The term genetics (Gk. Genesis – descent) was coined by Bateson (1906). Genetics is the study of principles and mechanism of heredity and variations. The resemblance amongst offspring is never 100% (except in monozygotic twins) due to reshuffling of chromosomes and their genes.

Table : 4-1

Father of genetics (classical genetics)	Mendel
Father of modern genetics / Animal genetics	Bateson
Father of experimental genetics /Drosophila genetics	Morgan
Father of human & physiological genetics	Garrod
Father of quantitative inheritance	Kolreuter
Father of Neurospora genetics	Dodge
Father of genetic Engineering	Paul Berg

Heredity

Heredity is the study of transmission of genetic characters and variations from one generation to the next. Heredity involves the transfer of chromosomes from parents to offspring or one individual to another. Therefore, chromosome is the base of heredity. The physical basis of heredity are genes while chemical basis of heredity is DNA.

Pre-Mendelian view points

Vapour theory (Pythogoras) : Different body parts produce minute particles.

Fluid theory : Empedocles, proposed that each body part produced a fluid. The fluid of different body parts of the two parents mixes up and is used in the formation of embryo.

Preformation theory : Malpighi believed that homunculus or miniature individual is present in sperm or egg. Antony Von Leeuwenhoek was first to observe human sperm.

Particulate theory : Maupertuis proposed that the body of each parent gives rise to minute particles. These particles unite together to form the daughter individual.

Encasement theory : Charles Bonnet and his supporters presumed that every female contains within her body miniature prototypes of all the creatures which would descend from her, one generation within the other, somewhat like a series of chinese boxes. This was named as encasement theory.

Theory of epigenesis : Wolff proposed that the germ cells contain definite but undifferentiated substances, which after fertilization, become organised into various complex body organs that form the adult. This idea was referred to as epigenesis.

Pangenesis theory : Proposed by Charles Darwin (1868) according to this theory every cell, tissue and organ of animal body produces minute invisible bodies, called gemmules or pangenes. They can produce offsprings.

Weismann theory of germplasm : August Weismann (1889) suggested the theory of continuity of germplasm. He described reproductive cells as germplasm and rest of the body as somatoplasm.

Pre-Mendelian theories of inheritance are also called theories of blending inheritance.

Evidences against blending theory

Under this concept, the progeny of a black and white animal would be uniformly grey. The further progeny from crossing the hybrids among themselves would be grey, for the black and white hereditary material, once blended, could never be separated again. Pattern of inheritance shown by atavism also speaks against blending theory. The traits of sex do not blend in unisexual organisms.

Basic features of inheritance

(i) Traits have two alternative forms.

(ii) Traits are represented in the individual by distinct particles which do not blend or change.

(iii) Traits may remain unexpected for one or more generations and reappear later unchanged.

(iv) Traits may remain together in one generation and separate in a later generation.

(v) One alternative of a trait may express more often than the other.

Variations

Variations are differences found in morphological, physiological and cytological behaviouristic traits of individuals belonging to same species race and family. They appear in offspring or siblings due to :

Reshuffling of genes/chromosomes by chance separation of chromosomes
Crossing over
Chance combination of chromosomes during meiosis and fertilization.

Types of variations

(1) Somatic variations : These variations influence the somatic or body cells. They appear after birth and are, also called acquired characters, modifications or acquired variations. Somatic variations are non-inheritable and usually disappear with the death of the individual. They are formed due to three reasons i.e., environmental factors, use and disuse of organs, and conscious efforts.

(2) Germinal variations : They are inheritable variations formed mostly in germinal cells which are either already present in the ancestors or develop a new due to mutations. Germinal variations are of two types :

(i) Continuous variations : They are fluctuating variations and also called recombinations because they are formed due to recombination of alleles as found in sexual reproduction. Darwin (1859) based his theory of evolution on continuous variations.

(ii) Discontinuous variations : They are mutations, which are ultimate source of organic variations. Discontinuous variations are caused by chromosomal aberrations, change in chromosome number and gene mutations. In pea seed coat colour changes gray to white is an example of spontaneous mutation.

Importance of variations

(1) Variations continue to pile up forming new species with time.

(2) They are essential in the struggle for existence.

(3) Adaptability is due to variations.

(4) Variations allow breeders to improve races of plants and animals.

(5) Discontinuous variations introduce new traits.

(6) Inbreeding between closely related organisms reduces variation.

Important terms used in inheritance studies

Gene : (Mendel called them factor) In modern sense an inherited factor that determines a biological character of an organism is called gene (functional unit of hereditary material).

Allelomorphs or alleles : Alleles indicates alternative forms of the same gene. e.g., Tall TT and dwarf tt are alternation forms of the same gene etc.

Gene locus : The specific place on a chromosome where a gene is located.

Wild and mutant alleles : An original allele, dominant in expression and wide spread in the population is called wild allele. An allele formed by a mutation in the wild allele, recessive in expression and less common in the population is termed as mutant allele.

Homozygous (Bateson and Saunders, 1902) : Both the genes of a character are identical is said to be homozygous or genetically pure for that character. It gives rise to offspring having the same character on self-breeding e.g., TT (Homozygous dominant) or tt (Homozygous recessive).

Heterozygous (Bateson and Saunders, 1902) : Both the genes of a character are unlike is said to be heterozygous or hybrid. Such organisms do not breed true on self fertilization e.g., Tt.

If we know the number of heterozygous pairs we can predict the following :

Number of types of gametes = 2ⁿ

Number of F₂ phenotype = 2ⁿ

(Where n is the number of heterozygous pairs).

Number of F₂ genotype = 3ⁿ

Genotype : The genotype is the genetic constitution of an organism. TT, Tt and tt are the genotypes of the organism with reference to these particular pairs of alleles.

Phenotype : External feature of organisms, colour and behaviour etc.

Pure line : Generations of homozygous individuals which produce offsprings of only one type i.e., they breed true for their phenotype and genotype.

Monohybrid, dihybrid and polyhybrid : When only one allelic pair is considered in cross breeding, it is called monohybrid cross. Similarly when two allelic pairs are used for crossing, it is called dihybrid cross and more than two allelic pairs in a cross are called polyhybrid cross.

Reciprocal cross : The reciprocal crosses involve two crosses concerning the same characteristics, but with reversed sexes.

Genome : Total set of genes (DNA instructions) in the haploid set of chromosomes and inherited as unit from parents to offspring is called genome.

Gene pool : All the genotypes of all organisms in a population form the gene pool.

F₁ Generation : F₁ or first filial (filus–son, filia–daughter; Bateson, 1905) generation is the generation of hybrids produced from a cross between the genetically different individuals called parents.

F₂ Generation (Bateson, 1905) : F₂ or second filial generation is the generation of individuals which arises as a result of inbreeding or interbreeding amongst individuals of F₁ generation.

Punnet square : It is a checker-board used to show the result of a cross between two organisms, it was devised by geneticist, R.C. Punnet (1927). It depicts both genotypes and phenotypes of the progeny.

Back cross : It is cross which is performed between hybrid and one of its parents. In plant breeding, back cross is performed a few times in order to increase the traits of that parent.

Test cross : It is a cross to know whether an individual is homozygous or heterozygous for dominant character. The individual is crossed with recessive parent. The ratio will be 50% dominant and 50% recessive in case of hybrid or heterozygous individual. In case of double heterozygote (e.g., RrYy) crossed with recessive (rryy) the ratio will be 1:1:1:1. Test cross helps to find out genotype of parents.

Self cross/selfing : It is the process of fertilization with pollen or male gametes of the same individual.

Observed Vs expected results : Experimental results confirm to the ones expected through the theory of probability if the size of the sample is small but they tend to approach the latter if the sample size is large.

Hybrid : The organism produced after crossing of two genetically different individuals is called hybrid.

Heredity and variations in sexual and asexual reproduction

Sexual reproduction : Variations are common in animals and plants which reproduce by sexual means. The reason for this is that the sexual reproduction is biparental, involves meiosis and fertilization, and the offspring receives some traits from father and some from mother.

