BT203_Genetics Unit 2

BT0203 Genetics and Cytogenetics

UNIT – I

Terminologies

Karyotype: A Karyotype is an array of chromosomes created by photographing the

metaphase chromosomes from one cell, cutting out the individual chromosomes from the

photograph and lining them up in order from largest to smallest, pairing the appropriate

homologous chromosomes. Chromosomes for karyotypes are often stained using special

procedures which create banding patterns the chromosomes, thus making pairing easier.

Diploid: A diploid nucleus contains two sets of chromosomes (two of each type of

chromosome).

Haploid: A haploid nucleus contains a single set of chromosomes.

Polyploidy: Polyploids contain more than two sets of chromosomes in each nucleus. For

example, bananas are triploid.

Homologous chromosomes: Homologous chromosomes are the same size, the same

shape, and have the same gene map. They are not necessarily genetically identical.

Genome; A genome is an individual organism’s total array of genetic information.

Gene pool: The gene pool is the total genetic diversity of a particular species.

Gene: A gene is a segment of DNA which controls the production of a particular

characteristic. More precisely, a gene is a recipe for the production of a specific kind of

protein.

Allele: Alleles are different forms of the same gene. For any gene, an individual may

possess only two alleles, and a gamete may possess only one. However, the gene pool of

a species may contain many alleles for any gene. Alleles are assigned symbols according

Rex Arunraj Department of Genetic EngineeringAssistant Prof.


to specific rules of convention. All alleles of a particular gene should be given versions of

the same symbol.

Locus (plural Loci): A gene’s locus is its position on a chromosome. All genes have

individual and unique locations characteristic of the species. What makes two organisms

members of the same species is that they have the same assortment of genes, arranged

according to the same gene map on their chromosomes. In other words, their various

genes have the same loci.

Multiple alleles: A gene has multiple alleles if there are more than two different alleles

for that gene in the gene pool. For example, there are three different alleles for the A-B-

O blood type gene in human populations (LA, LB and l).

Genotype: The genotype of an organism is the list of the symbols representing that

organism’s specific genetic constitution—in other words, a list of all the alleles the

individual carries for its genes. In actual usage, a stated genotype typically describes only

one or two genes at a time.

Phenotype: The phenotype is the actual physical expression of an organism’s traits.

Much of the phenotype is the product of the genotype, but environmental influence can

be very important as well. Geneticists discuss the heritability of traits. Heritability is the

expression of the degree to which a particular trait is controlled by heredity.

Homozygous; A homozygous individual has two identical alleles for the gene in

question. For example, BB, bb, AA, PP.

Heterozygous: A heterozygous individual has two different alleles for the gene in

question. For example, Bb, Aa, Pp.

Hemizygous: This term refers to the condition of a gene which is carried on an X or Y

chromosome in a male. Since there can be only one copy of such a gene in the cell, the



terms homozygous and heterozygous are inappropriate. Essentially, hemizygous means

that a gene is present in only one copy.

Complete dominance: If two alleles display complete , it is not possible to tell the

difference between the homozygous dominant individual and the heterozygous

individual. The recessive allele is hidden by the presence of the dominant allele.

Dominant: The dominant allele is the one which is displayed in the phenotype of the

heterozygote. In assigning allelic symbols, the convention is to assign a capital letter to

the dominant allele. Dominant traits can never skip generations in a pedigree, because

they can never be present but hidden—they always show.

Recessive: The recessive allele is the one which is hidden in the phenotype of the

heterozygote. The recessive allele is generally assigned a lower case letter symbol.

Recessive traits can skip generations in a pedigree.

Incomplete dominance: If two alleles show incomplete dominance, the phenotype of the

heterozygote is intermediate between the phenotypes of the two homozygotes. For

example, RR produces red flowers, R’R’ produces white flowers, and RR’ produces pink

flowers. Alleles showing incomplete dominance are typically assigned symbols which are

variations of capital letters.

Co-dominance: If two alleles show co-dominance, the phenotype of the heterozygote

expresses both of the alleles completely. For example, in the A-B-O blood group, the LA

and LB alleles show co-dominance. The heterozygote (Type AB) has all of the bloody

type characteristics of Type A blood as well as those of Type B blood. Again, co-

dominant alleles are generally assigned different versions of the same capital letter for

symbols.

Pseudodominance: Pseudodominance results when a particular genotype is lethal. For

example, curly sings in Drosophila (fruit flies). The heterozygote has curly wings, the



homozygous straight has straight wings (wild type), and the homozygous curly is lethal

(the eggs never hatch). Superficially, the effect looks like complete dominance (the curly

allele appears to be dominant), but upon closer examination is not. By convention, the

homozygous lethal allele is given a capital letter symbol and the pseudo-recessive allele

is given a lower case letter symbol.

Monohybrid cross: This is a mating between two individuals who are both heterozygous

for the one gene which you are following. Eg: Aa x Aa

Dihybrid cross: This is a mating between two individuals, both of whom are

heterozygous for the two genes you are following. Eg: AaBb x AaBb

Test cross: This is the mating between an individual of unknown genotype and a

homozygous recessive individual (eg, B- x bb) for the purpose of exposing hidden

recessive alleles in the unknown parent.

Back cross: Most literally, this is the mating between an offspring and one of its parents.

In practice, it is often a mating between an offspring and an individual of the same

genotype as one of the offspring’s parents.

Linkage: Genes which are carried on the same chromosome are considered to be linked.

When crosses using two linked genes are made, the two genes do not behave

independently, and results do not behave according to simple rules of statistics.

Independent Assortment: Genes which are not linked will behave independently of

each other, and will assort according to simple rules of probability.

Autosomal traits are traits carried on any of the chromosomes other than the sex

chromosomes.



Sex linked traits: This is a special type of linkage. Sex linked genes are carried either on

the X or the Y chromosome (in mammals or fruit flies—in birds, it would be the Z or W

chromosome).

X-linked genes are carried on the X chromosome. Since females have two copies of the

X chromosome and males have only one, rare recessive characteristics which are X-

linked will occur more often in males than in females. Also, since an XY zygote always

inherits its X from its female parent and its Y from the male parent, males inherit all of

their X-linked traits from their mothers.

Y-linked (holandric) genes are carried on the Y chromosome. Since Y chromosomes are

inherited exclusively through the male line, males inherit all Y-linked traits from their

fathers. Y-lined traits found in a father must appear in all of his sons, and all of their sons,

etc. In mammals, maleness is carried on the Y chromosome.

Sex influenced traits: Sex influenced traits are autosomal traits whose expression is

affected by gender. Usually, the alleles are influenced by the presence of certain

hormones which either increase or decrease the effects of the alleles. Eg, pattern baldness

in humans is sex influenced. The gene for this trait has two alleles, the bald allele and the

non-bald allele. The effectiveness of the bald allele is greatly increased in the presence of

high levels of the hormone testosterone. Since this hormone is found in much higher

levels in males than in females, the bald allele is dominant in males and recessive in

females.

Sex limited traits are autosomal traits whose expression is possible only in one of the

genders. These traits generally affect the primary and secondary sexual characteristics.

Eg, cryptorchidism is a condition in males in which one or both testes fail to descend into

the scrotum late in gestation. This characteristic is genetically controlled, by an

autosomal gene. A female can be genetically cryptorchid, but she can’t possibly express

the trait.



Pliotropy refers to genes with multiple effects. For example, the white spotting gene in

gerbils apparently also influences red blood cell count. Another simple example is that

genes which influence characteristics of the fingers will also influence characteristics of

the toes.



Mendelian Genetics

Gregor Johann Mendel is called the father of genetics. He was fond of gardening and

interested in plant hybridization and he performed a No. of experiments with pea plants.

With this experiments he was able to explain the inheritance of characters. His paper

“experiments in plant hybridization were published is 1866 to 1867 in the proceeding of

Natural History Society of Brunn. His work remained unnoticed for 33 years. His work

was recognized only in 1900 by Hugo de Veries – a Dutch biologist, Carl Correns, a

German botanist & Erich Von Tschermak an Austrian Botanist.

Mendel was not the first to conduct hybridization experiments, but just the extension of

the experiments conducted by earlier worker. Like Knight & Goss. There was another

scientist Kolreuter – A German Botanist performed experiments is tobacco similar

observations were made by a group of workers Garther, Dawis, Naudin etc., but they

could not figure out their results numerically as Mendel.

Reason’s for Mendel’s Success

1. The flower of pea plants are normally self fertilized.

2. The pea plant has contrasting characters.

3. Cross pollination was not very difficult.

4. The genes for the seven pairs of characters are located on seven separate

homologies pairs of chromosomes

5. Pure breeding varieties were available of easy to cultivate.

6. Short growth period & growth cycle.

7. He studied the inheritance of only one character at a time.

8. He maintained statistical records of the results, which helped him to derive

numerical ratio’s of significance.



CROSSING TECHNIQUE

Garden pea is self fertilizing; the anthers have to be removed and are called as

emasculation. This stigma is protected against any foreign pollen grain, and then they are

self pollinated.

Character selected by Mendel

Mendel selected seven characters with contrasting alternatives.

No. Character Alternatives

Dominant Recessive

1. Length of the stem Tall Dwarf

2. Position of the flower Axial Terminal

3. Colour of the pod Green Yellow

4. Shape of the pod Inflated Constricted

5. Shape of Seed Round Wrinkled

6. Colour of the Seed coat Coloured White

7. Colour of the cotyledon Yellow Green

Plants with one alternative trait were used as female and those with the other alternative

as male. Reciprocal crosses were also made.

T- tall female plant X t – dwarf male plant.

Reciprocal Cross

T- tall male plant X t –dwarf female plant.

The population obtained as a result of crossing plants exhibiting contrasting characters is

called the first fillal generation or F1 progeny. The progeny of F1 plants obtained due to

self pollinations is called second fillial generations or

F2. Similarly we can have F3, F4 etc.



Results of Mendel’s experiments

When tall plants were crossed with dwarf plants, all plants is the F1, generation were tall.

The plants used are the initial crosses are referred to as P1 & P2 (or) parents. When the

F1, plants are self fertilized, both tall & dwarf plants were obtained in the F2 generation.

The tall and dwarf plants were obtained in the ratio of 3:1 similar patterns were obtained

for other six pairs of character also. To summarize the pattern of inheritance in all the

seven cases.

(i) For any character the F1, individuals from crosses, between two different

varieties having alternative characters, showed only one of the traits & never the

other. This feature was expressed as dominant of one trait over another. The

trait which appeared is the F1 generation was called dominant and the other

which did not appear is the F1, population was called recessive.

(ii) It did not matter which parent variety provided the pollen which provided the

eggs the results were always the same in other words, the reciprocal crosses

gave the same results.

The determining agent responsible for each trait was called a factor.



Monohybrid Cross

Single character each controlled by a single pair of genes or alleles were considered such

crosses are known as monohybrid crosses the F2 ratio 3 :1 is known as monohybrid ratio.

Monohybrid Experiment

The crossing of two plants differing in one character is called monohybrid experiment. A

pure breeding plant is one that which retains a particular character for any number of

generations. A pure breeding tall & dwarf plants were treated as pants & were crossed.

These seeds were sown and a group of plants were raised these plants constituted the first

filial generations or F1, generation all the F1 plants were tall. The F1, plants crossed.

The seeds were collected to the next generation was raised. F2. In the F2 generation two

types of plants were found. They were tall and dwarf. He could raise 1064 plants in F2

generation 787 plants were tall and 277 plants were always 75% tall plants 25% dwarf

Plants.

Parents Tall Dwarf.

P TT tt

Gametes T X t

F1 Tt

F1 Selfing Tt X Tt

Gametes T t X T t

F2 TT Tt Tt tt

Phenotype 3:1

Genotype 1:2:1

The pure tall plant has two dominant alleles for

height TT, the pure dwarf plant two receive alleles


Gametes T t

T TT

Tall

Tt

Tall

t Tt

Tall

Tt

Dwarf


tt. During gamete formations, the alleles separate to enter two gametes. Hence each

gamete will contain only allele. The gametes produced by a homozygous tall plant

contain only one type of allele. Dominant - T, recessive – t. When a tall plant and dwarf

plant are crossed the gametes containing T- allele fuses with t- allele. The resulting F1,

plant is -Tt. It this plant, the domination allele T masks the effect of the recessive allele t.

The gametes of the F1 generation are 50% T, 50% t.



Dihybrid Experiment

The crossing of two plants differing in two characters is called dihybrid experiment. Two

characters are considered at a time (Colour & Shape). Colour of the cotyledon – (Yellow

& Green), seed shape – (Round & Wrinkled). Mendel selected a pure breeding yellow,

round (Dominant) and a Pure breeding green, wrinkled seed producing plant

( Recessive ).The F1 generation plants produced only yellow round seeds. The F1 plants

were self fertilized. In F2 generation four kinds of plants were produced.

They are

plants producing yellow, round seeds

Plants producing yellow, wrinkled seeds

Plants producing green, round seeds

Plants producing green, wrinkled seeds

Yellow (Y) is dominant over green (y); round seed shape (R) is dominant over wrinkled

(r). The dominant parent produce only one type of gamete and each gamete are carrying

one allele for colour (Y) & another allele for seed shape (R).

The F1 plants Yellow and Round YyRr

When F1 hybrid is selfed

Gametes YR Yr yR yr

YR YYRR

yellow round

YYRr

yellow round

YyRR

yellow round

YyRr

yellow round

Yr YYRr

yellow round

Yyrr

yellow wrinkled

YyRr

yellow round

Yyrr

yellow

wrinkled



yR YyRR

yellow round

YyRr

yellow round

yyRR

green round

yyRr

green round

yr Yyrr

Yellow wrinkled

Yyrr

Yellow wrinkled

yyRr

green round

Yyrr

green wrinkled

Dihybrid ratio: 9:3:3:1

Mendel’s Laws

Based on Mendel’s experiments results certain principles are framed. These are called

Mendel’s laws.

1. Law of dominance

2. Law of segregation or law of purity of gametes.

3. Law of independent assortment

Law dominance

Each organism is made of a bundle of character; each character is controlled by factors

or alleles or genes. Mendel’s law of dominance states that one factor in a pair may mask

or prevent the expression of the other. He called the variety that appeared in the F1

generation of a monohybrid cross as dominant variety and that which did not appear in

F1 generation to be recessive. A recessive factor freely expresses itself with the absence

of the dominant allele.

Law of segregation

Each character is controlled by a pair of alleles. The two alleles of a particular character

remain uncontaminated when they are inside the organism during gamete formation the

paired alleles segregated & enter different gametes. During gamete formation the alleles

of particular character separate and enter different gametes. This is the law of

segregation. This law is also called law of purity of gametes. These laws were

formulated based on monohybrid experiments.



Mendel made two innovations to the science of genetics:

developed pure lines

counted his results and kept statistical notes

Pure Line - a population that breeds true for a particular trait [this was an important

innovation because any non-pure (segregating) generation would and did confuse the

results of genetic experiments]

Results from Mendel's Experiments

Parental Cross F1 Phenotype F2 Phenotypic Ratio F2 Ratio

Round x Wrinkled Seed Round 5474 Round:1850 Wrinkled 2.96:1

Yellow x Green Seeds Yellow 6022 Yellow:2001 Green 3.01:1

Red x White Flowers Red 705 Red:224 White 3.15:1

Tall x Dwarf Plants Tall l787 Tall:227 Dwarf 2.84:1

Phenotype - literally means "the form that is shown"; it is the outward, physical

appearance of a particular trait

Mendel's pea plants exhibited the following phenotypes:

- round or wrinkled seed phenotype

- yellow or green seed phenotype

- red or white flower phenotype

- tall or dwarf plant phenotype

Dominant - the allele that expresses itself at the expense of an alternate allele; the

phenotype that is expressed in the F1 generation from the cross of two pure lines



Recessive - an allele whose expression is suppressed in the presence of a dominant allele;

the phenotype that disappears in the F1 generation from the cross of two pure lines and

reappears in the F2 generation

Mendel's Conclusions

The hereditary determinants are of a particulate nature. These determinants are called

genes.

Each parent has a allele pair in each cell for each trait studied. The F1 from a cross of two

pure lines contains one allele for the dominant phenotype and one for the recessive

phenotype. These two alleles comprise the allele pair.

One member of the allele pair segregates into a gamete, thus each gamete only carries

one member of the allele pair.

Gametes unite at random and irrespective of the other allele pairs involved.

Mendel's First Law - the law of segregation; during gamete formation each member of

the allelic pair separates from the other member to form the genetic constitution of the

gamete

Confirmation of Mendel's First Law Hypothesis

With these observations, Mendel could form a hypothesis about segregation. To test this

hypothesis, Mendel selfed the F2 plants. If his law was correct he could predict what the

results would be. And indeed, the results occurred has he expected.

From these results we can now confirm the genotype of the F2 individuals.



Phenotypes Genotypes Genetic Description

F2 Tall Plants 1/3 DD

2/3 Dd

Pure line homozygote dominant

Heterozygotes

F2 Dwarf Plants all dd Pure line homozygote recessive

Thus the F2 is genotypically 1/4 Dd : 1/2 Dd : 1/4 dd

This data was also available from the Punnett Square using the gametes from the F1

individual. So although the phenotypic ratio is 3:1 the genotypic ratio is 1:2:1

Mendel performed one other cross to confirm the hypothesis of segregation --- the

backcross. Remember, the first cross is between two pure line parents to produce an F1

heterozygote.

At this point instead of selfing the F1, Mendel crossed it to a pure line, homozygote

dwarf plant.

Backcross: Dd x dd

Backcross - the cross of an F1 hybrid to one of the homozygous parents; for pea plant

height the cross would be Dd x DD or Dd x dd; most often, though a backcross is a cross

to a fully recessive parent

Testcross - the cross of any individual to a homozygous recessive parent; used to

determine if the individual is homozygous dominant or heterozygous

So far, all the discussion has concentrated on monohybrid crosses.

Monohybrid cross - a cross between parents that differ at a single allele pair (usually AA

x aa)



Monohybrid - the offspring of two parents that are homozygous for alternate alleles of an

allele pair

Monohybrids are good for describing the relationship between alleles. When an allele is

homozygous it will show its phenotype. It is the phenotype of the heterozygote which

permits us to determine the relationship of the alleles.

Dominance - the ability of one allele to express its phenotype at the expense of an

alternate allele; the major form of interaction between alleles; generally the dominant

allele will make a gene product that the recessive can not; therefore the dominant allele

will express itself whenever it is present

Law of independent assortment

This law is based on dihybrid experiments. According to this law the alleles for each pair

of character separate independently from those of other character during gametes

formation. During gametes formations the allele Y may combine with the dominant allele

R or recessive allele -r of the other character.

To this point we have followed the expression of only one allele. Mendel also performed

crosses in which he followed the segregation of two alleles. These experiments formed

the basis of his discovery of his second law, the law of independent assortment. First, a

few terms are presented.

Dihybrid cross - a cross between two parents that differ by two pairs of alleles (AABB x

aabb)

Dihybrid- an individual heterozygous for two pairs of alleles (AaBb)

Again a dihybrid cross is not a cross between two dihybrids. Now, let's look at a dihybrid

cross that Mendel performed.

Parental Cross: Yellow, Round Seed x Green, Wrinkled Seed



F1 Generation: All yellow, round

F2 Generation: 9 Yellow, Round, 3 Yellow, Wrinkled, 3 Green, Round, 1 Green,

Wrinkled

At this point, let's diagram the cross using specific allele symbols.

Choose Symbol Seed Color: Yellow = G; Green = g

Seed Shape: Round = W; Wrinkled = w

The dominance relationship between alleles for each trait was already known to Mendel

when he made this cross. The purpose of the dihybrid cross was to determine if any

relationship existed between different allelic pairs.

Let's now look at the cross using our allele symbols.

Now set up the Punnett Square for the F2 cross.

Female Gametes

GW Gw gW gw

GW GGWW

(Yellow,

GGWw

(Yellow,

GgWW

(Yellow,

GgWw

(Yellow,



round) round) round) round)

Male

Gw

GGWw

(Yellow,

round)

GGww

(Yellow,

wrinkled)

GgWw

(Yellow,

round)

Ggww

(Yellow,

wrinkled)

Gametes

gW

GgWW

(Yellow,

round)

GgWw

(Yellow,

round)

ggWW

(Green,

round)

ggWw

(Green,ROUND)

gw

GgWw

(Yellow,

round)

Ggww

(Yellow,

wrinkled)

ggWw

(Green,

round)

ggww

(Green,

wrinkled)

The phenotypes and general genotypes from this cross can be represented in the

following manner:

Phenotype General Genotype

9 Yellow, Round Seed G_W_

3 Yellow, Wrinkled Seed G_ww

3 Green, Round Seed ggW_

1 Green, Wrinkled Seed ggww

The results of this experiment led Mendel to formulate his second law.

Mendel's Second Law - the law of independent assortment; during gamete formation the

segregation of the alleles of one allelic pair is independent of the segregation of the

alleles of another allelic pair

As with the monohybrid crosses, Mendel confirmed the results of his second law by

performing a backcross - F1 dihybrid x recessive parent.



Let's use the example of the yellow, round seeded F1.

Punnett Square for the Backcross

Female Gametes

GW Gw gW gw

Male

Gametes gw GgWw

(Yellow, round)

Ggww

(Yellow, wrinkled)

ggWw

(Green, round)

ggww

(Green, wrinkled)

The phenotypic ratio of the test cross is:

1 Yellow, Round Seed

1 Yellow, Wrinkled Seed

1 Green, Round Seed

1 Green, Wrinkled Seed

Testing for Independent Assortment

The Goodness-of-Fit Chi-Square Test

Clearly, we need some means of evaluating how likely it is that chance is responsible for

the deviation between the observed and the expected numbers. To evaluate the role of

chance in producing deviations between observed and expected values, a statistical test

called the goodness-of-fit chi-square test is used. This test provides information about

how well observed values fit expected values. Before we learn how to calculate the chi

square, it is important to understand what this test does and does not indicate about a

genetic cross.



