Laws of heredity

Gregor Johann Mendel was born in July 22, 1822 in Heinzendorf in

Austrian Silesia, where his father, Anton Mendel was the owner of a small farm. He
graduated from the Gymnasium in 1840. In the year 1846, Johann Gregor Mendel
attended courses of agriculture, pomiculture and viniculture at the Philosophical
Academy in Brunn. In the spring of 1856, he began experimental crossing of pea
varieties.

On February 8, 1865, he delivered his first lecture on pea experiments to

Brunn Naturals Science Society. In 1866 his paper “Experiments on plant
hybridization” published in volume 4 of the proceedings of the Natural Science
Society. In the same year, he began experiments with other plant species. In this
paper, Mendel proposed some basic genetic principles. But unfortunately his
remarkable piece of work remained unattended and unappreciated upto 1900. Mendel
failed to confirm his theory in other plants.
Rediscovery of mendel’s work

Mendel’s research paper remained dormant and unnoticed by the scientific

world until 1900. During these intervening thirty four years many developments
occurred in biology which prepared the way for the rediscovery of Mendel’s work.
It was in the beginning of 20th century that three botanists, namely Hugo de
Vries, working on Oenothera ; Correns working on Xenia, peas and maize and Von
Tschermak working on various flowering plants, independently drawn the
conclusions like Mendel. Later these botanists came across the research paper of
Mendel and rediscovered it in 1900. Mendel’s original paper was republished in
Flora, 89, 364 (1901). Bateson confirmed Mendel’s work by a series of hybridization
experiments.
Mendel’s Selection of the Experimental Plant

He selected garden pea as most suitable materials for his experiments,

because
1) it was easy to cultivate ;
(2) had short growth period and life-cycle ;
(3) had hermophrodite flowers
(4) had contrasting heritable characters, and
(5) easy to emasculate and pollinate
Mendel used a total of following seven pairs of characters :
1. The shape of the seed – Round and full : Irregularly-shaped and Wrinkled.
2. The colour of the cotyledons – Yellow : Green
3. The colour of the seed coat – Grey : White
4. The shape of the ripe pod – Simply inflated : Constricted between the seeds.
5. The colour of the unripe pods – Light to dark green : Yellow
6. The position of the flowers on the stem – Axial : Terminal.
7. The length of the stem – Tall or long : Short or dwarf.

Besides pea, Mendel has also used following plant species in his
hybridization experiments ; Zea mays, IpomoeaPhaseolus sp., Dianthus sp.
Mendel’s Experimental Observations and Results

To understand the observations and results of Mendel’s hybridization

experiments, one has to become familiar about few genetic terms, which have been
coined by W. Bateson between the years 1902-1909.

Parental (P) generation: The plants with unlike characteristics in which the
artificial cross is made are called parental (P) generation and the progeny obtained
from such a cross is called hybrid.
In genetical language, a hybrid can be defined as an individual which results
from the crossing of the two individuals differing atleast in one set of the character.
According to the number of pairs of contrasting characters in the parental
generation, the resultant hybrids are called as follows.
Monohybrids (have one pair of different character). Dihybrids (have two pairs of
different characters) or Polyhybrid (have more than two pairs of different
characters).

The process through which the hybrids are produced is called hybridization
and such a hybridization cross may be a monohybrid cross, dihybrid cross or
polyhybrid cross depending upon the kind of hybrid it produces. The resultant
hybrids of P generation is the first filial generation or F1. When F1 progeny is
allowed to self-fertilize or cross- fertilize among themselves, they produce the second
filial generation or F2. The subsequent generation which may be produced by self-
fertilization are called F3, F4, F5, F6, etc. Mendel’s observations of the hybrids of F1
revealed that hybrid plants of F1 contained the character of only one parent, none of
them displayed any intermediate character of both parents. Those characters which
were transmitted unchanged and expressed in the hybrid in the hybridization process
were called as dominant characters and those which became latent in the process
were called as recessive characters by Mendel. He tested each of the seven pairs of
contrasting characters for the phenomenon of the dominance and recessiveness and
found following characters as dominant.

Dominant and Recessive Characters is Pea

Character Dominant Recessive

Seed form Round or Smooth Wrinkle

Cotyledon colour Yellow Green

Seed coat Grey White
colour

Shape of pod Inflated Constricted

Colour of unripe pod Green Yellow
Flower position Axial Terminal
Stem length/plant Tall (long) Dwarf (Short)
height
 Mendel’s observations of F2 progeny further revealed the fact that the
recessive character which was depressed or concealed in F1 hybrids reappeared in the
F2 offspring in the definitely expressed average propagation of three to one, so that
among each four plants of F2, three displayed the dominant character and one the
recessive character. This 3:1 F2 ratio was observed to occur in the monohybrid cross
for each of seven pairs of characters. Mendels original F2 results for seven pairs of
character has been tabulated.

Results of Mendel’s Original Crosses for Seven Pairs of Characters.

S.No. Structure Character Dominant Recessive Ratio in

F2
1 Seed Form 5,474 1,850 2.96 : 1
Round Wrinkled

2 Cotyledon Colour 6,022 2,001 Green 3.01 : 1

Yellow

3 Seed coat Form 882 Inflated 299 2.95 : 1

Constricted

4 Seed coat Colour 705 Grey 224 5White 3.15 : 1

5 Unripe Colour 428 Green 152 Yellow 2.82 : 1

pods
6 Flowers Position 651 Axial 207 Terminal 3.14 : 1

7 Stem Length 787 277 Short 2.84 :

Long 1

Mendel’s explanations and predictions

According to Mendel, each male and female parent contained

a pair of such hereditary factors, and each parent passed only one factor of a
pair to their offsprings. Thus, each parental character was though not be
transmitted directly to the offsprings but by some indirect mean and that is
by hereditary factors which determine the characters. Further, he predicted
that each factor retained its individuality from generation and it was not
modified in the hybrid. The factors contributed by the parents united
randomly to produce the characters of a hybrid. Thus, indirectly, he
predicted about the reduction division during gametogenesis and the
physical hereditary mechanism, both were unknown to the scientific world at
that time.

Mendel’s Laws
Mendel postulated two laws, which are now called after his
name as Mendel’s laws of heredity. These are:
 1. Law of segregation
2. Law of independent assortment

The phenomenon of dominance has been considered

erroneously as the law of dominance in some of the text books of genetics.

Mendel’s Law of segregation(Purity of Gametes)

Mendel;s first law of inheritance, the law of segregation or law of purity of

gametes states that in a heterozygote a dominant and a recessive allele
remain together without contaminating or mixing with each other and
separate or segregate from each other during gametogenesis, so that, each
gamete receives only one allele either dominant or recessive. For example,
the F1 hybrids (Tt) of a monohybrid cross between tall (TT) and dwarf (tt)
pea plant have one dominant allele (T) for tallness and one recessive allele
(t) for dwarfness. The F1 hybrids by self- fertilization produce tall and
dwarf plants in the ratio of 3 : 1. It means that tall and dwarf alleles though,
remain together for long time but does not contaminate or mix with any one
and both alleles segregate to produce gametes which either having dominant
allele T or recessive allele t. These gametes unite to produce the 3 : 1
phenotypic ratio in F2.

Explanation - The law of segregation states that when a pair of contrasting

factors or genes or allelomorphs are brought together in a heterozygote (hybrid) the
two members of the allelic pair remain together without being contaminated and
when gametes are formed from the hybrid, the two separate out from each other
and only one enters each gamete.
Example - Pure tall plants are homozygous and, therefore/possess genes (factors)
TT; similarly dwarf possess genes tt. The tallness and dwarfness are two
independent but contrasting factors or determiners. Pure tall plants produce
gametes all of which possess gene T and dwarf plants t type of gametes.
During cross fertilization gametes with T and t unite to produce hybrids of
F1 generation. These hybrids possess genotype Tt. It means F1 plants, though tall
phenotypically, possess one gene for tallness and one gene for dwarfness.
Apparently, the tall and dwarf characters appear to have become contaminated
developing only tall character. But at the time of gamete formation, the genes T
(for tallness) and t (for dwarfness) separate and are passed on to separate
gametes. As a result, two types of gametes are produced from the heterozygote
in equal numerosity. 50% of the gametes possess gene T and other 50% possess
gene t. Therefore, these gametes are either pure for tallness or for dwarfness.
(This is why the law of segregation is also described as Law of purity of
gametes).

F1 Plants Tt X Tt
T t T t

Gametes unite at random and when gametes are numerous all possible
combinations can occur, with the result that tall and dwarf appear in the ratio of 3
:1. The results are often represented by Punnett square as follows: Law of
Independent Assortment

Definition: The inheritance of more than one pair of characters (two

pairs or more) is studied simultaneously, the factors or genes for each pair of
characters assort independently of the other pairs. Mendel formulated this law
from the results of a dihybrid cross.
Explanation: The cross was made between plants having yellow and round
cotyledons and plants having green and wrinkled cotyledons.
The F1 hybrids all had yellow and round seeds. When these F 1 plants
were self fertilized they produced four types of plants in the following
proportion
Yellow and round 9
(ii) Yellow and wrinkled 3
(iii) Green and round 3
(iv) Green and wrinkled 1
The above results indicate that yellow and green seeds appear in the ratio
of 9 + 3 : 3 + 1 = 3 : 1. Similarly, the round and wrinkled seeds appear in the ratio of
9 + 3 : 3 +1 = 12:4 or 3 :1. This indicates that each of the two pairs of alternative
characters viz. yellow-green cotyledon colour is inherited independent of the
round-wrinkled character of the cotyledons. It means at the time of gamete
formation the factor for yellow colour enters the gametes independent of R or r, i.e,
gene Y can be passed on to the gametes either with gene R or r.
Cytological explanation of the results: In the above experiment yellow
and round characters are dominant over green and wrinkled characters which can
be represented as follows:

(i) gene for yellow colour of cotyledons Y

(ii) gene for green colour of cotyledons y
(iii) gene for round character of cotyledons R
(iv) gene for wrinkled character of colyledons r
Therefore, plants with yellow and round cotyledons will have their
genotype YYRR and those with green and wrinkled cotyledons will have a
genotype yyrr. These plants will produce gametes with gene YR and yr
respectively. When these plants are cross pollinated, the union of these gametes will
produce F1 hybrids with YyRr genes. When these produce gametes all the four
genes have full freedom to assort independently and, therefore, there are possibilities
of four combinations in both male and female gametes.
(i) RY (ii) Ry (iii) rY (iv) ry
This shows an excellent example of independent assortment. These
gametes can unite at random producing in all 16 different combinations of genes,
but presenting four phenotypes in the ratio of 9: 3: 3: 1.
Dihybrid ratio : RR yy - Round, yellow seeded ; Rr yy - Wrinkled
and greed seeded
 The F1 hybrids all had yellow and round seeds. When these F 1 plants
were self fertilized they produced four types of plants in the following
proportion
Yellow and round 9
(ii) Yellow and wrinkled 3
(iii) Green and round 3
(iv) Green and wrinkled 1

The Testcross
Because some alleles are dominant over others, the phenotype of an organism
does not always reflect its genotype. A recessive phenotype (yellow) is only
expressed with the organism is homozygous recessive (gg). A pea plant with
green pods may be either homozygous dominant (GG) or heterozygous (Gg).
To determine whether an organism with a dominant phenotype (e.g. green
pod color) is homozygous dominant or heterozygous, you use a
testcross.

OR
 The breeding of an organism of unknown genotype with a homozygous
recessive. If all the progeny of the testcross have green pods, then the green pod
parent was probably homozygous dominant since a GG x gg cross produces Gg
progeny. If the progeny of the testcross contains both green and yellow phenotypes,
then the green pod parent was heterozygous since a Gg x gg cross produces Gg and
gg progeny in a 1:1 ratio. The testcross was devised by Mendel and is still an
important tool in genetic studies.
 Definitions of gene, allele, homozygous, heterozygous,
genome, phenotype, genotype, backcross and test cross.

Mendel Postulated that ‘Characters’ are determined by ‘elements’

found in the sex cells or gametes. The character and its determinant are
thus different and Bateson coined the word ‘factor’ for that which
determines a character. The Danish geneticist Johannsen recognised that
there is something in the gametes and in the fertilised egg that
determines a character and he proposed the word ‘gene’ for it.

Gene can be defined as the hypothetical unit of inheritance located

at a fixed position (i.e., the locus) on a chromosome which by interaction
with the other genes, the cytoplasm and the environment controls the
development of a character.

Allele

Allele is defined as alternate (or series) of forms of a gene situated

at the same locus of homologous chromosomes.

Homozygote and Heterozygote

Mendel recognised that a gamete can possess only one of a pair of

alleles, for example, either R or r and not both. An individual formed by
the union of like gametes is said to be pure. The British geneticist
Bateson introduced the term homozygote (homo= same; zygos = yolk)
for an organism in which the two genes at the same locus of homologous
chromosomes are identical.
The true-breeding round-seeded pea plant is formed by the union
of an egg with R and a sperm, also with R and is represented as RR, and
the true-breeding wrinkled-seeded plant formed by the union of an egg
and a sperm, each with r, is represented as rr.
An individual formed by the union unlike gametes is said to be a
hybrid. Bateson called this a heterozygote (hetro = different; zygos =
yolk) because the two genes at the same locus of homologous
chromosomes are not identical. The round seeds of the first hybrid
generation (i.e., F1) formed by the union of a gamete with R and another
gamete with r are therefore heterozygous and are represented as Rr.

Phenotype and Genotype

Johannsen clearly brought out the difference between the visible

character and the invisible gene that is responsible for the character.
He coined the world phenotype (pheno = appear) for the visible
character of an individual and the word genotype for the heredity of a
plant or animal.
The genotype of an individual can be determined by observing its
phenotype, but as two different genotypes may possess the same
phenotype because of the phenomenon of dominance, it can be
confirmed only by studying the ancestry or the progeny of the individual.
Thus, for example, in pea, two different genotypes, RR and Rr, have the
same phenotype, viz., round seeds, but wherease the former produces
nothing but round seed, the latter produces round seeds and wrinkled
seeds in the ratio of 3 round : 1 wrinkled.

Back cross and Test cross

Back cross is a cross between a hybrid and either of its parents

whereas test cross is a cross between hybrid and a recessive homozygote.
 Modification of monohybrid 3:1 phenotypic ratio due to

1) Incomplete dominance

2) Codominance

3) Lethal genes etc.

1. Incomplete dominance

Mendel always observed complete dominance of one allele over the other for all the seven
characters, which he studied, in garden pea. Later on cases of incomplete dominance were
reported. For example, in four 0’ clock plant (Mirabilis jalapa). There are two types of flowers
viz., red and white. A cross between red and white flowered plants produced plants with
intermediate flower colour i.e. pink colour in F1 and a modified ratio of 1 red: 2 pink: 1 White in
F2.
Parents Red flower x White
flower RR x rr
F1 Rr pink flower

F2 1 Red (Rr) : 2 Pink (RR) : 1 White (rr)

 CO-DOMINANCE

In the phenomenon of co-dominance, both dominant and recessive alleles lack

their dominant and recessive relationships and both have capability to express them
phenotypically, in the heterozygous condition. In a heterozygote of co-dominant nature,
the dominant and recessive traits occur side by side. The F1 heterozygotes produce a F2
progeny in the phenotypic and genotypic ratios of 1 : 2 : 1 like the incomplete
dominance. or

In case of codominance both alleles express their phenotypes in

heterozygote greater than an intermediate one. The example is AB blood group in
human. The people who have blood type AB are heterozygous exhibiting phenotypes
for both the IA and IB alleles. In other words, heterozygotes for codominant alleles are
phenotypically mixture to both parental types. The main difference between
codominance and incomplete dominance lies in the way in which genes act. In case
of codominance both alleles are expressed while in case of incomplete dominance
both alleles blend to make an intermediate one.

Example of Co-dominance
The best example of co-dominance is found in cattles. In one breed of cattle a
genotype of WW will be expressed phenotypically in white coat colour, while the
genotype of ww into a red coat colour. When a white-coated cattle is crossed with a red-
coated cattle, the F1 heterozygote are found to have a phentoype of redish gray or roan
colour. The roan coat of a F1 heterozygote has no hair of intermediate colour between
red and white, but rather has a mixture of red hair and white hairs. The F 1 heterozygotes
produce a F2 progeny of phenotypic and genotypic ratios of 1 : 2 : 1.

Lethal alleles:
 Gene, which causes the death of its carrier when in homozygous condition
is called lethal gene.

Mendel’s findings were based on equal survival of all genotypes. In

normal segregation ratio of 3:1 is modified into 2:1 ratio.

The recessive lethal allele kills the carrier individual only in homozygous
condition. They may be of two kinds (I) one which has no obvious phentoypic effect in
hetrozygotes and (ii) one which exhibits a distinctive phenotype when in heterozygous
condition.
The completely lethal genes usually cause death of the zygote, later in the
embryonic development or even after birth or hatching. Complete lethality, thus is the
case where no individual of a certain genotype attain the age of reproduction. However,
in many cases lethal genes become operative at the time the individuals become sexually
mature. Such lethal genes which handicap but do not destroy their possessor are called
subvital, sublethal or semilethal genes. The lethal alleles modify the 3 : 1 phenotypic
ratio into 2 : 1.

Examples of lethal alleles or complete lethality in plants

1. In snapdragons three types of plants occur : 1. Green plants with chlorophyll ; 2.
Yellowish green plants with carotenoids, usually are referred to as golden or auria
plants and 3. White plants without any chlorophyll. The homozygous green plants
have the genotype CC and the homozygous white plant has the genotype cc. The
auria plants have the genotype Cc because they are heterozygotes of green and white
plants. When two such auria plants are crossed, the F1 progeny has identical
phenotypic and genotypic ratio of 1 : 2 : 1 (viz., 1 green (CC) : 2 auria (Cc) : 1 white
(cc). But the white plants because lack chlorophyll pigment, therefore die to modify
the ratio of 1 : 2 : 1 into 1 : 2 or 2 : 1. In this case the homozygous recessive
genotype (cc) is lethal.
F1 heterozygote : Auria Auria
Cc x Cc
F2 : 1 CC : 2 Cc : 1 cc
Green Auria White (lethal)
Or 1 CC : 2 Cc or 1 : 2
2. In maize (Zea mays) the amount of chlorophyll is controlled by a recessive allele (g)
which exhibits a lethal effect in homozygous (gg) and in heterozygous condition
(Gg) has phenotype similar to homozygous condition for dominant gene GG. It
modifies 3 : 1 phenotypic ratio into 2 : 1.
F1 heterozygote : Green Green
Gg x Gg
F2 : 1 GG : 2 Gg : 1 gg
Green Green White (lethal) Or 1 GG : 2 Gg
 Lethal genes have been reported in both animals as well as plants. In mice
allele for yellow coat colour is dominant over grey. When a cross is made
between yellow and grey a ratio of 1:1 for yellow and gray mice was observed.
This indicated that yellow mice are always heterozygous. Because yellow
homozygotes are never born because of homozygous lethality. Such genes were
not observed by Mendel. He always got 3:1 ratio in F2 for single gene
characters.

In maize (Zea mays) the amount of chlorophyll is controlled by a recessive allele (g)
which exhibits a lethal effect in homozygous (gg) and in heterozygous condition
(Gg) has phenotype similar to homozygous condition for dominant gene GG. It
modifies 3 : 1 phenotypic ratio into 2 : 1.
F1 heterozygote : Green Green
Gg x Gg
F2 : 1 GG : 2 Gg : 1 gg
Green Green White(lethal)
1 GG : 2 Gg or 1 : 2 or 2 :1
 TYPES OF GENE ACTION
Interaction of two pairs of alleles

When an individual forms gametes, the two members of each pair of alleles
always separate from each other but the separation in one pair of alleles is independent
of the separation in any other pair of alleles. Gametes, therefore, always contain any one
allele of each of the several pairs of alleles found in an individual. At fertilisation, these
gametes recombine at random to give rise to new individuals.

When different pairs of alleles influence the same character of an individual, it is

likely that the expressions of these genes interact. As two different genes interact and
affect the same character, such a genetic interaction is said to be intergenic or nonallelic.
In nonallelic interactions different genes located on the same or different chromosomes
interact with one another for the expression of a single phenotypic trait of an organism.

Intergenic or nonallelic interactions may suppress or mask the action of a gene at

another locus or modify partially or completely the effect of another gene. This
nonallelic interaction is otherwise called epistasis.

Definition: - A kind of interaction between genes belonging to different pairs of alleles,

the dominant allele in one of the pairs preventing the dominant allele in the other pair
from expressing itself. Thus, the gene A may be epistatic over B. B is then said to be
hypostatic to A.

We shall now consider a few cases of independently transmitted pairs of alleles

that are not independent in their expression.

Intergenic Non-epistatic interaction (9: 3 : 3 : 1 Ratio)

A classical case of two pairs of alleles affecting the same characteristic and
producing in the F2 four different phenotypes in the ratio of 9 : 3 : 3 : 1 was discovered
in fowls by Bateson and Punnett.

Each breed of pultry possesses a characteristic type of comb. The Wyandotte

breed has a comb known as the ‘rose’ comb, the Brahma has a ‘pea’ comb, the Leghorn
has a ‘single’ comb and the Malay breed has a comb known as the ‘walnut’ comb. Each
of these breeds true. When, however, a rose-combed fowl is crossed with a pea combed
one, all the F1 birds show the walnut comb. When the F1 walnut combed birds are bred
together, there appears in the F2 9 walnut : 3 rose : 3 pea : 1 single comb.

These results can be interpreted as follows: The rose comb is due to a gene R and
the pea is due to another gene P. The walnut comb is due to the presence of both the
dominant genes, R and P and the single comb is due to their recessive alleles, r and p.
 The breeding behaviour of the different genotypes of the F2 is summarised

Parent RR PP x rrpp
Rose x rp

Rr pp(Walnut)

♂ RP Rp rP rp
♀
RP RRPP RRPp RrPP RrPp
(W) (W) (W) (W)

Rp RRPp RRpp RrPp Rrpp

(W) (R) (W) (R)

Rp RrPP RrPp rrPP rrpp

(W) (W) (P) (P)

Rp RrPp Rrpp rrPp rrPP

(W) (R) (P) (S)

9 Walnut: 3 Rose: 3 Pea: 1 Single

The above example depicts a case of non-epistatic intergenic interaction in which
two genes that determine the same character produce a new phenotype by mutual non-
epistatic interaction.

Difference between dominance and epistasis

The phenomenon of dominance involve intra-genic or inter-allelic gene

suppression, or the masking effect which one allele has upon the expression of another
allele at the same locus, while the phenomenon of epistasis involves inter-genic
suppression or the masking effect which one gene locus has upon the expression of
another. The classical phenotypic ratio of 9:3:3:1 observed in the progeny of dihybrid
parents becomes modified by epistasis into ratios which are various combinations of the
9:3:3:1 groupings.
 The term epistasis was coined by Bateson in 1909. Various types of
epistatic gene interaction are
1) Recessive epitasis (9:3:4)
2) Dominant epistasis (12:3:1)
3) Dominant and recessive (inhibitory) epistasis (13:3)
4) Duplicate recessive epistasis (9:7)
5) Duplicate dominant epistasis (15:1) and
6) Polymeric gene interaction (9:6:1).

Duplicate recessive epistasis (Complimentary gene action) 9:7

When recessive alleles at either of the two loci can mask the expression
of dominant alleles at the two loci, it is called duplicate recessive epistasis. This
is also known as complementary epistasis. The best example of duplicate
recessive epistasis if found for flower colour in sweet pea. The purple colour of
flower in sweet pea is governed by two dominant gene say A and B when these
genes are in separate individuals (Aabb or aaBB) and white (aabb) they produce
white flower. A cross between purple flower (AABB) and white flower (aabb)
strains produced purple colour in F1 intermating of F1 plants produced purple
and white flower plants in 9:7 ratio in F2 generation. Here the recessive allele
.a. is epistatic to B/b alleles and mask the expression of these alleles, another
recessive allele b is epistatic to A/a alleles and mask their expression.
Parents purple x White
AABB x aabb
AB ab
AaBb
Purple

♂ AB Ab aB ab
♀
AB AABB AABb AaBB AaBb
(P) (P) (P) (P)
Ab AABb AAbb AaBb Aabb
(P) (W) (P) (W)
aB AaBB AaBb aaBB aaBb
(P) (P) (W) (W)
Ab AaBb Aabb aaBb aabb
(P) (W) (W) (W)
 Ratio = 9 Purple : 7 white
Duplicate gene action (15:1) (Duplicate dominant epistasis)
When a dominant allele at either of two loci can mask the expression of
recessive alleles at the two loci, it is known as duplicate dominant epistasis. In
rice awn character is controlled by two dominant duplicate genes (A and B).
Presence of any of these two alleles can produce awn. The awnless condition
develops only when both these genes are in homozygous recessive state (aabb).
A cross between awned and awnless strains produced awned plants in F1.
Intermating of F1 plants produced awned and awnless plants in 15:1 ratio in F2
generation. The allele A is epistatic to a/b alleles and all plants having allele A
will develop awn. Another dominant allele B is epistatic to alleles a/b. An
individual with these allele also develop awn character.

Parents awned rice x awnless rice

AAbb x aaBB
AaBb
Awned rice
♂ AB Ab aB ab
♀
AB AABB AABb AaBB AaBb
(A) (A) (A) (A)
Ab AABb AAbb AaBb Aabb
(A) (A) (A) (A)
aB AaBB AaBb aaBB aaBb
(A) (A) (A) (A)
ab AABb AAbb AaBb Aabb
(A) (A) (A) (a)

Ratio = 15 awned : 1 awnless

Inhibitory
gene
interaction
(13:1)
In this type of epistasis, a dominat allele at one locus can mask the
expression of both (dominant and recessive) alleles at second locus. This is also
 known as inhibitory gene interaction. An example of this type of gene
interaction is found for anthocyanin pigmentation in rice. The green colour of
plants is governed by the gene I which is dominant over purple colour. The
purple colour is controlled by a dominant gene P. when a cross was made
between green (IIpp) and (iiPP) colour plants, the F1 was green. Intimating of
F1 plants produced green and purple plants in 13:3 ratio in F2.