Asexual reproduction : Those organisms which reproduce by asexual means e.g., bacteria, amoeba, euglena, rose etc. The asexual reproduction is monoparental, involves mitosis and the organism produced by it, inherits all the traits of its single parent. With the result, it is almost a carbon copy of the parent and is known as ramet. A group of ramets is called a clone.

Mendelian period

Gregor Johann Mendel first “geneticist”, also known as father of genetics was born on July 22, in 1822 in Silisian, a village in Heizendorf (Austria). In 1843, he joined Augustinian monastry at Brunn (then in Austria, now Brno Czechoslovakia). In 1856, Mendel got interested in breeding of Garden pea (Pisum sativum). He selected pure breeding varieties or pure lines of pea. Breeding experiments were performed between 1859 – 1864. The results were read out in two meetings of Natural History Society of Brunn in 1865 and published in 1866 in “Proceedings of Brunn Natural History Society” under the topic “Experiments in Plant Hybridisation”. Mendel died in 1884 without getting any recognition during his lifetime.

Rediscovery of Mendel’s work : In 1900, Hugo de Vries of Holland, Carl Correns of Germany and Erich von Tshermak of Austria came to the same findings as were got by Mendel. Hugo de Vries found the paper of Mendel and got it reprinted in ‘Flora’ in 1901. Correns converted two of the generalisations of Mendel into two laws of heredity. These are law of segregation and law of independent assortment.

Reasons for Mendel’s success

Method of working : He maintained the statistical records of all the experiments and analysed them. He selected genetically pure (pure breed line) and purity was tested by self-crossing the progeny for several generations.

Selection of material : Mendel selected garden pea as his experimental material because it has the following advantages.

It was an annual plant. Its short life–cycle made it possible to study several generations within a short period and has perfect bisexual flowers containing both male and female parts. The flowers are predominantly self-pollinating because of self-fertilization, plants are homozygous. It is, therefore, easy to get pure lines for several generations and also easy to cross because pollens from one plant can be introduced to the stigma of another plant by removing anthers (emasculation) and bagging. In addition to that there was one reason more for his success. He studied seven pairs of characters which were present on four different pairs of chromosomes.

Selection of traits : Mendel selected seven pairs of contrasting characters as listed in the table. Luckily all were related as dominant and recessive.

Table : 4-2 Seven pairs of contrasting characters in pea plant

S. No.	Character	Dominant	Recessive
(1)	Stem length	Tall	Dwarf
(2)	Flower colour	Violet	White
(3)	Flower position	Axial	Terminal
(4)	Pod shape	Inflated	Constricted
(5)	Pod colour	Green	Yellow
(6)	Seed shape	Round	Wrinkled
(7)	Seed colour	Yellow	Green

Mendel’s experiments

Monohybrid cross : Experiments with garden pea for single pair of contrasting characters.

Mendel crossed pure tall and dwarf plants. The plants belonged to F₁ generation all tall hybrid were self-pollinated. The plants of F₂ generation were both tall and dwarf, in approximate 3:1 ratio phenotypically and 1:2:1 genotypically.

Mendel’s explanation : Mendel explained above results by presuming that Tallness and dwarfness are determined by a pair of contrasting factors or determiners (now these are called genes). A plant is tall because it possesses determiners for tallness (represented by T) and a plant is dwarf because it has determiners for dwarfness (represented by t). These determiners occur in pairs and are received one from either parent. On the basis of this behaviour the tallness is described as dominant character and dwarfness as recessive (law of dominance). The determiners are never contaminated. When gametes are formed, these unit factors segregate so that each gamete gets only one of the two alternative factors. When F₁ hybrids (Tt) are self pollinated the two entities separate out and unite independently producing tall and dwarf plants (law of segregation). Monohybrid test cross ratio is 1:1.

Dihybrid cross (Crosses involving two pairs of contrasting traits).

Later on Mendel conducted experiments to study the segregation and transmission of two pairs of contrasting traits at a time. Mendel found that a cross between round yellow and wrinkled green seeds (P₁) produced only round and yellow seeds in F₁ generation, but in F₂ four types of combinations were observed. These are :

Round yellow 9 Parental combinations

Round green 3 Non-parental combinations

Wrinkled yellow 3 Non-parental combination

Wrinkled green 1 Parental combination

Thus the offsprings of F₂ generation were produced in the ratio of 9 : 3 : 3 : 1 phenotypically and 1 : 2 : 2 : 4 : 1 : 2 : 1 : 2 : 1 genotypically. This ratio is called dihybrid ratio.

Mendel’s explanation : Mendel explained the results by assuming that the round and yellow characters are dominant over wrinkled and green so that all the F₁ offsprings are round yellow. In F₂-generation since all the four characters were assorted out independent of the others, he said that a pair of alternating or contrasting characters behave independently of the other pair i.e., seed colour is independent of seed coat.

Therefore, at the time of gamete formation genes for round or wrinkled character of seed coat assorted out independently of the yellow or green colour of the seed. As a result four types of gametes with two old and two new combinations i.e., YR, Yr yR, yr are formed from the F₁ hybrid. These four types of gametes on random mating produce four types of offsprings in the ratio of 9:3:3:1 in F₂ generation (Law of Independent Assortment). Dihybrid test cross ratio is 1 : 1: 1 : 1.

Table : 4-3 Forked-line method showing formation of four types of gametes from a F₁ – dihybrid for seed colour and seed shape

Trihybrid cross : The offsprings shows 27 : 9 : 9 : 9 : 3 : 3 : 3 : 1 ratio is found in trihybrid cross. This suggests that a di, tri, or polyhybrid cross is actually a combination of respectively two, three or more monohybrid crosses operating together. Trihybrid test cross ratio is 1 : 1 : 1 : 1 : 1 : 1 : 1 : 1.

Mendel’s laws of inheritance

Mendel’s laws are still true because these take place in sexually reproducing organisms or parents are of pure breeding. He enunciated two major laws of inheritance i.e., law of segregation and law of independent assortment.

Law of segregation (Purity of gametes) : 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 as seen in monohybrid and dihybrid cross. That is why the law of segregation is also described as law of purity of gametes.

Law of independent assortment : If 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 out independently of the other pairs. Mendel formulated this law from the results of a dihybrid cross.

Interaction of genes

Genes interaction is the influence of alleles and non-alleles on the normal phenotypic expression of genes. It is of two types :

(1) Inter–allelic or intra–genic gene interaction : In this case two alleles (located on the same gene locus on two homologous chromosomes) of gene interact in such a fashion to produces phenotypic expression e.g., co-dominance, multiple alleles.

(i) Incomplete dominance or Blending inheritance (1: 2:1 ratio) : After Mendel, several cases were recorded where F₁hybrids were not related to either of the parents but exhibited a blending of characters of two parents. This is called incomplete dominance or blending inheritance.

Example : First case of incomplete dominance or blending inheritance was reported in 4-O’clock plant, (Mirabilis jalapa) by Carl Correns (1903) when plants with red flowers (RR) are crossed with plants having white flowers (rr) the hybrid F₁ plants (Rr) bear pink flowers. When these F₁ plants with pink flowers are self pollinated they develop red (RR), pink (Rr) and white (rr) flowered plants in the ratio of 1:2:1 (F₂ generation). Snapdragon or dog flower (Antirrhinum majus) is a other example of in complete dominance.

(ii) Codominance (1:2:1 ratio) : In codominance, both the genes of an allelomorphic pair express themselves equally in F₁ hybrids. 1:2:1 ratio both genotypically as well as phenotypically in F₂ generation.

Example : Codominance of coat colour in cattle, Codominance in andalusian fowl and Codominance of blood alleles in man.

Table : 4-4 Differences between incomplete dominance and codominance

Incomplete dominance	Codominance
Effect of one of the two alleles is more conspicuous.	The effect of both the alleles is equally conspicuous.
It produces a fine mixture of the expression of two alleles.	There is no mixing of the effect of the two alleles.
The effect in hybrid is intermediate of the expression of the two alleles.	Both the alleles produce their effect independently, e.g., I^A and I^B, Hb^Sand Hb^A.