The chi-square test cannot tell us whether a genetic cross has been correctly carried out,

whether the results are correct, or whether we have chosen the correct genetic explanation

for the results. What it does indicate is the probability that the difference between the

observed and the expected values is due to chance. In other words, it indicates the

likelihood that chance alone could produce the deviation between the expected and the

observed values.

When the probability calculated from the chi-square test is high, we assume that chance

alone produced the difference. When the probability is low, we assume that some factor

other than chance—some significant factor—produced the deviation.

To use the goodness-of-fit chi-square test, we first determine the expected results. The

chi-square test must always be applied to numbers of progeny, not to proportions or

percentages. The chi-square value is calculated by using the following formula:

χ2 = Σ (Oserved – Expected) 2 / Expected

where Σ means the sum of all the squared differences between observed and expected

divided by the expected values.

The next step is to determine the probability associated with this calculated chi-square

value, which is the probability that the deviation between the observed and the expected

results could be due to chance. This step requires us to compare the calculated chi-square

value with theoretical values that have the same degrees of freedom in a chi-square table.

The degrees of freedom represent the number of ways in which the observed classes are

free to vary. For a goodness-of-fit chi-square test, the degrees of freedom are equal to

n-1, where n is the number of different expected phenotypes. We are ready to obtain the

probability from a chi-square table (Table). The degrees of freedom are given in the left

hand column of the table and the probabilities are given at the top; within the body of the

table are chi-square values associated with these probabilities. First, find the row for the



appropriate degrees of freedom. Find where calculated chi-square value lies among the

theoretical values in this row. The theoretical chi-square values increase from left to right

and the probabilities decrease from left to right. Most scientists use the .05 probability

level as their cutoff value: if the probability of chance being responsible for the deviation

is greater than or equal to .05, they accept that chance may be responsible for the

deviation between the observed and the expected values. When the probability is less

than .05, scientists assume that chance is not responsible and a significant difference

exists. The expression significant difference means that some factor other than chance is

responsible for the observed values being different from the expected values.

For example,

Suppose we did a testcross for two pairs of genes, such as AaBb X aabb, and observed

the following numbers of progeny: 54 AaBb, 56 aabb, 42 Aabb, and 48 aaBb. Is this

outcome a 1:1:1:1 ratio? Not exactly, but its pretty close. Perhaps these genes are

assorting independently and chance produced the slight deviations between the observed

numbers and the expected 1:1:1:1 ratio. Alternatively, the genes might be linked, with

considerable crossing over taking place between them, and so the number of

nonrecombinants is only slightly greater than the number of recombinants.

How do we distinguish between the roles of chance and of linkage in producing

deviations from the results expected with independent assortment?

Testing for independent assortment between two linked genes requires the calculation of

a series of three chi-square tests. To illustrate this analysis, we will examine the data from

a cross between German cockroaches, in which yellow body (y) is recessive to brown

body (y+) and curved wings (cv) are recessive to straight wings (cv+). A testcross (y+y

cv+cv X yy cvcv) produced the following progeny:

63 y+y cv+cv brown body, straight wings

77 yy cvcv yellow body, curved wings

28 y+y cvcv brown body, curved wings



32 yy cv+cv yellow body, straight wings

Total = 200 total progeny

Testing ratios at each locus

To determine if the genes for body color and wing shape are assorting independently,

we must examine each locus separately and determine whether the observed numbers

differ from the expected (we will consider why this step is necessary at the end). At the

first locus (for body color), the cross between heterozygote and homozygote (y+y X yy)

is expected to produce half y+y brown and half yy yellow progeny; so we expect 100 of

each. We observe 63 + 28 = 91 brown progeny and 77 + 32 = 109 yellow progeny.

Applying the chi-square test to these observed and expected numbers, we obtain:

The degrees of freedom associated with the chi-square test are n- 1, where n equals the

number of expected classes. Here, there are two expected phenotypes; so the degree of

freedom is 2 - 1 = 1. Looking up our calculated chi-square value in Table, we find that

the probability associated with this chi-square value is between .30 and .20. Because the

probability is above .05 (our critical probability for rejecting the hypothesis that chance

produces the difference between observed and expected values), we conclude that there is

no significant difference between the 1:1 ratio that we expect in the progeny of the

testcross and the ratio that we observed.



We next compare the observed and expected ratios for the second locus, which

determines the type of wing. At this locus, a heterozygote and homozygote also were

crossed (cv+ cv X cvcv) and are expected to produce half cv+cv straight-winged

progeny and half cvcv curved-wing progeny. We actually observe 63 + 32 = 95 straight-

winged progeny and 77 + 28 = 105 curved-wing progeny; so the calculated chi-square

value is:

The degree of freedom associated with this chi-square value also is 2 - 1 = 1, and the

associated probability is between .5 and .3.We again assume that there is no significant

difference between what we observed and what we expected at this locus in the testcross.

Testing ratios for independent assortment



We are now ready to test for the independent assortment of genes at the two loci. If the

genes are assorting independently, we can use the multiplication rule to obtain the

probabilities and numbers of progeny inheriting different combinations of phenotypes:

The observed and expected numbers of progeny can now be compared by using the chi-

square test:

Here, we have four expected classes of phenotypes; so the degrees of freedom equal 4 - 1

= 3 and the associated probability is considerably less than .001. This very small

Probability indicates that the phenotypes are not in the proportions that we would expect

if independent assortment were taking place. Our conclusion, then, is that these genes

are not assorting independently and must be linked.

In summary, testing for linkage between two genes requires a series of chi-square tests: a

chi-square test for the segregation of alleles at each individual locus, followed by a test

for independent assortment between alleles at the different loci. The chi-square tests for

segregation at individual loci should always be carried out before testing for independent

assortment, because the probabilities expected with independent assortment are based on

the probabilities expected at the separate loci.



Gene Interaction

The expression of a single character by the interaction of more than one pair of alleles is

called gene interaction or interaction of genes.

The genes interaction is of two types namely

1. Non – allelic gene interaction

2. Allelic gene interaction

The genic interaction occurring between genes located in different rows of the same

chromosome or different chromosome is known as non-allelic gene interaction.

Allelic gene interaction

Incomplete dominance

In incomplete dominance both alleles of a character express their character in the F1

generation. So the F1 individual has mixture of character of both the parents.

Example : Mirabilis jalapa

When a homozygous red flowered (RR) 4’ o clock plant is crossed which a homozygous

white flowered plant (rr) a pink coloured variety is produced (Rr). This is due to the in

complete dominance of the allele R over its allele r. The expression of the two alleles (R

and r) in the same individual leads to the production of an individual with mixed

characters.

Eg: Mirabilis jalapa

Parents : Homozygous red flower X Homozygous White flowers

RR rr

Gametes: R r



F1 Generation: Rr (Pink Flower)

Codominance

In co-dominance, both alleles of a character an equally dominant and both of them

expressed their character in the F1 generation. None is masked.

Inheritance of coat colour in short horn cattle is another case of codominance

Parents (Red) X (White)

RR rr

F1 generation Rr

(Roan)

F1 selfed Rr X Rr

F2 generation RR Rr rr

Red Roan White

In short horn cattle, there are two colour of hair, red and white. Red colour is controlled

by R & White by r. When red & white are crossed, the F1 has roan colour having both

red & white hairs. This is because r allele also expresses its character in the F1

generation.



Non – allelic gene interaction

Epistasis

It is the prevention of the expression of one gene by another non-allelic gene. Epistasis

means stopping on inhibiting. The inhibiting gene is called epistatic gene, the inhibited

gene is called the hypostatic gene. This is counter part of dominance.

Dominance is intrallelic or intragenic

Epistasis is intergenic

Dominant Epistasis (12:3:1)

Dominant Epistasis the prevention of the expression, of a gene by a dominant non-allelic

gene..

Inheritance of colour pattern in case of dominant Epistasis.

Eg – Colour coats of dogs

One gene locus has a dominant epistatic inhibitor allele (I) of coat color pigment. The

allele I prevents the expression of hypostatic gene locus (B / b) and produces white coat

color. The alleles of hypostatic locus (BB, Bb, bb) express only when two recessive

alleles (ii) occur on the epistatic loci

iiBB / iiBb produce black and iibb produces brown coat color.

Parent : Ii Bb X Ii Bb

(White) (White)



Gamete IB Ib iB ib

Gameter IB Ib iB ib

IB IIBB

White

IIBb

White

IiBB

White

IiBb

White

Ib IIBb

White

Iibb

White

IiBb

White

Iibb

White

iB IiBB

White

IiBb

White

iiBB

Black

iiBb

Black

ib IiBb

White

Iibb

White

iiBb

Black

iibb

Brown

9:3:3:1 has become 12:3:1

Recessive Epistasis - 9:3:4

The prevention of the expression of a gene by a recessive non-allelic gene is called

recessive epistasis.

Eg- Coat color in mice

The common house mouse occurs in a number of coat colors, agouti, black, and albino.

The agouti is wild type.

When a homozygous agouti (BBAA) is crossed with homozygous albino (bbaa), the F1

all were agouti. When F1 was crossed the ratio was 9:3:4.

Parent Agouti (BBAA) X Albino(bbaa)



F1 Agouti(BbAa)

F1 gametes BA Ba bA ba

Gameter BA Ba bA ba

BA BBAA

Agouti

BBAa

Agouti

BbAA

Agouti

BbAa

Agouti

Ba BBAa

Agouti

Bbaa

Albino

BbAa

Agouti

Bbaa

Albino

bA BbAA

Agouti

BbAa

Agouti

bbAA

Black

bbAa

Black

ba BbAa

Agouti

Bbaa

Albino

bbAa

Black

bbaa

Albino

Hence the ratio of 9:3:3:1 become 9:3:4.

Duplicate Recessive genes (Complementary genes) – 9:7

Complementary genes may be defined as two or more non-allelic dominant genes interact

with one another to produce a character but one gene cannot produce that character in the

absence of the other. The action of these independent genes is complementary. IF both

loci have homozygous recessive alleles, both of them produce identical phenotypes.

Eg: Flower colour is Sweet Pea

Inheritance of flower colour is sweet pea, Lathyrutus ordoratus. Two varieties of pea

plants, one producing and flower the other white flower. The red Colour of the flower is

due to the presence of a pigment called anthocyanin. The anthocyanin is produced from a



colourless substance called chromogen by the action of an enzyme or activator. The

chromogen cannot be converted in to anthocyanin In the absence of the enzyme.

Gene C------ Chromogen

Gene A ---- Enzyme

Chromogen + Enzyme -- anthocyanin (Red)

Red flower is produce by the interaction of both dominant gene C and A. C&A cannot

give colour independently.

Parent White X White

CCaa ccAA

Gametes Ca cA

F1 generation CcAa (Red)

F1 Selfing CcAa (Red) X CcAa (Red)



Gametes CA Ca cA ca

Gametes CA Ca cA ca

CA CC AA

Red

CC Aa

Red

Cc AA

Red

Cc Aa

Red

Ca CCAa

Red

CCaa

White

CcAa

Red

Ccaa

White

cA CcAA

Red

CcAa

Red

ccAA

White

CcAa

White

ca CcAa

Red

Ccaa

White

ccAa

White

ccaa

White

Duplicate genes with cumulative effect (Supplementary genes) – 9:6:1

Two independent pair of dominant alleles interact in such a way that each dominant gene

produces its effect whether the other is present or not, but when the second dominant

gene is added to the first, a new character is expressed.

Ex: coat color in Duroc – jersey breed of pigs

Sandy – dominant S_, / R_ and White ss / rr

Parent Sandy (SSrr) X Sandy (ssRR)

F1 Red (SsRr)



F2 gametes SR Sr sR sr

Gametes SR Sr sR sr

SR SSRR

Red

SSRr

Red

SsRR

Red

SsRr

Red

Sr SSRr

Red

SSrr

Sandy

SsRr

Red

Ssrr

Sandy

sR SsRR

Red

SsRr

Red

ssRR

Sandy

ssRr

Sandy

sr SsRr

Red

Ssrr

Sandy

ssRr

Sandy

ssrr

White

Hence the Ratio 9:3:3:1 has become 9:6:1

Duplicate Dominant Genes (15:1)

Single character controlled by two or more pair of non-allelic genes independently. The

dominant alleles of both gene loci produce the same phenotype without cumulative

effect.

Eg- Seed case in Capsella – Triangular – T, or Top/ Oval - D

Any one dominant can produce triangular seed case, both recessive produce oval seed

case.

Parent TTDD X ttdd



(Triangular) (Top / Oval)

Gametes TD td

F1 Selfing TtDd (Triangular)

Gametes TD Td tD td

Gametes TD Td tD td

TD TTDD

Triangular

TTDd

Triangular

TtDD

Triangular

TtDd

Triangular

Td TTDd

Triangular

TTdd

Triangular

TtDd

Triangular

Ttdd

Triangular

tD TtDD

Tringular

TtDd

Tringular

ttDD

Tringular

TtDd

Tringular

td TtDd

Triangular

Ttdd

Triangular

ttDd

Triangular

ttdd

Oval / top

Ratio : 15: 1

(Triangular) (Oval)

Dominant and Recessive epistasis – 13:3

The dominant alleles of one gene locus (A) in homozygous (AA) or heterozygous (Aa)

condition and the homozygous recessive alleles (bb) of another gene locus (B) produce

the same phenotype.



Eg- In leghorn fowl the white color of feather is caused by the dominant genotype CCII,

similarly the white color of feathers of Plymouth rock is caused by the recessive genotype

ccii. When both white are crossed the f1 is white. F2 produces white and colored birds in

the ratio 13:3.

Parent CCII X ccii

Gametes CI ci

F1 CcIi (White)

F2 gametes CI Ci cI ci

Gametes CI Ci cI ci

CI CCII

White

CCIi

White

CcII

White

CcIi

White

Ci CCIi

White

CCii

Colored

CcIi

White

Ccii

colored

cI CcII

White

CcIi

White

ccII

White

ccIi

White

ci CcIi

White

Ccii

Colored

ccIi

White

ccii

White

Hence the ratio is 13:3

Back Cross : (TT X Tt)



The F1 individuals obtained in a cross are usually selfed to get the F2 progeny. They can

also be crossed with one of the other two parents from which they were derived. Such a

cross of F1 individual with either of the two parents is known as a backcross.

Test Cross : (Tt X tt) (1:1)

In such back crosses, when F1 is back crossed to the parent with recessive phenotype.

Penetrance :

The percentage of individual’s expressing the character for a particular genotype is called

Penetrance.

If all the individuals expressing the character for a particular genotype, the Penetrance is

called complete Penetrance.

If few individuals do not express the character even though they contain the necessary

gene, the Penetrance is incomplete Penetrance.

BB- Produce blue eyes – 90% of human beings. 10% people have white eyes even though

they contain the BB genes. Environmental factors influence Penetrance.

Expressivity

The variation in the degree of expression of a particular gene is called Expressivity.A

particular gene may produce varying degrees of expression in different

individuals.Expressivity is due to the influence of environmental factor on the genes.

Ex : Effect of temperature on length of wings in Drosophila .

Lethal Genes

A lethal gene kills its possessor. These are genes which not only provide certain

phenotypic traits but at the same time influence the viability of the organism.

Lethal Genes in mice



Yellow coat colour dominant gene Y

YY – Lethal effect

All yellow individuals are heterozygous (Yy)

Two yellow individuals are crossed.

Off springs 2Yy, 1yy

expected – 1YY : 2Yy: 1yy

(Lethal)

Pleiotropism

The production of many characters by a single pair of genes is called Pleiotropism

It is the multiple effect of a pair of genes.

Intermediate lethal genes

Partial effect in heterozygous condition.

Eg- Creepers – short, crooked legs - normally they creep.

Parent :creeper X creeper

Genotype Cc Cc

Gamete: C c

F1 Selfing

Homozygous creeper is missing

Balanced Lethal systems

Both Homozygotes (Dominant and recessive) die.


C C

C CC

Normal

CC

Creeper

C Cc

Creeper

Cc

Dies


MULTIPLE ALLELES

So for we have observed that a given phenotypic trait is being controlled by two

alternate forms of a gene (wild form) and the other being recessive (Mutant form) one

bring dominant (Wild form) at the other being recessive (Mutant form). If the wild form

mutates to give wise to the mutant form, there why not the wild form should mutate in

more than are way to give wise to many mutant forms, on why should not the mutant

form, mutate once again to give wise to other mutant forms. So a gene can haw more

than two allelomorphs which make a series of multiple alleles.

Definition

Multiple alleles are a set of three or more allelic form of a gene controlling the same

character located on the homologous chromosomes. In other words all the Mutant forms

of a single wild type gene constitute a service of multiply alleles.

A diploid individual possessor any two alleles of the allelic series and its gametes caries

only one allele.

Characters of Multiple Alleles.

Multiple alleles of a services always occupy the same locus in the chromosome.

Because all the alleles of multiple service occupy the seem locus in chromosome, on

enclosing over occurs within the same alleles of a multiple service.

Multiple alleles always influence the same character.

The wild type alleles is nearly always dominant while other mutant alleles may show

dominant or not.

When two mutant multiple alleles are crosses the phenotype is mutant type all not the

wild type

Example For Multiple Alleles :



1. ABO blood group

2. Rh blood group

3. Coat colour in Rabbit

4. Nature of wing in Drosophila

5. Self Sterility in tobacco.

6. Skin colour in Mice.

ABO Blood Grouping in Humans.

On the basis of presence or absense of antigens ABO blood groups have been

established by K. Landsteiner.

Antigen A A

B

2 Antigens 4 Blood groups AB

Antingen B O

They are called as ABO blood group or Landsteiner blood group.

A group person contain antigen A on RBC

B group person contain antigen B “ “

AB group person contain antigen A&B “ “

O group person contain No Antigen “ “



Different ABO Blood groups and the antigens & antitrades associated with them.

Blood group Antigen Antibody for

Antigen

A A B

B B A

AB A and B No antibody

O No Antigen A and B

Antigen A cannot Co- exist with antibody A

“ B “ “ “ “ B

The three alleles responsible for the inheritance of ABO blood group are.

LA Responsible For Synthesis of Antigen A

LB “ “ “ “ “ B

Lo Absence of Antigen

LA LB – produces both Antigen A & B.

Different ABO blood grupe on thein genotypes

Blood group possible genotypes

O LO LO

A LA LO or LA LA



B LB LO or LB LB

AB LA LB

Parent Father x Mother

(A group) (B group)

Genotype LA LA x LBLO

Gamete LA LB LO

F1 : LA LB (or) LA LO

Ex. AB A

1. A group x B group

2. A group x O group

3. B group x O group

4. AB x O

( Antigen (or) Agglutinogens present in RBC)

(Antibodies (or) Agglutinins present in the serum)

A B AB O

A - + - +

B + - - +

AB + + - +

O - - - -



RH bood group

There are two groups of human brings namely Rh- positive & Rh negative (Rh-)

Person Who Donates Blood is called as a Donor

Person Who Receives blood is called as a Recipient

In transfusion the blood of the donor and the recipient should be compatible while

testing the compatibility the reaction between the antigen of the doner’s RBC and the

antibodies of the recipient’s plasma alone are taken into consideration Antibodies in

blood group A will agglutinize RBC’s of blood group B on vice versa. AB blood

group will not agglutinize any other group science no antibodies are present. O blood

group should be able to agglutinize all other three groups.

Compatible donor and recipient

Blood group of donor Blood group of recipient

A A and AB

B B and AB

AB AB (Universal Reciepient)

O (Universal Donor) O, A, B, AB



Unit II

Introduction

E. Strasburger in 1875 discovered thread – like structures during cell division.

A given region of DNA with its associated proteins is called chromatin.

This is true for prokaryotic & Eukaryotic cells & even for viruses.

Packing of DNA into chromosome serves several important functions.

The chromosome is a compact form of the DNA that readily fits inside the cell.

Protects DNA from damage. (Naked DNA is unstable, chromosome DNA is

highly stable).

Each time a cell divides, only DNA packed can be efficiently transferred.

Chromosome provides a definite organization which helps is recombination.

Cell cycle – Four phases

G1 – Resting phase / pre DNA synthesis phase

S – DNA synthesis takes place / DNA synthesis phase inter phase

G2 – Resting phase following / Post DNA synthesis phase

M – Mitosis division / Mitotic phase Mitosis

Duration of different phases not only varies with the organism but with different

tissues in the same organism.

Majority of the associated proteins are small basic proteins called histones other

proteins are called non-histone proteins.

These are DNA binding proteins which regulate the transcriptions, replication

recombination of cellular DNA.

A human cell contains 3x10 9 bp per haploid set of chromosomes. The thickness

is 3.4A.

Diameter of a typical human cell nucleus is only 10-15 µ. Hence DNA has to be

compacted by several orders.

DNA with histones will form structures called nucleosomes.



The formation of nucleosomes is the first step in a process that allows the DNA to

be folded into much compact structure reducing their linear length by 10,000

times.

Prokaryotic cells have comparatively small genomes. Even then the DNA has to

be compacted.

Bacteria do not have histones or nucleosomes but have small basic proteins which

compact DNA.

In prokaryotic cell - single circular chromosome

In Eukaryotic cell - Multiple linear chromosome.

However, there are numerous examples of prokaryotic cells having multiple

chromosomes which are linear. But all eukaryotic cells have multiple linear

chromosomes.

Circular chromosomes require topoisomerases to separate the daughter molecules

after they have replicated, without which the few daughter molecules remain inter

locked after replication.

Ends of linear DNA of the have to be protected from enzyme which will degrade

the ends of the DNA.

Prokaryotes have one complete copy of their chromosome that is packed into a

structure called nucleoid.

They also frequently carry one or smaller independent circular DNAs called

plasmids.

Plasmids like chromosomal DNA are not essential for bacterial growth, but code

for desirable traits of bacteria.

The majority of eukaryotic cells are diploid i.e. they contain two copies of each

chromosome. The two copies of a given chromosome are called homologs, one

derived from each parent.

Not all cells in eukaryotic organisms are diploid. Some are haploid and some are

polyploid.

Haploid cells contain a single copy of each chromosome e.g. sperm and egg cells.