Parents awned rice x awnless rice

AAbxaaBB
AaBb
Awned rice
♂ IP Ip iP ip
♀
IP IIPP (G) IIPp (G) IiPP (G) IiPP (G)

Ip IIPp (G) IIpp (G) IiPp (G) Iipp (G)

iP IiPP (G) IiPp (G) IiPP (P) iiPp (P)

ip IiPp (G) Iipp (G) iiPp (P) Iipp (G)

Ratio = 13 Green : 3 Purple

Supplementary gene action. (Recessive epistasis) 9:3:4

Here one dominant gene has its own phenotypic effect and other
dominant gene has no effect of its own but its presence with the first gene
modified the phenotypic expression. Thus in supplementary gene action the
dominant allele of one gene is necessary for the development of the concerned
phenotype, while the other gene modifies the expression of the first gene.
Parents RR PP x rr pp
Purple Red
Gametes RP rp
F1 RrPp purple

♂ RP Rp RP Rp
♀
RP RRPP RRPp RrPP RrPP
(P) (P) (P) (P)
Rp RRPp RRpp RrPp Rrpp
(P) (W) (P) (W)
rP RrPp RrPp RrPP RrPp
(P) (P) (R) ®
rp RrPp Rrpp rrPp rrpp
(G) (W) (RP) (W)
Ratio = 9 Purple : 3 Red : 4 White
Additive factors (9:6:1) (Polymeric gene action)
In these two genes controlling a character produces identical phenotype
when they are alone i.e. with the homozygous recessive condition of the other
gene. But when both the genes are present together, their phenotype effect is
enhanced as if the effect of the two genes were cumulative or additives. It
should be noted that in this case both the genes show complete dominance. If
the two genes showing polymeric gene action, what will be the consequence. In
barley two completely dominant genes A and B affect the length of awns, the
thin needle like extension of lemma genes A and B alone (e.g. Aabb and aaBB
give gives rise to awn of medium length, the effect of A is the same as that of
B. But when both the genes A and B are present together they produce long
awn indicating the effect of A and B genes of awn length are added together.
Individual homozygous recessive for both these genes are awn less.
Parents AA BB x aa bb
Long awned x awnless
Aa Bb
Long awned
♂ AB Ab aB ab
♀
AB AABB AABb AaBB AaBB
(L) (L) (L) (L)
Ab AABb AAbb AaBb Aabb
(L) (A) (L) (A)
aB AaBB AaBb aaBB aaBb
(L) (L) (A) (A)
ab AaBb Aabb aaBb aabb
(L) (A) (A) (a)
Ratio = 9 Long awned: 6 Awned: 1 awnless

11. Dominant Epistasis (12:3:1)

 An example of dominant epistasis is found for fruit colour viz white,
yellow and green. White colour is controlled by dominant gene W and yellow
colour by dominant genes G. White is codominant over both yellow and green.
The green fruits are produced in recessive condition (wwgg). A cross between
plants having white and yellow fruits produced F1 with white fruits.
Intermating of F1 plants produced plants with white, yellow and green coloured
fruits in F2 was 12:3:1 ratio. Here W is dominant to w and epistatic to alleles G
and g. Hence it will mask the expression of G.g alleles. Hence in F2 plants with
W-G- (9:16) and W-gg (3:16) genotypes will produce white fruits; plants with
wwG-3/16 will produce yellow fruits and those with wwgg 1/16 genotype will
produce green fruits. Thus the normal dihybrid ration 9:3:3:1 is modified to
12:3:1 ratio in 1:2 generation. Similar type of gene interaction has been
reported for skin
colour in mice and seed coat colour in barley.

Parents White fruit x Yellow

fruit
WWgg x wwGG
WwGg White fruit

♂ WG Wg wG wg
♀
Wg WWGG WWGg WwGG WwGG
(W) (W) (W) (W)

Wg WWGg WWgg WwGg Wwgg

(W) (W) (W) (W)

WG WwGG WwGg wwGG WwGg

(W) (W) (Y) (Y)

Wg WwGg Wwgg wwGg wwgg

(W) (W) (Y)
(G)

Ratio = 12 White: 3 Yellow: 1 Green

 PLEIOTROPY

Until now we have observed that a specific gene has a specific effect upon a
specific phenotypic trait or in other words, each gene (allele) has its relation with a
single phenotypic trait. But a single gene often influences more than one phenotypic
trait. However, it may be that one gene may cause evidently well marked expression of
some phenotypic trait (major effect) than the others with less evident phenotype
(secondary effect). Most genes have their multiple effects and are called pleiotropic
genes. The phenomenon of multiple effect (multiple phenotypic expressions) of a single
gene is called pleiotropism.

Examples of Pleiotropism
1. In Drosophila the recessive gene for vestigial wings cause vestigial wings in
homozygous condition. However, careful observations show that other traits as well are
affected – (I) the tiny wig like balancer behind the wings : (ii) certain bristles ; (iii) the
structure of the reproductive organs ; (iv) egg production is lowered, and (v) longevity is
reduced.
2. In human, the gene for disease phenylketonuria has pleiotorpic effect and
produces various abnormal phentoypic traits, collectively called syndrome. For example,
the affected individuals secrete excessive quantity of amino acid phenylalanine in their
urine, cerebrospinal fluid and blood. They become short stature, mentally deficient, with
widely spaced incisors, with pigmented patches on skin, with excessive sweating and
with non-pigmented hairs and eyes.
 PHENOCOPY
The alteration of the phenotype, by nutritional factors or the exposure to
environmental stress during development, to a form imitating that characteristically
produced by a specific gene. Thus rickets due to a lack of vitamin D would be a
phenocopy of vitamin-D-resistant rickets.

PENETRANCE AND EXPRESSIVITY

The ability of a given gene or gene combination to be expressed phenotypically

to any degree is called penetrance. In other words, penetrance refers to the statistical
regularity with which a gene produces its effect when present in the requisite
homozygous (or heterozygous) state. It is of following two kinds:

1. Complete Penetrance

Most dominant and recessive genes in homozygous conditions and many

completely dominant genes even in heterozygous condition give their complete
phenotypic expressions. Such genes which always produce the expected phenotype are
called to have complete penetrance. If only 70 per cent of the individuals of a stock
homozygous for a certain recessive gene show the character phenotypically, the gene is
said to have 70 per cent penetrance.

Examples of Complete Penetrance

1. In pea, the alleles (RR) for red flowers and the alleles (rr) for white flowers
have complete penetrance in homozygous conditions.
2. In Drosophila the recessive alleles for vestigial wings in homozygous
conditions have complete penetrance.
3. In guinea pigs the dominant allele ‘B’ for black coat has complete penetrance
both in homozygous and heterozygous conditions.

2. Incomplete Penetrance
Some genes in homozygous as well as in heterozygous conditions fail to provide
 complete (cent per cent) phenotypic expression of them. Such genes are called to have
incomplete penetrance.

Examples of Incomplete Penetrance

1. Polydactyly in man is thought to be produced by a dominant gene P. the

normal condition with five digits on each limb is produced by the recessive genotype
(pp). some heterozygous individuals (Pp) are not polydactylus and therefore has a
penetrance of less than 70%.
2. In, man the tendency to develop diabetes mellitus (a condition in which there
is an excess of sugar in the blood) is controlled by certain genes. However, not everyone
carrying the genes for diabetes actually develops the conditions, for the genes have
incomplete penetrance.

EXPRESSIVITY

A trait though penetrant, may be quite variable in its phenotypic expressions. The
degree of effect produced by a penetrant genotype is called expressivity.
Example of expressivity

In man the polydactylous condition may be penetrant in the left hand (6 fingers)
and not in the right (5 fingers) ; or it may be penetrant in the feet and not in the hands.
 Multiple alleles

Multiple Alleles

So far, it has been observed that a given phenotypic trait of an individual depends
on a single pair of genes, each of which occupies a specific position called the gene locus,
on a homologous chromosome. Moreover, a particular gene has been found to occur in
two alternative forms. For example, a gene (L) for length of Drosophila wings may
occur in two alternative forms: a gene (L+) for normal development of wings and a gene
(L”) for vestigial wings. Because, most flies have normally developed wings, so, it can
easily, be concluded that gene L+ is the original form of gene from which the other form
of gene (L”) might have originated by certain mutational event at sometime in past. The
gene L+ for normal development of wings is called the normal or wild type allele of the
gene L and usually symbolized as L+, while the mutated gene L” for vestigial wings is
called L” reduced type or mutant allele of gene L. A fly with normal wings, thus has two
wild type alleles (L+L+) and the vestigial wings fly has two mutant alleles (L”L”). Both
of these allelic forms (L+ and L”) of gene L occur at corresponding positions on
genetically identical (homologous) chromosomes of same or different individual.

But, now there are ample evidences that a gene for a particular character, besides
occurring in two alternative forms or alleles may occur in several alternative forms or
alleles. All the variants or alleles of a given gene are supposed to be originated by
mutation of a single wild type gene. Out of several allelic forms of a gene, a given locus
may bear any one allele, so that, a diploid individual possesses any two alleles of the
allelic series. When any of the three or more allelic forms of a gene occupy the same
locus in a given pair of homologous chromosomes they are said to constitute a series of
multiple alleles. In other words, all the mutant forms of a single wild type gene constitute
a series of multiple alleles.

Blood Group

Multiple allelism also occur in man. The blood group inheritance in man can be
better understood by learning following aspects:

Multiple Allelic inheritance of A, B, AB and O Blood Types

. The gene controlling blood types has been labeled as I (after the name of immune traits)
or L (after the name of discoverer, Landsteiner). The L gene exists in three different
allelic forms : IA, IB and IO. The first two alleles produce characteristic antigens on the
surface of red blood cells or erythrocytes. Thus IA alleles specifies A antigen, IB allele B
antigen and IO allele specifies no antigen.
 The pedigree analysis has shown that alleles, IA and IB have dominance over
O
allele I . Likewise, the pedigree analysis of A and B parents revealed that children
have both A and B antigens and so it was concluded that the alleles IA and IB have
co- dominant relationship between them. The dominance hierarchy of this allelic-
series can symbolized as IA = IB >IO.

Characters of Multiple Alleles

The most important and distinguishing features of multiple alleles are summarized
below:
1. Multiple alleles of a series always occupy the same locus in the chromosome.
2. Because, all the alleles of multiple series occupy the same locus in chromosome,
therefore, no crossing-over occurs within the alleles of a same multiple allele
series.
3. Multiple alleles always influence the same character.
4. The wild type allele is nearly always dominant, while the other mutant alleles in
the series may show dominance or there may be an intermediate phenotypic
effect.
5. When any two of the mutant multiple alleles are crossed, the phenotype is mutant
type and not the wild type.
Symbols for Multiple Alleles

A capital letter is commonly used to designate the allele which is dominant to

all other alleles in the series. The corresponding lower case letter designates the allele
which is recessive to all others in the series. Other alleles which are intermediate in
their degree of dominance between these two extremes, are usually assigned the
lower case letter with some suitable super script.

Examples of Mulitple Allelism

Some of the characteristic cases of multiple alleles have been studied in

rabbits, guinea pigs, mice, Drosophila, man and certain plants, such as Nicotiana.

Coat colour in Rabbits

The coat of rabbit may have different colours as described below.

i) Full colour: The coat of the ordinary (wild type) rabbit is referred to as “agouti” or
full colour, in which individuals have banded hairs, the portion nearest the skin being
gray, succeeded by a yellow band, and finally a black or brown tip. The allele for full
colour may be represented by capital letter c+.
ii) Chinchilla: In some individuals, the coat lacking the yellow pigment and due to
the optical effect of black and gray hairs have the appearance of silvery-gray. The
allele for chinchilla is represented as, cch.
iii) Himalyan (Russian): The Himalyan type coat is white except for black
extremities (nose, ears, feet and tail). The condition in which black pigmentation is
confined to the ears, muzzle, feet and tail, is called acromelanism (Serra, 1965). In
Himalyan rabbits eyes remain pigmented. The allele for Himalyan coat is
represented by ch.
iv) Albino: The albino coat totally lacks in pigmentation and the eyes of a albino
also remain pink due to lack of pigment in iris of eye. The allele for albino is
represented by c.

Crosses of homozygous agouti (c+c+) and albino (cc) individuals produce a

uniform agouti F1 ; interbreeding of the F1 produces and F2 ratio of 3 agouti : 1
albino. Tow third of F2 agouti are found to be heterozygous by testcrosses. Thus, it is
a case of monohybrid inheritance, with agouti completely dominant to albino.
Likewise, crosses between chinchilla and agouti produce all agouti individuals in the
F1 and a 3 agouti : 1 chinchilla ratio in the F2. Such complete dominance of agouti
also occurs on Himalayan. Further crosses, real that cch allele for chinchilla, though is
recessive to c+ allele for agouti coat or skin, is incompletely dominant over
Himalayan (ch) and albino (c) alleles. Likewise, ch allele for Himalayan coat is
recessive to c+ (agouti) and cch (Chinchilla) but dominant over albino. The results
of all these crosses exhibit that c+ (agouti), cch (chinchilla), ch (Himalayan) and
c (albino) are allelic to each other and the alleles of this multiple allelic series have
following dominance hierarchy : c+ > cch > ch> c

P1 : Agouti x Albino P1 : Agouti x Chinchilla

c+c+ cc c+c+ cchcch

Agouti Agouti
F1: c+c F1: c+cch

F2: 1c+c+ : 2c+c : 1cc F2: 1c+c+ : 2c+cch : 1cchcch

3 Agouti : 1 Albino 3 Agouti : 1 Chinchilla

A monohybrid cross between agouti and A monohybrid cross between agouti and
albino rabbits. chinchilla rabbits.

P1 : Agouti x Himalayan P1 : Chinchilla x Himalayan

c+c+ chch cchcch chch

Agouti F1: light gray

F1: c+ch Cch ch
F2: 1c c : 2c+ch : 1chch
+ +
F2: 1c c : 2 cch ch : 1 ch ch
ch ch

1 Chinchilla : 2 Light gray : 1 Himalayan

3 Agouti : 1 Himalayan
A monohybrid cross between Chinchilla
A monohybrid cross between agouti and and himalayan rabbits. Showing
Himalayan (or Russian) rabbits. incomplete dominance of chinchilla (cch)
on Himalayan (ch)
P1 : Chinchilla x Albino P1 : Himalayan x Albino
cch cch cc chch cc

F1: light gray Himalayan

Cch c F1: ch c
F2: 1c c : 2 cch c : 1 c c
ch ch
F2: 1c c : 2ch c : 1c c
h h

1 Chinchilla : 2 Light gray : 1 Albino

3 Himalayan : 1 Albino
A monohybrid cross between Chinchilla
and albino rabbits. Showing incomplete A monohybrid cross between Himalayan
dominance of chinchilla (cch) on albino (c). and albino rabbits.

The possible phenotypes and their associated genotypes of this multiple

allelic series can be summarized.

The phenotypes and genotypes of multiple allelic series for coat colour in rabbit.
Phenotypes Genotypes
+ + + ch + h +
Full colour (Agouti) c c ,c c ,c c ,c c
Chinchilla cchcch
Light gray cch ch, cchc
Himalayan chch, chc
Albino cc

Self sterility in Nicotiana

multiple alleles have been associated with self-sterility or self-incompatibility

in several groups of plants. S elf-sterility is the phenomenon in which the pollen
grains from a plant fail to bring about fertilization in the ovules of the same plant. As
early as 1764 Kolreuter described self-sterility in tobacco, Nicotiana. In 1925 E. M.
East discovered self-sterility in Nicotiana is governed by alleles of multiple
allelic series of gene S.

Different alleles of this multiple allelic series were designated as S 1, S2, S3, S4, S5,
etc. None of the cross-fertilizing tobacco plants were homozygous, (i.e., S1S1 or S2S2)
but all plants were heterozygous (e.e., S1S2, S3S4, S5S6, etc.). When crosses were
attempted between different S1S2 plants, it was observed that pollen tubes did not
develop normally, but pollen from S 1S2 were effective on stigmas of plants with
other alleles, for example, S3S4.
When crosses were made between seed parents with S 1S2 and pollen parents with
S2S3, two kinds of pollen tubes were distinguished. Pollen grains carrying S 2 were not
effective, but the pollen grains carrying S3 were capable of fertilization. Thus, from the
cross S1S2 x S2S3, two kinds of progeny, S1S3 and S2S3, were produced. From a cross
S1S2 X S3S4, all the pollens were effective and four kinds of progeny resulted : S1S3,
S1S4, S2S3, and S2S4.
 Linkage – coupling and repulsion, complete and incomplete linkage.
LINKAGE
Every individual organism bears several heritable characters which are represented by
the innumerable genes present on the chromosomes. During meiosis, the chromosomes move
into the gametes as units, all the genes present on any given chromosome will segregate as a
group and move together from generation to generation. This tendency of the genes located on
the same chromosome, to stay together in hereditary transmission, is known as linkage. The
genes located on the same chromosome are called linked genes.
The principle of linkage was discovered by Bateson and Punnet in 1906 in the sweat pea,
plant, Lathyrus odoratus. However, linkage, as a concept was put forth by Thomas Hunt Morgan
in 1910 based on his experiment on Drosophila melanogaster.
Chromosome Theory of Linkage
Morgan, along with Castle formulated the chromosome theory of linkage. It has the
following postulates;
1. Genes are found arranged in a linear manner in the chromosomes.
2. Genes which exhibit linkage are located on the same chromosome.
3. Genes generally tend to stay in parental combination, except in cases of crossing
over.
4. The distance between linked genes in a chromosome determines the
strength of linkage. Genes located close to each other show stronger linkage than
that are located far from each other, since the former are less likely to enter into
crossing over.

Linkage: The tendency of two or more genes to stay together during inheritance is known as
linkage. Linkage is the consequence of the concerned gnes being located in the same
chromosome. Linked genes do not show independent segregation; as a consequence, the ratios
obtained in F2 and test cross generations are significantly different from the expected ratios of
9:3:3:1 and 1:1:1:1.

Example 1: Segregation of genes present on two different chromosomes

1
 Test cross ratio: 1:1:1:1

The two genes present on two different chromosomes, show independent assortment 50%
each of parental types and nonparental types obtained in F2 generation.

Example 2: Segregation of the genes present on same chromosome

2
Test cross ratio: 1:1

This is the example for complete linkage, where there is no crossing over and only parental types
are obtained.

Views of classical geneticists on linkage

Mendel could not notice the phenomenon of linkage because fortunately the seven pairs of
factors or alleles studied by him in pea were located on seven different pairs of chromosomes. It
was noticed and discovered by some other post-Mendelian geneticists who during their genetic
investigations came across to linked genes.
Fortunately Mendel took characters present on different chromosome, so based on
independent assortment he explained segregation of genes.
Three genes present on chr. 1 and the distance between the two was 50M.U.
Three genes present on Chr 4.
One gene on Chr 7.

The evolution of linkage concept took place by the views of following classical geneticists:

Coupling and Repulsion Hypothesis of Bateson and Punnett

Bateson and Punnett (1905-1908) formulated the ‘hypothesis of coupling and repulsion’ to
explain the unexpected F2 results of a dihybrid cross between a homozygous sweet pea
(Lathyrus odoratus).

Bateson and Punnett could not explain the exact reasons of coupling and repulsion, and
3
it was T.H. Morgan who while performing experiments with Drosophila, in 1910, found that
coupling or repulsion was not complete. He further suggested that the two genes are found in
coupling phase or in repulsion phase, because they are present on the same chromosome
(coupling) or on two different homologues chromosomes (repulsion). Such genes are then called
linked genes and the phenomenon of inheritance of linked genes is called linkage by Morgan.

The cross I revealed presence of more of parental genotypes indicating that the genes
are partially linked.

4
Same thing was revealed in cross II

Coupling phase: refers to the case where dominant alleles are on the same homologue
chromosome and both recessive alleles are on the other homologue chromosome.
Eg: F1 of cross I
Repulsion phase: refers to the case where each homologous chromosome has one dominant
and one recessive allele from the two genes (aB/Ab). Eg: F1 of cross II
Kinds of Linkage
The phenomenon of linkage is of following two kinds:

5
 1. Complete Linkage

When the linked genes are so closely located in chromosomes that they inherit
in same linkage groups for two or more generations in a continuous and regular fashion,
then, they are called completely linkage genes and the phenomenon of inheritance of
completely linked genes called complete linkage.

2. Incomplete Linkage

The linked genes do not always stay together because homologues non-sister
chromatics may exchange segments of varying length (which bearing many linked
genes) with one another during meiotic prophase, by the process of 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.

Linkage Groups
All the genes located on a particular chromosome, form a linkage group. Since, the genes
present on a particular chromosome have their alleles located on its homologous chromosome,
genes on a pair of homologous chromosomes. Hence, the number of linkage groups corresponds
to the number of haploid chromosomes found in a species.
Drosophila melanogaster has four linkage groups which can be distinguished into three
large and one small linkage groups corresponding to the four pairs of chromosomes. Twenty-
three linkage groups are present in humans corresponding to 23 pairs of chromosomes.
Pea plant has seven linkage groups, corresponding to the seven pairs of chromosomes.
Detection of Linkage
Compare the number of individuals observed in each class with those
expected on the basis of independent assortment and then to test the deviation
between these two values by chi-square test.

Coupling
In the condition is linked inheritance in which an individual heterozygous for two pairs of
genes receives the two dominant member from one parent and the two recessive members
from the other parent.
Repulsion is the condition is linked inheritance, in which an individual heterozygous for two
pairs of linked genes receives the dominant member of one pair and the recessive member of
the other pair from one parent and the reverse from the other parent
Linkage Map
Is a linear or circular diagram that shows the relative positions of genes on a chromosome as
determined by genetic analysis i.e frequencies of recombination.
The percent of occurrence of recombinants is more when the distance between two genes is
more.
This type of logic was suggested by A.H. Sturtevant, student of Morghan, suggested the relation
of percent recombinants to the distance between two genes.
One percent recombinants=one map units
Map Distance: It is the average number of cross over events that a chromosome has undergone
6
in the interval between two genetic markers.
Recombination Frequency: Is a measure of genetic linkage and used in the creation of genetic
linkage map.
Recombination frequency is the frequency with which a single chromosomal cross over will take
place between two genes during meiosis.
CentiMorghan (cM) is a unit that describes a recombination frequency.
Crossing over
Leading to recombination of linked genes is due to the exchange of corresponding
segments between the chromatids of homologous chromosomes and was first
observed by Belgian cytologist Janssens in 1909.
Linkage studies revealed the following
1. Genes that assort at random are non linked genes. Genes that do not
segregate at random are linked genes.
2. Linked genes are arranged in a lines fashion on the chromosome. Each
linked gene has a definite and constant order in its arrangement.
3. The distance between the linked genes determines the degree of
strength of linkage. Closely located genes show stronger linkage that the widely
located genes.
4. Linked genes do not always stay together, but are often exchanged
reciprocally by cross over.
Importance of linkage in breeding
When there is a close linkage between desirable and undesirable characters these
genes are inherited in blocks and not individually and recombination is practically nil.
In such cases linkage has to be broken by ' irradiation'.

Strength of linkage and recombination:

Two point:

The percentage of crossing over between two linked genes is calculated by test crosses in
which a F1 dihybrid is crossed with a double recessive parent. Such crosses because involve
crossing over at two points, so called two point test crosses. For example, a dihybrid having the
genotype AC/ac is test crossed with a double recessive parent (ac/ac), then among F 2 test cross
hybrids we may get 37% dominant genes at both gene loci (AC/ac), 37% recessive genes at both
gene loci (ac/ac), 13% dominant gene at first gene locus and recessive genes at both gene loci
(ac/ac), 13% dominant gene at first gene locus and recessive at the second gene locus (Ac/ac),
and 13% recessive gene at first gene locus and dominant gene at second gene locus (aC/ac).
The last two groups (i.e., 13% Ac/ac and 13% aC/ac) were produced by crossover gametes (13
+ 13) from the dihybrid and the distance between the loci A and C is estimated to be 26
centimorgans. Because, double crossovers usually do not occur between genes less than 5
centimorgans apart, so for genes further apart, the three point test crosses and used.

Three point test cross:

As three point test cross or trihybrid test cross (involving three genes) gives us
information regarding relative distance between these genes, and also shows us the linear order
in which these genes should be present on chromosome. Such a three point test cross may be
7
carried out if three points or gene loci on chromosome pair can be identified by marker genes. If,
in addition to genes A and C indicated above, a third marker genes B is located in fairly closed
proximity in the same linkage group, all three markers may be used together in conducting a
more precise analysis of the map distance and the relative position of three points.

Suppose that we testcross trihybrid individuals of genotype ABC/abc and find in

the progeny the following:
36% ABC/abc 9% Abc/abc 4% ABc/abc 1% AbC/abc
36% abc/abc 9% aBC/abc 4% abC/abc 1% aBc/abc

72% Parental : 18% Single : 8% Single : 2% Double

type crossover crossover bet- crossover
bet- ween A ween B and C.
and B. (region II)
(region I)

To find the distance A-B we must count all crossovers (both singles and doubles)
that occurred in region I = 18% + 2% or = 20% or 20 map units between the loci A and
B. To find the distance B-C we must again count all crossover (both singles and doubles)
that occurred in region II+8% + 2% = 10% or 10 map units between the loci B and C.

The A-C distance is therefore 30 map units when double crossovers are detected in a
three point linkage experiment and 26 map units when double crossovers are
undetected in the two-point linkage experiment above.

Without the middle marker (B), double crossovers would appear as parental
types and hence we underestimate the true map distance (crossover percentage). In this
case, the 2% double crossovers would appear with the 72% parental types, making a
total of 74% parental types and 26% recombinant types. Therefore, for any three linked
genes whose distances are known, the amount of detectable crossovers between the
two outer markers A and C when the middle marker B is missing ; (A-B crossover
percentage) plus (B-C crossover percentage) minus (2X double crossover percentage).

Interference and Coincidence

In higher organisms it has been found that one chiasma formation reduces the
probability of another chiasma formation in an immediately adjacent region of the
chromosome, probably because of physical inability of the chromatids to bend back
upon themselves within certain minimum distances. The tendency of one crossover to
interfere with the other crossover is called interference. The net result of this
interference is the observation of fewer double crossover types than would be expected
according to map distances, the strength of interference varies in different segments of
the chromosome and is usually expressed in terms of a coefficient of coincidence, or the
ratio between the observed and the expected double crossovers.

8
The coincidence is the complement of interference, so:
When interference is complete (1.0), no double crossovers will be observed and
coincidence becomes zero. When, interference decreases, coincidence increases.
Coincidence values ordinarily vary between 0 and 1. Coincidence is generally quite small for
short map distance. There is no interference across centromere.

Three Point Test Cross in Maize

The preparation of a linkage map can be exemplified by using an example from maize
involving three endosperm characters. These three characters are coloured aleurone (C)
versus colourless (c) aleurone, smooth seed (Sh) versus shrunked seed (sh) and normal or
non-waxy endosperm (Wx) versus waxy endosperm (wx). J. Sybenga (1972) has cited
following results of three point test cross in maize:

Results of a three point test cross in maize and calculation of

interference and coincidence. (after Sybenga (1972).

All three factors (genes) involve properties of the seed: the segregation can be
read on the cob of selfed F1 plant.

Parent: P1 c (colourless aleurone)-sh (shrunked seed)-wx (waxy

endosperm) P2 C (coloured aleurone)-Sh (smooth seed)-Wx (normal
endosperm)

c – sh – wx c – sh – wx
F1 : ----------------, test crossed with ---------------
C – Sh – Wx c – sh – wx

Types in testcross with numbers of seeds found (representing gametic ratios):

CShWx CShwx CshWx cShWx Cshwx cShwx cshWx cshwx Total

238 672 19 98 107 39 662 2198 6033

9
 Monofactorial segregation: C : c = 3036 : 2997
Sh : sh = 3047 : 2986 Slight shortage of
recessives Wx : wx = 3017 : 3016
Crossing over C-W : Cwx = 672 + 107
cWx = 98 + 662
1539 crossing over 1539 x 100 = 25.51%
6033
Crossing-over C-Sh : Csh = 19 + 107
cSh = 39 + 98
263 crossing over 263 x 100 = 4.36%
6033
Crossing-over Wx-Sh : Wxsh = 19 + 662
wxSh = 672 + 39
1392 crossing-over 1392 x 100 = 23.07%
6033

The greatest crossing-over percentage corresponds with the greatest distance and this
must be between the outer two loci. The order, therefore, is C-Sh – Wx. The sum of C-
Sh and Sh – Wx is 27.43 which is more than C-Wx estimated directly (25.51). The
difference is a result of double crossing-over.

Double crossing-over : Csh Wx = 19

cSh wx = 39
58 percentage 58 x 100 = 0.96 %
6033
The product of 23.07% and 4.36% = 1.01% is the expected double crossing-over
frequency. The difference is due to interference. The coincidence value C can be
calculated as 0.96 = 0.95 and the interference equals 1 – 0.95 = 0.05
1.01

The distance C-Wx can be estimated directly when double crossing-over is taken into
account, ie., the double crossing-over frequencies count twice:
Cwx = 672+107+2 x 19
cWx = 98=662=2 x 19
1655
1655 crossing-over ------- x 100 = 27.46%
6033

C Sh Wx

c sh wx

An example of a three-point-test in maize (compare table). The order of three genes c, sh

10
and wx can be given and the distance between them in per cent crossing over: this is the
beginning of a genetic map (after Sybenga, 1972).

