipid
glycolipid lipid raft plasma membrane
hydrophilic
DEFINITIONS
Match the definition below with its term from the list above.
10–1 Artificial phospholipid bilayer vesicle formed from an aqueous suspen-
sion of phospholipid molecules.
10–2 Small region of the plasma membrane enriched in sphingolipids and
cholesterol.
10–3 Any glycolipid having one or more sialic acid residues in its structure;
especially abundant in the plasma membranes of nerve cells.
10–4 Having both hydrophobic and hydrophilic regions, as in a phospholipid
or a detergent molecule.
10–5 The main type of phospholipid in animal cell membranes, with two fatty
acids and a polar head group attached to a three-carbon glycerol back-
bone.
10–6 Lipid molecule with a characteristic four-ring steroid structure that is an
important component of the plasma membranes of animal cells.
TRUE/FALSE
10–7 Although lipid molecules are free to diffuse in the plane of the bilayer,
they cannot flip-flop across the bilayer unless enzyme catalysts called
phospholipid translocators are present in the membrane.
10–8 All of the common phospholipids—phosphatidylcholine, phosphati-
dylethanolamine, phosphatidylserine, and sphingomyelin—carry a pos-
itively charged moiety on their head group, but none carry a net positive
charge.
10–9 Glycolipids are never found on the cytoplasmic face of membranes in
living cells.
210 Chapter 10: Membrane Structure
CH3 H H Figure 10–1 Icelike cage of water

O
molecules around a hydrophobic solute
C H (Problem 10–10).
H H H
H 3C H
CH3 O
O
H
H
2-methyl propane O
O
H
H
_ O O
δ H H H H
O O
H H H H
δ+ δ+
water 2-methyl propane in water
THOUGHT PROBLEMS
10–10 Hydrophobic solutes are said to “force the adjacent water molecules to
reorganize into icelike cages” (Figure 10–1). This statement seems para-
doxical because water molecules do not interact with hydrophobic sol-
utes. How could water molecules “know” about the presence of a hydro-
phobic solute and change their behavior to interact differently with one
another? Discuss this seeming paradox and develop a clear concept of
what is meant by an “icelike” cage. How does it compare to ice? Why
would such a cagelike structure be energetically unfavorable relative to
pure water?
10–11 When a lipid bilayer is torn, why does it not seal itself by forming a “hemi-
Problems p10.01/10.01
micelle” cap at the edges, as shown in Figure 10–2?
10–12 Five students in your class always sit together in the front row. This could
be because (1) they really like each other or (2) nobody else in your class
wants to sit next to them. Which explanation holds for the assembly of a
lipid bilayer? Explain your answer. If the lipid bilayer assembled for the
opposite reason, how would its properties differ?
10–13 Predict the properties of a lipid bilayer in which all of the hydrocarbon
chains were saturated. What would be the properties if all of the hydro-
carbon chains were unsaturated?
10–14 What is meant by the term “two-dimensional fluid”?
10–15 Margarine is made from vegetable oil by a chemical process. Do you sup-
pose this process converts saturated fatty acids to unsaturated ones, or
vice versa? Explain your answer.
10–16 Which one of the phospholipids listed below is present in very small
quantities in the plasma membranes of mammalian cells?
A. Phosphatidylcholine
B. Phosphatidylethanolamine
C. Phosphatidylinositol
D. Phosphatidylserine
E. Sphingomyelin
tear in bilayer
seal with hemi-micelle cap

Figure 10–2 A torn lipid bilayer sealed
with a hypothetical “hemi-micelle” cap
(Problem 10–11).
THE LIPID BILAYER 211
10–17 Many snake venoms contain enzymes that cause red blood cells to lyse.
Imagine that you have purified such an enzyme. When you add the puri-
fied enzyme to red blood cells, you find that in addition to cell lysis,
choline with a phosphate group attached to it is released, as well as dia-
cylglycerol (glycerol with two fatty acid chains attached). What molecule
is cleaved by the enzyme to cause cell lysis?
10–18 Predict which of the following organisms will have the highest percent-
age of unsaturated fatty acid chains in their membranes. Explain your
answer.
A. Antarctic fish
B. Desert iguana
C. Human being
D. Polar bear
E. Thermophilic bacterium
10–19 If lipid rafts form because membrane components such as sphingolipids
and cholesterol molecules preferentially associate with one another, why
do you think it is that they aggregate into multiple tiny rafts instead of into
a single large one?
10–20 The lipid bilayers found in cells are fluid, yet asymmetrical in the compo-
sition of the monolayers. Is this a paradox? Explain your answer.
10–21 Phosphatidylserine, which is normally confined to the cytoplasmic mon-
olayer of the plasma membrane lipid bilayer, is redistributed to the outer
monolayer during apoptosis. How is this redistribution accomplished?
CALCULATIONS
10–22 If a lipid raft is typically 70 nm in diameter and each lipid molecule has
a diameter of 0.5 nm, about how many lipid molecules would there be
in a lipid raft composed entirely of lipid? At a ratio of 50 lipid molecules
per protein molecule (50% protein by mass), how many proteins would O
be in a typical raft? (Neglect the loss of lipid from the raft that would be
required to accommodate the protein.) H N H
H H
DATA HANDLING
nitroxide
10–23 A classic paper studied the behavior of lipids in the two monolayers of radical
a membrane by labeling individual molecules with nitroxide groups,
which are stable free radicals (Figure 10–3). These spin-labeled lipids O
can be detected by electron spin resonance (ESR) spectroscopy, a tech- H

H
N H
H
nique that does not harm living cells. Spin-labeled lipids are introduced
into small lipid vesicles, which are then fused with cells, thereby transfer-
ring the labeled lipids into the plasma membrane. phospholipid 1
The two spin-labeled phospholipids shown in Figure 10–3 were incorpo-

rated into intact human red blood cell membranes in this way. To deter-
mine whether they were introduced equally into the two monolayers of
the bilayer, ascorbic acid (vitamin C), which is a water-soluble reduc-
ing agent that does not cross membranes, was added to the medium to
destroy any nitroxide radicals exposed on the outside of the cell. The ESR phospholipid 2
H
H
signal was followed as a function of time in the presence and absence of

O
ascorbic acid as indicated in Figure 10–4A and B.

H
H
A. Ignoring for the moment the difference in extent of loss of ESR signal,
offer an explanation for why phospholipid 1 (Figure 10–4A) reacts faster Figure 10–3 Structures of two nitroxide-
labeled lipids (Problem 10–23). The
with ascorbate than does phospholipid 2 (Figure 10–4B). Note that phos- nitroxide radical is shown at the top,
pholipid 1 reaches a plateau in about 15 minutes, whereas phospholipid and its position of attachment to the
2 takes almost an hour. phospholipids is shown below.
(A) PHOSPHOLIPID 1 – RED CELLS (B) PHOSPHOLIPID 2 – RED CELLS Figure 10–4 Decrease in ESR signal
intensity as a function of time in red cells
and red cell ghosts in the presence and
O
H N H
H H
absence of ascorbate (Problem 10–23).
H
H
(A and B) Phospholipid 1 and
N
– ascorbate
signal intensity (%)
H
H
100 100 phospholipid 2 in red cells. (C and D)
Phospholipid 1 and phospholipid 2 in
75 75 red cell ghosts.
− ascorbate
+ ascorbate
50 50
25 25
+ ascorbate
0 0
0 10 20 30 0 1 2 3
(C) PHOSPHOLIPID 1 – GHOSTS (D) PHOSPHOLIPID 2 – GHOSTS
− ascorbate − ascorbate
100 100
75 75
+ ascorbate + ascorbate
50 50
25 25
0 0
0 10 20 30 0 1 2 3
time (minutes) time (hours)
B. To investigate the difference in extent of loss of ESR signal with the two
phospholipids, the experiments were repeated using red cell ghosts that
had been resealed to make them impermeable to ascorbate (Figure
10–4C and D). Resealed red cell ghosts are missing all of their cytoplasm,
but have an intact plasma membrane. In these experiments, the loss of
ESR signal for both phospholipids was negligible in the absence of ascor-
bate and reached a plateau at 50% in the presence of ascorbate. What
do you suppose might account for the difference in extent of loss of ESR
signal in experiments with red cell ghosts (Figure 10–4C and D) versus
those with normal red cells (Figure 10–4A and B).
C. Were the spin-labeled phospholipids introduced equally into the two
monolayers of the red cell membrane?
10–24 Fluorescence resonance energy transfer
Problems (FRET) has been used to inves-
p10.04/10.04
tigate the existence of lipid rafts in living cells. To test for the presence
of lipid rafts by FRET, youProblem
use two different
A10-08 cell lines: one that expresses
a glycosylphosphatidylinositol (GPI)-anchored form of the folate recep-
tor, and one that expresses the transmembrane-anchored form. Folate
receptors can be made fluorescent by addition of a fluorescent folate
analog. Cells tagged in this way show variation in fluorescence intensity
over their surface because of chance variations in the density of labeled
receptors. This allows different densities of receptors to be analyzed by
examining different places in the same cell. The proximity of labeled
receptors can be determined by FRET, which depends on the distance
between receptors. The ratio of FRET to direct fluorescence gives differ-
ent expectations for dispersed receptors versus receptors that are clus-
tered together, as depicted in Figure 10–5.
A. Explain the basis for the difference between the graphs of these expecta-
tions.
THE LIPID BILAYER 213
(A) RANDOM DISTRIBUTION
high density low density
density
FRET/total
(B) NONRANDOM DISTRIBUTION
high density low density
density
FRET/total
Figure 10–5 Expectations for FRET between tagged folate receptors at different
densities of receptors (Problem 10–24). (A) Randomly distributed receptors. The
red dots in the box represent fluorescent receptors. (B) Receptors clustered in
microdomains. Circles represent microdomains such as lipid rafts. Red dots
represent fluorescent receptors.
Problems
B. The actual experiments showedp10.06/10.05
that transmembrane-anchored folate
receptors followed the expectations shown in Figure 10–5A, whereas
the GPI-anchored folate receptors followed those in Figure 10–5B. Do
these experiments provide evidence for the existence of lipid rafts in the
plasma membrane? Why or why not?
10–25 The asymmetric distribution of phospholipids in the two monolayers
of the plasma membrane implies that very little spontaneous flip-flop
occurs or, alternatively, that any spontaneous flip-flop is rapidly cor-
rected by phospholipid translocators that return phospholipids to their
appropriate monolayer. The rate of phospholipid flip-flop in the plasma
membrane of intact red blood cells has been measured to decide between
these alternatives.
One experimental measurement used the same two spin-labeled phos-
pholipids described in Problem 10–23 (see Figure 10–3). To measure the
rate of flip-flop from the cytoplasmic monolayer to the outer monolayer,
red cells with spin-labeled phospholipids exclusively in the cytoplasmic
monolayer were incubated for various times in the presence of ascorbate
and the loss of ESR signal was followed. To measure the rate of flip-flop
from the outer to the cytoplasmic monolayer, red cells with spin-labeled
phospholipids exclusively in the outer monolayer were incubated for
outside
various times in the absence of ascorbate and the loss of ESR signal was 100
followed. The results of these experiments are illustrated in Figure 10–6.

A. From the results in Figure 10–6, estimate the rate of flip-flop from the 50 inside
cytoplasmic to the outer monolayer, and from the outer to the cytoplas-
mic monolayer. A convenient way to express such rates is as the half-time
of flip-flop—that is, the time it takes for half the phospholipids to flip-flop
from one monolayer to the other.
B. From what you learned about the behavior of the two spin-labeled phos-
10
pholipids in Problem 10–23, deduce which one was used to label the 0 1 2 3 4 5 6 7
time (hours)
cytoplasmic monolayer of the intact red blood cells, and which one was
used to label the outer monolayer. Figure 10–6 Decrease in ESR signal
C. Using the information in this problem, propose a method to generate intensity of red blood cells containing
spin-labeled phospholipids in the outer
intact red cells that contain spin-labeled phospholipids exclusively in monolayer (outside) and cytoplasmic
the cytoplasmic monolayer, and a method to generate cells spin-labeled monolayer (inside) of the plasma
exclusively in the outer monolayer. membrane (Problem 10–25).
MEMBRANE PROTEINS
TERMS TO LEARN
bacteriorhodopsin membrane-associated protein
carbohydrate layer membrane-bending protein
cortex membrane protein
detergent multipass transmembrane protein
glycosylphosphatidylinositol single-pass transmembrane protein
(GPI) anchor spectrin
lectin transmembrane protein
lumen
DEFINITIONS
10–26 Protein that binds tightly to a specific sugar.
10–27 The outer coat of a eukaryotic cell, composed of oligosaccharides linked
to intrinsic plasma membrane glycoproteins and glycolipids, as well as
proteins that have been secreted and reabsorbed onto the cell surface.
10–28 Abundant protein associated with the cytosolic side of the plasma mem-
brane in red blood cells, forming a rigid network that supports the mem-
brane.
10–29 Protein whose polypeptide chain crosses the lipid bilayer more than
once.
10–30 Pigmented protein found in the plasma membrane of Halobacterium
halobium, where it pumps protons out of the cell in response to light.
10–31 The complicated cytoskeletal network in the cytosol just beneath the
plasma membrane.
10–32 Type of lipid linkage, formed as proteins pass through the endoplasmic
reticulum, by which some proteins are attached to the noncytosolic sur-
face of the membrane.
TRUE/FALSE
10–33 Whereas all the carbohydrate in the plasma membrane faces outward on
the external surface of the cell, all the carbohydrate on internal mem-
branes faces toward the cytosol.
10–34 Human red blood cells contain no internal membranes other than the
nuclear membrane.
10–35 Although membrane domains with different protein compositions are
well known, there are at present no examples of membrane domains that
differ in lipid composition.
THOUGHT PROBLEMS
10–36 Which of the arrangements of membrane-associated proteins indicated
in Figure 10–7 have been found in biological membranes?
10–37 Which one of the following statements correctly describes the mass ratio
of lipids to proteins in membranes?
MEMBRANE PROTEINS 215
3 6 NH2
COOH
P P
lipid
bilayer
CYTOSOL
COOH
1 2 5 8
Figure 10–7 A variety of possible

4 associations of proteins with a membrane
(Problem 10–36).
A. The mass of lipids greatly exceeds the mass of proteins.

B. The mass of proteins greatly exceeds the mass of lipids.
C. The masses of lipids and proteins are about equal.
D. The mass ratio of lipids to proteins varies widely in different membranes.
10–38 Name the three general types of lipid anchors that are used to attach pro-
teins to membranes.
10–39 Monomeric single-pass transmembrane Problems p10.08/10.07
proteins span a membrane with
a single helix that has characteristic chemical properties in the region
of the bilayer. Which of the three 20-amino-acid sequences listed below
is the most likely candidate for such a transmembrane segment? Explain
the reasons for your choice. (See Table 8, page 966, for one-letter amino
acid code; FAMILY VW is a convenient mnemonic for hydrophobic
amino acids.)
A. I T L I Y F G V M A G V I G T I L L I S
B. I T P I Y F G P M A G V I G T P L L I S
C. I T E I Y F G R M A G V I G T D L L I S
10–40 Consider a transmembrane protein complex that forms a hydrophilic
pore across the plasma membrane of a eukaryotic cell. The pore is made
of five similar protein subunits, each of which contributes a membrane-
spanning helix to form the pore. Each helix has hydrophilic amino CH3
acid side chains on one side of the helix and hydrophobic amino acid CH3 C CH3
CH2
side chains on the opposite side. Propose a possible arrangement of CH3
these five helices in the membrane. CH3 C CH3
CH2
CH2 H H
10–41 Why is it that membrane-spanning protein segments are almost always CH2
helices or barrels, but never disordered chains? CH2 H H
CH2 O
10–42 You are studying the binding of proteins to the cytoplasmic face of cul- CH2 CH2
tured neuroblastoma cells and have found a method that gives a good CH2 CH2
yield of inside-out vesicles from the plasma membrane. Unfortunately, CH2 O
your preparations are contaminated with variable amounts of right-side- CH2 CH2
CH2 CH2
out vesicles. Nothing you have tried avoids this problem. A friend sug- CH2 O
gests that you pass your vesicles over an affinity column made of lectin O 9–10
CH2
coupled to solid beads. What is the point of your friend’s suggestion? O S O CH2
O– Na+ OH
10–43 Detergents are small amphiphilic molecules—one end hydrophobic
and the other hydrophilic—that tend to form micelles in water. Examine
the structures of sodium dodecyl sulfate (SDS) and Triton X-100 in sodium (Na+) Triton X-100
dodecyl
Figure 10–8 and explain why the black portions are hydrophilic and the sulfate (SDS)
blue sections are hydrophobic.
Figure 10–8 The structures of SDS and
10–44 Why does a red blood cell membrane need proteins? Triton X-100 (Problem 10–43).
(A) (B) (C) (D)

CYTOSOL
EXTRACELLULAR
SPACE
protrusion
10–45 Glycophorin, a protein in the plasma membrane of the red blood cell, Figure 10–9 Bending of the plasma
normally exists as a homodimer that is held together entirely by intermembrane by cytosolic proteins (Problem
10–47). (A) Insertion of a protein “finger”
actions between its transmembrane domains. Since transmembrane into the cytosolic leaflet of the membrane.
domains are hydrophobic, how is it that they can associate with one (B) Binding of lipids to the curved
another so specifically? surface of a membrane-binding protein.
(C) Binding of membrane proteins to
10–46 Describe the different methods that cells use to restrict proteins to membrane lipids with large head groups.
specific regions of the plasma membrane. Is a membrane with many (D) A segment of the plasma membrane
anchored proteins still fluid? showing a protrusion.
10–47 Three mechanisms by which membrane-binding proteins bend a

membrane are illustrated in Figure 10–9A–C. As shown, each of these
cytosolic membrane-bending proteins would induce an invagination of
the plasma membrane. Could similar kinds of cytosolic proteins induce
a protrusion of the plasma membrane (Figure 10–9D)? Which ones?
Explain how they might work.
CALCULATIONS
10–48 In the membrane of a human red blood cell, the ratio of the mass of pro-
tein (average molecular weight 50,000) to phospholipid (average molec-
ular weight 800) to cholesterol (molecular weight 386) is about 2:1:1.
How many lipid molecules (phospholipid + cholesterol) are there for
every protein molecule?
10–49 Estimates of the number of membrane-associated proteins per cell and
the fraction of the plasma membrane occupied by such proteins provide
a useful quantitative basis for understanding the structure of the plasma
membrane. These calculations are straightforward for proteins in the
plasma membrane of a red blood cell because red cells are readily iso-
lated from blood and they contain no internal membranes to confuse
the issue. Plasma membranes are prepared, the membrane-associated
proteins are separated by SDS polyacrylamide-gel electrophoresis, and
then they are stained with a dye (Coomassie Blue). Because the intensity
of color is roughly proportional to the mass of protein present in a band,
quantitative estimates can be made as shown in Table 10–1.
A. From the information in Table 10–1, calculate the number of molecules
of spectrin, anion exchanger (AE1), and glycophorin in an individual red
blood cell. Assume that 1 mL of red cell ghosts contains 1010 cells and 5
mg of total membrane protein.
TABLE 10–1 Proportion of stain associated with three membrane-associated

proteins (Problem 10–49).
Protein Molecular weight Percent of stain
Spectrin 250,000 25
AE1 100,000 30
Glycophorin 30,000 2.3
MEMBRANE PROTEINS 217
Figure 10–10 Schematic diagram of AE1,

represented as a cylinder, in the plasma
membrane (Problem 10–49). AE1 was
originally identified as a prominent band
lipid (“band 3”) after the membrane proteins
bilayer from red cell ghosts were separated by
SDS polyacrylamide-gel electrophoresis;
it is often referred to as band 3 in the
literature.
6 nm AE1
B. Calculate the fraction of the plasma membrane that is occupied by AE1.

Assume that AE1 is a cylinder 3 nm in radius and 10 nm in height and is
oriented in the membrane as shown in Figure 10–10. The total surface
area of a red cell is 108 nmp10.10/10.10
Problems
2.
DATA HANDLING
10–50 Look carefully at the transmembrane anion exchanger (AE1) in
Figure 10–11A. Imagine that you could mark all the AE1 proteins spe-
cifically with a fluorescent group and measure their mobility by fluores-
cence recovery after photobleaching (FRAP). You photobleach a small
spot on the membrane and then measure the increase in fluorescence in
that spot as fluorescent AE1 molecules diffuse into it from neighboring
regions of the membrane. Sketch the recovery curve you would expect to
see with time (Figure 10–11B).
MEDICAL LINKS
10–51 Pythagoras forbade his followers to eat fava beans. Beyond the political
implications (Greeks voted with beans), there turns out to be a rational
basis for this proscription. In the Middle Eastern population, defective
forms of the gene encoding glucose 6-phosphate dehydrogenase (G6PD) (A)
are common. These mutant forms of the gene typically reduce G6PD AE1
activity to about 10% of normal. They have been selected for in the Middle
East, and in other areas of the world where malaria is common, because
they afford protection against the malarial parasite. G6PD controls the
first step in the pathway for NADPH production. A lower-than-normal
level of NADPH in red blood cells creates an environment unfavorable
for growth of the protist Plasmodium falciparum, which causes malaria.
AE1 100 nm
Although somewhat protected against malaria, G6PD-deficient indi-
viduals occasionally have other problems. NADPH is the principal agent (B)
required to keep the red cell cytosol in a properly reduced state, con-
stantly converting transient disulfide bonds (–S–S–) back to sulfhydryls BLEACH
(–SH HS–). When a G6PD-deficient individual eats raw or undercooked

fava beans, an oxidizing substance in the beans overwhelms the reduc-
fluorescence
? RECOVERY
ing capacity of the red cells, leading to a severe—sometimes life-threat-
ening—hemolytic anemia. How do you suppose eating fava beans leads
to anemia?
MCAT STYLE time
Passage 1 (Questions 10–52 to 10–56) Figure 10–11 Mobility of membrane

Cholesterol is transported in the bloodstream in lipoprotein particles. There are proteins (Problem 10–50). (A) Model of
red blood cell membrane. (B) Recovery of
several classes of lipoprotein particles, but one of the most important classes is fluorescence after photobleaching a red
called low-density lipoprotein (LDL) particles. LDL particles are composed of a cell plasma membrane containing AE1
single molecule of a large protein called apolipoprotein, as well as thousands of protein tagged with a fluorescent group.
cholesterol molecules and phospholipids. Part of the apolipoprotein forms the

surface of the particle and faces the aqueous phase, while other parts face the
hydrophobic interior of the particle where they interact with cholesterol and the
hydrophobic tails of the phospholipids. When cells need additional cholesterol,
they produce a receptor protein on the surface of their plasma membrane that
binds to apolipoprotein and initiates uptake of LDL particles into the cell via a
process called endocytosis.
10–52 What is the most likely reason that LDL particles are used to transport
cholesterol in the bloodstream?
A. Cholesterol in LDL particles easily inserts into lipid bilayers.
B. Cholesterol is largely insoluble in aqueous solutions.
C. Cholesterol is synthesized within LDL particles.
D. LDL particles can form lipid rafts in the lipid bilayer.
10–53 What best describes the class of protein that apolipoprotein belongs to?
A. Amphiphilic proteins
B. Cell-surface receptors
C. Membrane-associated proteins
D. Multipass transmembrane proteins
10–54 What is the most likely location of phospholipids in LDL particles?
A. Bound to hydrophilic domains of apolipoprotein
B. In the lipid bilayer that surrounds LDL particles
C. On the surface, interacting with the aqueous environment
D. Within the hydrophobic core of the particle
10–55 What kind of molecule on the surface of a cell would be most likely to
serve as a receptor for LDL particles?
A. A cholesterol-binding protein
B. A glycolipid
C. A phospholipid
D. A transmembrane protein
10–56 What conditions would be likely to trigger increased uptake of LDL parti-
cles by cells?
A. Increased glycolipid synthesis
B. Increased plasma membrane growth
C. Increased protein secretion
D. Increased protein synthesis
Chapter 11 219
Membrane Transport of Small CHAPTER
Molecules and the Electrical

