*frequency of recombination events = 2x recombination fraction

-1map unit = 1 recombinant chromatid

Chapter 1: Genetic Approach to Biology
1.2- Molecular basis of genetic information
Transcription: process of converting DNA into mRNA. The difference between RNA and
DNA is that the sugar in RNA is ribose not Deoxyribose. Also Thymine is not present in
RNA, rather Uracil is. Transcription takes place in the cell nucleus
- the transcript is not always a functional mRNA strand because many of the
nucleotides do not code for amino acids.
- Transcripts serve three main purposes
1) increases # of copies of genetic info available to cell
2) relieves traffic congestion as it leaves nucleus and enters cytoplasm.
3) Act as a control on how much of a protein is produced
Translation: production of amino acids based on a sequence of nucleotides in mRNA.
- every three amino acids (codon) codes for a specific amino acid.
- There are 64 different codon combinations however there are only 20 amino acids
- UAG= stop codon
- tRNA actually are the correspondence btw codons and amino acid formation.
tRNA have anticodons that compliment codons. They also have the amino acid
on there tail so when they meet up with appropriate codon the ribosomes act to
add the amino acid to the growing polypeptide.
Gene Regulation: occurs both in the attachment of the RNA polymerase to the beginning
of the DNA sequence that is to be read and in the initiation of its movement along the
DNA sequence.
1.3 Program of Genetic Investigation
Forward Genetics
1) genetic investigation that begins with observation of a variant phenotype
a)one such type of forward genetics involves looking for normal natural variation
within the phenotype (ex. Varying degrees of insecticide resistance in insects)
b) another form involves looking an abnormal variant in character for which all
individuals have a normal form.
-normal= wildtype variant=mutant type
2) make crosses between strains
3) note offspring ratios
4) identify genes
5)identify molecular and developmental differences between individuals of diff
genotypes
6) the last stage in forward genetics is to characterize the DNA of different variant alleles
Reverse Genetics
STARTS FROM AN OBSERVED NORMAL DNA SEQUENCE OF UNKNOWN
FUNCTION MANIPULATES THE GENE TO CREATE GENETICALLY
MODIFIED ORGANISMS, SUCH AS A KNOCKOUT, FACILITATING STUDY OF
WHAT THE GENE DOES AND HOW IT PARTICIPATES IN FORMATION OF A

PHENOTYPE
-ex) if dont want to induce mutations can study very similar species
1.4 Methodologies Used in Genetics
1) isolation of mutations affecting biological process under study
2) analysis of progeny of controlled matings
3) genetic analysis of cells biochemical processes
4) microscopic analysis
5)direct analysis of DNA
-genomics: study of structure, function, evolution of whole genomes
-bioinformatics: computational analysis of the content of genomes
Probing:
A) Southern Blotting (DNA)
1)piece of DNA is cut using restriction enzymes: produces many fragments
2) fragments are separated by size using gel electrophoresis
3)gel soaked in alkali to denature the fragments
4)fragments transferred to positively charged membrane that mirrors distance they
traveled during electrophoresis
5)Random DNA is added to adhere to all fragments but the one of interest
6)then treated with probe which is in excess so it will bind to a complimentary strand
B) Northern Blotting (RNA) similar process to southern blot
C) Western Blotting (protein)
1) run protein thru SDS polyacrylamidegel using electrophoresis
2) Run gel perpendicular to run protein out into a membrane
3) Block for nonspecific antibody sites
4) incubate with primary antibody for protein of interest
5) Use secondary antibody that recognizes tail of first one. With artificially
attached enzyme we can view if protein is present because it gives off
chemiluminescent light
1.6) Genes, the environment and the Organism
Model 1: Genetic Determination
-virtually all differences between species are determined by the differences in their
genomes (ie. A lion will never give birth to a lamb)
Model 2: Environmental Determination
-in this model the genes impinge on the system giving general signals for development,
but the environment determines the actual course of development
Model 3: Genotype-Environment Interaction

-what an organism will become critically depends on its present state and on the
environment it encounters during that moment
Developmental Noise
-genotypes are fixed throughout life while phenotypes are determined by the interaction
of a phenotype and a specific environment
-random events in development that lead to variation in phenotype
Chapter 2: Single Gene Inheritance
2.2 Mendels Law of Equal Segregation
-studied seven traits in Garden Peas: Pea color, pea shape, pod color, pod shape, flower
color, plant height and position of flowering shoot
-for each character he studied two contrasting phenotypes
MITOTIC CELL CYCLE:
1)Interphase: composed of G1, S and G2 phases. Cohesin is introduced during this phase
and it is a complex that includes 2 SMC proteins that are linked to 2 non SMC proteins
2)Prophase: cohesion still holds sister chromatids together and nuclear envelope breaks
down
3)Metaphase: condensin is introduced which further compacts the DNA together. Mitotic
spindle forms and bivalent attachment occurs at centromeres. Sister chromatids attach to
opposite kinetochore microtubules
4)Anaphase: cohesion is cleaved but condensin is still in contact and it holds DNA
compacted as segregation starts to occur
5) nuclear envelope reforms as telophase occurs and splits into two cells
MEIOSIS CELL CYLCE- gametes formed from meiocytes
Meiosis 1: homologous chromosomes separate
Meiosis 2: sister chromatids separate
Null alleles: the proteins encoded by them completely lack the function of the nonmutated protein
Leaky alleles: mutant alleles that have a reduced level of the protein function
Bakers Yeast Mating:
1) MATa and MATalpha cross
2) Become a diploid
3) Chromosomes undergo replication to become a meiocyte (tetrad)
4) Undergo meiosis to produce 4 haploid cells in the ascus
Haplosufficient: a heterozygote with one copy of mutant allele and one wild-type. The
wild-type still provides enough protein product for normal function
Haploinsufficient: When the wild-type doesnt provide enough protein for normal
function
Test Cross: cross of a heterozygous parent with a homozygous recessive parent (tester)
2.5 Sex-linked single gene inheritance patterns

-female is the homogametic sex (XX)

-male is the heterogametic sex (XY)
Sex-linkage: genes in differential regions
a) X-linkage: mutant alleles in the differential region of the X chromosome
b) Y-linkage: mutant alleles in the differential region of the Y chromosome
Pseudoautosomal Regions: The X and Y chromosomes have two short homologous
regions at each end
-sex-linked inheritance occurs at the differentiated regions
*in chickens and moths the female is the heterogametic sex(ZW) and the male,
homogametic (ZZ)
X-Linked Recessive Disorders:
1) Many more males than females show the phenotype because the female offspring
can only exhibit the phenotype if she inherits a copy from both mom and dad
2) None of the offspring of an affected male show the phenotype
3) None of the sons of an affected father show the phenotype
X-Linked Dominant Disorders:
1) Affected males pass on trait to all daughters but no sons
2) Affected heterozygous mothers pass trait to half of sons and half of daughters
Y-Linked Inheritance:
-only occurs in sons
Chapter 3: Independent Assortment of Genes
3.1- Mendels Law of Independent Assortment
-For two genes on different chromosomes: A/a;B/b
-For two genes on same chromosome: AB/ab
-If location is unknown: A/a*B/b
Mendels First Law: law of segregation. Each gamete only receives one copy of the
gene
Mendels Second Law: Different Gene pairs assort independently in gamete formation.
-Holds true for genes on different chromosomes
1-((1-(odds of desired genotype))^n)=% you are sure
Chi-Squared
-df=Number of variable-1
-if P<.05 then the results are not compatible with the hypothesis
Assumptions:
1)segregation laws in effect
2)assume all genotypes have same efficiency of fertilization

3)all fertilizations give rise to peas with equal probablility

Synthesizing Pure Lines:
Start with heterozygous gene pair (A/a). After selfing the progeny should be AA, aa,
and Aa. After selfing these f1 generations the total probability of heterozygosity
should once again be cut in half since the homozygous individuals from the F1 generation
will only produce Homozygous progeny
3.3-Chromosomal Basis of Independent Assortment
-carothers observed, through the experiments with grasshoppers, that chromosomes
segregate independently of eachother. She found this because there was a heteromorphic
pair of chromosomes and also a chromosome that wasnt paired at all. She observed that
the unpaired chromosome segregated with the other chromosomes with equal frequency.
Neurospora (Bread Mold):
1) MATa and MATA cross
2) Become a diploid
3) Chromosomes undergo replication to become a meiocyte (tetrad)
4) Undergo meiosis to produce 4 haploid cells in the ascus
5) Undergo extra mitosis to produce 8 ascospores
Recombination:
-a recombinant is defined as any meiotic product that has a new combination of alleles in
reference to the haploid genotypes that formed the meiocyte
3.4 Polygenic Inheritance
-polygenes are the interacting genes underlying hereditary continuous variation such as
skin color or height
3.5 Organelle Genes: Inheritance independent of the Nucleus
-Not all genes are found in the Nucleus.
-Some are found in the mitochondria, and in the chloroplasts of plants.
-mitochondria and chloroplasts are located in the cytoplasm and contain a
circular chromosome
-mtDNA(oxidative phosphorylation) and cpDNA(photosynthesis)
-Organelle Genes are inherited almost solely by the mother since the egg contributes a lot
of cytoplasm while the sperm contributes essentially none (Maternal Inheritance)
Cytohets: Cells containing mixtures of Mutant and Normal Organelle chromosomes
-by chance after the cells divide, distinct organelle types (mutant or Normal) will
segregate together into progeny cells causing completely mutant or normal progeny cells.
BOVERI
Studies with Sea Urchins:

Took a look at sea urchin eggs that are fertilized by 2 sperm. The centrosome that is
responsible for formation of mitotic spindles after fertilization are inherited only from the
father. So for 2 sperms there are 4 spindles formed. After the 54 chromosomes duplicate
to become 108 (18 from egg, 36 from two sperms) 27 each attach to 1 spindle. This led
to abnormal development. Boveri then did an experiment and fertilized the egg with two
sperm again. This time at fertilization he shook the culture which resulted in only 3
spindles formed. This would theoretically allow 36 chromosomes to attach to each
spindle pole. He found that most zygotes still developed abnormally but many more
developed normally than did those with 4 spindle poles.
Devised an experiment to test the probability that normal development would occur in 3
spindle case as compared to 4 spindle case. Made boards with 4 compartments and 3
compartments respectively. Got 54 balls and 3 balls had 1 specific #. Found that 11% of
the time with the 3 spindle situation each compartment had 1 of each type of ball. Never
got a successful trial with a 4 spindle situation. This helped him conclude that only a full
set of chromosome types could sustain normal development, and that each chromosome
had individual characteristics.
Chapter 4- Mapping Eukaryote Chromosomes By Recombination
Why Genetic Maps are important:
1) gene position is crucial for building complex genotypes
2) allows you to pinpoint a genes structure and function
3) useful in interpreting mechanisms of evolution
4.1- Diagnostics of Linkage
-found that some Genes are linked because when performing a self cross of a dihybrid,
they did not see a 9:3:3:1 phenotypic ratio that mendels second law suggests.
-also when he performed a cross of a dihybrid with a homo recessive tester he did not
observe a 1:1:1:1 ratio that independent segregation predicts
How crossover produces Recombinant:
-during meiosis when two dyads form a bivalent a chiasma occurs between two nonsister
chromatids.
Evidence that crossing over is a breakage and rejoining process:
-Plant was a dihybrid in the cis formation. One of the chromosomes was longer and had
a nob like structure while the other was shorter and normal. However they found that
after recombination the long chromosome no longer had the nob on it because it had
crossed over.
-must occur at a 4 chromatid stage because if it occurred when the chromosomes had
been unduplicated there could only ever be 2 genotypes when you isolate the tetrad (in
fungi). (4 is what is commonly seen)
-crossover can occur between 3 or even 4 chromatids
4.2- Mapping by Recombinant Frequency
-recombinant frequencies greater than 50% are never observed