Sex determination – chromosomal mechanism of sex determination and its types.

Genic balance theory of sex determination of Bridges.

Sex determination and Sex linkage:

Sex differentiation in living organisms into male and female causes
morphological, physiological and behavioral differentiation between the two sexes and
this phenomenon is called sexual dimorphism. In a large number of species of animals
and a small number of species of plants, eggs and sperms are produced by different
individuals, viz., females and males respectively. In most of the dioecious organisms,
females and males differ visibly in chromosomal constitution. The precise form of the
chromosomal differences between the sexes is not the same in different organisms.
Four types of sex chromosome mechanism or heterogamesis have been recognised and
they are the following:

1. Sex chromosome mechanism

a) Heterogametic Male - XX-XO type
The chromosome theory of sex determination was put forward by McClung
(1902), an American zoologist, who observed that the male grasshopper possessed and
odd number of chromosomes in contrast to the female which possessed an even
number. The proof for this was furnished by Wilson and Stevens (1905) who
demonstrated in bugs that chromosome distribution followed a course exactly similar to
that of sex distribution.

In the squash bug, Protenor, the females have 14 chromosomes and the males
have only 13 chromosomes in their somatic cells. In the females, 7 bivalents are formed
during meiosis. All the eggs have, therefore, 7 chromosomes each. In the males, one
odd, unpaired chromosome and 6 bivalents are seen during meiosis. The unpaired
chromosome passes undivided into one of the two daughter cells. Two kinds of sperms
in equal numbers are, therefore, formed, one kind with 7 chromosomes each and the
other kind with chromosomes each. An egg with 7 chromosomes fertilised by a sperm
with 7 chromosomes produces a female with 14 chromosomes and an egg with 7
chromosomes fertilised by a sperm with 6 chromosomes produces a male with 13
chromosomes. The odd chromosome of the male thus determines the sex and hence
called the sex-determiner or the sex chromosome or the ‘X’ chromosome. The other
chromosomes which are alike in females and males are called autosomes.

In grasshopers and bugs, the female is thus XX and the male is XO (using O to
indicate the absence of the X chromosome). Among plants, Dioscorea sinuata and
Vallisnaria spiralis are examples where the female is XX and the male XO.

XX-XY type
In many animals and plants, females and males have the same even number of
chromosomes, but, whereas in the females, the members of each pair of chromosomes
11
are alike, in the males the members of one pair of chromosomes are dissimilar in size or
form.

In Drosophila melanogaster the female has four pairs of chromosomes as follows:

(1) a pair of rod-shaped chromosomes, (2) a pair of V-shaped chromosomes, (3) a pair of
slightly longer V-shaped chromosomes, and (4) a pair of very short dot-like
chromosomes.

In the male Drosophila, there is only one rod shaped chromosome, the other
member of this pair being inverted J-shaped (i.e., hook-shaped, or like a rod with a bent
end). Wilson, who discovered this type of chromosome arrangement in 1905,
designated the unlike member of this pair in the male as the ‘Y’ chromosome and the
other member which is like the members of one pair in the female as the ‘X
chromosome.

All the eggs have one X chromosome and 3 autosomes each. Sperms are,
however, of tow kinds; one kind with one X chromosome and 3 autosomes each, and
the other kind with one Y chromosome and 3 autosomes each. Any egg fertilised by an
X- containing sperm produces a female and any egg fertilised by a Y-containing sperm
produces a male.

This type of sex determination in which the female has two X chromosomes and
the male one X and the Y chromosome is very widespread, being found in many
invertebrates including insects, in some fishes, in mammals including man and in many
dioecious plants like Melandrium album, M. rubrum, Humulus lupulus, rumex
angiocarpa, Salix, Smilax, Cannabis and Populus.

In human beings, 4 chromosomes are present in the somatic cells. Females have
22 pairs of autosomes and two X chromosomes. Males have 22 pairs of autosomes and
one X chromosome and one very short Y chromosome, considerably smaller than the X
chromosome. Each egg carriers 22 autosomes and an X chromosome. Sperms, however,
are of two kinds, one kind with 22 autosomes and a Y. The sex of a child is determined
at the time of fertilisation by the kind of sperm that happens to meet and penetrate the
egg, an X-bearing sperm producing a girl and a Y-bearing one, a boy.

b) Heterogametic Female – ZO-ZZ type

In all the above instances, the female is the homogametic sex because it
produces eggs, all of which are alike and the male is the heterogametic sex because it
produces two kinds of sperms. But there are instances where the female is the
heterogametic sex and the male is the homogametic one.

In a moth, Talaeoporia, the females have 59 chromosomes and the males have
60 chromosomes in their somatic cells. The eggs are of two kinds, one kind with 29
chromosomes and the other kind with 30 chromosomes. All the sperms have 30
chromosomes each. On fertilisation, an egg with 29 chromosomes gives rise to a female
and an egg with 30 chromosomes gives rise to a male.
12
 To distinguish this from the Protenor type, the sex chromosome found in the
male is designated at ‘Z’. The female is thus ZO (using O to denote the absence of one Z
chromosome) and the male is ZZ.

ZW-ZZ type
In birds, including the domestic fowl, certain insects, fishes and reptiles, the
female has an unlike pair of chromosomes, ZW, and forms eggs of two sorts, one with a
‘W’ chromosome and the other with a ‘Z’ chromosome. The male has like pairs of
chromosomes. On fertilisation, an egg with a W chromosome gives rise to a female and
an egg with a Z chromosome gives rise to a male.

Among plants, Fragaria elatior is one in which the female is ZW and the male is ZZ.

Balance theory of sex determination

A number of lines of evidence indicate that even in dioecious species, all

individuals have genes for both sexes. To quote Bridges, ‘Both sexes are due to the
simultaneous action of two opposed sets of genes, one set tending to produce the
characters called female and the other to produce the characters called male’. Which
sex actually develops is decided by the balance, i.e., by the preponderance of the
female- determining or of the male-determining genes. The sex chromosomes are
merely vehicles of genes which help in tilting the balance in one direction or another.

Support for the balance theory of sex determining comes from the work of
Bridges (1921) on Drosophila. Bridges observed some females of Drosophila
melanogaster with three X chromosome and three sets of autosomes (i.e., triploids).
When he crossed them with normal (diploid) males, he found that some of the progeny
and one or more chromosome less or more than the normal flies (i.e., aneuploids). His
results are given below:

X+A Y+A
2X + 2A 3X + 3A 2X + Y + 3A
Female Intersex
X+A 2X + 2A X + Y + 2A
Female Male
2X + A 3X + 2A 2X + Y + 2A
Superfemale Female
X + 2A 2X + 3A X + Y +3A
Intersex Supermale

Bridges found intersex, super females and supermales among the progeny.
Intersexes are sterile individuals intermediate between females and males but are
different from gynandromorphs which are typically female in certain portions of the
body and typically male in others. Superfemales and supermales are sterile individuals

13
which are very weak and very poor in viability.

Flies with two X chromosomes and two sets of autosomes are females but flies
with three X chromosomes and the same two sets of autosomes are superfemales.
2X + 2A Female
3X + 2A Superfemale

This shows that in Drosophla the X chromosomes carry genes that are
predominantly female-determining.

Flies with one X, one Y and two sets of autosomes are normal males but flies
with one X, one Y and three sets of autosomes are supermales.
1X + 1Y + 2A Male
1X + 1Y + 3A Supermale

This shows that the autosomes carry genes that are predominantly male-
determining.

Flies with two X chromosomes and two sets of autosomes are females. So also,
flies with two X, one Y and two sets of autosomes are females.
2X +2A Female
2X + 1Y + 2A Female

That individuals with two X, one Y and two sets of autosomes are female in spite
of the presence of the Y chromosome shows that the Y chromosome plays no positive
role in sex determination. That the Y chromosome does not determine maleness is also
shown by the fact that flies with one X chromosome and two sets of autosome (i.e., XO
flies) are males in spite of the absence of the Y chromosome. They are, however, sterile
showing thereby that the Y chromosome contains male fertility genes necessary for the
production of fertile male.

Bridges interpreted these results as follows:

Sex in Drosophila melanogaster is determined by the X chromosomes as well as

by the autosomes, the ratio of the number of X chromosomes to the number of sets of
autosomes being the deciding factor. In a normal (diploid) male, the X / A value is 1.00,
there being two X chromosomes and two sets of autosomes, and in a normal (diploid)
male, the X /A value is 0.50, there being only one X chromosome and two sets of
autosomes. Intersexes have an X /A value lying between 0.50 and 1.00. superfemales
have an X / A value exceeding 1.00 while supermale have an X / A value less than 0.50.
the relationship of chromosomes to sex determination in Drosophila melanogaster is
shown in Table.

14
 Relationship of chromosomes to sex in Drosophila

Chromosome constitution X/A Sex

X Y A
3X 2A 1.50 Superfemale
3X 3A 1.00 Female (triploid)
2X 2A 1.00 Female (diploid)
2X 1Y 2A 1.00 Female (diploid)
2X 1Y 3A 0.67 Intersex
2X 3A 0.67 Intersex
1X 1Y 2A 0.50 Male
1X 1Y 3A 0.33 supermale

Haplodiploidy

In hymenopterous insect such as ants, bees, sawflies and wasps, the fertilized
eggs develop into diploid females and the unfertilized eggs into haploid males. Male
haploidy is otherwise termed as arrhenotokous parthenogenesis. Thus haplodiploidy
mechanism is found to operate in some organisms in determining sex.

Single gene effect

In certain other organisms such as Chlamydomonas, Neurospora, Yeast,

Asparagus, Drosophila and several fishes, sex determination is the result of single gene
effect.

The Y Chromosome

Once different types of sex chromosomes could be distinguished, further

investigations by Wilson, Stevens, Montgomery, and others showed a wide variety of
chromosomal sex-determining mechanisms. The example of Protenor was essentially
the simplest, because only the presence of absence of a single chromosome was the
determining factor, and this was consequently called the XO-XX type. The male
Protenor, forming two types of gametes, with and without the X chromosome, and
designated as the heterogametic sex. The female, producing gametes all of the same
type, was termed homogametic.

It was soon found that the single X chromosome of the male in many species
pairs in meiosis with another chromosome, called the Y. Although similar in appearance
to the X in some cases, the Y is usually morphologically distinct. In Drosophila
metanogaster, for example, the Y has a J shape, as compared to the rod-shaped X. the
XY-XX system thus results in the same number of chromosomes in both males and
females. According to this terminology, if we designate a haploid set of autosomes
collectively by the letter A, the male produces two types of sperm, X + A and Y + A, and

15
the female one type, X +
A. Fertilization may, therefore, be of two kinds:

(X + A) + (X + A) = XX + 2A = female
sperm egg

(Y + A) + (X + A) = XY + 2A = male
sperm egg

In Drosophila melanogaster there are three autosomes in each haploid set,

yielding a total diploid number of eight chromosomes in each sex.

Sex linked inheritance – Cris cross inheritance – Reciprocal difference –

Holandric genes – Sex influenced and Sex limited inheritance –
Sex determination in plants – Melandrium, papaya and maize.

Sex linkage:

Red Eyed white eyed

F1 Both sexes – Red eye

F2 Red eye : White eye

3:2

F2 Female – Red eyed

16
 F2 males - ½ red eye: ½ white eye

White eye x red eye

F1
Females – red eye
Males – white eye

1) F1 females & males were different

2) F1 flies of both sexes from a cross between red eyed female and white eyed male
were red eyed.
3) Colour of eyes located in x chromosome.

In a culture of a normal wild type of Drosophila melanogaster with red eyes,

Morgan found in 1909 a single male individual in which the eyes were white. From this,
Morgan established a true breeding strain of white-eyed flies.

Morgan crossed a red-eyed female with a white eyed male and found that all
the first generation flies of both sexes were red eyed. When these were bred together
and an F2 generation obtained, it was found that three-fourths of the flies had red eyes,
and one fourth had white eyes, indicating that red and white eyes, indicating that red
and white eye colours are due to an allelic pair of genes of which red is the dominant.
All the F2 females, however, were red-eyed but of the F2 males, half were red-eyed and
half were white-eyed.

A reciprocal cross was made between a white-eyed female and a red-eyed male.
It was found that among the F1 offspring, all the females were red-eyed and all the
males were white-eyed.
The results were quite unexpected firstly because the phenotypes of the F1
females and males were different and secondly because all the F 1 flies of both sexes
from a cross between a red eyed female and a white-eyed male were red-eyed.

The F1 females seem to agree with out expectations that the heterozygotes will
exhibit the dominant character, viz., red colour of the eye. It is the white-eyed F1 males
that do not agree with our expectations. They do not seem to have received the gene
for red eye from their father. They seem to have received only the gene for white eye
from their mother.

The female and male flies are similar in having two pairs of V-shaped
chromosomes and one pair of dot like chromosomes. Whereas the females have one
pair of rod-shaped chromosome, the males have only one rod-shaped chromosome,

17
the other member of the pair being hook-shaped. The different results from the
reciprocal crosses could be explained only on the assumption that the gene for colour
of the eyes is located on the X chromosome and that the Y chromosome has no gene
for colour of the eyes.

Cytological studies revealed that the F1 males were like the male parent in
having one rod-shaped and one hook-shaped chromosomes besides the three pairs of
autosomes. The sons must have received the hook-shaped chromosome only form their
father. If, therefore, the hook-shaped chromosome of the father carried a gene for red
colour of the eye, the sons must have been red-eyed. The sons, however, were white-
eyed and it was therefore assumed that the Y chromosome carries no gene for red
colour of the eye. The X chromosome of the sons could be received only from their
mother. The sons could be white-eyed only if the gene for white eye is located on the X
chromosome of the white- eyed mother.

The daughters were like the mother in having two rod-shaped chromosomes
besides the three pairs of autosomes. The daughters could not have received more
than one X chromosome from the mother. The other X chromosome of the daughters
must have been received from the father. The daughters could be red-eyed only if the
gene for red eyes is located on the X chromosome of the father.

Because a white-eyed female crossed with a red-eyed male produces red-eyed

females and white-eyed males, this method of inheritance is often referred to as criss-
cross inheritance.

When the F1 flies were mated together, the following results were obtained in the F 2.
Red-eyed female 1
White-eyed female 1
Red-eyed male 1
White-eyed male 1

The F2 consisted of red-eyed and white-eyed individuals in equal numbers in both sexes.

Morgan concluded that the gene for eye colour is located on the X chromosome
and that the Y chromosome carries no gene for eye colour.

Sutton and Boveri hypothesized that genes are borne on chromosomes (i.e., the
chromosome theory of heredity) but it was Thomas Hunt Morgan (1910) who first
associated a particular gene (i.e., the gene for eye colour) with a particular
chromosome (i.e., X chromosome) visible in microscopic preparations and showed that
the gene for eye colour follows exactly the transmission of the X chromosome.
Eighteen other genes in Drosophila melanogaster follow the same method of
inheritance as white colour of the eye which indicates that these genes are also carried
on the X chromosome. As the gene is located on the X chromosome, it is called an X-

18
linked gene. This pattern of inheritance is called sex linkage.

Just as the eye colour in Drosophila whose gene is present only one the X
chromosome and not on the Y chromosome, hence called X-linked, there are genes
located only on the Y chromosome and its allele absent in the X chromosomes. Such
genes are called Y-linked or holandric genes. The gene responsible for hypertrichosis
causing hairy pinna (earlobes) in human beings is a Y-linked gene.

There are certain homologous regions on the X and Y chromosomes in which

both the alleles of a gene may be present as in the case of bobbed bristles (b) and its
allele (b+) for normal bristles. Such genes present both in the X and Y chromosomes are
called XY-linked genes. Genes for colour blindness, Xerodermia pigmentosum
(Pigmentation on the skin), Retinitis pigmentosa (pigmentation on the eye retina),
Nephritis etc., in human beings are XY- linked.

Sex-influenced character

All genes which are carried by the chromosomes are said to be sex-linked. All
known sex-linked genes lead to phenotypes which have nothing to do with sex.

Sex-influenced characters are characters which may be expressed differently in

the two sexes even when their genotypes are identical. The mere influence of the sex
of the individual may be sufficient to alter the phenotypic expression of a gene. The
most common expression of sex influence is that dominance is reversed between the
sexes. Genes determining sex influenced characters are borne on autosomes.

A typical example of a sex-influenced character is the presence of horns in

sheep. Both sexes of Dorset sheep are always horned while both sexes of Suffolk sheep
are always hornless. If Dorset and Suffolk are crossed, the F1 females are hornless while
the F1 males are horned. On interbreeding the F1 sheep, and F2 is obtained in which the
females show a ratio of 3 hornless: 1 horned and the males show a ratio of 3 horned: 1
hornless. Presence of horns may therefore be said to be a recessive character in
females but a dominant character in males.

P Horned female x Hornless male

HH hh
F1 Hh
Female, hornless Male, horned

F2 1 HH : 2Hh : 1 hh
Female Horned Hornless Hornless
Male Horned Horned Hornless

Reciprocal crosses show no differences because the gene is carried by the

19
 autosome.
Baldness in human beings is a sex-influenced character which is recessive in
females and dominant in males.

Sex-limited character

Sex-limited genes are genes that are present in both sexes of sexually
reproducing species but are expressed in only one sex and remain 'turned off' in the
other. In other words, sex-limited genes cause the two sexes to show different traits or
phenotypes, despite having the same genotype.
Sex-limited inheritance is an extreme type of sex influence in which a particular
phenotype can be expressed only in one sex. As genes for sex-limited characters are
borne on autosomes, all genotypes should occur with identical frequencies in both
sexes but the physiological differences between the sexes are such that certain
genotypes can be expressed only in one sex. Unlike sex influenced characters in which
gene is dominant in one sex and recessive in the other, sex-limited characters are
controlled by genes which have no visible influence at all in one sex either as a
homozygote or as a heterozygote.

In the yellow clove butterfly, Colias, females may be either yellow or white but
males are always yellow. White colour in the females depends on a dominant gene W
so that females of genotype WW or Ww are white while ww are yellow. Males are
yellow irrespective of whether they are WW, Ww or ww. The genotypes of the males
can be determined by mating them with yellow females and observing the colour of the
female progeny. If all the female progeny are white the male parent in WW. If the
female progeny are in the proportion of 1 white: 1 yellow, the male parent is Ww. If all
the female progeny are yellow, the male parent is w. Thus white colour is a sex-limited
character found only in the females.

In domestic poultry, cock-feathering is a character limited to the male sex. Hen-

feathering is due to a dominant gene H and cock-feathering is due to its recessive allele
h, but females with genotype hh are hen-feathered. The genotypes and their
corresponding phenotypes are as follows :

genotype Phenotype
Female Male
HH Hen-feathered Hen-feathered
Hh ” ”
Hh ” cock-feathered

Removal of the ovaries in hens with genotype hh results in cock-feathering. This

indicates that the female sex hormone inhibits the production of cock-feathering in
hens with genotype hh.

20
Sex reversal

In several species of plants that are normally bisexual, suppression of the male
or female structures has been observed in nature. The androecium getting converted
into petels in ornamental plants or carpels as in carrot and cabbage or pistils as in
maize, papaya and primrose has been observed. When the stamens get converted into
rudimentary organs without the pollen sac and pollen, they are called staminodes and a
similar conversion of the pistil into nonfunctional rudimentary organ is called the
pistillode. The phenomenon in which there is suppression of one sex at the expense of
the other is called sex reversal. The sex reversals are mostly due to physiological and
biochemical alterations involving sex hormones.

In maize, rarely it is observed that the male inflorescence called tassel bears
seeds due to sex reversal. The recessive gene ‘ba’ is responsible for barren plants and
another recessive gene ‘ts’ is responsible for tassel seed. Sex reversal in maize is due to
the genetic constitution of the plants.

21
 CYTOPLASMIC INHERITANCE

Introduction

The inheritance of most of the characters of an individual is governed by nuclear

genes. But in some cases, the inheritance is governed by cytoplasmic factors or genes. When
the transmission of characters from parents to offspring’s is governed by cytoplasmic genes,
it is known as “cytoplasmic inheritance” or extranuclear/extrachromosomal inheritance or
non-Mendelian inheritance. Extrachromosomal inheritance is defined as non- Mendelian
inheritance, usually involving DNA in replicating cytoplasmic organelles such as
mitochondria and plastids. The first case of cytoplasmic inheritance was reported by Correns
in 1909 in Mirabilis jalapa(four o’ clock) and by Baur in Pelargonium zonale in 1908.

Inheritance of most of the characters in eukaryotic organisms shows the following

characteristic features.
1. The contributions by both male and female parents are equal so that the results from
reciprocal crosses are identical.

2. Segregation produces the characteristic 3:1, 1:2:1 ratio in the monohybrid cross and a
typical 9:3:3:1in dihybrid crosses of F2 generation. These features of inheritance were first
demonstrated by Mendel; consequently, such an inheritance pattern is referred to as
“Mendelian inheritance”. It is universally accepted that genes showing Mendelian
inheritance are located in the chromosomes of eukaryotic nuclei. Therefore, Mendelian
inheritance pattern is regarded as a sufficient evidence for a gene to be located in the
chromosomes, such genes are termed as nuclear genes or more commonly simply as genes.

But some characters in several organisms do not show Mendelian inheritance or they
show a non Mendelian inheritance pattern. Characters showing non Mendelian inheritance
may be grouped under three broad categories: (1) those related to cellular structures and
patterns, (2) those produced by intracellular parasites, symbionts and viruses (3) those
associated with DNA containing cell organelles viz., mitochondria and chloroplasts. In
addition to these cases of non Mendelian inheritance, some characters in several organisms
exhibit a Mendelian inheritance pattern but the development of these characters in an
individual is markedly affected by the genotype of the maternal parent of the concerned
individual; such cases are classified as maternal effects.
Definitions:

In case of cytoplasmic inheritance generally the character of only one of the two
parents (usually the female parent) is transmitted to the progeny. Such inheritance is also
referred as extra nuclear inheritance, extrachromosomal inheritance and maternal
inheritance.

Genes governing the traits showing cytoplasmic inheritance are located outside the
nucleus and in the cytoplasm; hence they are referred to as plasma genes, cytoplasmic genes,
cytogenes, extranuclear genes or extra chromosomal genes.

The sum total of all the genes present in the cytoplasm of a cell is known as Plasmon,
while all the genes present in a plastid constitute a plastron.

Characteristics of cytoplasmic inheritance

1. Reciprocal differences: Reciprocal crosses show marked differences for the characters
governed by plasmagenes. To conduct reciprocal crosses, a female from strain A is mated
with a male from strain B and a male from strain A is mated with female from strain B. If sex
linkage were ruled out, differences in results of reciprocal crosses would indicate that one
parent (usually maternal) is exerting greater influence than the other on a particular trait. In
most cases, plasmagenes from only one parent, generally the female parent is transmitted, this
phenomenon is known as uniparental inheritance.

2. Lack of Mendelian segregation: lack of Mendelian segregation and characteristic

Mendelian ratios that depend on chromosomal transmission in meiosis would suggest
extrachromosomal inheritance. In general, F2, F3 and the subsequent generations do not show
segregation for a cytoplasmically inherited trait. This is because the F1 individuals generally
receive plasma genes from one parent only.

3. Irregular segregation in biparental inheritance: In some cases, plasma genes from both
the parents are transmitted to the progeny, this is known as biparental inheritance.
Biparental inheritance gives rise to irregular segregation ratios in the F1 generations of higher
plants.

4. Somatic segregation: Plasmagenes generally show somatic segregation during mitosis, a

feature of rare occurrence in the case of nuclear genes.
5. Association with organelle DNA: Several plasmagenes have been shown to be associated
with cp-DNA or mt-DNA.

6. Nuclear transplantation: If nuclear transplantation reveals a trait to be governed by the

genotype of cytoplasm and not by that of nucleus, cytoplasmic inheritance of the trait is
strongly indicated. In nuclear transplantation, nucleus of a cell is removed and replaced by a
nucleus of another genotype from a different cell.

7. Transfer of nuclear genome through back crosses: The nucleus of a variety or species
may be transferred into the cytoplasm of another species or variety through repeated back
crossing with the former, which is used as the recurrent male parent. Lines produced in this
way are known as alloplasmic lines since they have nuclei and cytoplasms from two different
species. A comparison of the various characters of alloplasmic lines with those of the
corresponding euplasmic line (lines having nuclei and cytoplasms from the same species)
demonstrates cytoplasmic effects, if any on these traits.

8. Mutagenesis: Some mutagens eg: Ethidium bromide are highly specific mutagens for
plasmagenes while nuclear genes are not affected by them.Induction of mutation by such
agents in a gene indicates it to be a plasmagene.

9. Lack of chromosomal location: In many organism, extensive linkage maps of nuclear

genes are available. If a gene is shown to be located in one of these linkage groups, it cannot
be a plasmagene. Failure to demonstrate the location of a gene in one of the linkage groups of
an organism is indicative of its cytoplasmic location.

10. Lack of association with a parasite, symbiont or virus: In many cases, a

cytoplasmically inherited character is associated with a parasite, symbiont or virus present in
the cytoplasm of the organism. Such cases cannot be regarded as cases of cytoplasmic
inheritance. Only those cytoplasmically inherited characters which are not associated with
parasites, symbionts or viruses can be regarded as governed by plasmagenes.

The known cases of true cytoplasmic inheritance are concerned with either
chloroplast or mitochondrial traits and are usually associated with their DNA. Such cases are
therefore often referred to as organellar inheritance, plastid inheritance and mitochondrial
inheritance.

Cases of Cytoplasmic Inheritance

1. Plastid inheritance- Variegations in Mirabilis jalapa

The inheritance pattern of plastid characters due to plasma genes located in plastid is
known as plastid inheritance. Carl Correns (1908) observed a differences in the results of
reciprocal crosses and was the first to describe deviations from Mendelian heredity. Different
shades of colour from white (albino) to dark green in the leaves of some plants were
investigated. Correns showed in studies of four-o’clock, that inheritance of certain traits
entirely from the seed parent. Colour differences were related to cytoplasmic plastids, most
important of which are chloroplasts which carry chlorophyll.

Variegation refers to the presence of white or yellow spots of variable size on the
green background of leaves. Variegation may be produced by some environmental factors,
some nuclear genes and in some cases, plasma genes. In Mirabilis jalapa, some of the
branches may have normal green leaves, while in the same plant, some other branches may
have only pale green or white leaves and still others may have variegated leaves. (1) Flowers
on branches with normal green leaves produce seeds that grow into plants with normal green
leaves irrespective of whether they are pollinated by pollen from branches with normal green,
variegated or white leaves. (2) Flowers on branches with white leaves produce seeds that
grow into plants with white leaves irrespective of whether they are pollinated by pollen from
branches with normal green, variegated or white leaves. (3)Flowers on branches with
variegated leaves produce seeds that grow into plants with normal green, whiteand variegated
leaves (Fig 1). Clearly, the phenotype of the progeny is same as that of the female parent,
except when the female parent is variegated. These results can be explained by assuming that
the plasmagenes producing variegation are located in plastids.