Properties of Membranes 11
PRINCIPLES OF MEMBRANE TRANSPORT IN THIS CHAPTER
TERMS TO LEARN PRINCIPLES OF MEMBRANE

active transport membrane transport protein TRANSPORT
channel passive transport
TRANSPORTERS AND ACTIVE
electrochemical gradient transporter
MEMBRANE TRANSPORT
DEFINITIONS CHANNELS AND THE
Match each definition below with its term from the list above. ELECTRICAL PROPERTIES OF
MEMBRANES
11–1 An aqueous pore in a lipid membrane, with walls made of protein,
through which selected ions or molecules can pass.
11–2 The movement of a small molecule or ion across a membrane due to a
difference in concentration or electrical charge.
11–3 General term for a membrane-embedded protein that serves as a carrier
of ions or small molecules from one side of the membrane to the other.
11–4 Movement of a molecule across a membrane that is driven by ATP
hydrolysis or other form of metabolic energy.
11–5 Driving force for ion movement that is due to differences in ion concen-
tration and electrical charge on either side of the membrane.
TRUE/FALSE
11–6 The plasma membrane is highly impermeable to all charged molecules.
11–7 Transport by transporters can be either active or passive, whereas trans-
port by channels is always passive.
THOUGHT PROBLEMS
11–8 Order the molecules on the following list according to their ability to
diffuse through a lipid bilayer, beginning with the one that crosses the
bilayer most readily. Explain your order.
1. Ca2+
2. CO2
3. Ethanol
4. Glucose
5. RNA
6. H2O
11–9 Why are the maximum rates of transport by transporters and channels
thought to be so different?
220 Chapter 11: Membrane Transport of Small Molecules and the Electrical Properties of Membranes
11–10 A simple enzyme reaction can be represented by the equation

E+S ES E+P
where E is the enzyme, S is the substrate, P is the product, and ES is the
enzyme–substrate complex.
A. Write a corresponding equation describing a transporter (T) that medi-
ates transport of a solute (S) down its concentration gradient.
B. The Michaelis–Menten equation for the simple enzyme reaction above is
[S]
rate = Vmax
[S] + Km
where “rate” is the initial rate of the reaction, Vmax is the maximum rate
of the enzyme-catalyzed reaction, [S] is the concentration of substrate,
and Km is the Michaelis constant. Write the corresponding Michaelis–
Menten equation for the process of solute transport by a transporter.
What do rate, Vmax, and Km mean in the equation for transport?
C. Would these equations provide an appropriate description for channels?
Why or why not?
11–11 Suppose a membrane contains a single passive transporter with a Km of
0.1 mM for its solute. How effective would the transporter be at equal-
izing the concentrations of solute across the membrane if the starting
concentrations were 0.01 mM inside and 0.05 mM outside? What if the
concentrations were 100 mM inside and 500 mM outside?
11–12 How is it possible for some molecules to be at equilibrium across a bio-
logical membrane and yet not be at the same concentration on both
sides?
CALCULATIONS
11–13 Brain cells, which depend on glucose for energy, use the glucose trans-
porter GLUT3, which has a Km of 1.5 mM. Liver cells, which store glucose
(as glycogen) after a meal and release glucose between meals, use the
glucose transporter GLUT2, which has a Km of 15 mM.
A. Calculate the rate (as a percentage of Vmax) of glucose uptake in brain
cells and in liver cells at circulating glucose concentrations of 3 mM (star-
vation conditions), 5 mM (normal levels), and 7 mM (after a carbohy-
drate-rich meal). Rearranging the Michaelis–Menten equation gives
rate [S]
=
Vmax [S] + Km
B. Although the concentration of glucose in the general circulation nor-

mally doesn’t rise much above 7 mM, the liver is exposed to much higher
concentrations after a meal. The intestine delivers glucose into the portal
circulation, which goes directly to the liver. In the portal circulation, the
concentration of glucose can be as high as 15 mM. At what fraction of the
maximum rate (Vmax) do liver cells import glucose at this concentration?
C. Do these calculations fit with the physiological functions of brain and
liver cells? Why or why not?
11–14 Cells use transporters to move nearly all metabolites across membranes.
But how much faster is a transporter than simple diffusion? There is suf-
ficient information available for glucose transporters to make a com-
parison. The normal circulating concentration of glucose in humans is 5
mM, whereas the intracellular concentration is usually very low. (For this
problem assume the internal concentration of glucose is 0 mM.)
A. At what rate (molecules/sec) would glucose diffuse into a cell if
there were no transporter? The permeability coefficient for glucose is
PRINCIPLES OF MEMBRANE TRANSPORT 221
3 × 10–8 cm/sec. Assume a cell is a sphere with a diameter of 20 μm. The

rate of diffusion equals the concentration difference multiplied by the
permeability coefficient and the total surface area of the cell (surface
area = 4 r2). (Remember to convert everything to compatible units so
that the rate is molecules/sec.)
B. If in the same cell there are 105 GLUT3 molecules (Km = 1.5 mM) in
the plasma membrane, each of which can transport glucose at a maxi-
mum rate of 104 molecules per second, at what rate (molecules/sec) will
glucose enter the cell? How much faster is transporter-mediated uptake
of glucose than entry by simple diffusion?
DATA HANDLING
11–15 Cytochalasin B, which is often used as an inhibitor of actin-based motility
systems, is also a very potent competitive inhibitor of d-glucose uptake
into mammalian cells. When red blood cell ghosts (red cells, emptied of
their cytoplasm) are incubated with 3H-cytochalasin B and then irradi-
ated with ultraviolet light, the cytochalasin becomes cross-linked to
the glucose transporter, GLUT1. Cytochalasin is not cross-linked to the
transporter if an excess of d-glucose is present during the labeling reac-
tion; however, an excess of L-glucose (which is not transported) does
not interfere with labeling. Why does an excess of d-glucose, but not
L-glucose, prevent cross-linking of cytochalasin to GLUT1?
11–16 Insulin is a small protein hormone that binds to receptors in the plasma
membranes of many cells. In fat cells, this binding dramatically increases
the rate of uptake of glucose into the cells. The increase occurs within
minutes and is not blocked by inhibitors of protein synthesis or of gly-
cosylation. These results suggest that insulin increases the activity of the
glucose transporter, GLUT4, in the plasma membrane, without increas-
ing the total number of GLUT4 molecules in the cell.
The two experiments described below suggest a possible mechanism
for this insulin effect. In the first experiment, the initial rate of glucose
uptake in control and insulin-treated cells was measured, with the results
shown in Figure 11–1. In the second experiment, the concentration of
GLUT4 in fractionated membranes from control and insulin-treated cells
was measured, using the binding of radioactive cytochalasin B as the
assay (see Problem 11–15), as shown in Table 11–1.
A. Deduce the mechanism by which glucose transport through GLUT4
increases in insulin-treated cells.
B. Does insulin stimulation alter either the Km or the Vmax of GLUT4? How
can you tell from these data?
30
+ insulin
rate of glucose uptake
(arbitrary units)
TABLE 11–1 Amount of GLUT4 associated with the plasma membrane 20

and internal membranes in the presence and absence of insulin
(Problem 11–16).
10
Bound 3H-cytochalasin B (cpm/mg protein) – insulin
Untreated cells Treated cells 0

Membrane fraction (– insulin) (+ insulin) 0 10 20
glucose (mM)
Plasma membrane 890 4480 Figure 11–1 Rate of glucose uptake into
Internal membranes 4070 80 cells in the presence and absence of
insulin (Problem 11–16).
TRANSPORTERS AND ACTIVE MEMBRANE

TRANSPORT
TERMS TO LEARN
ABC transporter P-type pump
antiporter symporter
Ca2+-pump (Ca2+ ATPase) transcellular transport
multidrug resistance (MDR) protein uniporter
Na+-K+ pump (Na+-K+ ATPase) V-type pump
DEFINITIONS
11–17 Large superfamily of membrane transport proteins that use the energy
of ATP hydrolysis to transfer peptides and a variety of small molecules
across membranes.
11–18 Type of ABC transporter protein that can pump hydrophobic drugs (such
as some anticancer drugs) out of the cytoplasm of eukaryotic cells.
11–19 Membrane carrier protein that transports two different ions or small
molecules across a membrane in opposite directions, either simultane-
ously or in sequence.
11–20 Transport of solutes across an epithelium, by means of membrane trans-
port proteins in the apical and basal surfaces of the epithelial cells.
11–21 Carrier protein that transports two types of solute across the membrane
in the same direction.
TRUE/FALSE
11–22 A symporter would function as an antiporter if its orientation in the
membrane were reversed; that is, if the portion of the protein normally
exposed to the cytosol faced the outside of the cell instead.
11–23 The co-transport of Na+ and a solute into a cell, which harnesses the
energy in the Na+ gradient, is an example of primary active transport.
11–24 In response to depolarization of the muscle cell plasma membrane, the
Ca2+-pumps in the sarcoplasmic reticulum (SR) use the energy of ATP
hydrolysis to move Ca2+ from the lumen of the SR to the cytosol to initiate
muscle contraction.
THOUGHT PROBLEMS
11–25 Which of the ions listed in Table 11–2 could be used to drive an electri-
cally neutral coupled transport of a solute across the plasma membrane?
Indicate the direction of movement of the listed ions (inward or outward)
and indicate what sort of ion would be co-transported to preserve electri-
cal neutrality.
Incidentally, there is a glaring intracellular deficiency of anions rela-
tive to cations in Table 11–2, yet cells are electrically neutral. What anions
do you suppose are missing from this table?
11–26 A transmembrane protein has the following properties: it has two bind-
ing sites, one for solute A and one for solute B. The protein can undergo a
conformational change to switch between two states: either both binding
sites are exposed exclusively on one side of the membrane, or both are
TRANSPORTERS AND ACTIVE MEMBRANE TRANSPORT 223
TABLE 11–2 A comparison of ion concentrations inside and outside a typical

mammalian cell (Problem 11–25).
Component Intracellular concentration Extracellular concentration
(mM) (mM)
Cations
Na+ 5–15 145
K+ 140 5
Mg2+ 0.5 1–2
Ca2+ 10–4 1–2
H+ 7 × 10–5 (10–7.2 M or pH 7.2) 4 × 10–5 (10–7.4 M or pH 7.4)
Anions
Cl– 5–15 110
exposed exclusively on the other side of the membrane. The protein can
switch between the two conformational states only if both binding sites
are occupied or if both binding sites are empty, but cannot switch if only
one binding site is occupied.
A. What kind of a transporter do these properties define?
B. Do you need to specify any additional properties to turn this protein into
a transporter that couples the movement of solute A up its concentration
gradient to the movement of solute B down its electrochemical gradient?
C. Write a set of rules like those in the body of this problem that defines the
properties of an antiporter.
11–27 A model for a uniporter that could mediate passive transport of glucose
down its concentration gradient is shown in Figure 11–2. How would you
need to change the diagram to convert the transporter into a pump that
transports glucose up its concentration gradient by hydrolyzing ATP?
Explain the need for each of the steps in your new illustration.
Figure 11–2 Hypothetical model

showing how a conformational change
in a transporter could mediate passive
transport of glucose (Problem 11–27). The
transition between the two conformational
glucose states is proposed to occur randomly and
gradient to be completely reversible, regardless of
binding-site occupancy.
11–28 Ion transporters are “linked” together—not physically, but as a conse-

quence of their actions. For example, cells can raise their intracellular
pH, when it becomes too acidic, by exchanging external Na+ for inter-
nal H+, using a Na+–H + antiporter. The change in internal Na+ is then
redressed using the Na -K+ pump.
+
A. Can these two transporters, operating together, normalize both the H+
and the Na+ concentrations inside the cell?
B. Does the linked action of these two pumps cause imbalances in either
the K+ concentration or the membrane potential?
Figure 11–3 The Na+-K+ pump (Problem

ADP Na+
11–29).
ATP
Na+
K+
P
K+
11–29 You have prepared lipid vesicles (spherical lipid bilayers) that contain
Na+-K+ pumps as the sole membrane protein. Assume for the sake of
simplicity that each pump transports one Na+ one way and one K+ the
other way in each pumping cycle, as illustrated in Figure 11–3. All of
the Na+-K+ pumps are oriented so that the portion of the molecule that
normally faces the cytosol faces the outside of the vesicle. Predict what
would happen under each of the following conditions.
A. The solution inside and outside the vesicles contains both Na+ and K+
ions, but no ATP.
B. The solution inside the vesicles contains both Na+ and K+ ions; the solu-
tion outside also contains both ions, as well as ATP.
C. The solution inside contains Na+; the solution outside contains Na+ and
ATP.
D. The solution is as in B, but the Na+-K+ pump molecules are randomly
oriented, some facing one direction, some the other.
CALCULATIONS
11–30 Microvilli increase the surface area of intestinal cells, providing more
efficient absorption of nutrients. Microvilli are shown in profile and cross
section in Figure 11–4. From the dimensions given in the figure, estimate
the increase in surface area that microvilli provide (for the portion of the
plasma membrane in contact with the lumen of the gut) relative to the
corresponding surface of a cell with a “flat” plasma membrane.
Figure 11–4 Microvilli of intestinal

epithelial cells in profile and cross
1 µm 0.1 µm section (Problem 11–30).
TRANSPORTERS AND ACTIVE MEMBRANE TRANSPORT 225
11–31 How much energy does it take to pump substances across membranes?
Or, to put it another way, since active transport is usually driven directly
or indirectly by ATP, how steep a gradient can ATP hydrolysis maintain
for a particular solute? For transport into the cell, the free-energy change
( Gin) per mole of solute moved across the plasma membrane is
Co
∆Gin = –2.3RT log + zFV
Ci
where R = the gas constant, 8.3 × 10–3 kJ/K mole

T = the absolute temperature in K (37°C = 310 K)
Co = solute concentration outside the cell
Ci = solute concentration inside the cell
z = the valence (charge) on the solute
F = Faraday’s constant, 96 kJ/V mole
V = the membrane potential in volts (V)
Since Gin = – Gout, the free-energy change for transport out of the cell is
Co
∆Gout = 2.3RT log – zFV
Ci
At equilibrium, where G = 0, the equations can be rearranged to the
more familiar form known as the Nernst equation.
RT Co
V = 2.3 log
zF Ci
For the questions below, assume that hydrolysis of ATP to ADP and
OUTSIDE INSIDE
Pi proceeds with a G of –50 kJ/mole; that is, ATP hydrolysis can drive CELL CELL
active transport with a G of +50 kJ/mole. Assume that V is –60 mV. +
A. What is the maximum concentration gradient that can be achieved by
the ATP-driven active transport into the cell of an uncharged molecule + K+
such as glucose, assuming that 1 ATP is hydrolyzed for each solute mol- +
ecule that is transported? +
B. What is the maximum concentration gradient that can be achieved by
+
active transport of Ca2+ from the inside to the outside of the cell? How
does this maximum compare with the actual concentration gradient +
observed in mammalian cells (see Table 11–2)? K+

+
C. Calculate how much energy it takes to drive the Na+-K+ pump. This +
remarkable molecular device transports five ions for every molecule of Na+ +
ATP that is hydrolyzed: 3 Na+ out of the cell and 2 K+ into the cell. The
pump typically maintains internal Na+ at 10 mM, external Na+ at 145 mM, +
internal K+ at 140 mM, and external K+ at 5 mM. As shown in Figure 11–5, +
Na+ is transported against the membrane potential, whereas K+ is trans-
+
ported with it. (The G for the overall reaction is equal to the sum of the
G values for transport of the individual ions.) +
D. How efficient is the Na+-K+ pump? That is, what fraction of the energy +
available from ATP hydrolysis is used to drive transport? Na+
+
DATA HANDLING plasma

membrane
11–32 If you have ever used the standard probe on a pH meter, you may well
Figure 11–5 Na+ and K+ gradients and
wonder how pH could possibly be measured in the tiny volumes inside direction of pumping across the plasma
cellular compartments. The recent development of pH-sensitive fluoro- membrane (Problem 11–31). Large letters
phores has simplified this difficult task immensely. One such fluorescent symbolize
Problemshigh concentrations
p11.07/11.05and small
indicator is a hydrophobic ester of SNARF-1, which can enter cells by letters symbolize low concentrations.
Both Na+ and K+ are pumped against
passive diffusion and then is trapped inside after intracellular enzymes chemical concentration gradients;
hydrolyze the ester bonds to liberate SNARF-1 (Figure 11–6). SNARF-1 however, Na+ is pumped up the electrical
absorbs light at 488 nm and emits fluorescent light with peaks at 580 gradient, whereas K+ is pumped down
nm and 640 nm. Emission spectra for SNARF-1 at pH 6.0 and pH 9.0 are the electrical gradient.
OR OH O– O
(CH3)2N O (CH3)2N O (CH3)2N O (CH3)2N O
pK = 7.5
HYDROLYSIS
O O O
O–
C O C O C O C
O
– – –
RO C O C O C O C
–
O SNARF-1 ester O SNARF-1 (HA) O SNARF-1 (A ) O SNARF-1 (A–)
dominant resonance structure
Figure 11–6 Structure of the ester and free forms of SNARF-1 (Problem 11–32). The blocking ester groups are shown as
R. The acid (HA) and salt (A–) forms of SNARF-1 are indicated. The two different resonance structures for the salt form of
SNARF-1 are shown in brackets.
shown in Figure 11–7. The pK of SNARF-1 is 7.5.

Problems
A. Explain why the ester of SNARF-1 diffuses p11.08/11.06
through membranes, whereas
the cleaved form stays inside cells.
B. Why do you think there are two peaks of fluorescence (at 580 nm and at
640 nm) that change so dramatically in intensity with a change in pH (see
Figure 11–7)? What features of SNARF-1 might be important in this?
C. What forms of SNARF-1 are present at pH 6.0 and what are their relative
proportions? At pH 9.0? The Henderson–Hasselbalch equation describ-
ing the dissociation of a weak acid is pH = pK + log ([salt]/[acid]).
D. Sketch an approximate curve for the SNARF-1 emission spectrum inside
a cell at pH 7.2. (All such curves pass through the point where the two
curves in Figure 11–7 cross.)
E. Why do you suppose indicators such as SNARF-1 that have emission
spectra with two peaks are preferred to those that have a single peak?
MEDICAL LINKS
11–33 CO2 is removed from the body through the lungs in a process that is
mediated by red blood cells, as summarized in Figure 11–8. Transport of
CO2 is coupled to the transport of O2 through hemoglobin. Upon release
of O2 in the tissues, hemoglobin undergoes a conformational change that
raises the pK of a histidine side chain, allowing it to bind an H+, which is
generated during hydration of CO2 by the action of the enzyme carbonic
anhydrase. This process occurs in reverse in the lungs when O2 is bound
to hemoglobin.
A. To what extent does the intracellular pH of the red blood cell vary during pH 6.0 pH 9.0
its movement from the tissues to the lungs and back, and why is this so?
B. In what form, and where, is the CO2 during its movement from the tissues
fluorescence
to the lungs?
C. How is it that the Cl––HCO3– exchanger operates in one direction in the
tissues and in the opposite direction in the lungs?
11–34 A rise in the intracellular Ca2+ concentration causes muscle cells to con-
tract. In addition to an ATP-driven Ca2+-pump, heart muscle cells, which
contract quickly and regularly, have an antiporter that exchanges Ca2+ for
550 600 650 700 750
extracellular Na+ across the plasma membrane. This antiporter rapidly
wavelength (nm)
pumps most of the entering Ca2+ ions back out of the cell, allowing the
cell to relax. Ouabain and digitalis, drugs that are used in the treatment of Figure 11–7 Emission spectra of
patients with heart disease, make the heart contract more strongly. Both SNARF-1 at pH 6.0 and pH 9.0 (Problem
drugs function by partially inhibiting the Na+-K+ pump in the membrane 11–32). SNARF-1 in solution at pH 6.0
or pH 9.0 was excited by light at 488 nm
of the heart muscle cell. Can you propose an explanation for the effects and the intensity of fluorescence was
of these drugs in patients? What will happen if too much of either drug is determined at wavelengths from 550 nm
taken? to 750 nm.
CHANNELS AND THE ELECTRICAL PROPERTIES OF MEMBRANES 227
IN TISSUES IN LUNGS
CO2 O2 CO2 O2
low O2, high CO2 high O2, low CO2
H+
N N
CO2 + H2O HCO3– + H+ CO2 + H2O HCO3– + H+
N N
HCO3– Cl– HCO3– Cl–
CHANNELS AND THE ELECTRICAL PROPERTIES Figure 11–8 Red blood cell-mediated
transport of CO2 from the tissues to the
OF MEMBRANES lungs (Problem 11–33). Low and high O2
and CO2 refer to their concentrations, or
TERMS TO LEARN partial pressures.
acetylcholine receptor metabotropic receptor
action potential myelin sheath
adaptation Nernst equation
AMPA receptor neuromuscular junction
aquaporin (water channel) neuron (nerve cell)
axon neurotransmitter
Ca2+-activated K+ channel NMDA receptor
channelrhodopsin oligodendrocyte
delayed K+ channel optogenetics
dendrite patch-clamp recording
depolarization resting membrane potential
excitatory neurotransmitter rapidly inactivating K+ channel
glial cell Schwann cell
inhibitory neurotransmitter selectivity filter
initial segment synapse
ion channel synaptic plasticity
K+ leak channel transmitter-gated ion channel
long-term depression (LDP) voltage-gated cation channel
long-term potentiation (LTP) voltage-gated K+ channel
mechanosensitive channel voltage-gated Na+ channel
membrane potential
DEFINITIONS
11–35 The adjustment in sensitivity of a cell or organism following repeated
stimulation that allows a response even when there is a high background
level of stimulation.
11–36 The long-lasting increase (days to weeks) in the sensitivity of certain syn-
apses in the hippocampus that is induced by a short burst of repetitive
firing in the presynaptic neurons.
11–37 Rapid, transient, self-propagating electrical signal in the plasma mem-
brane of a cell such as a neuron or muscle cell: a nerve impulse.
11–38 Quantitative expression that relates the equilibrium ratio of concentra-
tions of an ion on either side of a permeable membrane to the voltage
difference across the membrane.
11–39 A photosensitive ion channel that opens in response to light.
11–40 Voltage difference across a membrane due to the slight excess of posi-
tive ions on one side and of negative ions on the other (typically –60 mV,
inside negative, for an animal cell).
11–41 General term for a membrane protein that selectively allows cations
such as Na+ to cross a membrane in response to changes in membrane
potential.
11–42 That part of an ion channel structure that determines which ions it can
transport.
11–43 Insulating layer of specialized cell membrane wrapped around verte-
brate axons.
11–44 A K+-transporting ion channel in the plasma membrane of animal cells
that remains open even in a “resting” cell.
11–45 Long, thin nerve cell process capable of rapidly conducting nerve
impulses over long distances so as to deliver signals to other cells.
11–46 Transmembrane protein complex that forms a water-filled channel
across the lipid bilayer through which specific inorganic ions can diffuse
down their electrochemical gradients.
11–47 Specialized junction between a nerve cell and another cell, across which
the nerve impulse is transferred, usually by a neurotransmitter, which is
secreted by the nerve cell and diffuses to the target cell.
11–48 Small signaling molecule such as acetylcholine, glutamate, GABA, or gly-
cine, secreted by a nerve cell at a chemical synapse to signal to the post-
synaptic cell.
11–49 Technique in which the tip of a small glass electrode is sealed onto an
area of cell membrane, thereby making it possible to record the flow of
current through individual ion channels.
TRUE/FALSE
11–50 Transporters saturate at high concentrations of the transported molecule
when all their binding sites are occupied; channels, on the other hand,
do not bind the ions they transport and thus the flux of ions through a
channel does not saturate.
11–51 The membrane potential arises from movements of charge that leave ion
concentrations practically unaffected, causing only a very slight discrep-
ancy in the number of positive and negative ions on the two sides of the
membrane.
11–52 The aggregate current crossing the membrane of an entire cell indicates
the degree to which individual channels are open.
11–53 Transmitter-gated ion channels open in response to specific neurotrans-
mitters in their environment but are insensitive to the membrane poten-
tial; therefore, they cannot by themselves (in the absence of ligand) gen-
erate an action potential.
11–54 When an action potential depolarizes the muscle cell membrane, the
Ca2+-pump is responsible for pumping Ca2+ from the sarcoplasmic retic-
ulum into the cytosol to initiate muscle contraction.
THOUGHT PROBLEMS
11–55 According to Newton’s laws of motion, an ion exposed to an electric field
in a vacuum would experience a constant acceleration from the electric
driving force, just as a falling body in a vacuum constantly accelerates
due to gravity. In water, however, an ion moves at constant velocity in an
electric field. Why do you suppose that is?
11–56 What two properties distinguish an ion channel from a simple aqueous H+
pore?
11–57 You have prepared lipid vesicles that contain molecules of the K+ leak
channel, all oriented so that their cytosolic surface faces the outside of
the vesicle. Predict how K+ ions will move under the following conditions
and what sort of membrane potential will develop.
A. Equal concentrations of K+ ion are present inside and outside the vesicle.
B. K+ ions are present only inside the vesicle.
C. K+ ions are present only outside the vesicle.
11–58 If a frog egg and a red blood cell are placed in pure water, the red blood
cell will swell and burst, but the frog egg will remain intact. Although a
frog egg is about one million times larger than a red cell, they both have
nearly identical internal concentrations of ions so that the same osmotic
forces are at work in each. Why do you suppose red blood cells burst in
water, while frog eggs do not?
H+
11–59 Aquaporins allow water to move across a membrane, but prevent the
passage of ions. How does the structure of the pore through which the Figure 11–9 Rapid diffusion of H+ ions
water molecules move prevent passage of ions such as K+, Na+, Ca+, and by a molecular relay system involving
the making and breaking of hydrogen
Cl–? H+ ions present a different problem because they move by relay Problems
bonds betweenp11.10/11.09
adjacent water molecules
along a chain of hydrogen-bonded water molecules (Figure 11–9). How (Problem 11–59).
does the pore prevent the relay of H+ ions across the membrane?
11–60 Explain in 100 words or fewer how an action potential is passed along an
axon.
11–61 The neurotransmitter acetylcholine is made in the cytosol and then
transported into synaptic vesicles, where its concentration is more than
100-fold higher than in the cytosol. Synaptic vesicles isolated from neu-
rons can take up additional acetylcholine if it is added to the solution in
which they are suspended, but only in the presence of ATP. Na+ ions are
not required for acetylcholine uptake, but, curiously, raising the pH of
the solution in which the synaptic vesicles are suspended increases ace-
tylcholine uptake. Furthermore, transport is inhibited in the presence of
drugs that make the membrane permeable to H+ ions. Suggest a mecha-
nism that is consistent with all these observations.
11–62 Excitatory neurotransmitters open Na+ channels, while inhibitory neuro-
transmitters open either Cl– or K+ channels. Rationalize this observation
in terms of the effects of these ions on the firing of an action potential.
11–63 Acetylcholine-gated cation channels do not discriminate among Na+, K+,
and Ca2+ ions, allowing all to pass through freely. How is it, then, that
when acetylcholine receptors in muscle cells open there is a large net
influx principally of Na+?
CALCULATIONS
11–64 In a subset of voltage-gated K+ channels, the N-terminus of each subunit
acts like a tethered ball that occludes the cytoplasmic end of the pore
soon after it opens, thereby inactivating the channel. This “ball-and-
chain” model for the rapid inactivation of voltage-gated K+ channels
has been elegantly supported for the shaker K+ channel from Drosoph-
ila melanogaster. (The shaker K+ channel in Drosophila is named after
a mutant form that causes excitable behavior—even anesthetized flies
keep twitching.) Deletion of the N-terminal amino acids from the nor-
mal shaker channel gives rise to a channel that opens in response to
membrane depolarization but stays open (Figure 11–10A; 0 μM) instead
of rapidly closing as the normal channel does. A peptide (MAAVAGL-
YGLGEDRQHRKKQ) that corresponds to the deleted N-terminus can
partially inactivate the open channel at 50 μM and completely inactivate
it at 100 μM (Figure 11–10A).
(A) (B)
0 µM 21.4 nm
100 pA 50 µM
10 msec 100 µM
Is the concentration of free peptide (100 μM) required to inactivate Figure 11–10 Inactivation of voltage-
the defective K+ channel anywhere near the local concentration of the gated K+ channels (Problem 11–64).
(A) Patch-clamp recording of a defective
tethered ball on a normal channel? Assume that the tethered ball can shaker K+ channel in the absence and
explore a hemisphere [volume = (2/3) r3] with a radius of 21.4 nm, which presence of inactivating peptide. Current
is the length of the polypeptide “chain” (Figure 11–10B). Calculate the through the channel is indicated in
concentration for one ball in this hemisphere. How does that value com- picoamps (pA). (B) A “ball” tethered by a
“chain” to a normal channel.
pare with the concentration of free peptide needed to inactivate the
channel?
11–65 If the resting membrane potential of a cell is –70 mV and the thickness of
the lipid bilayer is 4.5 nm, what is the strength of the electric field across
the membrane in V/cm? What do you suppose would happen if you
applied this voltage to two metal electrodes separated by a 1 cm air gap?
11–66 The squid giant axon occupies a unique position in the history of our
understanding of cell membrane potentials and nerve action. Its large
size (0.2–1.0 mm in diameter and 5–10 cm in length) allowed electrodes,
large by modern standards, to be inserted so that intracellular voltages
could be measured. When an electrode is stuck into an intact giant axon,
the membrane potential registers –70 mV. When the axon, suspended in
a bath of seawater, is stimulated to conduct a nerve impulse, the mem-
brane potential changes transiently from –70 mV to +40 mV.
The Nernst equation relates equilibrium ionic concentrations to the
membrane potential.
RT C
V = 2.3 log o
zF Ci
For univalent ions and at 20°C (293 K),
Co
V = 58 mV × log
Ci
A. Using this equation, calculate the potential across the resting membrane
(1) assuming that it is due solely to K+ and (2) assuming that it is due
solely to Na+. (The Na+ and K+ concentrations in the axon cytosol and
in seawater are given in Table 11–3.) Which calculation is closer to the
measured resting potential? Which calculation is closer to the measured
action potential? Explain why these assumptions approximate the meas-
ured resting and action potentials.
B. If the solution bathing the squid giant axon is changed from seawater to
artificial seawater in which NaCl is replaced with choline chloride, there
is no effect on the resting potential, but the nerve no longer generates an
action potential upon stimulation. What would you predict would hap-
pen to the magnitude of the action potential if the concentration of Na+
TABLE 11–3 Ionic composition of seawater and of the cytosol in the

