BBCCT-121

CONCEPTS IN GENETICS
Indira Gandhi National
Open University
School of Sciences

Block

2
GENE MAPPING
UNIT 4
Genetic Definition of a Gene 65
UNIT 5
Microbial Genetics 78
UNIT 6
Linkage, Crossing Over and Mapping Techniques 97
 BLOCK 2: GENE MAPPING

Gene mapping helps us in determining the location of genes on chromosomes. It is an

important tool for locating a specific gene to a particular region of a chromosome and to
determine its relative distances between genes. Genetic maps are species-specific and
comprise of genomic markers. Uses of gene mapping identify genes responsible for
diseases. It also provides clues about which chromosome contains the desired gene and
precisely where the gene lies on that chromosome. Accordingly, it provides the basis for
map-based cloning of major genes involved in important agronomic traits. Mapping also
finds applications in study and research of heritable diseases such as cancer, forensics
for identification of potential suspects through DNA evidence, and much more.
In this Block on Gene Mapping you shall learn about complementation test, limitations of
cis-trans test, intragenic complementation, rII locus of phage T4 and concept of cistron.
Unit 5 is about mechanism of genetic exchange - conjugation, transformation and
transduction. Gene mapping in bacteria is also discussed in this unit. Unit 6 deals with
linkage and crossing over. Genetic mapping in eukaryotes, centromere mapping,
cytogenetic mapping, detection of linked loci by pedigree analysis in humans and somatic
cell hybridization for positioning genes on chromosomes is also discussed here.

Expected Learning Outcomes

After studying this block, you should be able to:

 define the unit of function, mutation and recombination at genetic level;
 explain cis - trans test of allelism (complementation test) and differentiate between
inter -and intra-genic complementation;
 describe the resolution of rII locus of T4 and the concept of cistron;
 compare recombination and complementation;
 explain the mechanism of transformation, conjugation and transduction;
 describe how bacterial gene transfer is used for gene mapping;
 explain linked transmission of genes and how linked genes are separated;
 indicate steps in genetic mapping and describe gene mapping in humans; and
 describe specialised mapping techniques in Neurospora and Drosophila;

We hope that after studying this block you will acquire understanding about gene mapping
techniques, its significance and limitations.

Wishing you success in this Endeavour!!

Unit 4 Genetic Definition of a Gene

UNIT 4
GENETIC DEFINITION OF A
GENE

Structure
4.1 Introduction 4.4 Seymour Benzer

Expected Learning Outcomes The Power of Phage Genetics

4.2 Evolution of Gene Concept Fine Structure Analysis of T4 rII

Locus.
4.3 Complementation Test
Concept of Cistron
Intragenic Complementation
Test 4.5 Summary

Limitations of cis-trans test 4.6 Terminal Questions

Complementation and 4.7 Answers

Recombination: A comparison
4.8 Further Readings

4.1 INTRODUCTION
Genetics is the study of heredity and variations. You have learnt in the
previous block that the science of Genetics began with the rediscovery of
Mendel‟s work on peas in 1900 although selection of crop plants and animals
was practiced from time immemorial on a purely hit and trial basis. Gregor J.
Mendel‟s paper on plant hybridization in 1866 suggested that particulate units
(„factors‟) controlled the inheritance of specific characters. Let us look at some
historical facts. In 1902 Sir Archibald E. Garrod investigated congenital
metabolic diseases in humans including alkaptonuria which is the easiest to
detect. He recognised that alkaptonuria is a single gene recessive disorder.
His concept of gene was one mutant gene – one defect (block) in metabolism.

In this unit you shall learn about the cis-trans test which delimits a gene. In
addition the unit deals with the fine structure analysis of the rII locus of phage
T4 by S. Benzer.
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Expected Learning Outcomes

After studying this unit, you should be able to:

 define the unit of function, mutation and recombination at genetic

level;

 explain cis - trans test of allelism (complementation test);

 differentiate between inter -and intra-genic complementation;

 point out the limitations of cis-trans test;

 indicate the power of Benzer‟s system for fine structure analysis of

the gene;

 describe the resolution of rII locus of T4 and the concept of cistron;

and

 compare recombination and complementation.

4.2 EVOLUTION OF GENE CONCEPT

The gene was considered prior to 1940s as an indivisible unit of function,
mutation and recombination unit, organised like beads on a string. Today we
know that a gene (cistron) is the structural as well as functional unit of genetic
information. It is made up of nucleotides which codes for a product (protein or
RNA). The sequence of DNA bases stores in a coded manner the information
to make specific proteins/ RNA. More importantly it is divisible by mutation and
recombination. Clarence Oliver in 1940 demonstrated recombination within the
lozenge gene of Drosophila. The gene as a unit of function is assessed by the
phenotype of the trans-heterozygote; all non complementing mutants are in
the same gene. The cis- trans test developed by E. B. Lewis is a test of
allelism.

It was in 1941 George W. Beadle and Edward L. Tatum modified Garrod‟s

concept of gene to one gene one enzyme hypothesis. The first attempt to
associate genotype with phenotype was made while investigating metabolic
pathways. It was hypothesized that each metabolic step is catalyzed by a
specific enzyme which in turn is encoded by a single gene. A mutation in the
gene alters the activity of the enzyme. Subsequently with the identification of
multimeric enzymes / proteins, the concept of gene changed to one gene-one
polypeptide. Now it is best defined as unit that codes for a protein or RNA.

4.3 COMPLEMENTATION TEST

With the acceptance of one gene one polypeptide hypothesis by the scientific
community, it was clear that gene is the biological unit of heredity. At that time
a gene was linked to cellular function by studying mutants with altered
phenotype. The major difficulty with this strategy was that there were many
mutants with similar phenotypes. In 1940s there was no way to know whether
mutations that give same phenotype are located on the same or different
66 genes.
Unit 4 Genetic Definition of a Gene

The gap was filled by Edward Lewis in 1942 by developing the

The Star eye mutation
complementation test (also known as "cis-trans" test) for functional allelism in
in Drosophila results in
Drosophila. Lewis observed that fruit flies carrying certain mutations in the cis irregular arrangement of
and trans configurations had different phenotypes. Initially he studied the Star- eye facets and facet
asteroid locus in which the results were not straight forward due to the partial hairs. The flies have
smaller eyes. It is lethal
dominance of Star mutation. Later he performed similar experiments with X-
in homozygotes.
linked eye color (white and apricot) mutants. Both mutations are recessive and
show up in homozygous females (and hemizygous males).

E. Lewis found that cis heterozygotes had wild type red eye colour while the
trans heterozygotes had light apricot colored eyes (Fig. 4.1). An organism that
carries the same genetic markers (say for eye color), but in different
arrangement which give rise to different phenotypes are said to exhibit position
effect (cis-trans position effect).

Fig. 4.1: The cis-trans position effect. E.B. Lewis (1918-

2004)

E.B. Lewis (1918-2004) was born in Pennsylvania. He joined Alfred

H. Sturtvant in 1939 at Caltech as a graduate student after obtaining
a B.A. degree in Biostatics from the University of Minnesota. The lab
environment gave him complete freedom to explore new genetic
phenomenon which led to the discovery of cis-trans effect in fruit
flies and fetched him a PhD. He is dubbed as the „scientific
grandson of Morgan‟.

His most famous work is on analysis of homeotic genes in

Drosophila. He isolated and studied many mutations in the Bithorax
locus and demonstrated their role in defining segment polarity.

Lewis shared the 1995 Nobel Prize in Medicine or Physiology with

Eric Wieschaus and C. Nüsslein-Volhard. He remained at Caltech
after joining as a faculty member.

Here you must note that the complementation or cis-trans test tells us whether
two mutations that produce same or similar phenotypes are situated in the
same gene or in two different genes. The two arrangements of a double
heterozygote are cis or coupling and trans or repulsion (Fig.4.2). In the former
both mutations are on the same chromosome while in the latter they are on
different chromosomes. The mutants are tested pair wise by crossing
organisms that are homozygous for each of the mutations or as in case of
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viruses by co-infecting the host cells with two mutants. The phenotype of the
trans-heterozygote indicates whether two mutations are on the same gene
(non-complementing) or they are on different genes (complementing). It is not
always easy to construct a cis heterozygote but serves a useful control for
recessive mutations.

Fig. 4.2: The two configurations of a double heterozygote.

A trans-heterozygote has a wild type phenotype if two mutations are in

different genes because the cell can still produce functional product which
does not happen in case the mutations are in the same gene (Fig.4.3).
Remember complementation is between products of the gene.

Cis Trans

Fig. 4.3: The trans-test distinguishes allelic from non allelic mutations.

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Unit 4 Genetic Definition of a Gene

SAQ 1
a) How does the concept of one gene one enzyme differ from one gene
one polypeptide?

b) How does complementation test distinguish allelic from non allelic

mutations?

4.3.1 Intragenic Complementation Test

In intragenic (or inter allelic) complementation mutant alleles of the same gene
complement each another. It is generally observed when mutations result in
amino acid substitution and a defective protein is synthesised by mutant
alleles. There are different ways by which mutant alleles of the same gene can
jointly or mutually rectify one another and have a wild type phenotype. One
common theme is that these proteins are active as oligomers. In that case the
mutant subunits combine to form an oligomer with partial or at times wild type
activity. At times a defective complex formed by one mutant gene product
may be stabilized by the other mutant gene product. It also occur when genes
encodes products with independently functioning domains.

4.3.2 Limitations of cis-trans Test

 Complementation test is applicable for regions of DNA that code for a
diffusable product and not for cis acting sites.

 The results of a trans-test are unambiguous if a mutation is recessive to

wild type. In other words the cis test for mutants on same or different
genes has a wild type phenotype. It is not informative in case of
dominant mutations or multigenic traits whose products interact such as
epistatic interactions.

 Alleles of the same gene may occasionally complement each other

through "intragenic complementation". This is sometimes observed
when the native proteins are oligomeric.

 Mutations in two separate genes may occasionally fail to complement

each other (non-allelic non-complementation). It is a rare phenomenon
that occurs when two gene loci encode physically interacting products as
in case of synaptic proteins (UNC-13 and UNC-64) of C.elegans.

 Polar mutation, a mutation that not only affects the function of the
mutated gene but also affects expression of one or more neighboring
genes. These genes are located downstream of the mutated gene (so
they are polar mutations). Complementation test cannot be used for
such mutations.

4.3.3 Complementation and recombination:

A Comparison
You have studied in the cell biology paper how recombinant gametes are
generated during meiosis I. One of these processes involves a physical
exchange of DNA (crossing over) between homologous chromosomes.
Recombination also occurs in microorganisms whenever partial diploidy is
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created (bacteria) or during co-infection of viruses. In this unit you have learnt
about complementation. Now we shall compare complementation and
recombination in table1. The following sections on fine structure analysis of rII
locus in phage T4 will use both these processes and so it is essential to
understand what information each one provides.

Table 4.1: A comparison of recombination and complementation

Recombination Complementation

Recombination results in exchange of Complementation is due to interaction of

genetic material between homologous gene products when present in a common
chromosomes. cytoplasm.