(2) Non–allelic or inter-genic gene interaction : Here two or more independent genes present on same or different chromosomes, interact to produce a new expression e.g., epistasis, complementary genes, supplementary genes, duplicate genes, inhibitory genes, lethal genes etc.

(i) Complementary genes (9 : 7 ratio) : The complementary genes are two pairs of nonallelic dominant genes (i.e., present on separate gene loci), which interact to produce only one phenotypic trait, but neither of them if present alone produces the phenotypic trait in the absence of other.

(ii) Supplementary genes (9 : 3 : 4 ratio) : Supplementary genes are two independent pairs of dominant genes which interact in such a way that one dominant gene will produce its effect whether the other is present or not. The second dominant when added changes the expression of the first one but only in the presence of first one. In rats and guinea pigs coat colour is governed by two dominant genes.

(iii) Epistasis (Inhibiting genes) : Epistasis is the interaction between nonallelic genes (Present on separate loci) in which one-gene masks, inhibits or suppresses the expression of other gene. The gene that suppresses the other gene is known as inhibiting or epistatic factor and the one, which is prevented from exhibiting itself, is known as hypostatic.

Dominant epistasis (12:3:1 or 13:3 ratio) : In dominant epistasis out of two pairs of genes the dominant allele, (i.e., gene A) of one gene masks the activity of other allelic pair (Bb). Since the dominant epistatic gene A exerts its epistatic influence by suppressing the expression of gene B or b, it is known as dominant epistasis. Example – Dominant epistasis in dogs

Similar phenomena have been seen in fruit colour in cucurbita as summer squash and coat colour in chickens.

Recessive epistasis (9:3:4 ratio) : Epistasis due to recessive gene is known as recessive epistasis, i.e., out of the two pairs of genes, the recessive epistatic gene masks the activity of the dominant gene of the other gene locus. The dominant A expresses itself only when the epistatic locus C also has the dominant gene if the epistatic locus has recessive gene c, gene A fails to express.

(iv) Duplicate genes ( ratio) : Sometimes two pairs of genes located on different chromosomes determine the same phenotype. These genes are said to be duplicate of each other. The dominant triangular fruit shape of Capsella bursa pastoris (shepherd’s purse) is determined by two pairs of genes, say A and B. If any of these genes is present in dominant form, the fruit shape is triangular. In double recessive forms the fruits are top shaped and thus we get a 15 (triangular) : 1 (top shaped) ratio in F₂ generation.

Example : Coat colour of mice.

(v) Collaborator genes : In collaboration two gene pairs, which are present on separate loci but influence the same trait, interact to produce some totally new trait or phenotype that neither of the genes by itself could produce.

Example : Inheritance of combs in poultry, where two genes control the development of comb.

Pleiotropic effect of genes

Lethal genes : Lethal factor were first of all reported in mice body by of French geneticst ‘Cuenot’. Certain genes are known to control the manifestation of some phenotypic trait as well as affect the viability of the organism. Some other genes have no effect on the appearance of the organism but affect the viability alone. These genes are known as lethals or semilethals depending upon their influence. Lethal factors in case of plants were reported first of all in snapdragons (Antirrhinum majus) by E. Baur (1907).

Dominant lethals : The dominant lethal genes are lethal in homozygous condition and produce some defective or abnormal phenotypes in heterozygous condition. Their most serious effect in heterozygous may also cause death. Following are the examples of dominant lethal genes.

Example – Yellow lethal in mice : A well known example of such lethals is from mice, given by Cuenot. He found that the yellow mice never breed true. Whenever the yellow mice were crossed with yellow mice, always yellow and brown were obtained in the ratio of 2:1. A cross between brown and brown mice always produced brown offsprings and a cross between brown and yellow produced yellow and brown in equal proportions. Yellow mice never present homozygous condition.

In 1917, Stiegleder concluded that yellow mice are heterozygous. The homozygous yellow (1/4^th of the total offsprings) dies in the embryonic condition. When there unborn ones are added to the 2:1 ratio of yellow and brown, these form typical 3:1 ratio. Cuenot suggested that gene Y has a multiple effect. It controls yellow body colour and has a dominant effect. It affects viability and acts as a recessive lethal. Other examples are Inheritance of sickle cell anaemia in man, Brachyphalangy, Huntington’s chorea in man.

Recessive lethals : The recessive lethals produce lethal effect only in homozygous condition. Their heterozygotes are normal. Therefore, recessive lethals remain unnoticed in the population but are established in the population because female are carrier for lethal gene. These are detected only when two heterozygous persons get married. Example : Tay Sach’s lethal

Qualitative inheritance : Qualitative inheritance or monogenic inheritance is that type of inheritance in which one dominant allele influences the complete trait, so that two such allele do not change the phenotype. Here dominant allele is monogene.

Quantitative/Polygenic inheritance : Quantitative inheritance or polygenic inheritance can be defined as, two or more different pairs of alleles which have cumulative effect and govern quantitative characters. The quantitative inheritance is due to incomplete dominance.

Examples : Ear size in maize, White spotting in mice, Grain colour in wheat.

Cytoplasmic / Extrachromosomal inheritance

The fact that nucleus contains the units of inheritance was proposed by Oscar Hertwig in 1870. The mechanism was clearly understood with the development of Mendel’s laws of inheritance. Further researchers proposed that cytoplasm also contains the hereditary material. The evidence for cytoplasmic inheritance was first presented by Correns in Mirabilis Jalapa and by Baur in Pelargonium zonale in 1908. The cytoplasm in such cases contain self perpetuating hereditary particles formed of DNA. These may be mitochondria, plastids or foreign organism, etc. The total self duplicating hereditary material of cytoplasm is called plasmon and the cytoplasmic units of inheritance are described as plasmagenes.

Criteria for cytoplasmic inheritance : The cases of cytoplasmic inheritance are found to exhibit maternal influence. The reason is very simple. Very little cytoplasm is contained in the sperm cell of an animal. Most of the cytoplasm is contributed to the zygote by the ovum or egg. Hence if there are hereditary units in the cytoplasm, these will be transmitted to the offsprings through the egg. The offspring, therefore will exhibit maternal influence. This could be explained further by following example :

(i) Maternal influence on shell coiling in snail.

(ii) Inheritance of sigma particles in Drosophila.

(iii) Breast tumour in mice.

(iv) Plastid inheritance in Mirabilis (4 O’ clock plant).

(v) Plastid inheritance in Oenothera.

(vi) Male sterility in plants – e.q. maize.

(vii) Inheritance of kappa particles in Paramecium.

(viii) Mitochondrial genetics – Sacromyces cerevieacae, Neurospora – crassa, Aspergillus nidulens.

Linkage

Introduction : “When genes are closely present link together in a group and transmitted as a single unit this phenomenon is called linkage”.

Theories of linkage

Sutton’s hypothesis of linkage (1903) : The number of groups of genes are equivalent to the number of chromosomes.

Morgan’s hypothesis of linkage (1910) : It was given by T. H. Morgan. According to him the genes of homologous parents enter in the same gamete and tend to remain together, which is opposite in heterozygous parents. Linked group are located on the same chromosome and distance between linked group of gene limits the grade of linkage.

Coupling and repulsion hypothesis : Proposed by Bateson and Punnet (1906) that dominant alleles tend to remain together as well with recessive alleles, called gametic coupling. If dominant and recessive alleles are present in different parents they tend to remain separate and called repulsion. When BBLL and bbll are crossed, the is BbLl and the test cross of it will show progeny in 7 : 1 : 1 : 7 ratio i.e., BbLl : Bbll : bbLl : bbll (coupling) when BBll is crossed with bbLL the is BbLl or the test cross progeny will show 1 : 7 : 7 : 1 ratio i.e., BbLl : Bbll : bbLl : bbll (repulsion). Coupled and repulsed genes are known as linked genes. Linkage has coupling phase and repulsion phase. In coupling phase both the linked genes have their dominant alleles in one chromosome and recessive alleles in other chromosomes. The heterozygotes with such constitution is called cis heterozygote. Cis-arrangement is a original arrangement. Which form two types of gametes as (AB) and (ab). In Human X–chromosomes carry 102 genes and Y chromosome carries 10 genes only.