squid giant axon (Problem 11–66).
Ion Cytosol Seawater
Na+ 65 mM 430 mM
K+ 344 mM 9 mM
in the external medium were reduced to a half or a quarter of its normal

value, using choline chloride to maintain osmotic balance?
11–67 The number of Na+ ions entering the squid giant axon during an action
potential can be calculated from theory. Because the cell membrane
separates positive and negative charges, it behaves like a capacitor. From
the known capacitance of biological membranes, the number of ions that
enter during an action potential can be calculated. Starting from a resting
potential of –70 mV, it can be shown that 1.1 × 10–12 moles of Na+ must
enter the cell per cm2 of membrane during an action potential.
To determine experimentally the number of entering Na+ ions during
an action potential, a squid giant axon (1 mm in diameter and 5 cm in
length) was suspended in a solution containing radioactive Na+ (specific
activity = 2 × 1014 cpm/mole) and a single action potential was propa-
gated down its length. When the cytoplasm was analyzed for radioactiv-
ity, a total of 340 cpm were found to have entered the axon.
A. How well does the experimental measurement match the theoretical cal-
culation?
B. How many moles of K+ must cross the membrane of the axon, and in
which direction, to reestablish the resting potential after the action
potential is over?
C. Given that the concentration of Na+ inside the axon is 65 mM, calculate
the fractional increase in internal Na+ concentration that results from the
passage of a single action potential down the axon.
D. At the other end of the spectrum of nerve sizes are small dendrites about
0.1 μm in diameter. Assuming the same length (5 cm), the same internal
Na+ concentration (65 mM), and the same resting and action potentials
as for the squid giant axon, calculate the fractional increase in internal
Na+ concentration that would result from the passage of a single action
potential down a dendrite.
E. Is the Na+-K+ pump more important for the continuing performance of a
giant axon, or of a dendrite?
11–68 Acetylcholine-gated cation channels at the neuromuscular junction
open in response to acetylcholine released by the nerve terminal and
allow Na+ ions to enter the muscle cell, which causes membrane depo-
larization and ultimately leads to muscle contraction.
A. Patch-clamp measurements show that young rat muscles have cation
channels that respond to acetylcholine (Figure 11–11). How many kinds
of channel are there? How can you tell?
B. For each kind of channel, calculate the number of ions that enter in one
millisecond. (One ampere is a current of one coulomb per second; one
pA equals 10–12 ampere. An ion with a single charge such as Na+ carries a
charge of 1.6 × 10–19 coulomb.)
Figure 11–11 Patch-clamp measurements

of acetylcholine-gated cation channels in
2 pA young rat muscle (Problem 11–68).
40 msec
DATA HANDLING
11–69 The shaker K+ channel in Drosophila opens in response to membrane
depolarization and thenProblems p11.12/11.11
rapidly inactivates via a ball-and-chain mecha-
nism (see Problem 11–64). The shaker K+ channel assembles as a tetramer
composed of four subunits, each with its own ball and chain. Do multiple
balls in the tetrameric channel act together to inactivate the channel, or
is one ball sufficient?
(A) MIXTURE OF K+ CHANNELS (B) PATCH-CLAMP RECORDINGS Figure 11–12 Analysis of the inactivation
of the shaker K+ channel (Problem 11–69).
toxin-sensitive
(A) Mixture of normal subunits with balls
1.0 – toxin + toxin (white) and toxin-resistant subunits
without balls (red). If the two subunits
current (µA)
were expressed in equal amounts, the
0.5 different forms of the K+ channel would
be present in the ratio 1:4:6:4:1; however,
higher levels of toxin-resistant subunit
0 mRNA were used, significantly skewing
0 50 100 0 50 100
the ratios in favor of channels with toxin-
time (msec) time (msec) resistant subunits. (B) Patch-clamp
recordings in the presence and absence
of scorpion toxin.
This question has been answered by mixing subunits from two dif-
ferent forms of the K+ channel: the normal subunits with balls, and scor-
pion-toxin-resistant subunits without balls (Figure 11–12A). Scorpion
toxin prevents the opening of normal (toxin-sensitive) K+ channels, but
not channels composed entirely of toxin-resistant subunits. Moreover,
hybrid channels containing even a single toxin-sensitive subunit fail to
open in the presence of toxin.
Mixed K+ channels were created by injecting a mixture of mRNAs for
the two types of subunit into frog oocytes. After expression, patches of
membrane containing several hundred channels were studied by patch-
clamp recording after subjecting the membranes to depolarization in the
absence or presence of scorpion toxin (Figure 11–12B).
A. Sketch the expected patch-clamp recordings, in the presence and
absence of toxin, for a pure population of K+ channels composed entirely
of toxin-resistant subunits without balls.
B. Sketch the expected patch-clamp recordings, in the presence and
absence of toxin, for a pure population of K+ channels composed entirely
of normal (toxin-sensitive) subunits with balls.
C. Sketch the expected patch-clamp recordings, in the presence and
absence of toxin, for a mixed population of K+ channels, 50% composed
entirely of normal (toxin-sensitive) subunits with balls and 50% com-
posed entirely of toxin-resistant subunits without balls.
D. The key observation in Figure 11–12B is that the final plateau values of
the currents in the absence and presence of toxin are the same. Does this
observation argue that a single ball can close a channel or that multiple
balls must act in concert? (Think about what the curves would look like if
one, two, three, or four balls were required to close a channel.)
11–70 A recording from a patch of membrane (Figure 11–13A) measured cur-
rent through acetylcholine-gated ion channels, which open when acetyl-
choline is bound (Figure 11–13B). To obtain the recording, acetylcholine
was added to the solution in the micropipette (Figure 11–13A). Describe
what you can learn about the channels from this recording. How would
the recording differ if acetylcholine were (a) omitted or (b) added only to
the solution outside the micropipette?
11–71 One important parameter for understanding any particular membrane
transport process is to know the number of copies of the specific trans-
port protein present in the cell membrane. To measure the number of
(A) (B)
micropipette
ion
2 pA
channels Figure 11–13 Analysis of acetylcholine-
gated ion channels (Problem 11–70). (A) A
10 msec
micropipette with a patch of membrane.
(B) Patch-clamp recording of current
CYTOSOL through acetylcholine-gated ion channels.
voltage-gated Na+ channels in the rabbit vagus nerve, you use a potent 200
toxin bound (pmol/g wet weight)

toxin, saxitoxin, a shellfish poison that specifically inactivates the volt- – unlabeled toxin
age-gated Na+ channels in these nerve cells. Assuming that each channel 150
binds one toxin molecule, the number of Na+ channels in a segment of
vagus nerve will be equal to the maximum number of bound toxin mol- 100
ecules.
You incubate identical segments of nerve for 8 hours with increas- + unlabeled toxin
50
ing amounts of 125I-labeled toxin. You then wash the segments to remove
unbound toxin and measure the radioactivity associated with the nerve
segments to determine the toxin-binding curve (Figure 11–14, upper 0
0 20 40 60 80
curve). You are puzzled because you expected to see binding reach a
I-toxin (nM)
125
maximum (saturate) at high concentrations of toxin; however, no distinct
end point was reached, even at higher concentrations of toxin than those Figure 11–14 Toxin-binding curves in
shown in the figure. After careful thought, you design a control experi- the presence and absence of unlabeled
ment in which the binding of labeled toxin is measured in the presence saxitoxin Problems p11.15/11.14
(Problem 11–71).
of a large molar excess of unlabeled toxin. The results of this experiment,
which are shown in the lower curve in Figure 11–14, make everything
clear and allow you to calculate the number of Na+ channels in the mem-
brane of the vagus nerve axon.
A. Why does binding of the labeled toxin not saturate? What is the point of
the control experiment, and how does it work?
B. Given that 1 gram of vagus nerve has an axonal membrane area of 6000
cm2 and assuming that the Na+ channel is a cylinder with a diameter of
6 nm, calculate the number of Na+ channels per square micrometer of
axonal membrane and the fraction of the cell surface occupied by the
channel. (Use 100 pmol as the amount of toxin specifically bound to the
receptor.)
MEDICAL LINKS
11–72 To make antibodies against the acetylcholine receptor from the electric
organ of electric eels, you inject the purified receptor into mice. You note
an interesting correlation: mice with high levels of antibodies against
the receptor appear weak and sluggish; those with low levels are lively.
You suspect that the antibodies against the eel acetylcholine receptors
are reacting with the mouse acetylcholine receptors, causing many of
the receptors to be destroyed. Since a reduction in the number of ace-
tylcholine receptors is also the basis for the human autoimmune dis-
ease myasthenia gravis, you wonder whether an injection of the drug
neostigmine into the mice might give them a temporary restoration of
strength, as it does for myasthenic patients. Neostigmine inhibits acetyl-
cholinesterase, the enzyme responsible for hydrolysis of acetylcholine in
the synaptic cleft. Sure enough, when you inject your mice with neostig-
mine, they immediately become very active. Propose an explanation for
how neostigmine restores temporary function to a neuromuscular syn-
apse with a reduced number of acetylcholine receptors.
11–73 The ion channels for neurotransmitters such as acetylcholine, serotonin,
GABA, and glycine have similar overall structures. Each class comprises
an extremely diverse set of channel subtypes, with different ligand affini-
ties, different channel conductances, and different rates of opening and
closing. Why is such extreme diversity a good thing from the standpoint
of the pharmaceutical industry?
MCAT STYLE
Numerous interesting drugs and toxins act on channels and transporters. One
such drug is scopolamine, which is used to treat vertigo, motion sickness, and
smooth muscle spasms. Scopolamine was first isolated from Jimson Weed, which
produces a beautiful white flower—often painted by Georgia O’Keeffe. Jimson
Weed is also called Sacred Datura, and was used by Native Americans for reli-
gious purposes because it can produce hallucinations and time distortions. Imag-
ine that you are the first to purify scopolamine and are trying to determine how
it works. You find that if you incubate isolated muscle cells with scopolamine,
subsequent addition of acetylcholine no longer causes membrane depolarization
and cell contraction, as it does in the absence of scopolamine.
11–74 Which one of the following statements best explains how scopolamine
might exert its effects?
A. It binds to the acetylcholine-gated Na+ channel and inhibits its opening.
B. It inhibits opening of the Ca2+ channel in the sarcoplasmic reticulum.
C. It inhibits transporters that import Na+ during an action potential.
D. It inhibits voltage-gated K+ channels during an action potential.
11–75 Scorpion -toxin, a component of scorpion venom, dramatically pro-
longs the change in membrane potential during the firing of a nerve
impulse (Figure 11–15). Which one of the following hypotheses best
explains how -toxin exerts its effects?
A. It accelerates the opening of voltage-gated K+ channels.
B. It opens the voltage-gated Na+ channel at a lower voltage.
C. It promotes the opening of a ligand-gated Na+ channel.
D. It slows the inactivation of the voltage-gated Na+ channel.
(A) CONTROL (B) + TOXIN Figure 11–15 Effects of scorpion -toxin

+45 on the duration of an action potential
(Problem 11–75).
0
mV
–75
1 msec seconds
11–76 Extracts from the African plant Stropthantus gratus were once used
to make poison arrowheads
Figurefor11-301
hunting. The active compound in the
extract is extremely toxic; when concentrated and placed on an arrow-
head, it can kill a hippopotamus. Imagine that you treat cells with the
compound and observeMCAT Problem 11-01
that they begin to swell and eventually burst.
Which one of the following actions would best explain the effects of the
toxic compound?
A. Closing of K+ leak channels.
B. Closing of Na+ channels.
C. Inhibition of glucose transporters.
D. Inhibition of the Na+-K+ pump.

Severe diarrhea associated with diseases such as cholera and dysentery was
the leading cause of infant mortality world-wide prior to 1980. Severe diarrhea
produces such a rapid fluid loss that the individual becomes so dehydrated that
organs fail. When it invades the gut, Vibrio cholerae, the bacterium responsible for
cholera, produces a toxin that hyperactivates the cystic fibrosis transmembrane
regulator (CFTR). Hyperactivation of CFTR increases the movement of Cl– ions
into the lumen of the intestine, which creates an ionic imbalance. As a result,
water rushes into the intestine from surrounding tissues, leading to diarrhea and
MCAT STYLE 235
rapid water loss. Mutations that inactivate CFTR cause cystic fibrosis. In this case,
decreased flow of Cl– to the lining of the lungs leads to an ionic imbalance that
dehydrates the mucus, which clogs the air passages.
11–77 Analysis of CFTR led to some surprises. Although CFTR is homologous
to ABC transporters, which use ATP hydrolysis to pump solutes in or out
of the cell, it shows unusual behavior. Which one of the following state-
ments would suggest that CFTR is not a typical member of the ABC trans-
porter family?
A. CFTR binds to Cl– to move the ions across membranes.
B. CFTR can move Cl– against a concentration gradient.
C. CFTR produces a robust Cl– current across membranes.
D. CFTR requires ATP to move C– across membranes.
11–78 The loss of fluids in severe diarrhea can be effectively treated by oral
rehydration therapy, which has been called one of the greatest medi-
cal advances of the twentieth century because it dramatically reduces
deaths caused by dehydration. Oral rehydration therapy is simple and
cheap: affected individuals drink a solution of 90 mM NaCl and 110 mM
glucose. Which of the following statements best describes how oral rehy-
dration therapy works?
A. Co-transport of Na+ and glucose via a symporter increases cell osmolar-
ity and water uptake.
B. Glucose powers the Na+-K+ pump, which pumps Na+ into the cell to
increase cell osmolarity.
C. In the gut, the low osmolarity rehydration solution drives water into cells
lining the intestine.
D. The salt and glucose in the rehydration solution kills Vibrio cholerae, nor-
malizing cell osmolarity.
Annulate Lamellae in a Human Oocyte.
These cytoplasmic whorls of double
membranes punctured by nuclear pore
complexes are found in many oocytes
and other cells, but their significance
and possible function(s) are still very
mysterious. Do they represent stockpiles
of nuclear membranes? Are the dark
blobs at the center stored maternal
mRNA? They are difficult to purify and
study biochemically, and compared to
other intracellular membranous structures
have received scant attention from cell
biologists.
Chapter 12 237
CHAPTER
Intracellular Compartments
and Protein Sorting 12
THE COMPARTMENTALIZATION OF CELLS IN THIS CHAPTER
TERMS TO LEARN THE COMPARTMENTALIZATION

cytoplasm organelle signal sequence OF CELLS
cytosol protein translocation sorting signal
gated transport signal patch vesicular transport THE TRANSPORT OF
lumen signal peptidase MOLECULES BETWEEN THE
NUCLEUS AND THE CYTOSOL
DEFINITIONS
THE TRANSPORT OF PROTEINS
Match the definition below with its term from the list above. INTO MITOCHONDRIA AND
12–1 Contents of a cell that are contained within its plasma membrane but, in CHLOROPLASTS
the case of eukaryotic cells, outside the nucleus.
PEROXISOMES
12–2 Protein sorting signal that consists of a specific three-dimensional
arrangement of atoms on the folded protein’s surface. THE ENDOPLASMIC RETICULUM
12–3 Contents of the main compartment of the cell, excluding the nucleus and
membrane-bounded compartments such as endoplasmic reticulum and
mitochondria.
12–4 Movement of proteins through nuclear pore complexes between the
cytosol and the nucleus.
12–5 Membrane-enclosed compartment in a eukaryotic cell that has a distinct
structure, macromolecular composition, and function.
12–6 Protein sorting signal that consists of a short continuous sequence of
amino acids.
TRUE/FALSE
12–7 The biological membranes that partition the cell into functionally dis-
tinct compartments are impermeable.
12–8 Like the lumen of the endoplasmic reticulum (ER), the interior of the
nucleus is topologically equivalent to the outside of the cell.
12–9 ER-bound and free ribosomes, which are structurally and functionally
identical, differ only in the proteins they happen to be making at a par-
ticular time.
12–10 Each signal sequence specifies a particular destination in the cell.
THOUGHT PROBLEMS
12–11 Discuss the following statement: “The plasma membrane is only a minor
component of most eukaryotic cells.”
238 Chapter 12: Intracellular Compartments and Protein Sorting
12–12 Is it really true that all human cells contain the same basic set of mem-
brane-enclosed organelles? Do you know of any examples of human cells
that do not have a complete set of organelles?
12–13 When cells are treated with drugs that depolymerize microtubules, the
Golgi apparatus is fragmented into small vesicles and dispersed through-
out each cell. When such drugs are removed, cells typically recover and
grow normally. If cells that have recovered from such treatment are
examined by electron microscopy, they are found to contain a perfectly
normal-looking Golgi apparatus. Does this mean that the Golgi appara-
tus has been synthesized anew from scratch? If not, how do you suppose
it might have happened?
12–14 Why do eukaryotic cells require a nucleus as a separate compartment
when prokaryotic cells manage perfectly well without?
12–15 What is the fate of a protein with no sorting signal?
12–16 Protein synthesis in a liver cell occurs nearly exclusively on free ribosomes
in the cytosol and on ribosomes that are bound to the ER membrane. (A
small fraction of total protein synthesis is directed by the mitochondrial
genome and occurs on ribosomes in the mitochondrial matrix.) Which
type of protein synthesis—in the cytosol or on the ER—do you think is
responsible for the majority of protein synthesis in a liver cell? Assume
that the average density and lifetimes of proteins are about the same in
all compartments. Explain the basis for your answer. Would your answer
change if you took into account that some proteins are secreted from
liver cells?
12–17 List the organelles in an animal cell that obtain their proteins via gated
transport, via transmembrane transport, or via vesicular transport.
12–18 Imagine that you have engineered a set of genes, each encoding a protein
with a pair of conflicting signal sequences that specify different compart-
ments. If the genes were expressed in a cell, predict which signal would
win out for the following combinations. Explain your reasoning.
A. Signals for import into nucleus and import into ER.
B. Signals for import into peroxisomes and import into ER.
C. Signals for import into mitochondria and retention in ER.
D. Signals for import into nucleus and export from nucleus.
12–19 If you think of the protein as a traveler, what kind of vehicle would best
describe the sorting receptor: a private car, a taxi, or a bus? Explain your
choice.
CALCULATIONS
12–20 A typical animal cell is said to contain some 10 billion protein molecules
that need to be sorted into their proper compartments. That’s a lot of pro-
teins. Can 10 billion protein molecules even fit into a cell? An average
protein encoded by the human genome is 450 amino acids in length, and
the average mass of an amino acid in a protein is 110 daltons. Given that
the average density of a protein is 1.4 g/cm3, what fraction of the volume
of a cell would 10 billion average protein molecules occupy? Consider a
liver cell, which has a volume of about 5000 μm3, and a pancreatic exo-
crine cell, which has a volume of about 1000 μm3.
12–21 The lipid bilayer, which is 5 nm thick, occupies about 60% of the volume
of a typical cell membrane. (Lipids and proteins contribute equally on a
mass basis, but lipids are less dense and therefore account for more of
the volume.) For liver cells and pancreatic exocrine cells, the total area
of all cell membranes is estimated at about 110,000 μm2 and 13,000 μm2,
THE COMPARTMENTALIZATION OF CELLS 239
respectively. What fraction of the total volumes of these cells is accounted

for by lipid bilayers? The volumes of liver cells and pancreatic exocrine
cells are about 5000 μm3 and 1000 μm3, respectively.
12–22 The rough ER is the site of synthesis of many classes of membrane
proteins. Some of these proteins remain in the ER, whereas others are
sorted to compartments such as the Golgi apparatus, lysosomes, and the
plasma membrane. One measure of the difficulty of the sorting problem
is the degree of “purification” that must be achieved during transport
from the ER to the other compartments. For example, if membrane pro-
teins bound for the plasma membrane represented 90% of all proteins in
the ER, then only a small degree of purification would be needed (and
the sorting problem would appear relatively easy). On the other hand, if
plasma membrane proteins represented only 0.01% of the proteins in the
ER, a very large degree of purification would be required (and the sorting
problem would appear correspondingly more difficult).
What is the magnitude of the sorting problem? What fraction of the
membrane proteins in the ER are destined for other compartments? A
few simple considerations allow one to answer these questions. Assume
that all proteins on their way to other compartments remain in the ER
for 30 minutes on average before exiting, and that the ratio of proteins to
lipids in the membranes of all compartments is the same.
A. In a typical growing cell that is dividing once every 24 hours, the equiva-
lent of one new plasma membrane must transit the ER every day. If the
ER membrane is 20 times the area of a plasma membrane, what is the
ratio of plasma membrane proteins to other membrane proteins in the
ER?
B. If in the same cell the Golgi membrane is three times the area of the
plasma membrane, what is the ratio of Golgi membrane proteins to other
membrane proteins in the ER?
C. If the membranes of all other compartments (lysosomes, endosomes,
inner nuclear membrane, and secretory vesicles) that receive membrane
proteins from the ER are equal in total area to the area of the plasma
membrane, what fraction of the membrane proteins in the ER of this cell
are permanent residents of the ER membrane?
DATA HANDLING
12–23 Although the vast majority of transmembrane proteins insert into mem-
branes with the help of dedicated protein-translocation machines, a few
proteins can insert into membranes on their own. Such proteins may
provide a window into how membrane insertion occurred in the days
before complex translocators had evolved.
You are studying a protein that inserts itself into the bacterial mem-
brane independent of the normal translocation machinery. This protein
has an N-terminal, 18-amino-acid hydrophilic segment that is located on
the outside of the membrane, a 19-amino-acid hydrophobic transmem-
brane segment flanked by negatively and positively charged amino acids,
and a C-terminal domain that resides inside the cell (Figure 12–1A). If
the protein is properly inserted in the membrane, the N-terminal seg-
ment is exposed externally where it can be clipped off by a protease,
allowing you to quantify insertion.
To examine the roles of the hydrophobic segment and its flanking
charges, you construct a set of modified genes that express mutant pro-
teins with altered charges in the N-terminal segment, altered lengths of
the hydrophobic segment, and combinations of the two (Figure 12–1B).
For each gene you measure the fraction of the total protein that is cleaved
by the protease, which is the fraction that was inserted correctly (Figure
12–1B). To assess the contribution of the normal membrane potential
(positive outside, negative inside), you repeat the measurements in the
presence of CCCP, an ionophore that eliminates the charge on the mem- (A)
N
brane (Figure 12–1B).
–
A. Which of the two N-terminal negative charges is the more important for OUTSIDE +
insertion of the protein in the presence of the normal membrane poten-
–
tial (minus CCCP)? Explain your reasoning.
B. In the presence of the membrane potential (minus CCCP), is the hydro-
phobic segment or the N-terminal charge more important for insertion
of the protein into the membrane? Explain your reasoning. INSIDE + –
C. In the absence of the membrane potential (plus CCCP), is the hydropho-
bic segment or the N-terminal charge more important for insertion of the
protein into the membrane? Explain your reasoning. C
THE TRANSPORT OF MOLECULES BETWEEN THE