A direct interaction with DNA during It does not involve direct interaction with
prophase-I of meiosis is a prerequisite DNA; the mutations persist present even
condition for generation of recombinants if they complement.
by crossing over.

Recombination is detected by examining It is assessed by the phenotype of the

the genotypes of the progeny transheterozygote and will occur in all
heterozygotes (next generation). The such cells if mutations are on different
frequency of recombination depends on genes.
the distance between the two mutations.

The results of recombination analysis The complementation test segregates

tell us whether the mutations are linked mutations as allelic or non allelic. The non
and their relative distance. allelic mutations may / may not be linked.

Recombination can take occur in any Regions of DNA that do not code for a
region of a chromosome irrespective of product (cis acting sites) cannot be
their ability to code for a diffusable analysed by complementation analysis.
product.

SAQ 2
a) Indicate whether the following statements are true or false:
i) The cis-trans test is a test for functional allelism.
ii) In cis heterozygote two mutations are on different chromosomes.
iii) The cis-trans test is applicable only to fruit flies.
iv) It is easier to generate a cis heterozygote as compared to a trans-
heterozygote.
v) Complementation occurs between products of genes.
vi) Recombination is a prerequisite condition to observe
complementation between genes.

b) Why complementation does not occur between cis acting sites?

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Unit 4 Genetic Definition of a Gene

4.4 SEYMOUR BENZER

Seymour Benzer was an American scientist born to polish Jewish immigrants
whose contributions range from physics to molecular biology to behavioural
genetics. He received his PhD in physics (1947) from Purdue University for
research on metalloid germanium for which he was awarded several patents.
His work was instrumental in the development of semiconductors for
transistors. In the same year he was appointed Professor in the department of
Physics at Purdue.

The following year (1948) he attended the phage course at Cold Spring Harbor
Laboratory and became increasingly interested in addressing fundamental
questions in biology. He then worked with leading biologists of the time and in
1961 published a detailed map of the rII locus of phage T4 and divided it into
two complementation groups (cistrons). Above all he defined linearity of the
gene and extended the work of earlier workers by demonstrating the basic unit
of mutation (base pair) and recombination (between two base pairs). In 1967
he joined Caltech and worked there till the end.

The third phase began with studies on behaviour using Drosophila as a model
system. He and his colleagues succeeded in isolating and studying the effect
of a range of behavioural mutants and their potential to study human diseases.
One of his novel approaches in the early phase was counter current
distribution to isolate these mutants. Benzer is regarded as the father of
neurogenetics.

S. Benzer (1921-2007) (Source:en.wikipedia.org)

The underlying theme of Benzer‟s work has been the use of simple assays,
clever tricks and the choice of organism to find specific answers to complex
biological problems. He reinvented himself many times and preferred to
venture into uncrowded fields. Benzer received many awards and prizes
including the Crafoord award of the Royal Swedish academy of Sciences
(1993) and the Albany Medical Center Prize in Medicine and Biomedical
Research (2000), however he never received the Nobel Prize.
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4.4.1 The power of Phage Genetics

Benzer and other investigators recognised the power of microorganisms and
A plaque is a clear region
chose to work with them. Not only it is much easier to generate large number
produced on agar plates
due to repeated cycles of of progenies in a short time but by simply plating them on a selective media it
phage multiplication and is possible to detect extremely rare recombinants facilitating detailed analysis.
destruction of bacterial Now let us evaluate the advantages of the rII system of T4 phage and its
cells. It has about 10 exploitation using clever strategies.
million phage particles.
Bacteriophage T4 is a lytic phage of E.coli. It infects the host, multiplies and
lyses the bacterial cell. On an agar surface plated with a lawn of bacteria, the
The „r‟ (rapid lysis)
application of diluted phage suspensions results in clear areas (plaques) of
mutants of T4 have been
mapped to three loci (r I, r characteristic morphology by repeated rounds of infection and lysis of
II and r III). neighbouring bacterial cells. A single plaque has approx. 10 million phage
particles so they can be observed with naked eye. Benzer worked with „r‟
These mutants have a
block in expressing lysis
(rapid lysis) mutants which are conditional lethal mutants. These phages
inhibition and form larger, form large, sharp-edged plaques on E.coli B (permissive host) but fail to grow
sharp edged plaques. on E.coli K lysogenic for λ (restrictive host). The wild type phages multiply in
The mutants can be both strains and form small plaques with rough edges (Fig. 4.4). The
distinguished by their
differences in plaque morphology and host range allow easy selection of
plating behaviour on
different bacteria and mutants; recombinants and complementation in co infections.
recombination.

Fig. 4.4: Plaque morphology and host range of wild type T4 and rII phages.

After initial attempts at mapping using recombination (two factor crosses) and
complementation analysis, Benzer shifted to a more efficient short cut method,
namely deletion mapping. These mutants are of non reverting type and by
using overlapping set of deletions the order of mutations can be determined
simply by qualitative „Yes or No‟ tests. This was another clever trick to map
more than 2000 mutants (reverting and non-reverting type) and generate an
exhaustive map of a small region of the phage genome.

4.4.2 Fine Structure Analysis of T4 rII Locus

Genetic mapping technique standardized in Drosophila by A.H. Sturtevant
has been used to map chromosomes in many different organisms. Benzer
reasoned out that this technique can also be extended to map mutations
inside the gene. It was known that recombination frequency is a function of
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Unit 4 Genetic Definition of a Gene

distance between two points. This was one of the reasons to shift to microbial
systems (Refer to section 4.2.1). He chose to study the internal structure of a
single gene (rII) in the phage chromosome by looking for mutants that are
altered in the same characteristic (altered plaque morphology) and perform
recombination experiments coupled with simple tricks to identify rare
recombinants.

The mutants were mapped by crossing them one against another in a liquid
culture of E.coli B. Coinfection provides opportunity for the progeny to
recombine and if mutations are on different parts wild type phages can be
generated which can now grow on E.coli K. In this way even a single
recombinant among a billion progeny can be detected simply by shifting from
E.coli B to E.coli K, allowing resolution of close by mutations. Recombination
in phages is reciprocal but it is not as complex as meiosis and can occur at
any time (including multiple times) of phage life cycle. But mapping thousands
of point mutations by this method is impractical.

Benzer overcame this problem by isolating a number of non reverting mutants

(deletions). These overlapping mutations have deletions of variable sizes
which divided the rII region into segments. The first set of point mutants were
then mapped to one of the seven large deletions (the “big seven”). Next each
point mutant was tested against these reference deletions. The recombination
test gives a negative result if the deletion overlaps the point mutation and a
positive result if it does not overlap. In this way a mutation is quickly located on
a segment of rII. This procedure is repeated with a second group of reference
mutants that divides the segment into smaller segments. The point mutants
were successfully mapped to 80 segments. Then mutations in a given
segment were tested against each other and based on recombination
frequency (RF) their order within the segment was established by three factor
crosses and then with the neighbouring segments. He showed that a gene
consisted of a linear array of sub elements that could mutate and recombine
with one another.

The above data has established the rII locus as the region responsible for
growth of phage on E.coli K which consists of many parts divisible by mutation
and recombination. What is not clear whether the region includes one gene or
many genes controlling host range? To verify this Benzer performed the cis -
trans test. It had been shown earlier that a rII mutant can grow normally on
E.coli K if the cell is coinfected with wild type phage.

Benzer crossed pairs of rII mutants (trans test) by infecting E.coli K cells. The
decision whether the phage will multiply or not occurs soon after infection
before there is any opportunity for recombination to take place. If there is
complementation and the phage develops normally then the two mutations are
in different functional units (non allelic) while absence of complementation
indicates that the mutations are in the same unit and no plaques are formed.
The test divided the rII locus into two parts (rII A and rII B) which he called
cistrons. The two cistrons function independently. 73
Block 2 Gene Mapping

In conclusion this was the most exhaustive fine structure analysis of a gene
using simple tricks and exploitation of the powerful phage system. He also
found that mutations do not occur with equal frequency at all sites; there are
mutational hot spots at which mutations occur more frequently.

4.4.3 Concept of Cistron

The term „cistron‟ was coined by Benzer to describe a region of DNA defined
by cis-trans test. The test provides a genetic definition of a gene – as a region
that codes for a diffusable product (protein or RNA). Generally mutations at
different sites in the cistron do not complement in trans. In other words they
are allelic mutations and are said to belong to the same complementation
group. A cistron is divisible by mutation and recombination as evident from
Benzer‟s fine structure analysis. The smallest unit of mutation is a base pair
and that of recombination is between two base pairs. The units of
recombination and mutation were called recon and muton, respectively.

SAQ 3
a) Deletion mapping is more efficient but less detailed than fine structure
mapping of genes (True or False)

b) A researcher identified three strains of rII (A, B and C). Co infection

studies were conducted in E. coli strain K (λ) and the following results
were obtained.

A × B = plaques obtained

A × C = plaques obtained

B × C = no plaques obtained

What can you conclude from the results?

i) Mutations in A, B and C are located in three different genes.

ii) Mutations in A and B are located in the same gene, while for C it is
on a different gene.

iii) Mutations in A and C are located in the same gene, but for B it is
on a different gene.

iv) Mutations in B and C are located in the same gene, while for A it is
on a different gene.

c) In order to map two spontaneously arising rII mutants you decide to use
a selective system. When the mutants were mixed and plated on E.coli
K, two plaques were produced. But coinfection on E.coli B produced
40,000 plaques. Calculate the map distance between them.

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Unit 4 Genetic Definition of a Gene

4.5 SUMMARY
 Genetics is the study of heredity and variations. Gregor Johann Mendel,
the father of genetics studied inheritance of characters in pea plants. He
called the hereditary units, “factors” that control the inheritance of
characters in successive generations.

 Gene is the basic structural and functional unit of genetic information. A

Gene (cistron) is a sequence of nucleotides that code for functional
protein or RNA. The sequence of DNA bases stores in a coded manner
the information to make specific proteins. More importantly it is divisible
by mutation and recombination.

 Edward Lewis developed the complementation test (also known as "cis-

trans" test) for functional allelism in Drosophila. The complementation
test is used to determine whether mutations that produce the same or
similar phenotypes are located in the same gene or in two different
genes. The phenotype of the transheterozygote helps to distinguish
allelic from non allelic mutations.

 The cis-trans test provides an unambiguous answer if the phenotype is

wild type for both allelic and non allelic mutations in cis configuration. It
serves as a control. In some situations like dominant mutations, gene
interaction and intragenic complementation, the test does not give clear
cut results.

 S. Benzer used a combination of recombination mapping, cis-trans test

and deletion mapping to map in great details the rII locus of phage T4.
He exploited the power of microbial systems and simple tricks to map
more than 2000 mutations in rII region.

 He coined the term „cistron‟ for the stretch of DNA defined by the cis-
trans test. A cistron encodes a diffusable product which could be protein
or RNA. Similarly the units of recombination and mutation were called
recon and muton, respectively.

4.6 TERMINAL QUESTIONS

1. Explain the cis-trans test for functional allelism.

2. What are the limitations of cis-trans test?

3. How do you construct cis- and trans- heterozygotes?

4. Indicate the reasons for Benzer‟s success in generating an exhaustive

map of rII locus in T4 phage.