Ovules, as well as somatic cells of mottled plants may carry both abnormal, nearly
colourless plastids and normal green chloroplasts in their cytoplasm. The variegated effect is
transmitted through maternal linegeneration after generations. Because the pollen of the four-
o’clock has little if any cytoplasm, its influence on variegation is negligible. A single plant
with green, white, and variegated branches or sectors may produce seed that perpetuates each
of the three types. Seeds borne on white branches contain only primordia for colourless
plastids, those on green branches only green, and those on variegated branches might contain
either colourlessor green chloroplast or a combination of both.

Conclusion:
 Variegation is thus a heredity character determined by stable, self-duplicating, extra
nuclear particles called plastids. Neither the nucleus of the female gamete nor the male
gamete is involved in the control of this type of heredity character.

Four o’clock plant

Fig 1: Plastid inheritance in Mirabilis jalapa (four o’clock) plant

2. Nuclear Cytoplasmic interaction - ‘iojap’ gene in maize

A classical example of interaction between nuclear and cytoplasm genotypes is that
between CMS and restorer genes. An unusual case of interaction between nuclear and
cytoplasmic genes is reported in maize. A type of variegation, called iojap, is produced by a
recessive nuclear gene ij; plants homozygous for this gene develop typical iojap variegation.
But once this variegation is produced by the nuclear gene ij, it shows a typical cytoplasmic
inheritance. The egg regularly contributes much more cytoplasm to the next generation than
does the sperm. It should therefore be expected that in cases of cytoplasmic inheritance,
differences between reciprocal crosses would result. Rhoades (1946) identified the ‘iojap’
gene (ijij) in maize located in chromosome VII controlling plastid inheritance in the plant.
The gene ‘Ij’ is responsible for the normal green colour of the plant.
 When normal green plants with IjIj are used as female and pollinated by pollen from
stripped with ijij, F1 plants are wholly green.

Green x stripped
IjIjijij
F1 Ijij Green
F23 green : 1 Iojap

When striped with ijij are pollinated by pollen from the normal green plants with IjIj
the F1 plants, all of which have the same genotype, Ijij are of 3 different phenotypes.

Stripped ijij X Green IjIj

F1IjijGreen, stripped or white

When plants with same genotype Ijij have different phenotype viz., normal green,
stripped or white, the differences can be attributed only the differences in plastids.

3. Cytoplasmic male sterility in Maize

In case of male sterility in maize, pollen grains of such male sterile are aborted. This
male sterility is transmitted only through the female and never by the pollen. When all of the
chromosomes of the male sterile line were replaced with chromosomes of normal plants, the
line still remained male sterile, showing thereby that male sterility in controlled by some
factors in the cytoplasm. It was later recognized that cytoplasmic male sterility in maize
results from alterations in the heredity units in the mitochondria (mitochondrial DNA) (Fig
2).
 Fig. 2 Cytoplasmic male sterility in Maize

4. Inheritance of Kappa particles in Paramecium

In Paramecium aurelia, two strains of individuals have been reported. One is called
as ‘Killer’ which secretes a toxic substance ‘paramecin’ and the other strain in known as ‘
sensitive’ and is killed if it comes in contact with the ‘paramecin’. In the cytoplasm of the
killer strain the kappa particles (cytoplasmic – DNA) are present and absent in sensitive
strains. The transmission of kappa particles is through cytoplasm but maintenance of kappa
particles and production of paramecin is controlled by dominant nuclear allele ‘K’.

We assume that the killer strains carry dominant allele ‘KK; and that sensitive ‘kk’.
When killers conjugate with sensitive under normal conditions and no cytoplasmic exchange
occurs. On conjugation, conjugants exchange their nuclear material so that ex-conjugants
resulted from conjugants ‘KK’ and ‘kk’ when conjugation is for normal time, then only
nuclear material is exchanged and therefore killer will produce killer daughters and sensitive
will produce sensitive daughters. But if the conjugation is for longer period, there will be
exchange of cytoplasm resulting in the inheritance of kappa particles by both the ex-
conjugants so that all the daughter paramecia produced are killers because all inherit the
kappa particles through the mixing of cytoplasm (Fig 3). Therefore, this trait is transmitted
through cytoplasmic heredity.
 Fig 3. Inheritance of kappa particles in Paramecium

5. Maternal Effects in Snail Shell coiling

The development of some characters in many organisms is either governed or

markedly influenced by the genotype of the female parent; this is known as maternal effect.
Although, such traits are governed by nuclear genes, they still show the following features:
(1) reciprocal differences in F1 (2) which, in most cases disappears in F2; and (3) a
considerably smaller variation in F2 as compared to that in F3. In some extreme cases, there
may be no phenotypic segregation in F2, but it does become evident only in the F3.

An extreme example of maternal effect is known in the snail Limnaea. In this snail,
the direction of coiling of its shell is controlled by a single nuclear gene D/d; the dominant
allele D produces right-handed coiling, while its recessive allele d produces left-handed
coiling. The direction of shell coiling in an individual is governed by the genotype of its
female parent and not by its own genotype.

1. Crosses between females with left handed coil (dd) and males with right-handed coil
(DD) produce F1 progeny (Dd) with left handed coil, since the genotype of the female
parent was dd. In F2, segregation of Dd produces three genotypes (DD, Dd, dd) in the
ratio 1:2:1. But all F2 snails exhibit right handed coiling since their female parents had
the genotype Dd, which directs right-handed coiling in the progeny. The F3 progeny
 from the F2 individuals with genotypes Dd and Ddill show right-handed coiling, while
those from dd F2 individuals will exhibit left -handed coiling; this produces the typical
3:1 in F3.
2. The reciprocal cross (female- DD X male-dd), on the other hand, produces right
handed coiling in the F1 (Dd) as well as in the F2. But in F3, a 3:1 ratio is obtained as
was the case in previous cross (Fig 4).
Thus inheritance of coiling of Limnaea shells shows
1. Reciprocal differences
2. No segregation in F2
3. Appearance of typical 3:1 ratio in F3

Fig. 4: Maternal effect on the direction of shell coiling in Limnaeasnail

Table: Differences between Mendelian and cytoplasmic inheritance

Sl. No. Mendelian Inheritance Cytoplasmic Inheritance
1 Governed by nuclear genes Governed by plasma genes
2 Exhibits distinct segregation pattern Does not exhibit distinct segregation
pattern
3 Reciprocal differences are not Reciprocal differences are observed
observed
4 Does not show maternal effects Show maternal effects
5 Genes can be easily mapped on Genes cannot be mapped on
chromosomes chromosomes
6 Nuclear genes are associated with Genes are associated with chloroplast
chromosomes DNA or mitochondrial DNA
 QUANTITATIVE TRAITS
Introduction:
Character or trait refers to any property of an individual showing heritable variation. It
may be morphological, physiological, biochemical and behavioural properties. The characters
which are governed by one or few genes are called qualitative or oligogenic traits. On the
other hand, some characters are governed by many genes and called as quantitative traits or
polygenic traits. The mode of inheritance of polygenic characters is termed as polygenic
inheritance or quantitative inheritance. Since in polygenic inheritance several genes (factors)
are involved, it is also known as multiple factor inheritance.

The phenotypic traits of the different organisms may be of two kinds, viz., qualitative
and quantitative. The qualitative traits are the classical Mendelian traits of kinds such as form
(e.g., round or wrinkle seeds of pea); structure (e.g., horned or hornless condition in cattle’s);
pigments (e.g., black or white coat of guinea pigs); and antigens and antibodies (e.g., blood
group types of man) and so on. Each qualitative trait may be under genetic control of two or
many alleles of a single gene with little or no environmental modifications to obscure the
gene effects. The organisms possessing qualitative traits have distinct (separate) phenotypic
classes and are said to exhibit discontinuous variations. The quantitative traits, however, are
economically important measurable phenotypic traits of degree such as height, weight, skin
pigmentation, susceptibility to pathological diseases or intelligence in man; amount of
flowers, fruits, seeds, milk, meat or egg produced by plants or animals, etc. The quantitative
traits are also called “metric traits” as they are subjected to measurements. They do not
show clear cut differences between individuals and forms a spectrum of phenotypes which
blend imperceptively from one type to another to cause continuous variations. In contrast to
qualitative traits, the quantitative traits may be modified variously by the environmental
conditions and are usually governed by many factors or genes (perhaps 10 or I00 or more),
each contributing such a small amount of phenotype that their individual effects cannot be
detected by Mendelian methods but by only statistical methods. Such genes which are non-
allelic and effect the phenotype of a single quantitative trait, are called “polygenes or
cumulative genes”. The inheritance of polygenes or quantitative traits is called quantitative
inheritance, multiple factor inheritance, multiple gene inheritance or polygenic inheritance.
Characteristic Features of Quantitative/Polygenic traits

The term polygene was coined by Mather in 1941. The important features of polygenic traits
are listed below.

1. Each polygenic character is controlled by many genes and each gene has small and
cumulative/additive effect on the expression of the trait.
2. These traits exhibit continuous variation. Hence they cannot be classified into clear
cut groups.
3. Effect of individual gene on the expression of the trait is not easily detectable in case
of polygenic characters and, therefore, such traits are also called as minor gene
characters.
4. The statistical analysis of polygenic variation is based on means, variances and co-
variances.
5. The traits are highly influenced by the environmental factors.
6. Generally, the expression of polygenic traits is governed by additive gene action, but
now cases are known where polygenic traits are governed by dominance and epistatic
gene action.
7. In case of pyogenic characters, metric measurements like weight, height, duration etc.
are possible.
8. Transgressive segregants are only possible for polygenic traits, the crosses between
two parents with mean values for polygenic characters produce transgressive
segregants.
9. The transmission of polygenic characters is generally low because of high amount of
environmental variation and influence.
10. The polygenes have pleiotropic effects; that is, one gene may modify or suppress
more than one phenotypic trait. A single allele may do only one thing chemically but
may ultimately affect many characters.

Qualitative traits

The easiest characters, or traits, to deal with are those involving discontinuous, or
qualitative, differences that are governed by one or a few major genes. Many such inherited
differences exist, and they frequently have profound effects on plant value and utilization.
Examples are starchy versus sugary kernels (characteristic of field and sweet corn,
respectively) and determinant versus indeterminant habit of growth in green beans. Such
differences can be seen easily and evaluated quickly, and the expression of the traits remains
the same regardless of the environment in which the plant grows. Traits of this type are
termed highly heritable. A qualitative trait is expressed qualitatively, which means that the
phenotype falls into different categories. These categories do not necessarily have a certain
order. The pattern of inheritance for a qualitative trait is typically monogenetic, which means
that the trait is only influenced by a single gene. The environment has very little influence on
the phenotype of these traits (Table 1).

Table 1: Differences between quantitative and qualitative traits

Sl. Qualitative traits Quantitative traits

No.
1 Each qualitative trait is governed by two Each quantitative trait is governed by
or many alleles of a single/few genes. many non-allelic genes or polygenes.
2 It deals with the inheritance of traits of It deals with the inheritance of traits of
kind, viz., form, structure, colour, etc. degree, viz., height, weight, number, etc.
3 Effect of each gene is detectable Effect of each gene is not detectable
4 Generally governed by non-additive Governed by additive genes
genes
5 The phenotypic expression of a gene is The phenotypic expression of polygenes
not influenced by environment. is highly influenced by environment
6 These traits can be classified into discrete
These traits can’t be classified distinct
phenotypic classes displaying
phenotypic classes and produce a
discontinuous variations spectrum of phenotypic classes exhibiting
continuous variations.
7 It concerns with individual matings and It concerns with a population of
their progeny organisms consisting of all possible kinds
of matings
8 Statistical analysis based on frequencies Statistical analysis is based on mean,
or ratios variances and covariances

Similarities between qualitative and quantitative traits

1. Both qualitative and quantitative characters are governed by genes. The former is
controlled by major or oligogenes and the later by minor or polygenes.
2. Both major and minor genes located on the chromosome.
3. Thepolygenes controlling the continuous variation exhibit segregation like major genes
controlling discontinuous variation.
4. Polygenes mutate like oligogenes.
5. Polygenes exhibit linkage like oligogenes.
Multiple Factor Hypothesis –Nilsson-Ehle (1908)

The multiple factor hypothesis was originally proposed by Yule in 1906. But the
experimental evidence for the existence of multiple factors was provided by Nilsson-Ehle in
1908; therefore, he is credited with the concept of multiple factor inheritance.

In studies on the inheritance of seed colour in wheat Nilsson-Ehle obtained 3:1, 15:1
and 63:1 ratio between coloured and white seeds from different crosses. It is clear from these
ratios that in these crosses the seed colour was governed by one (3:1 ratio in F2), two (15:1
ration in F2) and three(63:1 ration in F2) genes exhibiting duplicate interaction. However, on
close examination of the coloured seeds, Nilsson-Ehle found that there were marked
differences in the intensity of their colours. Therefore, the red seeds from the crosses showing
15:1 ratio turned out to be a 1:4:6:4:1 ratio on a closer examination. In order to explain the
1:4:6:4:1 ratio, Nilsson-Ehle made the following assumptions..

1. In crosses showing 15:1 ratio in F2, seed colour is governed by two genes.
2. One of the allele of each colour gene produces seed colour and is called positive
allele and denoted by capital letter, e.g., R1, R2, etc. The other allele of each colour
gene does not produce any colour and called negative allele denoted by
corresponding small letter, e.g., r1, r2 etc.
3. These genes do not show dominance so that the heterozygote for a colour gene, e.g.,
R1r1 is intermediate in colour between two homozygotes (R1R1 and r1r1). It may be
stated that two positive alleles of a colour gene (R1R1) produce twice the intensity of
red colour as that produced by a single positive allele (R1r1).
4. Each of the positive alleles have small, equal or almost equal and additive effect on
seed colour.
5. The effects of positive alleles of different colour genes are additive in the same
manner as those of the two positive alleles of single colour gene. Thus the intensity of
seed colour depends on the number of positive alleles of all the genes affecting the
seed colour.

According to this hypothesis, inheritance of seed colour in crosses exhibiting

1:4:6:4:1 ratio in the F2 may be explained as follows.
 The seed colour in such crosses is governed by two genes, say, R1 and R2. If a wheat
genotype having dark red seeds R1R1R2R2 is crossed with white seeded (r1r1 r2r2) genotype,
the F1 (R1r1R2r2) will have medium red seeds since it has two positive and two negative
alleles. In F2, one zygote will have four positive alleles (R1R1R2R2); such seeds will be dark
red in colour. In four seeds, three positive alleles will be present (R1r1R2R2and R1R1R2r2);
these seeds will be medium dark red. Six seeds will have two positive alleles with medium
red colour. In four other seeds one positive allele having light red colour. In the remaining
one seed zero positive alleles or four negative alleles (r1r1r2r2) thus seed colour will be white.
Thus the multiple factor hypothesis is able to explain the 1:4:6:4:1 ratio for seed colour in the
F2 generation of wheat crosses (Table 2& 3).Thus, a character is governed by several genes;
the positive alleles of all the genes governing the trait are similar to each other in their action
and their effects are additive or cumulative in nature. This is the essence of multiple factor
hypothesis.

Parents R1R1R2R2 Xr1r1r2r2

Dark red White
R1R2 r1r2

F1R1r1R2r2
Medium red

Table 2: Inheritance of seed colour in wheat

Female/Male R1 R2 R1r2 r1R2 r1r1
R1R2 R1R1R2R2 R1R1R2 r2 R1r1R2R2 R1r1R2r2
Dark Red Medium Dark Medium Dark Medium Red
Red Red
R1r2 R1R1R2r2 R1R1r2r2 R1r1R2r2 R1r1r2r2
Medium Dark Medium Red Medium Red Light Red
Red
r1R2 R1r1R2R2 R1r1R2r2 r1r1R2R2 r1r1R2r2
Medium Dark Medium Red Medium Red Light Red
Red
r1r1 R1r1R2r2 R1r1R2r2 r1r1R2r2 r1r1r2r2
Medium Red Light Red Light Red White

Table 3: Genotype &phenotype frequencies produced by segregation of two genes (R1&

R2) with additive effect on seed colour in wheat.
Genotype Frequency No.of positive alleles Phenotype Frequency
R1R1R2R2 1 4 Dark red 1
 R1R1R2r2 2 3 Medium Dark
red 4
R1r1R2R2 2 3 Medium Dark
red
R1r1R2r2 4 2 Medium red
R1R1r2r2 1 2 Medium red 6
r1r1R2R2 1 2 Medium red
R1r1r2r2 2 1 Light red
r1r1R2r2 2 1 Light red 4
r1r1r2r2 1 0 White 1
Examples of quantitative traits:

1. Skin Colour in Man: Another classical example of polygenic inheritance was given by
Davenport (1913) in Jamaica. He found that two pairs of genes, A-a and B-b cause the
difference in skin pigmentation between Negro and Caucasian people. These genes were
found to affect the character in additive fashion. Thus, a true Negro has four dominant genes,
AABB, and a white has four recessive genes aabb. The F1 offspring of mating of aabb with
AABB, are all AaBb and have an intermediate skin colour termed mulatto. A mating of two
such mulattoes produces a wide variety of skin colour in the offspring, ranging from skins as
dark as the original Negro parent to as white as the original white parent in F2 generation.

2.Height in Man: Skin colour in man is a rather simple example of polygenic inheritance
because only two pairs of genes are involved. The inheritance of height in man is a more
complex phenomenon involving perhaps ten or more pairs of genes. The character of tallness
is recessive to shortness, thus, an individual having the genotype of more dominant genes will
have the phenotype of shortness. Because, this quantitative trait is controlled by multiple
pairs of genes and is variously influenced by a variety of environmental conditions.

Transgressive Segregation:

Theappearance in F2 of individuals with a higher or lower intensity of a character than

those present in the parents involved in the cross or it is the phenomenon of appearance of
extreme phenotypes in F2 derived by crossing the two parents having intermediate phenotype
is termed as transgressive segregation, and such individuals are called transgressive
segregants.

Transgressivesegregants are produced when (1) the two parents involved in a cross
have intermediate phenotype (2) they have positive alleles of different genes affecting the
concerned quantitative trait. Segregation for these genes produces the two extreme
phenotypes (homozygotes) in F2, which transgress the parental limits for the character.
Example: Seed colour in wheat

If the parents having medium red seeds are crossed (R1R1r2r2 X r1r1R2R2) in F1 we get
medium red phenotype (R1r1R2r2). The segregation of these in F2 produce phenotype with
dark red and white. Clearly, these phenotypes, are either more or less intense than the seeds
colours of the two parents of F1, they are transgressive segregants.
 1. GENETIC MATERIAL - EXPERIMENTS

Introduction

Mendel in 1865 postulated that the development of characters in peas/organisms is

governed by particulate factors later these factors are called as genes.Sutton & Boveri in
1902, postulated that genes were located in chromosomesand proposed Chromosomal Theory
of Inheritance, today this is universally accepted. The chemical nature of genes became
evident through series of brilliant experiments conducted between 1928 to 1952.

Properties of genetic material:

Genetic material is the chemical of which genes are made up of. The chemical of which
genes are composed must possess the following properties.

1. Replication: The genetic material must be replicated with high fidelity so that
flawless copies are produced.
2. Distribution: The replicated copies must be distributed to the daughter cells with a
very high degree of precision.
3. Expression: The genetic material must be able to express itself by exercising a
control on the development of the character it governs.
4. Structure: The structure of genetic material must be able to store highly variable
information necessary for gene function.
5. Creation of variation: Replication/distribution of genetic material must allow errors
to occur in low frequency for the origin of new genetic variation by mutation.

Identification of Genetic Material:

In Eukaryotes, chromosomes are made up of NA & Proteins.Nucleic acids (DNA

&RNA) were discovered by Meischer in 1871, and called Nuclein.The term Nucleic acid was
coined by Altman in 1889. In eukaryotes the genes are located on chromosomes. Therefore, it
is certain that genes would be composed of DNA, RNA or protein. There was a prolonged
controversy as to which of these three is the genetic material.The process of identification of
genetic material began in 1928 with the experiments of Griffith and concluded in 1952 with
the studies of Hershey and Chase.Another ingenious experiment by Frankel- Conrat and
Singer in 1956 established that in some viruses RNA functions as genetic material.
 1.Frederick Griffith’sTransformation Experiment -1928

Griffith discovered the phenomenon of transformation by his studies on Streptococcus

pneumoniae ( Diplococcus pneumoniae ) which causes pneumonia in most of the
mammals.Different strains of S. pneumoniae produce either rough or smooth colonies when
they are cultured on a media in petriplate. The cells of strains producing smooth colonies on
media in a petriplate enclosed by polysaccharide capsule and are able to cause disease so
called virulent strains.The cells of strains producing rough colonies on media in a petriplate
are not enclosed by polysaccharide capsule and are not able to cause disease so called
avirulent strains. The virulent strain is further classified into several types e.g. II, III etc.,
based on the antigenic properties of their capsules.

To conduct the experiment Griffith used IIR & IIIS type

• The mice injected with III-S strain died but when injected with IIR strain lived.

• Then the mice were injected with heat killed IIIS no disease & all mice survived.

• Lastly when mice were injected with mixture of heat killed IIIS strain and live IIR
strain some mice died due to disease.Streptococcus cells isolated from dead mice
were of the type IIIS. Since all the cells of the heat killed IIIS type were dead, it was
postulated that some of the cells of IIR have changed into IIIS type due to the
influence of dead IIIS cells present in the mixture (Fig 1). This phenomenonwas
called transformation and the component of IIIS cells which induced conversion of
IIR to IIIS was called as transforming principle.

Transformation: It is a hereditary change in the traits of a cell/organism brought

about by an integration through recombination of a segment of DNA molecule into
the chromosome of that cell/organism.

Conclusion:

• He concluded that heat killed III-S bacteria somehow converted live avirulent IIR
cells into virulent IIIS cells which is known as phenomenon of transformation.

• The component of IIIS cells which induced conversion of IIR to IIIS was called as
transforming principle
 • This experiment of Griffith demonstrated transformation but didn’t give any hint at
the identity of transforming principle.

Fig 1. Experiments of Griffith usingStreptococcus

using pneumoniaein mice.
 2. In vitro Experiments ofAvery, MacLeod and McCarty-1944

Avery and his associates carried out the experiments of Griffith in vitro in place of
mice. The details are as follows.

1. Culture of IIR cells produced typical rough colonies on a media in petriplate.

2. Plating of heat killed IIIS or DNA isolated from IIIS cells did not produce colonies.

3. IIR cells when either mixed with heat killed IIIS or with DNA isolated from IIIS cells
and the mixture was plated on a culture medium containing antibodies specific for IIR
cells (anti-IIR) some IIIS colonies were obtained.

4. Anti-IIR was used for inactivating IIR cells so that the few transformed cells of IIIS
type may not be overrun/masked by IIR cells.

5. These findings clearly indicated that DNA is the transforming principle.But as DNA
extract contain traces of protein and RNA. So it is not clear whether
DNA/RNA/Protein is transforming Principle (Fig 2).

Further continued their Experiment….

• The extracts from heat treated IIIS cells were first treated with enzymes that
specifically destroyed protein (protease), RNA (Rnase) & DNA (Dnase)

• After the enzyme treatment, the treated extracts were mixed with live IIR cells.

• Encapsulated IIIS cells appeared in all of the cultures except those in which IIIS strain
extract had been treated with DNase, an enzyme that destroys DNA.

• These results suggested that DNA was the molecule responsible for transformation.
Fig 2. In vitroExperiments of Avery, MacLeod and McCarty

Conclusion:

• The findings of Oswald Avery, Colin Macleod and Maclyn McCarty’s presented
incontrovertible evidence for DNA to be the transforming principle. But in spite of
this, DNA could not be universally accepted as the genetic material as the underlying
molecular mechanism was not well understood.
 3. Experiments of Hershey & Chase -1952

Hershey & Chase studied the life cycle of T2 phage of E. coli; they clearly showed
that only the DNA component of T2 particles is transmitted to the progeny phage particles. T2
and most other Bacteriophages have hexagonal head & Tail. These phages are composed of
protein & DNA. Head coat and tail made up of protein while DNA is packed inside the head
coat. Viruses are obligate parasites so need living host to reproduce.

Bacteriopahges infect E. coli by coming in contact with the cell wall of bacteria
through their tail plate. The tail sheath contracts pushing part of the tail core through the
bacterial cell wall, which is already weakened by certain hydrolytic enzymes present in the
phage tail core.

Hershey and Chase labelled T2 DNA and Protein in two different experiments.DNA
contains phosphorus (P) but not sulphur (S), while protein contains S but no P.So labelled T 2
DNA with 32P, and proteins with 35S. In order to get 32P /35S labelled phage particles, E. coli
cells were grown for several generations on a culture medium containing32P /35S.These E.
coli cells were then infected with T2 phage.The progeny phage particles thus obtained were
labelled with either 32P /35S.

1. In the first experiment they used 32P labelled T2 phage to infect E. coli cells (grown on
normal media).Infection allowed for 10 minutes.The cells were then agitated in a
blender to separate the empty phage particles (called ghosts) remaining outside the
bacterial cells after infection. The bacterial cells were pelleted out through
centrifugation, separating them from the ghost and active phage particles. It was
32
observed that most of the P label/radioactivity was present in the infected E. coli
32
cells and ghosts and active phage particles contained very less fraction of total P
label. The progeny phage particles obtained after lysis of E. coli cells also contained
the 32P label. This clearly indicated that the 32P present in the phage DNA is injected
into E. coli cells and finally transmitted to progeny phages, and that DNA is
transmitted from one generation to the next.
2. In the second experiment they used 35S labelled T2 phage to infect E. coli cells (grown
on normal media).Infection allowed for 10 minutes. The cells were then agitated in a
blender to separate the empty phage particles (called ghosts) remaining outside the
bacterial cells after infection. The bacterial cells were pelleted out through
centrifugation, separating them from the ghost and active phage particles. It was
 35
observed that most of the S label/radioactivity was present in the ghosts and active
phage particles, and only a small fraction of it was present in the infected E. coli cells.
35
The progeny phage particles obtained after lysis contained negligible fraction of S
label (Fig 3). Clearly it is shown that almost all the proteins are left outside theE. coli
cells during the infection and that proteins are not transmitted from one phage
generation to the next.Hence in T2 phage DNA was proved to be genetic material.

Fig. 3. Experiments of Hershey & Chase using T2 phage and E. coli

Conclusion:

The experiment of Hershey & Chase clearly indicated that DNA of T2 phage is
transmitted from one generation to the next, while proteins are not transmitted. Hence DNA
is the Genetic material.The findings of Hershey & Chase led to the universal acceptance of
DNA as the genetic material.
 3. RNA as Genetic Material

In many viruses e.g., TMV (Tobacco mosaic virus) DNA is absent; these viruses are
composed of RNA and protein. In 1956, Gierer and Schramm, and Frankel-Conrat and Singer
independently demonstrated that RNA functions as genetic material in TMV. The proteins
and RNA of TMV can be separated chemically; when they are remixed under appropriate
conditions, they reassociate to produce active TMV particles.

1. In one experiment RNA isolated from TMV is used for infection on tobacco leaves.
Mosaic symptoms developed. In other experiment protein of TMV was used for
infection but no symptoms observed. Clearly, indicated that RNA fraction of TMV is
capable of producing the disease, and hence it appears to be the genetic material.
2. In another experiment, they produced hybrid TMV particles using proteins and RNA
from two different strains of virus. Strain A of TMV was isolated from tobacco, while
strain B Holmes rib grass virus was isolated from Plantago lanceolata. Strain Aand B
produce distinct symptoms on a certain tobacco variety.
3. Frankel-Conrat and Singer developed two types of hybrid virus particles by mixing
(1) RNA from Strain A and proteins from strain B (2) RNA from Strain B and
proteins from strain A. these hybrids were used for infection, when hybrid of first type
was used for infection, the disease symptoms of Strain A (TMV) developed, and the
virus particles isolated from the diseased leaves had proteins identical to strain A.
Similarly, when hybrid particles of second type was used for infection the disease
symptoms of Strain B (HRV) developed, and the virus particles isolated from the
diseased leaves had proteins identical to strain B (Fig 4).