-as recombination frequencies approach 50% it is difficult to distinguish whether they are
located far apart on the same chromosome or are on different chromosomes
Three-point Testcross
-cross a trihybrid with a triply recessive tester
*always use the sum of the two smallest distances and not the longest distance alone.
Because it doesnt take into account the double crossover
Without recombination frequencies we can see use inspection to see if three genes are
linked by the following observations:
A) Two genotypes at high frequency
B) Two at an intermediate frequency
C) Two at another intermediate frequency
D) Two rare frequency
Interference
-if double crossovers are independent then the frequency of double crossover would be
the product of the two single crossovers in the adjacent regions around the middle locus.
I=1-c.o.c=1-(observed # of double recomb)/(expected number of double recomb)
-expected number of double recomb is equal to the product of the two single crossovers
times the total # of progeny.
Ratios as diagnostics:
Monohybrid testcross: 1:1
Monohybrid selfed: 3:1
Dihybrid Testcrossed(independent): 1:1:1:1
Dihybrid selfed (independent) 9:3:3:1
Dihybrid testcrossed (linked) P:R:R:P
Trihybrid testcrossed (independent) 1:1:1:1:1:1:1:1
4.3 Mapping with Molecular Markers
-Loci of molecular heterozygosity are called molecular markers
-useful in detecting genes of interest because they act as milestones
1) Test cross a dihybrid (1 is a gene that is hybrid and the other is a molecular
marker)
2) Extract DNA and sequence it
3) and if you find a P:R:R:P ratio you can determine the distance of the gene alleles
from the molecular markers
SNPS-Single Nucleotide Polymorphisms
-there are theoretically 4 different alleles possible, however there are only ever 2 found in
a population.
1) Silent within genes: have no apparent effect on phenotype

2) SNP that causes a mutant phenotype showing single gene inheritance

3) SNP in polygenes that help to discover polygenes that contribute to a phenotype
showing continuous variation.
4) Intergenic SNPs: have no phenotypic effect but are useful as milestones
5) RFLPs: SNPs located at Restriction enzyme target sites
-Useful because dont require sequencing to determine location
A) select a restriction site with two morphs of a hybrid organism and then
southern blot it to examine differences
B) perform a test cross, extract DNA from progeny and add Restricition
enzymes
C) southern blot the DNA and look for recombinant frequency to use for
mapping
Mapping by SNP Haplotypes:
Haplotype: chromosomal segment defined by a series of SNPs generally between 10 and
50kbs
-This works by the concept of Linkage disequilibrium which is when cross-over does not
occur. You can determine where a gene is by recognizing if it is flanked by the same
SNPs over and over again and if it keeps showing the mutated gene. You can identify
where the gene is because SNPs are generally 1kb apart.
Simple Sequence Length Polymorphism (SLLPs)
-repeated short sections of DNA
-are of importance because the number of these repeats varies between individuals of a
population because of slippage events
1) Minisatellite markers- generally a repeating unit (15-100 bp long)
-the whole repetition lasts about 1-5 kb pairs in humans
-to recognize the difference you cut with restriction enzymes the flanking regions
of the minisatellite and use gel electrophoresis with a probe.
2) Microsatellite- repeating unit (2bp long)
-detect the size differences by using pcr to amplify the target SLLP and
plating it using gel electrophoresis using a probe
Making Human Genetic Map
1) need Markers that span the genome of unaffected individuals (about 1 marker for
every 3000 centimorgans) 1cm=1Mbp
2) location of these markers with respect to eachother must be mapped from normal
individuals
3) polymorphisms that span the genome must be chosen (pedigrees from affected
families needed)
4) Can pinpoint location of disease genes from markers by using pedigrees and
recombination analysis
4.5- Chi-square test to confirm Linkage
-in this case we actually test the hypothesis of no linkage. And if our value rejects the
hypothesis then we can infer linkage (null hypothesis)

-look at book to see how to find expected values

-df is calculated by taking the amount of variables in a row subtracting by 1 and then
multiplying it by the amount of variables in the column minus 1.
4.6-Lod Scores
Used to estimate distance of a certain gene to a marker when the sample size is very
small. Must assume the family is a dihybrid testcross where one hybrid is of the gene of
interest and the other is of a molecular heterozygosity.
-first find the probabilities of each gamete genotype if independent assortment is
assumed. Then you find probabilities of each genotype for certain RF values.
-you have to pick out which kids are Recomb and which are non recomb. Then you
multiply each probability of each kid by eachother and use the ratio of each specific
degree of linkage to the assumption of independent assortment. Whichever lod value is
the highest is most likely the accurate answer.
Mapping Function
1-(RFx2)=e^-m mx50=map units
Relationship of Physical and Recombinant Maps
The physical map shows a genes possible action at the cellular level, whereas the
recombination map contains info related to the effect of the gene at the phenotypic level.
From pedigree analysis we know of the area where the gene is because our markers flank
it, but not exact location. We know some gene functions on the physical map by work
with other organisms.
1) FIND MARKERS BY RANDOM CLONING OF SMALL DNA PIECES AND
SEQUENCING
2) SELECT MICROSATELLITES
3) FIND THOSE THAT SHOW VARIATION
4) MAKE A GENETIC MAP OF VARIABLE MARKERS
5) CHOOSE A MARKER SET TO COVER GENOME
6) DO PEDIGREE STUDIES ON AFFECTED FAMILIES
7) FIND MARKERS THAT FLANK THE DISEASE GENE
8) FIND MARKER SEQUENCES IN GENOME
9) DISEASE GENE MUST BE IN BETWEEN
Chapter 6-Gene Interaction
-Interacting Genes are members of the same pathway or a connected pathway.
-biosynthetic
-signal transduction
-developmental
6.1-Interactions between alleles of a single Gene: Variations of dominance
Complete dominance
-an allele is dominant when only one copy is present and it is expressed
Dominant mutants:

A) Null mutation: when the null mutation causes the body to not produce enough of a
given enzyme to allow the body to function
B) Dominant negative: aka as spoiler, happens when a mutant enzyme inhibits the
ability of the wild type enzyme
Incomplete Dominance:
When a heterozygote expresses an intermediate phenotype to the homozygous dominant
and homozygous recessive genotypes
Codominance:
when two separate alleles produce their own unique effect ex) blood types, there are three
types of alleles
sub-lethality:
-when lethality is expressed only in some but not all homozygous individuals.
6.2- Interaction of Genes in Pathways
Archibald Garrod- Discoverer of inborn error of metabolism
-Studied children with black feces and urine.
Found that they turned black because of oxidation of homogentisic acid.
-induced that tyrosine was involved in the formation of homogentisic acid in patients
with alkaptonuria.
-in 1902 he found 9 families with a total of 48 children that had 9 affected children.
-40% affection rate
-Also in 60% of the families parents were first cousins
-in England only 3% of all marriages were between first cousins
-He postulated that alkaptonuria was an inborn error of metabolism that would convert
tyrosine to homogentisic acid through fermentations.
-Like mendel no one gave any thought to Garrods studies for 39 years
Beadle and Tatums 1941 Paper experiment
-rediscovery of Garrods studies
-*asexual spores produce hyphae
1) mutagenized asexual spores of neurospora and crossed with wild type of
opposite mating type
2) grew haploid ascospores on complete media(glucose,malt, yeast extract,
inorganic salts) so they will grow no matter what
3) Took some of the ascospores and put them on minimal media
4) If it didnt grow on minimal media take the original ascospores and plate to
the following minimal medias
A) Minimal control
B) Minimal + amino acids

C) Minimal+ vitamins
D) Complete media
5) If it grew on amino acid media then that meant there is a mutation that doesnt
allow the fungi to produce amino acids
6) So they then plate the original ascospores into tubes that each have a different
single amino acid
Were able to conclude that biochemical reactions promoted by enzymes in metabolism
could be determined by a single gene
15 Mutants were obtained that could not grow without arginine
Possibilities:
1) All mutants represented the same DNA mutation
2) Each mutation occurred in a different gene
Used complementation test to determine what it was
Assume two different strains are each homozygous for a recessive trait that doesnt grow
argining
Cross the two strains together and see what happens
-if you cross them and the progeny still shows the same trait then there is no
complementation and the mutations must have occurred within the same gene
-if you cross them and the progeny shows wild type then it shows
complementation which means the mutations are on different genes
Method for Interpreting Results of Complimentation Test
-Put all the genes of interest around a circle and connect the genes that show no
complementation (this means that they are in the same complementation group)
-In the beadle and tatum group they found that there were 7 groups that behaved as a
single mendelian gene.
-members of three groups were analyzed
-each group had ornithine, citrrulline, and arginine separately to see if they would grow.
Make these inferences by making dihybrid self cross
9:3:3:1 = no gene interaction
9:7= genes in the same pathway
-no matter which wild-type is absent the same pathway fails
9:3:4= recessive epistasis (when a double mutant shows phenotype of one mutant but not
the other) results from genes being in the same pathway
12:3:1=Dominant epistasis
9:3:3= synthetic lethal because the double mutant is not viable and dies off
Suppressor: a mutant allele of a gene that reverses the effect of a mutation of another
gene, resulting in wild type or near wild type phenotype. These organisms that express
this mode of suppression are called psuedorevertants

Penetrance: the proportion of individuals with a given allele who exhibit the
corresponding phenotype. Has 3 Factors
1) Influence of environment
2) Influence of interacting genes
3) Subtlety of mutant phenotype
Expressivity: The degree to which a given allele is expressed at the phenotypic level.
Measures intensity

Chapter 5- Genetics of Bacteria and Their Viruses

-phages= nonliving because they have to parasitize bacteria and use their cell machinery
to propogate
5.1- Working with Microorganisms
Prototrophic- wild type bacteria, grow on minimal media
Auxotrophic-cells that need some sort of nutrient to grow
5.2- Bacterial Conjugation
Discovery: had two different ecoli strains that were both auxotrophs for different
nutrients. When plated on minimal media separately they couldnt grow. When they
were mixed however they regained the ability to grow. Some believed that some of the
cells would leak out substances that the other cells would absorb. This was ruled out by
the U shaped experiment which proved the cells needed to come into contact with
eachother.
Discovery of Fertility factor (F+)
One cell is always a donor (F+) and donates part of its genome to the recipient(F-)
-F+ is a plasmid which replicates in cytoplasm by rolling circle replication which reels
out a single stranded copy
-the replicated version then moves thru pore into recipient cell.
Hfr strains
-created by integration of plasmid F+ into chromosome
-when crossed with F- strains none of the F- strains gained the F+ factor, instead the
Chromosome of the Hfr stain inserts completely (RCR) into the donor cell and then
engages in recombination
Linear Transmission of Hfr
-used interrupted mating (put in blender after specific times) and found that certain genes
were always found to be transferred into F- strain first (exconjugants). This suggested
that the F plasmid always integrated into a specific point on the chromosome (O) so the
origin was always transferred into donor cell first and F last.
*there were different integration sites however and different strains because during
interrupted mating experiments sometimes different genes were transferred at different
times