Polyploid cells have more than 2 copies of each chromosome. In extreme case

there can be 100 or 1000 copies of each chromosome.



E.g. Megakaryocytes - 128 copies of each chromosome. The segregation of such

a large number of chromosomes is difficult.

No matter the number eukaryotic chromosomes are already contained with is a

membrane bound organelle called nucleus.

Size

A chromos is normally measured at mitotic metaphase.

0.25u - fungi & birds

30 u – plants

3 u – Drosophila

5 u – Man

Shape

Chromosome shape is observed at anaphase.

Primary constriction or centromere determines the shape of the chromosome.

This constriction can be terminal, sub – terminal or median in position.

This clear zone also called as kinetocore which divides the chromosome into 2

arms each one is called a chromosome arm.

The position of the centromere varies from chromosome and chromosome

providing different shapes.

Telocentric - Centromere on the proximal end

Acrocentric - Centromere at one end giving a very short arm and a long arm.

Submetacentric- Centromere occurs near the center forming two unequal arms.

Metacentric - Centromere occurs is the center and forming the equal arms.

Terms

Chromonemata

During mitotic prophase the chromosomal material becomes visible as very this

filaments, called chromonemata. A chromonema represents a chromatid at the early

stages of condensation. Chromonemata form the gene – bearing protions of the

chromosome. The chromonemata is embedded in a achromatic substance called matrix.



Matrix is enclosed in a sheath or pellicle both matrix and pellicle are non-genetic

material and appear only at metaphase when the nucleolus disappears.

Chromomeres

The chromomeres are bead – like accumulations of chromatin material that are

sometimes visible along interphase chromosomes. At metaphase the chromosomes are

highly coiled and the chromomeres are no longer visible.

Centromere

Consist of granules or spherules. Chromosome remains connected with the spherules of

the centromere. It is the region of the chromosome to which the spindle fibers of the

mitotic phase are attached. Chromosomes of most organisms contain only one centromere

and are known as monocentric chromosomes. Some species have diffuse centromeres

with microtubules attached along the length of the chromosome which are called

holocentric chromosomes.

Chromosomal abnormality

Chromosomes may break and fuse forming chromosomes without centromere (acentric

chromosomes). Chromosomes with two centromeres are called dicentric chromosomes.

Both types of those chromosomal aberations are unstable. The acentric chromosomes

remain in the cytoplasm since they cannot attach to mitotic spindle.

Telomeres

The chromosome extremities or terminal regions on either side are called telomeres. If

the chromosome breaks, the broken ends fuse due to lack of telomeres. A chromosome

however cannot fuse at the telomeric ends, since a telomere has a polarity which prevents

other segments from joining with it.

Secondary Constriction

Besides centromere (primary constriction) secondary constrictions can be observed in

some chromosomes. Such a constrictions if present is the distal region of the arm would



pinch off a small fragment called satellite. The satellite remains attached to rest of the

body by a thread of chromatin.

Karyotype and Idiogram

All the members of a species of plant or the animal are characterized by a set of

chromosomes which have certain constant characteristics. These characteristics include

the number of chromosomes, their relative size, and position of the centromere, length of

the arms, secondary constrictions and satellites. The term karyotype has been given to the

group of characteristics that identifies a particular set of chromosomes. A diagrammatic

representation of a karyotype (or morphological characteristics of the chromosomes) of a

species is called ideogram (Gr., idios = distinctive; gramma = something written).

Generally, in an ideogram, the chromosomes of a haploid set of an organism are ordered

in series of decreasing size. Sometimes an ideogram is prepared for the diploid set of

chromosomes, in which the pairs of homologues are ordered in a series of decreasing

size.

A karyotype of human metaphase chromosomes is obtained from their microphotographs.

The individual chromosomes are cut out of the microphotographs and lined up by size

with their respective partners. The technique can be improved by determining the so-

called centromeric index, which is the ratio of the lengths of the long and short arms of

the chromosome.

Some species may have special characteristics in their karyotypes; for example, the

mouse has acrocentric chromosomes, many amphibians have only metacentric

chromosomes and plants frequently have heterochromatic regions at the telomeres.

Uses of karyotypes

The karyotypes of different species are sometimes compared and similarities in

karyotypes are presumed to represent evolutionary relationship. A karyotype also

suggests primitive or advanced features of an organism.



Depending on their staining properties, the following two types of chromatin may be

distinguished in the interphase nucleus

Euchromatin

Portions of chromosomes that stain lightly are only partially condensed; this chromatin is

termed Euchromatin. It represents most of the chromatin that disperse after mitosis has

completed. Euchromatin contains structural genes which replicate and transcribe during

G1 and S phase of interphase. The Euchromatin is considered genetically active

chromatin, since it has a role in the phenotype expression of the genes. In Euchromatin,

DNA is found packed in 3 to 8 nm fibre.

Heterochromatin

In 1928, Heitz defined heterochromatin as those regions of the chromosome that remain

condensed during interphase and early prophase and form the so-called chromocentre.

Heterochromatin is characterized by its especially high content of reptititive DNA

sequences and contains very few, if any, structural genes (i.e., genes that encode

proteins). It is late replicating (i.e., it is replicated when the bulk of DNA has already

been replicated) and is not transcribed. It is thought that in heterochromatin the DNA is

tightly packed in the 30nm fibre.

Types of heterochromatin

In an interphase nucleus, usually there is some condensed chromatin around the

nucleolus, called perinucleolar chromatin, and some inside the nucleolus, called

intranucleolar chromatin. Both types of this heterochromatin appear to be connected and

together, they are referred to as nucleolar chromatin.

Dense clumps of deeply staining chromatin often occur in close contact with the inner

membrane of the nuclear envelope (i.e., with the nuclear lamina) and is called condensed

peripheral chromatin. Between the peripheral heterochromatin and the nucleolar

heterochromatin are regions of lightly staining chromatin, called dispersed chromatin. In

the condensed chromosomes, the heterochromatic regions can be visualized as regions



that stain more strongly or more weakly than the euchromatic regions, showing the so-

called positive or negative heteropyknosis of the chromosomes (Gr., hetero =

different+pyknosis = staining.).

Heterochromatin has been further classified into the following types:

Constitutive heterochromatin

In such a heterochromatin the DNA is permanently inactive and remains in the condensed

state throughout the cell cycle. This most common type of heterochromatin occurs around

the centromere, in the telomeres and in the C-bands of the chromosomes. In Drosophila

virilis, constitutive heterochromatin exists around the centromeres and such

Pericentromeric heterochromatin occupies 40per cent of the chromosomes. In many

species, entire chromosomes become heterochromatic and are called B chromosome,

satellite chromosomes or accessory chromosomes and contain very minor biological

roles. Such chromosomes comprising wholly constitutive heterochromatin occur in corn,

many phytoparastic insects and salamanders.

Constitutive heterochromatin contains short repeated sequence of DNA, called satellite

DNA. This DNA is called satellite DNA because upon ultracentrifugation, it separates

from the main component of DNA. Satellite DNA may have a higher or lower G + C

content than the main fraction. For in the mouse genome, constituting 10 per cent of the

total mouse DNA. The exact significance of constitutive heterochromatin is still

unexplained.

Facultative heterochromatin

Such type of heterochromatin is not permanently maintained in the condensed state; in

facultative heterochromatin one chromosome of the pair becomes either totally or

partially heterochromatic. The best known case is that of the X-chromosomes in the

mammalian female, one of which is active and remains euchromatic, whereas the other is

inactive and forms at interphase, the sex chromatin or Barr body (Named after its

discoverer, Canadian cytologist Murrary L.Barr). Barr body contains DNA which is not



transcribed and is not found in males. Indeed, the number of Barr bodies is always one

less than the number of X chromosomes (i.e., in humans, XXX female has two Barr

bodies and XXXX female has three Barr bodies).

Histones

Histones are very basic proteins, basic because they are enriched in the amino acids

arginine and lysine to a level of about 24 more present. Arginine and lysine at

physiological pH are cationic and can interact electrostatically with anionic nucleic acids.

Thus, being basic, histones bind tightly to DNA which is an acid. There are five types of

histones in the eukaryotic chromosomes, namely H1, H2A, H2B, H3 and H4.

One of the important discoveries that come from chemical studies is that the primary

structures of histones have been highly conserved during evolutionary history. For

example, histone H4 of calf and of garden pea contains only two amino acid differences

in a protein of 102 total amino acid residues. These organisms are estimated to have an

evolutionary history of at least 600 million years, during which they diverged

structurally. This conservation of structure suggests that over the eras, histones have had

a very similar and crucial role in maintaining the structural and functional integrity of

chromatin. Such an evolutionary conservation suggests that the functions of these two

histones involve nearly all of their amino acids so that a change in any position is

deleterious to the cell.

Histone H1 is the least rigidly conserved histone protein. It contains 210 to 220 amino

acids and may be represented by a variety of forms even within a single tissue. H1 is

present only once per 200 base pairs of DNA (in contrast to rest of the four types of

histones each of which is present twice) and is rather loosely associated with DNA. H1

histone is absent in yeast, Saccharomyces cerevisiae. Histones besides determining the

structure of chromatin play a regulatory role in the repression activity of genes.

Non-histones



In contrast to the modest population of histones in chromatin, non-histone proteins

display more diversity. In various organisms, number of non-histones can vary from 12 to

20. Heterogeneity of these proteins is not conserved in evolution as the histones. These

non-histones differ even between different tissues of the same organism suggesting that

they regulate the activity of specific genes.

About 50 per cent non-histones of chromatin have been found to be structural proteins

and include such proteins as action, and α and β tubulins and myosin. Although for

sometime these contractile proteins were thought to be contaminants, it is now believed

that they are vital ingredients of the chromosome, functioning during chromosome

condensation and in the movement of chromosomes during mitosis and meiosis. Many of

the remaining 50 per cent of non-histones include all the enzymes and factors that are

involved in DNA replication, in transcription and in the regulation of transcription. These

proteins are not as highly conserved among organisms, although they must carry out

similar enzymatic activities. Apparently they are not as important as the histones in

maintaining chromosome integrity.



Molecular organization of the eukaryotic genome

Three levels of genome organization

nuclear genome

mitochondrial genome

chloroplast genome

Genome size: It refers to the amount of DNA in a haploid cell.

Human genome includes 60 ~ 100,000 "genes" occupying <5% of DNA

The “C value paradox” – organism complexity does not correlate with genome size

Chromosome architecture at the microscopic level

DNA packaged into chromosomes.

Prokaryote DNA is usually circular,

smaller, and less elaborately folded &

structured. Eukaryotic DNA is complexed

with large amounts of protein to form

chromatin, is highly extended & tangled

during interphase but is condensed into

short thick chromosomes during mitosis.

Thus, eukaryotic chromosomes contain enormous amounts of DNA, which must be

packed correctly. Primary coiling of DNA is double helix.

A. Nucleosomes

1. Histones: small proteins rich in basic amino acid that bind to DNA --> chromatin;

similar from one eukaryote to another.

H1, H2A, H2B, H3, and H4



2. Nucleosomes: secondary coiling of DNA around histone core. 2o coiling of DNA

around histone core basic unit of DNA

packing, formed from DNA wound

around a protein core of 2 copies each of

four types of histone (H2A, H2B, H3,

and H4). Resembles beads along a

string; may control gene expression by

limiting access of transcription.

B. Higher Levels of DNA Packing

1. tertiary coiling of core + linker

forms "Solenoid":30-nm chromatin

fiber: consists of a tightly wound coil

with 6 nucleosomes / turn

2. Looped domain: each loop in

the 30-nm fiber contains 20,000

to 100,00 base pairs; looped

domains coil and fold further

compacting chromosome (as in

metaphase)



Interphase chromatin is much less condensed than mitotic chromatin

a. Heterochromatin: remains highly condensed, not actively transcribed

b. Euchromatin: less condensed, is actively transcribed

Polytene Chromosome

A giant chromosome produced by an endomitotic process in which, following synapsis of

the two homologues, multiple rounds of replication produce chromatids that remain

synapsed together in a haploid number of chromosomes. Large chromosome consisting of

many chromatids formed by rounds of endomitosis following synapsis of the two

homologues.

Polytene chromosomes have been studied mostly in Drosophila salivary glands, in which

chromosomes undergo 10 cycles of replication without separation of the daughter

chromosomes. This leads to 1024 identical strands of chromatin aligned side by side.

These chromosomes are easy to see with a light microscope because of their large size

and precise alignment. The chromosomes is seen as distinct alternating dark and light

bands. The dark bands correspond to more condensed regions of the chromatin, and the

light (interband) regions are less dense regions.

Can be 1000 times thicker than normal meiotic

chromosomes.

In each polytene chromosome, homologous

chromosomes are tightly paired.

Polytene chromosomes are joined together at their

centromeres by a proteinaceous structure called a

chromocenter.



When stained, polytene chromosomes show a banding pattern. Each band

contains an average of 30,000 bp, and may contain up to seven genes. Interbands

also contain genes.

In Drosophila, 5,000 bands have been observed.

Chromosome puffs are diffused uncoiled regions of the chromosome that are sites

of RNA transcription.

Balbiani ring is a large chromosomal puff.

Lampbrush Chromosomes

The lampbrush type of chromosome is characteristic of growing oocytes in the ovaries of

most animals with the exception of mammals and certain insects. The chromosomes are

greatly elongated diplotene bivalents, sometimes reaching lengths of a millimeter or

more.

Lampbrush chromosomes are exceedingly delicate structures and no further progress

beyond the pioneer studies of Flemming (1882) and Ruckert (1892) was possible until a

technique could be devised for dissecting them out of their nuclei and examining them in

a life-like condition, separated from the remainder of the nuclear contents. The largest

lampbrush chromosomes are to be found in growing oocytes of newts and salamanders.

These animals have big genomes, big chromosomes and big cells, so it is scarcely

surprising that they have good lampbrushes.

The best oocytes for lampbrush studies are the ones that make up the bulk of the ovary of

a healthy adult female at the time of year when the eggs are actively growing in

preparation for ovulation in the following spring. They are about 1 mm in diameter.

They have nuclei that are between a third and a half a millimeter in diameter, big enough



to see with the naked eye. It is not difficult to isolate these nuclei by hand and it is not

much more difficult to remove their nuclear envelopes and spill out their chromosomes.

A lampbrush chromosome is a meiotic half bivalent. This means that it must consist of

two chromatids. The entire lampbrush bivalent will therefore have a total of 4

chromatids. The chromosome appears as a row of granules of deoxyribonucleoprotein

(DNP), the chromomeres, connected by an exceedingly thin thread of the same material

(figure). Chromomeres are 1/4 to 2um in diameter and spaced 1 – 2 micrometers along

the length of the chromosome axis.

Figure

Chromomeres bearing pairs (L) or multiple pairs (LL) of lateral loops:

Sister loops of different lengths (L1): polarization of thickness along the lengths of loops

(P): Loops consisting of a single unit of polarization (P): Loops consisting of several

tandem units of polarization with the same or different directions of polarities (ppp).

Each chromomere has 2 or some multiple of 2 loops associated with it. The loops have a

thin axis of DNP surrounded by a loose matrix of ribonucleoprotein (RNP). The loops are

variable in length, and during the period of oogenesis when they are maximally



developed, they extend from 5 to 50 micrometers laterally from the chromosome axis,

which means that the longest loops in such a case would be 100 micrometers long.



Sex Determination

Genetic mechanism

Metabolically controlled

Hormonally controlled

Environmentally controlled

Genetically Controlled

a) Heterogametic Males.

The XY sex-determination system is the sex-determination system found in humans,

most other mammals, some insects (Drosophila) and some plants (Ginkgo). In this

system, females have two of the same kind of sex chromosome (XX), and are called the

homogametic sex. Males have two distinct sex chromosomes (XY), and are called the

heterogametic sex.

The XY sex determination system was first described independently by Nettie Stevens

and Edmund Beecher Wilson in 1905.

Some species (including most mammals) have a gene or genes on the Y chromosome that

determine maleness. In the case of humans, a single gene (SRY) on the Y chromosome

acts as a signal to set the developmental pathway towards maleness. Other mammals use

several genes on the Y chromosome for that same purpose. Not all male-specific genes

are located on the Y chromosome.

Other species (including most Drosophila species) use the presence of two X

chromosomes to determine femaleness. One X chromosome gives putative maleness. The

presence of Y chromosome genes are required for normal male development.

XX/X0 sex determination



The X0 sex-determination system is a system that grasshoppers, crickets, cockroaches,

and some other insects use to determine the sex of their offspring. In this system, there is

only one sex chromosome, referred to as X. Males only have one X chromosome (X0),

while females have two (XX). The zero (sometimes, the letter O) signifies the lack of a

second X chromosome. Maternal gametes always contain an X chromosome, so the sex

of the animals' offspring is decided by the male. Its sperm normally contain either one X

chromosome or no sex chromosomes at all.

In this variant of the XY system, females have two copies of the sex chromosome (XX)

but males have only one (X0). The 0 denotes the absence of a second sex chromosome.

The nematode C. elegans is male with one sex chromosome (X0); with a pair of

chromosomes (XX) it is a hermaphrodite.

b) Heterogametic females

ZW sex chromosomes

The ZW sex-determination system is a system that determines the sex of offspring in

birds, some fish, and some insects (including butterflies and moths). In the ZW system it

is the ovum that determines the sex of the offspring, in contrast to the XY sex-

determination system and the X0 sex-determination system. The letters Z and W are used

to distinguish this system from XY system. Males are the homogametic sex (ZZ), while

females are heterogametic (ZW). The Z chromosome is larger and has more genes, like

the X chromosome in the XY system.

It is unknown whether the presence of the W chromosome induces female features or the

duplication of the Z chromosome induces male ones; unlike mammals, no birds with a

double W chromosome (ZWW) or a single Z (Z0) have been discovered. It is possible

that either condition causes embryonic death, and both chromosomes are responsible for

gender selection.

In Lepidoptera (moths and butterflies), examples of Z0, ZZW and ZZWW females can be

found. This suggests that the W chromosome is essential in female determination in some



species (ZZW), but not in others (Z0). In Bombyx mori (the commercial silkworm), the

W chromosome carries the female-determining genes.

The ZW sex-determination system is found in birds and some insects and other

organisms. The ZW sex-determination system is reversed compared to the XY system:

females have two different kinds of chromosomes (ZW), and males have two of the same

kind of chromosomes (ZZ).

c) Haploidy

The Haplodiploid sex-determination system determines the sex of the offspring of many

Hymenopterans (bees, ants, and wasps), and coleopterans (bark beetles). In this system,

sex is determined by the number of sets of chromosomes an individual receives. An

offspring formed from the union of a sperm and an egg develops as a female, and an

unfertilized egg develops as a male. This means that the males have half the number of

chromosomes that a female has, and are haploid. This system produces a number of

peculiarities; chief among these is that a male has no father and cannot have sons, but he

has a grandfather and can have grandsons.

Unfertilized eggs develop into haploid individuals, which are the males. Diploid

individuals are generally female but may be sterile males. Thus, if a queen bee mates

with one drone, her daughters share ¾ of their genes with each other, not ½ as in the XY

and ZW systems.

After mating, fertile Hymenopteran females store the sperm in an internal sac called the

spermatheca. The mated female controls the release of stored sperm from within the

organ: If she releases sperm as an egg passes down the oviduct, the egg is fertilized.

Social bees, wasps, and ants can modify sex ratios within colonies to maximize

relatedness among members, and to generate a workforce appropriate to surrounding

conditions.

d) Sex balance theory or genic balance theory



Sex balance theory or genic balance theory states that the X chromosome determines the

sex of the individual and that sex is a dosage phenomena, where the ratio of the amount

of the X relative to the autosomes determines the sex. In addition, environmental effects

can influence the development of the intersex flies.

Further studies have shown that sex is ultimately determined by the locus sex-lethal on

the X chromosome, though several other loci on the X chromosome and the autosomes

are also needed for sex determination.

Environmental sex determination

The same chemical plays a unique role in the worm's sexual differentiation. The

planktonic, free-swimming Bonellia larvae are initially sexually undifferentiated. Larvae

which land on unoccupied sea-floor mature, over the period of years, into adult females.

But most larvae come in contact with the bonellin in the skin of an adult female, bonellin-

rich proboscis (The adult Bonellia female produces a vivid green pigment in its skin,

known as bonellin. This chemical, concentrated mostly in the proboscis) and are

masculinised by this exposure. The chemical causes these larvae to develop into the tiny

males, which cling to the female's body or are sucked inside it by the feeding tube, to

spend the remainder of their lives as parasites inside the female's genital sac, producing

sperm to fertilize her eggs and reliant on their host for all other needs.

The sex of a Green Spoonworm is thus determined by external, environmental factors

(the presence or absence of bonellin), not by internal, genetic factors (chromosomes), as

is the case with most other sexually-differentiated organisms. This environmental sex

determination helps Green Spoonworm populations respond to the availability of

burrows.

Hormonally controlled sex determination



Sex reversal in Hen

In birds only one gonad of a normal female develops into a functional ovary. The other

gonad remains rudimentary. If the functional ovary of a hen is destroyed, the rudimentary

gonad develops into a testis. Thus the female sex is reversed into male due to the

phenomenon called sex reversal.

During embryonic development the XY genotype stimulates the pituitary gland to

produce female hormones that cause the gonad of the hen to develop into an ovary. After

the development of the ovary the pituitary ceases to produce female hormones due to

inhibition of the pituitary by hormones produced by the ovary, thus acting as feed-back

system.

When the ovary of the hen is removed the steroid cells of the adrenal become active and

provoke the development of testis.

Sex Linked Inheritance

X-linked Inheritance

In animals with XY sex determining mechanism, the X chromosome has many loci,

many that have nothing to do with sex as such. The Y is usually smaller and possesses

fewer loci that are not the same loci as that on the X chromosome. Thus females that have

the same allele at a locus on the X chromosome are homozygous. Different alleles would

be heterozygous. Males, because they have only one X, are hemizygous and can have

only one allele at a locus. Because of this, one copy of a recessive allele will be expressed

in the phenotype in males.