In repulsion phase the normal alleles as well as mutant alleles lie in opposite chromosomes of the homologous pair, such heterozygote is called as trans heterozygote. It is not original arrangement, caused due to crossing over, which form two types of gametes as (Ab) and (aB).

Chromosomal hypothesis of linkage : It was given by Morgan and Castle. According to them linked genes are bound by chromosomal material and are transmitted as a whole.

Types of linkage

Depending upon the absence or presence of nonparental or new combination of linked genes, linkage has been found to be complete or incomplete.

Complete linkage (Morgan, 1919) : Such cases in which linked genes are transmitted together to the offsprings only in their original or parental combination for two or more or several generations exhibit complete linkage. In such cases the linked genes do not separate to form the new or non-parental combinations. This phenomenon is very rare. Some characteristics in males of Drosophila are found to exhibit complete linkage.

Incomplete linkage : In majority of cases, the homologous chromosomes undergo breakage and reunion during gametogenesis. During reunion the broken pieces of the chromatids are exchanged, producing some nonparental or new combinations. Therefore, the linkage is rendered incomplete. The phenomenon of interchange of chromosome segments between two homologous chromosomes is called crossing over. Incomplete linkage is very common and has been studied in almost all the organisms. Hutchinson discribe incomplete linkage in maize seed.

Linkage groups

All the genes which are linked with one another, form a linkage group. Since linked genes are present in the same chromosome, the number of linkage group in an animal or plant is equal to the haploid number of chromosomes present in its cells. This hypothesis was given by Sutton and was proved by experiments on Drosophila by T.H. Morgan.

Strength of linkage

The strength of linkage between any two pairs of linked genes of a chromosome depend upon the distance between them. Closely located genes show strong linkage, while genes widely located show weak linkages.

Strength of linkage µ.

Factor affected to linkage

Linkage is affected by the following factors :

Distance : Closely located genes show strong linkage while genes widely located show weak linkage.

Age : With increasing age the strength of linkage increases.

Temperature : Increasing temperature decreases the strength of linkage.

X-rays : X-rays treatment reduces the strength of linkage.

Significance of linkage

(i) It helps in maintaining the valuable traits of a newly developed variety.

(ii) It helps locating genes on chromosome.

(iii) It disallows the breeders to combine all the desirable traits in a single variety.

Crossing over

The process by which exchange of chromosomal segment take place is called crossing over. Crossing over may be defined as “the recombination of linked genes” brought about as a result of interchange of corresponding parts between the chromatid of a homologous pair of chromosomes, so as to produce new combination of old genes. The term was given by Morgan and Cattle. Janssen (1909) observed chiasmata during meiosis-I (Prophase). Morgan proposed that chiasmata lead to crossing over by breakage and reunion of homologous chromosomes. Crossing over results in new combination while non-cross over result in parental type, which leads to variations.

Crossing over and chiasma

There are two views extended to explain the relationship between crossing over and chiasma formation. They are summarised here under :

Chiasma type theory : According to Janssen, 1909 the act of crossing over is followed by chiasma formation. He suggests that the crossing over takes place at the pachytene stage and the chiasma appear at diplotene.

Classical theory : According to Sharp, 1934, crossing over is the result of chiasma formation. According to this view, the chiasma are organised at pachytene and crossing over takes place at diplotene stage. On the basis of evidence available from molecular biology, that is untenable and hence rejected.

Mechanism of crossing over

There are different views put forward to explain the mechanism of crossing over.

Copy choice hypothesis : According to Belling, 1928 the chromomeres represent the genes joined by interchromomeric regions. The chromomeres duplicate first and then the interchromomeric regions. The synthesis of these regions may occur in such a way that the chromomeres of the chromatid of a homologue get connected of the chromatid of the other homologue at a specific location. As a result, the adjacent chromatids of a pair of homologue are exchanged.

Precocity hypothesis : According to Darlington, the pairing of homologues occurs to avoid singleness of a chromosome. The pairing need of a chromosome could be nothing less than the replication of DNA. The crossing over takes place due to torsion on chromosome created by coiling of the two homologues around each other.

Cross over value : The percentage of crossing over varies in different materials. The frequency of crossing over is dependent upon the distance of two genes present on a chromatid.

Coincidence : Coincidence or coefficient of coincidence is inverse measure of interference and is expressed as the ratio between the actual number of double cross over and the expected number of such double cross. That is:

Factors controlling frequency of crossing over

Primarily, frequency of crossing over is dependent upon the distance between the linked genes, but a number of genetic, environmental and physiological factors also affect it. These are:

Temperature : High and low temperature increase the frequency of crossing over.

X-ray : Muller has discovered that exposure to X-ray and other radiations increases the frequency of crossing over.

Age : The frequency of crossing over decreases with increasing age in female Drosophila.

Chemicals : Certain chemicals which act as mutagens do affect the frequency of crossing over. Gene mutations may affect the frequency of crossing over. Some increase the frequency, whereas some may decrease it.

Sex : Crossing over in Drosophila males is negligible. Males of mammals also exhibit little crossing over. In silk-moth, crossing over does not occur in females.

Chiasmata formation : Chiasmata formation at one point discourages chiasmata formation and crossing over in the vicinity. This phenomenon is known as interference.

Inversions : Inversions of chromosome segments suppresses crossing over.

Distance : Distance between the linked genes is the major factor which controls the frequency of crossing over. The chances of crossing over between distantly placed genes are much more than between the genes located in close proximity.

Figure depicts that chance of crossing over between a and c are double as compared to the chances between a and b or b and c.

Nutritional effect : Crossing over frequencies are affected by concentration of metallic ions, such as calcium and magnesium.

Genotypic effect : Crossing over frequencies between the same two loci in different strains of the same species show variation because of numerous gene differences.

Chromosome structure effect : Changes in the order of genes on a chromosome produced by chromosomal aberrations usually act as cross over suppressors.

Centromere effect : Genes present close to the centromere region show reduced crossing over.

Interference : If there are two doubles crossovers, then one crossover tries to influence the other by suppressing it. This phenomenon is called as interference. Due to this phenomenon, the frequency of crossing over is always lower than the expected.

Significance of crossing over

This phenomenon is of great biological significance, which are as under:

(i) It gives evidence that the genes are linearly arranged on a chromosome. Thus, it throws light on the nature and working of the genes.

(ii) It provides an operational definition to a gene. It is deemed as the smallest heritable segment of a chromosome in the interior of which no crossing over takes place.

(iii) The crossing over is helpful in the chromosomal mapping. The percentage of crossing over is proportional to the distance between two genes.

(iv) It is the main cause of genetic variations. It’s occurrence during the act of meiosis produces variations in the heritable characters of the gametes.

(v) This phenomenon has also found it’s utility in breeding and evolving new varieties. The linkage of undesirable characters can be broken by temperature treatment, using X-ray or chemicals. Thus, new recombinants can be prepared.

Chromosomal maps

A linkage or genetic chromosome map is a linear graphic representation of the sequence and relative distances of the various genes present in a chromosome. A chromosome map is also called a linkage map or genetic map.

The percentage of crossing over between two genes is directly proportional to their distance. The unit of crossing over has been termed as by Haldane as centi Morgan (cM). One unit of map distance (cM) is therefore, equivalent to 1% crossing over. When chiasma is organised in between two gene loci, only 50% meiotic products shall be crossovers and 50% non-crossovers. Thus, the chiasma frequency is twice the frequency of cross over products i.e., chiasma % = 2 (cross over %) or crossover %= ½ (chiasma %).