5. How has the concept of gene changed from the pre 1940 era?

6. Explain deletion mapping by taking the example of non-reverting rII

mutants.
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4.7 ANSWERS
Self-Assessment Questions
1. a) The one gene one polypeptide concept takes into consideration
that enzymes may be coded by more than one gene as they are
assembled from different types of subunits.

b) A trans-heterozygote may either have a wild type or mutant

phenotype depending on whether the two mutations are non allelic
or allelic, respectively.

2. a) i) True

ii) False

iii) False

iv) False

v) True

vi) False

b) Complementation is between gene products and cis acting sites

do not code for a diffusable product.

3. a) True

b) Mutations in B and C are located in the same gene, while for A it is

on a different gene.

c) Number of recombinant plaques = 2X2 = 4 (Only wild type

recombinants grow on E.coli K and not double mutants)

Recombination frequency = 4 / 40,000 x 100 = 0.01 map units

Terminal Questions
1. The cis-trans test was developed by E. Lewis while investigating eye
color mutants in Drosophila. He observed cis-trans position effect and
showed that the phenotype of the trans-heterozygote indicates whether
two mutations are on the same gene (non-complementing) or they are
on different genes (complementing). Refer to section 4.3 for details.

2. Refer to subsection 4.3.2

3. A trans-heterozygote is obtained by crossing organisms homozygous for

one of the mutations or co-infecting the host with single mutants (as in
haploid viruses). A cis heterozygote can be created by selfing a trans-
heterozygote.

4. Benzer recognised the power of microorganisms for large scale work to

pick up rare recombinants / concentrated on the rII locus of phage T4
and exploited the conditional lethality of rII mutants and plaque
morphology / used deletion mutants to enhance the efficiency of his
work. Refer to section 4.4.2 for details.
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Unit 4 Genetic Definition of a Gene

5. The concept of gene has changed from an indivisible unit to one that
codes for a product (protein or RNA) and is divisible by mutation and
recombination. Refer to section 4.1 for details.
6. In deletion mapping each point mutant is tested against reference
deletions. The recombination test gives a negative result if the deletion
overlaps the point mutation and a positive result if it does not overlap. In
this way a mutation is quickly located on a segment of rII. Then this
procedure is repeated with a second group of reference mutants that
divides the segment into smaller segments. Refer to section 4.4.2 for
details.

4.8 FURTHER READINGS

1. Snustad, D.P and Simmons, M.J. Principles of Genetics, 3rd Ed, 2003,
John Wiley and sons, Inc.

2. Benzer, S., The fine structure of the gene, Scientific American,

Jan.1962. (Recommended reading)

3. Bonini, M. A tribute to Seymour Benzer, Genetics 180: 1265-73 (2008)

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UNIT 5
MICROBIAL
GENETICS

Structure
5.1 Introduction Mating using Different Donor
+
Strains (F ; Hfr and F‟)
Expected Learning Outcomes
Interrupted Mating Experiment
5.2 Genetic exchange in
and Temporal Gene Mapping
Bacteria
5.5 Transduction
Experimental Approaches
Generalized Transduction
A comparison with Eukaryotes
Specialized Transduction
5.3 Transformation
Gene mapping with
Griffith‟s Experiment
Generalized Transducing
Mechanism of Transformation Phages

Transformation and Gene 5.6 Summary

Mapping
5.7 Terminal Questions
5.4 Conjugation
5.8 Answers
Fertility (F) Factor
5.9 Further Readings

5.1 INTRODUCTION
Bacteria were known to reproduce only asexually by binary fission until the
discovery of genetic exchange by parasexual processes (conjugation,
transformation and transduction). These processes play an important role in
bacterial evolution as they allow them to acquire and efficiently spread new
characteristics such antibiotic resistance which confers adaptive advantages
in changing environmental conditions.

In this unit we shall learn about these three parasexual processes in

detail and how they have been used to map bacterial genes.
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Unit 5 Microbial Genetics

Expected Learning Outcomes

After studying this unit, you should be able to:

 explain the mechanism of transformation, conjugation and

transduction;

 compare bacterial genetic exchange processes with diploid organisms;

 highlight the salient features of fertility factor;

 describe how bacterial gene transfer is used for gene mapping;

 differentiate between F+ and Hfr mediated conjugal transfer;

 indicate the types of transduction and compare them, and

 define prophage, episome, merodiploids and sexduction.

5.2 GENETIC EXCHANGE IN BACTERIA

There are three genetic transfer processes (transformation, conjugation and
transduction) known in bacteria. They are parasexual processes, as
recombination occurs in absence of meiosis. In bacteria recombination usually
occurs between the main chromosome of the recipient and a DNA fragment
from another strain (donor). To initiate recombination a transfer of DNA from
donor to recipient is a prerequisite condition as they are haploid organisms.
The two products of genetic exchange may or may not be recovered. In order
to get recombinants in an exchange involving a circular chromosome and
linear DNA fragment, an even number of cross overs are required (Fig. 5.1a)
while a single exchange between two circular DNAs leads to integration of
donor DNA into the recipient chromosome (Fig.5.1b). The diploid state in
bacteria is partial (merodiploid) and generally transient.

Fig. 5.1: Recombination in bacteria (Adapted from Genetics: Snustad & Simmons). 79
 Block 2 Gene Mapping

The three processes can be easily distinguished by testing whether cell-cell

contact is required or not and nuclease sensitivity. Conjugation is nuclease
resistant and requires cell-cell contact; transformation is nuclease sensitive
and occurs without the need for cell-cell contact and finally transduction is
neither nuclease sensitive nor requires contact between donor and recipient
cells.

5.2.1 Experimental Approaches

Genetic exchanges in bacteria are studied using a variety of mutants in which
A selective media
the restoration to wild type phenotype after DNA transfer and recombination is
allows the growth of
specific microbial cells
checked in selective media. Bacterial growth is observed visually as colonies
and exclude the on semisolid agar based medium or as turbidity in liquid medium if they have
growth of other cells. acquired a new characteristic. Some commonly used mutants are antibiotic
For example, an resistant mutants (rifampicin resistant; Rifr), nutritional mutants (auxotroph;
ampicillin containing his- auxotroph grows only in a medium supplemented with histidine) or carbon
medium will allow only source mutants (fail to utilise certain carbon source). Other mutants include
r
amp cells to grow. colony morphology mutants, phage resistant and temperature sensitive (ts)
mutants.

5.2.2 A comparison with Eukaryotes

Most eukaryotes are diploid and both sexes produce haploid gametes by
meiosis in specialised cells. As you know recombinant gametes are formed by
crossing over between homologous chromosomes and independent
assortment of non homologous chromosomes during prophase I and
metaphase I of meiosis, respectively. The number of crossovers may be odd
or even and both recombinants are recovered.

SAQ 1
a) Name the three parasexual processes known in bacteria.
……………………………………………………………………………………
……………………………………………………………………………………
b) Indicate two ways to distinguish them.
……………………………………………………………………………………
……………………………………………………………………………………

5.3 TRANSFORMATION
Transformation is the uptake of free extracellular DNA fragment(s) from
surrounding medium by competent recipient cells. The donor DNA must
recombine with the homologous region of recipient„s main chromosome to
bring about heritable transformation. Bacterial species can even be induced to
take up DNA if they lack the ability to do so.

5.3.1 Griffith’s Experiment

Transformation was discovered by Frederick Griffith in Streptococcus
pneumoniae (Diplococcus pneumoniae) in 1928. It was the first kind of genetic
Fredrick Griffith exchange described in bacteria. The wild type strains of this bacterium are
(1879-1941) pathogenic in mammals and they form smooth (S strain) glistening colonies on
agar plates due to the presence of capsular polysaccharide. Mutations that
affect polysaccharide synthesis produce bacterial colonies with rough
80 morphology (R strains) and these strains are avirulent. In addition there were
Unit 5 Microbial Genetics

biochemically distinct variants of S type known (type II, type III, etc) which
could be easily distinguished immunologically. The conversion of smooth to
rough is type specific.

F.Griffith was a British bacteriologist who studied medicine and later

worked at the pathological lab of the ministry of health. He is known for
his discovery of pneumococcal transformation. His work later led to the
discovery of DNA as the genetic material.

He died in 1941 during a German bombing raid on London.

Griffith injected these bacteria into mouse and observed their effect after few
days. He used two strains; a type IIIS and a type IIR and injected live and /
heat killed bacteria. When he injected live type IIIS, heat killed type IIIS or type
IIR the results were as expected. The mouse that received live type IIIS died
and type IIIS bacteria could be isolated from its viscera. Those that were
injected live type II R or heat killed type IIIS survived and no living bacteria
could be isolated from the mouse.

Interestingly in one of his experiments Griffith found that mice were killed when
they were injected with a mixture of live type IIR and heat killed type III S. On
further analysis of dead mouse he could isolate live type IIIS bacteria. He
called this process transformation. Later similar results were reported by in
vitro studies and with partially purified extracts from heat killed S bacteria.
Finally the transforming principle was shown to be DNA by Ostwald T. Avery,
Colin M. Macleod and Maclyn McCarty in 1944.

Fig. 5.2: Griffith’s experiment on transformation.

81
 Block 2 Gene Mapping

Bacterial species like E. coli and Salmonella typhimurium are naturally non
transformable. One of the commonly used methods to induce competence in
these organisms was developed by M. Mandel and A. Higa (1970). In this
method, cells in early log phase are centrifuged and resuspended in cold
hypotonic CaCl2 solution. Next DNA is added which gets adsorbed to the cell
surface. The cells are then subjected to heat shock at 42o that promotes the
uptake of DNA. The transformed cells are selected by plating in appropriate
media.

5.3.2 Mechanism of Transformation

Naturally transformable bacteria possess an innate ability to develop a state
A number of bacterial
of “competence” such that they become capable of internalizing DNA. Here
species are known to
we shall take the example of a Gram positive spore forming bacteria, Bacillus
communicate via
subtilis to describe the mechanism of transformation (Fig. 5.3). In this species
secreted factors or
pheromones. These competence is established towards the end of log phase when the cells reach
factors trigger various high density.
responses such as
production of virulence
factors, competence,
sporulation and
secondary metabolism.

Fig. 5.3: Transformation in Bacillus subtilis.

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Unit 5 Microbial Genetics

Generally about 20% of cells become competent and remain in this state for
several hours. Actively growing cells secrete competence factor (pheromone)
which at high concentration induces the synthesis of a number of proteins
(Com proteins) needed for establishment of competent state. In a competent
culture some cells function as donors and release randomly cut DNA
fragments while the other cells are equipped to take up DNA and assimilate it.
Let us now elaborate the key steps of transformation:

 The recipient cell surface binds naked double stranded DNA, initially via
type IV pili.

 The bound DNA is then transferred to a DNA binding receptor which

lacks sequence specificity. Therefore DNA can be taken up even from
heterologous sources. The DNA – receptor complex changes slowly
from reversible to an irreversibly bound state.