In sex-linked inheritance, crosses are not reciprocal. The X-linked pattern is called the

criss-cross pattern of inheritance because fathers pass the trait to daughters who pass it on

to sons.



Transmission of X-linked genes

A. Females transmit an X chromosome both to sons and daughters; however, males

transmit their X chromosomes only to daughters and their Y chromosomes only to sons.

B. For rare X-linked dominant traits:

1. Twice as many females are affected as males.

2. Half the children of an affected female will be affected, regardless of sex.

3. All the daughters of an affected male will be affected but none of the sons.

C. For rare X-linked recessive traits:

1. Hemizygous males are affected, but only homozygous females are affected. The ratio

of affected males-to-females is >>1.

2. Affected males will transmit the gene to all daughters, who will usually be

heterozygous and therefore not affected, but to no sons.

3. Affected males in a pedigree are related to each other through nonaffected females;

e.g. grandfather—mother—son or maternal uncle—mother—son.

4. Homozygous recessive females can arise only from matings in which the father is

affected and the mother is heterozygous or homozygous.

D. Examples of X-linked recessive traits are:

1. Colorblindness.

2. Hemophilia.

3. Duchenne muscular dystrophy.

Color blindness: Half of the sons of a woman who is heterozygous for color blindness

will be color blind, irrespective of the genotype of their father. If the father is color blind,

half of the daughters will also be color blind. If the father has normal vision, none of the

daughters will be color blind. A normal vision man and a normal vision woman who is

heterozygous for color blindness will produce progeny with a 3:1 ratio of normal vision

to color blind, but all of the color blindness will be in male progeny. Note that all of this



is an oversimplification. There are actually various types of color blindness involving

loss of sensitivity to different colors of light.

Hemophilia: This disorder of blood clotting is X-linked and is common in the males of

European royal families because of frequent consanguineous matings within this limited

breeding pool. Pedigrees suggest that Queen Victoria of England was heterozygous for

classic hemophilia (hemophilia A), which involves a deficiency of clotting factor VIII.

Hemophilia B, with a deficiency of clotting factor IX, is also X-linked

Recessive means that disease only occurs when a person has two copies of the bad gene.

For autosomal recessive diseases, this usually means they must inherit the disease from

both parents, but this is not the case for X-linked recessive diseases. Men have only a

single X chromosome, so they have only one copy of any gene on the X chromosome.

Thus, a gene error on X definitely caused disease in men (who are XY), but women are

XX, and have two copies of the gene. X-linked recessive disorders are more likely to

occur than autosomal recessive disorders, because men have only one X chromosome,

whereas all people have 2 copies of each autosome.

Recessive diseases often occur in genes that produce an enzyme. In a carrier, who has

only one bad copy, there is often no disease, because the second gene can pull up the

slack, and maintain health. In some recessive diseases, a carrier gets a mild form of the

disease. For example, in X-linked recessive hemophilia, a female carrier has one bad

gene on chromosome X, but the good gene on the other X chromosome produces enough

of the good clotting enzyme to maintain health. The recessive disease only arises when

the male has no good gene on the other chromosome (because they get a Y instead of a

second good X).

Holandric or Y-linked Genetic Diseases



The Y chromosome is a sex-linked chromosome. Men are XY and women are XX, so

only men have a Y chromosome. The Y chromosome is very small and contains few

genes.

Y-linked transmission- The Y chromosome has a trivially simple inheritance pattern

because women are XX and men are XY. Only men have a Y chromosome and so the Y

is only passed from father to son. The Y chromosome is small and does not contain many

genes. There are few genetic diseases related to genes on Y.

Male sex determination: The main Y gene is called the SRY gene, which is the master

gene that specifies maleness and male features. It is the single gene that sets off the initial

cascade of hormone changes that make a person male. It is not the entire Y chromosome,

but just this gene that is necessary for maleness. There is evidence of this in rare diseases

where the SRY gene is missing. People who are genetically male with XY chromosomes,

but with a mutation or deletion of this SRY gene on the Y chromosome, will be female

despite having most of the Y chromosome. And people who are genetically female with

XX but also have a tiny piece of the Y chromosome with this gene; will become male

despite their female-like XX chromosomes.

Transmission of Y-linked genes (holandric inheritance)

A. Since males are hemizygous for Y-linked genes on the nonhomologous part of the Y

chromosome, all will be expressed.

B. Y-linked genes are transmitted from father to son, never to daughters.

C. There are no essential genes on the nonhomologous portion of the Y; however, the

gene for maleness is there, as is a locus concerned with male fertility.

Sex-limited traits

Sex-limited traits are traits that are expressed in one sex, but not in the other. Some

examples include milk yield in mammals, antlers in deer, and beards in humans.



Sex-influenced traits

Sex-influenced traits appear in both sexes but more so in one sex than another. Male

pattern baldness in humans is an example. The male hormone testosterone is needed for

full expression of baldness. Because of this hormone difference, the allele for baldness

behaves as a dominant trait in males (expressed when heterozygous), but behaves as a

recessive allele in females (must be homozygous to be expressed).



UNIT III

LINKAGE AND CROSSING OVER

Introduction:

Sutton and Bovery proposed that the genes are located on the chromosomes. Later other

workers also proved the same with sufficient evidences. During cell division (Mitosis or

Meiosis) each chromosome appears to behave as a unit and therefore it would be

expected that genes located on the same chromosome would move to the same pole

during cell division. As a consequence some genes would fail to show independent

assortment (segregation) and tend to be inherited together. Sutton expressed this

expectation in 1903 while propounding, the chromosome theory of inheritance. Bateson

and Punnet (1905) were the first to report a significant deviation from the expectation due

to independent assortment. They studied the flower colour in pea. A dominant gene 'B'

produces blue coloured flowers, while its recessive allele 'b' determines red flower.

Another dominant gene L governs elongate pollen grains, where as its recessive allele l

produces normal round pollen grains. When the F1 from the cross Blue elongate (BBLL)

X red round (bbll) was test crossed with red round (bbll), the progenies showed the ratio

of

7 blue elongate (BbLI)

1 blue round (Bbll)

1 red elongate (bbLI)

7 red round (bbll)

In the place of expected 1:1:1:1 ratio, it appears that the two dominant gene 'B' (blue

colour) and V (elongate pollen grain) have an affinity for each other so that they tend to

stay together during the transmission to the progenies. Bateson and Punnet called this

situation as "Coupling Phase". But when F1from the cross

Blue Round X Red Elongate

(BBll) (bbLL)

Blue Elongate (BbLI) was test crossed with red round (bbll), the progeny exhibited the

ratio of

1 blue elongate (BbLI)

7 blue round (Bbll)



7 red elongate (bbLI)

1 red round (bbll).

It appears as if the two dominant genes (B and L) repel each other in this case, so that

they tend to stay away from each other in the progeny. This situation was referred as

"Repulsion Phase".

Bateson and Punnet could not give suitable explanation for this situation and suggested

that there was a selective multiplication (through mitosis) of some types of gametes i.e.

BL and bl in the coupling phase and Bl and bL in the repulsion phase, after meiosis and

this gives rise to 7:1:1:7 and 1:7:7:1 ratio instead of 1:1:1:1 test cross ratio. Even though

this hypothesis is unstable and later disproved, but the terms 'coupling' and 'repulsion'

phase are widely used still to denote the linkage between two dominant genes an

recessive genes respectively.

Morgan’s View on Linkage

Later in 1919, Morgan worked with gene of Drosophila. and explained linkage. He

concluded that

1) Genes located on the same chrome tend to stay together during inheritance this

tendency is called linkage.

2) Genes are arranged in a linear fashion on the chromosome.

3) The intensity of the linkage between the two genes on the chromosome is inversely

proportional to the distance between the two linked genes on the chromosome.

4) Proposed that coupling and repulsion are the 2 aspects of linkage.



LINKAGE: The tendency of two or more genes to stay on the same chromosome is

called as linkage. Linkage is the consequence of genes located on the same chromosome.

Linked genes do not show independent assortment.

KINDS OF LINKAGE

T.H. Morgan and his co-workers by their investigation on the Drosophila and other

organisms have found two types of linkage, viz., complete linkage and incomplete

linkage.

1. Complete Linkage

The Complete linkage is the phenomenon in which parental combinations of characters

appear together for two or more generations in a continuous and regular fashion. In this

type of linkage genes are closely associated and tend to transmit together.

Example. The genes for bent wings (bt) and shaven bristles (svn) of the fourth

chromosome mutant of Drosophila melanogaster exhibit complete linkage.

Complete linkage in male Drosophila. In most of the organisms crossing-over takes

place both in males and females. But in male Drosophila and female silkworm, Bombyx

mori crossing-over takes place either very rarely or not at all. This becomes clear from

Morgan’s experimental results from Drosophila. In 1919, T.H. Morgan mated gray

bodied and vestigial winged (b+vg/b+vg) fruit flies with flies having black bodies and

normal wings (bvg+/bvg+). F1 progeny had gray bodies and normal long wings (b+vg/

bvg+), indicating thereby that these characters are dominant. When F1 males (b+vg/ bvg+),

were backcrossed (i.e., test crossed) to double recessive females (bvg/bvg or black

vestigial), only two types of progeny (one with gray bodies and vestigial wings, b +vg/bvg

and the other with black bodies and normal wings, to bvg+/bvg instead of four types of

phenotypes were obtained .

Parents: Gray, Vestigial X Black, Longb+vg/b+vg b+vg/b+vg

Gametes: (b+vg) (bvg+)



F1: All Gray, longb+vg/ bvg+

Test Cross : F1 male Gray, Long X Female Black, Vestigial b+vg/bvg+ bvg/bvg

Gametes : (b+vg)(bvg+) (bvg)

(Only two types of gametes due to complete linkage and lack of crossing over in male Drosophila)

Test cross ratio: 1Gray, vestigial 1Black, Long or 1:1.b+vg/ bvg bvg+/bvg

Here, the use of the testcross is very important. Because one parent (the tester)

contributes gametes carrying only recessive alleles, the phenotypes of the offspring

represent the gametic contribution of the other double heterozygote parent. So the

genetical analyst can concentrate on one meiosis and forget the other. This is in contrast

to the situation in an F1 selfing where there are two sets of meiotic divisions to consider

one for the F1 male parental gametes and one for the F1 female.

2. Incomplete Linkage

The linked genes do not always stay together because homologous non-sister chromatids

may exchange segments of varying length with one another during meiotic prophase.

This sort of exchange of chromosomal segments in between homologous chromosomes is

known as crossing over. The linked genes which are widely located in chromosomes and

have chances of separation by crossing over are called incompletely linked genes and the

phenomenon of their inheritance is called incomplete linkage.

Incomplete Linkage in female Drosophila. When F1 females of the Morgan’s classical

cross in Drosophila between gray, vestigial (b+vg/b+vg) and black, normal or long

(bvg+/bvg+) were test-crossed to double-recessive (bvg/bvg) males, all four types of

progeny were obtained in following ratio, showing occurrence of crossing-over:

Parents: Gray, Vestigial X Black, Long



b+vg/b+vg bvg+/bvg+

Gametes: (b+vg) (bvg+)

F1 : All Gray, longb+vg/ bvg+

Test Cross: F1 Female Gray, Long X Male Black, Vestigialb+vg/bvg+ bvg/bvg

↓ ↓

Gametes: (b+vg) (bvg+) = Non-crossovers (bvg) (b+vg+)(bvg) = Recombinants

Test cross ratio: 1. Gray, Vestigial; b+vg/bvg = 41.5% 83%parental combination 2. Black, Long; bvg+/bvg = 41.5% showing linkage

3. Gray, Long: b+vg+/bvg = 8.5% 17%recombinants due to 4. Black, Vestigial; bvg/bvg = 8.5% crossing over

Crossing Over

“The crossing over is a process that produces new combinations

(recombinations) of genes by interchanging of corresponding segments between non-

sister chromatids of homologous chromosomes” The chromatins resulting from such

interchanges of chromosomal parts are known as cross overs. The term crossing over

was coined by T.H.Morgan. In simple, Crossing over is the exchange of segments

between non-sister chromatids of homologous chromosomes.

Characteristics of Crossing Over

1. Crossing over or recombination occurs at two levels (i) at gross chromosomal

level, called chromosomal crossing over and (ii) at DNA level, called genetic

recombination.

2. A reciprocal exchange of material between homologous chromosomes in

heterozygotes is reflected in crossing over.

3. The crossing over results basically from an exchange of genetic material

between non-sister chromatids by break-and-exchange following replication.



4. The frequency of crossing over appears to be closely related to physical

distance between genes on chromosome and serves as a tool in constructing genetic maps

of chromosomes.

Types of Crossing Over

1. Somatic or Mitotic Crossing Over

When the process of crossing over occurs in the chromosomes of body or somatic cells of

an organism during the mitotic cell division it is known as somatic or mitotic crossing

over. The somatic crossing over is rare in its occurrence and it has no genetical

significance. The somatic or mitotic crossing over has been reported in the body or

somatic cells of Drosophila by Curt Stern.

2. Germinal or Meiotic Crossing Over

Usually the crossing over occurs in germinal cells during the gametogenesis in which the

meiotic cell division takes place. This type of crossing over is known as germinal or

meiotic crossing over. The meiotic crossing over is universal in its occurrence and is of

great genetic significance.

Mechanism of Meiotic Crossing Over

The process of crossing over includes following stages in it, viz., synapsis, duplication

chromosomes, crossing over and terminalization. The chromosomes which tend to

undergo recombination due to meiotic crossing over necessarily complete two functions:

1.99.7 per cent replication of DNA and 75 per cents synthesis of histones, both of which

take place prior to onset of prophase I, and 2. attachment of each chromosome by its both

ends (telomeres) to the nuclear envelope (i.e., to nuclear lamina) via the specialized

structure, called attachment plaques. This event occurs during the leptotene stage of

prophase I and though each chromosome at this stage is visually long and thin thread, but

contains material of two sister chromatids (i.e., two DNA molecules plus almost

duplicated amount of histones).

1. Synapsis



Synapsis or intimate pairing between the two homologous chromosomes (one maternal

and another paternal) is initiated during zygotene stage of prophase I of meiosis I.

Synapsis often starts when the homologous ends of the two chromosomes are brought

together on the nuclear envelope and it continues inward in a zipper-like manner from

both ends, aligning the two homologous chromosomes side by side. In other cases,

synapsis may begin in internal regions of the chromosomes and proceed towards the

ends, producing the same type of alignment. By synapsis each gene is, thus, brought into

juxtaposition (=being side by side) with its homologous gene on the opposite

chromosome. Thus, synapsis is the phase of prolonged and close contact of homologous

chromosomes due to attraction between two exactly identical or homologous regions or

chromomeres. The resultant pairs of homologous chromosomes are called bivalents.

2. Duplication of Chromosomes

The synapsis is followed by duplication of chromosomes (in pachytene). During this

stage, each homologous chromosome of bivalents splits longitudinally and forms two

identical sister chromatids which remain held together by an unsplitted centromere. The

longitudinal splitting of chromosomes is achieved by the separation of already duplicated

DNA molecules along with certain chromosomal proteins. At this stage each bivalent

contains four chromatids, so it is known as tetrad.

3. Crossing over by Breakage and Union

It is well evident that crossing over occurs in the homologous chromosomes only during

the four stranded or tetrad stage. Homologues continue to stay in synapsis for days during

pachytene stage and chromosomal crossing over occurs due to exchange of chromosomal

material between non-sister chromatids of each tetrad. In pachytene, the recombination

nodules become visible between synapsis chromosomes.

During the process of crossing over, two non-sister chromatids first break at the

corresponding points due to the activity of a nuclear enzyme known as endonuclease.

Then a segment on one side of each break connects with a segment on the opposite side

of the break, so that the two non-sister chromatids cross each other. At this stage 3 per



cent synthesis of DNA occurs to fill the gap. The fusion of chromosomal segments with

that of opposite one takes place due to the action of an enzyme known as ligase . The

crossing of two chromatids is known as chiasma (Gr., chiasma=cross) formation. The

crossing over, thus, includes the breaking of chromatid segments, their transposition and

fusion.

Chiasma frequency or percentage of crossing over. The crossing over may take place

at several points in one tetrad and may result in the formation of several chiasmata. The

number of chiasmata depends on the length of the chromosomes because the longer the

chromosome the greater the number of chiasmata. In a species each chromosome has a

characteristic number of chiasmata. The frequency by which a chiasmata occurs between

any two genetic loci has also a characteristic probability. The more apart two genes are

located on a chromosome, the greater the opportunity for a chiasma to occur between

them. The closer two genes are linked lesser the chances for a chiasma occurring between

them.

4. Terminalisation

After the occurrence of process of crossing over, the non-sister chromatids start to repel

each other because the force of synapsis attraction between them decreases. During

diplotene. desynapsis begins, synaptonemal complex dissolves and two homologous

chromosomes in a bivalent are pulled away from each other. During diakinesis,

chromosomes detaches from the nuclear envelope and each bivalent is clearly seen to

contain four separate chromatids with each pair of sister chromatids linked at their

centromeres, while non-sister chromatids that have crossed over are linked by chiasmata.

The chromatids separate progressively from the centromere towards the chiasma and the

chiasma itself moves in a zipper fashion towards the end of the tetrad. The movement of

chiasma is known as terminalisation. Due to the terminalisation the homologous

chromosomes are separated completely.

Kinds of Crossing Over



According to the number of chiasma following types of crossing over have been

described.

1. Single crossing over. When the chiasma occurs only at one point of the

chromosome pair then the crossing over is known as single crossing over. The single

crossing over produces two cross over chromatids and two non-crosses over chromatids.

2. Double crossing over. When the chiasmata occur at two points in the same

chromosome, the phenomenon is known as double crossing over. In the double crossing

over, the formation of each chiasma is independent of the other and in it four possible

classes of recombination occur. In the double crossing over following two types of

chiasma may be formed:

(i) Reciprocal chiasma. In the reciprocal chiasma the same two chromatids are

involved in the second chiasma as in the first. Thus, the second chiasma restores the order

which was changed by the first chiasma and it produces two non-cross over chromatids.

The reciprocal chiasma occurs in two strand double crossing over in which out of four

chromatids only two are involved in the double crossing over.

(ii) Complimentary chiasma. When both the chromatids taking part in the

second chiasma are different from those chromatids involved in the first chiasma is

known as complimentary chiasma. The complimentary chiasma produces four single

cross overs but no non-cross over. The complimentary chiasma occurs when three or four

chromatids of the tetrad undergo the crossing over.

3. Multiple crossing over. When crossing over takes place at more than two places in the

same chromosome pair then such crossing over is known as multiple crossing over. The

multiple crossing over occurs rarely.

Estimation of crossing over-frequency from a test cross:

No. of recombinant progeny is given by:

Frequency of crossing over = ...................................... X 100

Total no. of progenies

Factors affecting the recombination frequency



The frequency of recombination between two linked genes is affected by several factors.

They are (based on the data from Drosophila melanogaster)

Distance between genes: The frequency of cross over between two linked genes is

associated with the distance between their location in the chromosome. Thus crossing

over between two genes would increase with increase in the distance between them. As

location of genes on the chromosome is fixed, the frequency of recombination is expected

to be constant with little of variation. However, the frequency of recombination obtained

between genes may vary due to sampling error or due to other reasons like:

Sex: In Drosophila sp. heterogametic sex individuals show relatively lower frequency

than the homogametic individuals (XX).

Age of female: In general, the frequency of recombination shows progressive decline

with the aging in Drosophila.

Temperature: In Drosophila, lowest frequency of recombination is observed when the

females are cultured at 22 C and increased when temperature is increased

Nutrition: The presence of Ca and Mg in food reduces the recombination frequency in

Drosophila, while the removal of metallic ions (through the chelating agents present in

the diet) increases the recombination frequency. Increase in the recombination frequency

is noticed in the larvae starved during certain stages of development.

Chemicals: Injection of females with antibiotics like mytomycin D and actinomycin D

promotes recombination. Treatment of female- with alkylating agents such as

ethylmethane sulphonate (EMS), also show similar effects.

Radiation: Recombination frequency was increased when the females of Drosophila and

many organisms when irradiated with X-ray. Even the Drosophila male showed

recombination when treated with X-ray.

Plasmogenes: Certain genes present in Drosophila also affect the recombination

frequency. In Bajra, the gene trifton present in the cytoplasm shows reduced

recombination frequency.

Genotypes: Many genes are known to affect the occurrence as well as the rate of

recombination. Some genes affect the chromosome pairing and some other genes affect



the synapsis (after pairing). Genes for asynapsis is known in many crops. The C3G gene

of Drosophila (located on chromosome No. 3) prevents the recombination when present

in homozygous condition, while it promotes recombination at heterozygous condition. In

addition there are evidences to prove that the recombination is also affected by genetic

background, or modifying factors.

Chromosomal aberrations: In Drosophila, paracentric inversion reduces recombination

between genes located within the inverted segment. Translocations also reduce

recombination in the vicinity of breaks. Generally these chromosomal aberrations reduce

recombinations in the chromosomes in which they are located; they often increase the

recombination in other chromosomes present in the same cell. A similar effect of

inversion and translocation on recombination is also known in plants.

Distance from centromere: Centromeres tend to suppress recombination. Therefore

genes located near the centromere show relatively low recombination frequency than

those located away from the centromeres.

Crossing over frequency and cross over:

Crossing over is responsible for the recombination between linked genes. Crossing over

takes place during pachytene stage; each chromosome of a bivalent has two chromatids,

so that each bivalent has four chromatids (four-strand stage). During the process

(crossing over) a segment of one of the chromatid becomes attached with the homologous

segment of a non-sister chromatid and vise versa. It is assumed that breaks occur at

homologous points followed by reunion of the acentric segments. This produces X like

figure at the point of exchange of the chromatic segments is called as 'Chiasma'.