(B) percent inserted
NUCLEUS AND THE CYTOSOL construct – CCCP + CCCP
TERMS TO LEARN – – +
1. 99 99
inner nuclear membrane nuclear lamin nuclear transport +
nuclear envelope nuclear lamina receptor
2. 98 98
nuclear export receptor nuclear localization nucleoporin
nuclear export signal signal outer nuclear – –
+
3. 93 54
nuclear import receptor nuclear pore complex membrane
(NPC) Ran – – +
4. 65 0
DEFINITIONS
–
+
Match the definition below with its term from the list above. 5. 58 0
+
12–24 Sorting signal contained in the structure of macromolecules and com- –
6. 0 0
plexes that are transported from the nucleus to the cytosol through
nuclear pore complexes. +
7. 0 0
12–25 Large multiprotein structure forming a channel through the nuclear
envelope that allows selected molecules to move between nucleus and
cytoplasm. Figure 12–1 Insertion of a small protein into
the bacterial membrane (Problem 12–23).
12–26 Monomeric GTPase present in both cytosol and nucleus that is required (A) Normal orientation of the protein in
for the active transport of macromolecules into and out of the nucleus the membrane. (B) Mutant proteins used
through nuclear pore complexes. to investigate the contributions of the
N-terminal negative charges and length of
12–27 Fibrous meshwork of proteins on the inner surface of the inner nuclear the hydrophobic segment to membrane
insertion. The presence of negative charges
membrane. is indicated by –; green cylinders indicate
Problems
the p12.01/12.01
length of -helical, transmembrane
12–28 Protein that binds nuclear localization signals and facilitates the trans-
hydrophobic segments; deletions of the
port of proteins with these signals from the cytosol into the nucleus hydrophobic segments are shown as gaps.
through nuclear pore complexes. Percent inserted refers to the proportion
of the total protein whose N-termini are
12–29 Sorting signal found in proteins destined for the nucleus and which ena- sensitive to protease digestion.
ble their selective transport into the nucleus from the cytosol through the
nuclear pore complexes.
12–30 The portion of the nuclear envelope that is continuous with the endo-
plasmic reticulum and is studded with ribosomes on its cytosolic surface.
TRUE/FALSE
12–31 The nuclear membrane is freely permeable to ions and other small mol-
ecules under 5000 daltons.
12–32 To avoid the inevitable collisions that would occur if two-way traffic
through a single pore were allowed, nuclear pore complexes are special-
ized so that some mediate import while others mediate export.
THE TRANSPORT OF MOLECULES BETWEEN THE NUCLEUS AND THE CYTOSOL 241
nuclear Figure 12–2 Cross section through a

CYTOSOL
pore nuclear pore complex, showing continuity
of inner and outer nuclear membranes
nuclear
envelope (Problem 12–35).
NUCLEUS
12–33 Some proteins are kept out of the nucleus, until needed, by inactivating
their nuclear localization signals by phosphorylation.
12–34 All cytosolic proteins have nuclear export signals that allow them to be
removed from the nucleus when it reassembles after mitosis.
THOUGHT PROBLEMS
12–35 As shown in Figure 12–2, the inner and outer nuclear membranes form
a continuous sheet, connecting through the nuclear pores. Continu-
ity implies that membrane proteins can move freely between the two
nuclear membranes by diffusing through the bilayer at the nuclear pores.
Yet the inner and outer nuclear membranes have different protein com-
positions, as befits their different functions. How do you suppose this
apparent paradox is reconciled?
12–36 How is it that a single nuclear pore complex can efficiently transport pro-
teins that possess different kinds of nuclear localization signal?
12–37 How do you suppose that proteins with a nuclear export signal get into
the nucleus?
12–38 Your advisor is explaining his latest results in your weekly lab meeting.
By fusing his protein of interest to green fluorescent protein (GFP), he
has shown that it is located entirely in the nucleus. But he wonders if it is
a true nuclear protein or a shuttling protein that just spends most of its
time in the nucleus. He is unsure how to resolve this issue. Having just
read an article about how a similar problem was answered, you suggest
that he make a heterokaryon by fusing cells that are expressing his tagged
protein with an excess of cells that are not expressing it. You tell him that
in the presence of a protein synthesis inhibitor to block new synthesis of
the tagged protein, he can resolve the issue by examining fused cells with
two nuclei. He gives you a puzzled look and asks, “How does that help?”
You tell him what he has so often told you: “Think about it.”
A. How would examining the two nuclei in a heterokaryon answer the ques-
tion? What results would you expect if the protein were a true nuclear (A) NES-BSA
protein? What would you expect if it were a shuttling protein?
B. Why did you suggest that a protein synthesis inhibitor would be needed + cytoplasm
in this experiment? + GTP
12–39 Nuclear localization signals are not cleaved off after transport into the
nucleus, whereas the signal sequences for import into other organelles
(B)
are often removed after import. Why do you suppose it is critical that
nuclear localization signals remain attached to their proteins?
+ Crm1
12–40 To test the hypothesis that the directionality of transport across the + RanQ69L-GTP
nuclear membrane is determined primarily by the gradient of the Ran-
GDP outside the nucleus and Ran-GTP inside the nucleus, you decide
to reverse the gradient to see if you can force the import of a protein that
is normally exported from the nucleus. You add a well-defined nuclear Figure 12–3 Directionality of nuclear
export substrate, fluorescent BSA coupled with a nuclear export signal transport (Problem 12–40). (A) Exclusion
of fluorescent NES-BSA from the nucleus
(NES-BSA), to the standard permeabilized cell assay. Sure enough, it is in a standard import assay. (B) Import of
excluded from the nuclei (Figure 12–3A). Now you add Crm1, the nuclear fluorescent NES-BSA in the presence of
export receptor that recognizes the export signal, and RanQ69L-GTP, a Crm1 and RanQ69L-GTP.
mutant form of Ran that cannot hydrolyze GTP. With these additions, the
tagged BSA now enters the nuclei (Figure 12–3B). Unlike conventional
nuclear import, which concentrates imported proteins in the nucleus,
the concentration of NES-BSA in the nucleus in the import assay is no
higher than in the surrounding cytoplasm.
A. Why doesn’t NES-BSA accumulate to a higher concentration in the
nucleus than in the cytoplasm in these experiments?
B. In a standard nuclear import assay with added cytoplasm and GTP, pro-
teins with a nuclear localization signal accumulate to essentially 100% in
the nucleus. How is it that the standard assay allows 100% accumulation
in the nucleus?
CALCULATIONS
12–41 By following the increase in nuclear fluorescence over time in the forced
nuclear import experiments in Figure 12–3B, the authors were able to
show that nuclear fluorescence reached half its maximal value in 60 sec-
onds. Since the added concentration of NES-BSA was 0.3 μM, its concen-
tration in the nucleus after 60 seconds was 0.15 μM. Given that the vol-
ume of the nucleus is 500 fL and that each nucleus contains 3000 nuclear
pores, calculate the rate of import of NES-BSA per pore in this experi-
ment. (For this calculation, neglect export of NES-BSA.) Is your answer
physiologically reasonable? Why or why not?
12–42 A nuclear pore can dilate to accommodate a gold particle 26 nm in diam-
eter. If it could accommodate a spherical protein of the same dimensions,
what would the protein’s molecular mass (g/mole) be? [Assume that the
density of the protein is 1.4 g/cm3. The volume of a sphere is (4/3) r3.]
12–43 Assuming that 32 million histone octamers are required to package the
human genome, how many histone molecules must be transported per
second per nuclear pore complex in cells whose nuclei contain 3000
nuclear pores and are dividing once per day?
(A)
12–44 The nuclear pore complex (NPC) creates a barrier to the free exchange of
molecules between the nucleus and cytoplasm, but in a way that remains
mysterious. In yeast, for example, the central pore has a diameter of 35
nm and is 30 nm long, which is somewhat smaller than its vertebrate
counterpart. Even so, it is large enough to accommodate virtually all
components of the cytosol. Yet the pore allows passive diffusion of mol-
ecules only up to about 40 kd; entry of anything larger requires help from
a nuclear import receptor. Selective permeability is controlled by protein
components of the NPC that have unstructured, polar tails extending
into the central pore. These tails are characterized by periodic repeats of
(B) solution gel
the hydrophobic amino acids phenylalanine (F) and glycine (G).
At high enough concentration (50 mM), the FG-repeat domains of
30 s
these proteins can form a gel, with a meshwork of interactions between
the hydrophobic FG repeats (Figure 12–4A). These gels allow passive dif-
10 min
fusion of small molecules, but they prevent entry of larger proteins such
as the fluorescent protein mCherry fused to maltose binding protein
30 min
MBP-mCherry
Figure 12–4 FG-repeat gel and influx of proteins into the nucleus (Problem
12–44). (A) Cartoon of the meshwork (gel) formed by pairwise interactions 30 s
between hydrophobic FG repeats. For FG-repeats separated by 17 amino
acids, as is typical, the mesh formed by extended amino acid side chains
would correspond to about 4 nm on a side, which would be large enough 10 min
to account for the characteristic passive diffusion of proteins through
nuclear pores. (B) Diffusion of MBP-mCherry and importin-MBP-GFP
into a gel of FG-repeats. In each group, the solution is shown at left 30 min
and the gel at right. The bright areas indicate regions that contain the
fluorescent proteins. importin-MBP-GFP
(MBP) (Figure 12–4B). (The fusion to MBP makes mCherry too large to nucleoplasmin nuclear cytoplasmic
enter the nucleus by passive diffusion.) However, if the nuclear import preparation injection injection
receptor, importin, is fused to a similar protein, MBP-GFP, the importin-
intact
MBP-GFP fusion readily enters the gel (Figure 12–4B).
A. FG-repeats only form gels in vitro at relatively high concentration
(50 mM). Is this concentration reasonable for FG repeats in the NPC
core? In yeast, there are about 5000 FG-repeats in each NPC. Given the
dimensions of the yeast nuclear pore (35 nm diameter and 30 nm length), one tail
calculate the concentration of FG-repeats in the cylindrical volume of the
pore. Is this concentration comparable to the one used in vitro?
B. Is diffusion of importin-MBP-GFP through the FG-repeat gel fast enough
to account for the efficient flow of materials between the nucleus and
cytosol? From experiments of the type shown in Figure 12–4B, the dif-
heads only
fusion coefficient (D) of importin-MBP-GFP through the FG-repeat gel
was determined to be about 0.1 μm2/sec. The equation for diffusion is
t = x2/2D, where t is time and x is distance. Calculate the time it would
take importin-MBP-GFP to diffuse through a yeast nuclear pore (30 nm)
if the pore consisted of a gel of FG-repeats. Does this time seem fast tails only
enough for the needs of a eukaryotic cell?
DATA HANDLING
12–45 Before nuclear pore complexes were well understood, it was unclear Figure 12–5 Cellular location of injected
whether nuclear proteins diffused passively into the nucleus and accu- nucleoplasmin and nucleoplasmin
components (Problem 12–45). Schematic
mulated there by binding to residents of the nucleus such as chromo- diagrams of autoradiographs show the
somes, or whether they were actively imported and accumulated regard- cytoplasm and nucleus with the location
less of their affinity for nuclear components. of nucleoplasmin indicated by the red
A classic experiment that addressed this problem used several forms areas.
of radioactive nucleoplasmin, which is a large pentameric protein
involved in chromatin assembly. In this experiment, either the intact
protein or the nucleoplasmin heads, tails, or heads with a single tail were
injected into the cytoplasm of a frog oocyte or into the nucleus (Figure
12–5). All forms of nucleoplasmin, except heads, accumulated in the
nucleus when injected into the cytoplasm, and all forms were retained in
the nucleus when injected there.
A. What portion of the nucleoplasmin molecule is responsible for localiza-
tion in the nucleus?
B. How do these experiments distinguish between active transport, in
which a nuclear localization signal triggers transport by the nuclear pore
complex, and passive diffusion, in which a binding site for a nuclear
component allows accumulation in the nucleus?
pNL+
12–46 You have just joined a laboratory that is analyzing the nuclear transport nuclear protein EcoRI
machinery in yeast. Your advisor, who is known for her extraordinarily
clever ideas, has given you a project with enormous potential. In princi-
Gal1 NLS
ple, it would allow a genetic selection for conditional-lethal mutants in promoter present
the nuclear transport apparatus.
She gave you two plasmids. Each plasmid consists of a hybrid gene pNL–
under the control of a regulatable promoter (Figure 12–6). The hybrid
nuclear protein EcoRI
gene is a fusion between a gene whose product is normally imported into
the nucleus and the gene for the restriction nuclease EcoRI. The plasmid
Gal1 NLS
pNL+ contains a functional nuclear localization signal (NLS); the plasmid promoter absent
pNL– does not have an NLS. The promoter, which is from the yeast Gal1
gene, allows transcription of the hybrid gene only when the sugar galac-
tose is present in the growth medium. Figure 12–6 Two plasmids for
Following her instructions, you introduce the plasmids into yeast investigating nuclear localization in yeast
(Problem 12–46). Plasmids are shown
(in the absence of galactose) and then assay the transformed yeast in as linear molecules for clarity. Arrows
medium containing glucose and in medium containing galactose. Your indicate direction of transcription from the
results are shown in Table 12–1. You don’t remember what your advisor Problems
Gal1 promoter. p12.05/12.06
(A) + GTP – GTP

TABLE 12–1 Results of proliferation experiments with yeast carrying
plasmids pNL+ or pNL– (Problem 12–46).
Ran
Plasmid Glucose medium Galactose medium
pNL+ proliferation death
Ran
pNL– proliferation proliferation + importin
importin
told you to expect, but you know you will be expected to explain these
results at the weekly lab meeting.
Why do yeasts with the pNL+ plasmid proliferate in the presence of
glucose but die in the presence of galactose, whereas yeasts with the
pNL– plasmid proliferate in both media? (B) 1st incubation: 2nd incubation:
How might you use this system for a selection assay to isolate cells importin + substrate Ran + GTP
defective in nuclear transport?
12–47 Purified importin in the presence of Ran and GTP promotes uptake of
a labeled substrate into nuclei (Figure 12–7A). No uptake occurs in the
absence of GTP, and Ran alone is unable to promote nuclear uptake.
Importin by itself causes a GTP-independent accumulation of substrate
at the nuclear periphery, but does not promote nuclear uptake (Figure Figure 12–7 Effects of Ran, importin, and
12–7A). GTP on nuclear uptake of fluorescein-
To define the steps in the uptake pathway, you first incubate nuclei labeled substrate (Problem 12–47).
(A) Comparison of various combinations
with substrate in the presence of importin. You then wash away free of Ran andProblems 12.06/12.07
importin in the presence and
importin and substrate and incubate a second time with Ran and GTP absence of GTP. (B) Two-stage incubation
(Figure 12–7B). of cell remnants with importin and
A. Why do you think the substrate accumulates at the nuclear periphery, as substrate and then with Ran and GTP.
is seen in the absence of GTP or with importin alone in the presence of Circles are the nuclei; very light circles are
nuclei without bound substrate.
GTP?
B. To the extent these data allow, define the order of events that leads to
uptake of substrate into the nucleus.
12–48 The structures of Ran-GDP and Ran-GTP (actually Ran-GppNp, a stable
GTP analog) are strikingly different, as shown in Figure 12–8. Not sur-
prisingly, Ran-GDP binds to a different set of proteins than does Ran-
GTP.
To look at the uptake of Ran itself into the nuclei of permeabilized
cells, you attach a red fluorescent tag to a cysteine side chain in Ran
to make it visible. This modified Ran supports normal nuclear uptake.
Fluorescent Ran-GDP is taken up by nuclei only if cytoplasm is added,
whereas a mutant form, RanQ69L-GTP, which is unable to hydrolyze
GTP, is not taken up in the presence or absence of cytoplasm. To identify
the cytoplasmic protein that is crucial for Ran-GDP uptake, you construct
affinity columns with bound Ran-GDP or bound RanQ69L-GTP and pass
Ran-GppNp Ran-GDP
Figure 12–8 Structures of Ran-GDP and Ran-GppNp (Problem 12–48). The segment
of Ran shown in red displays two dramatically different conformations depending on
whether GDP or the GTP analog is bound.
Problems 12.07/12.08
cytoplasm through them. Cytoplasm passed over a Ran-GDP column no
TP
G
longer supports nuclear uptake of Ran-GDP, whereas cytoplasm passed
L-
P
69
D
over a column of RanQ69L-GTP retains this activity. You elute the bound
nQ
n-
Ra
Ra
proteins from each column and analyze them on an SDS polyacrylamide kd
gel, looking for differences that might identify the factor that is required 158
for nuclear uptake of Ran (Figure 12–9). 116
A. Why did you use RanQ69L-GTP instead of Ran-GTP in these experi- 97
ments? Could you have used Ran-GppNp instead of RanQ69L-GTP to 68
achieve the same purpose? 46

B. Which of the many proteins eluted from the two different affinity col-
umns is a likely candidate for the factor that promotes nuclear import of 27
Ran-GDP? 20
C. What other protein or proteins would you predict the Ran-GDP import 14
factor would bind in order to carry out its function? 7
D. How might you confirm that the factor you have identified is necessary 1 2
for promoting the nuclear uptake of Ran?
12–49 The broad-spectrum antibiotic leptomycin B inhibits nuclear export, but Figure 12–9 Proteins eluted from Ran-
GDP (lane 1) and RanQ69L-GTP (lane
how does it work? In the yeast S. pombe, resistance to leptomycin B can 2) affinity columns (Problem 12–48). The
arise by mutations in the Crm1 gene, which encodes a nuclear export molecular masses of marker proteins are
receptor for proteins with leucine-rich nuclear export signals. To look at shown on the left.
nuclear export directly, you modify the green fluorescent protein (GFP)
by adding a nuclear export signal (NES). In both wild-type and mutant
cells that are resistant to leptomycin B, NES-GFP is found exclusively Problems p12.08/12.09
in the cytoplasm in the absence of leptomycin B (Figure 12–10). In the

presence of leptomycin B, however, NES-GFP is present in the nuclei of
wild-type cells, but in the cytoplasm of mutant cells (Figure 12–10). Is this
result the one you would expect if leptomycin B blocked nuclear export?
Why or why not?
12–50 Frog oocytes are a useful experimental system for studying nuclear
export because they are large cells with large nuclei. It is easy (with prac-
tice) to inject oocytes with labeled RNA and to separate the nucleus and
cytoplasm to follow the fate of the injected label. You inject a mixture
of various 32P-labeled RNA molecules into the nucleus in the presence
and absence of leptomycin B to study its effect on nuclear export of RNA.
Immediately after injection and three hours later, you analyze total (T),
cytoplasmic (C), and nuclear (N) contents by polyacrylamide-gel elec-
trophoresis and autoradiography (Figure 12–11).
A. How good was your injection technique? Did you actually inject into the
nucleus? Did you rip apart the nuclear envelope when you injected the
RNAs? How do you know?
B. Which, if any, of the RNAs are normally exported from the nucleus?
C. Is the export of any of the RNAs inhibited by leptomycin B? What does
leptomycin B, nM 0 0 100
your answer imply about export of this collection of RNAs?
time, hr 0 3 3
T CN T CN T CN
mRNA
– leptomycin B + leptomycin B
NES-GFP NES-GFP U1 snRNA
U5 snRNA
U6 snRNA
DNA DNA
tRNA
wild type mutant wild type mutant
Figure 12–11 Effects of leptomycin B
Figure 12–10 Distribution of NES-GFP in S. pombe in the presence and absence of on export of various RNAs from frog
leptomycin B (Problem 12–49). Light areas in the NES-GFP panels show the position of oocyte nuclei (Problem 12–50). Total (T),
GFP. Light areas in the DNA panels result from a stain that binds to DNA and marks the cytoplasmic (C), and nuclear (N) fractions
position of the nuclei in the cells in the NES-GFP panels. are indicated.
THE TRANSPORT OF PROTEINS INTO