*Hfr single strands however after they have been copied and sent to a donor can only
recombine with donor genome by double stranded crossover.
Bacterial Recombination
Doesnt necessarily take place between two full chromosome sets. One, the F-, is full
and called the endogenote, while the other is derived from the Hfr and called the
exogenote. The cell is called a partial diploid or merozygote. Since the exogenote
usually dies after subsequent cell division only one recombinant product survives.
Chromosome Mapping using Recombination
-gotta have shown thru interrupted mating experiments that three different alleles were
incorporated into the F- bacteria that had recessive alleles in its own chromosome.
-then you have to select for stable exconjugants that picked up the last allele into its
chromosome so you can test it by recombination to the other donor alleles.
FPlasmid:
Sometimes the F plasmid can liberate itself from Hfr strain and sometimes it carries with
it some of the bacterial genome. Hence F plasmid
R plasmids
Plasmids of shigella genus that transfer drug resistant genes to similar bacterium species
-sometimes theses resistant genes are contained within transposons of the plasmid, hence
these can then be spread and transferred into other cells thru conjugation
5.3- Bacterial Transformation
-when bacteria adds fragments of DNA from an external medium. Not a recombination
event because the bacteria doesnt exchange DNA
-Frequency of Double transformants is equal to the product of the single transformants
for genes that are far apart. If genes are close the proportion of double will be greater
than that of the product of the singles
5.4- Bacteriophage Genetics
-contain chromosome covered by protein. Injects its DNA into the bacterial cell which
commandeers the cell machinery to produce more phage DNA and Phage protein. Then
when that is done the new phages break out of the Bacteria in a process called lysis
(virulent phages)
-clear plaques are formed when lysis occurs
Mapping phage chromosomes
h-r+ x h+r- RF=((h+r+) + (h-2r-rapid))/total plaques
5.5 Transduction
-when a phage carries genes from one bacterial cell to another
-discovered this because when they used two different auxotroph strains in a youtube
experiment they found that something was carrying the correct genes through the filter

and recombining to make a prototroph. These are temperant phages because they dont
kill the host
A) can either integrate into genome of bacteria (Prophage)
B) or just be harbored outside the nucleus (lysogenic)
Generalized Transduction
-when phage can carry any part of bacterial genome
-happens when a phage causes lysis of cell, sometimes it can incorporate some of the
broken up pieces of the bacterial cell genome in the phage head.
cotransductants when multiple genes are picked up into phage head and both
incorporated into a new bacterial cell genome. Greater percentage of cotransduction
means closer genetic markers
Chapter 7- DNA Structure and Replication
7.1- The Genetic Material
Discovery of Transformation
GRIFFITH: used cap S gene to prove it. S (capsule) strain kills mouse. R (nocapsule)
doesnt kill mouse. Heat killed S strain doesnt kill mouse. Heat killed S plus R kills
mouse
AVERY: postulated that DNA was genetic material. Deoxyribonuclease destroyed
transforming activity but ribonuclease and protease did not
HERSHEY and CHASE: labeled lytic bacteriophage (T2) protein with 35S and DNA
with 32P. found that as the phage mixes with host cells only the dna enters the cell.
Found that this was true because after the viral chromosome duplicates and produces new
phages that exit the cell the new phages only had the 32P still. No 35S
7.2- Structure of DNA
1) must allow faithful replication
2) must have informational content
3) must be able to change
-DNA is made up of linked nucleotides which are made of Phosphate, Deoxyribose and
Nitrogenous bases.
Wilkins/Franklin: photographed DNA- nucleotides linked together by 3,5
phosphodiester linkages Watson/Crick: found double helix shape held together by
hydrogen bonds on opposite bases that run in anti-parallel fashion. Chargaffs rule:
A=T, G=C, A+G (purine)=C+T(pyrimidine)
G-C pairs have 3 hydrogen bonds and A-T have 2.
- DNA is not symmetrical in all places there is a major and minor grooves which allow
different proteins and enzymes to have different accesses to the DNA

7.3- Semiconservative Replication

MESELSON: Strand Separation semi-conservative: tagged DNA with 15N. after one
generation DNA was HL after 2 generations some was LL and other was HL. Separated
by centrifuge found that there was no HH DNA(no conservative). If it was distributive
then all generations would be mixed HL
Replication Fork
-postulated that during replication there would be replication zippers. Cairns
incorporated tritiated thymine into the bacterial cells replication. He then lysed the cell
and placed it on photographic emulsion because tritium decays and emits a beta particle
that can be detected.
DNA Polymerases
-Polymerases added nucleotides to the 3 end of the growing chain. Synthesis is 5-3.
-the Substrates for DNA Polymerases are the triphosphate forms of the
deoxyribonucleotides.
7.4- Overview of DNA Replication
-DNA Pol I was shown not to be main polymerase in replication activity because it
moved to slow. DNA Pol III was shown to be the one that was present at most replication
forks.
-There is a leading and lagging strand. On the lagging strand DNA Pol forms short
segments (Okazaki)
-DNA Pols can extend a chain but cannot start one.
Solution: Primosome made up of RNA Polymerase Primase starts the replication then
DNA Pol III extends the chain.
Completion of DNA synthesis:
1) RNA primer must be removed to make the molecule completely DNA
2) The last RNA base is removed by a 5-3 exonuclease of DNA Pol 1
3) The gap is basically a primer template junction so DNA Pol I comes in and closes
the gap but it doesnt connect the 3OH to the 5 phosphate Resulting in Nick
4) so DNA ligase comes in and uses energy of ATP to form phosphodiester bond
7.5- The Replisome: A remarkable Replication Machine
Replisome coordinates activities at replication fork.
Pol III Holoenzyme- consists of 2 DNA Pol III and 2 B clamps that keep polymerase
attached to leading and lagging strands
Unwinding the Double Helix
Helicases- unwind the double stranded
Topoisomerases- (Gyrase) prevents overwinding of DNA.
Single Strand Binding Proteins- stabilize single stranded DNA
Replication Initiation (Prokaryotic)
1) DnaA proteins bind to AT rich sequence in oriC and unwind the double strands
2) Two Helicases (Dna B) unwind further in 5-3 direction

3) DNA Pol III Holoenzyme and primase is recruited to start replication

7.6 Replication in Eukaryotic Organisms
Eukaryotic Replisomes are much more complex than Prokaryotic because Eukaryotic
chromosomes consist in the cell as Chromatin and the basic unit of chromatin is the
Nucleosome (DNA wrapped around histones)
*thus the replisome not only has to Replicate the DNA but also disassemble the
nucleosome
- CAF-1 is a histone chaperone. During replication
PCNA is bound to a polymerase to facilitate synthesis of DNA. After Synthesis PCNA
unbinds to polymerase but still surrounds DNA. This signals CAF-1 to attach H3/H4
tetramer to start assembling nucleosome onto new DNA strand
Eukaryotic Origins of Replication
Unlike prokaryotic chromosomes each eukaryotic chromosomes have multiple origins of
replication. These origins are also AT rich sequences similar to the oriC of prokaryotic
cells
DNA Replication and Yeast Cell cycle
1) ORC binds replicator
2) ORC recruits Cdc6 and Cdt1 which are helicase loaders
3) Cdc6 and Cdt1 recruit Mcm helicase which pumps DNA thru in leftward manner
to unwind DNA by using energy in ATP bonds
*Assembly and activation of Pre-RC depends on cdks and ddks
-these levels are low in G1 phase but high in S phase
-high concentration of kinases activate existing Pre-RCs to begin replication but
prevent new assembly because they phosphorylate Cdc6 and Cdt1 which triggers
proteolysis of these proteins. Activates existing Pre-RC by phosphorylating Mcm 2-7.
-low concentration of kinases activates formation of new Pre-RCs but inhibits
activation of existing complexes
7.7- Telomeres and Telomerase: Replication Termination
-On the lagging strand there is an inherent problem with replication at chromosome ends.
Once the primer is degraded there is nowhere for the DNA Pol to bind and thus after
-Humans have telomere sequence of TTAGGG and the 3 end of the one strand far
outstretches the 5 end of the other strand
Telomerase is a ribonucleoprotein that uses a small RNA molecule as a template for
reverse transcription. It extends the 3OH of the DNA end by reverse transcription.
Telomerase translocates to the new end and repeats the process.
-now this 3 end is a center for Okazaki fragment processing. But there are telomere
binding proteins so there is always a 3 overhang
Telomere, cancer and aging
-somatic cells have no telomerase activity but germ cells do.

Chapter 8- Transcription and Processing

*-In prokaryotes Translation occurs while transcription is still happening. In Eukaryotes
Transcription happens in the nucleus but translation happens in the cytoplasm
8.1- RNA
Pulse-Chase Experiment:
Volkin and Astrachan showed that RNA is intermediate
1) Introduced radioactive isotope of uracil (red) so any RNA synthesized in the
cell would show up.
2) Then chased out the radioactive uracil with normal uracil and the radioactive
uracil is found in the cytoplasm
Properties of RNA
1) RNA is single stranded and more flexible so it can form 3-D shapes
2) RNA has ribose sugar that has hyrdroxyl group on 2 carbon as well
3) Uracil is in place of Thymine. It can also bind to Guanine
4) Some RNAs are ribozymes which mean they can catalyze biological reactions
Classes of RNA
1) mRNA- encodes information that makes polypeptide chains. Are the
intermediary between DNA and protein
2) tRNA- molecules that are responsible for bringing correct amino acid to mRNA
3) rRNA- major components of ribosomes, which guide assembly of amino acid
chain by mRNA and tRNA. (Most abundant in the cell)
4) snRNA- are small parts of ribonucleoprotein spliceosome that removes introns
5) miRNA- regulate the amount of protein produced by eukaryotic genes
6) siRNA- protect integrity of animal and plant genome. Inhibit production of
viruses and prevent spread of transposable elements
8.2 Transcription
-Transcription or adding of ribonucleotides always happens in 5-3 manner, so the
template strand is always read in 3-5.
-Only one strand is ever used as the template strand in any given Gene
-The nucleotide chain is elongated by RNA pol which also unwinds the DNA and rewinds
it after those nucleotides have been transcribed
Prokaryotes
Initiation
*we refer to genes in the 5-3 direction (nontemplate strand)
1) RNA pol binds to promoter sequence
-RNA pol Holoenzyme scans for promoter sequence.
-Two alpha subunits help assemble the holoenzyme
-B subunit is active in catalysis
-B subunit binds DNA
-w(omega) has roles in assembly and gene regulation