As a result of a cross over, two recombinant chromatids (involved in the cross over)

called cross over chromatids and two original chromatids (not involved in cross over) are

produced. The gametes carrying the recombinant chromatids (which carry the

recombination of linked genes) are called cross over types. Similarly the gametes

carrying non cross over chromatids which contain only the parental combination of

linked genes called non-cross over types. Therefore the frequency of crossing over



between two genes can be estimated as the frequency of recombinant progeny from a test

cross. This frequency is expressed as percentage and it is the frequency of cross over

between the two genes.

Relationship between chiasma and crossing over:

Chiasma is the X like configuration observed during diplotene or late pachytene, when a

homologous chromosome moves away from each other; it appears to be attached with

each other through chiasma. Two divergent theories have been proposed to explain the

relationship between chiasma and crossing over. They are: i) Classical theory (Two

plane theory)

ii) Chiasmatype theory. (One plane theory)

I. Classical Theory: Proposed by Sharp in 1934 also called as two-plane theory.

According to this theory, i) a chiasma is formed when chromatid of a chromosome comes

to associate with the non-sister chromatid of its homologous chromosomes, ii) chiasma

formation is the cause of crossing over, iii) each chiasma does not lead to crossing over

and iv) crossing over occurs during diplotene. The available experimental evidences did

not support this hypothesis, and it is only a historical significance.

II. Chiasmatype Theory: It is proposed by Janssens in 1909 and further expanded by

him in 1924. This is also called as one-plane theory. According to this theory, i)

chiasmata are produced due to crossing over, ii) crossing over occurs before diplotene,

iii) through out the entire bivalent, only sister chromatids are associated with each other

(where as in classical theory the association is only between non-sister chromatids to

produce chiasmata), iv) each chromatid is the consequence of crossing over event and v)

a 1:1 ratio is expected between frequencies of chiasmata and crossing over.

Most of the evidences support only the chiasmata type theory. Further, this theory is able

to explain several phenomena which are not explained by the classical theory. For

example, as per the classical theory three homologous chromosomes are required for a

trivalent configuration, which is not possible. But on the other hand as per the

chiasmatatype theory, only two chromosomes are required to pair in the different



segments to yield the same trivalent configuration. In maize, there is a close correlation

between the relative length of chromosomes determined on the basis of recombination

data and those estimated from the number of chiasmata

However, the most important evidence against the chiasmatatype theory comes from the

male Drosophila where chiasmata are present but there is no crossing over. Most likely

the chiasmata in male Drosophila are produced due to simple association between

homologous chromosomes, i.e. they are not chiasmata in real sense. However, chiasmata

theory is still universally accepted one.

Theories on crossing over:

Several models have been proposed to explain the molecular mechanism of crossing over

and these models may be grouped into two broad classes:

1. Breakage and reunion

2. Copy choice theory

1. Breakage and reunion: This theory was put forth by Darlington in 1932 and

explained in simple words as, "after synapsis, break occurs at identical points in one of

the two chromatids of each of the two homologues forming bivalent . The segments of

two non-sister chromatids reunite to produce cross over chromatids. This is well proved

by 1) by isotope labeling that revealed each cross over chromatids consisted of segments

from two non sister chromatids involved in crossing over. 2) as a rule, there is some DNA

synthesis associated with crossing over and this DNA synthesis most likely involved in

the repairing of chromatid breaks. Based on this theory, several models have been

proposed and these models are called as hybrid DNA models. AH these hybrid DNA

models are the modifications of either the White House model (1963) or the Holiday

model (1964). These two models differ in one important aspect i.e., Whitehouse proposed

that single strand breaks occurs in the strands having opposite polarity, While according

to Holiday it occurs in the strands having the same polarity. Hence, Holiday model is

relatively simpler and more attractive.



2.Duplication theory:

This theory is mostly based on the mechanism of crossing over proposed by Belling in

1933. He postulated that i) genes present in a chromosome are the first to be replicated ii)

they are subsequently connected with each other through the synthesis of the remaining

parts (other than genes of the chromosome) and iii) The homologous chromosome are

likely to be coiled with each other so that the newly produced copies of genes present in a

segment of one chromosome would be adjacent to those of the neighbouring segment of

the homologous chromosome . As a result, the new copies of genes present in a segment

of one chromosome may sometimes become joined with those of the neighbouring

segment of the homologous chromosome, which gives rise to cross over or recombinant

chromatids. According to this theory, chromosome replication, or at least replication of

the segment involved in crossing over, must occur after synapsis, which is contrary to the

known facts, hence this postulate appears to be unrealistic.

3. Copy-choice Model

In 1955 Laderberg proposed a modification in Belling's hypothesis to explain the same

unusual features of recombination in bacteria. This modification is commonly known as

copy-choice theory. According to this theory, during DNA replication, a DNA molecule

serves as a template (for the DNA molecule to synthesis new DNA molecule) upto a

certain distance after which the homologous DNA molecule is used as the template.

Consequently the newly produced DNA molecule is a recombinant.

This theory requires two important pre-requisites viz.,

i) chromosome replication takes place after synapsis and

ii) DNA replication is conservative.

But this is contrary to the fact in eukaryotic chromosome replication occurs before

synapsis and DNA replication is universally semi-conservative. Therefore the copy-

choice model is not acceptable, however, in some prokaryotes the DNA replication

appears to be copy-choice type.



4. Cross- over theory: According to this theory, an endonuclease produces single-strand

nicks at identical points in the two homologous DNA molecules in strands having the

same polarity. The two strands of each DNA molecule separate from each other upto

certain distance from the point of 'nick'. The free strands now pair with the intact strands

of the homologous DNA molecule during the unwinding of chromatids.

This produces a "hybrid DNA molecule". The two nicks present in the molecules are

sealed by DNA ligase. Then the hybrid DNA molecule undergoes reorientation to form

X-shaped figure; one end of this X rotates to 180°. Such hybrid DNA molecules have

been actually isolated from bacteria and photographed with the help of electron

microscope. An endonuclease now induces nicks in the two intact (not cut earlier) strands

of the hybrid molecules; this yields two recombinant DNA molecules. Each recombinant

molecule has a nick which is finally sealed by DNA ligase. Most of the evidences are in

favour of this model since almost all the enzymes and proteins involved in the process are

known to occur in bacteria.

LNKAGE GROUPS AND LINKAGE MAPPING

All the linked genes form a linkage group. The genes that are linked may be represented

on a straight line in the same order in which they are normally present in the concerned

chromosome. In such representation, the distance between any two neighbouring genes is

proportional to the frequency of recombination between them.Such a diagrammatic

representation depicting the linked genes and the recombination frequencies between

them is known as linkage map or genetic map or chromosome map.

Thus for preparing a chromosome map the following two information are required: i)

the frequency of recombination between linked genes, ii) the order or sequence of these

genes in the chromosomes.

As we have already seen the recombination frequency between any two genes can be

estimated from appropriate test crosses or F2 progenies. These percentages of

recombination frequencies are used as map units for construction of linkage maps. A map

unit is defined as that distance in a chromosome which permits one percent of



recombination between the linked genes. A map unit is also referred as Morgan (1

centiMorgan = 1 map unit).

It should be noted that a map unit is an imaginary distance and it does not represent the

actual distance between two linked genes in the chromosome. Therefore a map unit does

not have a unit of measurement like A, u., mm etc. Since the map units are the

recombination frequencies between two linked genes, they are likely to be influenced by

several factors.

DETERMINATION OF THE SEQUENCE OF LINKED GENES:

The sequence of linked genes can be determined by studying the test crosses for three

genes at a time. The data from a test cross involving three genes will provide the

information on the order of the three genes in the chromosome as well as their

recombination frequencies between them. To begin with a three point test cross involving

any two of these three linked genes and a new gene expected to be linked with them is

studied to map the new gene. In this manner each new gene is mapped by ordering it in

three-point test cross with two already mapped genes.

In such studies it is desirable to include only those genes show less than 20% preferably

10% or less recombination with each other to avoid confusion due to double and triple

cross overs. If the crossing over between the genes in test cross is more than 20% the

linkage maps would not be very reliable. In addition the number of test cross progeny

should be sufficiently large to yield reliable recombination frequencies and to avoid the

effects of sampling error. The number of linked group in a species is a rule, equal to its

gametic chromosome number (n). Each linkage group of a species is to be assigned to a

specific chromosome of that species with the help of chromosomal aberrations. In general

the relative length of linkage group of a species correspond closely with the relative

lengths of the chromosomes in which they are located.

These linkage maps provide the information on i) the genes that are linked together and

ii) the frequencies of recombination that may be expected between them. Sometimes the



distance between any two genes in a linkage group may exceed 50% or even 100 %, but

that does not mean that they show recombination beyond 50%, the recombination

between two linked genes cannot exceed 50% which is the frequency expected in

independent assortment. However, there is a 1:1 correspondence between map distance

and the observed recombinant frequency upto 20 map units. But there is a progressive

decline in the frequency of observed recombination for every additional map unit

distance beyond 20 map units. .

DETERMINATION OF GENE SEQUENCE:

Now using this data the gene sequence can be obtained by comparing the genotypes of

the parental and double cross over types. In a double crossing over simultaneous cross

over occurs on both sides of the genes located in the middle. In the figure the gene 'sh' is

located between 'C and 'Wx..In this case the double cross over produced will be between

'C and 'Sh' and 'Sh' and 'Wx'. Hence, parental types are CShWx and cshwx and the double

cross over types are CshWx and cShwx. So the comparison between parental and double

cross over shows that only the gene located in the middle is shifted. This property of the

double cross overs is used for determining the sequences of the genes, which are linked.

But in the given table, the parental types are CWxSh and cwxsh and the double cross

overs are CWxsh and cwxSh, i.e. the allele 'sh' have been shifted their position in the

double cross overs as compared to parental types. Therefore the gene 'sh' must be located

between 'c' and 'wx'.

ALTERNATE PROCEDURE TO FIND THE ORDER OF THE GENES:

An alternate procedure for determining the gene frequency involves the comparison of

recombination frequencies. If the genes 'c\ 'sh' and 'wx' are placed in the order of c-sh-wx

single cross over between 'c' and 'sh 'which yield the cross over gametes viz., Cshwx and

cShWx. In addition to the above frequencies between 'c' and 'Sh' and between 'sh' and

'wx', double cross over viz., CshWx and cShwx are also produced due to simultaneous

crossing over between genes 'c' and 'sh' and 'sh' and 'wx', therefore these double cross

over frequencies should also be included to estimate recombination frequencies between

'c' and 'sh' as well as between 'sh' and 'wx'.



Therefore the frequency of recombination between 'C and 'Sh' will be the sum of the

frequencies of

Cwxsh ... 1.7

cWxSh ... 1.8

Total . 3.5 +freq. of Double cross overs

Similarly single cross over between 'sh' and 'wx' will produce the cross over types viz.,

CShwx and cshWx and the sum of these frequencies will be their recombination between

'sh' and 'wx' i.e.

CShwx ... 9.2

cshWx ... 8.9

Total ... 18.1 + Double cross overs

Now let us consider that crossing over are between 'c' and 'wx' i.e. two genes located on

either sides of the gene 'sh'. In such case the crossing over between *c' and 'sh' and 'sh'

and 'wx' would produce recombination between 'c' and 'wx'.

Therefore the recombination frequency between C and wx = frequency of c and sh +

frequency of sh and wx (It should be noted that double cross over does not lead to

recombination between 'c' and 'wx' although two events of crossing over occur between

them).

A map distance of around 90 units is expected to show to 50% recombination, Thus,

i) a map distance is the frequency of recombination upto 20 map units

ii) linked genes would show independent segregation if they are more than 80 map

units apart and

iii) maximum recombination observed between two linked gene is 50%. ■

COEFFICIENT OF INTERFERRENCE:

As a rule the observed frequencies of double cross overs are less than the expected

values. This is because" the occurrence of crossing over in one region of a chromosome

interferes with its occurrence in the neighbouring segment, this is called as interference.

It may be expected that the intensity of interference would decrease as the point of



second crossing over becomes farther from that of the first one. Therefore the coefficient

of coincidence will be lower when the concerned genes are located close to each other.

The intensity of interference may be estimated as coefficient of interference = 1 -

coefficient of coincidence.

Interference=%of observed double cross overs / %of expected cross overs



Constructing a Genetic Map with Two-Point Testcrosses

Genetic maps can be constructed by conducting a series of testcrosses between pairs of genes and examining the recombination frequencies between them. A testcross betweentwo genes is called a two-point testcross or a two-point cross for short. Suppose that we carried out a series of two-point crosses for four genes, a, b, c, and d, and obtained the following recombination frequencies:

Gene loci in testcross Recombination frequency (%)a and b 50a and c 50a and d 50b and c 20b and d 10c and d 28

We can begin constructing a genetic map for these genes by considering the recombination frequencies for each pair of genes. The recombination frequency between a and b is 50%, which is the recombination frequency expected with independent assortment. Genes a and b may therefore either be on different chromosomes or be very far apart on the same chromosome; so we will place them in different linkage groups with the understanding that they may or may not be on the same chromosome:

The recombination frequency between a and c is 50%, indicating that they, too, are in different linkage groups. The recombination frequency between b and c is 20%; so thesegenes are linked and separated by 20 map units:

The recombination frequency between a and d is 50%, indicating that these genes belong to different linkage groups, whereas genes b and d are linked, with a recombinationfrequency of 10%. To decide whether gene d is 10 map units to the left or right of gene b, we must consult the c-to-d distance. If gene d is 10 map units to the left of gene b, then the distance between d and c should be 20 m.u. + 10 m.u. = 30 m.u. This distance will be only approximate because any double crossovers between the two genes will be missed and the recombination frequency will be underestimated. If, on the other hand, gene d lies


Linkage group 1

Linkage group 2

a

b

Linkage group 1

Linkage group 2

a

b c

20 mu


to the right of gene b, then the distance between gene d and c will be much shorter, approximately 20 m.u. - 10 m.u. = 10 m.u. By examining the recombination frequency between c and d, we can distinguish between these two possibilities. The recombination frequency between c and d is 28%; so gene d must lie to the left of gene b. Notice that the sum of the recombination between d and b (10%) and between b and c (20%) is greater than the recombination between d and c (28%). (This is what was meant by saying thatrecombination rates—i.e., map units—are approximately additive.) This discrepancy arises because double crossovers between the two outer genes go undetected, causing anunderestimation of the true recombination frequency. The genetic map of these genes is now complete:


Linkage group 1

Linkage group 2

a

b c

20 mu10 mu

d


Linkage and Recombination between Three Genes

Genetic maps can be constructed from a series of testcrosses for pairs of genes, but this approach is not particularly efficient, because numerous two-point crosses must be carried out to establish the order of the genes and because double crossovers are missed. A more efficient mapping technique is a testcross for three genes (a three-point testcross, or threepoint cross).With a three-point cross, the order of the three genes can be established in a single set of progeny and some double crossovers can usually be detected, providing more accurate map distances.

Consider what happens when crossing over takes place among three hypothetical linked genes. Figure illustrates a pair of homologous chromosomes from an individual that is heterozygous at three loci (AaBbCc). Notice that the genes are in the coupling configuration; that is, all the dominant alleles are on one chromosome ( A----- B----- C ) and all the recessive alleles are on the other chromosome ( a---- b---- c ). Three types of crossover events can take place between these three genes: two types of single crossovers (Figure ) and a double crossover (Figure). In each type of crossover, two of the resulting chromosomes are recombinants and two are nonrecombinants.

Notice that, in the recombinant chromosomes resulting from the double crossover, the outer two alleles are the same as in the nonrecombinants, but the middle allele is different. This result provides us with an important clue about the order of the genes. In progeny that result from a double crossover, only the middle allele should differ from the alleles present in the nonrecombinant progeny.

Gene Mapping with the Three-Point Testcross



To examine gene mapping with a three-point testcross, we will consider three recessive mutations in the fruit fly Drosophila melanogaster. In this species, scarlet eyes (st) arerecessive to red eyes (st+), ebony body color (e) is recessive to gray body color (e+), and spineless (ss)—that is, the presence of small bristles—is recessive to normal bristles(ss+). All three mutations are linked and located on the third chromosome.

We will refer to these three loci as st, e, and ss, but keep in mind that either recessive alleles (st, e, and ss) or the dominant alleles (st+, e+, and ss+) may be present at each locus. So, when we say that there are 10 m.u. between st and ss, we mean that there are 10 m.u. between the loci at which these mutations occur; we could just as easily say that there are 10 m.u. between st+ and ss+. To map these genes, we need to determine their order on the chromosome and the genetic distances between them. First, we must set up a three-point testcross, a cross between a fly heterozygous at all three loci and a flyhomozygous for recessive alleles at all three loci. To produce flies heterozygous for all three loci, we might cross a stock of flies that are homozygous for normal alleles at all three loci with flies that are homozygous for recessive alleles at all three loci:

The order of the genes has been arbitrarily assigned because at this point we do not know which is the middle gene.

Additionally, the alleles in these heterozygotes are in coupling configuration (because all the wild-type dominant alleles were inherited from one parent and all the recessivemutations from the other parent), although the testcross can also be done with genes in repulsion. In the three-point testcross, we cross the F1 heterozygotes with flies that are homozygous for all three recessive mutations. In many organisms, it makes no differencewhether the heterozygous parent in the testcross is male or female (provided that the genes are autosomal) but, in Drosophila, no crossing over takes place in males. Becausecrossing over in the heterozygous parent is essential for determining recombination frequencies, the heterozygous flies in our testcross must be female. So we mate female F1flies that are heterozygous for all three traits with male flies that are homozygous for all the recessive traits:

The progeny produced from this cross are listed in FIGURE. For each locus, two classes of


P

F1

st+ e+ss+st+ e+ss+

st e ss

st e ss

X

st+ e+ ss+

st e ss

st+ e+ss+

st essst ess

Xst ess

female

male


progeny are produced: progeny that are heterozygous, displaying the dominant trait, and progeny that are homozygous, displaying the recessive trait. With two classes of progenypossible for each of the three loci, there will be 2 3 = 8 classes of phenotypes possible in the progeny. In this example, all eight phenotypic classes are present but, in some three-point crosses, one or more of the phenotypes may be missing if the number of progeny is limited. Nevertheless, the absence of a particular class can provide important information about which combination of traits is least frequent and ultimately the order of the genes, as we will see.

To map the genes, we need information about where and how often crossing over has occurred. In the homozygous recessive parent, the two alleles at each locus are the same; and so crossing over will have no effect on the types of gametes produced; with or without crossing over, all gametes from this parent have a chromosome with three recessive alleles ( --st--- e---- ss-- ). In contrast, the heterozygous parent has different alleles on its two chromosomes; so crossing over can be detected. The information that we need for mapping, therefore, comes entirely from the gametes produced by the heterozygous parent. Because chromosomes contributed by the homozygous parent carry only recessive alleles, whatever alleles are present on the chromosome contributed by the heterozygous parent will be expressed in the progeny. As a shortcut, we usually do not write out the complete genotypes of the testcross progeny, listing instead only the alleles expressed in the phenotype (as shown in Figure), which are the alleles inherited from the heterozygous parent.

Determining the gene order The first task in mapping the genes is to determine their order on the chromosome.In Figure aove, we arbitrarily listed the loci in the order st, e, ss, but we had no way of knowing which of the three loci was between the other two. We can now identify the middle locus by examining the double-crossover progeny. First, determine which progeny are the nonrecombinants— they will be the two most-numerous classes ofprogeny. (Even if crossing over takes place in every meiosis, the nonrecombinants will comprise at least 50% of the progeny.) Among the progeny of the testcross in Figure, themost numerous are those with all three dominant traits

and those with all three recessive traits


st+ e+ ss+

st e ss


Next, identify the double-crossover progeny. These should always be the two least-numerous phenotypes, because the probability of a double crossover is always less than the probability of a single crossover. The least-common progeny among those listed in Figure are progeny with red eyes, gray body, and spineless bristles( st+ e+ ss ) andprogeny with scarlet eyes, ebony body, and normal bristles ( st e ss+ ); so they are the double-crossover progeny.

Three orders of genes are possible: the eye-color locus could be in the middle ( e st ss ), the body-color locus could be in the middle ( st e ss ), or the bristle locus could be in the middle ( st ss e ). To determine which gene is in the middle, we can draw the chromosomes of the heterozygous parent with all three possible gene orders and then see if a double crossover produces the combination of genes observed in the doublecrossoverprogeny. The three possible gene orders and the types of progeny produced by their double crossovers are:

The only gene order that produces chromosomes with alleles for the traits observed in the double crossovers (st+ e+ ss and st e ss+) is the third one, where the locus for bristle shape lies in the middle. Therefore, this order (-- st--- ss--- e-- ) must be the correct sequence of genes on the chromosome. With a little practice, it’s possible to quickly determine which locus is in the middle without writing out all the gene orders. The phenotypes of the progeny are expressions of the alleles inherited from the heterozygous parent. Recallthat, when we looked at the results of double crossovers, only the alleles at the middle locus differed from the nonrecombinants. If we compare the nonrecombinant progeny with double-crossover progeny, they should differ only in alleles of the middle locus.

Let’s compare the alleles in the double-crossover progeny st+ e+ ss with those in the nonrecombinant progeny st+ e+ ss+ . We see that both have an allele for red eyes (st+) and both have an allele for gray body (e+), but the nonrecombinants have an allele for normal bristles (ss+), whereas the double crossovers have an allele for spineless bristles(ss). Because the bristle locus is the only one that differs, it must lie in the middle.We would obtain the same results if we compared the other class of double-crossover progeny ( --st ---e –ss+ --) with other nonrecombinant progeny ( --st—e—ss-- ). Again the only trait that differs is the one for bristles. Don’t forget that the nonrecombinants and the double crossovers should differ only at one locus; if they differ in two loci, the wrong classes of progeny are being compared.

Determining the locations of crossovers



When we know the correct order of the loci on the chromosome, we should rewrite the phenotypes of the testcross progeny with the loci in the correct order so that we can determine where crossovers have taken place ( FIGURE ). Among the eight classes of

progeny, we have already identified two classes as nonrecombinants ( st+ ss+ e+ andst ss e ) and two classes as double crossovers ( st+ ss e+ and st ss+ e ). The other fourclasses include progeny that resulted from a chromosome that underwent a single crossover: two underwent single crossovers between st and ss, and two underwent singlecrossovers between ss and e.