MITOCHONDRIA AND CHLOROPLASTS
TERMS TO LEARN
chloroplast mitochondrial precursor SAM complex
inner membrane protein stroma
intermembrane space outer membrane thylakoid
matrix space OXA complex TIM complex
mitochondria porin TOM complex
mitochondrial hsp70 protein translocator
DEFINITIONS
12–51 Membrane-enclosed organelles, about the size of bacteria, that carry out
oxidative phosphorylation and produce most of the ATP in eukaryotic
cells.
12–52 Part of a multisubunit protein assembly that is bound to the matrix side
of the TIM23 complex and acts as a motor to pull the precursor protein
into the matrix space.
12–53 Multisubunit protein assembly that transports proteins across the mito-
chondrial outer membrane.
12–54 The matrix space of a chloroplast.
12–55 The membrane of a mitochondrion that encloses the matrix and is folded
into cristae.
12–56 Central subcompartment of a mitochondrion, enclosed by the inner
mitochondrial membrane.
12–57 Flattened sac of membrane in a chloroplast that contains the protein
subunits of the photosynthetic system and of the ATP synthase.
12–58 Protein encoded by a nuclear gene, synthesized in the cytosol, and sub-
sequently transported into mitochondria.
TRUE/FALSE
12–59 The TOM complex is required for the import of all nucleus-encoded
mitochondrial proteins.
12–60 The two signal sequences required for transport of nucleus-encoded pro-
teins into the mitochondrial inner membrane via the TIM23 complex are
cleaved off the protein in different mitochondrial compartments.
12–61 Import of proteins into mitochondria and chloroplasts is very similar;
even the individual components of their transport machinery are homol-
ogous, as befits their common evolutionary origin.
THOUGHT PROBLEMS
12–62 To aid your studies of protein import into mitochondria, you treat yeast
cells with cycloheximide, which blocks ribosome movement along
mRNA. When you examine these cells in the electron microscope, you
are surprised to find cytosolic ribosomes attached to the outside of the
mitochondria. You have never seen attached ribosomes in the absence
THE TRANSPORT OF PROTEINS INTO MITOCHONDRIA AND CHLOROPLASTS 247
of cycloheximide. To investigate this phenomenon further, you prepare

mitochondria from cells that have been treated with cycloheximide and
then extract the mRNA that is bound to the ribosomes associated with
the mitochondria. You translate this mRNA in vitro and compare the
protein products with similarly translated mRNA from the cytosol. The
results are clear-cut: the mitochondria-associated ribosomes are trans-
lating mRNAs that encode mitochondrial proteins.
You are astounded! Here, clearly visible in the electron micrographs,
seems to be proof that protein import into mitochondria occurs during
translation. How might you reconcile this result with the prevailing view
that mitochondrial proteins are imported only after they have been syn-
thesized and released from ribosomes?
12–63 You have made a peptide that contains a functional mitochondrial import
signal. Would you expect the addition of an excess of this peptide to affect
the import of mitochondrial proteins? Why or why not?
12–64 Components of the TIM complexes, the multisubunit protein translo-
cators in the mitochondrial inner membrane, are much less abundant
than those of the TOM complex. They were initially identified using
a genetic trick. The yeast Ura3 gene, whose product is an enzyme that
is normally located in the cytosol where it is essential for synthesis of
uracil, was modified so that the protein carried an import signal for the
mitochondrial matrix. A population of cells carrying the modified Ura3
gene in place of the normal gene was then grown in the absence of ura-
cil. Most cells died, but the rare cells that grew were shown to be defec-
tive for import into the mitochondrial matrix. Explain how this selection
identifies cells with defects in components required for import into the
mitochondrial matrix. Why don’t normal cells with the modified Ura3
gene grow in the absence of uracil? Why do cells that are defective for
mitochondrial import grow in the absence of uracil?
12–65 Mitochondria normally provide cells with most of the ATP they require
to meet their energy needs. Mitochondria that cannot import proteins
are defective for ATP synthesis. How is it that cells with import-defective
mitochondria can survive at all? How do they get the ATP they need to
function?
12–66 Describe in a general way how you might use radiolabeled proteins and
proteases to study import processes in isolated, intact mitochondria.
What sorts of experimental controls might you include to ensure that the
results you obtain mean what you think they do?
12–67 If the enzyme dihydrofolate reductase (DHFR), which is normally located
in the cytosol, is engineered to carry a mitochondrial targeting sequence
at its N-terminus, it is efficiently imported into mitochondria. If the mod-
ified DHFR is first incubated with methotrexate, which binds tightly to
the active site, the enzyme remains in the cytosol. How do you suppose
that the binding of methotrexate interferes with mitochondrial import?
12–68 Why do mitochondria need a special translocator to import proteins
across the outer membrane, when the membrane has already has large
pores formed by porins?
CALCULATIONS
12–69 The vast majority of mitochondrial proteins are imported through the
outer membrane by the multisubunit protein translocators known as
TOM complexes. Since the number of TOM complexes in yeast mito-
chondria is known (10 pmole/mg of mitochondrial protein), it is pos-
sible to calculate whether a co-translational mechanism could account
for the bulk of mitochondrial protein import. If mitochondrial proteins

were all imported co-translationally, then 10 pmol of TOM complexes
would need to import 1 mg of protein each generation. Given that mito-
chondria double every 3 hours and that the rate of protein synthesis is 3
amino acids per second (which is therefore the maximum rate of import
through a single TOM complex), how many milligrams of mitochondrial
protein could 10 pmol of TOM complexes import in one generation? (On
average, an amino acid has a mass of 110 daltons.)
DATA HANDLING
12–70 Barnase is a 110-amino-acid bacterial ribonuclease that is often used
as a model for studies of protein folding and unfolding. It forms a com-
pact folded structure that has a high energy of activation for unfolding
(about 85 kJ/mole). Can such a protein be imported into mitochondria?
To the N-terminus of barnase, you add 35, 65, or 95 amino acids from the
N-terminus of pre-cytochrome b2, all of which include the cytochrome’s
mitochondrial import signal. N35-barnase is not imported, N65-barnase
is imported at a low rate, and N95-barnase is imported very efficiently
into isolated mitochondria. None of these N-terminal extensions have
any measurable effect on the stability of the barnase domain. If these
proteins are denatured before testing for import, they are all imported at
the same high rate. How do you suppose that longer N-terminal exten-
sions facilitate the import of barnase?
12–71 Are proteins imported into mitochondria as completely unfolded poly-
peptide chains, or can the translocation apparatus accommodate fully
or partially folded structures? That is, is the protein sucked up like a noo-
dle, or is it swallowed whole, as a python devours its prey? It is possi-
ble to engineer cysteine amino acids into barnase and then cross-link
them to make disulfide bonds either between C5 and C78 or between
C43 and C80 (Figure 12–12). Import of N95-barnase (see Problem 12–70)
was tested in the presence and absence of disulfide cross-links at these
two positions. Its import was unaffected by either cross-link. By contrast,
import of N65-barnase was blocked by the C5–C78 cross-link but unaf-
fected by the C43–C80 cross-link. Do these results allow you to distin-
guish between import of extended polypeptide chains or of folded struc-
tures? Why or why not?
12–72 Tim23 is a key component of the mitochondrial TIM23 protein transloca-
tor complex. The N-terminal half of Tim23 is hydrophilic, while the C-ter-
minal half is hydrophobic and probably spans the membrane four times,
as suggested by the hydropathy plot in Figure 12–13A. To determine the
arrangement of Tim23 in mitochondrial membranes, a protease was
added to intact mitochondria or to mitoplasts, which are mitochondria
barnase (C5–C78) barnase (C43–C80)
Figure 12–12 Structures of barnase

S
C-terminus S C-terminus molecules with disulfide bonds
S S
between cysteines at positions 5 and
78 (C5–C78) or between positions 43
N-terminus N-terminus and 80 (C43–C80) (Problem 12–71).
PEROXISOMES 249
(A) (B) Figure 12–13 Arrangement of Tim23

hydrophobic in mitochondrial membranes (Problem
mitochondria + + –
mitoplasts – – + 12–72). (A) Hydropathy plot for Tim23.
protease – + + (B) Sensitivity of Tim23 in mitochondria
and mitoplasts to digestion with a
hydrophilic protease.
50 100 150 200 antibodies
specific for
residue number
C-terminus
antibodies
specific for
N-terminal
half
1 2 3
from which the outer membranes have been removed. The mobility of
Tim23 was detected on SDS polyacrylamide gels by immunoblotting
with antibodiesProblems
specific forp12.16/12.13
the N-terminal half or for the extreme C-ter-
minus of Tim23 (Figure 12–13B). Normal-sized Tim23 was present in
both mitochondria and mitoplasts (as shown for mitochondria in Figure
12–13B), but Tim23 was partially digested when mitochondria and mito-
plasts were treated with a protease (Figure 12–13B).
A. Is Tim23 an integral component of the inner or outer mitochondrial
membrane? Explain your reasoning.
B. To the extent the information in this problem allows, diagram the
arrangement of Tim23 in mitochondrial membranes.
PEROXISOMES
TERMS TO LEARN
peroxin peroxisome
DEFINITIONS
12–73 Small membrane-bounded organelle that uses molecular oxygen to oxi-
dize organic molecules.
12–74 One of several proteins that are involved in protein import into peroxi-
somes.
TRUE/FALSE
Decide whether this statement is true or false, and then explain why.
12–75 Peroxisomes are found in only a few specialized types of eukaryotic cell.
THOUGHT PROBLEMS
12–76 Catalase, an enzyme normally found in peroxisomes, is present in nor-
mal amounts in cells that do not have visible peroxisomes. It is possible
to determine the location of catalase in such cells using immunofluo-
rescence microscopy with antibodies specific for catalase. Fluorescence
micrographs of normal cells and peroxisome-deficient cells are shown
in Figure 12–14. Where is catalase located in cells without peroxisomes
(Figure 12–14B)? Why does catalase show up as small dots of fluores-
cence in normal cells (Figure 12–14A)?
(A) (B) Figure 12–14 Location of catalase in cells

as determined by immunofluorescence
microscopy (Problem 12–76). (A) Normal
cells. (B) Peroxisome-deficient cells. Cells
were reacted with antibodies specific for
catalase, washed, and then stained with
a fluorescein-labeled second antibody
that is specific for the catalase-specific
antibody. The two panels are at the
same magnification; a 10 μm scale bar is
10 µm
shown in (B).
12–77 Cells with functional peroxisomes incorporate 9-(1 -pyrene)nonanol

(P9OH) into membraneProblems
lipids. Exposure of such cells to ultraviolet (UV)
12.19/12.14
light causes cell death because excitation of the pyrene moiety generates
reactive oxygen species, which are toxic to cells. Cells that do not make
peroxisomes lack a critical enzyme responsible for incorporating P9OH
into membrane lipids. How might you make use of P9OH to select for
cells that are missing peroxisomes?
DATA HANDLING
12–78 You have isolated two mutant cell lines that lack typical peroxisomes.
When you test these cells for peroxisomal enzymes, you find that cata-
lase activity is virtually the same as in normal cells. By contrast, acyl
CoA oxidase activity is absent in both mutant cell lines. To investigate
the acyl CoA oxidase deficiency, you perform a pulse-chase experiment:
you grow cells for 1 hour in medium containing 35S-methionine, then
transfer them to unlabeled medium and immunoprecipitate acyl CoA
oxidase at various times after transfer (Figure 12–15). You observe two
forms of oxidase in normal cells, but only one in the mutant cell lines. To
clarify the relationship between the 75 kd and 53 kd forms of the oxidase,
you isolate mRNA from wild-type and the mutant cell lines, translate it
in vitro, and immunoprecipitate acyl CoA oxidase. All three sources of
mRNA give similar levels of the 75 kd form, but none of the 53 kd form.
A. How do you think the two forms of acyl CoA oxidase in normal cells are
related? Which one, if either, do you suppose is the active enzyme?
B. Why do the mutant cells have only the 75 kd form of acyl CoA oxidase,
and why do you think it disappears during the pulse-chase experiment?
If you had done a similar experiment with catalase, do you suppose it
would have behaved the same way?
12–79 Proteins that are imported into the peroxisome matrix using a C-termi-
nal tripeptide signal are recognized by the cytosolic receptor Pex5, which
docks at a complex of proteins in the peroxisomal membrane. Delivery by
a cytosolic receptor distinguishes peroxisomal import from import into
mitochondria, chloroplasts, and the endoplasmic reticulum. Moreover,
unlike import into those organelles, peroxisomes can import fully folded
proteins and protein oligomers. You wish to distinguish three potential
modes of action for Pex5: (1) it delivers its cargo to the peroxisomal mem-
brane but remains cytosolic; (2) it enters the peroxisome along with its
cargo; or (3) it cycles between the cytosol and the peroxisomal matrix.
normal CHO cells mutant 1 mutant 2
chase (hours) 0 1 3 8 24 0 1 3 8 24 0 1 3 8 24
75 kd
53 kd
Figure 12–15 Pulse-chase experiments
with normal Chinese hamster ovary (CHO)
cells and mutant cells (Problem 12–78).
PEROXISOMES 251
Figure 12–16 Mechanism of Pex5-mediated peroxisomal import (Problem (A) CONSTRUCT

12–79). (A) Modified Pex5. (B) Expectations for different mechanisms of
Pex5-mediated peroxisomal import. (C) Analysis of transfection of the cleavage
modified Pex5 gene into cells. “WCE” stands for whole-cell extract, “P” for site
FLAG tag
pellet, and “S” for supernatant. Proteins were detected by immunoblotting
Pex5
using mAb1 or mAb2, followed by reaction with a second antibody that
binds mAb1 and mAb2 and also carries bound horseradish peroxidase,
which then converts an added precursor into a light-emitting molecule that peptide
can be detected on photographic film. Black bands correspond to sites
where the film has been exposed by the light-emitting molecule. In (B),
uncleaved Pex5 (upper band) and cleaved Pex5 (lower band) are separated (B) THEORETICAL
by a larger distance than they are in the experiment (C) for clarity.
WCE P S WCE P S
cytosolic
To define the mechanism for Pex5-mediated import, you modify

the Pex5 gene to encode an N-terminal peptide segment that includes a imported
cleavage site for a protease localized exclusively to the matrix of the per-
oxisome (Figure 12–16A). Immediately adjacent to the cleavage site is a cycling
sequence of amino acids (the so-called FLAG tag) that can be recognized
mAb2 mAb1
by commercial antibodies. One antibody, mAb2, binds the FLAG tag in
any context, whereas another, mAb1, binds the FLAG tag only when it is (C) EXPERIMENTAL
at the N-terminus and thus will detect only cleaved Pex5. By preparing a WCE P S WCE P S
whole-cell extract (WCE) and fractionating it into a pellet (P), which con-
tains the peroxisomes, and supernatant (S), which contains the cytosol,
mAb2 mAb1
you can distinguish the three possible mechanisms of Pex5-mediated
import—cytosolic, imported, cycling—by using the mAb1 and mAb2
antibodies, as shown in Figure 12–16B.
When you express the modified Pex5 gene in cells, prepare cell frac-
tions, separate the proteins by electrophoresis, and react them with mAb1
and mAb2 antibodies, you obtain the results shown in Figure 12–16C. Problems p12.22/12.16
A. Explain the theoretical results (Figure 12–16B) expected for each of the
three possible mechanisms of Pex5-mediated import.
B. Based on the results in Figure 12–16C, how does Pex5 mediate the import
of proteins into the matrix of the peroxisome? Explain your reasoning.
C. Pex5-mediated import into peroxisomes resembles most closely import
into what other cellular organelle?
MEDICAL LINKS
12–80 Primary hyperoxaluria type 1 (PH1) is a lethal autosomal recessive dis-
ease caused by a deficiency of the liver-specific peroxisomal enzyme
alanine:glyoxylate aminotransferase (AGT). About one-third of PH1
patients possess significant levels of AGT protein and enzyme activity.
Analysis of these patients shows that their AGT contains two critical sin-
gle amino acid changes: one that interferes with peroxisomal targeting
and a second that allows the N-terminus to form an amphiphilic helix
with a positively charged side. Where do you suppose this mutant AGT is
found in cells from these patients? Explain your reasoning.
12–81 Trypanosomes are single-celled parasites that cause sleeping sick-
ness when they infect humans. Trypanosomes from humans carry the
enzymes for a portion of the glycolytic pathway in a peroxisomelike
organelle, termed the glycosome. By contrast, trypanosomes from the
tsetse fly—the intermediate host—carry out glycolysis entirely in the
cytosol. This intriguing difference has alerted the interest of the pharma-
ceutical company that employs you. Your company wishes to exploit this
difference to control the disease.
You decide to study the enzyme phosphoglycerate kinase (PGK)
because it is in the affected portion of the glycolytic pathway. Trypano-
somes from the tsetse fly express PGK entirely in the cytosol, whereas
trypanosomes from humans express 90% of the total PGK activity in gly-
cosomes and only 10% in the cytosol. When you clone PGK genes from
trypanosomes, you find three forms that differ slightly from one another. probe oligo 1 oligo 2 oligo 3
Exploiting these small differences, you design three oligonucleotides source
H F H F H F
of mRNA
that hybridize specifically to the mRNAs from each gene. Using these oli-
gonucleotides as probes, you determine which genes are expressed by
trypanosomes from humans and from tsetse flies. The results are shown
in Figure 12–17.
gene 3
A. Which PGK genes are expressed in trypanosomes from humans? Which gene 1
are expressed in trypanosomes from tsetse flies?
gene 2
B. Which PGK gene probably encodes the glycosomal form of PGK?
C. Do you think that the minor cytosolic PGK activity in trypanosomes
from humans is due to inaccurate sorting into glycosomes? Explain your
answer.
Figure 12–17 Hybridization of specific
THE ENDOPLASMIC RETICULUM oligonucleotide probes (oligos) to
mRNA isolated from trypanosomes
TERMS TO LEARN from humans (H) and tsetse flies (F)
(Problem 12–81). The intensity of the
BiP microsome bands on the autoradiograph reflects
calnexin multipass transmembrane protein Problemsof12.20/12.17
the concentrations the mRNAs.
calreticulin polyribosome
co-translational post-translational
dolichol protein glycosylation
endoplasmic reticulum (ER) rough ER
ER lumen Sec61 complex
ER resident protein signal-recognition particle (SRP)
ER retention signal single-pass transmembrane protein
ER signal sequence smooth ER
ER tail-anchored protein SRP receptor
free ribosome start-transfer signal
glycoprotein stop-transfer signal
GPI anchor translocon
membrane-bound ribosome unfolded protein response
DEFINITIONS
12–82 Region of the ER not associated with ribosomes.
12–83 Type of lipid linkage by which some proteins are bound to the mem-
brane.
12–84 Labyrinthine membrane-enclosed compartment in the cytoplasm of
eukaryotic cells, where lipids are synthesized and membrane-bound
proteins and secretory proteins are made.
12–85 Ribonucleoprotein complex that binds an ER signal sequence on a par-
tially synthesized polypeptide chain and directs the polypeptide and its
attached ribosome to the ER.
12–86 Hydrophobic amino acid sequence that halts translocation of a polypep-
tide chain through the ER membrane, thus anchoring the protein chain
in the membrane.
12–87 Describes import of a protein into the ER before the polypeptide chain is
completely synthesized.
12–88 Short amino acid sequence on a protein that keeps it in the ER.
12–89 Cellular action triggered by an accumulation of misfolded proteins in the
ER.
12–90 The protein translocator that forms a water-filled pore in the ER
THE ENDOPLASMIC RETICULUM 253
membrane, allowing passage of a polypeptide chain as it is being synthe-

sized by membrane-bound ribosomes.
12–91 Any protein with one or more oligosaccharide chains covalently linked to
amino acid side chains.
12–92 Ribosome in the cytosol that is unattached to any membrane.
TRUE/FALSE
12–93 The signal peptide binds to a hydrophobic site on the ribosome causing a
pause in protein synthesis, which resumes when SRP binds to the signal
peptide.
12–94 Nascent polypeptide chains are transferred across the ER membrane
through a pore in the Sec61 protein translocator complex.
12–95 In multipass transmembrane proteins, the odd-numbered transmem-
brane segments (counting from the N-terminus) act as start-transfer sig-
nals and the even-numbered segments act as stop-transfer signals.
12–96 The ER lumen contains a mixture of thiol-containing reducing agents
that prevent the formation of S–S linkages (disulfide bonds) by maintain-
ing the cysteine side chains of luminal proteins in reduced (–SH) form.
THOUGHT PROBLEMS
12–97 Explain how an mRNA molecule can remain attached to the ER mem-
brane while the individual ribosomes translating it are released and
rejoin the cytosolic pool of ribosomes after each round of translation.
12–98 Why are cytosolic hsp70 chaperone proteins required for import of pro-
teins into mitochondria and chloroplasts, but not for co-translational
import into the ER?
12–99 Where would you expect to see microsomes in an electron micrograph of
a liver cell?
12–100 Compare and contrast protein import into the ER and into the nucleus.
List at least two major differences in the mechanisms and speculate on
why the nuclear mechanism might not work for ER import and vice versa.
12–101 Four membrane proteins are represented schematically in Figure 12–18.
The boxes represent membrane-spanning segments and the arrows rep-
resent sites for cleavage of the signal sequence. Predict how each of the
mature proteins will be arranged across the membrane of the ER. Indicate
(A)
N 1 2 C
(B)
+ –
1 C
Figure 12–18 Distribution of the
N
membrane-spanning segments in
proteins to be inserted into the ER
(C) membrane (Problem 12–101). Boxes
– +
1 2 3 4 represent membrane-spanning segments
N C
and arrows indicate sites at which signal
sequences are cleaved. The pluses and
(D) minuses indicate the charged amino acids
at the ends of some transmembrane
N 1N 2 3 4 5 C segments.
COOH Figure 12–19 Arrangement of a

multipass transmembrane protein in the
1 3 5 ER membrane (Problem 12–102). Blue
hexagons represent covalently attached
CYTOSOL
oligosaccharides. The positions of
positively and negatively charged amino
acids flanking the second transmembrane
segment are shown.
ER LUMEN
2 4 6
NH2
clearly the N- and C-termini relative to the cytosol and the lumen of the
ER, and label each box as a start-transfer or stop-transfer signal.
12–102 Examine the multipass transmembrane protein shown in Figure 12–19.
What would you predict would be the effect of converting the first hydro-
phobic transmembrane segment to a hydrophilic segment? Sketch the
arrangement of the modified protein in the ER membrane.
12–103 Why might it be advantageous to add a preassembled block of 14 sugars
to a protein in the ER, rather than building the sugar chains step-by-step
on the surface of the protein by the sequential addition of sugars by indi-
vidual enzymes?
12–104 Outline the steps by which misfolded proteins in the ER trigger synthesis
of additional ER chaperone proteins. How does this response benefit the
cell?
12–105 All new phospholipids are added to the cytosolic leaflet of the ER mem-
brane, yet the ER membrane has a symmetrical distribution of different
phospholipids in its two leaflets. By contrast, the plasma membrane,
which receives all its membrane components ultimately from the ER, has
a very asymmetrical distribution of phospholipids in the two leaflets of
its lipid bilayer. How is the symmetry generated in the ER membrane,
and how is the asymmetry generated and maintained in the plasma
membrane?
DATA HANDLING
12–106 Translocation of proteins across rough microsomal membranes can be
judged by several experimental criteria: (1) the newly synthesized pro-
teins are protected from added proteases, unless detergents are present
to solubilize the lipid bilayer; (2) the newly synthesized proteins are glyc-
osylated by oligosaccharide transferases, which are localized exclusively
to the lumen of the ER; (3) the signal peptides are cleaved by signal pepti-
dase, which is active only on the luminal side of the ER membrane.
Use these criteria to decide whether a protein is translocated across
rough microsomal membranes. The mRNA is translated into protein in
a cell-free system in the absence or presence of microsomes. Samples
of newly synthesized proteins are treated in four different ways: (1) no
treatment, (2) addition of a protease, (3) addition of a protease and deter-
gent, and (4) disruption of microsomes and addition of endoglycosidase
H (endo H), which removes N-linked sugars that are added in the ER. An
electrophoretic analysis of these samples is shown in Figure 12–20.
A. Explain the experimental results that are seen in the absence of micro-
somes (Figure 12–20, lanes 1 to 4).
B. Using the three criteria outlined in the problem, decide whether the
experimental results in the presence of microsomes (Figure 12–20,
lanes 5 to 8) indicate that the protein is translocated across microsomal
MICROSOMES MICROSOMES Figure 12–20 Results of translation of a

ABSENT PRESENT pure mRNA in the presence and absence
TREATMENT
of microsomal membranes (Problem
protease – + + – – + + – 12–106). Treatments of the products
detergent – – + – – – + – of translation before electrophoresis
are indicated at the top of each lane.
endo H – – – + – – – +
Electrophoresis was on an SDS
polyacrylamide gel, which separates
proteins on the basis of size, with lower
molecular weight proteins migrating
farther down the gel.
1 2 3 4 5 6 7 8
membranes. How would you account