Before DNA enters the cell one of its strands is randomly degraded by a
membrane bound endonuclease.

 Single stranded DNA finally enters assisted by the receptor and an

uptake system. It is protected from nuclease action by coating with
single strand DNA binding proteins.

 The donor DNA fragment undergoes homologous recombination with

the recipient‟s chromosomal DNA. The exchange is non reciprocal as
the recipient is genetically altered and the other product of
recombination is degraded.

5.3.3 Transformation and Gene Mapping

Transformation is used for gene mapping in some bacterial species. In order
to map genes excess of randomly fragmented donor DNA is mixed with
competent recipient cells. In most species, transformation takes place at a
frequency of about 1 transformed cell per 103 cells.

The basic principle of mapping is that if two genes a and c are far apart on the
donor chromosome such that they are invariably present on two different DNA
fragments then the probability of simultaneous transformation (co-
transformation) of recipient (a-c-) into wild type is roughly the multiplication of
their individual probabilities (10-6) or one a+ c+ transformant per 106 recipient
cells. However, if the two genes are very close to each other (a and b) such
that they are more likely to be part of a single donor fragment, then the
frequency of co-transformation is nearly the same as the frequency of single-
gene transformation (one transformants per 103 recipients). To map, we have
to measure co transformation frequency of various gene pairs. For example, if
genes a and b can be co-transformed; genes b and c can be co-transformed
but not genes a and c, then the gene order must be a b c.

In order to obtain unambiguous cotransformation frequencies, the

competence of the cell population must be high otherwise it has to be
supported by other tests such as dilution test. A quantitative dilution test
measures single and double transformation as a function of DNA
concentration (Fig.5.4 ). At high DNA concentration single transformation
frequency declines linearly as the DNA concentration deceases. In case of
double transformation, the decline is almost identical to single transformation
83
Block 2 Gene Mapping

for linked markers but the slope is much steeper if the genes are far apart and
more likely to be present on separate fragments.

Fig. 5.4: Dilution test for cotransformation.

SAQ 2
a) Define competence.

b) What was the key finding of F. Griffith?

c) Dilution test is used ……………………………………………..

5.4 CONJUGATION
The process of conjugal transfer was discovered by Joshua Lederberg and
Edward L. Tatum in 1946. They used multiply auxotrophic strains (nutritional
mutants) of E.coli and demonstrated the appearance of prototrophs in mixed
cultures at a relatively higher frequency (1x 10-7) than expected from
simultaneous reversion of multiple markers (Fig.5.5). Strain A or B alone did
not yield prototrophs.

Strain A (58-161) Strain B (W 677)

(metbiothr+ leu+) (met+bio+thr leu)

The mixed cultures of E.coli are plated on a minimal medium

(would not support the growth of either strain) and incubated for
few hours.

They obtained few prototrophs (met+ bio+ thr+ leu+)

Fig. 5.5: Discovery of conjugation in E.coli.

84
Unit 5 Microbial Genetics

The prototrophs were genetically stable as they could be further sub- cultured
on minimal medium. This led them to suggest that some sort of assortment of
genetic material between the two strains has occurred and named it
conjugation. They eliminated the possibility of transformation by showing that
extracted DNA from either strain failed to transform the other strain.

The proof for cell-cell contact in conjugation came in 1950 when Bernard
Davis demonstrated it with the U-tube experiment. Later William Hayes
discovered that gene transfer was unidirectional (from donor to recipient).
The donor strain harbors the sex (fertility) factor which is almost always
transferred to the recipient in an F+ x F- mating (infectious fertility).

In 1958 Joshua Lederberg shared with George Beadle and Edward L. Tatum
the Nobel Prize in Physiology or Medicine for his contributions concerning
genetic recombination in bacteria.

5.4.1 Fertility (F) Factor

The fertility factor is a self transmissible, relatively large (100kb), low copy
number (1-2 copies /cell) plasmid that encodes all functions needed for its
transfer during conjugation (Fig.5. 6). Many of these genes (tra genes) are
clustered on one side of the F factor (not shown). The tra region has genes for
pilus biogenesis & assembly; mating pair stabilization; nickase, DNA helicase,
coupling factor and to control entry of multiple F factors.

Fig. 5.6: A simplified map of the fertility factor.

A donor strain may have the F factor integrated into the main chromosome by
recombination. The IS (insertion) sequences (IS2 and IS3) and Tn1000 serve
as portable regions of homology. The term „episome’ applies to such DNA
elements that have a dual existence; maintained either free autonomously
replicating molecules or integrated into the main chromosome. F factor has
multiple origins of replication but only OriT is the origin of conjugal transfer. It
replicates by rolling circle mode and transfers the displaced single strand to
the recipient. 85
Block 2 Gene Mapping

5.4.2 Mating using different donor strains (F+;

Hfr and F′)
In this subsection we shall learn about the outcome of mating with three
variants of donor strains, beginning with F+ (Fig. 5.7). The process is initiated
by cell-cell contact via sex pilus expressed on the surface of donor. DNA
replication precedes transfer of F factor. It is initiated by introducing a nick at
OriT and as DNA replicates the displaced single strand is simultaneously
transferred to the recipient. In the recipient the single strand is circularized and
converted to duplex DNA. The transfer of F factor is very efficient under lab
conditions and therefore after mating the recipient becomes a donor and the
donor remains F+. In this type of mating the transfer of chromosomal markers
is very inefficient. You may recall Lederberg and Tatum‟s experiment in which
they reported prototrophs for chromosomal genes. Can you think how this
could have happened?

Occasionally an F plasmid integrates into the main chromosomes generally by

utilizing portable regions of homology such as IS sequences. This donor strain
is called Hfr (high frequency recombination). As the name suggest an Hfr
strain transfers chromosomal markers at high frequency. There are multiple
potential sites on the E.coli chromosome where integration can occur thereby
generating different Hfr donors (Hfr H, Hfr C, Hfr b, etc).The Hfr strains also
differ in the orientation of the F factor integration which determines whether
the direction of transfer is clockwise or counterclockwise.

A mating between Hfr strain and F- results in the transfer of part of the F factor
and chromosomal markers close to the point of its integration in the main
bacterial chromosome. In this case also replication is initiated by nicking at
OriT and as it proceeds the displaced 5′ end enters the recipient cell. All steps
of conjugation remain the same as described earlier. A significant feature of
this mating is that the recipient remains a recipient because it requires around
100 min to transfer the entire bacterial chromosome and then the remaining
part of the F factor will enter the recipient. Generally the matings are
spontaneously broken and therefore recipient does not become a donor.

-
A gal mutant is The fate of the linear DNA fragment transferred depends on whether it
unable to utilise recombines with the homologous chromosomal markers and survives with
galactose as a new characteristics (gal- becomes gal+) in a selection medium or is degraded
carbon source. because it is not autonomously replicating.

The F factor can also excise from an Hfr strain by recombination. Sometimes
excision is not precise and it carries along with chromosomal markers and
may at times leave behind few F factor genes. The modified F- factors are
called F′ (F-prime) factors and they also serve as donors in conjugation. In an
F′ x F- mating, the bacterial genes incorporated in the F′ plasmid are
transferred at high frequency. The transfer of bacterial genes by a sex factor is
called sexduction. These genes may not always recombine with the host
chromosome for their survival as the F′ factor is autonomously replicating. In
such a situation stable partial diploidy for these genes is created. Following
mating the recipient becomes a donor. F′ factors are extremely useful tools to
determine dominance relationships, gene mapping and complementation
analysis.
86
Unit 5 Microbial Genetics

+ -
Fig. 5.7: Conjugation in E.coli (F x F ) (Adapted from Brooker).

87
Block 2 Gene Mapping

SAQ 3
i) Define the term episome.

ii) How does an F+ strain transfer chromosomal markers to

the recipient cell?

5.4.3 Interrupted Mating Experiment and Temporal

Gene Mapping
-
The F strain is not In late 1950s Elie L. Wollman and F. Jacob designed an interrupted mating
able to synthesise experiment to map bacterial genes. It is based on the logic that bacterial DNA
-
leucine (leu ) &
-
threonine (thr ). It moves from the donor to the recipient at a constant rate and the kinetics of
cannot utilise transfer can be followed by deliberate interruption of mating. The map
-
galactose (gal ) and generated is in units of time (temporal map). Let us now briefly describe their
-
lactose (lac ) as
carbon sources. In
experiment in the flow chart diagram (Fig.5.8) and then look at the results in
addition it is Fig. 5.9. The F- strain used is thr leu- gal- lac- tons azis strr.
sensitive to phage
Hfr H (thr+ leu+ gal+ lac+ tonr azir strs) x Fthrleugallactons azis strr)
s
T1 (ton ) and azide
s
(azi ) but resistant
to streptomycin
r
(str ). Mix the parental suspension and take aliquots at regular intervals

Agitate in a blender to disrupt mating

Dilute and plate on a medium lacking threonine (thr) and

leucine (leu) and containing strptomycin (str)

Only thr+ leu+ and strr recombinants will grow in this medium.
Leucine and threonine are among the earliest to be transferred
(selection markers) and strs is a counter selection marker that
eliminates the donor

The leu+ thr+ recombinants are replica plated onto selective

media (containing galactose or lactose or phage T1 or azide) to
test for unselected markers (gal+, lac+, tonr and azir)

Each of the unselected markers appears first in the recipient at

a characteristic time and then reaches a final frequency (Fig.
5.8). The genes that are further apart have a lower plateau than
those that are closer to the origin of transfer (OriT), due to
spontaneous separation of mating pairs

88 Fig. 5.8: Interrupted mating experiment (Wollman & Jacob).

Unit 5 Microbial Genetics

Results of Elie L. Wollman and F. Jacob interrupted mating experiment

designed to map bacterial genes is shown in Fig. 5.9..

+ +
Fig. 5.9: Kinetics of transfer of unselected markers among thr leu
recombinants.

The zero point is set on the map at the thr locus because it is the gene closest
to the point of insertion of the F factor described by William Hayes (Hfr H). It
transfers chromosomal DNA in “clockwise” direction to the F- strain. It takes
eight minutes before thr+ and leu+ recipients are detected. All donor cells do
not start transferring DNA at the same time, so the number of recombinants
increases with time. The times on the temporal map indicates minimum time
that must elapse between contact of donor and recipient before that gene is
first transferred.

The large size of the E. coli genome makes it difficult to map all genes using a
single Hfr strain. Therefore a number of Hfr strains with different integration
sites and orientation are used. Finally the maps are combined by aligning at
overlaps to generate a complete map. The map is circular and in E.coli it is
divided into 100 minutes.

SAQ 4
a) Explain the terms selected, counter selected and unselected markers.

b) Which of the following is not a protein or group of proteins required for

conjugation?

i) Pilin
ii) The origin of transfer
iii) Coupling protein
iv) Nickase

89
Block 2 Gene Mapping

5.5 TRANSDUCTION
Transduction is the process of moving bacterial genes from one bacterial cell
(donor) to another bacterium (recipient) by a bacteriophage. It was discovered
accidently by Norton D. Zinder and J. Lederberg (1952) while attempting to
induce sexual mating in Salmonella typhimurium. They used two auxotrophic
strains for their experiment; strain LA-2 was met- his- phe+ trp+ and LA-22 was
met+ his+ phe- trp-. The two strains were mixed and incubated in an amino acid
free medium. Few colonies appeared at a frequency of 1 in 100, 000 cells
suggesting some kind of genetic exchange.