To determine where the crossovers took place in these progeny, compare the alleles found in the single-crossover progeny with those found in the nonrecombinants, just as we did for the double crossovers. Some of the alleles in the singlecrossover progeny are derived from one of the original (nonrecombinant) chromosomes of the heterozygous parent, but at some place there is a switch (due to crossing over) and the remaining alleles are derived from the homologous nonrecombinant chromosome. The position of the switch indicates where the crossover event took place. For example, consider progeny with chromosome st+ ss e . The first allele (st+) came from the nonrecombinant chromosome st+ ss+ e+ and the other two alleles (ss and e) must have come from

the other nonrecombinant chromosome st ss e through crossing over(figure).

This same crossover also produces the st ss+ e+ progeny. This same method can be used to determine the location of crossing over in the other two types of singlecrossover progeny. Crossing between ss and e produces st+ ss+ e and st ss e+ chromosomes (figure).

Calculating the recombination frequencies

Next, we can determine the map distances, which are based on the frequencies of recombination. Recombination frequency is calculated by adding up all of the recombinant progeny, dividing this number by the total number of progeny from the cross, and multiplying the number obtained by 100%. To determine the map distances accurately, we must include all crossovers (both single and double) that take place between two genes. Recombinant progeny that possess a chromosome that underwent crossing over between the eye-color locus (st) and the bristle locus (ss) include the singlecrossovers ( st+ / ss e and st / ss+ e+ ) and the two double crossovers ( st+ / ss / e+ and



st / ss+ / e ). There are a total of 755 progeny; so the recombination frequency between ss and st is:

st–ss recombination frequency =(50+52+5+ 3)/755 X 100 = 14.6%

The distance between the st and ss loci can be expressed as 14.6 m.u.

The map distance between the bristle locus (ss) and the body locus (e) is determined in the same manner. The recombinant progeny that possess a crossover between ss and e arethe single crossovers st+ ss+ / e and st ss / e+ , and the double crossovers st+ / ss / e+ and st / ss+ / e . The recombination frequency is:

ss–e recombination frequency =

(43+41+5+3)/755 X 100 = 12.2%

Thus, the genetic distance between ss and e is 12.2 m.u.

Finally, calculate the genetic distance between the outer two loci, st and e. Add up all the progeny with crossovers between the two loci. These progeny include those with a single crossover between st and ss, those with a single crossover between ss and e, and the double crossovers ( st+ / ss / e+ and st / ss+ / e ). Because the double crossovers have two crossovers between st and e, we must add the double crossovers twice to determine the recombination frequency between these two loci:

st– e recombination frequency =

(50+52+43+41+(2X5)+(2X3)) / 755 X100 = 26.8%

Notice that the distances between st and ss (14.6 m.u.) and between ss and e (12.2 m.u.) add up to the distance between st and e (26.8 m.u.). We can now use the map distances to draw a map of the three genes on the chromosome.

InterferenceThe degree to which one crossover interferes with additional crossovers in the same region is termed the interference.

Coefficient of coincidence, which is the ratio of observed double crossovers to expected double crossovers.


st ss e

26.8 mu

14.6 mu 12.2 mu


Unit-IV

Mutation

A Gene mutation is abrupt inheritable qualitative or quantitative change in the

genetic material of an organism. Since in most organisms genes are segments of DNA

molecule, so a mutation can be regarded as a change in the DNA sequence which is

reflected in the change of sequence of corresponding RNA or protein molecules. Such a

change may involve only one base/base pair or more than one base pair of DNA.

Mutation occurs in a random manner, ie.., they are not directed according to the

requirements of the organism. Most mutations occur spontaneously by the environmental

effect; however, they can be induced in the laboratory either by radiations, physical

factors or chemicals (called mutagens.) A unicellular organism is more subjected to

environmental onslaughts since it is at the same time a somatic or germ cell. In

multicellular organisms the germ cells are distinct cells, and are relatively protected from

the environment. Mutation has a significant role to play in the origin of species or

evolution.

HISTORICAL BACKGROUND

Hugo de vries was the first hybridist who used the term “mutation” to describe the

heritable phenotypic changes of the evening primrose, Oenothera lamarckiana. Many

mutations described by de vries in Oenothera lamarckiana, are known to be due to

variation in chromosome number or ploidy and chromosomal aberrations (viz gross

mutations). The first scientific study of mutations was started in 1910, when Morgan

and his work was in fruit fly, Drosophila melanogaster and reported white eyed male

individuals among red eyed male individuals. The discovery of white eyed mutants in

Drosophila was followed by Morgan and his co-workers and other geneticists.

Consequently about 500 mutants of Drosophila have been reported by geneticists all over

the world.

Mutants occur frequently in the nature and have been reported in many organisms, e.g.,

drosophila, mice and other rodents, rats, rabbits, guinea pigs and man. In the drosophila,

mutation causes white and pink eyes, black and yellow body colours, and vestigial wings.



In rodents the mutations are responsible for black, white and brown coats. In man the

mutations cause variation in hair colour, eye colour, skin pigmentation and several

somatic malformations. Various genetical diseases of human beings such as haemophilia,

colour blindness, phenylketonuria etc., form other examples for mutation in human

beings.

How does a mutation act? Any change in sequence of nucleotides in the DNA will

result in the corresponding change in the nucleotide sequence of mRNA. This may result

in alignment of different RNA molecules on mRNA (during protein synthesis). Thus the

amino acid sequence and hence the structure and properties of the enzyme formed will be

changed. This defective enzyme structural protein may adversely affect the trait

controlled by the protein. In consequence a mutant phenotype makes its expression.

KINDS OF MUTATIONS

1. Classification of Mutation According to Type of Cells

According to their occurrence in somatic and germinal cells following types of mutations

have been classified:

Somatic Mutations. The mutations occurring in non-reproductive body cells are known

as somatic mutations. The genetical and evolutionary consequences body cells are

insignificant, since only single cells and their daughter cells are involved. If, however, a

somatic mutation occurs early during embryonic life, the mutant cells may constitute a

large proportion of body cells and the animal body may be a mosaic for different type of

cells. Somatic mutations have been often related with malignant

(cancerous) growth. Examples of somatic mutation have been reported in Oenothera

lamarckiana (Hugo de Vries) and several other cases including man. In man somatic

mutation causes several fatal diseases such as paraoxysmal nocturnal hemoglobinura,

unilateral retinoblastoma and heterochromia of the iris.



Gametic mutations. The mutations occurring in gamete cells (e.g., sperms and Ova) are

called gametic mutations. Such mutations are heritable and of immense genetical

significance. The gametic mutations only form the base for the natural selection.

2. Classification of Mutations According to the Size and Quality

According to size following two types of mutations have been recognized:

Point mutation. When heritable alterations occur in a very small segment of DNA

molecule, i.e., a single nucleotide or nucleotide pair, then this type of mutations are

called “point mutations”. The point mutations may occur due to following types of

subnucleotide change in the DNA and RNA.

1. Deletion mutations. The point mutation which is caused due to loss or

deletion of some portion (single nucleotide pair) in a triplet codon of a cistron

or gene is called deletion mutation. Deletion mutations have been frequently

reported in some bacteriophages.

2. Insertion or addition mutation. The point mutations which occur due to

addition of one or more extra nucleotides to a gene or cistron are called

insertion mutations. The insertion mutations can be artificially induced by

certain chemical substances called mutagens such as acridine dye and

proflavin. A proflavin molecule, it is believed, insert between two successive

bases of a DNA strand, thereby stretching the strand lengthwise. At

replication, this situation would allow the insertion of an extra nucleotide in

the complementary chain at the position occupied by the proflavin molecule.

The mutations which arise from the insertion or deletion of individual nucleotides and

cause the rest of the message downstream of the mutation to be read out phase, are called

frameshift mutations. They result in the production of an incorrect, hence, inactive

protein, due to which the death of the cell may occur.

3. Substitution mutation. A point mutation in which a nucleotide of a triplet is



replaced by another nucleotide, is called substitution mutation. The

substitution mutation affects only a particular triplet codon. Such an altered

code word (triplet codon) may designate a different amino acid and may result

in the production of a protein with a single amino acid substitution. The

substitution mutations alter the phenotype of an organism variously are of

great genetical significance. They may be of following:

(i) Transition. When a purine (e.g., ademine) base of a triplet codon of a cistron is

substituted by another purine base (e.g., guanine) or a pyrimidine (e.g., thymine) is

substituted by another pyrimidine base, (e.g., cytosine) then such kind of substitution is

called transition. The transitional substitution mutations occur due to tuatomerization.

Tautomerization

In a DNA molecule, normally, the purine, adenine (A) is linked to the pyrimidine,

thymine (T), by two hydrogen bonds, while the purine guanine (G) is linked to the

pyrimidine, cytosine (C) by three hydrogen bonds. Besides the common molecular

configurations, each DNA base may have some altered uncommon molecular

configuration.

Such uncommon forms of DNA bases are generated by single proton shifts and are called

rare states of tautomers. A tautomeric shift is believed to occur when the amino (NH2)

form of adenine is changed to an imino (NH) form. Similarly, a tautomeric shift may

occur in thymine changing it form the keto (C = 0) form to the rare enol (COH) form.

When a base occurs in its rare or tatuomeric state, it cannot be linked to its normal

partner. However, a purine, such as adenine can in its rare state forms a bond with

cytosine (besides thymine), provided the cytosine is in its normal state.

.

3. Transversion. The substitution mutation when involves the substitution or

replacement of a purine with a pyrimidines of vice versa then type of substitution



mutation is called transversion mutation. The existence of transversion mutation was first

of all postulated by E. Freese in 1959. We have still poor information about the

mechanism of induction, identification and charectirization of trnsversion mutations.

Moreover, it is extremely difficult to recognize transversion mutations genetically.

However, they can be recognized only by amino acid substitution in proteins.

Effects of physical Conditions on Nucleotide Sequence

High temperature and low pH value are known to affect depurination or loss of purine

bases. The removal of a purine from a strand of DNA leaves a gap at that. At the time of

replication, it would be possible for any of the four bases to insert in the complementary

strand would contain a transversion.

B. Multiple mutations or gross mutations. When changes involving more than one

nucleotide pair, or entire gene, then such mutations are called gross mutations, the gross

mutations occur due to rearrangements of genes within the genome and may be of the

following types:

1. The rearrangements of genes may occur within a gene. Two mutations within the same

functional gene can produce different effects depending on gene whether they occur in

the cis or trans position.

2. The rearrangements of gene may occur in number of genes per chromosome, they may

cause different types of phenotypic effects over the organisms.

3. Due to movement of a gene locus new type of phenotypes may be created, especially

when the gene loci may take place due to following method:

(i) Translocation: Movement of a gene may take place to a non-homologous

chromosome and this is known as translocation.

(ii) Inversion. The movement of a gene within the same chromosome is called

inversion.

3. Classification of Mutation According to the Origin

According to the mode of origin, following two kinds of mutations have been recognized.



(1) Spontaneous mutations. The spontaneous mutations occur suddenly in the

nature and their origin is unknown. They are also called “background mutation” and

have been reported in many organisms such as, Oenothera, maize, bread molds,

microorganisms (bacteria and viruses), Drosophila, mice, man, etc.

(2) Induced mutations. Besides naturally occurring spontaneous mutations, the

mutations can be induced artificially in the living organisms by exposing them to

abnormal environment such as radiation, certain physical conditions (i.e., temperature)

and chemicals. The substances or agents which induce artificial mutations are called

mutagens or mutagenic agents.

Mutagenic agents. The mutagenic agents are of the following kinds:

A. Radiations. The radiations which are important in mutagenesis are of two

categories: one type is ionizing radiations such as X-rays and gamma rays; alpha and

beta rays; electrons, neutrons, protons and other fast moving particles. The second type is

non-ionizing radiations such as ultraviolet and visible light. Both types of radiations

induce mutations by following methods:

(i) Ionizing radiations as mutagens. Relatively little is known about the mechanism by

which ionizing radiations cause mutation. As, familiar that matter composed of atoms and

atoms, in their turn, are made up of a positively charged atomic nucleus (with neutrons,

protons) and a surrounding constellation of negatively charged electrons. The charges of

atomic particles remain so balanced that normal is electrically neutral. When ionizing

radiations pass through matter, they dissipate their energy in part through the ejection of

electrons from the outer shell of atoms and the loss of these balancing, negatively

charged particles (electrons) leaves atoms which are no longer neutral but are positively

charged. The positively-charged atom is called ion. The ejected electrons move at high

speed, knock other electrons free from their respective atoms and when their energy is

dissipated, become attach to other atoms and convert the ion into negatively charged ions.

To achieve their stable configuration (i.e., neutral charge) ions undergo many chemical



reactions and during these chemical reactions ionizing radiation is thought to cause

mutation.

Further, ionizing radiations cause breaks in poly-sugar phosphate backbone of DNA and,

thus, causing chromosomal mutations such as break, deletion, addition, inversion and

translocation. During breakage of DNA molecule due to ionizing radiation the active role

of oxygen is predicted. Because, oxygen is important in the formation of H2O2 and HO2

in irradiated water and these products may induce breaks in DNA molecule.

(ii) Non-ionized radiations as mutagens: The ultraviolet (UV) light is a non-ionizing

radiation which may cause mutation. The most effective wave length of ultraviolet for

inducing mutations is about 2,600 Ao. This is a wave length that is best absorbed by DNA

and a wave length at which proteins absorb little energy. When a substance absorbs

sufficient energy from the ultraviolet light, some of their electrons are raised to higher

energy levels, a state called excitation. The excited molecule becomes reactive and

mutated and is called photoproducts.

Dimerization: The ultraviolet radiation produces several effects on DNA, one being the

formation of chemical bonds between two adjacent pyrimidines molecules in a

polynucleotide and particularly, between adjacent thymine residues. As the two thymine

residues associate, or dimerize to form a dimer, their position in the DNA helix becomes

so displaced that they can no longer from hydrogen bonds with the opposing purines and

thus regularity of the helix becomes resorted. Thus, dimerization interferes with the

proper base pairing of thymine with adenine, may result in thymine’s pairing with

guanine. This will produce a T-A to C-G transition.

B.Temperature as mutagen. The rate of all chemical reactions is influenced by

temperature. It is reported that the rate of mutation is increased due to increase in

temperature. For example, an increase of 10o C temperature increases the mutation rate

two or three fold. Temperature probably affects both thermal stability of DNA and the

rate of reaction of other substances with DNA.



C. Chemical mutagens. Many chemical substances have been responsible to increase

the mutability of genes. The ability of chemicals to induce mutation was first of all

demonstrated by Auerbach and Robson in 1947 using mustard gas and related

compounds as the nitrogen and sulphur mustards, mustard oil and chloracetone in

experiments with male Drosophilae melanogaster. Since then many chemical compound

which are ordinarily considered to be non-toxic have been found to be mutagenic in

certain specific situations. Any chemical substance that affects the chemical environment

of chromosomes is likely to influence, at least indirectly, the stability of DNA and its

ability to replicate without error. A chemical mutation can cause mutation only when it

enters in the nucleus of the cell. It can affect the chromosomal DNA by following two

ways:

(1) Direct gene change. Certain chemical mutagens affect DNA directly. They affect the

constituents of DNA only when DNA is not replicating. For example, nitrous acid

converts adenine into hypoxanthine and cytosine to uracil by deamination. Like the

nitrous acid, nitrogen mustard, formaldehyde, epoxides, dimethyl and diethyl sulphonate,

methyl and ethyl methanesulphonate (MMS and EMS) and nitrosoguadine (NG) also

have direct mutagenic effect on the DNA molecule.

(2) Copy error. Certain chemical compounds, called base analogues (e.g., 5-

bromouracil, 2-aminopurine, etc.)Closely resemble with certain DNA bases and are,

therefore, act as mutagens. During analogues such as urethane triazine, caffeine (in

coffee, tea and soft drinks), phenol and carcinogens, chloride is mutagenic for many

organisms, as they are the compounds which bind calcium and, thus, interfere with the

integrity of the chromosome structure.

Effect of Chemical Mutagens on Nucleotide Sequence

(a) Alteration in Resting Nucleic Acid

Deamination: Some chemical substances such as nitrous acid causes transitional

mutation due to oxidative deamination of DNA base, where the amino group is replaced



by hydroxyl (OH) group by the chemical mutagen. Thus, adenine is deaminated into

hypoxanthine by nitrous acid. Similarly, deamination converts cytosine to uracil, which

has pairing properties similar to thymine and in such a case G: C pair would be changed

into A: T pair.

Alkylating agents: Some alkylating agents carry one, two or more alkyl groups in a

reactive form and act as strong mutagens. Example of some most extensively studied

alkylating agents include diethylsulphate (DES), dimethyl sulphate (DMS), methyl

methane sulphonate (MMS), ethylethane sulphonate (EMS). These mutagens produce

mutations in the following ways:

(1) They add ethyl methyl groups to guanine. This makes guanine the base

analogue to adenine.

(2) They remove the alkylated guanine. This is known as depurination. The loss

of the base produces gaps in the DNA chain which may be filled with a wrong base, thus,

producing mutation.

(3) The gap may also produce a deletion, causing mutation.

(b) Alteration during Replication of Nucleic Acid

1. Base analogues. Certain chemical substances have molecular structure similar to the

usual DNA bases that, if they are available, such analogues may be incorporated into a

replicating DNA stand. For example, 5-bromouracil (5BU) or its nucleoside 5-

bromodeoxyuridine (5-BUdR) in its usual (keto) form is a structural analogue of

thymine (5-methyl uracil) and it will substitute for thymine. Thus, an A-T pair becomes

and remains A-BU. There is some in vitro evidence to indicate the BU immediately

adjacent to an adenine in one of DNA strands causes the latter to pair with guanine. But,

in its rare (enol) state, 5BU behaves similar to the tautomer of thymine and pairs with

guanine. This converts A: T to G: C.

2-Aminopurine (2-AP) is another base analogue which is relatively undifferentiated

purine that apparently can pair with cytosine and thymine. It is thought that 2-AP acts by

“switching” pyrimidines: for example, it may be incorporated opposite thymine during



one round of replication and then pair with a cytosine at the next round to produce an AT

GC transition.

2. Inhibition of precursors of nucleic acid:There are some mutagents which interfere

with the synthesis of nitrogen bases of nucleic acids such as purines or pyrimidines.

Often lack of one base either causes breaks or causes pairing mistakes. For example,

azaserine (a potent alkylating agent) is an inhibitor of pyrimidine synthesis. However,

urethane induced chromosome breaks are inhibited by thymine

4. Classification of Mutation According to the Direction

According to their mode of direction following types of mutations have been recognized:

(A) Forward mutations. In an organism when mutations create change from wild type to

abnormal phenotype, then that type of mutations are known as forward mutations. Most

mutations are forward type.

(B) Reverse or back mutations. The forward mutations are often corrected by error

correcting mechanism, so that an abnormal phenotype changes into wild type phenotype.

They may be of the following types:

(i) Single site mutation. Some reverse mutations change only one nucleotide in the gene

and are called single site mutations. For example, due to forward mutation the adenine

is changed into guanine and backward mutation change guanine into adenine:

Forward reverse

Adenine Guanine Adenine

(ii) Mutation suppressor. When a mutation occurs at a different site from the site where

already primary mutation occurred and that mutated gene reverse the effects of primarily

mutated gene, then such (secondary) mutations are called mutation suppressors. They

may be of following types:



(a) Extragenic suppressor. The extragenic suppressor mutation occurs in a different

gene from that of the mutant gene. In E.coli, a gene called rec A (rec for

recombination) is known which is necessary for recombination and is found to repair

ultraviolet-induced thymine dimmers of a gene by a process called post replication

recombinational repair .

(b) Intergenic suppressor. The intergenic suppessor mutation occurs in a different

nucleotide within the same gene and shifts the reading frame back into register.

(c) Photoreactivation. In photoreactivation type reverse mutation reversal of ultraviolet

induced thymine dimers takes place by specific enzymes in the presence of visible light

waves. During ultraviolet radiation a particular enzyme is selectively bound to the

bacterial DNA. During photoreactivation the enzyme is activated by visible light and that

cleaves the pyrimidine or purine dimers into monomers and restores their original forms.

(d) Excision repair or Dark reactivation. In an ultraviolet (UV) induced mutations, the

reverse mutation may also occur in the absence of light. According to Howard Flanders

and Boyce (1964) dark reactivation includes following stages: (i) An enzyme possibly

endonuclease makes a cut in the polynucleotide strand, on either side of the dimer which

may be formed due to ultraviolet radiation and excises a short, single strand segment of

the DNA. (ii) Another enzyme, possibly exonuclease widens the gap produced by the

action of the endonuclease. (iii) DNA polymerase resynthesizes the missing segment,

using the remaining opposite strand as a template; and (iv) the final gap is closed by some

enzymatic rejoining process, (i.e., DNA ligase).

5. Classification of mutation According to Magnitude of Phenotypic Effect

According to their phenotypic effects following kinds of mutations may occur:

1. Dominant mutations. The mutations which have dominant phenotypic expression are

called dominant mutations. For example, in man the mutation disease aniridia (absence

of iris of eyes) occurs due to dominant mutant gene.



2. Recessive mutations. Most types of mutations are recessive in nature and so they are

not expressed phenotypically immediately. The phenotypic effects of mutations of a

recessive gene are seen only after one or more generations, when the mutant gene is able

to recombine with another similar recessive gene.

3. Isoalleles. Some mutations alter the phenotype of an organism so slightly that they can

be detected only by special techniques. Mutant genes that give slightly modified

phenotypes are called isoalleles. They produce identical phenotypes in homozygous or

heterozygous combinations.

4. Lethal mutations. According to their effects on the phenotype mutations may be

classified as lethals, subvitals and supervitals. Lethal mutations result in the death of the

cells or organisms in which they occur. Subvital mutations reduce the chances of

survival of the organism in which they occur. Supervital mutations, in contrast, cause

the improvement of biological fitness under certain conditions.