Problems for the migration of the proteins in
p12.25/12.20
Figure 12–20, lanes 5, 6, and 8?
C. Is the protein anchored in the membrane, or is it translocated all the way
through the membrane?
12–107 Things are not going well. You’ve just had a brief but tense meeting with
your research advisor that you will never forget, and it is clear that your
future in his lab is in doubt. He is not fond of your habit of working late
and sleeping late; he thinks it is at the root of your lack of productivity
(as he perceives it). A few days after the meeting, you roll in at the bright
and early hour of noon, to be informed by your student colleagues that
your advisor is “looking all over for you.” You are certain the axe is going
to fall. But it turns out he is excited, not upset. He’s just heard a seminar
that reminded him of the note you’d left on his desk the month before,
describing a selection scheme you had crafted (late one night, of course)
for isolating mutants in the ER translocation machinery. You are com-
pletely flabbergasted.
You quickly settle on the details for the selection, which involves fus-
ing an ER import signal to the N-terminus of the His4 gene product. His4
is a cytosolic enzyme that converts histidinol to histidine. Yeast strains
that are defective for an early step in the histidine biosynthetic pathway
can grow on added histidinol, if His4 is present. You decide to look for
temperature-sensitive (ts) mutants, which are normal at 24°C but defec-
tive at 37°C. Using a strain that expresses His4 with an ER import signal,
you select for cells that grow on histidinol at 30°C and screen them for
ones that die at 37°C. The first mutant you isolate is in the Sec61 gene
(later shown to encode a principal component of the translocator through
which ribosomes insert nascent proteins across the ER membrane.) You
are back in your advisor’s good graces!
A. Why is it that normal cells with the modified His4 cannot grow on histidi-
nol, whereas cells with a defective ER-import apparatus can?
B. Why did the selection scheme set out to find ts mutants? Why was selec-
tion applied at the intermediate temperature of 30°C, rather than at 24°C
or 37°C?
12–108 A classic paper describes a genetic method for determining the organi-
zation of a bacterial protein in the membrane of E. coli. The hydropathy
plot of the protein in Figure 12–21 indicated three potential membrane-
spanning segments. Hybrid fusion proteins of different lengths, some
with internal deletions, were made with the membrane protein at the
N-terminus and alkaline phosphatase at the C-terminus (Figure 12–22).
Alkaline phosphatase is easy to assay in whole cells and has no significant
Figure 12–21 Hydropathy plot of a
hydropathy index
more hydrophobic
membrane protein (Problem 12–108). The
three hydrophobic peaks indicate the
0 positions of three potential membrane-
spanning segments.
more hydrophilic
0 50 100 150 200 250 300
amino acid number
hydrophobic stretches. Moreover, when it is on the cytoplasmic side of

the membrane its activity is low, and when it is on the external side of
the membrane (in the periplasmic space) its activity is high. The assayed
levels of alkaline phosphatase activity are indicated (HIGH or LOW) in
Figure 12–22. Problems p12.28/12.21
A. How is the protein organized in the membrane? Explain how the results
with the fusion proteins indicate this arrangement.
B. How is the organization of the membrane protein altered by the deletion?
Are your measurements of alkaline phosphatase activity in the internally
deleted plasmids consistent with the altered arrangement?
12–109 Mitochondria and peroxisomes, as opposed to most other cellular mem-
branes, acquire new phospholipids via phospholipid exchange proteins.
One such protein, PC exchange protein, specifically transfers phosphati-
dylcholine (PC) between membranes. Its activity can be measured by
mixing red blood cell ghosts (intact plasma membranes with cytoplasm
removed) with synthetic phospholipid vesicles containing radioactively
labeled PC in both monolayers of the vesicle bilayer. After incubation
at 37°C, the mixture is centrifuged briefly so that ghosts form a pellet,
whereas the vesicles stay in the supernatant. The amount of exchange is
determined by measuring the radioactivity in the pellet.
Figure 12–23 shows the result of an experiment along these lines,
using labeled (donor) vesicles with an outer radius of 10.5 nm and a
bilayer 4.0 nm in thickness. No transfer occurred in the absence of the
exchange protein, but in its presence up to 70% of the labeled PC in the
vesicles could be transferred to the red cell membranes.
Several control experiments were performed to explore the reason
why only 70% of the label in donor vesicles was transferred.
original
constructs alkaline phosphatase activity
34
1 HIGH
55
2 HIGH
105
3 LOW
131
4 LOW
225
5 HIGH
310
6 HIGH Figure 12–22 Structures of hybrid
proteins used to determine the
internally deleted organization of a membrane protein
derivatives (Problem 12–108). The membrane protein
105 (orange segment) is at the N-terminus
3* HIGH and alkaline phosphatase (blue segment)
131 is at the C-terminus of the protein. The
4* HIGH
inverted V indicates the site from which
225
5* LOW amino acids were deleted from modified
310 hybrid proteins. The most C-terminal
6* LOW amino acid of the membrane protein is
numbered in each hybrid protein. The
deletion of activity of alkaline phosphatase in each
residues 68–103 hybrid protein is shown on the right.
1. Five times as many membranes from red cell ghosts were included in the 100
incubation: the transfer still stopped at the same point.
from vesicles (percent)

+ PC exchange protein
labeled PC removed
80
2. Fresh exchange protein was added after 1 hour: it caused no further
transfer. 60
3. The labeled lipids remaining in donor vesicles at the end of the reaction
were extracted and made into fresh vesicles: 70% of the label in these 40
vesicles was exchangeable.
20 – PC exchange protein
When the red cell ghosts that were labeled in this experiment were
used as donor membranes in the reverse experiment (that is, transfer of 0
0 0.5 1.0 1.5
PC from red cell membranes to synthetic vesicles), 96% of the label could time (hours)
be transferred to the acceptor vesicles.
A. What possible explanations for the 70% limit do each of the three control Figure 12–23 Transfer of labeled PC from
experiments eliminate? donor vesicles to red cell membranes by
B. What do you think is the explanation for the 70% limit? (Hint: the area of PC exchange protein (Problem 12–109).
the outer surface of these small donor vesicles is about 2.6 times larger
than the area of the inner surface.) Problems p12.30/12.23
C. Why do you think that almost 100% of the label in the red cell membrane
can be transferred back to the vesicle?
MCAT STYLE
You are studying a transcription factor that controls entry into the cell cycle. It is
present in the nucleus in nondividing cells, but rapidly exits the nucleus when
cells are stimulated to begin cell division. You hypothesize that the transcription
factor represses expression of genes that drive entry into the cell cycle, and that
transport of the repressor out of the nucleus is a critical step that helps initiate the
cell cycle. While searching for the signals that control this step, you find that the
protein is phosphorylated by a protein kinase at a specific serine. Since phosphor-
ylation of proteins is an important mode of regulation, you investigate further. To
do so, you create a mutant version of the transcription factor in which the serine
that is the target for phosphorylation is changed to an alanine, which cannot be
phosphorylated. When you express this mutant version of the transcription factor
in the cell, you find that very little of it can be detected in the nucleus.
12–110 Transport of the transcription factor into the nucleus most likely requires:
I. Binding to a nuclear import receptor
II. Direct participation of Ran-GDP
III. Involvement of the Sec61 complex
A. I
B. I and II
C. I and III
D. I, II, and III
12–111 Which one of the following hypotheses best explains how phosphoryla-
tion regulates nuclear localization of the transcription factor?
A. Phosphorylation causes a conformational change that exposes a nuclear
export signal.
B. Phosphorylation near a nuclear export signal blocks binding of a nuclear
export factor.
C. Phosphorylation near a nuclear localization signal blocks binding of a
nuclear import factor.
D. Phosphorylation promotes binding of the transcription factor to Ran-
GTP.
12–112 How might you test the best hypothesis from the previous question?
A. Addition of a nuclear import signal to the mutant transcription factor
should allow it to accumulate in the nucleus.
B. Deletion of the nuclear export signal from the mutant transcription fac-
tor should allow it to accumulate in the nucleus.
C. Deletion of the Ran-GDP binding site from the mutant transcription fac-
tor should allow it to accumulate in the nucleus.
D. Inactivation of the Ran-GTPase should allow the normal—but not the
mutant—transcription factor to enter the nucleus.

When cells are broken open, the endoplasmic reticulum (ER) fragments into
spherical vesicles coated with ribosomes, which are called rough microsomes.
These rough microsomes can be purified and used to form a lipid bilayer that
separates two liquid-filled chambers. One side of the lipid bilayer is coated with
ribosomes and corresponds to the cytosolic side of the ER, while the other cor-
responds to the luminal side of the ER. The chambers on each side of the mem-
brane can be monitored to detect conductance of salt ions across the membrane.
No conductance can be detected across the rough microsomal membrane as it is
isolated from cells. However, treatment of the ribosome-coated side of the mem-
brane with puromycin, a compound that causes release of growing peptide chains
from ribosomes, causes a large increase in conductance. Further treatment with
high-salt buffers causes the membrane to be impermeable to ions again.
12–113 Association of ribosomes with the endoplasmic reticulum requires:
I. Signal-recognition particle
II. Chaperones
III. Protein translation
A. I
B. I and III
C. II and III
D. I, II, and III
12–114 Which one of the following statements best explains the increase in con-
ductance caused by puromycin?
A. Premature termination of peptide synthesis by puromycin leads to
improperly folded proteins that activate the unfolded protein response.
B. Release of the growing peptide exposes the water-filled pore that is used
for translocation of proteins across the ER membrane.
C. Release of the peptide allows the signal-recognition particle to open a
channel for translocation of peptides across the ER membrane.
D. Treatment with puromycin opens channels in the membrane that are
normally used to release Ca2+ from the ER.
12–115 Why does washing the membrane with a buffer containing high concen-
trations of salt block the conductance of the microsomal membrane?
A. High salt causes the Ca2+ channel to close.
B. High salt increases the resistance of the membrane.
C. High-salt removal of ribosomes closes the pore.
D. High salt shuts off the unfolded protein response.
Chapter 13 259
CHAPTER
Intracellular Membrane Traffic 13

THE MOLECULAR MECHANISMS OF MEMBRANE IN THIS CHAPTER
TRANSPORT AND THE MAINTENANCE OF THE MOLECULAR MECHANISMS
COMPARTMENTAL DIVERSITY OF MEMBRANE TRANSPORT
TERMS TO LEARN AND THE MAINTENANCE OF
COMPARTMENTAL DIVERSITY
adaptor protein COPI-coated vesicle Rab effector
ARF protein COPII-coated vesicle Rab protein TRANSPORT FROM THE ER
cargo dynamin Sar1 protein
THROUGH THE
clathrin lumen SNARE protein (SNARE)
clathrin-coated vesicle NSF transport vesicle GOLGI APPARATUS
coated vesicle phosphatidylinositide t-SNARE
TRANSPORT FROM THE
coat-recruitment (PIP) v-SNARE
GTPase Rab cascade TRANS GOLGI NETWORK TO
LYSOSOMES
DEFINITIONS TRANSPORT INTO THE
Match each definition below with its term from the list above. CELL FROM THE PLASMA
MEMBRANE: ENDOCYTOSIS
13–1 General term for a membrane-enclosed container that moves material
between membrane-enclosed compartments within the cell. TRANSPORT FROM THE TRANS
13–2 Any of a large family of monomeric GTPases present in the plasma mem- GOLGI NETWORK TO THE CELL
brane and organelle membranes that confer specificity on vesicle dock- EXTERIOR: EXOCYTOSIS
ing.
13–3 A protein that mediates binding between the clathrin coat and trans-
membrane proteins, including transmembrane cargo receptors.
13–4 Cytosolic GTPase that binds to the neck of a clathrin-coated vesicle and
helps it to pinch off from the membrane.
13–5 The protein that catalyzes the disassembly of the helical domains of
paired SNARE proteins.
13–6 Coated vesicle that transports material from the plasma membrane and
between endosomal and Golgi compartments.
13–7 The coat-recruitment GTPase responsible for both COPI coat assembly
and clathrin coat assembly at Golgi membranes.
13–8 Protein that facilitates vesicle transport, docking, and membrane fusion
once it is bound by an activated Rab protein.
13–9 General term for a member of the large family of proteins that catalyze
the membrane fusion reactions in membrane transport.
13–10 The interior space of a membrane-enclosed compartment.
13–11 General term for a transport vesicle that carries a distinctive cage of
proteins covering its cytosolic surface.
260 Chapter 13: Intracellular Membrane Traffic
13–12 The coat-recruitment GTPase responsible for COPII coat assembly at the
ER membrane.
TRUE/FALSE
13–13 In all events involving fusion of a vesicle to a target membrane, the cyto-
solic leaflets of the vesicle and target bilayers always fuse together, as do
the leaflets that are not in contact with the cytosol.
13–14 Complementary Rab proteins on transport vesicles and target mem-
branes bind to one another to allow transport vesicles to dock selectively
at their appropriate target membranes.
THOUGHT PROBLEMS
13–15 In a nondividing cell such as a liver cell, why must the flow of membrane
between compartments be balanced, so that the retrieval pathways
match the outward flow? Would you expect the same balanced flow in a
gut epithelial cell, which is actively dividing?
13–16 The diagram in Figure 13–1 shows the various intracellular compart-
ments involved in the biosynthetic-secretory, endocytic, and retrieval
pathways.
A. Label the various compartments in the diagram.
B. Indicate on the arrows whether the indicated flow is part of the biosyn-
thetic-secretory pathway, the endocytic pathway, or a retrieval pathway.
13–17 Discuss the following analogy: “Cargo receptors competing to be trans-
ported by the coated-pit system can be compared to skiers joining a
cable-car network. Entry is permitted to ticket holders only, but there
is no guarantee of who is found with whom in a particular cable car,
although all travelers, hopefully, will reach the next station.”
13–18 The clathrin coat on a vesicle is made up of numerous triskelions that
form a cage 60–200 nm in diameter, composed of both pentagonal and
hexagonal faces, just like C60 fullerene (Figure 13–2). Sketch the location
of an individual triskelion in the clathrin-coated vesicle in Figure 13–2B.
CYTOSOL EXTRACELLULAR SPACE
Figure 13–1 The intracellular

compartments in the biosynthetic-
secretory, endocytic, and retrieval
pathways, with flow between
compartments indicated (Problem 13–16).
THE MOLECULAR MECHANISMS OF MEMBRANE TRANSPORT 261
(A) (B) (C) Figure 13–2 Structure of a clathrin coat

(Problem 13–18). (A) A triskelion subunit.
(B) A clathrin-coated vesicle. (C) A C60
fullerene.
25 nm
triskelion clathrin-coated vesicle fullerene (C60)
At what point in its structure does a triskelion have to be most flexible to

accommodate changes in size of the vesicle? At what point does it have to
be most flexible to fit into both the pentagonal and hexagonal faces?
13–19 Yeast, and many other organisms, make a single type of clathrin heavy
chain and a single type of clathrin light chain; thus, they make a single
kind of clathrin Problems
coat. How isp13.02/13.02
it, then, that a single clathrin coat can be
used for three different transport pathways—Golgi to late endosomes,
plasma membrane to early endosomes, and immature secretory vesicles
to Golgi—that each involves different specialized cargo proteins?
13–20 Imagine that ARF1 protein was mutated so that it could not hydrolyze
GTP, regardless of its binding partners. Would you expect COPI-coated
vesicles to form normally? How would you expect transport mediated by
COPI-coated vesicles to be affected? If this were the only form of ARF1 in
a cell, would you expect it to be lethal? Explain your answers.
13–21 How can it possibly be true that complementary pairs of specific SNAREs
uniquely mark vesicles and their target membranes? After vesicle fusion,
the target membrane will contain a mixture of t-SNAREs and v-SNAREs.
Initially, these SNAREs will be tightly bound to one another, but NSF can
pry them apart, reactivating them. What do you suppose prevents target
membranes from accumulating a population of v-SNAREs equal to or
greater than their population of t-SNAREs?
13–22 Viruses are the ultimate scavengers—a necessary consequence of their
small genomes. Wherever possible, they make use of the cell’s machin-
ery to accomplish the steps involved in their own reproduction. Many
different viruses have membrane coverings. These so-called enveloped
viruses gain access to the cytosol by fusing with a cell membrane. Why
do you suppose that each of these viruses encodes its own special fusion
protein, rather than making use of a cell’s SNAREs?
CALCULATIONS
13–23 For fusion of a vesicle with its target membrane to occur, the membranes
have to be brought to within 1.5 nm so that the two bilayers can join
(Figure 13–3). Assuming that the relevant portions of the two mem-
branes at the fusion site are circular regions 1.5 nm in diameter, calculate vesicle
the number of water molecules that would remain between the mem-
branes. (Water is 55.5 M and the volume of a cylinder is r2h.) Given that
an average phospholipid occupies a membrane surface area of 0.2 nm2, target membrane 1.5 nm
how many phospholipids would be present in each of the opposing mon-
olayers at the fusion site? Are there sufficient water molecules to bind
to the hydrophilic head groups of this number of phospholipids? (It is Figure13–3 Close approach of a vesicle
estimated that 10–12 water molecules are normally associated with each and its target membrane in preparation
phospholipid head group at the exposed surface of a membrane.) for fusion (Problem 13–23).
GTP COPI Figure 13–4 Normal pathway for

ARF-GDP ARF-GTP COPI-coated formation of COPI-coated vesicles
(cytosol) (membrane) vesicles (Problem 13–24). The small GTPase ARF
carries a bound GDP in its cytosolic
GDP form. In response to a guanine nucleotide
GEF-catalyzed exchange factor (GEF), ARF releases GDP
and picks up GTP. Binding of GTP causes
a conformational change that exposes
DATA HANDLING a fatty acid tail on ARF, which promotes
binding of ARF-GTP to the membrane.
13–24 When the fungal metabolite brefeldin A is added to cells, the Golgi appa- COPI subunits bind to ARF-GTP to form
ratus largely disappears and the Golgi proteins intermix with those in COPI-coated vesicles.
the ER. Brefeldin-A Problems
treatment alsop13.05/13.04
causes the rapid dissociation of some
Golgi-associated peripheral membrane proteins, including subunits of
the COPI coat. These observations imply that brefeldin A prevents trans-
port involving COPI-coated vesicles by blocking the assembly of coats,
and thus the budding of transport vesicles. In principle, brefeldin A
could block formation of COPI-coated vesicles at any point in the nor-
mal scheme for assembly, which is shown in Figure 13–4. The following
observations identify the point of action of brefeldin A.
1. ARF with bound GTP S (a nonhydrolyzable analog of GTP) causes
COPI-coated vesicles to form when added to Golgi membranes. Forma-
tion of vesicles in this way is not affected by brefeldin A.
2. ARF with bound GDP exchanges the GDP for GTP when added to
Golgi membranes. This exchange reaction does not occur in the presence
of brefeldin A. The essential component in the Golgi membrane is sensi-
tive to trypsin digestion, suggesting that it is a protein.
Given these experimental observations, how do you think brefeldin A
blocks formation of COPI-coated vesicles?
13–25 Small GTPases are generally active in the GTP-bound state and inactive
when the GTP is hydrolyzed to GDP. In the absence of a GTPase-activat-
ing protein (GAP), small GTPases typically hydrolyze GTP very slowly.
The mechanism by which a GAP stimulates GTP hydrolysis is known for
the small GTPase Ras. When Ras-GAP binds to Ras, it alters the confor-
mation of Ras and provides a critical, catalytic arginine “finger” that sta-
bilizes the transition state for GTP hydrolysis, thereby stimulating hydrol-
ysis by several orders of magnitude.
During assembly of COPI-coated vesicles, ARF1—a small GTPase—
binds to ARF1-GAP, which locks ARF1 into its active catalytic conforma-
tion but does not supply the catalytic arginine. Since COPI subunits also
bind to ARF1, you wonder if they might affect GTP hydrolysis. To test this
possibility, you mix ARF1, ARF1-GAP, and COPI subunits in various com-
binations and measure GTP hydrolysis (Table 13–1).
How would you interpret these results? How might you further test
your conclusions?
13–26 SNAREs exist as complementary partners that carry out membrane
fusions between appropriate vesicles and their target membranes. In this
way, a vesicle with a particular variety of v-SNARE will fuse only with a
TABLE 13–1 Rates of GTP hydrolysis by various combinations of

ARF1, ARF1-GAP, and COPI subunits (Problem 13–25).
Components added Rate of GTP hydrolysis
ARF1 0
ARF1 + ARF1-GAP 1
ARF1 + COPI subunits 0
ARF1 + ARF1-GAP + COPI subunits 1000
THE MOLECULAR MECHANISMS OF MEMBRANE TRANSPORT 263
(A) strain A strain B (B)
v t 100
pro-Pase protease
t v
75
alkaline phosphatase
DOCKING
(% maximum)
50
pro-Pase protease
25
FUSION
0
Pase SNARE strain A vt t t vt v v vt t v – vt
combinations strain B vt t vt t v vt v v t vt –
experiment 1 2 3 4 5 6 7 8 9 10 11
membrane that carries the complementary t-SNARE. In some instances, Figure 13–5 SNARE requirements for
however, fusions of identical membranes (homotypic fusions) are known vesicle fusion (Problem 13–26).
(A) Scheme for measuring the fusion of
to occur. For example, when a yeast cell forms a bud, vesicles derived vacuolar vesicles. (B) Results of fusions
from the mother cell’s vacuole move into the bud where they fuse with of vesicles with different combinations of
one another to form a new vacuole. These vesicles
Problems carry both v-SNAREs
p13.06/13.05 v-SNAREs and t-SNAREs. The SNAREs
and t-SNAREs. Are both types of SNAREs essential for this homotypic present on the vesicles of the two strains
are indicated as v (v-SNARE) and
fusion event?
t (t-SNARE).
To test this point, you have developed an ingenious assay for fusion
of vacuolar vesicles. You prepare vesicles from two different mutant
strains of yeast: strain B has a defective gene for vacuolar alkaline phos-
phatase (Pase); strain A is defective for the protease that converts the
precursor of alkaline phosphatase (pro-Pase) into its active form (Pase)
(Figure 13–5A). Neither strain has active alkaline phosphatase, but when
extracts of the strains are mixed, vesicle fusion generates active alkaline
phosphatase, which can be easily measured (Figure 13–5).
Now you delete the genes for the vacuolar v-SNARE, t-SNARE, or both
in each of the two yeast strains. You prepare vacuolar vesicles from each
and test them for their ability to fuse, as measured by the alkaline phos-
phatase assay (Figure 13–5B).
What do these data say about the requirements for v-SNAREs and
t-SNAREs in the fusion of vacuolar vesicles? Does it matter which kind of
SNARE is on which vesicle?
13–27 You wish to identify the target proteins that are bound by NSF and its two
accessory proteins. You incubate purified NSF and its accessory proteins
with a crude detergent extract of synaptic membranes, and then add
NSF-specific antibodies that are attached to beads. By centrifuging the
mixture, you can readily separate the beads, and any attached proteins,
from the rest of the crude extract. The proteins attached to the beads can
be analyzed by SDS polyacrylamide-gel electrophoresis. When the incu-
bation is carried out in the presence or absence of ATP, you find that NSF
alone is present on the beads. If you incubate in the presence of ATP S, a
nonhydrolyzable analog of ATP, the beads bring down NSF, its accessory
proteins, syntaxin, SNAP25, and synaptobrevin. What is the substrate for
NSF and its accessory proteins? Why does the experiment work when
ATP S is present, but not in the presence or absence of ATP?
13–28 The Sec4 gene of budding yeast encodes a small GTPase that plays
an essential role in the secretion pathway that forms the daughter
bud. Normally, about 80% of the Sec4 protein is found on the cyto-
solic surface of transport vesicles and 20% is free in the cytosol. When
temperature-sensitive Sec4 mutants of yeast (Sec4ts) are incubated at

high temperature, growth ceases and small vesicles accumulate in the
daughter bud.
To define the role of Sec4 in secretion, you engineer two specific Sec4
mutants based on the way other small GTPases work. One mutant, Sec4-
cc , lacks two cysteines at its C-terminus, which you expect will prevent
attachment of the fatty acid required for membrane binding. The second
mutant, Sec4N133I, encodes an isoleucine in place of the normal aspara-
gine at position 133; you expect that this protein will be locked into its
active state, even though it should not be able to bind GTP or GDP.
You find that Sec4-cc binds GTP but remains entirely cytosolic with
none bound to vesicles. When expressed at high levels in yeast that also
have a normal Sec4 gene, it does not inhibit their growth. In contrast,
Sec4N133I is located almost entirely on vesicles, and when it is expressed
at high levels in normal yeast, it completely inhibits growth, and the yeast
are found to be packed with small vesicles.
A. Do you think Sec4 is required for formation of vesicles, for vesicle fusion
with target membranes, or for both? Based on its function, would you
guess it was analogous to mammalian ARF, Sar1, or Rab proteins?
B. Using your knowledge of the way the analogous mammalian protein
works, outline how you think normal Sec4 functions in vesicle formation
and fusion. Why is some Sec4 free in the cytosol of wild-type cells? How
does removal of the C-terminal cysteines prevent Sec4-cc from carry-
ing out its function?
C. Why do you think expression of Sec4N133I inhibits growth of the yeast
that also express normal Sec4?
TRANSPORT FROM THE ER THROUGH THE GOLGI