Conclusion:

It is evident that from these findings that only RNA of TMV has the capacity to
produce disease, and that type of proteins present in the virus particles is determined by the
RNA. Clearly, RNA is the genetic material in TMV.
Fig 4. Experiments of Frankel-Conrat and Singer on tobacco mosaic virus (TMV)
 2. GENETIC MATERIAL- DNA STRUCTURE

Introduction

There are two types of nucleic acids, viz., deoxyribose nucleic acid (DNA) and ribose
nucleic acid (RNA). Nucleic acids are found in the cells of all living organisms. The DNA is
mainly found in the chromosomes in the nucleus, while RNA is mostly found in the
ribosomes in the cytoplasm. In most of the organisms, DNA is the genetic material. However,
in some virusesRNA is the genetic material.

The Composition of Nucleic Acids

The remarkable properties of the nucleic acids, which qualify these substances to
serve as the carriers of genetic information, have claimed the attention of many investigators.
The groundwork was laid by pioneer biochemists who found that nucleic acids are long
chainlike molecules called polymers. The monomer units of nucleic acids (NA) are
nucleotides, and the polymer is known as a "polynucleotide". Each nucleotide consists of
three types of molecules: (1)5-carbon sugar (pentose) (2) a cyclic nitrogen-containing
compound called bases (3) phosphate group(Fig 1).

Phosphate
Nitrogenous
Base

Pentose
Sugar

Fig 1. Composition of a Nucleotide

1. Pentose Sugar: The pentose sugar present in DNA is “2-deoxyribose’’ (thus the
name deoxyribonucleic acid). The oxygen atom present at the second carbon of ribose
is missing in deoxyribose.The sugar in RNA is “ribose” (thus ribonucleic acid). The
carbon atoms are numbered 1', 2', 3', 4', and 5' to distinguish from the numbering of
the atoms of the purine and pyrimidine rings. The hydroxyl groups on the 5'- and 3'-
carbons of the pentose sugar link to the phosphate groups to form the phosphodiester
 linkage forming the DNA backbone, while the1'carbon is always occupied by one of
the four organic bases.
2. Organic (Nitrogen) bases: The organic bases present in the NA are heterocyclic
compounds containing nitrogen in their rings, hence they are also called nitrogenous
bases. DNAcontains four different bases called, adenine (A), guanine (G), cytosine
(C) and thymine (T). RNA also generally contains adenine, guanine, cytosine but has
uracil (U), in place of thymine. These five bases (A, G, C, T, U) are grouped into two
classes (1) purine (A, G) and (2) pyrimidine (C, T, U).
1. Purine bases (A, G): these, bases are six-membered and five membered double ring
structures. In adenine (-NH2) group is present at position 6, but in guanine a keto (=O)
group is found at position 6 and an -NH2 group is attached at position 2. The N
present at the 9th position of purines participates in linkage with the 1'C of the
pentose.
2. Pyrimidine bases (C, T, U): these bases are six-membered single ring structures,
which is similar to a benzene ring. The pyrimidine bases contain a keto oxygen(=O) at
position 2. In cytosine, an amino (-NH2) group is also present at position 4; in uracil, a
keto (=O) group is present at the fourth carbon, while thymine is essentially 5-methyl
uracil. All the pyrimidines, therefore, contain an -H atom at position 1', which is
involved in their linkage with the 1'C of pentose(Fig 2& Table 2).

It may be pointed out that both the purines (A, G) are present in both DNA and RNA,
while only one of the pyrimidine (C) is normally present in the both the NA. Of the
remaining two pyrimidines, T is normally found in DNA, while U is found in RNA. These
bases occur in keto and enol tautomeric forms. But the nucleic acid bases are primarily in
their keto tautomeric form.

Nucleosides: In nucleic acids, organic bases are linkedwiththe pentose molecules forming
nucleosides. Linkage between ribose sugar and the four different bases produce
ribonucleosides, while that between deoxyribose sugar and the four different bases produce
deoxyribonucleosides; both ribonucleosides and deoxyribonucleosides together are called
nucleosides (Table 1).

Nucleotides: A nucleotide is formed when a phosphate group is attached to either the 3' C or
the 5'C of the pentose residue of a nucleoside. The nucleotides formed by four different
ribonucleosides are known as ribonucleotides or ribotides and that of DNA formed by four
different deoxyribonucleosides are called deoxyribonucleotides or deoxyribotides (Table 1).
The nucleotides participating in DNA replication have the phosphate groups are ordinarily
attached to the 5'C.

Table 1. The various bases, nucleosides and nucleotides commonly found in DNA &
RNA

Base Deoxyriboside Deoxyribotide Rriboside Ribotide (base

(base + (base + (base + ribose + ribose sugar
deoxyribose deoxyribose sugar sugar) + 5'phosphate)
sugar) + 5' phosphate)
Adenine Deoxyadenosine Deoxyadenylic acid Adenosine Adenylic acid
Guanine Deoxyguanosine Deoxyguanylic acid Guanosine Guanylic acid
Cytosine Deoxycytidine Deoxycytidylic Cytidine Cytidylic acid
acid
Thymine Thymidine Thymidylic acid -- --
Uracil -- -- Uridine Uridylic acid

3. Phosphate group: The phosphate group is a phosphorous atom with four oxygen
atoms bonded to it. A phosphate moiety joins 5' C of one and the 3'C of the
neighbouring pentose to produce the “phosphodiester linkage”.

Fig 2. The Purine and Pyrimidine bases

Table 2: Differences between Pyrimidines and purines

Pyrimidines Purines
These are single ring (six member) These are double ring (nine member)
compounds. compounds.
C,T,U. A,G
They occupy less space in DNA structure. They occupy more space in DNA structure.

Deoxyribose is linked at position 3 of Deoxyribose is linked at position 9 of purine.

pyrimidine.

The Watson and Crick DNA Double Helix

Thecorrect structure of DNA was first deduced by J. D. Watson and F. H. C. Crick in

1953. Their double –helix model of DNA structure was based on the two major evidence.

1. When the composition of DNA from many different organisms was analysed by E.
Chargaff and colleagues, it was observed that the concentration of thymine was
always equal to the concentration of adenine and the concentration of guanine was
always equal to the concentration of cytosine. This strongly suggested that thymine
and adenine as well as cytosine and guanine were present in DNA with some fixed
interrelationship and these relationships are generally called as Chargaff’s rule. The
total concentration of pyrimidines (T, C) always equal to the concentration of purines
(A, G). however, the (T + A)/(C + G) ratio was found to vary widely in DNAs of
different species.
2. The X-ray crystallographic data on DNA structure from the studies of Franklin,
Wilkins and their co-workers revealed that DNA was highly ordered, multi-stranded
structure with repeating subunits spaced every 3.4Åalong the axis of the molecule.

On the basis of Chargaff’s chemical data, Wilkins and Franklin’s X-ray diffraction
data, Watson and Crick proposed double-helix model of DNA, which soon became
universally accepted.

The main features of the Watson and Crick double-helix model of DNA

1. A DNA molecule is made up of two polynucleotide strands or chains wound around

each other.
2. Each polynucleotide strand consists of sequence of nucleotides linked together by
phosphodiester bonds between their sugar and phosphate residues.
3. The backbone of each consists of alternating deoxyribose and phosphate groups.
4. The phosphate group bonded to the 5' carbon atom of one deoxyribose is covalently
bonded to the 3' carbon of the next.
5. The two strands are "antiparallel"; that is, one strand runs 5′ to 3′ while the other
runs 3′ to 5′ that is, they have the opposite chemical polarity. Antiparallel orientation
of the two strands is essential for the formation of hydrogen bonds between the pairs
of DNA bases. The opposite polarity of the strands is also very important in the
mechanism of DNA replication.
6. The base paring is highly specific, adenine in one strand always pairs with thymine in
the corresponding position of the other strand and vice-versa. Similarly, guanine in
one strand is always paired with cytosine in the opposite strand and vice-versa.
Adenine and thymine linked by two hydrogen bonds, and guanine and cytosine form
three hydrogen bonds.
7. Once the sequence of bases in one strand of a DNA double helix is known, the
sequences of the bases in other strandcan be easily deduced because of specific base
pairing. Two strands of DNA double helix are thus said to be “complementary” (not
identical). The formation of hydrogen bonds between A and T, and between G and C
is known as complementary base pairing, and these base pairs are called
complementary base pairs or Watson and Crick base pairs.
8. The two strands of a DNA molecule are coiled together in a right handed helix
forming the DNA double helix.
9. The diameter of the helix is 20 Å. The pitch length (the length of helix required to
complete one turn) is 34 Å. In each DNA strand the bases occur at a regular interval
of 3.4 Å so that about 10 bases are present in one pitch of DNA double helix.
10. The path taken by the two backbones forms a major (wider) groove and a minor
(narrower) groove (Fig 3).
Fig 3. The double helix model of DNA molecule as proposed by Watson and Crick

Structure of RNA:

It contains ribose sugar, nitrogen bases and phosphate group. The nitrogen bases
include adenine, guanine, cytosine and uracil. In DNA thymine is present in place of uracil
and deoxyribose sugar is found in place of ribose sugar. In RNA, the pairing occurs between
adenine and uracil. It has usually single strand (Fig 4). However, some viruses have double
stranded RNA.

Fig. 4: RNA structure

Table 3: Differences between DNA & RNA

S. No. Particulars DNA RNA

1 Pentose Sugar deoxyribose ribose
2 Base Adenine, guanine, cytosine Adenine, guanine, cytosine
and thymine and uracil
3 Pairing AT and GC AU and GC
4 Number of strands Generally, double stranded Generally, single stranded
5 Function Genetic material Generally,non genetic
(mRNA, tRNA, rRNA). IN
some viruses, it serves as
genetic material
6 Origin Replication of pre-existing Through transcription of
DNA DNA.
The genetic RNA by
replication of RNA.
7 Location Mostly in chromosomes some In chromosomes and
in mitochondria and ribosomes
chloroplasts
8. Types A, B, C, Z forms mRNA, tRNA, rRNA
 3. GENETIC MATERIAL- DNA REPLICATION
Introduction
Living organisms perpetuate their kind through reproduction. This may be simple duplication
(cell fission) as in bacteria or complex modes of sexual reproduction as in higher plants and animals.
In all cases, however, reproduction entails the faithful transmission of the genetic information of the
parents to the progeny. Since the genetic information is stored in DNA, the replication of DNA is
central to all of biology.

The three models for DNA replication:

There were three basic models for DNA replication that had been proposed by the
scientific community after the discovery of DNA's structure. These models are discussed
below.

1. Dispersive replication: The old DNA molecule would break into several pieces, each
fragment would replicate and the old and new segments would recombine randomly
to yield progeny DNA molecules. Each progeny molecule would have both old and
new segments along its length i.e., two DNA molecules that are mixtures, or
“hybrids,” of parental and daughter DNA. In this model, each individual strand is a
patchwork of original and new DNA.
2. Conservative replication: In this model, DNA replication results in one molecule
that consists of both original (old) DNA strands (identical to the original DNA
molecule) and another molecule that consists of two new strands (with exactly the
same sequences as the original molecule).
3. Semiconservative replication: In this model, the two strands of DNA unwind from
each other, and each acts as a template for synthesis of a new, complementary strand.
This results in two progeny DNA molecules with one original strand and one new
strand (Fig 1).

Dispersive Replication
S

Semiconservative Replication

Conservative Replication
Fig 1.. Models of DNA Replication

Evidence for semiconservative replication –The “Meselson-Stahl

Stahl Experiment”
Experiment

The evidence for semiconservative replication of DNA was presented by Meselson

Meselson-
Stahl in 1958. They grew E. coli cells for many generations in a medium containing heavy
15
isotope of nitrogen, N. The bases in DNA contain nitrogen; thus, the DNA of cells grown
on medium containing 15N will have a greater density (1.724 g/cm3) than the DNA of cells
grown on a medium containing 14N (1.710 g/cm3); therefore these DNA molecules are called
“heavy” and“light” DNA molecules respectively. Since molecules of different densities can
be separated by a procedure called equilibrium density-gradient
gradient centrifugation, wherein
they form distinct bands (Fig 2). In density gradient centrifugation, a heavy salt solution such
as 6 M CsCl is centrifuged at very high speeds (30,000 – 50,000 rpm) for 48 -72
72 hours; this
leads to formation of a linear gradient of increasing density from the top to the bottom of
centrifuge tube. This gradient is formed due to the sedimentation of salt molecules towards
the bottom of the tube due to centrifugal force. When DNA is centrifuged in such a solution,
it will move to a position where the density of salt solution is the same as that of DNA. This
technique is highly sensitive and is able to separate molecules having only slightly different
densities (e.g., 1.710 and 1.724 g/cm3).

Fig 2. Equilibrium
quilibrium density-gradient
densit centrifugation with heavy and light bands
15
Meselson and Stahl took cells that had been grown in medium containing N for
DNA), and transferred them to medium containing 14N.
several generations (contained heavy DNA),
After allowing the cells to grow iin the presence of 14N for varying periods of time, the DNA
was extracted and analysed in CsCl equilibrium density gradient. The results of their
experiments aree only consistent with semiconservative replication, excluding both
conservative and dispersive model of DNA replication. All the DNA isolated from cells after
one generation of growth in medium containing14N had a density halfway between the
densities of “heavy” DNA and “light” DNA. This intermediate density is generally referred to
14
as “hybrid” density. After two generations of growth in medium containing N, half of the
DNA was of “hybrid” density and half of “light” density. The same two bands were
recovered in the DNA isolated after three cell generations, although the hybrid band was
relatively lower in intensity than the light band. These results are precisely those predicted by
the Watson and Crick semiconservative mode of replication. One generation of
semiconservative replication of a parental double helix containing 15N in medium containing
14 15
N would produce two progeny DNA molecules both of which had N in one strand (old
strand) and 14N (new strand) in other strand. Such DNA molecules would have intermediate
density called “hybrid” DNA molecules. One more round of semiconservative replication of
these DNA molecules: (1) half of the molecules would have one “heavy” and one “light”
strand (intermediate density), while (2) the remaining half would have both “light” strands
(light density). These molecules would be obviously form one “hybrid” and one “light” band
(Fig 3). On the third round of replication in14N medium, the “hybrid” molecule would
produce half “hybrid” and half “light” molecules, while all the molecules obtained from
“light” DNA molecules would be “light”; this is the reason for the lower intensity of
intermediate band after three cell generations.

Conservative replication would not produce any DNA molecules with “intermediate”
density; after one generation of conservative replication of “heavy” DNA in light medium,
half of the DNA would still be “heavy” and the other half would be “light”. If replication
were dispersive, Meselson and Stahl would have observed single band of DNA after every
cell generation. Meselson and Stahl results are inconsistent with either of these possibilities.
Subsequent studies have verified Meselson and Stahl’s conclusion that DNA replication is
semiconservative.
Fig 3.. Meselson and Stahl experiment on semiconservative replication of DNA

Types of DNA

DNA can be classified in various ways based on

1. Number of base pair per turn

2. Coiling pattern
3. Location
4. Structure
5.Nnucleotide sequence
 6. Number of strands
1. Number of base per turn: Depending upon the nucleotide base per turn of the helix, tilt
of the base pair and humidity of the sample, the DNA can be observed in four different forms
namely A,B, C and D.

2.Coiling pattern: On the basis of coiling pattern of the helix DNA is of two types viz., right
handed and left handed. Most of the DNA molecules are right handed i.e. coiling of helix is
in the right direction. It is also called positive coiling. All the four forms of DNA viz., A, B,
C and D are right handed. The Z DNA has left handed double helical structure. This DNA is
considered to be associated with gene regulation (Table 1).

The vast majority of the DNA molecules present in the aqueous protoplasm of living
cells almost certainly exists in the B-form (Watson-Crick double helix). The B-form is the
confirmation that DNA takes under physiological conditions (aqueous solutions containing
low concentrations of salts). However, DNA is not a static, invariant molecule i.e., DNA
molecule exhibit a considerable amount of conformational flexibility. The structure of DNA
molecules change as a function of their environment. In high concentrations of salts or in a
dehydrated state, DNA exists in the A-form.

3.Location:Based on the location in the cell DNA is of three types. viz., chromosomal DNA,
cytoplasm DNA and promiscuous DNA. Chromosomal DNA is found in chromosomes and
are called as chromosomal DNA or nuclear DNA. Cytoplasmic DNA is found in the
cytoplasm especially in mitochondria and chloroplasts. Such DNA plays an important role in
cytoplasmic inheritance and has circular structure.Some DNA segments with common base
sequence are found in the chloroplasts, mitochondria and nucleus. This suggests that some
DNA sequences move from one organelle to other. Such DNA is referred to as promiscuous
DNA.

Table 1: Differences between different types of DNA

Sl. No. Characteristics A-DNA B-DNA C-DNA Z-DNA

1 Coiling Right handed Right handed Right handed Left handed
2 Pitch length 28Å 34 Å 31Å 60Å
3 Base pairs per 11 10 (10-10.6) 9.33 12
turn
4 Diameter 26Å 20Å 19Å 18Å
5 Vertical rise per 2.56Å 3.38Å 3.32Å 3.71Å
 base pair
6 Sugar phosphate Regular Regular Regular Zig-zag
backbone

DNA Replication:

The semi conservative mode of DNA replication was postulated by Watson and Crick
along with the double helix model of DNA. The main features of this mode of DNA
replication are as follows:

 A progressive separation of the two strands of a DNA molecule.

 Complementary base pairing of the bases located in the single stranded regions thus
produced with the appropriate free deoxyribonulceotides.
 Formation of phosphodiester linkages between the neighbouring
deoxyribonucleotides that have base paired with the single stranded regions, thereby
producing regions the new strand.
 This ensures that the base sequence of the new strands are strictly complementary top
those of the old strands.
 The base sequence of a newly synthesized strand is dictated by the base sequence of
the old strand, since the old strand serves as a template for the synthesis of the new
strand.

DNA replication proposed by Watson and Crick. According to this method, both the
strands of parental DNA separate from one another. Each old strand synthesizes a new strand.
Thus, each of the two resulting DNA has one parental and one new strand. This method of
DNA replication is universally accepted because there are several evidences in support of
semi conservative method and it consists of several steps.
1. Initiation of Replication:DNA replication begins at a unique and fixed point on the
chromosome. This unique site is known as “origin”.
2. Unwinding of strands: The two stands of DNA double helix unwind. The opening of
DNA stands take’s places with the help of DNA unwinding enzymes. Two enzymes,
DNA gyrase and DNA helicase induce the unwinding of the complementary strands
of DNA double helix; this is called melting. Certain proteins called single-stranded
binding proteins (ssbp) bind to single stranded regions thus produced; so that they
remain single stranded as two strands of DNA molecule have high tendency of re-
joining.
2.1 Melting in the origin region generates two forks (Y) in the DNA duplex which are
called replication forks (RF). The replication forks may be associated with the plasma
lemma in prokaryotes, and to the nuclear envelope in eukaryotes. A replication fork
produces an ‘eye’ in linear DNA molecule and θ structure in circular DNA.
3. Formation of RNA Primer: Synthesis of RNA primer is essential for initiation of
DNA synthesis. DNA polymerase has an absolute requirement for a free 3' –OH of
pre-existing polynucleotide for the initiation of DNA replication. RNA primer is
synthesized by the enzyme primase near the origin which initiates transcription of the
strand oriented in the 3' 5'; this generates 10-60 nucleotide long RNA primer.
The free 3'-OH of this RNA primer provides initiation point for DNA polymerase for
the sequential addition of deoxyribonucleotides.
4. Synthesis of DNA on primer: After formation of RNA primer, DNA synthesis starts
on the RNA primer. Deoxyribose nucleotides are added to the 3' end position of RNA
primer by DNA polymerase. Further, DNA polymerase progressively adds
deoxyribonucleotides to the free 3' -OH of this growing polynucleotide chain so that
replication of the 3'5' strand of the DNA molecule is continuous (growth of new
strand in 5'3' direction).
4.1 Replication of the second strand (5'3' strand) of the DNA molecule is
discontinuous,and it begins somewhat later that of the 3' 5’ strand. Therefore 5'3'
stand is called “lagging strand” and 3'5' strand is called “leading strand”.
4.2 When replication of the 3'5' strand has progressed for some time; primase initiates the
synthesis of RNA primer on the 5'3' strand at a point close to the replication fork.
The3'-OH of this primer provides the initiation point for DNA polymerase to catalyse
replication of the ‘lagging strand’. Obviously, the replication of lagging strand
proceeds in the direction opposite to that of leading strand.However, the new strand is
 synthesized from the 5'3' in the case of both leading and lagging strands of DNA
molecule.
4.3 After some more time of replication of leading strand, primase again initiates the
transcription of lagging strand. The primer thus formed provides the free3'-OH for the
replication of lagging strand. Clearly, the replication of lagging strand generates small
nucleotide fragments called “Okazaki fragments” (after R. Okazaki who first
identified). Okazaki fragments are about 1,000 -2,000 nucleotide long in E. coli.
5. Removal of RNA Primer: The RNA primers associated with newly synthesized
DNA strands/Okazaki fragments are digested by DNA polymerase I in prokaryotes.
This enzyme also catalyses the filling of gaps so generated in the new strands.
6. Union of Okazaki Fragments. The discontinuous fragment (Okazaki fragments) are
joined to make continuous strands. The union of Okazaki fragments takes place with
the help of a joining enzyme called polynucleotide “ligase”, which catalyses
formation of phosphodiester bonds betweenthe neighbouring nucleotides of the
adjacent fragments (Fig 4).

The replication may take place either in one direction or in both the directions from
the point of origin.

DNA Polymerases in Prokaryotes

A total of 5 different DNAPs have been reported in E. coli

• DNAP I: functions in repair and replication

• DNAP II: functions in DNA repair

• DNAP III: principal DNA replication enzyme

• DNAP IV: functions in DNA repair

• DNAP V: functions in DNA repair

OR
Fig.4. A generalized model of DNA replication
Gene Expression-Protein Synthesis-Transcription
Role of mRNA, tRNA and rRNA
THE CENTRAL DOGMA OF BIOLOGY

The central dogma of Biology was proposed by Crick in 1953 which indicates the flow of
genetic information from DNA to protein. The information present in the DNA (in the form of
base sequence) is transferred to DNA via replication catalysed by DNA polymerase and to RNA
via transcription carried out by DNA dependent RNA polymerase. The information from RNA
(in the form of base sequence) is transferred to protein (in the form of amino acid sequence) via
translation. This information does not flow in the reverse direction, that is, from protein to RNA
to DNA. This flow of information from DNA to RNA and RNA to protein is known as “central
dogma of Biology”. However, in some viruses where ever the RNA is the genetic material
(TMV), the RNA produces new copies of itself with the help RNA replicase and the process is
referred to replication. Genetic RNA of some other viruses e.g., RSV, serves as a template for
the production of a complementary copy of DNA and referred to reverse transcription catalysed
by reverse transcriptase enzyme (Fig 1). In simple terms, sequence information is transferred
from nucleic acid to nucleic acid, and from nucleic acid to protein. However, there is no
evidence so far that the amino acid sequence information from proteins is converted by cells into
DNA, RNA or proteins.
Fig 1:: The direction of flow of genetic information among DNA, RNA and protein

The central dogma of molecular biology is that genetic information flows from DNA to DNA during
chromosome replication, from DNA to RNA during transcription, and from RNA to protein during
translation.

Transcription involves the synthesis of an RNA transcript complementary to one strand of DNA of a gene.

Translation is the conversion of information stored in the sequence of nu

nucleotides
cleotides in the RNA transcript
into the sequence of amino acids in the polypeptide gene product, according to the specifications of the
genetic code.

DNA, Genes, Protein and Polypeptides:

• DNA contains genes, sequences of nucleotide bases
• These Genes code for polypeptides (proteins)
• Proteins are used to build cells and do much of the work inside cells
• Proteins are made of amino acids linked together by peptide bonds
 • 20 different amino acids exist
• Amino acid chains are called polypeptides
• DNA Begins the Process:DNA is found inside the nucleus
• Proteins, however, are made in the cytoplasm of cells by organelles called ribosomes
• Ribosomes may be free in the cytosol or attached to the surface of rough ER

TYPES OF RNA MOLECULES: Five different classes of RNA molecules play essential roles
in gene expression. messenger RNAs(mRNAs) are the intermediaries that carry genetic
information from DNA to the ribosomes where proteins are synthesized. Transfer RNAs
(tRNAs) are small RNA molecules that function as adaptors between amino acids and the
codons in mRNA during translation. Ribosomal RNAs (rRNAs) are structural and catalytic
components of the ribosomes, the intricate machines that translate nucleotide sequences of
mRNAs into amino acid sequences of polypeptides. Small nuclear RNAs (snRNAs) are
structural components of spliceosomes, the nuclear organelles that excise introns from gene
transcripts. Micro RNAs (miRNAs) are short 20- to 22-nucleotide single-stranded RNAs that
are cleaved from small hairpin-shaped precursors and block the expression of complementary or
partially complementary mRNAs by either causing their degradation or repressing their
translation. All five types of RNA—mRNA, tRNA, rRNA, snRNA, and miRNA—are produced
by transcription. Unlike mRNAs, which specify polypeptides, the final products of tRNA, rRNA,
snRNA, and miRNA genes are RNA molecules. Transfer RNA, ribosomal RNA, snRNA, and
miRNA molecules are not translated.

Messenger RNA: Messenger RNA (mRNA) copies DNA’s code & carries the genetic
information to the ribosomes.
• Long Straight chain of Nucleotides
• Made in the Nucleus
• Copies DNA & leaves through nuclear pores
• Contains the Nitrogen Bases A, G, C, U ( no T )

Ribosomal RNA (rRNA): Ribosomal RNA (rRNA), along with protein, makes up the
ribosomes.
• rRNA is a single strand 100 to 3000 nucleotides long
• Globus in shape
 • Made inside the nucleus of a cell
• Associates with proteins to form ribosomes
• Site of protein Synthesis

Transfer RNA (tRNA): Transfer RNA (tRNA) transfers amino acids to the ribosomes where
proteins are synthesized.
• Clover-leaf shape
• Single stranded molecule with attachment site at one end for an amino acid
• Opposite end has three nucleotide bases called the anticodon

Different types of RNA with their features:

Features Ribosomal RNA Messenger RNA Transfer RNA (tRNA)

(rRNA) (mRNA)
1. Percentage of cell’s About 80 About 5 About 15
total RNA
2. Length of molecule Variable Longest Shortest
3.Shape of molecule Greatly coiled Linear Clover leaf-
leaf like, folded
into L-shape
L
4. Types Six Numerous About 60
5. Role Form greater part of Carry information from Carry amino acids to
ribosome’s DNA mRNA codons
TRANSCRIPTION AND TRANSLATION
The expression of genetic information occurs in two steps: transcription and translation. During
transcription, one strand of DNA of a gene is used as a template to synthesize a complementary
strand of RNA, called the gene transcript. For example, the DNA strand containing the
nucleotide sequence AAA
is used as a template to produce the complementary sequence UUU in the RNA transcript.
During translation, the sequence of nucleotides in the RNA transcript is converted into the
sequence of amino acids in the polypeptide gene product. This conversion is governed by the
genetic code, the specification of amino acids by nucleotide triplets called codons in the gene
transcript. For example, the UUU triplet in the RNA transcript shown in Figure 11.1 specifies the
amino acid phenylalanine (Phe) in the polypeptide gene product. Translation takes place on
intricate macromolecular machines called ribosomes, which are composed of three to five RNA
molecules and 50 to 90 different proteins. However, the process of translation also requires the
participation of many other macromolecules.