6. Classification of Mutation According to Consequent Change in Amino Acid

Sequence.

1. Missence mutations. They change the meaning of a codon, changing one amino acid

into another.

2. Temperature sensitive mutations or Ts mutations. If the substitution produces a

protein that is active at one temperature (typically 30o C) and inactive at a higher

temperature (usually 40- 42o C).

3. Nonsense or chain termination mutations. They arise when a codon for an amino

acid is mutated into a termination codon (UAG, UAA or UGA), resulting in the

production of a shorter protein.



Since, temperature-sensitive and chain termination mutations exhibit the mutant

phenotype only under certain conditions, they are called conditional mutations; they are

the most versatile and useful mutations.

4. Silent mutations. They change nucleotide but not the amino acid sequence because

they affect the third position of the codon, which is usually less important in coding. This

is a silent mutation because it leaves the protein sequence unchanged.

7. Classification of Mutation According to the types of Chromosomes.

According to the types of chromosomes, the mutations may be of following two kinds:

1. Autosomal mutations. This type of mutation occurs in autosomal chromosomes.

2. Sex chromosomal mutations. This type of mutation occurs in sex chromosomes.

Mutation rate

The frequency with which genes mutate spontaneously is called mutation rate. Most

genes are relatively stable and mutation is a rate. The great majority of genes have

mutation rate of 1X10-5 to 1 X 10-5, viz, one gamete in 100,000 to one gamete in million

would contain a mutation at a given locus. Mutations occur much more frequently in

certain regions of the gene than in others. The favoured regions are called hot spots.



CHROMOSOME ABERRATIONS

Changes in Structure of Chromosomes

The changes in the genome involving chromosome parts, whole chromosomes, or whole

chromosome sets are called chromosome aberrations or chromosome mutations.

Chromosome mutations have proved to be of great significance in applied biology-

agriculture (including horticulture), animal husbandry and medicine.

Chromosome mutations are inherited once they occur and are of the following types:

A. Structural changes in chromosomes:

1. Changes in number of genes

(a) Loss: deletion

(b) Addition: Duplication

2. Changes in gene arrangement:

(a) Rotation of as group of genes 180° within one chromosome:

inversion

(b) Exchange of parts between chromosomes of different pairs:

translocation.

B. Changes in number of chromosomes:

1. Loss, or gain, of a part of the chromosome set (aneuploidy)

2. Loss, or gain, of whole chromosome set (euploidy)

(a) Loss of an entire set of chromosomes (haploidy)

(b) Addition of one or more sets of chromosomes (polyploidy)

Both types of changes (structural and numerical) in chromosomes can be detected not

only with a microscope (i.e., cytologically) but also by standard genetic analysis. This

gave birth to a hybrid science, called Cytogenetics which attempts to correlate cellular

events, especially those of chromosomes, with genetic phenomena.

A. Structural Changes in Chromosomes

For better understanding of the abnormalities of chromosome structure, let us consider

two important features of chromosome behaviour : (1) During prophase I of meiosis,



homologous regions of chromosomes show a great affinity for pairing and they often go

through considerable contortions in order to pair. This property results in many curious

structures observed in cells containing one normal chromosome set plus an aberrant set.

(2) structural changes usually involve chromosome breakage; the broken chromosome

ends are highly “reactive” or “stickly”, showing strong tendency to join with broken

ends.

Types of Structural Changes in Chromosome

Structural changes in chromosome may be of following types: 1. deficiency or deletion

which involves loss of a broken part of a chromosome; 2.duplication involves addition

of a part of chromosome (i.e., broken segment becomes attached to a homolog which,

thus, bears one block of genes in duplicate); 3. inversion in which broken segment

reattached to original chromosome in reverse order, and 4. translocation in which the

broken segment becomes attached to a non-homologous chromosome resulting in new

linkage relations.

Further, structural abnormalities can occur in both homologous chromosomes of a pair in

only one of them. When both homologous chromosomes are involved, these are called

structural homozygotes, e.g., deletion homozygote, duplication homozygote, etc. When

only one homologous chromosome is involved, it is called structural heterozygote.

1. Deletion (or Deficiency)

The simplest result of breakage is the loss of a chromosome. Portions of chromosomes

without a centromore (called acentric fragments) lag in anaphase movement and are lost

from reorganizing nuclei or digested by nucleases. Such loss of a portion of a

chromosome (and of some genes) is called deletion. The chromosomes with deletions

can never revert to a normal condition. If gametes arise from the cells having a deleted

chromosome, this deletion is transmitted to the next generation. Further, a deletion can be

terminal or intercalary (insterstitial). In terminal deletion a terminal section of a

chromosome is absent and it is resulted by only one break. While in the intercalary

deletion, an intermediate section or portion of chromosome is lost and it is caused by two



breaks – one on either end of the deleted region. Thus, in the latter case, the chromosome

is broken into three pieces, the middle one of which is lost and the remaining two pieces

get joined again.

In general, if a homozygous deletion is made, it is lethal. Even individuals heterozygous

for deletion (deletion in one of the homologous chromosomes) may not survive.

However, smaller deletion in heterozygous condition can be tolerated by the organisms.

If meiotic chromosomes in such heterozygotes are examined, the region of deletion can

be detected by the failure of the corresponding segment on the normal chromosome to

pair properly; so a “deletion-loop” results. The cytological studies of pairing between

normal and deleted chromosomes have helped a lot in finding out the relative position of

genes in chromosomes.

TERMINAL DELETION INTERCALARY DELETION

Genetical effects of deletion.

Deletion of some chromosome regions produce their own unique phenotypes. A good

example of this is a dominant notch wing mutation in Drosophila. In fact, this is a small

deletion and acts as a recessive lethal in this regard. Further, in the presence of a deletion,

a recessive allele of the normal homologous chromosome will behave like a dominant

allele, i.e., it will be phenotypically expressed, this phenomenon is called

pseudodominance.

The phenomenon of pseudodominance exhibited by deficiency heterozygotes has been

utilized for the location of genes on specific chromosomes and in preparing cytological

maps in Drosophila, maize, bacteriophage and other organisms. Such cytological maps

are often used to verify the genetic maps (based on linkage analysis) of these organisms.


a b c d e f g h

a b c d e f g h a b c d e f g h

c d e f g h a b e f g h


Examples of pseudodominance (deletion)

Human babies missing a portion of the short arm of chromosome 5 (autosome) have a

distinctive cat-like cry; hence, the French name “cridu chat” (cry of the cat) syndrome

(first described by Lejeune et al., 1963). They are also mentally retarded (IQ below 20),

have malformation in the larynx, moon faces, saddle noses, small mandibles

(micrognathia), malformed low-set ears and microcephally (small head).

2. Duplication

The presence of a part of a chromosome in excess of the normal complement is known as

duplication. Thus, due to duplication some genes are present in a cell in more than two

does. If duplication is present only on one of two homologous chromosomes, at meiosis

the chromosome bearing the duplicated segment forms a loop to maximize the

juxtaposition (during pairing) of homologous regions.

Extra segments in a chromosome may arise in a varsity of ways such as follows:

1. Tandem duplication. In this case the duplicated regions are situated just by the side of

the normal corresponding section of the chromosome and the sequences of genes are the

same in normal and duplicated region. For example, if the sequence of genes in a

chromosome is ABC. DEFGH (The full stop depicts the Centro mere) and if the

chromosomal segment containing the genes DEF is duplicated, the sequence of genes in

tandem duplication will be ABC. DEF DEFGH.

2. Reverse tandem duplication. Here, the sequence of genes in the duplicated region of

a chromosome is just the reverse of a normal sequence. In the above mentioned example,

therefore, the sequence of genes due to reverse tandem duplication will be ABC. DEF

FEDGF.

3. Displaced duplication. In this case the duplicated region is not situated adjacent to the

normal section. Depending on whether the duplicated portion is on the same side of the



centromere as the original section or on the other side, the displaced duplication can be

termed either homobranchial or heterobranchial.

Example. Homobranchial duplication =ABC.DEFG. DEFH

Heterobranchial duplication = A.DEFB.C.DEFGH

4. Transposed duplication. Here, the duplicated portion of chromosome becomes

attached to a non-homologous chromosome. For example, if ABC.DEFGH and

LMNOPQ.RST represent the gene sequences of two nonhomologous chromosomes, a

transposed duplication will result into chromosomes with gene sequence ABC.GH and

LMN DEF.OPQ.RST. Such a transposed duplication may be either interstitial (e.g.,

LMN. DEFOPQ.RST) or terminal (i.e., LMN OPQ.RST DEF).

5. Extra-chromosomal duplication. In the presence of centromere the duplicated part of

a chromosome act independent chromosome.

Genetical effects of duplication. Due to duplication, there occur unequal crossing over

which results in deletion and reduplication which produce distinct phenotypes as shown

by the following examples :

Duplications of Drosophila lead to following phenotypic effects : (1) a reverse repeat in

chromosome 4 causes eyeless dominant (Ey); (2) a tandem duplication in chromosome 3

causes confluens (Co) resulting in thickened veins, and (3) another duplication causes

hairy wing (Hw)

Genetic redundancy, of which duplication is one type, may protect the organism from the

effects of a deleterious recessive gene or from an otherwise lethal deletion.

3. Inversion

Inversion involves a rotation of a part of a chromosome or a set of genes by 180° on its

own axis. It essentially involves occurrence of breakage and reunion. The net result of

inversion is neither a gain nor a loss in the genetic material but simply a rearrangement of



the gene sequence. An inversion can occur in the following way: suppose that the normal

order of segments within a chromosome is 1-2-3-4-5-6; breaks occur in regions 2-3 and

5-6 and broken piece is reinserted in reverse order. This results in an inverted

chromosome having segments 1-2-5-4-3-6.

An inversion heterozygote has one chromosome in the inverted order and its homologue

in the normal order. The location of the inverted segment can be detected cytologically in

the meiotic nuclei of such heterozygotes by the presence of an inversion loop in the

paired homologs. The location of the centromere relative to inverted segment determines

the genetic behaviour of the chromosomes. If the centromere is not included in the

inversion it is called paracentric inversion and when inversion includes the centromere

it is called pericentric inversion. Homologous chromosomes, with identical in meiosis.

However, crossing over in inversion heterozygotes produce deletions, duplications and

other curious configurations.

Advantage of inversions. Fertility of inversion homozygotes and sterility of inversion

heterozygotes lead to establishment of two group (or varieties) which are mutually fertile

but do not breed well with the rest of the species. Both varieties evolve in different

directions and later become reproductively isolated species. There is plenty of cytological

evidence to prove that such evolutionary mechanisms have and are operating in

Drosophila and a number of other orgasisms.

4. Translocation

The shifting or transfer of a part of a chromosome or a set of genes to a non-

homologous one, is called translocation. There is no addition or loss of genes during

translocations, only a rearrangement (i.e., change in the sequence and position of a gene).

Translocations may be of following three types.

1. Simple translocation. They involve a single break in a chromosome. The

broken piece gets attached to one end of a nonhomologous chromosome.

2. Shift translocation. In this type of translocation, the broken segment of one

chromosome gets inserted interstitially in a nonhomologous chromosome.



3. Reciprocal translocations. In this case, a segment from one chromosome is

exchanged with a segment from another nonhomologous one, so that in reality two

translocation chromosomes are simultaneously achieved.

Outcomes of reciprocal translocation. The exchange of chromosome parts between

nonhomologous chromosomes creates new linkage relationships. Such translocations also

drastically change the size of a chromosome as well as the position of its centromere. For

example, a large metacentric chromosome is shortened by one-half in length to an

acrocentric one, where as the small chromosome becomes a large one. Two types of

translocations have been recognized: homozygous and heterozygous. The translocation

homozygotes may have normal meiosis and in fact, are difficult to detect cytologically

unless morphologically dissimilar chromosomes are involved, or banding patterns differ

markedly. The translocation heterozygotes produce both translocated and normal

chromosomes and exhibit characteristic cytological and genetical effects. Thus,

translocation heterozygotes are marked by considerable degree of meiotic irregularity.

B.Changes in number of chromosomes (ploidy)

Polyploidy

Any organism with more than more than two genomes (2x) is called a polyploidy. Many

plant genera include species whose chromosome numbers constitute a euploid series. For

example the rose genus Rosa includes species with the somatic numbers 14, 21, 28, 35

and 56. These numbers are the multiples of 7. Therefore, this is a euploid series of the

basic monoploid number 7, which gives diploid, triploid, tetraploid, pentaploid,

hexaploid and octaploid species. Except diploids, rest of these belongs to polyploidy

category. Ploidy levels higher than tetraploid are not commonly encountered in natural

populations, but our most important crops and ornamental flowers are polyploids, e.g.,

wheat (hexaploid 6x), strawberries (octaploid, 8x), many commercial fruits and

ornamental plants. Generally, polyploidy is common in plants (more common in

monocots) but rare in animals.

Types of polyploidy



These are the three different kinds of polyploids: (i) autopolyploids, (ii) allopolyploids,

(iii) autoallopolyploids.

(a) Autopolyploids

The autopolyploids are those, polyploids, which consist of same basic set of

chromosomes multiplied. For example, if a diploid species has two similar sets of

chromosomes or genomes (AA), an autotriploid will have three similar genomes (AAA),

and an autotetraploid, will have four such genomes (AAAA).

(i) Origin and production of autopolyploids

The autopolyploids may occur in nature or may be produced artificially. When they are

found in nature, their autopolyploidy is deduced by their meiotic behaviour. Some of

common examples of autotriploid crop plants, which are mainly produced by artificial

methods, are seedless varities of watermelons, sugar beet, tomato, grapes and banana.

Similarly, many important crop plants include autotetraploids such as rye (Secale

cereale), corn (Zea mays), red clover (Trifolium pretense), berseem (Trifolium

alexandrium), marigolds (Tagetes), snapdragons (Antirrhinum), Phlops, grapes, apples,

Oenothera lamarckiana (which was recognized as mutation by Hugo de vries).

Induced autopolyploidy

The autopolyploidy have been induced in many plant and animal cells by artificial means

such as chemical (e.g., chloral hydrate, colchicines, sulphanil amide, mercury chloride,

hexachlocyclohexane, etc,), radioactive substances, e.g., radium and X-ray) and

temperature shocks. These inducers usually disturb the mitotic spindle and cause non-

segregation of already duplicated chromosomes, during cell divisions.

Colchicine

Colchicine is a drug (i.e., an alkaloid obtained from the corms of plants- Colchicum

autmunale and C.luteum) and its aqueous solution is found to prevent the formation and

organization of spindle fibres, so the metaphase chromosomes of the affected cells (called

C-metaphase or colchicine metaphase) do not move to a metaphase plate and remain



scattered in the cytoplasm. Even the process of cytokinesis is prevented by colchicines

and with duplications of chromosomes the number goes on increasing. As colchicine

interferes with spindle formation, its effects are limited to divided and meristematic cells.

(ii) Effects of autopolyploidy

Autopolyploidy results in gigantism of plant cells, i.e., leaves, flowers and fruits of an

polyploid are larger in size than a diploid plant. For example, the size of lower epidermis

of leaf of a tetraploid Saxifraga pencylvanica was found greater than the diploids. Some

of the significant effects of autopolyploidy are as follows : (1) With the increase of cell

size, the water content increases which leads to a decrease in osmotic pressure. This

results into loss of resistance against frost, etc, (2) Due to lower rate of cell division, the

plants growth rate in decreases. This leads to a decrease in auxin supply and a decrease

in respiration. (3) Due to slow growth rate, the time of blooming of an autopolyploid is

delayed. (4) At higher ploidy level, such as autooctoploids, the adverse effects become

highly pronounced and lead to the death of the plants. Polyploid varieties with an even

number of genomes (e.g., tetraploids) are often fully fertile whereas those with an odd

number (e.g., triploids) are highly sterline.

Uses of induced polyploidy

Since in the induced polyploids, the fertility level and seed set are low, so seedless fruits

can be produced by using triploids as in case of seedless watermelons which were

produced by using triploids as in case of seedless watermelon which were produced by a

Japanese scientist, Dr. Hitoshi Kihara. These triploids are obtained from seeds by

colchicines treatment.

Allopolyploids

When the polyploidy results due the doubling of chromosome number in a F1 hybrid

which is derived from two distinctly different species, then it is called allopolyploidy and

the resultant species is called an allopolyploid. Let A represent a set of chromosomes

(genome) in species X; and let B represent another genome in a species Y. The F1

hybrids of these species then would have one A genome and another B genome. The



doubling of chromosomes in the F1 hybrids will give rise to allotetraploids with two A

and B genomes.

Raphanobrassica is a classical example of allopolyploidy or amphipolyploidy. In 1927, a

Russian geneticist, G.D.Karpechenko performed a cross between radish (Raphanus

sativum, 2n = 18) and cabbage (Brassica oleracea, 2n = 18) and in F1 got sterile (diploid)

hybrids. Among these sterile F1 hybrids, he found certain fertile plants which were found

to contain 36 chromosomes. These fertile tetraploids were called Raphanobrassica.

Synthesized Allopolyploids

To find out the origin of naturally occurring allopolyploids some cytogeneticists

produced certain allopolyploids in laboratory by employing artificial means. Common

hexaploid wheat and tetraploid cotton furnish two such examples.

Triticum spelta

Triticum spelta is a hexaploid wheat which was artificially synthesized in 1946 by

E.S.McFadden and E.R.Sears and also by H.Kihara. They crossed an emmer wheat,

Triticum dicoccoides, (tetraploid : 2n = 28) with goat grass, Aegilops squarrosa (diploid ;

2n = 14) and doubled the chromosome number in the F1 hybrid. This artificially

synthesized hexaploid wheat was found to be similar to the primitive wheat T.spelta.

When the synthesized hexaploid wheat was crossed with naturally occurring T.spelta, the

F1 hybrid was completely fertile. This suggested that hexaploid wheat must have

originated in the past due to natural hybridization between tetraploid wheat and goat

grass followed by subsequent chromosome doubling.

Triticale

Triticale (Triticosecale Wittmack) is the first man made cereal which has been developed

in recent years and is cultivated on about one million hectares of land throughout the

Globe for the commercial use. Triticale is an artificial allopolyploid which has been

derived by crossing wheat (Triticum) and rye (Secale). Depending upon whether Triticum

is a tetraploid (2n = 4x =28) or hexaploid (2n = 4x = 42), one would get hexaploid



triticale (2n = 6x = 42) or octaploid triticale (2n = 8x = 56), respectively. In each case,

only diploid rye (2n = 4x = 14) was used.

Segmental allopolyploids

Different genomes of some allopolyploids are not quite different from each other.

Consequently in these polyploids chromosomes belonging to different genomes do pair

together to some extent. This indicates that segments of chromosomes and not the whole

chromosomes are homologous. Therefore, such allopolyploids are called segmental

allopolyploids .The segmental allopolyploids are intermediate between autopolyploids

and allopolyploids and can be identified by their peculiar meiotic behaviour.

Phenotypic Effects of Polyploidy

The increase in the genome’s size beyond diploid level is often caused following

detectable phenotypic characteristics in a polyploidy organism:

(i) Morphological effect of polyploidy.

The polyploidy is invariably related with gigantism. The polyploidy plants have been

found to contain large-sized pollen grains, cells, leaves, stomata, xylem, etc. The

polyploid plants are more vigorous than diploids.

(ii) Physiological effect of polyploidy.

The ascorbic acid content has been reported to be higher in

tetraploid cabbages and tomatoes than in corresponding diploids. Likewise corn meal of a

tetraploid maize seed contains 40 per cent more than vitamin A than cornmeal from a

diploid plant.

(iii) Effect on fertility of polyploidy.

The most important effect of polyploidy is that it reduces the fertility of polyploid plants

in variable degrees.

(iv) Evolution through polyploidy.

Interspecific hybridization combined with polyploidy offers a mechanism whereby new

species may arise suddenly in natural populations.



Extra-nuclear inheritance by cellular Organelles

Cytoplasmic extra nuclear genes or DNA molecules of plastids, mitochondria,

chloroplasts have a characteristic pattern of inheritance which does not resembles genes

of nuclear chromosomes and are hence called as non-Mendelian, non-chromosomal, extra

chromosomal, cytoplasmic and extra nuclear inheritance..

Chloroplasts and mitochondria are organelles that contain their own DNA and protein

synthesizing apparatus. A widely held theory concerning their origin proposes that they

were once infectious endosymbiotic prokaryotes that involved such as dependence on the

gene products of the host that they are no longer able to function autonomously.

(a) Chloroplast inheritance in variegated four o’ clock plant. The cytoplasmic or extra

nuclear inheritance of colour in plant by plasticides was first of all described by C.

Correns in 1908 in the four o’ clock plant, Mirabilis Jalapa. In contrast to other higher

plants, Miabilis contains three types of leaves and parts: (1) Full green leaves or branches

having chloroplast, (2) White (pale) leaves and branches having no chloroplast, (3)

Variegated branches having leucoplast in white (pale) areas and chloroplast in green

patches. Because the chlorophyll pigment of chloroplast is related with photosynthesis of

food and leucoplasts are incapable to perform photosynthesis, so the white or pale parts

of plant survive by receiving nourishments from green parts. Correns reported that

flowers in green branches produced only green offspring’s, regardless of the genotype

and phenotype of pollen parent and likewise, flowers from the white or pale branches

produced only white or pale seedings regardless of genotype and phenotype of pollen

parent. The plants from the white or pale seedings die because the lack chlorophyll and

cannot carry photosynthesis. Correns further reported that flowers from the variegated

branches yielded mixed progeny of green, white (pale) and variegated plants in widely

varying ratios.

The irregularly of transmission from variegated branches could be understood by

considering cytoplasmic genes (plasmagenes) of plastids. A study of the egg during

oogenesis in Mirabilis reveals that the ooplasm contains plastids like cytoplasm of other



plant cells. If the egg cell is derived from green plant tissues, its ooplasm will contain

coloured plastids; if derived from white plant tissues, its ooplasm will contain white

plastids; if derived from variegated tissues, its cytoplasm may contain coloured plastids

only, white plastids only or a mixture of coloured and white plastids. A study of the

pollenogenesis, however, reveals that pollen contains very little cytoplasm which in most

cases is devoid of plastids. Without the plastids, pollen cannot affect this aspect of the

offspring’s phenotype.