-Sigma 70 binds to the -35(TTGACAT) and -10 (TATAAT) sequence and unwinds
the DNA
2) after core enzyme begins RNA synthesis the sigma 70 unbinds
Elongation
-adds to 3 end of transcript
Termination
Transcription continues beyond protein coding region resulting in 3 UTR
Rho-Independent- 3UTR is GC rich (40 bases) followed by at least 6 Us.
This results in a loop formation and RNA Pol stops processing as it backtracks to
stabilize the RNA-DNA hybrid.
Rho-Dependent- Termination signal is mainly C rich with less Gs and very little Us.
Upstream from this are is a rut site which binds Rho protein. This protein uses energy
from ATP hydrolysis to help the RNA Pol disassociate from the RNA
8.3 Eukaryotic Transcription
-More complicated because there are more eukaryotic genes. There is more non-coding
regions in eukaryotic genomes. The presence of the Nucleus means transcription and
translation are spatially separated.
-RNA Pol I used for rRNA
-RNA Pol III used to transcribe tRNA
-RNA Pol II used for regular genes. First makes pre-mRNA which is spliced to regular
mRNA
Initiation:
-RNA Pol II and GTFs (TFIIA,B,D etc) form pre-initiation complex
1) TBP which is part of TFIID binds TATA box (-30)
2) this recruits the other GTFs and the RNA Pol II core enzyme
3) Carboxyl Terminal Domain of B subunit of RNA Pol is phosphorylated by GTFs and
ends the initiation phase
4) the GTFs remain at promoter so new RNA Pol II can come and simultaneously
transcribe the same gene
Elongation
-large subunit of RNA Pol II has sequence of 7 amino acids repeated 52 times
-serines are located at position 2, 5, and 7 of sequence
1) phosphorlation of serines unloads GTFs and loads TFIIS and hsPT5 (xscritpion
elongation factors)
2) serines are dephosphorylated
3) serine 2 is phosphorylated which loads splicing factors
4) serine 5 is phosphorylated which loads capping factors
Co transcriptional Processing
1) When nascent RNA strand emerges from RNA Pol a cap (7-methylguanosine) is
added to 5 end to protect from degredation by interaction with CTD

2) When RNA Pol hits termination sequence AAUAAA at 3 end it disaccociates and
adds 150-200 Adenine nucleotides
8.4- Functional RNAs
Small Nuclear RNAs (snRNAs)
Spliceosome Splicing (Pre-mRNA mRNA)
-happens while RNA is still being transcribed and after the cap has been added.
1) -U1 binds to 5 splice site (requires SR Protein)
2) U2 binds branch point (A)
3) U4, U5, U6 join in as a complex
4) U1, U4, U6 fall off
5)U5 brings 5 splice site into contact with internal A and U2
6) 3 splice site is cleaved and exons join together
Self-Splicing
-this suggests that RNA is kind of an enzyme since it can catalyze the removal of its own
intron
Small interfering RNAs (siRNA)
Injected dsRNA into elegans which was complimentary to a neurological gene.
-the ds RNA somehow silenced the gene. One strand was sense RNA (coding) the other
was complimentary or antisense
1)dicer binds to dsRNA and chops it up into siRNA (short interfering RNA)
2) siRNA get taken up by RNA induced silencing complex (RISC) and get broken into
single strands
3)the RISC and singlestranded RNA find the mRNA of the gene and break down the
mRNA causing it to not be able to translate into the required protein.
-Conserved in both plants and animals
CHAPTER 9: Proteins and their Synthesis
tRNA- Translate the codon of the mRNA into corresponding amino acid which is brought
by the tRNA to the ribosome
rRNA- major component of ribsome that assemble amino acids to form proteins
-these functional RNAs are much higher in concentration in the cell than there mRNA
counterparts because they are much more stable.
9.1- Protein Structure
-amino acid have N-terminus and C-terminus which combine to form peptide bonds.
Primary structure: Linear sequence of amino acids
Secondary Structure: Local region of polypeptide chain (alpha helix, B-pleated sheat)
Tertiary Structure: Folding of secondary structure
Quaternary Structure: combination of 2 or more folded tertiary structures
Globular Proteins=compact
Fibrous Proteins= More linear

9.2- Colinearity of Gene and Protein

-correspondence between the linear sequence of the gene and that of the polypeptide
9.3- The Genetic Code
Proof that codons are three letters long
Suppressor Mutations:
1)intragenic: where a mutation at a different point on the same gene can reverse
the first mutation(2 missense mutations can restore the structure and function sometimes)
2)intergenic: a mutation on a different gene can reverse the first mutation(change
the way the mRNA template is read) changes a specific tRNA-amino acid anticodon to a
stop anticodon that can read the mutated stop codon on mRNA.
1) Silence Mutation: where one base is switched but leads to no change in protein
sequence. UUU -> UUA because both code for same AA
2) Missense Mutation: causes change in one specific amino acid in sequence
3) Nonsense: forms a premature stop codon which produces an incomplete
polypeptide chain
4) Frameshift: insertion of a base pair that alters the reading frame
5) Deletion: one base pair is deleted which causes shift in reading frame
Cracking the Code
-use Polynucleotide Phosphorylase (RNA Synthesizing enzyme) and nucleotides to make
synthetic RNA strands. By using specific Nucleotides you can make any sequence you
want and observe which type of protein subsequently forms. (in vitro in ecoli)
Stop Codon
UAG (amber), UAA(ochre), UGA(opal) are all stop codons. Found UAG stop codon by
analyzing mutants of T4 phage that had shorter head proteins. All that was needed to
form these mutants was a single mutation that would form a premature UAG codon
9.4-tRNA the adaptor
Ribosomes
1) a charged tRNA (aminoacyl, amino acid attached to cca-3) comes into contact
with traveling ribosome at A- site
2) the amino acid with tRNA still attached is connected to growing peptide held at Psite
3) the tRNA now without the amino acid is sent to the E-site
Aminoacyl Transferases: attach the amino acids to the 2 OH(class1) or the
3OH(class2) of the ribose of the tRNA. There are 20 different kinds, 1 for each amino
acid
Wobble
1)certain charged tRNAs can bring their specific amino acids to one of several codons
because of a loose kind of base pairing at 3 end of codon and the 5 end of anticodon
Isoaccepting tRNA: has same anticodon as other tRNAs but differ in nucleotides
elsewhere in the molecule. They pair with the same codons as the normal tRNAs

9.5- Ribosomes
-protein synthesis takes place when mRNA and tRNA molecules associate with
ribosomes
-consists of 2 subunits which each are made up of protein and rRNA
Prokaryotes: 30s (16sRNA, 21 proteins), 50s(23sRNA,5sRNA, 31 proteins) = 70s
Eukaryote: 40s(18sRNA, 33proteins), 60s(28sRNA, 5.8sRNA, 5sRNA, 49 proteins)=80s
Decoding center-small subunit which makes sure the anticodon and codon match
Peptidyl-transferase center- in the large subunit. where the peptide from the A site gets
added to the growing polypeptide in the P site
-Both act as ribozymes because they are made up of entirely RNA and not protein
Translation
Prokaryotic Initiation
1) 3 end of 16s rRNA of 30s subunit binds with shine-Delgarno sequence
(AGGAGGU) of mRNA to position initiator codon in P site
2) IF3 binds to 30s subunit to keep 50s from associating
3) IF1 and IF2 recognize eachother and make sure only N-formylmethionine
initiator tRNA enters P-site (mRNA, tRNA and 30 s form initiation complex)
4) 50s subunit associates with the Initiation complex and releases the initiation
factors
Eukaryotic Initiation
1) mRNA is transported to the cytoplasm with many proteins coating it
2) eIF4 A,B,G remove the proteins
3) eIF4 A,B,G associate with the 5Cap and with the 40s subunit and initiator tRNA
to form initiation complex
4) because eukaryotic mRNA is sometimes base paired with itself the complex
unwinds the mRNA
5) AUG codon is found and aligned with tRNA
6) 60s subunit attaches
7) Initation factors disassociate
Elongation
1) EFTu + tRNA-amino acid (ternary complex) enters the A site on the traveling
ribosome. (In eukaryotes, eEF1 is analogous to EFTu.)
2) Peptidyl transferase (part of 23S ribosomal RNA) attaches the incoming amino
acid to the polypeptide.
EF-G + GTP allows translocation of the incoming charged tRNA from the ribosomal A
site to the P site and the ribosome to the next codon
Termination
1) RF1 (class1) recognizes UAA and UAG
2) RF2 (class 1) recognizes UGA and UAA
3) RF3 assists RF1 and RF2

-tripeptides in the RFs recognize the stop codons and place a water molecule in the
peptidyl transferase center which causes disassociation of the polypeptide chain from the
tRNA in the P site
9.6-The Proteome
-complete set of proteins that can be expressed by genetic material of organism
25000 genes but over 100000 proteins in body
Alternative Splicing
Allows creation of multiple types of proteins by the same gene
Post-Translational Events
Protein folding- happens after the protein is released from ribosome and it is called the
native conformation
Chaperones (GroE) are a class of proteins that assist in the folding of other proteins
Modification of Protein side chains
Kinases-attach phosphate groups to hydroxyl groups of serine, threonine, and tyrosine
Phosphates are negatively charge and thus cause a change in protein conformation
Phosphatase- removes phosphates
Ubiquitin- adding ubiquitin to amine of lysine residues targets protein for degradation by
a protease known as 26S proteasome
Protein Targeting
Most proteins have a series of 15 amino acids at the N terminal of their chain that targets
them for where they belong in the cell
Chapter 10-Gene Regulation in Bacteria and their Viruses
10.1 Gene Regulation
-Regulation in Bacteria is very much the same as it is in eukaryotes
-bacterial cells have evolved to shut down transcription of certain enzymes that are not
needed at a given time so they dont expend unnecessary energy.
Genetic Switches in General
1)RNA Pol binds to promoter sequence
Positive Regulation
-If activator binds activator site which is just downstream of promoter sequence then
transcription occurs
-If activator doesnt bind then transcription does not occur
Negative Regulation
-If repressor binds operator(just upstream of promoter) then transcription does not
occur
-If repressor doesnt bind then transcription does occur

-these regulatory proteins (repressor, activator) each have two sites: DNA-binding
domain, and an allosteric site
-Allosteric effectors bind to allosteric site and turn the regulatory proteins either on or
off. In activators binding of effector allows activator to bind to DNA. In repressors the
binding of the allosteric site inhibits repressor from binding to DNA. In both cases it
allows for transcription
Lactose Operon: i-CAP-P-O+1-ZYA
Operon- DNA that has multiple genes coded into same mRNA molecule and that
includes regulatory sites.
*I gene is not considered part of operon
Lac Regulatory Circuit
-Z codes for B-galactosidase which breaks down lactose into glucose and galactose
-Y codes for permease which transports lactose into the cell
-A codes for transacetylase which has nothing to do with metabolism of lactose
Regulatory Components
1) I codes for lac repressor protein which blocks this transcription
2) Lac promoter site coded by P
3) Lac operator site which is bound by lac repressor protein
*Inducer=effector Lactose and its analogs can bind to lac repressor protein and inhibit it
from binding to DNA causing transcription of operon genes
10.2 Discovery of Lac system: Negative Control
-used radioactively labeled amino acids to show that the addition of an inducer would
provide translation of three different enzymes (ZYA)
Evidence for operator and Repressor
-used IPTG which acts like lactose but cannot be broken down by B-galactosidase
-Found that several different mutations that altered expression of structural genes and
wanted to see if they were dominant or recessive. Since bacteria are haploid they needed
to insert an F plasmid into the cell so it would be diploid for the structural genes.
-Found that the Z+ Y+ wild types were dominant
Constitutive Mutations also found two regulatory mutations that caused structural genes
to be expressed regardless of whether there was inducer present Oc (cis acting) and II+ is dominant over I- but Is is dominant over I+
-Used DNase to show that the operator sequence was a specific sequence because it
would cleave everything not bound to the repressor and the repressor protein covered up
the operator sequence protecting it from degredation
Polar Mutations- mutations in one operon gene that show that other genes are not
transcribed. Proves that the genes are all part of the same transcript