Accordingly, Sturtevant, 1911 prepared the first chromosomal map. Infact this map is a line representation of a chromosome where the location of genes has been plotted as points at specific distances. These distances are proportional to their crossing over percentage. Suppose there are three genes on a chromosome say, A B and C which could be arranged as A, B, C, A, C, B or B, A, C. A three point test cross confirms as to which gene is located in the centre. By determining the crossing over value between A and B, B and C as also between A and C, the linkage maps can be prepared. Broadly speaking, a chromosomal map can be prepared from the following results of crossing over between the genes A, B and C :

(i) 4% crossing over taking place between A and B. (ii) 9% crossing over taking place between A and C.

Hence the genes be located as above and there should be 13% crossing over between B and C and the genes may be arranged as under :

If there is 5% crossing over between B and C, the genes are arranged in the following manner and there should be 9% crossing over between A and C.

Uses of chromosomal map

(i) Finding exact location of gene on chromosomes.

(ii) Knowing recombination of various genes in a linkage group of chromosomes.

(iii) Predicting result of dihybrid and trihybrid cross.

Nucleic acids

Two types of nucleic acids are found in the cells of all living organisms. These are DNA (Deoxyribonucleic acid) and RNA (Ribonucleic acid). The nucleic acid was first isolated (reported) by Friedrich Miescher in 1869 from the nuclei of pus cells and was named nuclein. The term nucleic acid was given by Altman (1899).

Term was given by Zacharis, which is found in the cells of all living organisms except plant viruses,where RNA forms the genetic material and DNA is absent. In bacteriophages and viruses there is a single molecule of DNA, which remains coiled and is enclosed in the protein coat. In bacteria, mitochondria, plastids and other prokaryotes, DNA is circular and lies naked in the cytoplasm but in eukaryotes it is found in nucleus and known as carrier of genetic information and capable of self replication. Isolation and purification of specific DNA segment from a living organism achieved by Nirenberg. H.Harries is associated with DNA-RNA hybridization technique.

Chemical composition

The chemical analysis has shown that DNA is composed of three different types of compound.

(i) Sugar molecule : Levene identified a five carbon sugar, ribose in nucleic acid in 1910. It is represented by a pentose sugar the deoxyribose or 2-deoxyribose which derived from ribose due to the deletion of oxygen from the second carbon.

(ii) Phosphoric acid : H₃PO₄ that makes DNA acidic in nature.

(iii) Nitrogeneous base : Kossel demonstrated the presence of two pyrimidines (cytosine and thymine) and two purines (adenine and guanine) in DNA and he was awarded Nobel Prize in 1910. These are nitrogen containing ring compound. Which classified into two groups:

(a) Purines : Two ring compound namely as Adenine and Guanine.

(b) Pyrimidine : One ring compound included Cytosine and Thymine. In RNA Uracil is present instead of Thymine.

Nucleosides : Nucleosides are formed by a purine or pyrimidine nitrogenous base and pentose sugar. DNA nucleosides are known as deoxyribosenucleosides.

Nucleotides : In a nucleotide, purine or pyrimidine nitrogenous base is joined by deoxyribose pentose sugar (D), which is further linked with phosphate (P) group to form nucleotides.

Table : 4-5

Nitrogenous base	Nucleoside (Base + Sugar)	Nucleotide (Base + Sugar + Phosphate)
DNA Adenine = A	Deoxyadenosine	Deoxyadenosine monophosphate or Adenine deoxyribose nucleotide
Guanine = G	Deoxyguanine	Deoxygunine monophosphate or Guanine deoxyribose-nucleotide
Thyamine = T	Thymidine	Deoxythymidine monophosphate or Thymidine deoxyribose nucleotide
Cytosine = C	Deoxycitidine	Deoxycytidine monophosphate or Cytosine deoxyribose nucleotide
RNA Adenine = A	Adenosine	Adenosine monophosphate or Adenine ribose nucleotide
Guanine = G	Guanosine	Guanosine monophosphate or Guanine ribose nucleotide
Uracil = U	Uridine	Uridine monophosphate or Uracil ribose nucleotide
Cytosine = C	Cytidine	Cytidine monophosphate or Cytosine ribose nucleotide

Watson and Crick’s model of DNA

In 1953 James Watson and Francis Crick suggested that in a DNA molecule there are two such polynucleotide chains arranged antiparallal or in opposite directions i.e., one polynucleotide chain runs in 5^’®3^’ direction, the other in 3^’® 5^’direction. It means the 3^’ end of one chain lies beside the 5^’end of other in right handed manner.

Important features

(i) The double helix comprises of two polynucleotide chains.

(ii) The two strands (polynucleotide chains) of double helix are anti-parallel due to phosphodiester bond.

(iii) Each polynucleotide chain has a sugar-phosphate ‘backbone’ with nitrogeneous bases directed inside the helix.

(iv) The nitrogenous bases of two antiparallel polynucleotide strands are linked through hydrogen bonds. There are two hydrogen bonds between A and T, and three between G and C. The hydrogen bonds are the only attractive forces between the two polynucleotides of double helix. These serve to hold the structure together.

The two polynucleotides in a double helix are complementary. The sequence of nitrogenous bases in one determines the sequence of the nitrogenous bases in the other. Complementary base pairing is of fundamental importance in molecular genetics.

Erwin Chargaff (1950) made quantitative analysis of DNA and proposed “base equivalence rule” starting that molar concentration of A = T & G º C or =1& which is constant for a species. Sugar deoxyribose and phosphate occur in equimolar proportion.

Ten base pairs occur per turn of helix (abbreviated 10bp). The spacing between adjacent base pairs is 3.4Å. The helix is 20Å (19.8Å) in diameter and DNA molecule found 360^o in a clockwise.

Forms of DNA

Five different morphological forms of DNA double helix have been described. These are A, B, C, D and Z forms. Most of these forms (except B, and Z) occur in rigidly controlled experimental conditions. Watson and crick model represents commonest form, Biotic-form (B-form or B-DNA) of DNA. Some DNA forms are inter convertible also. The differences in these DNA forms are associated with :

(i) The numbers of base pairs, present in each turn of DNA helix.

(ii) The pitch or angle between each base pair.

(iii) The helical diameter of DNA molecule.

(iv) The handedness of double helix. Which is mentioned in table.

Table : 4-6 Comparison of different types of DNA

Characters	A-DNA	B-DNA	C-DNA	D-DNA	Z-DNA
Base pair per turn of the helix	11	10	9.33	8	12
Tilt of pairs () base	20.2⁰	6.3⁰	-7.8⁰	-16.7⁰	7 Å
Axial rise (h)	2.56 Å	3.37 Å	3.32 Å	3.03 Å	3.7 Å
Helical diameter	23 Å	20 Å	19 Å	–	18 Å
Handedness of the double helix	Right handed	Right handed	Right handed	Right handed	Left handed

Promiscuous DNA : Special type of DNA which makes movement between mitochondria, chloroplast and nucleus. It was discovered in 1983 in cambridge university in maize. It was later reported in yeast, mungbean, spinach and peas.

Repetitive DNA : Multiple copies of DNA having same or almost same base pair sequence constitute repetitive DNA. In higher organisms 20% – 90% DNA is of this type.

Satellite DNA : In some eukaryotes small highly repetitive DNA sequences have been found called satellite DNA, which differ in base composition.

Characteristics of DNA

Denaturation or melting : The phenomenon of separating of two strand of DNA molecule by breaking of hydrogen bond at the temp. 90⁰C. In prokaryotes and human mitochondria G º C are more because this melting point is more. In eukaryotes the amount of A = T are more because melting point is less.

Renaturation or annealing : Separated strands reunite to form double helix molecule of DNA by cooling at the room temp. i.e., 25⁰C.

These properties help to form hybrid from different DNA or with RNA.

Evidences of DNA as the genetic material

The following experiments conducted by the molecular biologists provide direct evidences of DNA being the genetic material.