To test for conjugation or transformation they grew the two strains in the two
arms of the Davis U tube separated by a sintered glass filter in a medium
containing DNase (Fig.5.10).

Fig. 5.10: Zinder and Lederberg’s experiment.

The filter allows the exchange of media, DNA and virus but not bacterial cells.
Surprisingly prototrophs still appeared but only in one arm of the U-tube. The
results could not be explained by transformation (DNase sensitive) or
conjugation (requires cell-cell contact).

Further work revealed that one of the bacterial strains used carried a
temperate virus (P22) in a latent form. Upon induction it released transducing
particles that could pass through the filter and infect the other strain. The
bacterial genes are then assimilated into the main chromosome by
90 recombination.
Unit 5 Microbial Genetics

Phage particles that carry bacterial DNA are transducing phages. They
are produced due to errors in the phage life cycle and can transfer bacterial
genes to a recipient. . Depending on whether a phage carries only specific or
any part of the bacterial genome they are classified into specialized and
generalized phages respectively. In the following subsections we shall learn
how generalised and specialised transducing phages are produced during the
phage life cycle.

5.5.1 Generalized Transduction

Bacteriophage P1is one of the best studied generalised transducing phage. It

has double stranded DNA genome (90Kb) packaged into phage head. When
it infects an E. coli cell the phage DNA is released into the host cell where it
replicates. The replicated DNA is packaged into phage head by introducing
cuts at specific sites (pac sites) on DNA. Most phage particles carry only
phage DNA.

Occasionally phage particles package fragments of bacterial chromosomal

DNA by making cuts at sites that resemble the phage pac sites (pseudo pac).
This results in transducing phage particles that carry 90Kb of host DNA and no
phage DNA. Such packaging errors are rare (1 in 500). A P1 lysate generally
has 109 infectious phages / ml which means it has 106-107 transducing particles
/ ml.

The bacterial DNA fragment can be derived from any part of the chromosome
(hence the name generalized transduction). It takes 52 transducing phages to
cover the entire bacterial chromosome (4369/ 90). Therefore the lysate has
enough transducing particles covering the entire genome.

When a transducing phage infects new host cell it transfers only chromosomal
DNA which may either recombine with the recipient‟s chromosomal DNA or is
degraded. Such an infection produces no phage particles. The frequency of
transduction for any given bacterial gene is about 1 per 106 phage particles.

5.5.2 Specialized Transduction

Specialized transduction is characterised by transfer of only certain regions of

bacterial chromosome between bacteria through a phage. The markers
transferred by a given virus depend on specific point of its integration in the
host chromosome which varies from one virus to another. Thus different
phages carry different parts of the bacterial chromosome. The specialized
transducing phages are produced due to imprecise excision (illegitimate
recombination) of the viral DNA upon induction. The phage head packages
both phage as well as bacterial DNA fragments as a hybrid chromosome.

Bacteriophage lambda (λ) is the best understood specialized transducing

phage of E.coli. It has linear double stranded DNA genome with 12 base pair
91
Block 2 Gene Mapping

cohesive / sticky ends that facilitate circularization and play a role in packaging
of DNA into phage head. Phage λ is a temperate phage which can enter
either a lytic or lysogenic cycle. In the latter decision it first circularizes and
then integrates into host DNA by site-specific recombination using
homologous attachment sites (attB on E.coli and attP on phage DNA).

Fig. 5.11: Lysogenic cycle of phage lambda (Adapted from Genetics: Snustad &
Simmons).

During lysogeny very few phage genes are expressed (prophage) and it
multiplies with the host chromosome and does not produce new virus particles
(Fig.5.11). This may continue for several generations until conditions favor
induction such as exposure to UV or change in nutrient status. Then the virus
excises and enters the lytic cycle. Phage DNA excision is essentially the
reverse of the site-specific integration process and generally yields intact
circular phage and bacterial chromosome. At times excision is imprecise
(recombination occurs at a site other than the original attachment site) and
results in transducing phages carrying chromosomal markers present on
either side of the integration site (gal or bio locus) and it may leave few phage
genes. Such a phage is defective (λ dgal or λ dbio) and requires a wild type
helper virus for infection.

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Unit 5 Microbial Genetics

The excised phages express their genes in a defined sequence to finally

assemble mature particles which are released by lysis of the host cell. Most of
the virus particles in the lysate have normal λ and only few transducing
particles (1in 105 or 106 particles). Such a lysate is a low frequency
transduction (LFT) lysate.

The infection of the defective transducing phage and helper phage initiates
another cycle. The defective phage (λ dgal) may either recombine with the
homologous chromosomal marker on the bacterial chromosome (gal-) or
integrate along with the helper DNA forming an unstable double lysogen
(Fig.5.11). In the former case a stable gal+ transductant is generated while in
the latter the double lysogen excises to enter the lytic cycle and produces a
lysate that has both normal and defective phages at almost equal frequency
(high frequency transduction or HFT lysate).

Fig. 5.12: Specialised transduction (Adapted from Genetics: Snustad & Simmons).

5.5.3 Gene Mapping using Generalised

Transducing Phage
Generalised transducing phages can be used to determine if two genes are
close to each other on the bacterial chromosome. The basic procedure is to
select the phenotype of one of the genes in a transduction. Among the
transductants that have inherited the first gene are screened for the
inheritance of the second unselected gene. The frequency of co transduction
(%) gives a measure of their relative distance although it is not very accurate.
The co transduction frequencies can be converted to physical distance in kilo
base (Kb) by using Wu’s formula:

C= (1-d / L) 3

93
Block 2 Gene Mapping

C: Co transduction frequency (expresses as decimal; Ex: 50%=0.5)

d: distance between the selected and unselected marker in minutes
L: Size of the transducing fragment in minutes (P1 carries 90Kb=2min)
Once d is calculated in minutes, then it is converted to kb (45kb is ~1 min).

The two factor strategy can be extended to 3 or more genes in a cross to

determine gene order.

SAQ 5
a) What is a phage?

b) Name a generalized and specialised transducing phage and their hosts.

5.6 SUMMARY
 DNA can be transferred between bacteria by three parasexual
processes viz, conjugation, transformation and transduction. The
transfer of genetic material is unidirectional (donor to recipient) and
homologous recombination occurs between donor DNA fragment(s) and
the main chromosome of the recipient.

 These three processes can be easily distinguished by testing whether it

is dependent on cell-cell contact and nuclease sensitivity.

 Transformation was the first kind of genetic exchange described in

bacteria by F. Griffith (1928). It is the uptake of naked single stranded
DNA fragment(s) from surrounding medium by competent recipient cells
followed by recombination between region(s) of homology with the
recipient„s main chromosome.

 Conjugation is a mating process between a donor (F+, Hfr or F‟) strain

and a recipient (F-). The transfer of single stranded DNA is
unidirectional and recombination occurs in the recipient. The latter may
or may not become a donor depending upon the donor strain.

 Transduction is the process of moving bacterial genes from one bacterial

cell (donor) to another bacterium (recipient) by a bacteriophage.

 The bacteriophages are classified into generalized transducing and

specialised phages depending on whether they can transfer any part of
bacterial the chromosome or only few genes, respectively. Transducing
phages are produced due to errors in the phage life cycle.

 All three gene transfer processes have been exploited to map bacterial
genes.

5.7 TERMINAL QUESTIONS

1. Compare the following pairs:
a) Specialised and generalised transduction.
b) Hfr x F- Vs F+ X F- mating
2. The genes, A, B, G, H, I and T were tested in all possible pairs for co-
transduction with phage P1. The following pairs were found to co-
transduce: G and H; G and I; T and A; I and B; A and H. What is the
94 order on these genes on bacterial chromosome?
Unit 5 Microbial Genetics
3. Given that bacteriophage λ has a genome size of 50Kb, what is the
approximate genetic length of prophage in minutes? (Hint: The genetic
map of E. coli is divided into 100 minutes).
4. In E.coli the following mating was carried out:
Hfr lac+ gal+ trp+ his + Str-s x F- lac- gal- trp- his- str-r
Which medium will select for (a) lac+; (b) his + recombinants?
5. a) A transformation experiment is carried out using a donor that is
A+B+C+ and the recipient is A-B-C-. A+ transformants are selected
and tested further. Of these 64% are B+ and none are C+. Also B+
cells are selected, and 8% are also C+. What is the gene order?
b) A student prepared competent bacterial culture for transformation.
On testing he obtained 8,000 colonies on incubating with
8picogram of DNA, What is the transformation efficiency (colonies
/ microgram of DNA)?

5.8 ANSWERS
Self-Assessment Questions
1. a) Transformation, conjugation and transduction.
b) Test nuclease sensitivity and need for cell-cell contact.
2. a) Competence is the ability of certain bacterial species to take up
naked DNA from the surrounding medium at a high frequency. In
Gram positive bacteria it occurs at high cell density.
b) Griffith found that something released from the heat killed bacteria
can transform the live bacteria and the change was heritable.
Refer to section 5.3.1for details.
c) Dilution test is used to measure co-transformation.
3. a) DNA elements such as F factor which can exist either as free
autonomously replicating molecules or integrated into the main
chromosome are called episome.
b) A culture of F+ cells invariably has few Hfr cells which are
responsible for transfer of chromosomal markers.

4. a) Selected marker: It is originally present in the Hfr parent and is

indispensable for survival of recombinant F- progeny in the
selection medium.
Counter selected marker: It is present in the F- progeny and
prevents (counter selects) the survival of the Hfr parent in the
selection medium.
Unselected marker: It is also present originally in the Hfr parent
and is used to select recombinants in prototrophs.
b) Option (ii)
5. a) Phages are viruses that infect bacteria.
b) Generalised transducing phage: P22 of Salmonella typhimurium
Specialised transducing phage: Phage λ of Escherichia coli
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Block 2 Gene Mapping

Terminal Questions
1. a) Generalised transduction: These phages transduce all regions of
bacterial chromosome; arise due to packaging errors; a
transducing phage particle has only bacterial chromosomal
fragment; viral DNA integration is not a prerequisite; they are
useful in mapping bacterial genes.
Specialised transduction: A specialised transducing phage carries
only chromosomal genes close to the site of integration; arise due
to excision errors; the phage particles packages both viral and
bacterial genes as a hybrid chromosome; phage must integrate as
a prophage; can at best be used to map att sites and close by
markers.
b) Hfr x F- : Chromosomal markers are transferred at high
frequency; recipient remains a donor.
F+ x F-: Chromosomal markers are transferred at low frequency;
recipient becomes a donor.
2. Logic: Cotransduction indicates close linkage like G and H are close but
not H and I. Map is:
B-I-G-H-A-T or T-A-H-G-I-B
3. E. coli genome has 4.6 x 106 base pairs = 100 min on the map.
Therefore, 50Kb is approximately 1minute.
4. a) Medium has streptomycin, lactose, histidine and tryptophan.
b) Medium has streptomycin, tryptophan, glucose (or any other).
5. a) Gene order: A------B---------C [1st selection: B is nearer to A;
2nd selection: B and C co transform and not B and A].
b) Given 8000 colonies / 8pg of DNA; 109 colonies / μg of DNA
Terminal Que. No.2 to 5 is taken from Genetics by Hartl & Jones;
Microbial Genetics by Maloy, Cronan, Jr. and Freifelder.