Transcription: The RNA molecules that are translated on ribosomes are called messenger
RNAs (mRNAs). In prokaryotes, the product of transcription, the primary transcript, usually is
equivalent to the mRNA molecule In eukaryotes, primary transcripts often must be processed by
the excision of specific sequences and the modification of both termini before they can be
translated. Thus, in eukaryotes, primary transcripts usually are precursors to mRNAs and, as
such, are called pre-mRNAs. Most of the nuclear genes in higher eukaryotes and some in lower
eukaryotes contain non-coding sequences called introns that separate the expressed sequences or
exons of these genes. The entire sequences of these split genes are transcribed into pre-mRNAs,
and the non-coding intron sequences are subsequently removed by splicing reactions carried out
on macromolecular structures called spliceosomes.
• The process of copying the sequence of one strand of DNA, the "template strand"
• mRNA copies the template strand
• Requires the enzyme RNA Polymerase
• During transcription, RNA polymerase binds to DNA and separates the DNA strands
• RNA Polymerase then uses one strand of DNA as a template to assemble nucleotides
into RNA
• Promoters are regions on DNA that show where RNA Polymerase must bind to
begin the Transcription of RNA called the TATA box
• Specific base sequences act as signals to stop Called the termination signal.
 RNA Polymerase
mRNA Synthesis: RNA synthesis occurs by a mechanism that is similar to that of DNA
synthesis except that (1) the precursors are ribonucleoside triphosphates rather than
deoxyribonucleoside triphosphates,(2) only one strand of DNA is used as a template for the
synthesis of a complementary RNA chain in any given region, and (3) RNA chains can be
initiated de novo, without any requirement for a preexisting primer strand. The RNA molecule
produced will be complementary and antiparallel to the DNA template strand and identical,
except that uridine residues replace thymidines, to the DNA nontemplate strand. If the RNA
molecule is an mRNA, it will specify amino acids in the protein gene product. Therefore, mRNA
molecules are coding strands of RNA. They are also called sense strands of RNA because their
nucleotide sequences “make sense” in that they specify sequences of amino acids in the protein
gene products. An RNA molecule that is complementary to an mRNA is referred to as antisense
RNA. This terminology is sometimes extended to the two strands of DNA. However, usage of
the terms sense and antisense to denote DNA strands has been inconsistent. Thus, we will use
template strand and nontemplate strand to refer to the transcribed and nontranscribed strands,
respectively, of a gene. The synthesis of RNA chains, like DNA chains, occurs in the 5_ → 3_
direction, with the addition of ribonucleotides to the 3_-hydroxyl group at the end of the chain..
The reaction involves a nucleophilic attack by the 3_-OH on the nucleotidyl (interior)
phosphorus atom of the ribonucleoside triphosphate precursor with the elimination of
pyrophosphate, just as in DNA synthesis. This reaction is catalyzed by enzymes called RNA
polymerases. The overall reaction is as follows: n(RTP) DNA template (RMP)n _ n(PP) RNA
polymeraseP) where n is the number of moles of ribonucleotide triphosphate, (RTP) consumed,
ribonucleotide monophosphate, (RMP) incorporated into RNA, and pyrophosphate
(PP)produced.

RNA polymerases bind to specific nucleotide sequences called promoters, and with the help of
proteins called transcription factors, initiate the synthesis of RNA molecules at transcription start
sites near the promoters. The promoters in eukaryotes are typically more complex than those of
prokaryotes. A single RNA polymerase carries out all transcription in most prokaryotes, whereas
five different RNA polymerases are present in eukaryotes, with each polymerase responsible for
the synthesis of a distinct class of RNAs. RNA synthesis takes place within a locally unwound
segment of DNA, sometimes called a transcription bubble, which is produced by RNA
polymerase.The nucleotide sequence of an RNA molecule is complementary to that of its DNA
template strand, and RNA synthesis is governed by the same base-pairing rules as DNA
synthesis, but uracil replaces thymine. As a result, the origin of RNA transcripts can be
determined by studying their hybridization to DNAs from different sources such as the
chromosome(s) of the cell, viruses, and other infectious organisms (see Problem-Solving Skills:
Distinguishing RNAs Transcribed from Viral and Host DNAs).

Transcription and RNA Processing in Eukaryotes: Although the overall process of RNA
synthesis is similar in prokaryotes and eukaryotes, the process is considerably more complex in
eukaryotes. In eukaryotes, RNA is synthesized in the nucleus, and most RNAs that encode
proteins must be transported to the cytoplasm for translation on ribosomes. There is
evidence suggesting that some translation occurs in the nucleus; however, the vast majority
clearly occurs in the cytoplasm. Prokaryotic mRNAs often contain the coding regions of two or
more genes; such mRNAs are said to be multigenic. In contrast, many of the eukaryotic
transcripts that have been characterized contain the coding region of a single gene (are
monogenic). Nevertheless, up to one-fourth of the transcription units in the small worm
Caenorhabditis elegans may be multigenic. Clearly, eukaryotic mRNAs may be either
monogenic or multigenic. Five different RNA polymerases are present in eukaryotes, and each
enzyme catalyzes the transcription of a specific class of genes. Moreover, in eukaryotes, the
majority of the primary transcripts of genes that encode polypeptides undergo three major
modifications prior to their transport to the cytoplasm for translation.
1. 7-Methyl guanosine caps are added to the 5_ ends of the primary transcripts.
2. Poly(A) tails are added to the 3_ ends of the transcripts, which are generated by
cleavage rather than by termination of chain extension.
3. When present, intron sequences are spliced out of transcripts.
The 5_ cap on most eukaryotic mRNAs is a 7-methyl guanosine residue joined to the initial
nucleoside of the transcript by a 5_-5_ phosphate linkage. The 3_ poly(A) tail is a polyadenosine
tract 20 to 200 nucleotides long. In eukaryotes, the population of primary transcripts in a nucleus
is called heterogeneous nuclear RNA (hnRNA) because of the large variation in the sizes of
the RNA molecules present. Major portions of these hnRNAs are noncoding intron sequences,
which are excised from the primary transcripts and degraded in the nucleus. Thus, much of the
hnRNA actually consists of pre-mRNA molecules undergoing various processing events before
leaving the nucleus. Also, in eukaryotes, RNA transcripts are coated with RNAbinding proteins
during or immediately after their synthesis. These proteins protect gene transcripts from
degradation by ribonucleases, enzymes that degrade RNA molecules, during processing and
transport to the cytoplasm. The average half-life of a gene transcript
in eukaryotes is about five hours, in contrast to an average half-life of less than five minutes in E.
coli. This enhanced stability of gene transcripts in eukaryotes is provided, at least in part, by their
interactions with RNA-binding proteins.

Interrupted Genes in Eukaryotes: Exons and Introns: Most of the well-characterized genes
of prokaryotes consist of continuous sequences of nucleotide pairs,which specify colinear
sequences of amino acids in the polypeptide gene products. However, in 1977, molecular
analyses of three eukaryotic genes yielded a major surprise. Studies of mouse and rabbit _-globin
(one of two different proteins in hemoglobin) genes and the chicken ovalbumin (an egg storage
protein) gene revealed that they contain noncoding sequences intervening between coding
sequences. They were subsequently found in the nontranslated regions of some genes. They are
called introns (for intervening sequences.) The sequences that remain present in mature mRNA
molecules (both coding and noncoding sequences) are called exons (for expressed sequences).

Removal of Intron Sequences by RNA Splicing: Most nuclear genes that encode proteins in
multicellular eukaryotes contain introns. Fewer, but still many, of the genes of unicellular
eukaryotes such as the yeasts contain introns. Rare genes of archaea and of a few viruses of
prokaryotes also contain introns. In the case of these “split” genes, the primary transcript
contains the entire sequence of the gene, and the intron sequences are excised during RNA
processing.
The non-coding introns are excised from gene transcripts by three different mechanisms as
detailed below.
1. The introns of tRNA precursors are excised by precise endonucleolytic cleavage and ligation
reactions catalyzed by special splicing endonuclease and ligase activities.
2. The introns of some rRNA precursors are removed autocatalytically in a unique reaction
mediated by the RNA molecule itself. (No protein enzymatic activity is involved.)
3. The introns of nuclear pre-mRNA (hnRNA) transcripts are spliced out in two-step reactions
carried out by complex ribonucleoprotein particles called spliceosomes.

mRNA Processing: After the DNA is transcribed into RNA, editing must be done to the
nucleotide chain to make the RNA functional.
Introns, non-functional segments of DNA are snipped out of the chain
mRNA Editing: Exons, segments of DNA that code for proteins, are then rejoined by the
enzyme ligase.
• A guanine triphosphate cap is added to the 5” end of the newly copied mRNA
• A poly A tail is added to the 3’ end of the RNA, The newly processed mRNA can then leave
the nucleus
mRNA Transcript: mRNA leaves the nucleus through its pores and goes to the ribosomes.

*****
Genetic Code-Translation-Formation of Polypetide Chain
The Genetic Code: The genetic code is a nonoverlapping code, with each amino acid plus
polypeptide initiation and termination specified by RNA codons composed of three nucleotides.
• A codon designates an amino acid
• An amino acid may have more than one codon
• There are 20 amino acids, but 64 possible codons
• Some codons tell the ribosome to stop translating
• Use the code by reading from the center to the outside Example: AUG codes for Methionine

An interesting aspect of protein synthesis is that the DNA and ribosomes are located at different
sites in the cell. Location of instruction centre (DNA) and manufacturing centre (ribosomes) at
different sites in a cell is advantageous. If both were in the nucleus, the manufacturing centre
would be far away from the energy sources and raw materials; and if both were in the cytoplasm,
the information centre would be exposed to respiratory breakdown. The nuclear
envelope preserves stability of the DNA by protecting it from respiratory destruction. The
message in the DNA in the form of genes (codes) are, permanent, authentic master documents
from which working copies are prepared in the form of mRNAs, as and when required by the
cell. The complex process by which the information in RNA is decoded into a polypeptide is one
of the exciting discoveries the genetic code. With only four biochemical letters (A, G, C, U) a
one letter code could not unambiguously encode 20 amino acids. A two letter code could encode
only 16 amino acids. So, a triplet code based on 3 biochemical letters or nucleotide bases could
make 4 x 4 x 4 = 64 codons. This will be required to code for 20 or so different amino acids.
The discovery of the genetic code became possible through the contribution of many scientists
like Francis Crick, Seveno Ochoa, Maxell Ninenberg, Hargobind Khorana and J. H. Malther in
the 1960s. Ninemberg and Khorana shared the Nobel Prize in 1968.
Characteristics of Genetic Code
The genetic code is a triplet code: Three adjacent bases, termed as codon, specify one amino
acid
Non-overlapping: Adjacent codons do not overlap
No punctuation: The genetic code is comma less
The genetic code is universal i.e., a given codon specifies the same amino acid in all protein
synthesising organisms
The genetic code is degenerate: it lacks specificity and one amino acid often has more than one
code triplet. Each codon codes for only one amino acid, none for more than one. Three of the 64
codons, names UAA, UAG and UGA do not specify any amino acid but signal the end of the
message. They are called nonsense or terminator codons. The codons AUG and GUG are
called the initiation or start codons as they begin the synthesis of polypeptide.

In the above figure, the sequence of nucleotides in the triplet codons of RNA is indicated; each triplet
specifies a particular amino acid

Codons and Anticodons: The 3 bases of an anticodon are complementary to the 3 bases
of a codon Example: Codon ACU and anticodon UGA.
•

Protein Synthesis: The production or synthesis of polypeptide chains (proteins).

 Two phases: Transcription & Translation.
 mRNA must be processed before it lealeaves
ves the nucleus of eukaryotic cells for
Translation process.

Translation: Translation is the process of decoding the mRNA into a polypeptide

chain.. Ribosomes read mRNA three bases or 1 codon at a time and construct the proteins.
proteins
Transcription occurs when DNA acts as a template for mRNA synthesis. Translation occurs
when the sequence of the mRNA codons determines the sequence of amino acids in a protein.
Three main steps of Translation are as follows.

Ribosome: Made of a large and small subunit; Small subunit attaches to large ribosomal subunit.
Composed of rRNA (40%) and proteins (60%)
(60%).. Have two sites for tRNA attachment --- P and A

A ribosome has two binding sites for tRNA molecules. One is called A site (acceptor or
aminoacyl)) and the other is termed P site (peptidyl). These sites span across the larger and
smaller subunits of the ribosome. The A site receives the tRNA amino acid complex. The tRNA
leaves from P site, after releasing its amino acid. However, the first tRNAamino acid complex
directly enters the P site of the ribosome.

A eukaryotic ribosome has a groove at the junction of the two subunits. From this groove, a
tunnel extends through the large subunit and opens into a canal of
the endoplasmic reticulum. The polypeptides are synthesized in the groove between the two
ribosomal subunits and pass through the tunnel of the large subunit into the endoplasmic
reticulum. While in the groove, the developing polypeptide is protected from the cellular
enzymes.
The smaller subunit forms a cap over the larger subunit. The larger subunit attaches to the
endoplasmic reticulum by two glycoproteins named ribophorin I and II. The function of the
ribosome is to hold the mRNA, tRNA and the associated enzymes controlling the process in
position, until a peptide bond is formed between the adjacent amino acids.

Mechanism of Protein Synthesis

The events in protein synthesis are better known in bacteria than in eukaryotes. Although these
are thought to be similar in the two groups there are some differences. The following description
refers mainly to protein synthesis in bacteria on the 70S ribosome. Protein synthesis is a highly
complex and an elaborate process and involves the following steps:

Activation of Amino Acids

It is the step in which each of the participating amino acid reacts with ATP to form amino acid
AMP complex and pyrophosphate. The reaction is catalyzed by a specific amino acid activating
enzyme called aminoacyl-tRNA synthetase in the presence of Mg2+. There is a separate
aminoacyl tRNA synthetase enzyme for each kind of amino acid. Much of the energy released by
the separation of phosphate groups from ATP is trapped in the amino acid AMP complex. The
complex remains temporarily associated with the enzyme. The amino acid AMP enzyme
complex is called an activated amino acid. The pyrophosphate is hydrolyzed to two in organic
phosphates (2pi)
Activatiing enzyme Mg 2+
Amino acid + ATPAmino acid AMP enzyme complex + ppi
Activation of Amino Acids
Charging of tRNA
It is the step in which the amino acid AMP-enzyme complex joins with the amino acid binding
site of its specific tRNA, where its COOH group bonds with the OH group of the terminal base
triplet CCA. The reaction is catalyzed by the same enzyme, aminoacyl tRNA synthetase. The
resulting tRNA-amino acid complex is called a charged tRNA. AMP and enzyme are released.
The released enzyme can activate and attach another amino acid molecule to another tRNA
molecule. The energy released by change of ATP to AMP is retained in the amino acid-tRNA
complex. This energy is later used to drive the formation of peptide bond when amino acids link
together and form a polypeptide
Amino acid AMP Enzyme complex +t RNA
The tRNA amino acid complex moves to the ribosomes, the site of protein synthesis.
Activation of Ribosome
It is the step in which the smaller and the larger subunits of ribosome are joined together. This is
brought about by mRNA chain. The latter joins the smaller ribosomal subunit with the help of
the first codon by a base pairing with an appropriate sequence on rRNA. The combination of the
two is called initiation complex. The larger subunit later joins the small subunit, forming active
ribosome. Activation of ribosome by mRNA requires proper concentration of Mg ++
Assembly of Amino Acids (Polypeptide Formation)
It is the step in which the amino acids are assembled into a polypeptide chain. It involves 3
events: initiation, elongation and termination of polypeptide chain
Initiation of Polypeptide Chain
The mRNA A chain has at its 5 end an “initiator” or “start” codon (AUG or GUG) that signals the
beginning of polypeptide formation. This codon lies close to the P site of the ribosome. The
amino acid formylmethionine (methionine in eukaryotes) initiates the process.
process It is carried by
tRNA having an anticodon UAC which bonds with the initiator codon AUG of mRNA. Initiation
factors (IF1, IF2 and IF3) and GTP promote the initiation process.
The large ribosomal subunit now joins the small subunit to complete the ribosome.
ribosom At this stage,
GTP is hydrolysed to GDP. The ribosome has formylmethionine bearing tRNA at the P site.
Later, the formylmethionine is changed to normal methionine by the enzyme deformylase in
prokaryotes. If not required, methionine is later separated ffrom rom the polypeptide chain by a
proteolytic enzyme aminopeptidase.

Elongation of Polypeptide Chain

Chain:
Three elongation factors (EF Tu, EF Ts and EF G) assist in the elongation of the polypeptide
chain. A charged tRNA molecule along with its amino acid, proline, for example, enters the
ribosome at the A site. Its anticodon GGA locates and binds with the complementary codon CCU
of mRNA chain by hydrogen bonds. The amino acid methionine is transferred from its tRNA
onto the newly arrived proline tRNA complex where the two amino acids join by a peptide bond.
The process is catalyzed by the enzyme peptidyl transferase located on the ribosome. In this
process, the linkage between the first amino acid and its tRNA is broken, and the -COOH group
now forms a peptide bond with the free -NH2 group of the second amino acid. Thus, the second
tRNA carries a dipeptide, formylmethionineproline.
The energy required for the formation of a peptide bond comes from the free energy released by
separation of amino acid (formylmethionine or methionine) from its tRNA. The first tRNA, now
uncharged, separates from mRNA chain at the P site of the ribosome and returns to the mixed
pool of tRNAs in the cytoplasm. Here, it is now available to transp
transport
ort another molecule of its
specific amino acid. Now the ribosome moves one codon along the mRNA in the 3 direction.
With this, tRNAdipeptide complex at the A site is pulled to the P site. This process is called
translocation. It requires GTP and a transl
translocase protein called EF-G G factor. The GTP is
hydrolysed to GDP and inorganic phosphate to release energy for the process At this stage, a
third tRNA molecule with its own specific amino acid, arginine, for example arrives at the A site
of the ribosome and binds with the help of anticodon AGA to the complementary codon UCU of
the mRNA chain. The dipeptide formylmethionineproline is shifted from the preceding tRNA on
the third tRNA where it joins the amino acid arginine again with the help of peptidyl transferase
transfe
enzyme. The dipeptide, thus, becomes a tripeptide, formylformyl-methionine-proline
proline-arginine. The
second tRNA being now uncharged, leaves the mRNA chain, vacating the P site. The
tRNAtripeptide complex is translocated from A site to P site The entire process involving arrival
of tRNA-amino
amino acid complex, peptide bond formation and translocation is repeated. As the
ribosome moves over the mRNA, all the codons of mRNA arrive at the A site one after another,
and the peptide chain grows. Thus, the amino acids are linked up into a polypeptide in a
sequence communicated by the DNA through the mRNA. A polypeptide chain which is in the
process of synthesis is often called a nascent polypeptide The growing polypeptide chain always
remains attached to its original riboso
ribosome,
me, and is not transferred from one ribosome to another.
Only one polypeptide chain can be synthesized at a time on a given ribosome.
The above figure shows, that charged tRNA arriving at the A site, reading its codon on the
mRNA; Amino acid of tRNA at P site is ready to be transferred to the amino acid of tRNA at A
site; Amino acids are joined by peptide bond and tRNA is discharged from P site and Peptide
chain-carrying tRNA is translocated to P site, making A site free to receive another charged
tRNA.

Termination and Release of Polypeptide Chain:

At the terminal end of mRNA chain there is a stop, or terminator codon (UAA, UAG or UGA). It
is not joined by the anticodon of any tRNA amino acid complex. Hence, there can be no further
addition of amino acids to the polypeptide chain.
The linkage between the last tRNA and the polypeptide chain is broken by three release factors.
(RF 1, RF 2 and RF 3) and GTP. The release is catalyzed by the peptidyl transferase enzyme, the
same enzyme that forms the peptide
ptide bonds. The ribosome jumps off the mRNA chain at the stop
codon and dissociates into its two subunits. The completed polypeptide (amino acid chain)
becomes free in the cytoplasm.
The ribosomes and the tRNAs on release from the mRNA can function again in the same manner
and result in the formation of another polypeptide of the same protein.

Modification of Released Polypeptide

The just released polypeptide is a straight, linear exhibiting a primary molec
molecule,
ule, structure. It may
lose some amino acids from the end with the help of a peptidase enzyme, and then coil and fold
on itself to acquire secondary and tertiary structure. It may even combine with other
polypeptides, to have quaternary structure.
The proteins
teins synthesized on free polysomes are released into the cytoplasm and function as
structural and enzymatic proteins. The proteins formed on the polysomes attached to ER pass
into the ER channels and are exported as cell secretions by exocytosis after packaging
pac in the
Golgi apparatus.
Polysome Formation
When the ribosome has moved sufficiently down the mRNA chain towards 3 end, another
ribosome takes up position at the initiator codon of mRNA, and starts synthesis of a second
molecule of the same polypeptidetide chain. At any given time, the mRNA chain will, therefore,
carry many ribosomes over which are similar polypeptide chains of varying length, shortest near
the initiator codon and longest near the terminator codon. A row of ribosomes joined to the
mRNA molecule, is called a polyribosome, or a polysome. Synthesis of many molecules of the
same polypeptide simultaneously from one mRNA molecule by a polysome is called
translational amplification.
Energy Used for Protein Synthesis
One GTP is hydrolysed to GDP as each successive amino acid-tRNA complex attaches to the A
site of the ribosome. A second GTP is broken down to GDP as the ribosome moves to each new
codon in the mRNA. One ATP is hydrolysed to AMP during amino acid activation. Thus, the
formation of each peptide bond uses 3 high-energy molecules, one ATP and two GTP.

*****
 Gene Regulation
All the genes are not expressed at all the time, particular genes will express depending
on requirement. If all the genes are expressed there is wastage of energy resource. It means to
say there is necessity of putting ‘on’ or ‘off’ of the genes which is nothing but Gene
Regulation.

Regulation may be at:

i) Synthesis of primary RNA transcript

ii) Post transcriptional processing of mRNA(hnRNA)
iii) mRNA degradation
iv) Translation
v) Post transcriptional modification of proteins
vi) Protein degradation

If the regulation is at transcription level, more energy will be conserved. There are set of
genes which are always expressed in all the cells and in all organisms. These genes are called
as Constitutive genes (House keeping genes). Ex: Genes coding for tRNA, rRNA and
Ribosomal proteins.

Gene regulation is defined as the control of gene activity i.e protein production that
permits the function of only those genes whose products are required in a given cell at a
given time.

There are two types of Regulation

1) Induction:

E.coli utilizes carbon for growth. Main carbon source is Glucose, Lactose, Fructose,
Galactose and Arabinose. If E.coli is placed in medium containing glucose, it will not
transcribe the gene which is essential for breakdown of lactose sugar in to glucose and
galactose. If bacteria are transferred into another medium containing lactose, genes which are
responsible for synthesis of enzymes for breakdown of lactose will be switched on or turned
on. Normally they are not expressed but induced to express and hence called Inducible genes.
The process by which the expression of genes is turned on in response to a substance in the
environment is called Induction. Genes involved are inducible genes. Process is induction
and products of such regulation are called inducible enzymes. Substance or molecules
responsible for induction are called Inducers.

Induction will change /turn on the system which is normally off, this is the case of
Catabolism.

2) Repression:

Normally gene will be expressing but making them to switch off which is the case of
Anabolism or biosynthetic pathway. Ex: Tryptophan synthesis in E.coli. It has been
identified that there are five genes which are involved in synthesis of five enzymes which are
required for synthesis of tryptophan. When tryptophan is supplied externally, it will switch
off its genes which are required for Tryptophan synthesis. In this case normally genes are
‘on’, but external supply will make ‘off’, hence called as Repression.

OPERON: The basic concepts

 Mechanism of Gene Regulation was first described by Jacob and Monad (1961) by using
Operon model and received the Nobel Prize in 1965.
 ‘Operon’ is a group of structural genes whose transcription is regulated by coordinated
action of of a Regulator, a Promoter and an Operator.

 A cluster of functionally related genes can be under coordinated control by a single on-off
“switch”. The regulatory “switch” is a segment of DNA called an operator usually
positioned within the promoter.
 Regulator gene codes for protein called as repressor which will bind to the operator and
avoid the transcription process by not allowing occupation of promoter site by RNA
polymerase (operon switched off). Promoter present next to Regulator which is a site for
RNA polymerase binding. Regulator gene producing repressor protein is determined by
presence /absence of effector molecule in the environment.

I) NEGATIVE GENE REGULATION: Repressible and Inducible operons are two

Types of Negative Gene Regulation.

1) Lac operon: Lac operon in E.coli i.e operon involved in lactose (milk sugar-a
disaccharide) utilization for Inducible type of Regulation.

a) Lactose absent, repressor active, operon off

In the absence of lactose, the repressor protein produced from Regulator gene will
bind to the operator site, which will not allow the transcription of structural genes. Lac
operon is ‘turned off’.
b) Lactose present, repressor inactive, operon on

In the presence of Lactose in the environment, which acts as inducer binds to the
repressor molecule and makes inactive and hence can not bind to the operator. Therefore
RNA polymerase binds to promoter and transcription of structural gene proceeds.

2) Trp operon: The trp operon is a repressible operon

a) Tryptophan absent, repressor inactive, operon on:

In the absence of tryptophan, regulator gene produces inactive repressor which can
not bind to the operator, thus facilitating RNA polymerase to bind to promoter and
transcription of structural genes required for tryptophan biosynthesis proceeds.
b) Tryptophan present, repressor active, operon off:

In the presence of tryptophan, the excess tryptophan acts as corepressor/effector

molecule which converts the inactive repressor to active repressor which will bind to operator
and thus not allowing RNA polymerase to bind to promoter and hence no structural gene
transcription. This type of system is repressible system of gene regulation.

Note:

• Inducible enzymes usually function in catabolic pathways; their synthesis is induced

by a chemical signal

• Repressible enzymes usually function in anabolic pathways; their synthesis is

repressed by high levels of the end product

• Regulation of the trp and lac operons involves negative control of genes because
operons are switched off by the active form of the repressor

II) POSITIVE GENE REGULATION

• Some operons are also subject to positive control through a stimulatory protein, such
as catabolite activator protein (CAP), an activator of transcription

• When glucose (a preferred food source of E. coli) is scarce, CAP is activated by

binding with cyclic AMP

• Activated CAP attaches to the promoter of the lac operon and increases the affinity of
RNA polymerase, thus accelerating transcription
 • When glucose levels increase, CAP detaches from the lac operon, and transcription
returns to a normal rate.

• CAP helps to regulate other operons that encode enzymes used in catabolic pathways.

Note: In negative control------The product of the regulator gene is Repressor. Genes are
expressed unless association of a specific regulator protein, called Repressor with the
operator.

In positive control-------Regulator gene product is activator. Genes are not

transcribed unless a specific protein termed as activator binds to a segment of DNA in the
promoter gene or to RNA polymerase which enables transcription initiation.
 MUTATION

Introduction :
 The sudden heritable changes in the charecters of an organisms is called as mutation.
 Individuals showing these heritable changes are known as mutants.

History :
 The term mutation was introduced by Hugo de VRIES in 1900 to describe sudden
heritable changes in Oenothera.
 Seth Wright discovered a male lamb with unusually short legs. This lamb served as
the source of dominant short-legged trait for the development of the ‘Ancon’ breed of
sheep.
 Sysytematic studies began in 1910 with the discovery of the white eye mutant of
Drosophila by Morgan.
 H.J. Muller discovered the mutagenic action ox X-rays in 1927 in Drosophila.
 In 1929 Stadler described the mutagenic effects of X-rays in barley.