Mitotic segregation. Variegated branches of Mirabilis Jalapa produce three types of

eggs: some contain only white chloroplasts, some contain only green chloroplasts and

some contain both types of chloroplasts. In the subsequent mitotic divisions, some form

of cytoplasmic segregation occurs that segregate the chloroplast types into pure cell lines,

thus, producing the variegated phenotype in the progency individual. This process of

sorting might be described as “mitotic segregation” of this is a pure extra nuclear

phenomenon. In mitotic segregation since both segregation and recombination of

organelle genotype takes place, so it is called cytoplasmic segregation and

recombination (its acronym is CSAR).

(b) Cytoplasmic male sterlity (CMS). In maize and many other plants, cytoplasmic

control of male sterility is known. In such cases, if the female parent is male sterile

(having plasmagene for male sterility), the F1 progeny would always be male sterile,

because the cytoplasm is mainly derived from the egg which is obtained from the male

sterile female parent.

(c) Cytoplasmic genetic male sterility. In certain plants, though the male sterility is

fully controlled by the cytoplasm, but a restorer gene if present in the female parent in

the nucleus, will restore fertility. For example, if the female parent is male sterile (due to

plasmagene of male sterility) then the nuclear genotype of the male parent will determine

the phenotype of F1 progeny. Thus, if male sterile female parent contains recessive

nuclear genotype rr of restorer gene and male parent is RR, having homozygous

dominant restorer genes. Their F1 progeny would be male fertile Rr. However, if the male



parent is male fertile rr, the F1 progeny would be male sterile rr. If the F1 male fertile

heterozygote (Rr) is test crossed with male fertile progeny with 50 per cent male fertile

and 50 per cent male sterile will be obtained.

Since, in maize expression of male sterility depends on an interaction between nuclear

and extra chromosomal genes. Male sterile lines can bear seeds only after cross-

pollination. For this reason they are useful in raising hybrid seeds, especially on large

scale.

Later on, in maize the following four types of cytoplasms have been recognized: the

normal (N) cytoplasm and three types of male sterile cytoplasms (T, C and S). The recent

studies of mitochondria in these cytoplasm revealed that the factors responsible for

cytoplasmic male sterility are located in mitochondrial DNA (mt DNA) and mt DNA of

N, T, C and S cytoplasms are found to be different. The cytoplasmic male sterility (CMS)

of C and S type can be reversed by nuclear storer genes, however, the CMS-T cannot.

Parents

Male sterile Male fertile

F1

(O) Male fertile (O)

Intercrossor selfing

Male fertile Male fertile


,,


F2

25% 50% 25% Male fertile -Pure Male fertile- hybrid Male sterile -pure

Inheritance pattern of genetic male sterility



UNIT – V

GENETIC TRANSFER

Recombination, however, is undoubtedly important in the evolution of bacteria just as it

is in the evolution of eukaryotes. Three different processes have evolved that mediate

transfer of genetic material from one bacterium to another, making possible the

subsequent recombination events. The most obvious difference between these three

processes is the most of transfer of DNA from one cell to another. (1) Transformation

involves the uptake of naked DNA molecules from one molecule from one bacterium (the

donor cell) by another bacterium (the recipient cell). (2) Transduction occurs when

bacterial genes are carried from a donor cell to a recipient cell by a bacteriophoge. (3)

Conjugation is the process during which DNA from a donor or male cell is transferred to

a recipient or female cell through a specialized sex plus or “conjugation tube”.

TRANSFORMATION

Transformation was discovered in pathogenic strains of Diplococcus pneumoniae by

Griffith in 1928. The details of Avery, Macleod, and McCarty’s (1944) proof that the

“transforming principle” (the cellular component mediating transformation) is DNA.

The uptake of DNA molecules by recipient bacteria is an active, energy-requiring

process. It does not involve passive entry of DNA molecules through permeable cell

walls and membranes (although this type of uptake of DNA molecules may be induced

by experimental manipulations of bacteria in the laboratory, such as in the Escherichia

coli recombinant DNA cloning experiments). Thus, transformation does not occur

“naturally” in all species of bacteria, only in those species possessing the enzymatic

machinery involved in the active uptake and recombination processes. Most of the studies

on transformation have been done with three species, D. pneumoniae, Bacillus subtilis,

and Haemophilus influenzae. Even in the species, all cells in the given population are not

capable of active uptake of DNA. Only Competent cells, which possess a so-called



“competence factor” (probably a cell-surface protein or enzyme involved in binding or

in taking up DNA), are capable of serving as recipients in transformation. The proportion

of bacteria in a culture that are in the physiologically competent state varies with growth

conditions and the stage of the growth curve (becoming maximal in late log-phase).

Transformation involves the uptake of "naked" DNA (DNA not incorporated into

structures such as chromosomes) by competent bacterial cells. Cells are only competent

(capable of taking up DNA) at a certain stage of their life cycle, apparently prior to the

completion of cell wall synthesis. Genetic engineers are able to induce competency by

putting cells in certain solutions, typically containing calcium salts. At the entry site,

endonucleases cut the DNA into fragments of 7,000-10,000 nucleotides, and the double-

stranded DNA separates into single strands. The single-stranded DNA may recombine

with the host's chromosome once inside the cell. This recombination replaces the gene in

the host with a variant - albeit homologous - gene. DNA from a closely related genus

may be acquired but, in general, DNA is not exchanged between distantly related

microbes. Not all bacteria can become competent. While transformation occurs in nature,

the extent to which it contributes to genetic diversity is not known.

The process of transformation can be divided into several stages: (1) reversible binding

of doublestranded DNA molecules to receptor sites on the cell surface; (2) irreversible



uptake of the donor DNA (at which time the donor DNA becomes resistant to DNase in

the medium); (3) conversion of the doublestranded donor DNA molecules to single-

stranded molecules by nucleotic degradation of one strand; (4) integration (covalent

insertion) of all or part of the single strand of donor DNA into the chromosome of the

recipient; and (5) the segregation and phenotypic expression of the integrated donor gene

or genes in the recombinant (“transformed”) cell. Steps (2) and (3) may well be

coincident effects of a single process. One attractive model, for which there is supporting

evidence in the case of Pneumococcus, proposes that a specific exonuclease (or DNA

“translocase”) pulls one strand of donor DNA into the cell using energy derived from the

degradation of the complementary strand. Whether degradation of the complementary

strand or DNA actually occurs during uptake or immediately after uptake is uncertain.

Moreover, considerable evidence suggests that these processes may vary in different

species.

The first three steps in transformation-binding, uptake, and degradation of one strand of

the double stranded DNA – are not specific for homologous DNA. In fact, competent

bacteria will carry out these three processes equally with other foreign DNAs. However,

the integration, or DNA recombination step, is specific for homologous DNA. This is

not to say that the integration of segments of heterologous (foreign) DNA never occur,

however, it does so at frequencies very much lower than the frequencies observed using

homologous DNA. Although very small fragments of DNA taken up by competent cells,

a minimum length of about 500 nucleotide-pairs appears to be required for integration to

occur. During integration, a single strand (either strand, the previous degradation of one

strand is at random) of donor DNA is physically inserted into the recipient chromosome,

replacing a segment of one strand of the recipient chromosome). In most transformation

experiments, donor DNA fragments are about 20,000 nucleotide-pairs (or about 1/200 of

the total chromosome) in length. This means that mapping experiments can be done using

transformation only if the genetic markers employed are located close together on the

host chromosome.

Genetic mapping by transformation



If two genes are far apart on the chromosome, they will never be carried on the same

molecule of transforming DNA. Thus, double transformants for the two genes (say a to a+

and b to b+, using an a+ b+ donor and an a b recipient) will require two independent

transformation events (uptake and integration of one DNA molecule carrying a+ and

another molecule carrying b+). The probability of two such independent events occurring

together will equal the product of the probability of each occurring alone. Since

transformation of any single marker occurs with allow frequency, double independent

transformation events of this type will be extremely rare. If, on the other hand, two genes

are closely linked, they may be carried on a single molecule of transforming DNA. In this

case, double transformants can be formed by the uptake and integration of one molecule

of donor DNA carrying both genes. Thus, if two genes or genetic markers are very

closely linked, double transformants may be formed at a frequency approaching the

frequency of single transformants in comparable single-marker experiments. The

frequency with which two genetic markers are cotransformed can thus be used as a crude

estimate of the linkage distance between them. Genetic markers can also be ordered be

means of three-factor transformation experiments using the same rationale as in three-

factor transduction, conjugation, or sexduction experiments.

TRANSDUCTION

Principle

Transduction is another method for transferring genes from one bacterium to another; this

time the transfer is mediated by bacteriophages (bacterial viruses, also called phages). A

bacteriophage infection starts when the virus injects its DNA into a bacterial cell. The

bacteriophage DNA may then direct the synthesis of new viral components assembled in

the bacterium. Bacteriophage DNA is replicated and then packaged within the phage

particles. Early in the infective cycle the phage encodes an enzyme that degrades the

DNA of the host cell. Some of these fragments of bacterial DNA are packaged within the

bacteriophage particles, taking the place of phage DNA. The phage can then break open

(lyse) the cell. When released from the infected cell, a phage that contains bacterial genes

can continue to infect a new bacterial cell, transferring the bacterial genes. Sometimes

genes transferred in this manner become integrated into the genome of their new bacterial



host by homologous recombination. Such transduced bacteria are not lysed because they

do not contain adequate phage DNA for viral synthesis. Transduction occurs in a wide

variety of bacteria and is a common mechanism of gene transfer.

Transduction, discovered by N.Zinder and J. Lederberg in 1952, occurs when a

bacteriophage particle carries a segment of the chromosome from one bacterium (the

donor) to another bacterium (the recipient), facilitating subsequent recombination of the

genetic markers of the two cells. Two very different types of transduction are known. (1)

In generalized transduction, a random segment of bacterial DNA is “wrapped up” during

phage maturation or along with phage chromosome in a few “progeny” particles, called

transducing particles. Generalized transducing phages can therefore transport any gene

of the donor cell to the recipient cell. Since all the genes of the donor are represented in a

population of these transducing particles (although any one transducing phage contains

only one segment of host DNA, representing 1/100 to 1/50 of the total donor of the

chromosome), this type of transduction was named “generalized” transduction. (2) In

specialized transduction (also called restricted transduction), a recombination event

involving the host chromosome and the phage chromosome occurs, producing a phage

chromosome containing a segment a bacterial DNA. Specialized transducing particles

thus always contain both phage and bacterial DNA. Specialized transduction is so

named because a given virus transduces only genetic markers of the host that are located



in one small region of the bacterial chromosome. Bacteriophage lambda, the best-known

specialized transducing phage, for example, usually mediates transduction of the gal and

bio genes of E.coli.

GENERALIZED TRANSDUCTION

Bacteriophages have been classified into two types on the basis of their interactions with

the bacterial cell. Virulent phages always multiply and lyse the host cell after infection.

Temperate phages have a choice between two life-styles after infections. They can either

(1) enter the lytic cycle, during which they reproduce and lyse their host cells just like

virulent phages, or, alternately, they can (2) enter the lysogenetic pathway during which

their chromosomes are integrated into the chromosomes of the host and replicate like any

other segments of the host chromosomes. Generalized transduction is mediated by some

virulent bacteriophages whose chromosomes are not integrated at specified attachment

sites on the host chromosome. Generalized transducting particles are produced during the

lytic cycles of these phages. Of the generalized transducing phages, E.coli phage P1,

Salmonella phage P22, and Bacillus subtilis phages PBS1 and SP10 have been

extensively used for genetic fine structure maping.

After a transducing phage injects a fragment of DNA into a recipient cell, that DNA may

either (1) be integrated into the host chromosome in a manner similar to the integration of

transforming DNA, except that the integrated segment is double-stranded, or (2) remain

free in the cytoplasm. If it is not integrated, it will not replicate and will be transmitted to

only one progeny cell during each cell division. The genes located on the transduced

chromosome fragments may be expressed, even they are not integrated. Cells carrying

nonintegrated transducing fragments are called abortive transductants.

Gene mapping by transduction

Transducing particles are produced at a low frequency. Only one out of 105-107 of the

“progeny” particles present in a lysate contains bacterial DNA. Thus, the probably of the

cell being doubly transduced for markers carried in two different transducing particles in

negligible. (If cells are simultaneously infected with 100 or more phage particles, they are



rapidly killed by a process called “lysis-from-without”, which apparently results from

simply punching too many holes in the cell membrane.) The cotransduction of two or

more genetic markers therefore indicates that the markers are relatively closely linked,

and the frequency of cotransduction of any two markers is indicative of the degree of

linkage between them. Occasionally, genetic markers can be ordered by cotransduction

patterns. If (1) markers a+ and b+ are cotransduced, (2) markers b+ and c+ are

cotransduced, but (3) markers a+ and c+ are not cotranduced, then the order of the three

markers must be a+-b+-c+. More frequently, however, three-factor transduction

experiments must be used to unambiguously order genetic markers.

SPECIALIZED TRANSDUCTION

Specialized transduction is mediated by temperate bacteriophages whose chromosomes

are able to integrate at one or a few specified attachment sites on the host chromosome.

The chromosomes of temperate phages of this type are thus capable of both (1)

autonomous replication (replication independent of the replication of the host

chromosome) and (2) integrated replication (replication as a segment of the host

chromosome). As such, they are examples of genetic elements called episomes.

Integration of the chromosome of a specialized transducing phage, such as the coliphage

lambda, involves a recombination event between the circular intercellular form of the

phage chromosome and the circular bacterial chromosome at specific attachments sites

on the two chromosomes. This site specific recombination event results in the covalent

linear insertion of the phage chromosome and the circular bacterium chromosome at

specific attachment sites on the two chromosomes. This site specific recombination event

results in the covalent linear insertion of the page chromosome into the chromosome

bacterium. In its integrated state, the phage chromosomes are called a prophage. The

lytic genes of the virus, those involved in viral reproduction and lysis of the host, are

repressed (turned off) when the chromosome is in the prophage state. (The mechanism by

which the prophased genes is repressed). A bacterium harboring a prophage is said to be

lysogenic, the prophage-host relationship is called lysogeny. A lysogenic cell is immune

to secondary infections by the same virus (homologous to the prophage), because the



lytic genes of the infecting virus will be repressed just as those of the prophage are

repressed.

Temperate phages undergo rare (about one in105 cell divisions) spontaneous transitions

from the lysogenic or prophage state to the lytic state. Such transitions can be also

induced, for example, by irradiation with ultraviolet light. During the switch from the

lysogenic state to lytic growth, the prophage is excised from the host chromosome and

commences replicating autonomously. The excision process is site specific, like the

integration process. The site-specific integration and excision processes are catalyzed by

enzymes encoded by phase genes.

The excision process is usually very precise in cutting out the phage chromosome in

exactly the form in which it existed prior to its integration. Occasionally, however, the

excision event occurs at a site other than the original attachment site. When this happens,

a portion of the phage chromosome is left in the host chromosome and a portion of the

bacterial chromosome is excised with the phage DNA. Such “mistakes” during prophage

excision are responsible for the formation of specialized transducing particles. Only host

genes located close to the site of prophage insertion can be excised with the phage DNA

and packaged in “phage” particles. Thus specialized transduction is restricted to the

transfer of genes located within a short distance on each side of the prophage attachment

site. Phage lambda integrates between the gal genes (required for the utilization of



galactose as an energy source) and the bio genes (essential for the synthesis of biotin) on

the E.coli chromosome; lambda thus usually only transduces gal and bio markers.

Specialized tansducing phage O 80, on the other hand, integrates near the E.coli trp genes

(required for the synthesis of the amino acid trytophan) and transduces trp markers.

If specialized transducing particles are formed during prophage excision, only phage

lysates produced by induction of lysogenic cells should have transducing activity. This is

indeed the case. If bacteria are infected by specialized transducing phages under

conditions where only lytic infections occur, no transducing particles are present in the

phage lysates. The frequency of transducing particles in lysates produced by induction of

induction of lysogenic cells is about one in 106 progency particles.

CONJUGATION

Conjugation was discovered in 1946 by J. Lederberg and E.L. Tatum (1958 Nobel Prize

co recipients). During conjugation, DNA is transferred from a donor cell to a recipient

cell through a specialized intercellular connection, or conjugation tube, that forms

between them. The donor and recipient cells are sometimes referred to as male and

female cells, respectively). The transfer of genetic information is thus a one-way transfer

during conjugation, just as in transformation and transduction, rather than a reciprocal

exchange of genetic material. Cells that have the capacity to serve as donors during

conjugation are differentiated by the presence of specialized cell-surface appendages

called F pili. The synthesis of these F pili is controlled by several (nine, based on current

data) genes that are carried by a small circular molecule of DNA or “minichromosome”

(about 94,500 nucleotide-pairs long) called on F factor (for fertility factor; also called

“sex factor” and “F plasmid”). Cells carrying an F factor (donor cells) form conjugation

tubes and initiate DNA transfer making contact with cells not carrying an F factor,

called F - cells (recipient cells).

The F factor can exist in two different states: (1) the autonomous state, in which it

replicates independently of the chromosome, and (2) the integrated state, in which it is



covalently inserted into the host chromosome like any other set of chromosomal genes.

The F factor is thus, like the chromosomes of specialized transducing phages, an example

of a class of genetic elements called episomes.

A donor cell containing the F factor in the autonomous state is called an F+ cell. When

an F+ donor cell conjugates with an F- recipient cell, only the autonomous F factor is

transferred. Both exconjugants (cells that have been involved in conjugation) become F+

because the F factor replicates during transfer. Thus, mixing a population of F+ cells with

a population of F - cells results in virtually all the cells in the new population becoming

F+.

The F factor can integrate into the host chromosome at any one of many sites by a

mechanism that appears analogous to the integration of the chromosomke of a specialized

transducing phage, namely, a site-specific recombination event. The integration of the F

factor is believed to be mediated by short DNA sequences called IS elements. A cell

carrying a integrated F factor is called an Hfr. In the integrated state, the F factor

mediates the transfer of a chromosome of the Hfr cell to a recipient (F -) cell. Usually,

only a portion of the Hfr chromosome is transferred before the cells separate, thus

breaking the chromosome is transferred.



The mechanism of transfer of DNA from a donor cell to a recipient cell during

conjugation appears to be the same whether just the F factor is being transferred, as in F+

by F – matings, or the chromosome is being transferred, as in Hfr by F – matings. Transfer

is believed to be initiated by an endonucleolytic nick in one strand at a specific site (the

“origin” of transfer) on the F factor. The 5’ end of the nicked strand is then transferred

through the conjugation tube into the recipient cell. Transfer is believed to be coupled to

rolling circle replication, with the circular strand being replicated in the donor cell and

the displaced stand being replicated in the recipient cell as it is transferred. Because the

origin of transfer is within the integrated F factor, one portion of the F factor is

transferred from an Hfr cell to an F - cell prior to the sequential transfer of chromosomal

genes. The remaining part of the F factor, however, is the last segment of DNA to be

transferred. Thus, in Hfr by F – matings, the recipient F – cell acquires a complete F factor

(thus becoming an Hfr donor) only in those rare cases when an entire Hfr chromosome,

with its integrated F factor, is transferred.

Gene mapping by conjugation

Subsequent studies with different Hfr strains revealed similar fixed transfer sequences,

although different Hfr’s initiated transfer from different sites on the chromosome. It is

now clear that the F factor can integrate at many different sites in the circular E .coli

chromosome, and the site of integration determines the origin of transfer characteristic of

a given Hfr. The transfer of a complete chromosome from an Hfr to an F – cell takes

from 90 to 100 minutes, depending on the strain. Chromosome transfer appears to

proceed at a fairly constant rate. Thus, the time interval between the transfer of any two

markers (easily determined by interrupted mating experiments) is a good estimate of the

physical distance separating the markers on the chromosome. It has therefore proven

convenient to use the minute, representing the time interval between the transfer of

markers in interrupted mating experiments, as the standard unit for measuring linkage in

E. coli. A map distance of 1 minute corresponds to the length of the segment of the

chromosome transferred in 1 minute during conjugation. The standard E. coli linkage

map is thus divided into minute intervals from 0 (arbitrarily set at the thrA gene) to 100

minutes on the basis of interrupted mating experiments.



Linkage relationships can also be determined from uninterrupted mating experiments.

Consider, for example, the Hfr H by F – cross, with the matings being allowed to proceed

uninterrupted for 1-2 hours. If thr+ leu+ str –r recombinants are selected and scored for

the presence of other segregating markers by replicating, what will the frequencies of the

donor (say azi-s, T1-s, lac+, and gal+ markers be among the recombinants? Will these

donor markers all be present with the same frequency? The frequencies of these donor

markers are observed to vary, with the frequency of the marker decreasing as a function

of its distance from the selected (thr+ and leu+) donor markers. The frequency will, in

fact, be identical to the plateau frequencies observed in the interrupted mating

experiment. Donor markers azi-s, T1-s,lac+, and gal+ will occur among thr+ leu+ str-r

recombinants with percentage frequencies of 90, 80, 40, and 25, respectively. The farther

a marker is from the selected donor marker (in the HfrH experiment, thr+ and leu+), the

lower its frequency among the recombinants. The marker frequency gradient is caused by

two major factors: (1) the approximately constant probability per unit time of

spontaneous rupture of the conjugation tube and the chromosome and (2) the decreasing

probability that any two donor markers will be incorporated into the recipient

chromosome by a single pair of recombination events (incorporation of a donor fragment

into a recipient chromosome always requires two recombination events) as the distance

separating the two markers increases. Although uninterrupted conjugation experiments of

this type can be used to determine linkage relationships, interrupted mating experiments

are simpler and more direct. Thus, when a new mutation is identified, its approximate

location is usually first determined by interrupted conjugation mapping. Its exact location

is then usually determined by transduction mapping.


BT203_Genetics Unit 2

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