5.9 FURTHER READINGS

1. Trun, N and Trempy, J. Fundamental bacterial genetics, 1st Ed, 2004,
Blackwell Science Ltd.
2. Maloy, S.R, Cronan, Jr. J.E and Freifelder, D. Microbial Genetics, 2nd
Ed, Narosa publishing house.
3. Brooker, R.J. Genetics: Analysis and Principles, 3rd Ed, 2009, McGraw-
Hill.
4. Snustad, D.P and Simmons, M.J. Principles of Genetics, 3rd Ed, 2003,
John Wiley and sons, Inc.

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Unit 6 Linkage, Crossing Over and Mapping Techniques

UNIT 6
LINKAGE, CROSSING OVER
AND MAPPING
TECHNIQUES

Structure
6.1 Introduction Centromere Mapping with
Ordered Tetrads
Expected Learning Outcomes
Cytogenetic Mapping in
6.2 Linkage and crossing over
Drosophila using Deletion and
Discovery of linkage Duplication Mutants

Linkage vs Independent 6.4 Gene Mapping in Humans

Assortment
Human Pedigree Analysis
Physical Basis of Separation of
Somatic Cell Hybridization
Linked Genes
6.5 Summary
Genetic or Recombination
Mapping in Eukaryotes 6.6 Terminal Questions

6.3 Specialised Mapping 6.7 Answers

Techniques
6.8 Further Readings

6.1 INTRODUCTION
The chromosomal theory of heredity provided the cellular basis for Mendel’s
laws of heredity. But it raised an equally important question, viz, we expect to
have more genes than the limited number of chromosomes in any organism’
so why linked (group) transmission is not observed? It was therefore difficult to
explain why gene pairs only assort independently until the first departure from
independent assortment of two genes pairs was reported by William Bateson,
Edith Rebecca Saunders and Reginald Crundall Punnett in 1905.
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Block 2 Gene Mapping

Even before deviations from Mendel’s results were reported, Hugo de Vries in
1903 proposed the theory of factor exchange during meiosis His idea did not
gain acceptance until Morgan’s experiments with X-linked markers in
Drosophila supported it. Subsequently Alfred H. Sturtevant invented the
technique of genetic mapping based on recombination frequencies.

This unit deals with transmission of linked genes; physical basis of separation
of linked genes; genetic mapping in eukaryotes including humans and
specialised mapping techniques.

Expected Learning Outcomes

After studying this unit, you should be able to:

 Explain linked transmission of genes;

 Distinguish independent assortment from linkage in genetic

crosses;

 explain how linked genes are separated;

 indicate steps in genetic mapping;

 describe specialised mapping techniques in Neurospora and

Drosophila;

 appreciate the problems of gene mapping in humans; and

 describe gene mapping in humans.

6.2 LINKAGE AND CROSSING OVER

The rediscovery of Mendel’s work was followed by Sutton-Boveri’s hypothesis
(1902) which provided a cellular basis for his principles, if it is assumed that
Mendelian factors (genes) are present on chromosomes. But it raised an
equally important question that is, ‘there are more genes than the limited
number of chromosomes in any organism’. At that time it was difficult to
explain why gene pairs only assort independently. Even experiments
conducted around that period in peas and other organisms behaved in a
typical Mendelian fashion.

In the absence of the expected group transmission (linkage) some biologists

believed that there may be a complete breakdown of chromosomes during
meiosis into individual factors (genes) and subsequently they reassembled
independently. In other words factors on the same chromosome also
assort independently. This and other possible explanations prevailed till the
first departure from the 9:3:3:1 ratio was reported in 1905. It was later
demonstrated experimentally that crossing over is responsible for generation
of recombinant gametes in case of linked genes.
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Unit 6 Linkage, Crossing Over and Mapping Techniques

6.2.1 Discovery of Linkage in Sweet Peas

(Lathyrus Odoratus)

The first deviation from independent assortment of two genes pair was
reported by William Bateson, Edith Rebecca Saunders and Reginald
Crundall Punnett in 1905 (Fig. 6.1). They crossed two varieties of sweet
peas (Lathyrus odoratus) that differed in flower colour (purple or red) and
pollen length (long or round). All F1 plants had purple flower and round pollen
indicating purple flower, long pollen is dominant over red flower and round
pollen, respectively. The F2 plants obtained by F1xF1 selfing had more parental
types (89%) and fewer recombinants (11%). At that time they could not explain
these results and suggested preferential multiplication of certain gametes after
meiosis, rather than chromosomal exchange.

Fig. 6.1: Non Mendelian assortment of two gene pairs in Lathyrus odoratus.

Now if the same cross was repeated with true breeding parents having purple
flowers, round pollen and red flower, long pollen, the F1 results are identical
but in F2, the dominant class is again parental types which incidentally were
the recombinants in the preceding experiment. They coined the term coupling
and repulsion to explain these results. They said A and B tend to enter the
same gamete if they come from the same parent (AB/ab; coupling or cis) or
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Block 2 Gene Mapping

enter different gametes if they come from different parents (Ab /aB; repulsion
or trans-configuration). The slash (virgule) notation is used for linked genes.

Edith Saunders William Bateson Reginald Punnet

The discovers of linked transmission

6.2.2 Linkage vs independent Assortment

Genes on the same chromosome may show complete or incomplete
linkage. Generally linkage is incomplete; only few cases of complete linkage
are known. On the other hand independent assortment occurs when genes
pairs considered are present on non homologous chromosomes or they are
far apart on the same chromosome (as you will learn in this unit). Two genes
are said to be syntenic if they are on the same chromosome regardless of
whether they show independent assortment or linkage.

A test cross (a heterozygote crossed is with homozygous recessive parent) is

used to distinguish whether two or more genes are linked (complete /
incomplete) or they assort independently. If two genes pairs are linked then
the test cross results of a double heterozygote depends on their configuration
(cis Vs trans) and the extent of linkage (Fig. 6.2).

Fig. 6.2: Test cross results for linked markers.

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Unit 6 Linkage, Crossing Over and Mapping Techniques

A test cross for linked genes (crosses I and II) will produce only two types of
progenies in equal proportion if linkage is complete whereas incomplete
linkage results in four types of progenies in unequal proportions (parental >
recombinants). In addition, the configuration of gene pairs dictates the classes
which will be parental or recombinants although the recombination frequency
will remain unchanged in cis or trans-configuration (Fig.6.2). It is rare to
encounter complete linkage; one such example is of Drosophila males.

Another possibility is that gene pairs assort independently. You know this can
happen when A & B are on non homologous chromosomes or if they are far
apart on the same chromosome. A double heterozygote (AaBb) when test
crossed will produce four types of progenies in equal proportion and it is
immaterial whether A & B come from the same or different parent(s).

6.2.3 Physical Basis of Separation of Linked Genes

Hugo de Vries in 1903 proposed the theory of factor exchange even before
deviations from Mendel’s results were reported. He suggested that in
prophase I of meiosis the paired homologous chromosomes may exchange
material. This idea did not gain acceptance until Morgan’s experiments with X-
linked markers in Drosophila supported it. He crossed white eyes, miniature
wings males with wild type females and F1 progeny flies were intercrossed or
F1females were test crossed. In the former cross F2 males are scored as their
phenotype is dependent on the meiotic events in the female parent and in the
latter phenotype of both sexes are scored. Morgan obtained approximately
37% recombinants and suggested that they arise due to crossing over in the
F1 females.

A definitive proof that recombination is associated with a physical exchange of

segments between homologous chromosomes was first demonstrated by
Harriet Creighton and Barbara McClintock (1931). They used two linked
markers (controlling kernel color and texture) on chromosome 9 of maize; one
of the homolog had a heterochromatic knob attached in the short arm and
translocation from chromosome 8 at the other arm. These ends served as
visible markers that could be seen under the microscope. The recombinants
obtained carried only one of the abnormal cytological markers confirming
chromosome exchange. Similar experiments with structurally abnormal X-
chromosomes in Drosophila were also reported by Curt Stern in 1933 and
arrived at the same conclusion.

SAQ 1
i) Define the terms linkage and syntenic genes

ii) Indicate two ways of generating recombinant gametes.

a) ………………… b) …………………..

6.2.4 Genetic or Recombination Mapping in Eukaryotes

The technique for relative mapping of genes on chromosomes was invented
by Alfred H. Sturtevant. He envisaged that the strength of linkage between
genes is related to their relative distance; closer the genes stronger the
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 Block 2 Gene Mapping

linkage. The frequency of recombinants is therefore dependent on their

relative distance. The genetic maps are also called linkage or
chromosome maps.

Alfred H. Sturtevant was an American geneticist who

is credited for constructing the first genetic map in
1913. He realized that if frequency of crossing over
was related to distance as proposed by Morgan, then
one could use this information to map genes on a
chromosome. He succeeded in mapping a number of
X-lined genes in Drosophila. He received his PhD
degree (1914) from Columbia University and in 1928
he moved along with Morgan to California institute of
technology and stayed in the famous ‘fly room’ till his
end. Throughout his life he worked with T.H. Morgan
on Drosophila.
Source: (a) britanica.com/ biography/ Sturtevant (b)
www.caltech.edu

Alfred Henry Sturtevant (1891-1970)

Cross between A three point cross is used for genetic mapping as it provides a better estimate
individuals who are of the distance between genes by scoring even the double crossovers (DCO).
heterozygous for three Let us analyse the data of a three point cross and learn how it was used to
markers and those who map genes on Drosophila X chromosome (Bridges and Olbrycht, 1926). The
are either homozygous F1 females were crossed to male’s mutant for all three characters.
recessive or hemizygous P: + ec + / + ec + x sc + cv / Y
at these loci.

F1: + ec + / sc + cv x sc ec cv / Y
Note: sc –scute (certain thoracic bristles missing); cv- crossveinless (cross
vein missing); ec- echinus (rough eyes)

F2 (eight phenotypic classes)

Table 6.1: F2 results of a three point cross

S.No F2 phenotypes Observed numbers

1 Echinus 8576

2 Scute; crossveinless 8808

3 Crossveinless 716

4 Scute; echinus 681

5 echinus; crossveinless 1002

6 Scute 997

7 Scute; echinus; crossveinless 4

8 Wild type 1

Total 20,785
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Unit 6 Linkage, Crossing Over and Mapping Techniques

Now how do we analyse this data and calculate the distance between them.
Let us work on it step by step.

Step (I): The most informative classes in mapping are those that are
maximally represented (non crossover or parental) and least represented
(DCO). The parental classes tell the linkage phase (cis Vs trans) of the alleles
in the F1 parent and the DCO is used to determine the gene order.