Classification of Mutations:
 Based on direction of mutation:
a) Forward mutation
b) Backward mutation

 Based on source / cause of mutation:

a) Spontaneous mutation
b) Induced mutation

 Based on dominance relationship:

a) Dominant mutation
b) Recessive mutation
c) Co dominant mutation
d) Incompletely dominant mutation

 Based on tissue of origin:

a) Somatic mutation
b) Germinal mutation

 Based on effect on survival:

a) Lethal mutation
b) Sub –lethal mutation
 c) Sub –vital mutation
d) Vital mutation
e) Super vital mutation

 Based on character or trait affected:

a) Morphological mutation
b) Biochemical mutation

 Based on the site of mutation or on cytological

a) Chromosomal mutation
b) Gene or point mutation
c) Cytoplasmic mutation

 Based on visibility:
a) Macro mutation
b) Micro mutation

Methods of inducing mutations :

Mutagens
Mutations can be induced by a number of agents; the agents capable of inducing mutations
are called mutagens. Mutagen is a natural or human-made agent (physical or chemical) which
can alter the structure or sequence of DNA. The different mutagenic agents may be classified
into the following two broad groups:
1) Physical mutagens
2) Chemical mutagens
Physical mutagens
The different types of radiations having mutagenic properties are known as physical
mutagens. The radiations may be a part of the electromagnetic spectrum having shorter
wavelength and higher energy than visible light (eg: uv rays, X rays, gamma rays and cosmic
rays) or may be particulate radiations produced by the decay of radio isotopes.
Radiations
Radiation was the first mutagenic agent known; its effects on genes were first reported in the
1920's. Radiation itself was discovered in 1890's: Roentgen discovered X-rays in 1895,
Becquerel discovered radioactivity in 1896, and Marie and Pierre Curie discovered
radioactive elements in 1898. These three discoveries and others led to the birth of atomic
physics and our understanding of electromagnetic radiation.
Radiations are grouped into two classes depending on the kind of effects they have on the
atoms in their path:
1. ionizing and
2. non – ionizing radiations
Non – ionizing radiations
Ultraviolet rays are the only non ionizing radiation with mutagenic properties. The wave
o
length ranges from 100 – 3900 A And they are specifically absorbed by puriines and
pyrimidines present in DNA. The maximum absorption of UV rays by DNA as well as by
pyrimidines, particularly thymine occurs at the wavelength of 254 nm, which is also the most
mutagenic wavelength of UV. The mutagenic action of uv is the consequence of both its
direct and indirect effects on DNA. The direct effect of uv on DNA is of two types: formation
of (1) pyrimidine dimmers and pyrimidine hydrates.
Ionizing radiations
Ionizing radiations are so called because they cause ionization in the atoms present in their
path. There are two types of ionizing radiations: (1) particulate and (2) non particulate
radiations. Particulate radiations consist of high energy atomin particles generated due to
radioactive decay. The non particulate ionizing radiations are represented by X rays and
gamma rays which are high energy radiations composed of photons.
The genetic effects of radiations may be (1) direct or (2) indirect. The direct effect of
radiations is produced due to ionizations directly in the DNA molecule, while their indirect
effect is produced through ionizations ini molecules other than DNA and is believed to be
mediated by free radical formation.

Sources of radiation
Natural sources of radiation produce so-called background radiation. These include cosmic
rays from the sun and outer space, radioactive elements in soil and terrestrial products (wood,
stone) and in the atmosphere (radon). One's exposure due to background radiation varies with
geographic location.
In addition, humans have created artificial sources of radiation which contribute to our
radiation exposure. Among these are medical testing (diagnostic X-rays and other
procedures), nuclear testing and power plants, and various other products (TV's, smoke
detectors, airport X-rays).
Chemical mutagens
The first report of mutagenic action of a chemical was in 1942 by Charlotte Auerbach, who
showed that nitrogen mustard (component of poisonous mustard gas used in World Wars I
and II) could cause mutations in cells. Since that time, many other mutagenic chemicals have
been identified and there is a huge industry and government bureaucracy dedicated to finding
them in food additives, industrial wastes, etc. It is possible to distinguish chemical mutagens
by their modes of action; some of these cause mutations by mechanisms similar to those
which arise spontaneously while others are more like radiation in their effects.
1. Base analogs
These chemicals structurally resemble purines and pyrimidines and may be incorporated into
DNA in place of the normal bases during DNA replication:
Eg:5-bromouracil, 2-aminopurine, 5-chloro uracil, 5-iodo uracil
• bromouracil (BU)--artificially created compound extensively used in research.
Resembles thymine because it has Br in the 5 position instead of methyl group and
has the same effect on its base pairing behavior as that of –CH 3 in the same position
and therefore 5 BU behaves like thymine and usually pairs with adenine.
• aminopurine --adenine analog which can pair with T or (less well) with C; causes A:T
to G:C or G:C to A:T transitions. Base analogs cause transitions, as do spontaneous
tautomerization events.
2. Chemicals which alter structure and pairing properties of bases
There are many such mutagens; some well-known examples are:
 • nitrous acid--formed by digestion of nitrites (preservatives) in foods. It causes C to U,
me C to T, and A to hypoxanthine deaminations. Hypoxanthine in DNA pairs with C
and causes transitions. Deamination by nitrous acid, like spontaneous deamination,
causes transitions.
• nitroso guanidine, methyl methane sulfonate, ethyl methane sulfonate-chemical
mutagens that react with bases and add methyl or ethyl groups. Depending on the affected
atom, the alkylated base may then degrade to yield a baseless site, which is mutagenic
and recombinogenic, or mispair to result in mutations upon DNA replication.
3. Intercalating agents /Acridine dyes:
Acridine orange, proflavin, ethidium bromide (used in labs as dyes and mutagens). All are flat,
multiple ring molecules which interact with bases of DNA and insert between them. This insertion
causes a "stretching" of the DNA duplex and the DNA polymerase is "fooled" into inserting an extra
base opposite an intercalated molecule. The result is that intercalating agents cause frameshifts.
4. Agents altering DNA structure
This is used as a "catch-all" category which includes a variety of different kinds of agents.
These may be:
• --large molecules which bind to bases in DNA and cause them to be noncoding--we
refer to these as "bulky" lesions (eg. NAAAF)
• --agents causing intra- and inter-strand crosslinks (eg. psoralens--found in some
vegetables and used in treatments of some skin conditions)
• --chemicals causing DNA strand breaks (eg. peroxides)

What these agents have in common is that they probably cause mutations not directly but by
induction of mutagenic repair processes.

Detection of mutation :
The occurrence of mutational event at the gene level is detected by the altercation it brings
about in the phenotypic expression of one or more traits of the concerned organism.
Therefore the efficiency of detection of mutations will depend largely on the availability of
techniques for an easy and rapid scoring of the mutant phenotypes in very large populations.
Scoring of some types of mutations in certain organisms is relatively easy. For example,
mutations for antibiotic resistance in bacteria are simply detected by plating the bacterial cells
on a medium containing a lethal concentration of the concerned antibiotic (selective
medium); the colonies that develop on such a medium will be produced by cells resistant to
the antibiotic. The medium lacking the antibiotic is called the non selective medium.
Number of colonies on the selective medium
Frequency of mutant cells (%) = -------------------------------------------------- X 100
Number of colonies on the non selective medium
Detection of morphological mutations in eukaryotes requires examination of each individual
of the population for the mutant phenotype; this is not only tedious requiring time, but is also
a source of errors in the data. Therefore, elaborate procedures for mutation detection have
been developed in some eukaryotes . eg; Drosophila, maize etc. These procedures employ
specific markers to facilitate the identification of chromosomes from the treated or irradiated
individuals. Clearly these techniques detect only germinal mutations. In drosophila, several
special genetic stocks have been constructed for the detection of lethal and visible mutations
in X chromosomes and in autosomes; the two genetic stocks most commonly used for
mutation detection in X chromosome are (1) CIB and (2) Attached X stocks.
CIB Technique :
This method was invented by Muller and used for the unequivocal demonstration of
mutagenic action of X rays. In this method, females containing one normal X-chromosome
and another X-chromosome (CIB) containing extra 3 genes are used for the analysis. Out of
the 3 extra genes, one gene suppresses crossover (c), the other is a recessive lethal (L) in
heterozygous condition, and the last gene is semidominant marker, Bar (B) gene.
Females containing CIB chromosome are called as CIB stock drosophila. The normal males
are exposed to mutagenic source for a fixed period and then mated to the CIB stock
drosophila. Males containing CIB chromosome will die due to the effect of lethal genes,
whereas norm ill males and females both normal and with CIB will survive.
Females with CIB chromosomes and identified by barred phenotype are selected and crossed
to normal males. In this next generation 50% of males (which have received the CIB gene)
will die.
If mutation has occurred in normal X chromosome then even the normal male (without CIB
gene) will die. If no mutation has occurred all the other 50% of males will survive. The
frequency of lethal mutations can be accurately scored in large samples. This technique is
simple, rapid and there is little chance of an error in scoring. However, it is suitable for the
scoring of sex linked recessive lethal only.
Concept of Gene: Classical and Modern
Concepts: Gene as Cistron, muton and recon
The hereditary units which are transmitted from one generation to the next generation are
called genes. A gene is the fundamental biologic unit, like the atom which is the fundamental
physical unit. Mendel while explaining the result of his monohybrid and dihybrid crosses, first of
all conceived of the genes as particulate units and referred them by various names such as
hereditary factors or hereditary elements. But this concept about the gene was entirely
hypothetical and remained ignorant about the physical and chemical nature of gene.
Even before the rediscovery of Menders laws in 1900, it was already established that
chromosomes have a definite role in the inheritance because it was found that chromosomes
were the only link between one generation and the next generation and a diploid chromosome set
consists of two morphologically similar sets, one is derived from the mother and the other from
the father at fertilization. Later on, a parallel behavior among chromosomes and genes was
discovered.

Earlier workers proposed various hypotheses to explain the nature of genes. For instance,
De Vries postulated one gene one character hypothesis according to which a particular trait of an
individual is controlled by a particular gene. Bateson and Punnett proposed the presence or
absence theory. According to them, in a cross the character which dominates the other has a
determiner, while, the recessive character has no such determiner. But all the theories were
discarded by Morgan, who produced the particulate gene theory in 1926. He considered genes as
corpuscles, which are arranged in a linear order on the chromosomes and appear like beads on a
string. Each gene was supposed to be different from ail others. The particulate theory of gene
was widely accepted and supported by cytological observations. But, the discovery of DNA
molecule as a sole carrier of genetic information base altogether discarded the Morgan's theory.
Therefore, before defining the gene it will be advisable to consider both the classical as well as
modern definitions of gene.
Changing Concept of Gene

The concept of gene has been the focal point of study from the beginning of twentieth century to
establish the basis of heredity. The gene has been examined from two main angles, i.e., (1)
genetic view, and (2) biochemical and molecular view. These aspects are briefly described
below:

(1) A Genetic View: The genetic view or perspective of gene is based mainly on the Mendelian
inheritance, chromosomal theory of inheritance and linkage studies. Mendel used the term
factors for genes and reported that factors were responsible for transmission of characters from
parents to their offspring. Sutton and Boveri (1903) based on the study of mitosis and meiosis in
higher plants established parallel behaviour of chromosomes and genes. They reported that both
chromosomes and genes segregate and exhibit random assortment, which clearly demonstrated
that genes are located on chromosomes. The Sutton- Boveri hypothesis is known as chromosome
theory of inheritance.

Morgan based on linkage studies in Drosophila reported that genes are located on the
chromosome in a linear fashion. Some genes do not assort independently because of linkage
between them. He suggested that recombinants are the result of crossing over. The crossing over
increases if the distance between two genes is more. The number of linkage group is the same as
the number of chromosomes. The chromosome theory and linkage studies reveal that genes are
located on the chromosomes. This view is sometimes called as bead theory. The important points
about the bead theory are given below:
1. The gene is viewed as a fundamental unit of structure, indivisible by crossing over. Crossing
over occurs between genes but not within a gene.
2. The gene is considered as a basic unit of change or mutation. It changes from one allelic form
to another, but there are no smaller components within a gene that can change.
3. The gene is viewed as a basic unit of function. Parts of a gene, if they exist, cannot function.

The chromosome has been viewed merely as a vector or transporter of genes and exists simply to
permit their orderly segregation and to shuffle them in recombination. The bead theory is no
more valid for any of the above three points. Now evidences are available which indicate that:
(1) a gene is divisible (2) part of a gene can mutate, and (3) part of a gene can function.

The Gene is Divisible

Earlier it was believed that gene is a basic unit of structure which is indivisible by crossing over.
In other words, crossing over occurs between genes but not within a gene. Now intragenic
recombination has been observed in many organisms which indicates that a gene is divisible. The
intragenic recombination has following two main features.

1. It occurs with rare frequency so that a very large test cross progeny is required for its
-6
detection. Benzer expected to detect a recombination frequency as low as 10 , the lowest he
-4
actually found was 10 (0.01 x 2 = 0.02%).
2. The alleles in which intragenic recombination occurs are separated by small distances within a
gene and are functionally related.

Examples of intragenic recombination include bar eye, star asteroid eye and lozenge eye
in Drosophila. The bar locus is briefly described below. Lozenge eye and star asteroid have been
discussed under pseudo alleles.

Bar Eye in Drosophila

The first case of intragenic recombination was recorded in Drosophila for bar locus which
controls size of eye. The bar locus contains more than one unit of function. The dominant bar
gene in Drosophila produces slit like eye instead of normal oval eye. Bar phenotype is caused by
tandem duplication of 16A region in X chromosome, which results due to unequal crossing over.
The flies with different dose of 16A region have different types of eye as follows:
1.Single 16A region → Wild type oval eye
2.Double 16A region → Bar eye small in size
3.Triple 16A region → Double bar or ultrabar eye very small in size

The homozygous bar eye (B/B) produced both wild and ultra bar types though at a low frequency
which indicated intragenic recombination in the bar locus but the frequency was much higher
than that expected due to spontaneous mutations.

Part of a Gene Can Function

It was considered earlier that gene is the basic unit of function and parts of gene, if exist, cannot
function. But this concept has been outdated now. Based on studies on rll locus of T4 phage,
Banzer (1955) concluded that there are three sub divisions of a gene, viz., recon, muton
and cistron. These are briefly described below:

Recon: Recons are the regions (units) within a gene between which recombinations can occur,
but the recombination cannot occur within a recon. There is a minimum recombination distance
within a gene which separates recons. The map of a gene is completely linear sequence of
recons.
Muton: It is the smallest element within a gene, which can give rise to a mutant phenotype or
mutation. This indicates that part of a gene can mutate or change. This disproved the bead theory
according to which the entire gene was a mutate or change.

Cistron: It is the largest element within a gene which is the unit of function. This also nocked
down the bead theory according to which entire gene was the unit of function. The name cistron
has been derived from the test which is performed to know whether two mutants are within the
same cistron on in different cistrons. It is called cis-trans test which is described below.
Cis – Trans Test : When two mutations in trans position produce mutant phenotype, they are in
the same cistron. Complementation in trans position (appearance of wild type) indicates that the
mutant sites are in different cistrons. There is no complementation between mutations within a
ciston.

It is now known that some genes consist of only one cistron; some consist of two or even more.
For example, the mutant miniature (m) and dusky (dy) both decrease wing size in Drosophila and
map in the same part of X chromosome. But when brought together in dy +/+m heterozygote, the
phenotype is normal which indicates that the locus concerned with wing size is composed of at
least two cistrons.

(2) A Biochemical View: It is now generally believed that a gene is a sequence of nucleotides in
DNA which controls a single polypeptide chain. The different mutations of a gene may be due to
change in single nucleotide at more than one location in the gene. Crossing over can take place
between the altered nucleotides within a gene. Since the mutant nucleotides are placed so close
together, crossing over is expected within very low frequency. When several different genes
which affect the same trait are present so close that crossing over is rare between them, the term
complex locus is applied to them. Within the nucleotide sequence of DNA, which represents a
gene, multiple alleles are due tomutations at different points within the gene.

Fine Structure of Gene: Benzer (1955) divided the gene into recon, muton and cistron which
are the units of recombination, mutation and function within a gene. Several units of this type
exist in a gene. In order words, each gene consists of several units of function, mutation and
recombination. The fine structure of gene deals with mapping of individual gene locus. This is
parallel to the mapping of chromosomes. In chromosome mapping, various genes are assigned
on a chromosome, whereas in case of a gene several alleles are assigned to the same locus. The
individual gene maps are prepared with the help of intragenic recombination. Since the
frequency of intragenic recombination is extremely low, very large population has to be grown to
obtain such rare combination. Prokaryotes are suitable materials for growing large population. In
Drosophila, 14 alleles of lozenge gene map at four mutational sites which belong to the same
locus (Green, 1961). Similarly, for rosy eye in Drosophila, different alleles map at 10 mutational
sites of the same locus.

Genes can be classified in various ways. The classification of genes is generally done on the
basis of (1) dominance, (2) interaction, (3) character controlled, (4) effect on survival, (5)
location, (6) movement, (7) nucleotide sequence, (8) sex linkage, (9) operon model, and (10) role
in mutation. A brief classification of genes on the basis of above criteria is presented below

Classification and brief description of A brief description

genes Classification of genes
Based on Dominance
Dominant genes Genes that express in the F1
Recessive genes Genes whose effect is suppressed in F1
Based on Interaction
Epistatic gene A gene that has masking effect on the other gene
controlling the same trait.
Hypostatic gene A gene whose expression is masked by another gene
governing the same trait
Based on Character Controlled
Major gene A gene that governs qualitative trait. Such genes have
distinct phenotypic effects.
Minor gene A gene which is involved in the expression of
quantitative trait. Effect of such genes cannot be
easily detected.

More about Genes: There are some genes which are different from normal genes either in terms
of their nucleotide sequences or functions. Some examples of such genes are split gene, jumping
gene, overlapping gene and pseudo gene. A brief description of each of these genes is presented
below:

Split Genes: Usually a gene has a continuous sequence of nucleotides. In other words, there is
no interruption in the nucleotide sequence of a gene. Such nucleotide sequence codes for a
particular single polypeptide chain. However, it was observed that the sequence of nucleotides
was not
continuous in case of some genes; the sequences of nucleotides were interrupted by intervening
sequences. Such gene with interrupted sequence of nucleotides is referred to as split genes or
interrupted genes. Thus, split genes have two types of sequences, viz., normal sequences and
interrupted sequences.

1. Normal Sequence. This represented the sequence of nucleotides which are included in the
mRNA which is translated from DNA of split gene These sequences code for a particular
polypeptide chain and are known as exons.

2. Interrupted Sequence: The intervening or interrupted sequence of split gene are known as
introns. These sequences do not code for any peptide chain. Moreover, interrupted sequences
are not included into mRNA which is transcribed from DNA of split genes. The interrupted
sequences are removed from the mRNA during processing of the same. In other words, the
intervening sequences are discarded in mRNA as they are non-coding sequences. The coding
sequences or exons are joined by ligase enzyme.

The first case of split gene was reported for ovalbumin gene of chickens. The ovalbumin gene
has been reported to consist of seven intervening sequences.. Later on interrupted sequences
(split genes) were reported for beta globin gene of mice and rabbits, tRNA genes of yeast and
ribosomal genes of Drosophila.

The intervening sequences are determined with the help of R loop technique. This technique
consists of hybridization between mRNA and DNA of the same gene under ideal conditions, i.e.,
at high temperature and high concentration of formamide. The mRNA pairs with single strand of
DNA. The non-coding sequences or intervening sequences of DNA make loop in such pairing.
The number of loops indicates the number of interrupted sequences and the size of loop indicates
length of the intervening sequence. These loops can be viewed under electron microscope. The
ovalbumin gene has seven interrupted sequences (introns) and eight coding sequences (exons).
The beta globin gene has been reported to have two intervening sequences, one 550 nucleotides
long and the other 125 nucleotides long.
The intervening sequences are excised during processing to form mature mRNA molecule. Thus,
about half of the ovalbumin gene is discarded during processing. Earlier it was believed that
there is colinearity (correspondence) between the nucleotide sequence and the sequence of amino
acids which it specifies. The discovery of split genes has disproved the concept of colinearity of
genes. Now colinearity between genes and their products is considered as a chance rather than a
rule. Split genes have been reported mostly in eukaryotes.

Jumping Genes: Generally, a gene occupies a specific position on the chromosome called locus.
However in some cases a gene keeps on chaining its position within the chromosome and also
between the chromosomes of the same genome. Such genes are known as jumping genes or
transposons or transposable elements. The first case of jumping gene was reported by Barbara
Mc-Clintock in maize as early as in 1950. However, her work did not get recognition for a long
time like that of Mendel. Because she was much ahead of time and this was an unusual finding,
people did not appreciate it for a long time. This concept was recognized in early seventies and
McClintock was awarded Nobel Prize for this work in 1983.
Later on transposable elements were reported in the chromosome of E. coli and other
prokaryotes. In E.coli, some DNA segments were found moving from one location to other
location. Such DNA segments are detected by their presence at such a position in the nucleotide
sequence, where they were not present earlier. The transposable elements are of two types, viz,
insertion sequence and transposons.

1. Insertion Sequence. There are different types of insertion sequences each with specific
properties. Such sequences do not specify for protein and are of very short length. Such
sequence has been reported in some bacteria bacteriophages and plasmids.

2. Transposons. These are coding sequences which code for one or more proteins. They are
usually very long sequences of nucleotides including several thousand base pairs.
Transposable elements are considered to be associated with chromosomal changes such as
inversion and deletion. They are hot spots for such changes and are useful tools for the study of
mutagenesis. In eukaryotes, moving DNA segments have been reported in maize, yeast and
Drosophila.

Overlapping Genes: Earlier it was believed that a nucleotide sequence codes only for one
protein. Recent investigations with prokaryotes especially viruses have proved beyond doubt that
some nucleotide sequences (genes) can code for two or even more proteins. The genes which
code for more than one protein are known as overlapping genes. In case of overlapping genes,
the complete nucleotide sequence codes for one protein and a part of such nucleotide sequence
can code for another protein. Overlapping genes are found in tumor producing viruses such as φ
X 174, SV 40 and G4, in virus φ X 174 gene A overlaps gene B. In virus SV 40, the same
nucleotide sequence codes for the protein VP 3 and also for the carboxyl – terminal end of the
protein VP2. In virus G4, the gene A overlaps gene B and gene E overlaps gene D. The gene of
this virus also contains some portions of nucleotide sequences which are common for gene A and
gene C.

Pseudo genes: There are some DNA sequences, especially in eukaryotes, which are non-
functional and defective copies of normal genes. These sequences do not have any function.
Such DNA sequences or genes are known as pseudo genes. Pseudo genes have been reported in
humans, mouse and Drosophila. The main features of pseudo genes are given below :
1. Pseudo genes are non functional or defective copies of some normal genes. These genes are
found in large numbers.
2. These genes being defective cannot be translated.
3. These genes do not code for protein synthesis, means they do not have any significance.
4. The well known examples of pseudo genes are alpha and beta globin pseudo genes.
*****
 Variation in Chromosome Structure

There are four types of structural aberrations of chromosomes

1. Deletions/deficienciies
2. Duplications
3. Inversions
4. Translocations

1. Deficiencies (Deletions): A deficiency is the loss of a segment of a

chromosome or a whole chromosome. Since loss of whole chromosomes will
be dealt in separate chapter on numerical aberrations of chromosomes here
we are restricted to the treatment of deficiencies of chromosome segments
only. The presence of a deficiency in one of the two chromosomes in a
homologous pair results in the presence of some specific gens only on one of
the two chromosomes of the pair. This condition in a deficiency heterozygote
is termed hemizygous condition.
Types of deficiencies: deficiencies can be either
1. terminal deletion or
2. Intersititial deficiency

Terminal deletion can result from asingle break, the latter will need two
breaks folloed by fusion of the broken ends.

Terminal deficiencies will survive, only when the broken end heals up and
gives rise to a telomere. It is bnelieved that healing can take place if
ultraviolet radiation causes the break.

Origin:

1. Spontaneous
2. Irradiation through x rays and neutrons
3. Through gametocidal chromosome
Deletion or Deficiency homozygote: Both the chromosomes of a
homologous pair carry deletion.
Normal pair of chromosomes ( . ) represents centromere
12345.6789
12345.6789
Terminal Deletion homozygote:

12345.67
12345.67
Interstitial deletion homozygote

125.6789
125.6789

Deletion or deficiency heterozygote: Of the two chromosomes of a

homologous pair one chromosome is normal and the other carry deletion

Normal pair of chromosomes ( . ) represents centromere

12345.6789
12345.6789
Terminal Deletion heterozygote:

12345.6789
12345.67
Interstitial deletion heterozygote

1234 5.6789
12 5.6789

Meiosis and breeding behavior of deficiency heterozygotes:

A deficiency may occur in homozygous or heterozygous condition. In
homozygous condition, deficiencies may not survive, unless it is a very
small deficiency, when it behaves like a recessive allele for the gene
located on that locus. Cytologically the deficiencies in homozygous
condition cannot be easily detected unless with the help of missing bands
in salivary gland chromosomes. However, in deficiency heterozygote, a
characteristic buckling in salivary gland chromosome or a loop in the
pachytene chromosomes will indicate the presence of deficiency. In case
of terminal deficiency, instead of a loop an unpaired stretched segment of
a chromosome is observed.

Significance:
Unless the deficiency is very small, deficiencies are usually lethal to both
pollen and ovules.
In heterozygous condition, in the absence of a dominant allele due to
deletion recessive allele is expressed. This phenomenon is called pseudo
dominance.
 Duplications

A gain of a segment of a chromosome in the genome is called a duplication.

Origin:

The duplications can originate spontaneously and can also be produced artificially

1. Natural origin
2. Origin from inversion and translocation heterozygotes
3. Through irradiation treatments
4. Unequal crossing over

Types of duplications:

Normal chromosome

Eg: A B C D . E F G H I

DUPLICATED SEGMENT IS F G

1. Tandem duplication: the duplicated segment is present adjacent to the

original segment in the same direction

Eg: A B C D . E F G F G H I

2. Reverse tandem duplication: here duplication segment is present adjacent

to the original segment but in reverse direction
Eg: A B C D . E F G G F H I
 3. Displaced duplication: the duplicated segment is present on the same
chromosome but at different location
a) Homobrachial displaced duplication: duplicated segment is present on the
same arm of the chromosome but not adjacent to the original segment
Eg: A B C D . E F G H F G I

b) Heterobrachial displaced duplication: duplicated segment is present on the

arm of the same chromosome
Eg: A B F G C D . E F G H I

4. Transposed duplication: duplicated segment is present on the non

homologous chromosome

Duplication homozygote: Both the chromosomes of a homologous pair carry

duplication

Eg: ABCD.EFGFGHI

ABCD.EFGFGHI

Duplication heterozygote: in a pair of homologous chromosome one chromosome

carries duplication and other chromosome is normal

ABCD.EFGHI

ABCD.EFGFGHI

MEIOSIS:

Duplicated segment will often form a loop during meiosis due to non availability
of a segment to pair with it. Such a loop can be seen in pachytene chromosomes
of high plants or in salivary gland chromosomes of a duplication heterozygote.

Crossing over in a bivalent carrying duplication in one of the two chromosomes

may lead to different consequences.
Significance:

1. The duplications of chromosomes are not deleterious to the organism like the
deficiency, but, they usually protect the organism from the effect of a deleterious
recessive gene or from an otherwise lethal deletion.