10.3- Catabolite Repression of the lac Operon: Positive Control

Catabolite= breakdown product
-Lactose is really broken down because it makes glucose which the cell uses for energy,
so when glucose and lactose are present the cell will not go through energy expensive
process of breaking down lactose
1) CAP is activator protein
2) cAMP is effector protein
3) High glucose doesnt allow ATP to form into cAMP so it doesnt bind to CAP
which then cant bind to Activation site and thus transcription does not occur
*CAP binding site and Lac operator are both two fold in symmetry because it allows
binding of the regulatory proteins, which are normally composed of multiple identical
subunits, to have more sites to interact with the DNA
10.4- Dual Positive and Negative Control: The Arabinose Operon
(C-0-Initiatior or activator site-P-BAD)
-BAD are functional genes that break down arabinose
-C codes for activator protein
-arabinose is inducer or effector
-In order for transcription to occur both arabinose-activator protein complex and CAPcAMP complex must bind to initiator region.
-If arabinose is absent then Activator protein (C) binds to both I site and O site and forms
a loop inhibiting transcription.
10.5- Metabolic Pathways and Additional Levels of Regulation: Attenuation
(O-attenuator region-EDCBA) Repressor gene is elsewhere in the genome
-the functional genes are in order of which they act in the metabolic pathway of
synthesizing tryptophan
attenuation- when regulation acts after transcription initiation has already occurred
Tryptophan Operon: R-P-O-1/2/3/4-E/D/C/B/A
-transcription occurs when tryptophan levels are low
1) trp repressor protein binds trp then binds operator sequence which turns off
transcription 70 fold
2) attenuation 8-10 fold
low levels: ribosomes stall at attenuation region 1 at UGG (trp codons) sites so region 3
snaps back on region 2(palindromic). 4 left free and transcription continues
high levels: ribosomes dont stall at region 1, but continues behind RNA Pol. After 3
transcribed it cant snap back on 2 cause of ribosome so 4 snaps back on 3 and causes rho
independent termination.
10.6- Lamba Phage: Lytic or Lysogenic
Int-cIII-N-PL-CI-PRM-OR3-OR2-OR1-PR-Cro-PRE-CII
1) Host is infected by phage. Transcription occurs at PL and PR promoters.

-PL allows transcription of N gene which keeps RNA pol transcribing to the left
(CIII) and even allows transcription of genes to the right (CII)
-CII encodes activator protein which binds to another leftward promoter (PRE)
which allows transcription of CI lamba repressor protein which prevents lytic
growth by binding to OR1 and OR2. It also allows transcription of Integrase gene
2) If resources are abundant then CRII is degraded and Cro, which is transcribed
from PR, binds to OR3 which blocks transcription of Prm which helps transcribe
CI lamba repressor. This causes lytic cycle to propogate
Lysogenycell damage creates DNA s.s. gapsactivation of RecA
proteinauto-cleavage of CI proteinlysis
Binding of these regulator proteins bind throught helix turn helix motifs.
Their specific affinities to DNA sites has to do with the specific amino acids that are
in these recognition helixs.
10.7-Alternative Sigma Factors Regulate Large Sets of Genomes
-Under stress bacterium form spores that are heat and dessication resistant
-The bacterium divides asymmetrically with the small forespore being nurtured by the
rest of the cell (mother cell) which ends up dying at the end of the process
-in normal bacterial cells sigma A and H are active
-During sporulation sigma F is activated in the forespore which activates group of 40
genes one of which activtes Pro-sigmaE which is sigma factor in the mother. It also
activates Sigmas-K and G.
*the activation of these sigmas allows coordinated transcription of different sets of genes,
regulons
Chapter 11- Regulation of Gene Expression in Eukaryotes
11.1- Overview of Transcriptional Regulation in Eukaryotes
-basic mechanism at work involves molecular signals inside or outside the cell lead to
binding of regulatory proteins to DNA
-Eukaryotic DNA is packaged into nucleosomes (DNA wrapped in Histones)
*ground state of prokaryotes is on, in contrast to Eukaryotic where transcription can only
occur if regulatory proteins are present
11.2 Lessons from Yeast: The Gal System
7-10-1-2- in between each set of genes is UAS (upstream Activator Sequence)
7,10 are transcribed to the left, while 1 and 2 are to the right
-GAL1,2,7,10 genes encode enzymes that help breakdown galactose into glucose
-GAL4 is the key regulator protein

Gal4 Protein has separable DNA binding domain and Activator Domain
-Found this through experiment in which they were able to bind the binding domain to
the Gal4 site without the activator domain and transcription was turned off. They made
this site upstream from LacZ gene which if transcribed would bind to a certain substrate
and produce a fluorescent color. Also showed that the Gal4 activator domain could bind
to LexA site and have the same effect.
*shows that activator domain helps recruit transcription machinery to the promoter
GAL4 is physiologically regulated
-When galactose is low in the cell Gal80 protein binds to activator domain of Gal4 and
inhibits transcription
-When galactose is high in the cell Gal3 occupies Gal80 and keeps it from binding to
Gal4 thus allowing transcription to occur.
-Gal4 activator domain also can directly interact with TBP which is bound to TFIID.
This brings RNA Pol closer and promotes transcription. Gal4 also interacts with
Mediator complex which in turn interacts with RNA Pol II, thus further enhancing
likelihood of transcription.
-Since Mediator Complex neither binds to protein nor is part of transcription machinery it
is a Coactivator
11.3 Dynamic Chromatin and Eukaryotic Gene Regulation
Each histone contains 150bp of DNA wrapped twice around it
Chromatin Remodeling and Gene Activation
-Structure of Nucleosome: Octamer of two of each: 2A,2B,3,4. Each of the pieces of
the histone has its N-terminal end sticking out from the nucleosome. Histone Tails
-Lysines at specific positions on the tails can be molecularly modified to signal for
remodeling
-Acetylation of lysines signals the histones for remodeling and gene expression
-Histone Actetyltransferases are the enzyme that are responsible for this
-ex) GCN5- binds to regulatory sequences on DNA and acetylates histone tails
-Histone Deacetylases remove acetyl groups
-Tup1- deacteylase that is recruited by repressor Mig1 so it is a corepressor
11.4- Enhancer Action
-Transcriptional levels are finely adjustable due to the clustering of binding sites into
enhancers
-the binding of many regulatory proteins to many binding sites in enhancer leads to
formation of enhanceosome, which is a large protein complex that acts
synergistically(greater than additive) to activate transcription.
B-Interferon Enhanceosome
-antiviral gene
1) transcription factors bind to same face of DNA 100bp upstream from TATA box to
form enhanceosome

2) They Recruit GCNG which acetylates Nucleosomes, and then leaves

3) This recruits CBP, a coactivator, which also recruits transcriptional machinery. CBP
adds more acetyltransferase activity
4) RNA Pol II holoenzyme is recruited, and so is SWI-SNF chromatin remodeling
complex which moves aside nucleosome to expose TATA box
5) TBP binds and allows start of transcription
Control of Yeast Mating type
-yeast is haploid and there are two types a and alpha. They mate with eachother and
each cell type is determined by the MAT locus
MATa locus
-encodes for a1 regulatory protein which only has effect in diploid cells. In haploid cells
MCM1 regulates expression of structural genes
MATalpha locus
-encodes for alpha1 acts in accordance with MCM1 to express alpha specific genes.
-alpha2 represses transcription of a-specific genes
Diploid Yeast
-a1 protein finally has part to play. It binds with alpha2 and binds upstream of haploid
specific genes to regulate them.
Enhancer Blocking insulators
-Because enhancers are sometimes thousands of bps away from the genes which they
regulate it could cause the transcription of more nearby genes. Thus insulators have
evolved to stop such promiscuous activity. When positioned between an enhancer and
promoter it blocks transcription
11.5- Genomic Imprinting
-When only one copy of the gene is expressed even when there are two alleles present,
thus creating monoallelic inheritance
-Paternal imprinting: when only the allele from the mother is expressed (H19)
-Maternal Imprinting: When only the allele from the father is expressed (igf2)
The other gene is turned off by methylation of that site
-Because the actually DNA sequence is not changed this is an example of epigenetic
inheritance
-most imprinting genes are located in clusters as is the H19 and the igf2. Between these
two genes is the Imprinting control Region (ICR)
-in the paternal allele these site is methylated and doesnt allow binding of
regulatory protein CTCF. This means the igf2 gene is transcribed and the H19 is not
-in the maternal allele ICR is not methylated and CTCF can bind and transcribe
H19 but not igf2
11.6- Chromatin Domains and Their Inheritance
YEAST: switching mating types

-There are 3 locations on yeast chromosome with mating type genes

-MAT: can be either a or alpha
-HML is always alpha
-HMR is always a
1) HO endonuclease recognizes sequence adjacent to MAT locus and creates double
strand break
2) 5-3 resection occurs deleting the MAT gene on one of the strands
3) if a type was original at MAT then invasion will occur with HML. If alpha was original
at MAT then invasion will occur at HMR
PEV
-Position effect Variegation- when certain genes are rearranged to become closer to the
heterochromatic region and are thus silenced is some cells and consequently in the
products of their cell division. This for example led to patches of red and white on fly
eyes
Genes necessary for heterochromatin formation
Su(Var) genes: when mutated these genes reduce the spread of heterochromatin
-ex) Heterochromatin 1 gene (HP-1)
Ex) Histone Methyl Transferase (HMTase)
Barrier Insulators: stop spreading of heterochromatin by binding Histone
Acetyltransferases
Dosage Compensation
1) X-inactivation: When one of the female sex X chromosomes has its histones
methylated and also its DNA
2) Hypertranscription: happens in drosophilia when MSL complex binds along
entire length of males X chromosome and it has acetyltransferase activity
3) Hypotranscription: When both X chromosomes on the female are semi methylated
Chapter 12: Genetic Control of Development
12.1-The Genetic Approach to Development
Drosophila- very fast life cycle, fast development (7 days), and cytogenetics (study of
function of cell) and its previous genetic analysis (morgan)
-abnormality in body plan of a mutant is identifiable even in larvae stage
12.2- Genetic Toolkit for Drosophila Development
Housekeeping genes: Genes that encode proteins that function in essential processes in
all cells of the body
Toolkit Genes: Genes that are concerned with building and development of organs and
tissues
Homeotic Genes and Segmental Identity
Homeotic Genes: When one body part is replaced by another normal body part