Step (II): In this case the phenotype of the parental classes is echinus and
scute and crossveinless. So the F1 female inherited one homolog with two wild
type alleles (sc+; cv+) and one mutant gene (echinus, ec) whereas the other
homolog she had two mutants (sc and cv) and ec+ . Together the parental
classes account for 17384/ 20785= 83.64% of the progeny flies.

Step (III): The next step is to find out which of the three genes is in the middle.
To know the gene order, compare the parental and DCO classes. Draw the
heterozygous F1 parent and place one by one each of these in the middle and
perform a DCO. The one which gives the phenotype of the least represented
classes is the gene in the middle. The effect of a DCO is to interchange the
middle pair of alleles between the homologs.

The gene order is found to be:

sc---ec----cv or cv------ec------sc

Step (IV): Once the gene order is known we have to work with middle four
classes mentioned in Table 6.1 (3-6). They are the single cross over (SCO) The map distance
classes; 3 and 4 is SCO between sc-ec and 5 and 6 is SCO between ec-cv. between genes in a
genetic map is related to
the frequency of crossing
over between the genes
Step (V): Now we are ready to calculate map distance between sc and ec and during meiosis.
ec and cv.

The % of recombinants between ec and ec are: (716+681) / 20785 = 6.72%

and between ec and cv are: (1002+997) / 20785 = 9.62%

Step (VI): Remember that DCO recombinant classes result from two
exchanges one in each of the chromosome region of the three genes.
Therefore, the recombination frequency between the two genes must include
the DCO frequency (5/ 20785=0.02%).

Step (VII): The unit of distance in a genetic map is called a map unit (mu) or
centi- Morgan (cM) in honor of T.H. Morgan and over short distances it is
equal to 1% recombination (which means 2% crossover, as every cross over
generates two recombinants and two parental chromatids).

The distance between two genes on the genetic map actually refers to the
average number of crossovers between them. It has two important effects-(i)
formation of chiasmata in late prophase (counted cytologically) and (ii)
recombination of the flanking alleles (scored as recombinants in the next
generation).
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 Block 2 Gene Mapping

Therefore, the map distance between sc-ec is 6.72+0.02= 6.74 cM and

between ec-vv is 9.62 +0.02= 9.64cM

Genetic map is sc--------------ec---------------------cv

6.74cM 9.64cM

Genetic map distances are additive and they are build by adding
recombination frequencies obtained by considering close by genes. In the
cross analysed above the distance between sc and cv is (6.74 + 9.64)
=16.38cM. An important consequence of this is that although maximum
recombination frequency in a cross cannot exceed 50% but genetic maps can
be more than 50 map units.

The next question is to find out whether the two crossovers in a DCO class
occur independently. To begin with let us calculate the expected and observed
frequency of DCO.

From the data given in table 1, total number of flies scored is 20,785. You
know that the frequency of recombination between sc and ec is 0.0674 and
between ec and cv is 0.0964.

If two crossovers occur independently between the three gene pairs then the
expected frequency of DCO is the product of their individual frequencies:

0.0674 x 0.0964 = 0.00650

This corresponds to 20,785 x 0.00650 = 135 flies

But, the number of flies in DCO classes was only 5 (5/ 20,785= 0.00024)

These results suggest that crossover in one region interferes with another
crossover in its vicinity (chromosome interference).
Heterochromatin is
transcriptionally inactive Interference = (1- coefficient of coincidence)
condensed chromatin. It
may be either constitutive The coefficient of coincidence is the ratio of observed number of double
/ facultative. The recombinant chromosomes by expected number.
frequency of crossing
over especially in highly Coefficient of interference is = 0.00024 / 0.0065 or 5 /135
condensed regions is
much is lower than = 0.037 =3.7%
euchromatin.
Interference = (1-0.037) = 0.963

Thus due to interference the expected DCO are prevented 96.3% of the time.
In general the degree of interference depends on the distance between the
genetic markers and the species studied.

The recombination mapping technique described here can also be extended

to map autosomal markers. In this case the triply heterozygous parent is test
crossed and then the progeny are segregated into eight classes for
determining the relative map distance. The number of linkage groups is equal
to the haploid chromosome number of a species.

The genetic mapping technique suffers from inherent limitations as it depends

104 on crossing over to produce recombinants. It is well known that crossing over
Unit 6 Linkage, Crossing Over and Mapping Techniques

is not uniform along the length of chromosomes (euchromatin >

heterochromatin) or between chromosomes; it differs among sexes (females >
males) and between species. There are regions in chromosomes where it
occurs with much higher frequency (hot spots). Therefore map distances are
relative and may differ considerably from the actual physical map (base
sequence) although they are generally collinear.

SAQ 2
i) How many linkage groups are there in humans?

ii) Genetic maps can be more than 50 map units although the maximum
frequency of recombination never exceeds 50%. Explain.

iii) What is meant by the term chromosome interference?

6.3 SPECIALISED MAPPING TECHNIQUES

In the previous section you learnt about linkage and independent assortment.
We also discussed how genetic mapping is done in eukaryotes. In this section
we shall discuss centromere mapping in Neurospora crassa and cytogenetic
mapping using deletions and duplications in Drosophila melanogaster. It is
possible to map centromere in Neurospora by simply analyzing the products of
meiosis as they are arranged in an ordered array that reflects the sequence in
which they were formed. Similarly Drosophila larvae have large polytene
chromosomes in their interphase nuclei. These chromosomes have a
consistent pattern of light and dark bands which have been extensively
mapped allowing investigators to pinpoint the position of deletions and
duplications. Let us now consider centromere mapping in detail.

6.3.1 Centromere Mapping with Ordered Tetrads

The bread mold Neurospora crassa is a heterothallic filamentous fungi (forms
filaments of different mating types). It grows as a branching mass of haploid
thread like hyphae called a mycelium and forms asexual spores (conidia).
These spores can give rise to more hyphae or participate in fertilisation with
spores of different mating types (A and a). Both mating types form
protoperithecium in which many fertilized cells (2N) undergo meiosis shortly
after they are formed. The zygote is contained in a sac like ascus and
constraints of space make the products of meiosis to align in a linear fashion
exactly the way they were produced. The four haploid products then undergo
a round of mitosis forming eight cells (octad); two genetically identical
adjacent spores. The eight nuclei are finally partitioned into individual cells
(ascospores), which can be carefully removed and germinated to determine
their phenotype as there is no dominance effect.

You know that centromeres do not have a phenotype and so it is mapped in

relation to close by markers. If we consider a single heterozygous marker (Aa),
there are two possible segregation patterns depending on whether there was
no crossover between the gene and centromere or there was a crossover.
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 Block 2 Gene Mapping

Let us have a closer look at the two segregation patterns depicted in Fig.6.3.
When there is no crossover between the gene and centromere, the alleles A
and a separate in the first meiotic division followed by sister chromatid
separation at meiosis II. Each of these four products then enters a round of
mitosis. The resultant octad preserves the order in which these events
occurred; it is called 4:4 (AAAAaaaa or aaaaAAAA / M-1 / first division
segregation (FDS) pattern. But if there is a crossover between the
centromere and the gene, then the two alleles will move to the same cell at the
end of meiosis I. The two alleles will segregate only at meiosis II. The resultant
pattern after a round of mitosis is 2:2:2:2 (AAaaAAaa or aaAAaaAA) or 2:4:2
(AAaaaaAA or aaAAAAaa) pattern. It is also called M-II / second division
segregation pattern (SDS). The random alignment of homologous
chromosomes at metaphase accounts for two patterns for FDS and four for
SDS.

The maximum
frequency possible for
second division
segregation pattern is
2/3 (66.7%).
Fig. 6.3: Segregation patterns in Neurospora crassa.

The frequency of asci with SDS allows us to map the gene with respect to the
centromere. The map distance (cM) between a gene and its centromere is
calculated using the formula given below:

1 Asci with second division segregation patterns

Map distance = X X 100
2 Total number of asci

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Unit 6 Linkage, Crossing Over and Mapping Techniques

Furthermore, in each cell in which crossing-over takes place, two of the

chromatids are recombinant and two are non recombinant. By convention,
map distance refers to the frequency of recombinant meiotic products rather
than to the frequency of cells with crossovers.
Centromere mapping can be extended to more than one heterozygous pairs
and one can even determine from the segregation pattern whether they are
unlinked; present on same or opposite sides of the same chromosome.

SAQ 3
In Neurospora crassa tetrad analysis was performed to map gene A with
respect to centromere. A total of 300 asci were analysed; out of which 258 had
FDS pattern and the rest showed SDS. What is distance of gene A from the
centromere?

6.3.2 Cytogenetic Mapping with Deletion and

Duplication Mutants in Drosophila
Cytogenetic mapping allows us to map genes in relation to cytological markers
on chromosomes such as light and dark bands. It involves staining of
chromosomes with stains like Giemsa followed by microscopic observation of
the chromosome morphology and number. These visual studies of
chromosomes are simple and helpful tools to identify major chromosomal
aberrations like changes in chromosome number and structure (deletion,
duplications, etc).

Chromosomal deletions and duplications (insertions) have been extremely

useful in finding and mapping genes on the cytological maps of Drosophila
chromosomes. Over the years flies with these aberrations are maintained and
readily made available to researchers. The primary advantage with Drosophila
is the presence of large polytene chromosomes in interphase nuclei of larval
salivary glands and few other tissues. These chromosomes produce a
consistent pattern of bands and inter bands which can be easily visualised in
stained preparations under the light microscope. Calvin Bridges made
accurate cytological maps of polytene chromosomes which are used even
today. The banding pattern is characteristic of each Drosophila species.
Chromosomal changes like deletions and insertions of DNA segments can be
identified by studying the change in banding pattern and its number.

In deletion mapping a deletion that unmasks a recessive mutation in a

heterozygote (it has a mutant gene on one homolog and a deletion which may
or may not include a wild type of the gene being investigated) lacks a wild type
copy of the mutant gene. The gene under consideration is therefore present
within the boundaries of the deleted region. By using smaller deletions in that
region the gene of interest can be mapped to a given band. On the other
hand if the deletion does not include the wild type of the gene then the
recessive mutation is masked.

In duplication mapping a duplication which masks a recessive mutation carries

a wild type copy of the mutant gene. This allows us to localise the gene within
the boundaries of the duplication. Using a panel of deletions and duplications
the gene for white eye color was mapped on the X-chromosome by
cytogenetic mapping.
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Block 2 Gene Mapping

SAQ 4
What is the basic difference in the principle of deletion and duplication
mapping?

6.4 GENE MAPPING IN HUMANS

In humans gene mapping by traditional methods involving controlled mating
experiments is not feasible and they produce fewer progenies. In this section
we shall learn about pedigree analysis and somatic cell hybridization to map
genes on chromosomes and establish linkage where possible.

6.4.1 Human Pedigree Analysis

One of the earliest alternatives used to study linkage in humans is pedigree
analysis in which the inheritance of particular characters is analysed in a
family. In a multi locus analysis it is possible to look for group transmission;
calculate the strength of linkage and then construct a chromosome map. In
general the study of linkage of X- linked genes was simpler than the genes on
autosomes.