Bacterial transformation or Griffith’s Experiments : Griffith (1928) injected into mice with virulent and smooth (S-type, smooth colony with mucilage) form of Diplococcus pneumoniae. The mice died due to pneumonia. No death occurred when mice were injected with nonvirulent or rough (R-type, irregular colony without mucilage) form or heat- killed virulent form. However, in a combination of heat killed S-type and live R-type bacteria, death occurred in some mice. Autopsy of dead mice showed that they possessed S-type living bacteria, which could have been produced only by transformation of R-type bacteria. The transforming chemical was found out by O.T.Avery, C.M. Mc. leod and M. Mc. Carty (1944). They fractionated heat-killed S-type bacteria into DNA, carbohydrate and protein fractions. DNA was divided into two parts, one with DNAase and the other without it. Each component was added to different cultures of R-type bacteria. Transformation was found only in that culture which was provided with intact DNA of S-type. Therefore, the trait of virulence is present in DNA. Transformation involves transfer of a part of DNA from surrounding medium or dead bacteria (donor) to living bacteria (recipient) to form a recombinant.

Evidence from genetic recombination in bacteria or bacterial conjugation : Lederberg and Tatum (1946) discovered the genetic recombination in bacteria from two different strains through the process of conjugation. Bacterium Escherichia coli can grow in minimal culture medium containing minerals and sugar only. It can synthesize all the necessary vitamins from these raw materials. But its two mutant strains were found to lack the ability to synthesize some of the vitamins necessary for growth. These could not grow in the minimal medium till the particular vitamins were not supplied in the culture medium.

Mutant strain A : It (used as male strain) had the genetic composition Met^– , Bio^–, Thr⁺, Leu⁺, Thi⁺. It lacks the ability to manufacture vitamins methionine and biotin and can grow only in a culture medium which contains these vitamins in addition to sugar and minerals.

Mutant strain B : It (used as female strain or recipient) has a genetic composition Me⁺⁺, Bio⁺, Thr^–, Leu^–, Thi^–. It lacks the ability to manufacture threonine, leucine and thionine and can grow only when these vitamins are added to the growing medium.

These two strains of E.coli are, therefore, unable to grow in the minimal culture medium, when grow separately. But when a mixture of these two strains was allowed to grow in the same medium a number of colonies were formed. This indicates that the portion of donor DNA containing information to manufacture threonine, leucine and thionine had been transferred and incorporated in the recipient’s genotype during conjugation.

This experiment of Lederberg and Tatum shown that the conjugation results in the transfer of genetic material DNA from one bacterium to other. During conjugation a cytoplasmic bridge is formed between two conjugating bacteria.

Evidence from bacteriophage infection : Hershey and Chase (1952) conducted their experiment on T₂ bacteriophage, which attacks on E.coli bacterium. The phage particles were prepared by using radioisotopes of S³⁵ and P³² in the following steps :

(i) Few bacteriophages were grown in bacteria containing ³⁵S. Which was incorporated into the cystein and methionine amino acids of proteins and thus these amino acids with ³⁵S formed the proteins of phage.

(ii) Some other bacteriophages were grown in bacteria having ³²P. Which was restricted to DNA of phage particles. These two radioactive phage preparations (one with radioactive proteins and another with radioactive DNA) were allowed to infect the culture of E.coli. The protein coats were separated from the bacterial cell walls by shaking and centrifugation.

The heavier infected bacterial cells during centrifugation pelleted to bottom. The supernatant had the lighter phage particles and other components that failed to infect bacteria. It was observed that bacteriophages with radioactive DNA gave rise to radioactive pellets with ³²P in DNA. However in the phage particles with radioactive protein (with ³⁵S) the bacterial pellets have almost nil radioactivity indicating that proteins have failed to migrate into bacterial cell. So, it can be safely concluded that during infection by bacteriophage T₂, it was DNA, which entered the bacteria. It was followed by an eclipse period during which phage DNA replicates numerous times within the bacterial cell. Towards the end of eclipse period phage DNA directs the production of protein coats assembly of newly formed phage particles. Lysozyme (an enzyme) brings about the lysis of host cell and release, the newly formed bacteriophages. The above experiment clearly suggests that it is phage DNA and not protein, which contains the genetic information for the production of new bacteriophages. However, in some plant viruses (like TMV), RNA acts as hereditary material (being DNA absent).

DNA replication

Watson and Crick suggested a very simple mechanism of DNA replication or DNA transcription on the basis of its double helical structure. During replication the weak hydrogen bonds between the nitrogeneous bases of the nucleotides separate so that the two polynucleotide chains of DNA also separate and uncoil. The chains thus separated are complementary to one another. Because of the specificity of base pairing, each nucleotide of separated chains attracts it complementary nucleotide from the cell cytoplasm. Once the nucleotides are attached by their hydrogen bonds, their sugar radicals unite through their phosphate components, completing the formation of a new polynucleotide chain.

The method of DNA replication is semi-discontinuous and described as semi-conservative method, because each daughter DNA molecule is a hybrid conserving one parental polynucleotide chain and the other one newly synthesized strand. DNA replication occur in S-phage in cell cycle.

Mechanism of DNA replication

The entire process of DNA replication involves following steps in E.coli :

Recognition of the initiation point : First, DNA helix unwind by the enzyme “Helicase” which use the energy of ATP and replication of DNA begin at a specific point, called initiation point or origin where replication fork begins.

Unwinding of DNA : The unwinding proteins bind to the nicked strand of the duplex and separate the two strands at DNA duplex. Topoisomerase (Gyrase is a type of topoisomerase in E.coli) helps in unwinding of DNA.

Single stranded binding protein (SSB) : Which remain DNA in single stranded position and also known as helix destabilising protein (HDP).

RNA Priming : The DNA directed RNA polymerase now synthesizes the primer strands of RNA (RNA primer). The priming RNA strands are complementary to the two strands of DNA and are formed of 50 to 100 nucleotides.

Formation of DNA on RNA primers : The new strands of DNA are formed in the 5^¢ ® 3^¢ direction from the 3¢®5¢ template DNA by the addition of deoxyribonucleotides to the 3^¢ end of primer RNA.

Addition of nucleotide is done by DNA polymerase III. The leading strand of DNA is synthesized continuously in 5^¢®3^¢ direction as one piece. The lagging strand of DNA is synthesized discontinuously in its opposite direction in short segments. These segments are called Okazaki fragments.

Excision of RNA primers : Once a small segment of an okazaki fragment has been formed. The RNA primers are removed from the 5^¢ by the action of 5^¢®3^¢ exonuclease activity of DNA polymerase I.

Joining of okazaki fragments : The gaps left between Okazaki fragments are filled with complimentary deoxyribonucleotide residues by DNA polymerase-I. Finally, the adjacent 5^¢ and 3^¢ ends are joined by DNA ligase.

DNA polymerase enzymes

There are three DNA polymerase enzymes that participate in the process of DNA replication.

(i) DNA polymerase-I : This enzyme has been studied in E. coli in detail. It possesses a sulphydryl group, single interchain disulphide and one zinc molecule at the active site. DNA polymerase-I was discovered by Kornberg and his colleagues in 1955. It was considered to carry out DNA replication and also participates in the repair and proof reading of DNA by catalyzing the addition of mononucleotide units (the deoxyribonucleotide residues) to the free 3^¢ -hydroxyl end of DNA chain. A pure DNA polymerase-I can add about 1,000 nucleotide residues per minute per molecule and catalyses 5^¢®3^¢ exonuclease activity and removes nucleotide residues of primer RNA at 3^¢.

(ii) DNA polymerase-II : The biological role of polymerase II is not yet known.

(iii) DNA polymerase-III : This enzyme was discovered by T. Kornberg and M.L. Gefter (1972). It is the most active enzyme and responsible for DNA chain elongation.

DNA repair : When DNA damaged by mutagen, a system is activate to repair damage DNA. Say for example UV light induced thymidine dimers in DNA and repair mechanism of that DNA called photoreactivation. Many enzyme involved in repair mechanism in which endonuclease (Chemical knives) cut the defective part of DNA then gap is filled with DNA polymerase I and finally DNA ligase seals that repaired part.