-Can cause loss of homeotic gene function where it normally acts

-can also cause homeotic gene function where it normally doesnt act
Hox genes: genes that affect identity of segments and their appropriate appendages
-Located in two Gene Complexes
1) Bithorax complex- contains 3 genes
2) Antennapedia complex- contains 5 genes
-Able to study hox genes and their functions by being able to make cDNA clones of the
mRNA transcripts (in situ hybridization) and the protein they encode and using visual
technology to monitor them during embryo development after injecting them into fixed
embryos
*In absence of Hox genes, segments will form but will all have the same identity
Homeobox
-the cluster of hox genes into complexes suggests that they are due to tandem duplication
of an ancestral gene. Proven by fact that they all have similar sequences and hybridize to
eachother. This region of similarity is dubbed Homeobox. This box encodes a protein
domain of 60 Amino acids and is known as the Homeodomain
*-Because the Hox proteins have a Helix-turn Helix motif similar to lac repressor and
Cro proteins it was found that they were DNA binding proteins
-Thus Hox genes bind to regulatory regions of other genes to activate or repress their
expression
*Homeodomains are astoundingly similar between species suggesting that they play a
fundamental role in development of most animals
12.3- Defining the Entire Toolkit
-other toolkit genes are important for the correct development of larvae and embryos
-they are also genes that code for transcription factors and thus have effect on succeeding
set of genes. Can also affect gene regulation indirectly as signaling pathway proteins.
-maternal required genes-genes with products provided by the female to the egg. Only
needs one mutant type from the female to show expression of phenotype in offspring.
*-mutant offspring arise form a homozygous mutant mother
Embryonically active
1) Maternal Required Genes- Bicoid: effect embryo development
Zygotically acive
2) Gap genes: affect formation of continuous blocks of segments
3) Pair-Rule genes: act as double segment periodicity. Mutants miss part of each
pair
4) Segment Polarity genes: Affect patterning within each segment
4b) Hox genes: do not affect number but appearance
5) Genes for segment specific features

12.4-Spatial Regulation of Gene Expression and Development

-requires much of the same transcription machinery as physiological control but just more
of them.
Example)
*Combinations of maternal-effect and gap proteins control individual pair-rule stripe
formation. Thus the eve stripe 2 element contains multiple sites for maternal bicoid
protein and the hunchback, giant, and kruppel gap proteins.
-bicoid and hunchback activate expression over wide range, while kruppel and
giant repress expression to just a few cells wide.
Making Segments Different
Hox proteins from Ubx gene and abdominal-a gene
Ubx is expressed in abdominal segments 1-7 and abdominal-a in 2-7
-these proteins bind to distal-less gene to repress expression in the abdomen and promote
expression in the thorax. They do this in collaboration with 2 segment-polarity genes
known as Slp and En, and two other genes, extradenticle and homothorax
12.5-Post Transcriptional Regulation of Gene Expression in Development
-Transcriptional regulation is not only means of restricting expression of gene products.
Alternative Splicing is another mechanism
-Regulatory sequences in RNA are recognized by splicing factors, mRNA binding
proteins, and miRNAs.
RNA splicing and Sex determinism in Drosophila
Dsx (double sex gene) when mutated causes males and females to develop ambiguously
Dsx^m contains a c terminus of 150 amino acids not found in Dsx^f. the Male version
represses certain genes in males that the female version activates.
-These male and female versions are examples of alternative splicing of the same original
transcript by tra gene alternative splicing factor
Regulation of mRNA translation and cell lineages in C. Elegans
-simple construction, rapid life cycle and transparency has made it a valuable model.
-In blastomere stage mRNA of toolkit genes is present in all cells, but translation of these
mRNAs differ in each cell.
glp-1 mRNA is only translated in ABa and ABp anterior cells
-the posterior cells have GLD-1 protein which binds to the SCR (spatially controlled
region) of the glp-1 mRNA to repress expression of it
miRNA control of developmental timing in C. elegans and other species
-Development is temporally ordered as well as spatially
Mutations of heterochronic genes alter the timing of events
-the regulatory molecules that arise from these genes (lin-4, let-7) are actually not
proteins but RNA products. These RNA products are complimentary to 3 untranslated
regions of developmentally regulated genes such as lin-41. Mutations in this gene cause
precocious development from larvae into adult C. elegans

12.6- Many Roles of Individual Toolkit Genes

Shh gene is expressed in ZPA (zone of polarizing activity) region. Mutations in this gene
caused some mice to grow up cyclopic.
12.7- Development and Disease
Polydactyly- development of extra digits on hand or feet due to mutation in the Shh
gene.
These mutations are not in coding region rather in the regulatory sequences upstream
from the coding region.
1) Because they are mutations in cis-acting regulatory elements the phenotypes are
dominant
2) Also other gene functions are likely to be completely normal
Holoprosencephaly- mutations in coding region of Shh gene. Since Shh is a ligand, this
disease is not exclusively caused by mutation to Shh gene.
Chapter 13- Genome and Genomics
13.1 The Genomics Revolution
The construction of genomes of species can help aid in identifying disease causing genes
-need PCR and bacterial plasmids to clone the genes of interest
The Shotgun Method:
Logic is sequence first, map later
1) Cut genome into random fragments
2) Make library of cloned fragments
3) sequence reads of clones by using primers
4) map the sequence reads by finding homologous overlapping segments from
different clones to form contigs
5) Use paired end reads. If one read is from a certain contig and the other is from a
second contig then this insert spans the gap and forms a scaffold
Ordered Clone Method
Logic is map first, sequence later
1) Use restriction enzymes and order large insert clones by overlap fingerprints to
create a physical map
2) Select clones with minimal overlap
3) Divide into subclones by further treating with restriction endonucleases
4) Sequence subclones
5) Assemble subclones to create genome sequence
13.3 Bioinformatics: Meaning from Genome Sequencing
-study of information hidden in the genome
Information includes:

1) Sequences that encode proteins

2) Sequences that encode RNA
3) Sequences that are binding sites that govern time and place of their actions
How to deduce Proteome
1) ORFs- there are 3 possible reading frames on each strand so there are 6 possible
reading frames all together. Computer scans area where DNA would produce a
mRNA where there is 5 start codon and 3 stop codon.
2) cDNA- create cDNA clones that are complimentary to mRNA. These cDNA will
only be made of exons and they can anneal to genomic DNA and will show only
where exons are and from this you can deduce where the introns are
3) ESTs: Expressed Sequence Tags are cDNAs that are only made of the 5 and 3
ends of transcripts and they can thus be used to find where the boundaries of
transcripts are
4) BLAST: BLASTn are commonly known sequences that code for certain proteins
and computers use this knowledge to look for areas in genomic DNA where there
is similarity to previously known sets of Data. BLASTp is the same thing but is
amino acid sequence instead.
5) Codon Bias
13.4-Human Structure of the Genome
-Transposable elements and repetitive sequences make up 45% of genomic DNA
-only 3% of DNA are exons and only half of those encode for polypeptides because
introns are much larger than exons
-3 splice variants per gene on average thus there are 3 fold greater proteins than genes
Pseudogenes: genes that have acquired mutations and activity that make them inactive.
13.5 Comparative Genomics
-Genes are conserved over long periods of time because mutations to genes that decrease
fitness are selected out
Homologs- closely related genes
-orthologs- homologs at same genetic locus inherited from common ancestor
-paralogs- homologs that are at different sites due to gene duplication
Synteny- where the order of genes within variously sized blocks is the same between two
species
Comparative genomics with Chimpanzee
-35 million single nucleotide differences (1.06%)
-5 million insertions or deletions from 1b-15kb (3% difference) lie outside coding region
-29% of orthologous proteins are completely identical
-80 genes present in ancestor have lost function in humans
-170 duplicated genes in humans and 90 in chimps
13.6 Functional Genomics and Reverse Genetics
Transcriptome- sequence and expression pattern of all transcripts

Proteome- sequence and expression pattern of all proteins

Interactome- interactions between proteins and RNA, proteins and DNA, and between
proteins
Microarrays to study Transcriptome
-microarrays are chips of several DNA sequences. It is exposed to a sample of labeled
RNA from the cell. RNA complimentary to certain DNA sequences will hybridize and
indicate which genes are actively transcribed under given conditions
Two-Hybrid test to study interactome
1) Place Gal-4 binding domain gene next to DNA of protein of interest into one yeast
vector (bait)
2) Place Gal-4 activation domain next to another DNA of protein of interest into
another yeast vector (target)
3) Finally look for activation of transcription of Gal-4 regulated reporter gene (lacZ)
Chromatin Immunoprecipitation Assay to study interactome
1) Cross link proteins to DNA
2) Break chromatin into small pieces
3) Add antibody to target protein and purify
4) Reverse cross links to separate protein and DNA
Reverse Genetics
-start with observed normal DNA, mRNA, or protein and induce changes into it and see
effects that it has
Phenocopying
1) RNA interference: naturally protects cells from foreign DNA. dsRNA is made
(introduce reverse repeat into genome) with sequences homologous to gene of
interest. The RISC then degrades any mRNA that is complimentary to the dsRNA
2) Chemical Genetics:
A) Using Forward Genetics: add one compound per well to yeast colonies
-find compound that produces phenotype of interest
-Identify protein target of compound
B) Using Reverse Genetics: Find Protein of interst
- screen for compounds that bind to protein
- treat cells with molecule that binds to protein
- assay for phenotype

Chapter 14- Transposable Elements

-can insert into genes and inactivate them
-account for 50% of our genomic material

14.1 Discovery of Transposable Elements

-Barbara McClintock discovered that in Maize chromosome 9 would very frequently
break at a specific locus. Required 2 Factors:
1) Ds for disassociation was located at the site of the break
2) Ac for activation was needed to activate the break but was located at a different
site. Also activates the transposition of Ds.
-postulated that they were mobile elements when she found that Ac of different plants
were located to different locations
Unstable phenotype- resulted from movement of Ds away from the C gene
Autonomous Elements- elements like Ac that do not require other elements for their
mobility
Nonautonomous elements- elements like Ds that do require other elements for their
mobility
14.2 Transposable Elements in Prokaryotes
IS elements (insertion sequence elements)
- All IS elements encode protein transposase
- All begin and end with short inverted repeats
Transposons
1) Composite: contain variety of genes that reside between two identical IS elements
that are oriented in opposite directions. The IS elements cannot transpose on their
own without the genes inbetween because of a mutation in their inverted repeats
2) Simple: flanked by IR sequences that do not code for transposase. Instead
transposase gene is included in the group of genes that are flanked by the IR.
Mechanism of Transposition:
1) Replicative: when a new copy of the transposable element is generated by
transposition event. This events produces a cointegrate which is the fusion
together of 2 plasmids before they get separated. This allows the replication of the
Transposable element
2) Conservative: Cut and paste method, where the transposable element is removed
from one place in the genome and placed in a new position.
14.3 Transposable Elements in Eukaryotes
Class 1: Retrotransposons
-Studying HIS4 mutants in yeast. Found that 2 of the mutants of HIS4 were unstable
phenotypes that were more than 1000x likely to revert back to wild-type than were the
other mutants. Found that the cause was a large insertion into the HIS4 gene. It did not
look like IS elements however instead looked like animal retrovirus.
Provirus: double stranded DNA copy of retroviral genome
Contains:
1) Gag gene: involved in maturation of viral genome
2) Pol gene: encodes the reverse transcriptase gene
3) Env gene: encodes structural protein that surrounds viral Genome