APPARATUS
TERMS TO LEARN
cis face O-linked glycosylation
cis Golgi network (CGN) proteoglycan
cisternal maturation model trans face
complex oligosaccharide trans Golgi network (TGN)
Golgi apparatus (Golgi complex) vesicle transport model
high-mannose oligosaccharide
DEFINITIONS
13–29 The hypothesis that new cisternae form continuously at the cis face of the
Golgi and then migrate through the stack as they mature.
13–30 Molecule consisting of one or more glycosaminoglycan chains attached
to a core protein.
13–31 The side of the Golgi stack at which material enters the organelle.
13–32 Chain of sugars attached to a glycoprotein that is generated by initially
trimming the original oligosaccharide attached in the ER and by then
adding other sugars.
13–33 Membrane-enclosed organelle in eukaryotic cells in which proteins and
lipids transferred from the ER are modified and sorted.
13–34 Chain of sugars attached to a glycoprotein that contains many mannose
residues.
13–35 Meshwork of interconnected cisternae and tubules on the side of the
Golgi stack at which material is transferred out of the Golgi.
TRANSPORT FROM THE ER THROUGH THE GOLGI APPARATUS 265
TRUE/FALSE
13–36 There is one strict requirement for the exit of a protein from the ER: it
must be correctly folded.
13–37 All of the glycoproteins and glycolipids in intracellular membranes have
their oligosaccharide chains facing the lumenal side, and all those in the
plasma membrane have their oligosaccharide chains facing the outside
of the cell.
13–38 The Golgi apparatus confers the heaviest glycosylation of all on pro-
teoglycan core proteins, which are converted into proteoglycans by the
addition of one or more O-linked glycosaminoglycan chains.
THOUGHT PROBLEMS
13–39 How is it that soluble proteins in the ER can be selectively recruited into
vesicles destined for the Golgi?
13–40 Isn’t quality control always a good thing? How can quality control in the
ER be detrimental to cystic fibrosis patients?
13–41 The C-terminal 40 amino acids of three ER resident proteins—calnexin,
calreticulin, and HMG CoA reductase—are shown in Figure 13–6. Decide
for each protein whether it is likely to be a transmembrane protein or a
soluble one. Explain your answers.
13–42 If you were to remove the ER retrieval signal from protein disulfide
isomerase (PDI), which is normally a soluble resident of the ER lumen,
where would you expect the modified PDI to be located?
13–43 The KDEL receptor must shuttle back and forth between the ER and the
Golgi apparatus in order to accomplish its task of ensuring that soluble
ER proteins are retained in the ER lumen. In which compartment does
the KDEL receptor bind its ligands more tightly? In which compartment
does it bind its ligands more weakly? What is thought to be the basis
for its different binding affinities in the two compartments? If you were
designing the system, in which compartment would you have the high-
est concentration of KDEL receptor? Would you predict that the KDEL
receptor, which is a transmembrane protein, would itself possess an ER
retrieval signal?
13–44 When the KDEL retrieval signal is added to rat growth hormone or
human chorionic gonadotropin, two proteins that are normally secreted,
the proteins are still secreted, but about six times more slowly. If the
C-terminal L in the signal is changed to V, the proteins are once again
secreted at their normal rate. By contrast, bona fide ER resident proteins
rarely, if ever, are secreted from the cell; they are usually captured and
returned very efficiently. How is it, do you suppose, that normal resident
proteins with a KDEL signal are efficiently retained in the ER, whereas
secreted proteins to which a KDEL signal has been added are not
efficiently retained? Is this what you would expect if the KDEL signal and
the KDEL receptor accounted entirely for retention of soluble proteins in
the ER?
Calnexin C-terminus
...KDKGDEEEEGEEKLEEKQKSDAEEDGGTVSQEEEDRKPKAEEDEILNRSPRNRKPRRE
Calreticulin
...KQDEEQRLKEEEEDKKRKEEEEAEDKEDDEDKDEDEEDEEDKEEDEEEDVPGQAKDEL
Figure 13–6 C-terminal amino acids of
HMG CoA reductase proteins that are residents of the ER
...PGENARQLARIVCGTVMAGELSLMAALAAGHLVKSHMIHNRSKINLQDLQGACTKKTA (Problem 13–41).
membrane-spanning Figure 13–7 The membrane-spanning

segment domains of normal and mutant VSV
pMS20 K S S I A S F F F I I G L I I G L F L V L R G proteins (Problem 13–46). Plasmid
numbers indicate the number of amino
pMS18 K S S I A S F F F I I G L - - G L F L V L R acids in the membrane-spanning
segment; for example, pMS20 contains
pMS16 K S S I A S F F F I I G - - - - L F L V L R
the wild type, 20-amino-acid segment.
pMS14 K S S I A S F F F I I - - - - - - F L V L R Dashed lines indicate amino acids
that are missing in the other plasmids.
pMS12 K S S I A S F F F I - - - - - - - - L V L R Highlighted letters indicate the basic
pMS8 K S S I A - - - - - - - - - - - - F L V L R amino acids that flank the membrane-
spanning segment.
pMS0 K - - - - - - - - - - - - - - - - - - - - R
13–45 Cells have evolved a set of complicated pathways for addition of carbo-
Problems
hydrates to proteins, p13.10/13.07
implying that carbohydrates serve important func-
tions. List at least three functions that carbohydrates on proteins are
known to carry out.
DATA HANDLING
13–46 The vesicular stomatitis virus (VSV) G protein is a typical membrane gly-
coprotein. In addition to its signal peptide, which is removed after import
into the ER, the G protein contains a single membrane-spanning segment
that anchors the protein in the plasma membrane. The membrane-span-
ning segment consists of 20 uncharged and mostly hydrophobic amino
acids that are flanked by basic amino acids (Figure 13–7). Twenty amino
acids arranged in an helix are just sufficient to span the 3-nm thickness
of the lipid bilayer of the membrane.
To test the length requirements for membrane-spanning segments,
you modify a cloned version of the VSV G protein to generate a series of
mutants in which the membrane-spanning segment is shorter, as indi-
cated in Figure 13–7. When you introduce the modified plasmids into
cultured cells, roughly the same amount of G protein is synthesized from
each mutant as from wild-type cells. You analyze the cellular distribution
of the altered G proteins in several ways.
1. You examine the cellular location of the modified VSV G proteins by
immunofluorescence microscopy, using G-protein-specific antibodies
tagged with fluorescein.
2. You characterize the attached oligosaccharide chains by digestion
with endoglycosidase H (Endo H), which cleaves off N-linked oligosac-
charides until the first mannose is removed in the medial portion of the
Golgi apparatus (see Figure 13–8, Problem 13–47).
3. You determine whether the altered VSV G proteins retain the small
C-terminal cytoplasmic domain (which characterizes the normal G pro-
tein) by treating isolated microsomes with a protease. In the normal VSV
G protein, this domain is sensitive to protease treatment and is removed.
The results of these experiments are summarized in Table 13–2.
A. To the extent these data allow, deduce the intracellular location of each
altered VSV G protein that fails to reach the plasma membrane.
B. For the VSV G protein, what is the minimum length of the membrane-
spanning segment that is sufficient to anchor the protein in the mem-
brane?
C. What is the minimum length of the membrane-spanning segment that is
consistent with proper sorting of the G protein? How is it that transmem-
brane segments shorter than this can make it to the Golgi apparatus, but
then not be able to exit?
13–47 You have isolated several mutant cell lines that are defective in their abil-
ity to add carbohydrate to exported proteins. Using an easily purified
TABLE 13–2 Results of experiments characterizing the cellular distribution of

G proteins from normal and mutant cells (Problem 13–46).
Plasmid Cellular location Endo H treatment Protease treatment
pMS20 plasma membrane resistant sensitive
pMS12 intracellular +/– resistant sensitive
pMS8 intracellular sensitive sensitive
pMS0 intracellular sensitive resistant
protein that carries only N-linked complex oligosaccharides, you have

analyzed the sugar monomers that are added in the different mutant
cells. Each mutant is unique in the kinds and numbers of different sugars
contained in its N-linked oligosaccharides (Table 13–3).
A. Arrange the mutants in the order that corresponds to the steps in the path-
way for processing N-linked oligosaccharides (Figure 13–8). (Assume
that each mutant cell line is defective for a single enzyme required to
construct the N-linked oligosaccharide.)
B. Which of these mutants are defective in processing events that occur in
the ER? Which mutants are defective in processing events that occur in
the Golgi?
C. Which of the mutants are likely to be defective in a processing enzyme
that is directly responsible for modifying N-linked oligosaccharides?
Which mutants might not be defective in a processing enzyme, but rather
in another enzyme that affects oligosaccharide processing indirectly?
TABLE 13–3 Analysis of the sugars present in the N-linked oligosaccharides

from wild-type and mutant cell lines defective in oligosaccharide processing
(Problem 13–47).
Cell line Man GlcNAc Gal NANA Glc
Wild type 3 5 3 3 0
Mutant A 3 5 0 0 0
Mutant B 5 3 0 0 0
Mutant C 9 2 0 0 3
Mutant D 9 2 0 0 0
Mutant E 5 2 0 0 0
Mutant F 3 3 0 0 0
Mutant G 8 2 0 0 0
Mutant H 9 2 0 0 2
Mutant I 3 5 3 0 0
Abbreviations: Man = mannose; GlcNAc = N-acetylglucosamine; Gal = galactose;
NANA = N-acetylneuraminic acid, or sialic acid; Glc = glucose. Numbers indicate the number
of sugar monomers in the oligosaccharide.
glucosidase I UDP (2) GlcNAc transferase II
Golgi Golgi UDP (3) galactose transferase

glucosidase II mannosidase I GlcNAc transferase I mannosidase II (3) NANA transferase
CMP
ER mannosidase UDP UDP

5 UDP + 3 CMP
Asn Asn Asn Asn Asn Asn
1 2 3 4 5
high-mannose Endo H- Endo H- complex

oligosaccharide sensitive resistant oligosaccharide
ER LUMEN GOLGI LUMEN

KEY:
= N-acetylglucosamine (GlcNAc) = mannose (Man) = glucose (Glc) = galactose (Gal) = N-acetylneuraminic acid (sialic acid, or NANA)
13–48 Two extreme models—vesicle transport and cisternal maturation—have Figure 13–8 Oligosaccharide processing
been proposed to account for the movement of molecules across the in the ER and the Golgi apparatus
(Problem 13–47).
polarized structure of the Golgi apparatus. In the vesicle transport model,
the individual Golgi cisternae remain in place as proteins move through
them (Figure 13–9A). By contrast, in the cisternal maturation model, the
individual Golgi cisternae move across the stack, carrying the proteins
with them (Figure 13–9B). Transport vesicles serve critical functions in
both models, but their roles are distinctly different.
Problems Describe the roles of
p13.11/13.08
the transport vesicles in each of the two models. Comment specifically
on the roles of vesicles in the forward movement of proteins across the
Golgi stack, in the retention of Golgi resident proteins in individual cis-
ternae, and on the return of escaped ER proteins to the ER.
(A) VESICLE TRANSPORT MODEL
(B) CISTERNAL MATURATION MODEL
cisternae
vesicular
ER tubular cluster CGN cis medial trans TGN
Figure 13–9 Two models for the

movement of molecules through the
Golgi apparatus (Problem 13–48).
(A) The vesicle transport model. (B) The
cisternal maturation model. In (B), the
individual cisternae have been separated
for illustration purposes. ER, endoplasmic
reticulum; CGN, cis Golgi network;
TGN, trans Golgi network.
TABLE 13–4 Addition of galactose to VSV G protein after fusion of

VSV-infected cells with uninfected cells (Problem 13–49).
Infected cells Uninfected cells Precipitate Supernatant
Mutant cells Wild-type cells 45% 55%
Mutant cells Mutant cells 5% 95%
Wild-type cells Wild-type cells 85% 15%
The percentage of radioactivity in the precipitate indicates the fraction of
GlcNac-labeled G protein that has acquired galactose.
13–49 One early test of the vesicle transport and cisternal maturation models
(see Figure 13–9) looked for the movement of a protein between Golgi
cisternae. This study made use of mutant cells that cannot add galactose
to proteins, which normally occurs in the trans compartment of the Golgi
(see Figure 13–8). The mutant cells were infected with vesicular stomatitis
virus (VSV) to provide a convenient marker protein, the viral G protein.
At an appropriate point in the infection, an inhibitor of protein synthesis
was added to stop further synthesis of G protein. The infected cells were
then incubated briefly with a radioactive precursor of GlcNAc, which is
added only in the medial cisterna of the Golgi (see Figure 13–8). Next,
the infected mutant cells were fused with uninfected wild-type cells to
form a common cytoplasm containing both wild-type and mutant Golgi
stacks. After a few minutes, the cells were dissolved with detergent and
all the VSV G protein was captured using G-protein-specific antibodies.
After separation from the antibodies, the G proteins carrying galactose
were precipitated, using a lectin that binds galactose. The radioactivity in
the precipitate and in the supernatant was measured. The results of this
experiment along with control experiments (which used mutant cells
only or wild-type cells only) are shown in Table 13–4.
A. Between which two compartments of the Golgi apparatus is the move-
ment of proteins being tested in this experiment? Explain your answer.
B. If proteins moved through the Golgi apparatus by cisternal maturation,
what would you predict for the results of this experiment? If proteins
moved through the Golgi via vesicular transport, what would you pre-
dict?
C. Which model is supported by the results in Table 13–4?
MEDICAL LINKS
13–50 Processing of N-linked oligosaccharides is not uniform among species.
Most mammals, with the exception of humans and Old World primates,
occasionally add galactose—in place of an N-acetylneuraminic acid—
to a galactose, forming a terminal Gal( 1–3)Gal disaccharide on some
branches of an N-linked oligosaccharide. How does this explain the pre-
ferred use of Old World primates for production of recombinant proteins
for therapeutic use in humans?
TRANSPORT FROM THE TRANS GOLGI NETWORK TO

LYSOSOMES
TERMS TO LEARN
acid hydrolase lysosomal storage disease M6P receptor protein
autophagosome lysosome vacuole
autophagy
DEFINITIONS
13–51 Digestion of obsolete parts of the cell by the cell’s own lysosomes.
13–52 Very large, fluid-filled vesicle found in most plant and fungal cells, typi-
cally occupying more than 30% of the cell volume.
13–53 Membrane-enclosed organelle in eukaryotic cells that contains digestive
enzymes, which are typically most active at the acidic pH found in the
lumen.
TRUE/FALSE
13–54 Lysosomal membranes contain a proton pump that utilizes the energy of
ATP hydrolysis to pump protons out of the lysosome, thereby maintain-
ing the lumen at a low pH.
13–55 Late endosomes are converted to mature lysosomes by the loss of distinct
endosomal membrane proteins and a further decrease in their internal
pH.
13–56 If cells were treated with a weak base such as ammonia or chloroquine,
which raises the pH of organelles toward neutrality, M6P receptors would
be expected to accumulate in the Golgi because they could not bind to
the lysosomal enzymes.
THOUGHT PROBLEMS
13–57 How does the low pH of lysosomes protect the rest of the cell from lyso-
somal enzymes in case the lysosome breaks?
13–58 Imagine that an autophagosome is formed by engulfment of a mito-
chondrion by the ER membrane. How many layers of membrane would
separate the matrix of the mitochondrion from the cytosol outside the
autophagosome? Identify the source of each membrane and the spaces
between the membranes.
13–59 The principal pathway for transport of lysosomal hydrolases from the
trans Golgi network (pH 6.6) to the late endosomes (pH 6) and for the
recycling of M6P receptors back to the Golgi depends on the pH differ-
ence between those two compartments. From what you know about M6P
receptor binding and recycling and the pathways for delivery of material
to lysosomes, describe the consequences of changing the pH in those
two compartments.
A. What do you suppose would happen if the pH in late endosomes were
raised to pH 6.6?
B. What do you suppose would happen if the pH in the trans Golgi network
were lowered to pH 6?
13–60 Melanosomes are specialized lysosomes that store pigments for even-
tual release by exocytosis. Various cells such as skin and hair cells then
take up the pigment, which accounts for their characteristic pigmenta-
tion. Mouse mutants that have defective melanosomes often have pale normal mouse Mocha mouse
or unusual coat colors. One such light-colored mouse, the Mocha mouse
(Figure 13–10), has a defect in the gene for one of the subunits of the
Figure 13–10 A normal mouse and
adaptor protein complex AP3, which is associated with coated vesicles the Mocha mouse (Problem 13–60). In
budding from the trans Golgi network. How might the loss of AP3 cause a addition to its light coat color, the Mocha
defect in melanosomes? mouse has a poor sense of balance.
TRANSPORT FROM THE TRANS GOLGI NETWORK TO LYSOSOMES 271
(A) (B) (C) Figure 13–11 Pigmentation defects in

Ashen mice (Problem 13–61). (A) Normally
pigmented mice and pale Ashen mice.
(B) A melanocyte from a normal mouse.
(C) Melanocytes from an Ashen mouse.
normal and Ashen mice normal melanocyte Ashen melanocytes
DATA HANDLING
13–61 More than 50 different genes are known to affect coat color in mice. Three
of them—Dilute, Leaden, and Ashen—are grouped together because of
their highly similar phenotypes. Although these mice have normal mela-
nosomes in their melanocytes, the pigment in the melanosomes is not
delivered correctly to hair cells, giving rise to pale coats, as shown for
Ashen mice in Figure 13–11A. Dilute mice lack an unconventional myo-
sin heavy chain, MyoVa, which interacts with a microtubule-based trans-
port motor. Ashen mice carry a mutation in the gene for the Rab protein,
Rab27a, which associates with melanosomes. Leaden mice are miss-
ing melanophilin (Mlph), which is a modular protein with individual
domains that bind to MyoVa, to Rab27a, and to actin filaments in the cell
cortex.
Melanocytes from normal mice have a characteristic branch mor-
phology (Figure 13–11B) and normally discharge their melanosomes
near the tips of the branches. As shown in Figure 13–11C, melanocytes
from Ashen mice have a normal morphology but their melanosomes sur-
round the nucleus. Melanocytes from Dilute mice and Leaden mice have
the same appearance as those from Ashen mice. Try to put these observa-
tions together to formulate a hypothesis to account for the normal deliv-
ery of melanosomes to the tips of the melanocyte branches, and for the
defects in melanosome function in these mice.
MEDICAL LINKS
13–62 Patients with I-cell disease are missing the enzyme GlcNAc phospho-
transferase, which catalyzes the first of the two steps required for addi-
tion of phosphate to mannose to create the M6P marker (see Figure
13–12). In the absence of the M6P marker, the M6P receptor cannot bind
to the protein and deliver it to a lysosome. How do you suppose that the
lysosomes in some cells from these patients—liver cells, for example—
acquire a normal complement of lysosomal enzymes?
13–63 Patients with Hunter’s syndrome or with Hurler’s syndrome rarely live
beyond their teens. These patients accumulate glycosaminoglycans in
lysosomes due to the lack of specific lysosomal enzymes necessary for
their degradation. When cells from patients with the two syndromes are
fused, glycosaminoglycans are degraded properly, indicating that the
cells are missing different degradative enzymes. Even if the cells are just
cultured together, they still correct each other’s defects. Most surprising
of all, the medium from a culture of Hurler’s cells corrects the defect in
Hunter’s cells (and vice versa). The corrective factors in the media are
inactivated by treatment with proteases, by treatment with periodate,
which destroys carbohydrate, and by treatment with alkaline phos-
phatase, which removes phosphates.
Figure 13–12 Synthesis of M6P marker on

UDP UMP a lysosomal hydrolase (Problem 13–64).
GlcNAc GlcNAc
CH2OH CH2O P CH2O P
O O GlcNAc O
OH OH OH OH OH OH
OH O GlcNAc OH O GlcNAc OH O
phosphotransferase phosphoglycosidase
α -D-mannose mannose 6-phosphate
A. What do you suppose the corrective factors are? Beginning with the
donor patient’s cells, describe the route by which the factors reach the
medium and subsequently enter the recipient cells to correct the lysoso-
mal defects.
Problems
B. Why do you suppose p13.14/13.12
the treatments with protease, periodate, and alka-
line phosphatase inactivate the corrective factors?
C. Would you expect a similar sort of correction scheme to work for mutant
cytosolic enzymes?
13–64 Children with I-cell disease synthesize perfectly good lysosomal
enzymes, but they are secreted outside the cell instead of being sorted
to lysosomes. The mistake occurs because the cells lack GlcNAc phos-
photransferase, which is required to create the M6P marker that is essen-
tial for proper sorting (Figure 13–12). In principle, I-cell disease could
also be caused by deficiencies in GlcNAc phosphoglycosidase, which
removes GlcNAc to expose M6P (Figure 13–12), or in the M6P receptor
itself. Thus, there are three potential kinds of I-cell disease, which could
be distinguished by the ability of various culture supernatants to correct
defects in mutant cells. Imagine that you have three cell lines (A, B, and
C), each of which derives from a patient with one of the three hypotheti-
cal I-cell diseases. Experiments with supernatants from these cell lines
give the results below.
1. The supernatant from normal cells corrects the defects in B and C but
not the defect in A.
2. The supernatant from A corrects the defect in Hurler’s cells, which are
missing a specific lysosomal enzyme, but the supernatants from B and C
do not.
3. If the supernatants from the mutant cells are first treated with phos-
phoglycosidase to remove GlcNAc, then the supernatants from A and C
correct the defect in Hurler’s cells, but the supernatant from B does not.
From these results, deduce the nature of the defect in each of the
mutant cell lines.
TRANSPORT INTO THE CELL FROM THE PLASMA

MEMBRANE: ENDOCYTOSIS
TERMS TO LEARN
caveola macropinocytosis
caveolin multivesicular body
clathrin-coated pit neutrophil
early endosome phagocytosis
endocytic vesicle phagosome
endocytosis pinocytosis
endosome maturation receptor-mediated endocytosis
ESCRT protein complexes recycling endosome
late endosome transcytosis
low-density lipoprotein (LDL) transferrin receptor
macrophage
TRANSPORT INTO THE CELL FROM THE PLASMA MEMBRANE: ENDOCYTOSIS 273
DEFINITIONS
13–65 General term for the process by which cells take up macromolecules,
particulate substances, and even other cells into membrane-enclosed
vesicles.
13–66 Complex vesicle with invaginating buds and internal vesicles involved in
the maturation of early endosomes into late endosomes.
13–67 Phagocytic cell—derived from a hematopoietic stem cell—that ingests
invading microorganisms and plays an important role in scavenging
senescent cells and apoptotic cells.
13–68 Type of endocytosis in which soluble materials are taken up from the
environment and incorporated into vesicles for digestion.
13–69 Invagination that forms from lipid rafts at the cell surface and buds off
internally to form a pinocytic vesicle.
13–70 Region of plasma membrane of animal cells that is covered with the pro-
tein clathrin on its cytosolic face; it will bud off from the membrane to
form an intracellular vesicle.
13–71 Process by which macromolecules bind to complementary transmem-
brane receptor proteins, accumulate in coated pits, and then enter the
cells as receptor–macromolecule complexes in clathrin-coated vesicles.
13–72 One of a family of structural proteins in caveolae that are unusual because
they extend multiple hydrophobic loops into the membrane from the
cytosolic side, but do not cross the membrane.
13–73 Membrane-enclosed compartment just beneath the plasma membrane,
to which external molecules are first delivered by endocytosis.
13–74 Specialized form of endocytosis in which a cell uses large endocytic vesi-
cles to ingest large particles such as microorganisms and dead cells.
TRUE/FALSE
13–75 Any particle that is bound to the surface of a phagocyte will be ingested
by phagocytosis.
13–76 Like the LDL receptor, most of the more than 25 different receptors
known to participate in receptor-mediated endocytosis enter coated pits
only after they have bound their specific ligands.
13–77 All the molecules that enter early endosomes ultimately reach late
endosomes, where they become mixed with newly synthesized acid
hydrolases and end up in lysosomes.
13–78 During transcytosis, vesicles that form from coated pits on the apical sur-
face fuse with the plasma membrane on the basolateral surface, and in
that way transport molecules across the epithelium.
THOUGHT PROBLEMS
13–79 A macrophage ingests the equivalent of 100% of its plasma membrane
each half hour by endocytosis. What is the rate at which membrane is
returned by exocytosis?
(A) MICROGRAPH (B) DRAWING Figure 13–13 A coated pit about to bud
from the membrane (Problem 13–80).
(A) An electron micrograph.
C (B) A schematic drawing.
A B
F
D
E
100 nm
13–80 The electron micrograph in Figure 13–13A is illustrated schematically by

the drawing in Figure 13–13B. Name the structures that are labeled in the
drawing.
13–81 Caveolae are thought to form from lipid rafts, which are patches of the
plasma membrane that are especially rich in cholesterol and glycosphin-
golipids. Caveolae may collect cargo proteins by virtue of the lipid com-
Problems
position of their membrane, p13.16/13.13
rather than by assembly of a cytosolic protein
coat. What might you predict would be a characteristic of the structure of
transmembrane proteins that collect in caveolae?
13–82 Iron (Fe) is an essential trace metal that is needed by all cells. It is
required, for example, for the synthesis of the heme groups that are part
of cytochromes and hemoglobin. Iron is taken into cells via a two-com-
ponent system. The soluble protein transferrin circulates in the blood-
stream, and the transferrin receptor is a membrane protein that is con-
tinually endocytosed and recycled to the plasma membrane. Fe ions (A) HRP UPTAKE
bind to transferrin at neutral pH but not at acidic pH. Transferrin binds
(pmol/hour/106 cells)
to the transferrin receptor at neutral pH only when it has bound an Fe 2

ion, but it binds to the receptor at acidic pH even in the absence of bound
HRP uptake
iron. From these properties, describe how iron is taken up, and discuss
the advantages of this elaborate scheme. 1
CALCULATIONS 0
0 10 20 30 40
13–83 Cells take up extracellular molecules by receptor-mediated endocytosis HRP in medium (μM)
and by fluid-phase endocytosis. A classic paper compared the efficien-
cies of these two pathways by incubating human cells for various peri-
ods of time in a range of concentrations of either 125I-labeled epidermal (B) EGF UPTAKE
growth factor (EGF), to measure receptor-mediated endocytosis, or 20
horseradish peroxidase (HRP), to measure fluid-phase endocytosis. Both
(pmol/hour/106 cells)
16
EGF and HRP were found to be present in small vesicles with an internal
125I-EGF uptake
radius of 20 nm. The uptake of HRP was linear (Figure 13–14A), while 12
that of EGF was initially linear but reached a plateau at higher concentra-
tions (Figure 13–14B). 8
A. Explain why the shapes of the curves in Figure 13–14 are different for 4
HRP and EGF.
B. From the curves in Figure 13–14, estimate the difference in the uptake 0
0 20 40 60 80
rates for HRP and EGF when both are present at 40 nM. What would the EGF in medium (nM)
difference be if both were present at 40 μM?
C. Calculate the average number of HRP molecules that get taken up by Figure 13–14 Uptake of HRP and EGF
each endocytic vesicle (radius 20 nm) when the medium contains 40 μM as a function of their concentration in the
HRP. [The volume of a sphere is (4/3) r3.] medium (Problem 13–83).
TRANSPORT INTO THE CELL FROM THE PLASMA MEMBRANE: ENDOCYTOSIS 275
D. The scientists who did these experiments said at the time, “These calcula-
tions clearly illustrate how cells can internalize EGF by endocytosis while
excluding all but insignificant quantities of extracellular fluid.” What do
you think they meant?
13–84 A ligand for receptor-mediated endocytosis circulates at a concentration
of 1 nM (10–9 M). It is taken up in coated vesicles with a volume of 1.66 ×
10–18 L (about 150 nm in diameter). On average, there are 10 of its recep-
tors in each coated vesicle. If all the receptors were bound to the ligand,
how much more concentrated would the ligand be in the vesicle than it
was in the extracellular fluid? What would the dissociation constant (Kd)
for the receptor–ligand binding need to be in order to concentrate the
ligand 1000-fold in the vesicle? (You may wish to review the discussion of
Kd in Problem 3–86.)
13–85 The recycling of transferrin receptors has been studied by labeling the
receptors on the cell surface and following their fate at 0°C and 37°C. A
sample of intact cells at 0°C was reacted with radioactive iodine under
conditions that label cell-surface proteins. If these cells were kept on
ice and incubated in the presence of trypsin, which destroys the recep-
tors without damaging the integrity of the cell, the radioactive transfer-
rin receptors were completely degraded. If the cells were first warmed
to 37°C for 1 hour and then treated with trypsin on ice, about 70% of the
initial radioactivity was resistant to trypsin. At both temperatures, most (A) BINDING
of the receptors were not labeled and most remained intact, as apparent 0.2 normal
(μg/g cell protein)

normal
from a protein stain.
LDL bound
A second sample of cells that had been surface-labeled at 0°C and 0.1 JD
incubated at 37°C for 1 hour was analyzed with transferrin-specific anti-
bodies, which identify transferrin receptors via their linkage to Fe–trans- FH
ferrin complexes. If intact cells were reacted with antibody, 0.54% of the 0.0
0 50 100 0 50 100
labeled proteins were bound by antibody. If the cells were first dissolved
in detergent, 1.76% of the labeled proteins were bound by antibody. (B) INTERNALIZATION
A. When the cells were kept on ice, why did trypsin treatment destroy the (μ g/g cell protein)
LDL internalized
1.0
labeled transferrin receptors, but not the majority of receptors? Why did normal normal
most of the labeled receptors become resistant to trypsin when the cells
0.5
were incubated at 37°C?
B. What fraction of all the transferrin receptors is on the cell surface after a FH JD
1-hour incubation at 37°C? Do the two experimental approaches agree? 0.0