Evidence in support of semiconservative mode of DNA replication (Meselson and Stahl’s experiment)

(1) Meselson and Stahl (1958) cultured (Escherichia coli) bacteria in a culture medium containing N¹⁵ were isotopes of nitrogen. After these had replicated for a few generations in that medium both the strands of their DNA contained N¹⁵ as constituents of purines and pyrimidines. When these bacteria with N¹⁵ were transferred in cultural medium containing N¹⁴, it was found that DNA separated from fresh generation of bacteria possesses one strand heavier than the other. The heavier strand represents the parental strand and lighter one is the new one synthesized from the culture indicating semiconservative mode of DNA replication. circular form of replication on as characteristic of prokaryotes is theta replication discovered by J. Cairns.

(2) Evidence from Taylor’s experiment on Vicia faba (Broad Bean) root tips using autoradiography technique and further he used tritiated thymidine (H³-tdR).

(3) Evidence from Cairn’s autoradiography experiment in bacteria. He used tritiated thymidine (H³-tdR).

RNA is found in the cytoplasm and nucleolus. Inside the cytoplasm it occurs freely as well as in the ribosomes. RNA can also be detected from mitochondria, chloroplasts and associated with the eukaryotic chromosomes. In some plant viruses RNA acts as hereditary material.

Structure of RNA

More commonly RNA is a single stranded structure consisting of an unbranched polynucleotide chain, but it is often folded back on itself forming helices. DNA is a double stranded structure and its two polynucleotide chains are bounded spirally around a main axis. It is made up by :

(1) Sugar : Ribose

(2) Phosphate : In the form of H₃PO₄.

(3) Nitrogenous base : Two types:

(a) Purine,

(b) Pyramidine

(i) Purine is further divided into Adenine and Guanine.

(ii) Pyramidin divided into Cytosine and Uracil.

Types of RNA

RNA can be classified into two types.

(1) Genetic RNA : Which established by Conrat. In most of the plant viruses, some animal viruses and in many bacteriophages DNA is not found and RNA acts as hereditary material. This RNA may be single stranded or double stranded.

(2) Nongenetic RNA : In the all other organisms where DNA is the hereditary material, different types of RNA are nongenetic. The nongenetic RNA is synthesized from DNA template.

In general, three types of RNAs have been distinguished :

Messenger RNA or Nuclear RNA (mRNA) : mRNA is a polymer of ribo-nucleotide as a complementary strand to DNA and carries genetic information in cytoplasm for the synthesis of proteins. For this reason only, it was named messenger RNA (mRNA) by Jacob and Monod (1961) is 5% of total RNA. It acts as a template for protein synthesis and has a short life span.

Ribosomal RNA (rRNA) : rRNA constitutes redundant nature upto 80% of total RNA of the cell. It occurs in ribosomes, which are nucleoprotein molecules.

Inside the ribosomes of eukaryotic cells rRNA occurs in the form of the particles of four different dimensions. These are designated 28S, 18S, 5.8S and 5S.

The 28S and 5S molecules occur in large subunit (60S subunit) of ribosome, whereas 18S molecules is present in the small subunit (40S subunit) of ribosome. In prokaryotic cells there are only 23S, 16S and 5S rRNA are found. Which are synthesized in Nucleolus / SAT region.

Transfer RNA (tRNA) : The transfer RNA is a family of about 60 small sized ribonucleic acids which can recognize the codons of mRNA and exhibit high affinity for 21 activated amino acids, combine with them and carry them to the site of protein synthesis. tRNA molecules have been variously termed as soluble RNA or supernatant RNA or adapter RNA. It is about 0-15% of RNA of the cell.

tRNA molecules are smallest, containing 75 to 80 nucleotides. The 3^¢ end of the polynucleotide chain ends in CCA base sequence. This represents site for the attachment of activated amino acid. The end of the chain terminates with guanine base. The bent in the chain of each tRNA molecule contains a definite sequence of three nitrogenous bases, which constitute the anticodon. It recognizes the codon on mRNA.

Most accepted model for t-RNA structure is clover leaf model, which way given by Robert Holley (1965) along with H.G. Khorana and Nirenberg (for yeast alanyl t-RNA) and for this work, they were awarded Nobel prize in 1968.

Four different region or special sites can be recognised in the molecule of tRNA.These are :

Amino acid attachment site : It occurs at the 3^¢ end of tRNA chain and has OH group combines with specific amino acid in the presence of ATP forming amino acyl tRNA.

Site for activating enzymes : Dihydrouridine or DHU loop dictate activation of enzymes.

Anticodon or codon recognition site : This site has three unpaired bases (triplet of base) whose sequence is complementary with a codon in mRNA.

Ribosome recognition site () : This helps in the attachment of tRNA to the ribosome.

Other types of RNA

Small nuclear RNA (snRNA) : It is a small sized RNA present in the nucleus. SnRNA takes part in splicing (U1 and U2), rRNA processing (U3) and mRNA processing.

Small cytoplasmic RNA (scRNA) : It is small sized RNA occurring free in the cytoplasm. It helps in taking and binding a ribosome to endoplasmic reticulum for producing secretory proteins.

Genetic code

Defined as structure of nitrogen bases(nucleotides) in mRNA molecule which contain the information for the synthesis of protein molecule. It is discovered by frame shift mutation by Crick.

Codon is the sequence of nitrogen bases (nucleotides) in mRNA, which codes for a single amino acid. Nirenberg and Mathaei (1961) experimentally proved that a single amino acid is determined by a sequence of three nitrogen bases which is known as triplet code. Khorana has got Nobel prize on genetic code.

Salient Features

Triplet : A single amino acid is specified by a sequence of three nucleotides in mRNA i.e., called codon. Due to triplet nature, it consist 64 codon.

Universal : A codon specifies the same amino acid in all organisms from viruses to human beings.

Commaless : There is no pause, so it reads continously.

Non-overlapping : No overlapping between adjacent nucleotide.

Initiation codon : The synthesis of polypeptide chain initiated by initiation codon, which located beginning the cistron i.e., AUG or GUG, which codes to methionine and valine amino acid respectively.

Termination codon : Termination is done by codon. These are UAA, UGA or UAG which does not code to any amino acid. These are also called nonsense codon.

Degeneracy : A single amino acid may be specified by many codon i.e., called degeneracy. Degeneracy is due to the last base in codon, which is known as wobble base. Thus first two codon are more important to determining the amino acid and third one is differ without affecting the coding i.e., known wobble hypothesis, (proposed by Crick) which establishes a economy of tRNA molecule and put forwarded by Crick. Degeneracy of genetic code was discovered by Berrfield and Nirenberg.

Table : 4-7 The Genetic Code Dictionary

Second Letter

First Letter

U

UUU

UUC

UUA

UUG

UCU

UCC

UCA

UCG

UAU

UAC

UAA

UAG

UGU

UGC

UGA

UGG

Third Letter

CUU

CUC

CUA

CUG

CCU

CCC

CCA

CCG

CAU

CAC

CAA

CAG

CGU

CGC

CGA

CGG

AUU

AUC

AUA

AUG

ACU

ACC

ACA

ACG

AAU

AAC

AAA

AAG

AGU

AGC

AGA

AGG

GUU

GUC

GUA

GUG

GCU

GCC

GCA

GCG

GAU

GAC

GAA

GAG

GGU

GGC

GGA

GGG

Central dogma

Central dogma of molecular biology proposes a unidirectional or one way flow of information from DNA to RNA (transcription) and from RNA to protein (translation). The concept was given by Watson and Crick.

As mentioned above the first step of central dogma is transcription (synthesis of mRNA from DNA), but in case of reverse transcription DNA is synthesizes from RNA in retrovirus. That concept is given by Temin and Baltimore in Rous sarcoma virus, also known as teminism or reverse transcription and enzyme catalyze this reaction is reverse trancriptase or RNA dependent DNA polymerase. For this work, Temin, Baltimore and Dulbecco were given Nobel prize (1975).