4) Flanked by LTRs or long terminal repeats

Similar to Ty elements but Ty elements dont have Env gene so they cannot leave the
cell, rather they just insert into different parts of the genome within the same cell.
Ty elements transpose through RNA intermediate:
Placed galactose inducible promoter near Ty element and also inserted an intron into its
coding region. Found that the new site where the Ty element was inserted lacked the
intron meaning that it had been spliced from the pre-mRNA transcript of the original Ty
element.
Ex of class I element: Copia like elements of Drosophila
Class 2: DNA transposons
-similar to mechanisms of prokaryotic transposition
P elements in Drosophila
Contains transposase gene with 4 exons and 3 introns flanked by short IRs.
Female M x Male P causes hybrid dysgenesis
Reciprocal cross produces viable offspring
-this happens because the female cytoplasm (mitochondria) contains a repressor that
represses transcription of the transposase of the p element.
Using P Elements to insert Genes
Insert a gene into P element that has had transposase gene deleted but still has the IR
sequences that bind transposase. Add this plasmid along with a normal P element
plasmid into multinucleated embryo. The developing fly will not show the phenotype
you are looking for but its offspring will because the P-elements mobilize only in germ
cells.
14.4 The Dynamic Genome: More Transposable Elements than imagined
C-Value Paradox- when the size of the genome does not correspond to the complexity of
the organism
Transposable Elements in the Human Genome
1) LINEs (autonomous)- Long interspersed Elements: similar to retrotransposons
because they encode reverse transcriptase but do not include flanking LTRs
2) SINEs (nonautonomous)- short interspersed elements that do not include gene for
reverse transcriptase. Instead are mobile because of reverse transcriptase of
LINEs elsewhere in the genome. Ex) alu
3) DNA transposons
Insert into Introns and Exons
-those inserted into introns have no negative effect because they are spliced out of the
mRNA product
-those inserted into exons are subject to negative selection

Chapter 15: Mutation Repair and Recombination

Mutation and Recombination are two major processes that are responsible for genetic
variation.
15.1- Phenotypic Consequences of DNA Mutations
Point Mutations: mutation to a single base pair or small number of adjacent base pairs
1) Base Substitutions
A) Transition: replacement of base by another base of the same
category
B) Transversion: replacement of base by another base of a
different category
2) Insertion/ deletion
Synonymous: mutation changes one codon for an amino acid into another codon that
codes for the same amino acid (silent)
Nonsynonymous: condon for one amino acid is changed into codon for another amino
acid (missense)
Nonsense: condon for an amino acid is changed into a stop codon
Conservative: mutation that encodes for a different amino acid of similar chemical
structure
Nonconservative: mutation that encodes for a different amino acid not of a similar
chemical structure
15.2 Molecular Basis of Spontaneous Mutations
Fluctuation Test
Used to see if mutations to make bacteria resistant to phage attack were induced by the
phage or were spontaneous
-if spontaneous than each culture should show various number of colonies that are
resistant
Used the idea of replica-plating
Mechanism of Spontaneous Mutation
1) Errors in DNA Replication
Transitions: can occur because tautomers of nitrogenous bases can occur. Normally they
are present in the cell as ketones
Cytosine and Adenine can tautomerize into iminos (2 H bonds)
Guanine and thymine can tautomerize into enols (3 H Bonds)
Transversions: occur when purine-purine pairs form or pyrimidine-pyrimidine pairs
form
Framshifts-caused by slip mispairing during DNA synthesis
2) Spontaneous Lesions
Depurination- the loss of a purine base due to the interruption of the glycosidic linkage
between the base and the deoxyribose

Deamination causes cytosine to become uracil and thus a transition to occur

-can also cause 5-methylcytosine to become thymine which is hard to correct
Tri Nucleotide Repeat Diseases
Ex) Fragile X syndrome: normal CGG repeats in the FMR-1 gene contain 6-54 repeats
but diseased contain hundreds of repeat caused by slipped mispairing
15.3 The Molecular Basis of Induced Mutations
1) incorporation of base analogs
-5-Bromo Uracil, which is analog of thymine, when it changes to ionized form pairs with
guanine
-2-amino purine, which is analog of adenine still pairs with thymine, but when it is
protonated it will pair with cystine
2)Specific Mispairing- altering a base so that it forms a specific mispair
-EMS and NG add alkyl groups to many positions on all 4 bases
-most commonly it adds ethyl to Guanine forming, O-6-Ethylguanine which pairs with
thymine instead of cytosine
-also adds ethyl to thymine forming O-4-ethylthymine which pairs with guanine
3)intercalating agents
-Proflavin, Acridine Orange, ICR compounds, are planar molecules that can slip between
stacked nitrogen bases causing insertion or deletion of a single nucleotide pair.
4)Base Damage
A) UV light causes adjacent pyrimidines to form a cyclobutyl ring, making specific base
pairing impossible and thus blocks replication at this point
B)Ionizing radiation can cause water molecules to form radicals, or hydroxide ions, or
hydrogen peroxide, which can lead to degredation products of bases causing mutations
-can also directly cause breakage of N-glycosidic linkage sites and cause apurinic or
apyrimidinic sites
5)Bulky addition products
-such as aflatoxin, bind covalently to DNA leads to breakage of bond between sugar and
base
AMES Test
-Ames found that not all carcinogens directly cause mutations, rather that their metabolite
products directly caused mutations.
1) broke down rat liver to extract enzymes.
2) added them to S. typhimurium strains that needed histidine to grow. Plated them on
plates lacking histidine.
3) added enzymes and potential carcinogens to the plate.
-if the bacterium would form colonies knew that mutations were formed causing
revertants and thus because mutagenicity and carcinogenicity are correlated could
postulate that certain compounds were likely carcinogens

15.4 Biological Repair Mechanism

Error Free Repair:
Direct Reversal of Damaged DNA:
-CPD Photolyase, with the help of UV light, can split a cyclobutyl photodimer back into
its original bases
-alkyltransferases can remove certain alkyl groups that have been added to bases by DMS
and NG. it does this by transferring the methyl group to the cysteine residue in its active
site.
Base Excision Repair: targets non-bulky damage to bases
Methylation, deamination, oxidation, loss of DNA base
1) Enzyme Uracil Glycosylase cleaves at glycosidic bond btw base and sugar
2) AP Endonuclease comes and cleaves abasic site on both ends of backbone
3) Deoxyribophosphodiesterase then removes a part of the backbone of the DNA
4) DNA Pol acts on 3OH side and fills in the gap and ligase seals the nick
Nucleotide Excision Repair- corrects bulky adducts that distort the DNA helix
A) Global Genomic Repair- activated by stalled replication forks
1) Recognition complex recognizes photodimer
2) Attracts TFIIH
3) XPD, XPB, subunits of TFIIH are helicases that unwind DNA
4) RPA, single stranded binding protein, stabilizes the structure
5) The DNA is excised 15-24 bases upstream and 2 bases downstream
6) Bypass polymerase fills in the gap with help from PCNA, ligase seals the nick
B) TCNER- activated by stalled transcription
1) CSA, CSB recognize stalled polymerase
-all other steps are the same TFIIH comes in, CSA, CSB and RNA pol leave
Mismatch Repair- Post Replication Repair
-error rate in replication is 10^-5. Proofreading reduces this to 10^-7, and mismatch
repair further reduces it to 10^-9
A) MutS scans hemimethylated DNA and detects a bulge in structure
B)MutL is recruited which in turn activates MutH endonuclease which cuts a nick in the
strand that contains an adenine methylation site
C) UvrD binds at the nick and unwinds using helicase activity
D) single strand binding proteins bind to stabilize
E) An exonuclease then comes in and removes the strand containing the mismatch
F) DNA Pol III fills in the gap and ligase seals it
Error Prone Repair
DNA Translesion Synthesis
1) DNA Pol is synthesizing new strand then comes across a thymine dimer on
template
2) It cant synthesize past this so a Rad6 adds Ubiquitin to PCNA causing its
conformation to change

3) Bypass DNA Pol comes in and clamps to PCNA. through evolution has learned
to just put down As
4) After putting on just two bases it leaves and DNA Pol comes back to finish
because Rad 18 removes ubiquitin
5) The thymine dimer that was on the template strand is then fixed by nucleotide
excision repair
Repair of Double Strand Breaks
Non Homologous End Joining: NHEJ
-predominant form of repair of dsDNA breaks in multicellular organisms
-occurs if replication has not occurred. It can happen because there are many repetitive
non expressive sequences in a multicellular organisms genome
1) Ku70/80 recognizes ds breaks and binds the broken ends
2) Proteins are recruited that trim the strands back
3) DNA ligase IV, XRCC4 comes in and connects the two strands
Homologous Recombination- Synthesis-dependent strand annealing (SDSA)
Similar to homologous recombination during meiosis. However this repair mechanism
occurs between sister chromatids and is error free. Dmc1 is not involved in this process
-replicated by conservative mechanism creating single strands
1) Enzymes trim back 5 ends
2) resulting 3 overhangs are coated with Rad 51
3) Rad 51 DNA filament then begins search for sister chromatids to fill in the gaps
15.5 Mechanism of Homologous Recombination
-recombination has to occur at meiosis I or else the homologs will not align properly
leading to progeny cells getting either too many or too few chromosomes. Dont develop
properly. Called NONDISJUNCTION
1) Spo11 contains a tyrosine group which attacks the DNA backbone and covalently
bonds to the 5 Phosphate. Unlike topoisomerase it doesnt RELIGATE
2) MRX complex (analogous to RecBCD) processes the break and forms 3 overhangs
3) Rad51 and Dmc1 (homologs to RecA) coat 3 overhangs and promote strand exchange
btw sister chromatids. Rad51 involved in meiosis and mitosis while Dmc1 is only
involved in meiosis
2 important factors
1) There are more fewer chiasmata formed than there are breaks by spo11
2) Meiotic crossover has several different genetic outcomes
15.6- Cancer: an important Phenotypic Consequence of Mutation
Mutations in Cancer Cells
1) Mutations in onco genes: gain of function dominant mutations. Mutation only
needs to be present in one allele. Normal gene is a proto-onco gene
2) Mutations in tumor suppressor genes: loss of function recessive mutations.
Causes encoded gene to lose its function

Ras Onco Protein: Single base pair substitution converts glycine into valine at AA
number 12. Normal Ras protein cycles between active GTP bound state and inactive
GDP bound state. The missense mutation produces an oncoprotein that always binds
GTP, that constantly promotes cell proliferation
P53 tumor suppressor gene: Undamaged gene prevents the cell cycle until the DNA is
repaired, and it also induces apoptosis.
Chapter 16- Large Scale Chromosomal Changes
1) Changes in chromosome number
2) Changes in chromosome structure
16.1 Changes in Chromosome Number
Abberant Euploidy- the changes in whole chromosome sets
Euploidy- is multiples of the basic set of chromosomes
Monoploid- when a normally diploid organism only has one set of chromosomes
(different from haploid)
-includes male bees,wasps
Polyploids
-Autopolyploids: multiple chromosome sets originating from same species
-autotetraploids can be induced by treating diploids with colchicine
-allopolyploids: sets from different species. Homeologous because they are partly
homologous
-can be induced by breeding 2 different diploid plant species to produce an
amphidiploid. The sterile F1 is then selfed to produce the amphidiploids
Agricultural Applications
1) Monoploids can be derived from anthers of plants. Useful if a desired trait is
normally a recessive allele in a diploid. Process involves cold treatment of a cell
destined to become a pollen and it grows into an embryoid
2) Autotetraploids are made because they have increased size
Aneuploidy-changes in parts of chromosome sets
Trisomic- 2n+1
Monosomic-2n-1
Nullisomic-2n-2
Disomic-n+1 (for haploids)
-cause of aneuploidy is nondisjunction, which is abnormal segregation
-normal disjunction is thought to be related to the presence of crossovers (chiasma)
Turner Syndrome: Monosomic for the X chromosome, represented as XO
-sterile females
Klinefelter Syndrome: XXY trisomic males who have low IQ and are sterile
Down Syndrome: Trisomy at chromosome 21