One of the earliest reported and best understood examples of linkage

between autosomal genes in human pedigrees was of gene controlling ABO
blood group and nail-patella syndrome (Fig. 6.4).

Fig.6.4: A human pedigree showing linkage between ABO blood groups and
nail-patella syndrome.

Pedigree analysis of nail-patella syndrome in humans indicates

that it is due to an autosomal dominant mutation and
heterozygotes have a mutant phenotype. The unaffected
individuals are homozygous recessive. The salient features of the
pedigree showing linkage between nail patella syndrome and ABO
108 alleles are:
Unit 6 Linkage, Crossing Over and Mapping Techniques

(a) In the second generation, each person (except II-8) with nail-patella
syndrome (NPS I) also has blood group B indicating linkage. The unaffected
individual means that the affected parent is heterozygous.

(b) The linkage between NPS and B indicates that the genotype of the female
(I-2) is NPS I- B / + O (repulsion heterozygote) and the male is +O /+ O.

(c) The woman (I-2) produced four types of gametes which fused with the
single type of male gamete to produce four types of offsprings (three
recombinants; II-5, II-8 and II-14 and 8 non recombinants; P>R). A rough
estimate of frequency of recombination is 3/11 = 27%

(d) In generation III, child (III-3) is a recombinant; unaffected (+/ +) with B

blood group (B/O). Since these early studies the map distance has been
revised and is now estimated to be 10cM. The genes are located near the tip
of the long arm of chromosome 9.

At present differences in DNA sequences are used as molecular genetic

markers in pedigrees that are segregating for a particular gene. The members
of the pedigree are analysed both for the presence or absence of the markers
along with the phenotype investigated. The linkage between the marker and
the gene is calculated using statistical techniques.

Genetic mapping by pedigree analysis is not free from problems, for instance
genes that cause genetic disorders are very rare; most of the genes of interest
are recessive, so they are masked in heterozygous state and family records
are generally incomplete. Our inability to control matings and fewer numbers
of progenies further makes analysis difficult.

6.4.2 Somatic Cell Hybridization

Somatic cell hybrids are formed by fusing cells from two different cell lines in
the presence of Sendai virus or polyethylene glycol such as mouse-human
somatic cell hybrids. The hybrid initially has complete set of chromosomes
from both sources but as it divides it loses chromosomes derived from one
species (in this case human). After few divisions the chromosome numbers
are stabilised with each hybrid retaining one or few human chromosomes. The
loss of chromosomes is random therefore a panel of hybrids are available.
These are used for assigning specific genes to a human chromosome. HGPRT stands for
hypoxanthine, guanine
The selection of hybrids from large number of unfused cells or same species phosphoribosyl
fusions is facilitated by using cell lines that will not survive in HAT transferase.
(hypoxanthine, aminopterin and thymidine) medium and only hybrid cells TK is an abbreviation for
survive. These cell lines are either HGPRT- (mouse cells) or TK-(human cells). thymidine kinase.
The hybrid survives because it can salvage hypoxanthine and thymidine when
denovo pathway is inhibited by aminopterin.

Once the hybrid is selected it is characterised by cytogenetic techniques which

helps to distinguish human and mouse chromosomes and identifying the
human chromosomes retained in a given hybrid line. Using a panel of cell
lines and whether they express a given gene product, for example thymidine
kinase, one looks for the chromosome common to all cell lines expressing the
gene product of interest. If a cell line lacks that chromosome then the gene
product is missing. So the gene is present on that chromosome.
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Block 2 Gene Mapping

With the advent of molecular techniques it is possible to locate the position of

genes on specific chromosomes by using fluorescent labelled DNA specific
probes and assess the chromosome to which it hybridises (in situ
hybridization). It circumvents the need for expressed products. Further if two
DNA markers are repeatedly found on the same chromosome, then it means
that they are linked. You will learn these techniques in molecular biology and
recombinant DNA technology courses.

SAQ 5
Indicate the difficulties faced in mapping human genes.

a) ………………………… b) ……………………….

6.5 SUMMARY
 The first departure from independent assortment of two genes pairs was
reported by William Bateson, Edith R. Saunders and Reginald C.
Punnett in 1905 while investigating the inheritance of flower colour and
pollen shape in sweet peas.

 Genes on the same chromosome are more likely to be inherited

together. Linkage is generally incomplete. Recombinant gametes are
produced as a result of crossing over between homologous
chromosomes during meiosis I.

 A test cross of F1 dihybrid yields more parental than recombinants in

case of linked genes. In addition, the configuration of gene pairs dictates
the classes which will be parental or recombinants although the
recombination frequency will remain unchanged in cis or trans-
configuration.

 The technique for relative mapping of genes on chromosomes was

invented by Alfred H. Sturtevant. He envisaged that the strength of
linkage between genes is related to their relative distance; closer the
genes stronger the linkage. The frequency of recombinants is therefore
dependent on their relative distance.

 A recombination frequency of 1% means that only one out of 100

offspring was the combination of two genes different from that in their
parents. 1% recombination = 1map unit (cM)

 The maximum recombination frequency in a cross cannot exceed 50%

but genetic maps can be more than 50 map units because they are
constructed by adding map distances of close by markers.

 Neurospora crassa forms ordered octad in which the products of meiosis

are aligned in the way they were formed. The segregation pattern of
various asci is used to map centromere with respect to a close by gene
using the relation:
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Unit 6 Linkage, Crossing Over and Mapping Techniques
1 Asci with second division segregation patterns
= X X100
2 Total number of asci
 Chromosomal deletions and duplications have been extremely useful in
finding and mapping genes on the cytological maps of Drosophila
chromosomes. Chromosomal changes like deletions and insertions of
DNA segments can be identified by studying the change in banding
pattern, especially in large polytene chromosomes.

 In humans gene mapping by traditional methods involving controlled

mating experiments is not feasible and they produce fewer progenies.
Some of ways used for gene mapping include pedigree analysis,
somatic cell hybridization and a host of molecular biology tools (in situ
hybridisation, DNA sequencing).

6.6 TERMINAL QUESTIONS

1. Explain how centromere is mapped with respect to a close by gene in
Neurospora crassa.

2. How will you ascertain whether the double heterozygote is in the

coupling or repulsion phase?

3. Genetic mapping of three recessive mutations affecting body colour,

wing shape and bristle morphology in Drosophila was carried out. The
mutants had black body colour (b), dumpy (dp) wings and hooked (hk)
bristles at the tip. In a cross between dumpy female and black body
colored, hooked male, all F1 were wild type for all three characters. The
test cross results of an F1 female with a dumpy, black, hooked male gave
the following results:

Wild type 169

black 19

black, hooked 301

dumpy, hooked 21

hooked 8

hooked, dumpy, black 172

dumpy, black 6

dumpy 304

Total 1000

a) Construct a genetic map and give the map distance between them.

b) Determine the coefficient of coincidence and the extent

of interference.

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Block 2 Gene Mapping

4. Explain how somatic cell hybridization is used to localise genes to

specific chromosomes.

5. What is the principle of HAT selection?

6.7 ANSWERS
Self Assessment Questions
1. i) Linkage: When two genes are close by on the same chromosome
(linked) they tend to be transmitted together more often and are
said to exhibit linkage.

Syntenic genes: Two genes are syntenic if they are on the same
chromosome regardless of whether they show independent
assortment or linkage.

ii) a) Independent assortment; b) Crossing over

2. i) 23 (haploid chromosome number of a species)

ii) Genetic maps can be more than 50 map units because they are
constructed by adding recombination frequencies of markers
across shorter intervals (to avoid multiple crossovers).

iii) Chromosome interference is the tendency of one cross over to

inhibit another cross over in its vicinity.

3. Given: Total number of asci: 300; FDS= 258 (86%) and SDS = 42 (14%)

Distance of gene A from centromere = ½ x 42 / 300 =0.07 or 7mu

OR

You know in a cross between centromere and gene A, only half the
chromosomes are recombinant (SDS).
Therefore, length in mu = 14 / 2 = 7mu

4. A deletion that unmasks a recessive mutation in a heterozygote or a

duplication which masks a recessive mutation carries a wild type copy of
the mutant gene. In either case the gene of interest is located within the
boundaries of the deletion or duplication.

5. a) Controlled matings are not possible

b) Small number of progenies.

Terminal Questions
1. Neurospora crassa forms ordered tetrads. The tetrads are analysed by
growing them separately and then segregated into FDS and SDS
tetrads. The map distance s calculated by the formula:
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Unit 6 Linkage, Crossing Over and Mapping Techniques
(1/2)(Asci with second 
division segregation patterns)
x 100
Totalnumber of asci

Refer to section 6.3.1 for details.

2. a) Test cross b) Genotype of the parents

3. Step 1: The linkage phase is dp-hk+-b+ / dp+-hk-b (known from the non
crossover classes in test cross / from the phenotype of the parents.

Step 2: Gene order (by comparing parental Vs DCO classes) is dp – b-

hk

Step 3: The frequency of single crossover (SCO) between dp and b is

(169+ 172) =341 progeny flies. The map distance between dp and b is
341/ 1000 x100= 34.1%. Similarly the frequency of SCO between b and
hk is (21+19) = 40 / 1000 x 100= 4%.

Step 4: The proportion of observed number of DCO is (8+6) =14 /1000 x

100 = 1.4%

Step 5: Add DCO % to SCO % to calculate relative map distance. The

distance between dp and b is 34.1+1.4 =35.5 cM and between b and hk
is 4+1.4= 5.4cM.

Genetic map is dp-------------------------------b------------hk

35.5cM 5.4cM

Step 6: Expected number of DCO is 0.355 x 0.054 =

0.01917 (1.917%)

Coefficient of coincidence = 1.4 / 1.917 =0.73

Interference = (1-0.73) = 0.27

4. Refer to subsection 6.4.2 for details.

5. HAT medium allows selection of rare somatic hybrids by eliminating both

unfused cells and fused cells of the same kind such as mouse-mouse.
The medium has hypoxanthine, aminopterin and thymidine. All cells in
this medium are unable to synthesise nucleotides by de novo route due
to the presence of aminopterin but the hybrid cells survives because it is
able to salvage hypoxanthine and thymidine while unfused cells fail to do
so as they are either HGPRT- or TK-.

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6.8 FURTHER READINGS

1. Johnson, George B. Hoe scientists think: Twenty experiments that have
shaped our understanding of Genetics and Molecular Biology, 1996;
Wm. C. Brown publisher. Recommend chapters 3-5.

2. Strickberger, M.W. Genetics, 3rd Ed (1976), Macmillan

Publishing Company, New York.

3. Snustad, D.P and Simmons, M.J. Principles of Genetics, 3rd

Ed (2003), John Wiley & sons, Inc.

4. Hartl, D.L and Jones, E.W. Essential Genetics, 4th Ed;

Jones and Bartlett Publishers.

5. Pierce, B. Genetics: A conceptual approach, 6th Ed. W.H. Freeman

and Company.

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