0 50 100 0 50 100
MEDICAL LINKS (C) REGULATION

cholesterol synthesis
0.4
13–86 Cholesterol is an essential component of the plasma membrane, but FH JD
(nmol/hr)
people who have very high levels of cholesterol in their blood (hyper-
cholesterolemia) tend to have heart attacks. Blood cholesterol is carried 0.2
in the form of cholesterol esters in low-density lipoprotein (LDL) parti- normal normal
cles. LDL binds to a high-affinity receptor on the cell surface, enters the 0.0
cell via a coated pit, and ends up in lysosomes. There its protein coat is 0 50 100 0 50 100
LDL (μ g/mL) LDL (μ g/mL)
degraded, and cholesterol esters are released and hydrolyzed to choles-
terol. The released cholesterol enters the cytosol and inhibits the enzyme
Figure 13–15 LDL metabolism in normal
HMG CoA reductase, which controls the first unique step in cholesterol cells and in cells from patients with
biosynthesis. Patients with severe hypercholesterolemia cannot remove severe familial hypercholesterolemia
LDL from the blood. As a result, their cells do not turn off normal choles- (Problem 13–86). (A) Surface binding of
terol synthesis, which makes the problem worse. LDL. Assays at 4°C allow binding but not
internalization. (B) Internalization of LDL.
LDL metabolism can be conveniently divided into three stages exper- Problems
After p13.19/13.15
binding at 4°C, the cells are warmed
imentally: binding of LDL to the cell surface, internalization of LDL, to 37°C. Binding and uptake of LDL can
and regulation of cholesterol synthesis by LDL. Skin cells from a normal be followed by labeling LDL either with
person and two patients suffering from severe familial hypercholester- ferritin particles, which can be seen by
electron microscopy, or with radioactive
olemia were grown in culture and tested for LDL binding, LDL internaliza- iodine, which can be measured in
tion, and LDL regulation of cholesterol synthesis. The results are shown in a gamma counter. (C) Regulation of
Figure 13–15. cholesterol synthesis by LDL.
A. In Figure 13–15A, the surface binding of LDL by normal cells is compared

with LDL binding by cells from patients FH and JD. Why does binding by
normal cells and by JD’s cells reach a plateau? What explanation can you
suggest for the lack of LDL binding by FH’s cells?
B. In Figure 13–15B, internalization of LDL by normal cells increases as the
external LDL concentration is increased, reaching a plateau 5-fold higher
than the amount of externally bound LDL. Why does LDL enter cells from
patients FH or JD at such a slow rate?
C. In Figure 13–15C, the regulation of cholesterol synthesis by LDL in
normal cells is compared with that in cells from FH and JD. Why does
increasing the external LDL concentration inhibit cholesterol synthesis
in normal cells, but affect it only slightly in cells from FH or JD?
D. How would you expect the rate of cholesterol synthesis to be affected if
normal cells and cells from FH or JD were incubated with cholesterol
itself? (Free cholesterol crosses the plasma membrane by diffusion.)
13–87 What is wrong with JD’s metabolism of LDL? As discussed in Problem
13–86, JD’s cells bind LDL with the same affinity as normal cells and in
almost the same amounts, but the binding does not lead to internaliza-
tion of LDL. Two classes of explanation could account for JD’s problem:
1. JD’s LDL receptors are defective in a way that prevents internaliza-
tion, even though the LDL-binding domains on the cell surface are unaf-
fected.
2. JD’s LDL receptors are entirely normal, but there is a mutation in the
cellular internalization machinery such that loaded LDL receptors can-
not be brought in.
To distinguish between these explanations, JD’s parents were stud-
ied. It is known that an autosomal gene encodes the receptor that binds
LDL. Thus, each parent must have donated one defective gene to JD. JD’s
mother suffered from mildly elevated blood cholesterol. Her cells bound
only half as much LDL as normal cells, but the bound LDL was internal-
ized at the same rate as in normal cells. JD’s father also had mild hyper-
cholesterolemia, but his cells bound even more LDL than normal cells.
Of the bound LDL, less than half the label could be internalized; the rest
remained on the cell surface.
The association of this family’s LDL receptors with coated pits was
studied by electron microscopy (EM), using LDL that was labeled with
ferritin. The results are shown in Table 13–5.
A. Why does JD’s mother have mild hypercholesterolemia? Based on the
LDL binding and internalization studies, and on the EM observations,
decide what kind of defective LDL receptor gene she passed to JD.
TABLE 13–5 Distribution of LDL receptors on the surface of cells from

JD and his parents as compared with normal individuals (Problem
13–87).
Number of LDL receptors
Individual In pits Outside pits
Normal male 186 195
Normal female 186 165
JD 10 342
JD’s father 112 444
JD’s mother 91 87
TRANSPORT FROM THE TRANS GOLGI NETWORK TO THE CELL EXTERIOR: EXOCYTOSIS 277
B. Why does JD’s father have mild hypercholesterolemia? Based on the LDL
binding and internalization studies, and on the EM observations, decide
what kind of defective LDL receptor gene he passed to JD.
C. Can you account for JD’s hypercholesterolemia from the behavior of the
LDL receptors in his parents? In particular, how is it that JD binds nearly
a normal amount of LDL, but has severe hypercholesterolemia?
D. At the beginning of this problem, two possible explanations—defec-
tive receptor or defective internalization machinery—were proposed to
account for the lack of internalization by JD’s LDL receptors in the face
of nearly normal LDL binding. Do these studies allow you to decide
between these alternative explanations?
TRANSPORT FROM THE TRANS GOLGI NETWORK

TO THE CELL EXTERIOR: EXOCYTOSIS
TERMS TO LEARN
constitutive secretory pathway regulated secretory pathway
default pathway secretory vesicle
exocytosis synaptic vesicle
DEFINITIONS
13–88 Specialized class of tiny secretory vesicles that store neurotransmitter
molecules.
13–89 Pathway for exocytosis that operates continuously in all cells.
13–90 Membrane-enclosed organelle in which molecules destined to be
exported are stored prior to release.
13–91 Process involving fusion of vesicles with the plasma membrane.
13–92 Pathway for exocytosis that operates mainly in cells specialized for secret-
ing products rapidly on demand.
TRUE/FALSE
13–93 When a foreign gene encoding a secretory protein is introduced into a
secretory cell that normally does not make the protein, the alien secre-
tory protein is not packaged into secretory vesicles.
13–94 Once a secretory vesicle is properly positioned beneath the plasma
membrane, it will immediately fuse with the membrane and release its
contents to the cell exterior.
THOUGHT PROBLEMS
13–95 In a cell capable of regulated secretion, what are the three main classes of
protein that must be separated before they leave the trans Golgi network?
13–96 You are interested in exocytosis and endocytosis in a line of cultured liver
cells that secrete albumin and take up transferrin. To distinguish between
these events, you tag transferrin with colloidal gold and prepare ferritin-
labeled antibodies that are specific for albumin. You add the tagged
transferrin to the medium, and then after a few minutes you fix the cells,
prepare thin sections, and react them with ferritin-labeled antibodies
against albumin. Colloidal gold and ferritin are both electron-dense and
therefore readily visible when viewed by electron microscopy; moreover,

they can be easily distinguished from one another on the basis of size. extracellular space
A. Will this experiment allow you to identify vesicles in the exocytic and
endocytic pathways? How?
B. Not all the gold-labeled vesicles are clathrin-coated. Why?
13–97 What would you expect to happen in cells that secrete large amounts of cytosol
protein through the regulated secretory pathway, if the ionic conditions
in the ER lumen could be changed to resemble those of the trans Golgi 100 nm
network?
13–98 Dynamin was first identified as a microtubule-binding protein, and its Figure 13–16 Electron micrograph of a
sequence indicated that it was a GTPase. The key to its function came nerve terminal from a shibire mutant fly at
elevated temperature (Problem 13–98).
from neurobiological studies in Drosophila. Shibire mutant flies, which
carry a mutation in the dynamin gene, are rapidly paralyzed when the
temperature is elevated. They recover quickly once the temperature is
lowered. The complete paralysis at the elevated temperature suggested
that synaptic transmission between nerve and muscle cells was blocked.
Electron micrographs of synapses of the paralyzed flies showed a loss of
synaptic vesicles and a tremendously increased number of coated pits
relative to normal synapses (Figure 13–16).
Suggest an explanation for the paralysis shown by the shibire mutant
flies, and indicate why signal transmission at a synapse might require
dynamin.
DATA HANDLING
13–99 Proteins without special signals are transported between cisternae in
Golgi stacks and onward to the plasma membrane via the nonselective
constitutive secretory pathway, or the default pathway, as it is commonly
known. This transport is also sometimes referred to as bulk transport
because the Golgi contents do not become concentrated in the vesicles.
Given that transport in clathrin-coated vesicles is so highly concentrat-
ing, you are skeptical that no concentration occurs in the default pathway
for secretion.
To determine whether vesicles in the default pathway concentrate
their contents, you infect cells with vesicular stomatitis virus (VSV) and
follow the viral G protein, which is transported by the default pathway.
Your idea, an ambitious one, is to compare the concentration of G pro-
tein in the lumen of the Golgi stacks with that in the associated transport
vesicles. You intend to measure G-protein concentration by preparing
thin sections of VSV-infected cells and incubating them with G-protein-
specific antibodies tagged with gold particles. Since the gold particles are
visible in electron micrographs as small black dots, it is relatively straight-
forward to count dots in the lumens of transport vesicles (fully formed
and just budding) and of the Golgi apparatus. You make two estimates
of G-protein concentration: (1) the number of gold particles per cross-
sectional area (surface density) and (2) the number of gold particles per
linear length of membrane (linear density). Your results are shown in
Table 13–6.
Do the vesicles involved in the default pathway concentrate their con-
tents or not? Explain your reasoning.
13–100 Insulin is synthesized as a pre-pro-protein in the cells of the pancreas.
Its pre-peptide is cleaved off after it enters the ER lumen. To define the
cellular location at which its pro-peptide is removed, you have prepared
two antibodies: one that is specific for pro-insulin, and one that is spe-
cific for insulin. You have tagged the anti-pro-insulin antibody with a red
fluorophore and the anti-insulin antibody with a green fluorophore, so
you can follow them independently in the same cell. When you incubate
TRANSPORT FROM THE TRANS GOLGI NETWORK TO THE CELL EXTERIOR: EXOCYTOSIS 279
TABLE 13–6 Relative densities of G protein in Golgi and vesicle lumens

and membranes (Problem 13–99).
Source of Golgi Site measured Parameter Mean
measured density
Uninfected cells Whole Golgi Surface density 5/μm2
Infected cells Whole Golgi Surface density 271/μm2
Infected cells Golgi buds and Surface density 233/μm2
vesicles
Infected cells Golgi cisternal Linear density 6/μm
membranes
Infected cells Golgi buds and Linear density 4/μm
vesicles
a pancreatic cell with a mixture of your two antibodies, you obtain the
results shown in Table 13–7. In what cellular compartment is the pro-
peptide removed from pro-insulin?
13–101 Polarized epithelial cells must make an extra sorting decision since their
plasma membranes are divided into apical and basolateral domains,
which are populated by distinctive sets of proteins. Proteins destined for
the apical or basolateral domains seem to travel there directly from the
trans Golgi network. One way to sort proteins to these domains would
be to use a specific sorting signal for one group of proteins, which would
then be actively recognized and directed to one domain, and to allow the
rest to travel via a default pathway to the other domain.
Consider the following experiment to identify the default pathway.
The cloned genes for several foreign proteins were engineered by recom-
binant DNA techniques so that they could be expressed in the polarized
epithelial cell line MDCK. These proteins are secreted in other types of
cells, but are not normally expressed in MDCK cells. The cloned genes
were introduced into the polarized MDCK cells, and their sites of secre-
tion were assayed. Although the cells remained polarized, the foreign
proteins were delivered in roughly equal amounts to the apical and baso-
lateral domains.
TABLE 13–7 Fluorescence associated with various compartments

of cells after reaction with fluorescent antibodies directed against
pro-insulin and insulin (Problem 13–100).
Compartment Fluorescence
cis Golgi network Red
Endoplasmic reticulum Red
Golgi cisternae Red
Immature secretory vesicles Yellow
Lysosomes None
Mature secretory vesicles Green
Mitochondria None
Nucleus None
trans Golgi network Red
A. What is the expected result of this experiment, based on the hypothesis

that targeting to one domain of the plasma membrane is actively signaled
and targeting to the other domain is via a default pathway?
B. Do these results support the concept of a default pathway as outlined
above?
13–102 Neurons are difficult to study because of their excessively branched struc-
ture and long, thin dendrites, as shown in Figure 13–17. Fluorescently
tagged antibodies are powerful tools for investigating certain aspects of
neuron structure. Synaptic vesicles, for example, were shown to be con-
centrated in the presynaptic cells at nerve synapses in this way. A culture
of neurons was first exposed for 1 hour to a fluorescently tagged antibody
specific for the lumenal domain of synaptotagmin, a transmembrane
protein that resides exclusively in the membranes of synaptic vesicles.
The culture was then washed thoroughly to remove all synaptotagmin
antibodies. When the culture was examined by fluorescence microscopy,
dots of color from the synaptotagmin-specific antibody were found to
mark the positions of the synaptic vesicles in the nerve terminals. Figure 13–17 A hippocampal neuron
If antibodies do not cross intact membranes, how do you suppose the (Problem 13–102).
synaptic vesicles get labeled? When the procedure was repeated using
an antibody specific for the cytoplasmic domain of synaptotagmin, the
nerve terminals did not become labeled. Explain the results with the two
different antibodies for synaptotagmin.
Figure 13-23
13–103 The original version of the SNARE hypothesis suggested that the ATP-
dependent disassembly of SNAREs by NSF provided the energy neces-
sary for membrane fusion, and thus that NSF should act at the last step in Problem 13-118
secretion. More recent evidence suggests that NSF acts at an earlier step
to prime the vesicle for secretion, and that SNAREs alone are sufficient
to catalyze membrane fusion at the last step in secretion. It is important
to know what really happens, and you have the means at hand to answer
the question. Second prize goes to Keiran Boyle in the
Using a whole-cell patch-clamp protocol (FigureDepartment13–18A), you of Cell & Developmental Biolog
can diffuse cytosolic components into the cell through the pipet and
at University College London. His wonderf
assay exocytosis by changes in capacitance, which is a measure of the
increase in the area of the plasma membrane. To control image,
preciselywhich
the looks like an aerial view of
2+ road chemical
timing of vesicle fusion, you enclose Ca in a photosensitive networkvesicle
at fusion
night, shows a cultured
Figure 13–18 Analysis of the role of NSF in
(Problem 13–103).
hippocampal
“cage,” from which it can be released with a flash of light. In response to neuron
(A) Whole-cell in the
patch-clamp. early
(B) Responsesstages of
Ca2+ release, there is a rapid burst of vesicle fusion (indicated by a to the first flash-mediated release of
rapid rise in capacitance), followed by a longer, slower fusion process Ca2+. (C) Reponses to the second flash-
(Figure 13–18B, –NEM). The initial burst represents the fusion of vesi- mediated release of Ca2+. Secretion was
2+ 2+ measured as an increase in membrane
cles that were just waiting for the Ca trigger. Because Ca is rapidly capacitance (in femtoFarads, f F) in the
removed from the cell, the procedure can be repeated with a second flash absence (–NEM) or presence (+NEM) of an
of light 2 minutes later; it yields the same rapid and slow components inhibitor of NSF.
(Figure 13–18C, –NEM).
(A) (B) FIRST FLASH (C) SECOND FLASH
− NEM – NEM
patch
membrane capacitance
pipet
+ NEM
(secretion)
+ NEM
200 f F
200 f F
0 5 10 0 5 10
cell time (seconds) time (seconds)
MCAT STYLE 281
To test the role of NSF in vesicle fusion, you first diffuse N-ethylma-
leimide (NEM) into cells to inhibit NSF. (The name “NSF” stands for
NEM-sensitive factor.) You then repeat the flash protocols as before. In
response to the first flash, the rapid component is unaffected, but the
slow component is decreased (Figure 13–18B, +NEM). In response to the
second flash, both components are inhibited (Figure 13–18C, +NEM).
A. What do you suppose the slow component of the fusion process repre-
sents?
B. Why does inhibition of NSF affect the slow component after both flashes,
but inhibit the rapid component only after the second flash?
C. Which of the alternatives for the role of NSF in vesicle fusion—acting at
the last step or an early step—do these experiments support? Explain
your reasoning.
D. Propose a model for the molecular role of NSF in fusion of secretory
vesicles.
MEDICAL LINKS
13–104 Antitrypsin, which inhibits certain proteases, is normally secreted into the
bloodstream by liver cells. Antitrypsin is absent from the bloodstream of
patients who carry a mutation that results in a single amino acid change
in the protein. Antitrypsin deficiency causes a variety of severe problems,
particularly in lung tissue, because of uncontrolled protease activity. Sur-
prisingly, when the mutant antitrypsin is synthesized in the laboratory,
it is as active as the normal antitrypsin at inhibiting proteases. Why then
does the mutation cause the disease? Think of more than one possibility
and suggest ways in which you could distinguish among them.
MCAT STYLE
Ricin is one of the most toxic substances known: less than 2 mg injected into the
bloodstream will kill an adult human. Ricin is produced by the castor bean plant
as a 65 kd protein heterodimer composed of an A chain and a B chain. The B chain
is a lectin that binds to carbohydrates on the cell surface. The A chain is an enzyme
that modifies a highly conserved site in rRNA, leading to inhibition of translation.
After entering the cell, ricin eventually ends up in the lumen of the endoplasmic
reticulum (ER), and from there it moves into the cytosol, where it inactivates ribo-
somes.
13–105 What is the most likely mechanism by which ricin enters the cell?
A. Binding to clathrin proteins
B. Entry through pore complexes
C. Interaction with SNARE proteins
D. Internalization via endocytosis
13–106 Which one of the following is required in order for ricin to be delivered to
the ER?
A. N-ethylmaleimide-sensitive factor (NSF)
B. Golgi-derived COPI-coated vesicles
C. Mannose 6-phosphate (M6P) receptors
D. The Sar1 monomeric GTPase
13–107 Which one of the following describes the most likely scenario for how
ricin gets into the cytosol?
A. Ricin has a signal sequence that allows it to be transported across the ER
membrane into the cytosol.
B. Ricin is packaged into vesicles that form vesicular tubular clusters, which
release proteins into the cytosol.

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132 Chapter 6: How Cells Read the Genome: From DNA to Protein

same, although 11 of the 15 have very similar sequences. Your advisor is

Passage 2 (Questions 6–103 to 6–105)

Control of Gene Expression 7

TERMS TO LEARN AN OVERVIEW OF GENE CONTROL

DEFINITIONS TRANSCRIPTION REGULATORS

genes; some represent modified forms of a protein that migrate to dif-

NUCLEUS CYTOSOL Figure 7–2 Six steps at which the

the analysis. The surrogate mothers are

arranged left to right in the same order

MAJOR GROOVE MAJOR GROOVE

cytosine H guanine thymine adenine

MINOR GROOVE MINOR GROOVE

Figure 7–8 Domains of the lambda

repressor monomers repressor dimer DNA-binding site

(A) MyoD–HLH HETERODIMER (B) PHOSPHO–MyoD/Id

HLH MyoD Id MyoD

TABLE 7–1 Nucleotide sequences of selected and amplified DNAs

(C) CAP–CAP (D) (A5N5)4–CAP

(A) rhodamine fluorescein (B) EMISSION SPECTRA (C) EXCHANGE KINETICS

fluorescein + AP-1 DNA

relative fluorescence at 603 nm

TRANSCRIPTION REGULATORS AS GENE SWITCHES

GLUCOSE LACTOSE OPERON ACTIVITY Figure 7–15 Arrangement of binding sites

CAP Lac repressor

CAP RNA polymerase

biotin Figure 7–17 Stimulation of β-globin gene

How does this experiment distinguish between the DNA looping

Figure 7–18 Proliferation of E. coli

β−galactosidase (arbitrary units)

(A) MAP OF THE Gal1-Gal10 LOCUS Figure 7–20 Analysis of SAGA

(A) YEAST SILENT MATING-TYPE LOCI

(B) INSERTION OF Ura3 GENES NEAR Hml

THOUGHT PROBLEMS cI gene Cro gene

Figure 7–24 An illustration of

of the body. It first appears about 2 hours after fertilization at a uniform

Figure 7–25 Binding sites for transcription

Figure 7–26 Expression of transcription repressors and

You make flies containing these novel genetic constructs integrated

position of C-terminus in mutant receptors Figure 7–28 Effect of C-terminal deletions

a mating type. In a/ diploid cells, the 2 repressor collaborates with the

Figure 7–29 Binding of transcription

A. In the presence of a1, is 2 present in two forms with different binding

MECHANISMS THAT REINFORCE CELL MEMORY

(A) Figure 7–30 Pedigrees reflecting maternal

exons 1 & 2 exon 3

Figure 7–31 Effects of methylation on transcription of the -globin gene (Problem

You create several different -globin constructs that are unmethylated

lines containing individual constructs, verify their methylation patterns,

Why do you suppose there is such a critical position-dependence for reg-

1 kb HeLa F9 Figure 7–33 Effects of mutations in

which has a molecular mass of 48 kilodaltons, is synthesized by the intes-

Intestine Liver Intestine Liver Figure 7–34 Location of the sequence

Figure 7–35 Effects of mutations on the regulation of -tubulin mRNA

(A) Tra2 mRNA (B) Fem3 mRNA

long poly A FBF1 NOS

REGULATION OF GENE EXPRESSION BY

mouse Figure 7–39 Conservation of piRNA

B A B A B A B A B A B A Figure 7–41 Analysis of expression