-females are more likely to be fertile than males. Incidence is related to maternal age
-Pautau syndrome (13) and Edwards Syndrome (18) also occur
XXX: trisomy which results in phenotypically normal, fertile females
Gene Balance
-Abberant Euploidy leads to increase in size of organism but proportions and shape
remains the same
-Abberant Aneuploidy tends to lead to malformation in size and shape
-normal physiology depends on the proper ratio of gene products and aneuploidy in
humans can result in haplo-abnormal or triplo-abnormal
*the reason monosomy is more severe than trisomy is because any deleterious recessive
alleles present on a monosomic autosome are automatically expressed
16.2 Changes in Chromosome Structure
Unbalanced:
1) lost chromosome segment
2) duplicated chromosome segment
Balanced:
3) inversion of segment
4) translocation of segment
-can either occur by:
A) chromosome breakage
B) crossover between repetitive DNA regions (Non-allelic homologous Recombination)
-Chromosome rearrangements have to result in chromosomes with a centromere and 2
telomers
-chromosomes without centromeres (acentric) will not be dragged to either pole at
anaphase
-chromosomes lacking a telomere will not replicate properly
Deletions:
Intragenic deletions: small in size and are equivalent to null mutations. Never revert back
to wild-type
Multigenic Deletions also occur and are more severe
Pseudodominance when recessive alleles show dominance because their counterpart
allele on the other homolog has been deleted
-ex) Williams syndrome, Cri du Chat syndrome
-animal sperm are functional with deletions, while pollen normally are not
Duplications
-tandem duplication- when the region is inserted next to itself
-insertional duplication- when region is inserted elsewhere in the genome
Inversions
Paracentric: centromere is outside the inversion

-results in formation of dicentric bridge and produces an acentric fragment

Pericentric: centromere is within the inversion
-does not result in creation of a bridge but results in same products
Reciprocal Translocations
When two segments trade trade acentric fragments
-create two types of segregations
1) Adjacent 1: (T1+N2) and (N1+T2)
2) Alternate: (N1+N2) and (T1+T2) both complete and viable
-these two are equal in number and thus half the gametes are viable and half are not
(Semi-Sterility)
Robertsonian Translocation
-when the translocation produces an almost complete extra copy of the chromosome
(Down Syndrome)
Chapter 17- Population Genetics
17.1 Variation and its Modulation
Allele Frequency
p+q= fA/A+ fa/a +fA/a=1
q=1-p
Protein Polymorphisms
Immunological
-there are n(n-1)/2 different ways of combining n different things two at a time
Amino Acid Sequence
-use gel electrophoresis to plate differences in protein structure
DNA Structure and Sequence Polymorphism
Chromosomal
-extra chromosomes, inversions, reciprocal translocations are observed in many
populations
Restriction site Variation
-causes Restriction Fragment Length Polymorphisms (RFLPs)
Tandem Repeats
-A person or population could be polymorphic for Variable Number Tandem Repeats
(VNTRs)
Complete Sequence Variation
-variation in single nucleotide (single nucleotide Polymorphisms)
1) -sequence of coding regions can be translated to reveal exact amino acid sequence
differences
-this is superior to electrophoresis studies of proteins because electrophoresis
studies can only show how many or which AAs are different but not what the difference
is.

2) sequence variation is studied in sequences that dont change protein sequence, such as
regulatory sequences, introns etc. this is important because it is believed that most
evolutionary variation is due to variations in these sequences
-variation in intron sequence (silent mutations) are much more common because
variations in amino acid sequence are commonly selected out
17.2- Effect of Sexual Reproduction on Variation
Hardy-Weinberg Equilibrium:
p^2+2pq+q^2=1
Heterozygosity
-can be used as measure of genetic variation
Haplotype-combination of alleles of different genes on the same chromosomal homolog
Inbreeding and Assortative Mating
Inbreeding- when mating btw relatives is more common than by chance
Enforced outbreeding- when mating between relatives is less common than would occur
by chance
Positive assortative mating- mating of like with like
Negative Assortative mating- mating with unlike partners
17.3 Sources of Variation
Three sources of Variation
1) mutation
2) Recombination
3) Immigration of genes
4) Genetic Drift
-Increase in mutant frequency= nonmutant frequency x mutation rate
Variation from migration
M=deltap/P-po P=allele frequency of donor

po=original freq of recipients

17.4 Selection
Darwinian Fitness: Probability of survival and rate of reproduction of a phenotype or
genotype
Two types of selection
Frequency independent: the fitness of a genotype does not depend on the how rare or
frequent it is in the population
Frequency dependent: fitness of a type changes as it becomes more or less frequent
Viability: probability of survival

17.5-Balanced Polymorphism
overdominance: when a heterozygote is more fit than either homozygote
-selection favors an allele when it is rare
underdominance: when a heterozygote is less fit than either homozygote
-selection favors an allele when it is common, not rare
Genetic Drift: Random change in allele frequencies
Founder effect: form of genetic drift when a single generation of sampling of a small
number of colonizers from the original large population

Chapter 19-Evolutionary Genetics

-mechanism of evolution starts with the variation that exists between organisms of a
specific specie. Thus there is a different rate of survival between individuals
19.1- Darwinian Revolution
1) principle of variation- variation in morphology, physiology, behavior
2) principle of heredity- offspring resemble parents more than other individuals
3) Principle of selection- some variants are more successful at surviving than others
Explains two things:
Phyletic evolution- successive change of form and function of single continuous line
Diversification- there have existed many different contemporaneous species having
different forms and living in different ways
-these are the consequences of heritable evolution
19.2- A Synthesis of Forces: Variation and Divergence of Populations
Example of the finches on the Galapagos islands is explained by diversity and
adaptation
Variation within populations: caused by mutation, migration, balancing
Variation between populations: caused by inbreeding, genetic drift, incompatibility,
directional selection (basically things that cause homozygosity)
Population will retain variation in alleles and not differentiate from local inbreeding if:
m>1/N or u>1/N
19.3-Multiple Adaptive Peaks
-arise if intermediate genotypes have higher fitness (Ab/Ab, aB/aB, AB/ab)
-not every difference is an adaptive difference (African Rhinos have 2 horns, Indian have
1)
Exploration of Adaptive Peaks
Random drifts cause a population under selection to ascend an adaptive peak erratically.
-random drift can enhance selection and also counteract it.

-there are always unfavorable alleles at certain loci that are fixed because selection
intensity is insufficient to overcome random drift to fixation.
-when there are 5 different changes and they can happen in any order there are:
5x4x3x2x1= 120 possible orders.
Sign epistasis: when fitness advantage/disadvantage depends on the mutations previously
fixed
19.4-Genetic Variation
Heritability of Variation
-in general behavioral traits have lower heritabilitys than morphological traits
Canalized Characters: when there is substantial genetic variation but very little
morphological variation
-genetic differences are revealed through phenotypes when you place the variants
in stressful environments
Variation within and between populations
-in general different human populations show similar allele frequencies for polymorphic
genes
19.5 Mutation and Molecular Evolution
1/3 of all protein encoding loci are polymorphic
-this DNA variation has three effects:
1) they may be deleterious, reducing probability of survival
2) they may increase fitness by increasing efficieny
3) they may have no effect on fitness (neutral mutations)
-include mutations that have no effect, and those that have a fitness selection that
is lower than the reciprocal of population size
Purifying Selection on DNA
-if you plot number of nucleotide differences btw 2 species that diverged from common
ancestor then against time since divergence then their slopes should be mu or mutation
rate.
-we see that synonymous substitutions have much higher mutation rate than do nonsynonymous substitutions
-is expected because non-synonymous have deleterious effect and are selected
out(purifying selection)
19.6- Relating Genetic to Functional Change: Protein Evolution
Signature of Positive Selection on DNA sequences
-comparing synonomous and non-syn mutations within a species against synonomous and
non-syn mutations between species can detect adaptive evolution of proteins
-if species differences exceeds the number of polymorphisms than there has been
adaptive selection
Morphological Evolution

Melanism is an example
-eumelanin forms black and brown pigments
phaeomelanin forms yellow or red
-amounts of these two types of pigments are controlled by two genes MC1R and agouti
protein
-Agouti is an antagonist and binds to MC1R to inhibit production of eumelanin
-dark mice have mutations in MC1R region that makes it constitutively active bypassing
regulation by agouti protein
Gene Inactivation
Example: different cave populations of fish separately evolved to lose pigmentation and
eyes in underground caves.
-found to have mutations in the Oca2 gene.
What might account for repeated inactivation of Oca2 gene?
1) Mutations appear to cause no serious defects compared to other pigmentation
genes
2) Oca2 region is really large and is more susceptible to mutation
19.7- Regulatory Evolution
There are many animals that have special coloring patterns. This means that
pigmentation-gene regulation must evolve by some mechanism that does not disrupt
pigmentation-protein function
-Mutations in regulatory sequences provide a mechanism for changing one aspect of gene
expression while preserving the role of pleiotropic proteins in other developmental
processes.
19.8-Origin of New Genes
-Old DNA from old genes must be preserved while new genes with new functions are
evolving. Where do new genes come from?
1) Polyploidy- frequent polyploidy
2) Duplications: has three consequences
a. Polypeptide may simply increase in production
b. General function of sequence is maintained
c. New segment may diverge dramatically and take on new function
3) Imported DNA-when genes from totally unrelated species are incorporated into
host genome
a. Cellular organelles have been obtained in this way (mitochondria,
chloroplasts. Evidence for their extracellular origin is based on
experiments that show that the DNA-RNA code is different in
mitochondria than it is for nuclear genes.
b. Horizontal Transfer- Within a genome DNA can be transferred by
transposable elements. Can be transferred between genes by retroviruses

19.9- Genetic evidence of common Ancestry in Evolution

-we can observe common evolutionary origin through:
a) anatomical differences
b) genome and protein differences
-If there was no selection divergence within genes would result in loss of similarity and
inability to decipher if the two organisms are related. However mutation rates are
generally not high enough to cause loss of similarity and most new mutations cause
deleterious effects and are selected against.
19.10 Process of Speciation
Species-when a group of one species cannot exchange genes with another organism
Most new species form as a result of geographical isolation.
-a single migrant between populations is sufficient to prevent populatins from
fixing at alternative alleles by genetic drift alone
-selection toward different adaptive peaks will not succeed in causing complete
divergence unless it is extremely strong
In order to for populations to diverge enough there has to be a strong enough barrier to
stop migration from happening. Such spatially separated populations are allopatric
-enough separation will lead to two different enough species that even if they were to
come into contact and mate it would cause severe physiological, developmental and
behavioral problems. They become biologically isolated.
Two biologically isolating mechanisms:
1) Prezygotic Isolation-occurs when there is failure to form zygotes
2) Postzygotic isolation- results from failure of fertilized zygotes to contribute
gametes to future generations (common in animals)