2. Some duplications are useful in the evolution of new genetic material. In an

organism with duplications, because the old genes can continue to provide for the
present requirements of the organism, the superfluous genes may be free to
mutate to new forms without a loss in immediate adaptability.

3. Large duplications can reduce the fertility as a result of meiotic complication,

and in this way reduce their own probability of survival.

4. Relocation of chromosomal material without altering its quantity may result in

an altered phenotype, this is called position effect.
 INVERSIONS

An inversion is an intra-chromosomal aberration in which a segment is inverted

180 degrees resulting in inversion of genetic information contained the segment. For
example if a chromosome has segments in the order of 1-2-3-4-5-6 and breaks occur in
regions 2-3 and 5-6 and the broken piece (3-4-5-) is reinserted in reverse order, then the
inverted chromosome will have segments in order of 1-2-5-4-3-6, such as shown in the
figure 21.5:

The origin of an inversion (after Stansfield, 1969).

In a diploid organism, when out of two homologous chromosomes one

chromosome undergoes the inversion, then, it is called inversion heterozygote. During
synapsis of such a homologous pair having inversion heterozygoe, the synapsis
configuration attempts to maximize the pairing between homologous regions in the two
chromosomes. This is usually accomplished by a characteristic inversion loop in one of
the chromosome.
The inversion involves two chromosomal breakages and two rejoining within a
chromosome.
Types of inversions

The inversions are of two types:

i) Pericentric inversions – When the inverted segment of chromosome includes or

contains centromere, then such inversions are called heterobranchial or pericentric
inversions. If crossing over occurs within the loop of a pericentric inversion, the resulted
chromatids include half on the chromatids with duplications and deficiencies forming
nonfunction. The other half of the chromatids form functional gametes: ¼ gametes have
normal chromosome order, ¼ gametes have the inverted arrangement.

ii) Paracentric inversions – When the inverted segment includes no centromere and the
centromere remains located outside the segment, then such type of inversion is called
homobranchial or paracentric inversion. Crossing over within the inverted segment of a
paracentric inversion, produces a diacentric chromosome contains two centromeres and
forms a bridge from one pole to the other during first meiotic anaphase. When
anaphase chromosomes separate towards poles, this bridge breaks somewhere along its
length and the resulting fragments contain duplications and/ or deficiencies. The
acentric chromosome because lacks in centromere and fails to move to either pole and
so, is not included in the meiotic products. Such, breakage-fusion bridge cycles of
crossing over of paracentric inversions are most common in maize. The meiotic products
includes half non-functional, ¼ functional normal and ¼ functional inverted
chromosomes.

Genetic significance of inversions

i) There is change in order of arrangements of genetic information.

ii) This leads to change in linkage relationship among the genes present in inverted
segment.
iii) There is problem of pairing of homologous chromosomes, due to which there is
suppression of crossing over in the inverted region.
iv) Paracentric inversion leads to formation of diacentric chromosomes resulting in
bridge breakage fusion cycle.
v) Pericentric inversion can alter the morphology of the inverted chromosomes.
Meiotic behavior:

Paracentric Inversion

Only 50% gametes are viable and other 50% are nonviable because of deletion and duplication.

Pericentric Inversion

Only 50% gametes are viable and other 50% are nonviable because of deletion and duplication.

Because of genetic variability and sterility can lead to crossability barrier which in turn lead to speciation
in course of evolution. Eg.: Drosophila melanogaster.

TRANSLOCATION

It involves a non-homologous chromosomes and it is a chromosomal structural

change involving change in position of a segment of chromosome by movement to non-
homologous chromosomes within a genome.
Different types
1. Shift translocation : Different types
a) Intra chromosomal shift: Transfer of segment within the same arm,
OR Transfer of segment to different arm of the same chromosome
 b) Inter chromosomal shift: Transfer of segment to inter calary position
of another chromosome
2. Simple :There is a single break and transfer of segment to the end of non-
homologous chromosomes, however it is not possible
3. Reciprocal: It is very common. It involves exchange of broken
chromosomal fragments between two non-homologous chromosomes
4. Complex

Genetic significance of Heterozygotic Translocation:

1. Translocation leads to change in linkage group.

2. Change in relationship between loci of chromosome
3. It can lead to change in Karyotype of genome
4. It has lead to speciation ie lead to generation of new species. This type of species found
in nature frequently in heterozygous genomes.
This is observed in Oenothera lamarhiana, Rhoeo discolor, Tradenscantia

Translocation involves the shifting of a part of one chromosome to another non-

homologous chromosome. If two non-homologous chromosomes exchange parts, which
need not be of the same size, the result is a reciprocal translocation. The reciprocal
translocation may be of following types:

1. Translocation homozygote - In homozygotic translocation normal meiosis occur

and cannot be detected cytologically. Genetically they are marked by altered
linkage group by the fact that a gene with new neighbours may produce a
somewhat different effect in its new location (position effect).

Homozygotic and heterozygotic

translocations (after De Robertis, Saez and
Nowinski 1970)
 2. Translocation Heterozygote – In heterozygotic translocation a considerable
degree of meiotic irregularity occur. During meiosis, an individual which is
heterozygous for a reciprocal translocation must form a cross-shaped
configuration in order to affect pairing of all homologous segments. This cross-
shaped configuration often opens out into a ring as chiasmata terminalize. The
meiotic products (gametes) are of three types –normal balanced and
unbalanced gametes as have been illustrated in the fig.(attached in the different
file)

Meiosis in heterozygotic translocation (after Sutton 1965).

There are three types of separation in heterozygotic translocation

Adi I: Two non homologous centromeres move to similar pole

Adj II: Homologous centromeres move to one pole.

Adj III: Alternate separation:

In Adj I and Adj II separation there is complete sterility while Adj III separation only functional gametes
are produced.

Some other structural aberrations

1. Ring chromosomes: Produced when both ends of chromosomes are damaged.

2. Diacentric chromosomes: Produced by fusion of two damaged chromosomes

3. Isochrmosomes: Produced by misdivision of centromere ie vertical division of centromere,

because of this, isochrmosomes with Same genetic content are produced.
Variation in chromosome number: Euploid, Aneuploid
Types of changes in Chromosome Number
The somatic chromosome number of any species, whether diploid or polyploid, is
designated as 2n, and the chromosome number of gametes is denoted as n. An individual
carrying the gametic chromosome number, n, is known as haploid. A monoploid, on the
other hand, has the basic chromosome number, x. In a diploid species, n= x; one x
constitutes a genome or chromosome complement. The different chromosomes of a
single genome are distinct from each other in morphology and/ or gene content and
homology; members of a single genome do not show a tendency of pairing with each
other. Thus, a diploid species has two, a triploid has 3 and a tetraploid has 4 genomes and
so on.

Individuals carrying chromosome numbers other than the diploid (2x, and not 2n)
number are known as heteroploids, and the situation is known as heteroploidy.

The change in chromosome number may involve one or a few chromosomes of

the genome; this is known as aneuploidy.

Heteroploidy that involves one or more complete genomes is known as euploidy.

By definition, therefore, the chromosome numbers of euploids are an exact

multiple of the basic chromosome number of the concerned species.

A summary of the terms used to describe heteroploidy (variation in chromosome number)

Term Type of change Symbol*

Heteroploid A change from 2x -
A. Aneuploid One or a few chromosomes extra or 2n ± few
missing from 2n
Nullisomic One chromosome pair missing 2n – 2
Monosomic One chromosome missing 2n – 1
Double monosomic One chromosome from each of two
different chromosome pairs missing 2n – 1 – 1
Trisomic One chromosome extra 2n + 1
Double trisomic One chromosome from each of two
different chromosome pairs extra 2n + 1 + 1
Tetrasomic One chromosome pair extra 2n + 2

B. Euploid Number of genomes or copies of a

genome more than two x
Monoploid One copy of a single genome n
Haploid Gametic chromosome complement
Polyploid More than 2 copies of one genome or 2
 copies each of 2 or more genomes**
1. Autoployploid Genomes identical with each other 3x
Autotriploid Three copies of one genome 4x
Autotetraploid Four copies of one genome 5x
Autopentaploid Five copies of one genome 6x
Autohexaploid Six copies of one genome 8x
Autooctaploid Eight copies of one genome
2. Alloployploid Two or more distinct genomes
AABB (generally each genome has two (2x1 + 2x2)**
copies)** (2x1+ 2x2 + 2x3)**
Allotetraploid Two distinct genomes (2x1+2x2+2x3+2x4)**
Allohexaploid Three distinct genomes
Allooctaploid Four distinct genomes
*2n = Somatic chromosome number (and complement) and n = gametic chromosome
number (and complement) of the species, whether diploid or polyploid.
X = The basic chromosome number (and complement) or genomic number.
x1, x2, x3, x4 = Distinct genomes from different species.
** In general, this condition occurs; other situations may also occur.

Aneuploid individuals from which one chromosome pair is missing (2n – 2) are
termed as nullisomics, while those lacking a single chromosome (2n – 1) are known as
monosomics. A double monosomic individual has two chromosomes missing, but the
two chromosomes belong to two different chromosome pairs (2n – 1 – 1). An individual
having one extra chromosome (2n + 1) is known as trisomic, and that having two extra
chromosomes each belonging to a different chromosome pair is called double trisomic
(2n + 1 +1). When an individual has an extra pair of chromosomes, it is known as
tetrasomic (2n + 2). The detailed terminology describing aneuploidy in very complex,
The breeder is generally concerned with monosomics and trisomics, and in some
situations, with nullisomics and tetrasomics.
In euploids, the chromosome number is an exact multiple of the basic or genomic
number. Euploidy is more commonly known as polyploidy. When all the genomes
present in a polyploidy species are identical, it is known as autopolyploid and the
situation in termed as autopolyploidy. In the case of allopolyploids, two or more distinct
genomes are present. Euploids may have 3 (tripoid), 4 (tetraploid), 5 (pentaploid), 6
(hexaploid), 7 (heptaploid), 8 (octaploid) or more genomes making up their somatic
chromosome number.
In case of autopolyploidy, they are known as autotriploid autotertaploid,
autopentaploid, autohexaploid, autoheptaploid, autooctaploid and so on, while in the case
of allopolyploidy they are termed as allotriploid, allotetraploid, allopentaploid,
allohexaploid, alloheptaploid, allooctaploid etc.

Amphidiploid is an allopolyploid that has two copies of each genome present in it

and, as a consequence, behaves as a diploid during meiosis. A segmental allopolyploid
contains two or more genomes, which are identical with each other, except for some
minor differences.
 Origin of Haploids
These are saprophytes having karyotypic number and genetic constitution.
Origin of haploids
Spontaneous: Parthenogenic development of egg or any other cell of embryosac into
embryo. Eg. Maize
Artificial: Irradiation through X-rays and gama rays leading to production of haploids. Eg:
N.tobaccum, N. glutinosa, N.rustica, T.aestivum, T.persicum
Temperature shock
Delay in pollination leads to production of haploids, depends on the receptivity of the
stigma and pollen.
Wide Hybridization
S.tuberosum X S. phereja
2n=4x=48 ↓ 2n=2x=24
2n=2x=24(Dihaploid)
Halpoid inducing genes
Maize: ig
Barley: ‘hap’ gene (haploid initiator gene)
Semigamy: eg cotton
Chrmosome Elimination
Eg. Barley (Hordeum volgare X H. bulbosum) Wheat X H. bulbosum
↓ ↓
Haploid wheat
Uses
1. Mutation studies
2. Production of homozygous lines
3. Haploids are used to find out the basic chromosome number.

Polyploids – Auto and Allopolyploids

AUTOPOLYPLOIDY

In autopolyploidy are included triploidy, tetraploidy and higher levels of ploidy.

Autopolyploids are produced directly or indirectly through chromosome doubling, which
is briefly considered as follows.

Origin and production of Doubled Chromosome Numbers

Cells/ individuals having doubled chromosome numbers may originate in one of

the following several ways:

(1) Spontaneous,
(2) Due to treatment with physical agents,
(3) Regeneration in vitro,
(4) Colchicine treatment, and
(5) Other chemical agents.

(1) Spontaneous – Chromosome doubling occurs occasionally in somatic tissues and

unreduced gametes are also produce in low frequencies.

(2) Physical Agents – Heat or cold treatments, and X–ray or gamma-ray irradiation
may produce polyploids in low frequencies. Tetraploid branches were produced in
Datura in response to cold treatment. Exposure of maize (Z.mays) plants or ears to
a temperature of 38-45OC at the time of the first division of zygote produces 2-5
per cent tetraploid progeny. Heat treatment has been successfully used in barley
(H.vulgare), wheat (T.aestivum), rye (S.cereale) and some other crop species.

(3) Regeneration in vitro – Polyploidy is a common feature of the cells cultured in

vitro. Some of the plants regenerated from callus and suspension cultures may be
polyploids. Plants of various ploidy have been regenerated from callus cultures of
Nicotiana, Datura, rice (O. sativa) and several other species.

(4) Colchicine treatment – Colchincine treatment is the most effective and the most
widely used treatment for chromosome doubling. It has been used with great
success in a large number of crop species belonging to both dicot and monocot
groups. Pure colchicine is C22H25O6N. It blocks spindle formation and thus
inhibits the movement of sister chromatids to the opposite poles. The resulting
restitution nucleus includes all the chromatids; as a result, the chromosome
number of the cell is doubled.

(5) Other chemical agents – several other chemical have polyploidizying effect.
Notable among them are, 8- hydroxyquinoline and nitrous oxide. These
chemicals are much less effective than colchicine and are not commonly used.
Morphological and Cytological Features of Autopolyploids

Morphological features of polyploids vary to some extent from one species to the
other. Some general features are summarized below.

1. Polyploids have larger cell size than diploids. Guard cells of stomata are
larger, and the number of stomata per unit area is lower in polyploids than in
diploids.
2. Pollen grains of polyploids are generally larger than those of the
corresponding diploids.
3. Polyploids are generally slower in growth and later in flowering.
4. Polyploids usually have larger and thicker leaves, and larger flowers and
fruits, which are usually less in number than the diploids.
5. Polyploids generally show reduced fertility due to irregularities during
meiosis and due to genotypic imbalance.
6. In many cases, autopolyploidy leads to an increase in general vigour and
vegetative growth. But in some cases polyploids are smaller and weaker.
7. Different species have different levels of optimum ploidy. For sugarbeet
(Beta vulgaris), the optimum level is 3x, while for Timothy it is between 8-
10x.
 Autopolyploid crop species

Somatic Somatic
chromosome number chromosome number
Common name Scientific name
(2n) of the cultivated of related wild
form species
Potato Solanum tuberosum 48 (4x) 24 (2x) form of
S. tuberosum
Coffee Coffea arabica 44 (4x) 22,66,68
Alfalfa Medicago sativa 32 (4x) 14,16,32
Banan Musa sapientum 33 (3x)
a (M.paradisiaca)
Sweet Ipomoea batatas 90 (6x) 22
potato
-

Application of Autopolyploidy in Crop Improvement

Autopolyploidy has found some valuable applications in crop improvement.

These are briefly summarised below.

Triploids: Triploids are produced by hybridization between tetraploid and diploid

strains. They are generally highly sterile, except in a few cases. This feature is useful in
the production of seedless watermelons. In certain species, they may be more vigorous
than the normal diploids, e.g., in sugarbeets.

Seedless watermelons are grown commercially in Japan. They are produced by crossing
tetraploid (4x, used as female) and diploid (x, used as male) lines. The triploid plants do
not produce true seeds; almost all the seeds are small, white rudimentary structures like
cucumber seeds. But a few normal sized seeds may occur, which are generally empty.
For good fruit setting, pollination is essential. For this purpose, diploid lines are planted
in the ratio 1 diploid: 5 triploid plants.

Triploid sugarbeets (B. vulgaris) produce larger roots and more sugar per unit area than
diploids, while tetraploids produce smaller roots lower yields than diploids. Apparently,
3x is the optimum level of poloidy in sugarbeets. Triploid sugarbeet varieties have been
grown commercially in Europe and Japan. Seed production of triploid sugarbeet is
difficult because the beet flower is small. Triploid seed may by produced in one of the
following two ways: (1) using 4x plants as females and 2x as male or (2) using 4x as
male and 2x as female. Commercial triploid sugarbeet seed in produced by interplanting
4x and 2x lines in the ratio 3 : 1. Triploid sugarbeet may give 10-15 per cent higher yields
than diploids.

A triploid (3x) clone of tea (Camelia assamica) has been recently released by the Tea
Research Association, India, for cultivation in the Northern parts of the country. The
triploid cultivar, TV 29, produces larger shoots and , thereby, biomass, yields more cured
leaf per unit area and is more tolerant to drought than the available diploid cutlivars.

ALLOPOLYPLOIDY

Allopolyploids have genomes from two or more species. Several of our crop
plants are allopolyploids. Production of allopolyploids has attracted considerable
attention; the aim almost always was the creation of new species. Some success has been
obtained as is evident from the emergence of Triticale as a new crop species in some
areas, and the promise shown by some other allopolyploids, e.g., Raphanobrassica and
some allopolyploids of forage grasses.

Some genera, which contain allopolyploid species, and one or more crop species.
The crop species themselves may be allopolyploid or diploid. Genera like Triticum,
Brassica and Gossypium have both diploid and allopolyploid crop spices.

Gametic Cultivated
Scientific name Common name chromosome / Wild**
number (n)
A. sativa Cultivated oats 14 C
B. oleracea Cabbage 9 (C) C
B. juncea Rai, Indian mustard 18 C
Rape
B. napus Asiatic (desi) cotton 19 C
Gossypium arboreum Asiatic cotton 13 (A2) C
G. herbaceum Wild American cotton
G. thurberi Sea island (Egyptian) cotton 13 (A1) W
American upland cotton 13 (D1) W
G. barbadense Cultivated barley
G. hirsutum Noble canes 26 (A2D2) C
Hordeum vulgare Indian canes C
Saccharum officinarum Indan canes 26 (A1D1) C
S. barberi Kans (wild canes) C
7 C
S. sinense C
S. spontaneum 40 W

41,45,46,58,62
58,59
20-64

* Letters within parentheses denote the symbols used for genomes present in the species.
**C = Cultivated; W = Wild.
 Role of polyploidy in evolution of crops – Wheat, Cotton, Tobacco and Brassica
Role of Allopolyploidy in Evolution:
It is estimated that about one-third of the Angiosperms are polyploids, and by far
the vast majority of them are allopolyploids.

The identification of parental diploid species is primarily based on pairing

between the chromosomes of the diploid and the allopolyploid species. When the
chromosomes of a diploid species pair with some of those of the allopolyploid species,
homology between chromosomes of the two species is apparent. This homology suggests
that the diploid species may be one of the parental species of the allopolyploid.

We shall briefly consider the possible evolutionary history of some important

allopolyploid crop species, viz., wheat, tobacco, cotton and Brassica.

Evolution of Bread Wheat (Triticum aestivum). Evolutionary history of wheat has been
the most extensively investigated, and is perhaps the least understood. Identity of the
diploid species contributing the three genomes (A, B, and D genomes) of T. aestivum has
been investigated by many workers, more notably by Sears, Kihara and others.

Triticum monococcum x Aegilops speltoids

n=7 n=7
AA BB
AB Sterile.
Spontaneous
chromosome
doubling.
Tetraploid emmer wheats
T. Tauschii x AA BB
(n = 7, DD) (n = 14) (T. dicoccum)
AMPHIDIPLOID
(T. dicoccum)

ABD Sterile

SPONTANEOUS
CHROMOSOME
DOUBLING.
AA BB DD Hexaploid wheat.
(n = 21)
Amphidiploid.
Triticum aestivum
Evolution of Nicotiana tabacum. N. tabacum (n = 24) is most likely an amphidiploid
from the cross N. sylvestris(AA) x N. tomentosa (BB); both the species are diploid with n
= 12. The interspecific hybrids N. tabacum x N. sylvestris and N. tabacum x N. tomentosa
produce 12 II and 12 I at metaphase I. This indicates a homology between chromosomes
of N. tabacum and those of N. sylvestris and N. tomentosa. The amphidiploid from the
cross N. sylvestris x N. tomentosa is similar to N. tabacum in many characteristics, which
further supports the above conclusion. The species N. tabacum has undergone
considerable differentiation during its evolutionary history, mostly due to the
accumulation of gene mutations and, to some extent, due to the loss of some duplicated
segments of the two genomes.

Evolution of Gossypium hirsutum. The 9 old World and the 8 New World species of
Gossypium have n = 13, but the chromosomes of the New World species are smaller than
those of the Old World species. Three other species, G. hirsutum, G. barbadense and
G. tomentosum (wild Hawaii cotton), have n = 26; in these species, 13 chromosomes are
relatively larger than the remaining 13. A possible origin of G. hirsutum is from the cross
between Asiatic cotton G. arboreum x G. thurberi (American wild cotton), followed by
chromosome doubling of the inter specific hybrid. According to a more recent scheme,
G. hirsutum has originated from the cross G. herbaceum (AA) x G. raimondii(BB),
followed by chromosome doubling of the F1.

Evolution of Amphidiploid Brassica Species: The origin of amphidiploid Brassica species

is presented based on the famous U’s Triangle proposed by N. U in 1935. According to
this scheme, B. juncea (n = 18) is an amphidiploid from B. nigra (n =8) x B. campestris (n
= 10), B. napus (n = 19) is an amphidiploid from the cross B. oleracea (n =
9) x B. campestris (n = 10), and B. carinata (n = 17) is an amphidiploid from the cross B.
nigra (n = 8) x B. oleracea (n = 9). The synthetic allopolyploids produced according to
the above scheme resemble the natural amphidiploids, cross easily with them, and the
hybrids between the synthetic and natural amphidiploids are reasonably fertile.

B.nigra
n=8
(BB)*

B.carinata B.juncea
n = 17
n = 18
(BB CC) (AA BB)

B.oleracee B.campestris
B.napus n = 10
n=9
n = 19 (AA)
(CC)
(AA CC)
 TRITICALE: MAN MADE CEREAL
Triticale (X Triticosecale), a new man made cereal
Triticale is the first man made crop, in so far as it resulted as an artificial allopolyploid derived by
crossing wheat and rye (Secale) Depending on whether tetraploid (2n=4x=28) or hexaploid
(2n=6x=42) wheat is utilized for the synthesis, one would get hexaploid triticale (2n=6x=42) or
octoploid triticale (2n=8x=56) respectively. In each case, only diploid rye (2n=2x=14) was used.
The derivations are shown in the following picture.

At present only the hexaploid triticales are considered to have the potential of becoming new
crop. The hexaploid triticales which are initially produced as amphiploids are called primary
triticales. These primary triticales have many drawbacks like shriveled grains, meiotic instability,
poor yield and preharvest sprouting.

Types of Aneuploids and their origin

ANEUPLOIDY

Of the various anecuploids, monosomics (in polyploid species, such as, tobacco,
wheat and oats) and trisomics [in diploid species, e.g., maize, bajra, tomato, rye, pea,
spinach, etc.] are most commonly used in genetic studies.

Nullisomics are viable in a few highly polyploid species only, e.g., wheat and
oats; they are not viable even in tobacco. Aneuploids are usually less vigorous than their
diploid progenitors. Another characteristic of aneuploids is their high sterility resulting
form irregular meiosis.

Monosomics

A monosomic is an individual that lacks one chromosome of the normal

complement of somatic cells (2n – 1).

If the lost chromosome is one that is not absolutely essential for the organism, it
may survive but if the lost chromosome is one that is very important, it may not live.

Loss of one chromosome in normal diploid plants may result in lethality. Thus,
for example, monosomics are inviable in Datura sp. Polyploid plants, however, have
been found to tolerate the loss of one chromosome. Twenty-four different monosomics,
each lacking a single different chromosome of the normal complement, have been
isolated in Nicotiana tabacum which is a tetraploid with 2n = 48. These 24 monosomics
are morphologically distinct from each in haploid wheat (2n = 42), 21 different
monosomics have been isolated.

Monosomics produce two kinds of gametes, one kind with n chromosomes and
the other kind with n –1 chromosomes. When selfed, monosomics, therefore, produce
normal (i.e., disomic), monosomic and nullisomic offspring.

Nullisomics

A nullisomic is an individual that lacks both members of one specific pair of

chromosomes (2n-2).

Nullisomics are inviable in some species like Nicotiana tabacum, but in other
species like Triticum aestivum, they are viable. In the Chinese Spring variety of wheat,
Sears established 21 nullisomic lines (2n = 40), each lacking a single pair of
chromosomes of the normal complement of the somatic cells. Different nullisomics are
morphologically different from one another and from the normal Chinese Spring. They
are reduced in size and vigour and are highly sterile. On selfing, they produce only
nullisomics as their gametes contain only n – 1 (i..e, 20) chromosomes each.
Trisomics

A trisomic is an individual with one chromosome more than the normal

complement of the somatic cells ( 2n +1).

In general, an extra chromosome does not produce so striking effect as a missing

one. In wheat, trisomics ( 2n = 43) occur but they are nearly indistinguishable from
normal plants (i.e., disomes with 2n = 42. Blakeslee has isolated 12 different trisomics in
Datura sp. (2n = 24), each having in triplicate a single different chromosome of the
normal set. These trisomics differ morphologically from one another and from the
diploid form.

Although trisomics give rise to two kinds of gametes, one kind with n
chromosomes an the other kind with n + 1 chromosomes, they tend to be somewhat more
stable genetically than monosomics.

Tetrasomics

A tetrasomic is an individual with two chromosomes more than the normal

complement of the somatic cell ( 2n +1).

In a normal tetrasomic, two units of the same chromosome will be found besides
the normal diploid number. If two different chromosomes (say chromosome No. 1 and
chromosome No.2) are present besides the normal diploid number, it is called a double
trisomic (2n + 1 + 1). During meiosis a quadrivalent is formed besides the bivalents in a
tetrasomic, while two trivalents and two bivalents are formed in a double trisomic.

Tetrasomic produce gametes with n + 1 chromosome and when crossed with

normal diploids (2n), they produce high frequency of trisomics.

Origin and Production

Spontaneous. Aneuploids originate spontaneously at a low frequency. The earlier cases

of aneuploidy were produced spontaneously in experimental populations. Meiotic
irregularities lead to the formation of n + 1 and n- 1 gametes, e.g., in Datura about 0.4 per
cent of pollen is likely to be n + 1; these gametes give rise to 2n + 1 and 2n –1,
respectively, progeny following fertilization.

Autotriploid Plants. The best sources of aneuploids are triploid plants. Distribution of
chromosomes at the first meiotic anaphase of triploids is irregular leading to the
production of a whole range of aneuploids in the progeny.

Asynaptic And Desynaptic Plants. In asynaptic and desynaptic plants, few to all
chromosomes are present as univalents at metaphase I of meiosis. In the progeny of such
plants, a relatively high frequency of aneuploids occur.
Translocation Hetrozygotes. A 3 : 1 disjunction of the ring or the chain of four
chromosomes in a translocation heterozygote would produce one n + 1 and one n –1
gamete. As a result, a variable frequency of aneuploids are found in the progeny of
translocation heterozygotes.

Tetrasomic Plants. Tetrasomic (2n + 2) plants would produce n + 1 gametes in

considerable frequencies. Therefore, when they are crossed with normal disomic (2n)
plants, they produce a high frequency of trisomics, where possible, tetrasomics may be
maintained for the production of trisomics.

Double Trisomy - In a diploid organism when two different chromosomes are

represented in triplicate, the double trisomy is resulted. A double trisomic has the
chromosomal formula 2n + 1 + 1.