Transcription

Formation of mRNA from DNA is called as Transcription. It is heterocatalytic function of DNA. Template of DNA called sense strand (Master Strand) is involved. The segment of DNA involved in transcriptions is cistron, which have a promoter region where initiation is start and terminator region where transcription ends. Enzyme involved in transcription is RNA polymerase-II. Which consist five polypeptide (constitute core enzyme) and (sigma factor). Sigma factor recognise promoter site while remaining core enzyme takes part in chain elongation. After transcription, DNA molecule reassociates to form its original structure. In eukaryotes hn RNA (heterogenous nuclear RNA) which consist exon (coded region) and introns (non coded region or intervening sequences) formed in nucleus and diffuse in cytoplasm is also known as split gene which goes to transcription changes for removing the introns and later formed mRNA.

It consist three phenomenon

(1) Initiation : Initiation start with help of (sigma) factor of RNA polymerase enzyme. At the cap region which have 7 methyl guanosine residue at the 5^¢.

(2) Elongation : Elongation is done by core enzyme, which moves along the sense strand.

(3) Termination : In prokaryotes termination is done by rho factor while in eukaryotes poly A tail is responsible for termination at the 3^¢.

Translation or Protein synthesis

Formation of protein from mRNA is called translation is also known as polypeptide synthesis or protein synthesis. It is unidirectional process. The ribosomes of a polyribosome are held together by a strand of mRNA. Each eukaryotic ribosome has two parts, smaller 40S subunit (30S in prokaryotes) and larger 60S subunit (50S in prokaryotes).

Larger subunit has a groove for protection and passage of polypeptide, site A (acceptor or aminoacyl site), enzyme peptidyl transferase and a binding site for tRNA. The smaller subunit has a point for attachment of mRNA. Along with larger subunit, it forms a P-site or peptidyl transfer (donor site).

There are binding sites for initiation factors, elongation factors, translocase, GTPase, etc. The raw materials for protein synthesis are amino acids.mRNA, tRNAs and amino acyl tRNA synthetases.

Amino acids : Twenty types of amino acids and amides constitute the building blocks of proteins.

mRNA : It carries the coded information for synthesis of one (monocistronic) or more polypeptides (polycistronic). Its codons are recognised by tRNAs.

tRNAs : They picks up specific amino acid from amino acid pool and carrying over the mRNA strand.

Amino Acyl tRNA Synthetases : The enzymes are specific for particular amino acids and their tRNAs.

Activation of Amino Acids : An amino acid combines with its specific aminoacyl tRNA synthetase enzyme (AA-activating enzyme) in the presence of ATP to form aminoacyl adenylate enzyme complex (AA-AMP-E).

Pyrophosphate is released. Amino acid present in the complex is activated amino acid. It can attach to CCA or 3^¢ end of its specific tRNA to form aminoacyl or AA-tRNA (charged tRNA / adaptor molecule).

Amino Acid (AA) + ATP + Aminoacyl tRNA Synthetase (E)

AA-AMP-E + tRNA AA—tRNA + AMP + Enzyme.

Initiation : It is accomplished with the help of initiation factors. Prokaryotes have three initiation factors – IF₃, IF₂ and IF₁. Eukaryotes have nine initiation factors – eIF₁, eIF₂, eIF₃, eIF_4A, eIF_4B, eIF_4C, eIF_4D, eIF₅, eIF_6,,mRNA attaches itself to smaller subunit of ribosome with its cap coming in contact with 3^¢ end of 18 S rRNA (16S RNA in prokaryotes).

It requires eIF₂ (IF₃in prokaryotes). The initiation codon AUG or GUG comes to lie over P-site. It produces 40S – mRNA complex. P-site now attracts met tRNA (depending upon initiation codon). The anticodon of tRNA (UAC or CAC) comes to lie opposite initiation codon. Initiation factor eIF₃ (IF₂ in prokaryotes) and GTP are required. It gives rise to 40S-mRNA – tRNA^Met. Methionine is nonformylated (tRNA) in eukaryotic cytoplasm and formylated (tRNA) in case of prokaryotes.

The larger subunit of ribosome now attaches to 40S-mRNA-tRNA^Met complex to form 80S mRNA -tRNA complex. Initiation factors eIF₁ and eIF₄ (A, B and C) are required in eukaryotes and IF₁ in prokaryotes. Mg²⁺ is essential for union of the two subunit of ribosomes. A-site becomes operational. Second codon of mRNA lies over it.

Elongation/chain formation : A new AA-tRNA comes to lie over the A site codon by means of GTP and elongation factor (eEF₁ in eukaryotes, EF-Tu and EF-Ts in prokaryotes). Peptide bond (–CO.NH–) is established between carboxyl group (–COOH) of amino acid of P-site and amino group (–NH₂) of amino acid at A-site with the help of enzyme peptidyl transferase/synthetase.

Connection between tRNA and amino acid of P-site and A-site tRNA comes to bear a dipeptydl. Free tRNA of P-site slips away. By means of translocase (eEF₂in eukaryotes and EF-G in prokaryotes) and GTP, ribosome moves in relation to mRNA so that peptidyl carrying tRNA comes to lie on P-site and a new codon is exposed at A-site.Incorporation of an amino acid in polypeptide chain thus requires one ATP and two GTP molecules. Peptide formation and translocation continue uninterrupted till the whole m-RNA code is translated into polypeptide. In a polyribosome, when a number of ribosomes are helping in translation of same mRNA code, the ribosome nearest the 5^¢ end of mRNA carries the smallest polypeptide and the one towards the 3^¢ end the longest. Of course, ultimately the whole polypeptide is formed by each.

Termination : Polypeptide synthesis stops when a nonsense or termination codon [UAA, (ochre), UAG (Amber) or UGA (opal)] reaches A-site. It does not attract any AA-tRNA, P-site tRNA seperates from its amino acid in the presence of release factor eRF₁ in eukaryotes (RF₁for UAG and UAA, RF₂ for UAA and UGA in prokaryotes). The completed polypeptide is released, mRNA and ribosome separate. The two subunits of ribosome also dissociate with the help of dissociation factor.

Modification : Formylated methionine present at the beginning of polypeptide in prokaryotes and organelles is either deformylated (enzyme deformylase) or removed from chain (enzyme exopeptidase). Initially the polypeptide is elongated having only primary structure. As soon as the polypeptide comes out the groove of larger ribosome sub-unit, it forms -helix (secondary structure) which coils further forming a number of linkages (tertiary structure). Two or more polypeptides may get associated to become -pleated which then coil to produce tertiary and quaternary structure.

Genes expression and its regulation

Gene expression in prokaryotes

Gene expression refers to the molecular mechanism by which a gene expresses a phenotype by synthesizing a protein or an enzyme. Which determines the character. The gene contains the blue print or the information for the protein or an enzyme.

The category includes mechanism involved in the rapid turn-on and turn-off gene expression in response to environmental changes. Regulatory mechanism of this type are very important in microorganisms, because of the frequent exposure of these organisms to sudden changes in environment.

Gene concept can be studied by operon model. Operon are segment of genetic material which function as regulated unit that can be switched on and switched off, which was given by French scientists. Jacob and Monod (1961) working at Pasteur institute. They were studying lactose utilization in mutants of E.coli. An operon consists of one to several structural genes (three in lac operon and five in tryptophan operon of Escherichia coli, nine in histidine operon of Salmonella typhimurium), an operator gene a promoter gene a regulator gene, a repressor and inducer or corepressor. Operons are of two types, inducible and repressible.

(1) Inducible operon system /lac operon system : An inducible operon system is that regulated genetic material which remains switched off normally but becomes operational in the presence of an inducer. It occurs in catabolic pathways. The components are :

Structural genes : They are genes, which produce mRNAs for forming polypeptides/proteins/enzymes. Lac operon of Escherichia coli has three structural genes-Z (produces enzyme -galactosidase for splitting lactose/galactoside in to glucose and galactose) Y (produces enzyme galactoside permease required in entry of lactose/galactoside) and A (produces enzyme galactoside acetylase/transacetylase without any function in E.coli). The three structural genes of lac operon produce a single polycistronic mRNA. The three enzymes are, however, produced in different concentration.

Operator gene (O) : It gives passage to RNA polymerase when the structural genes are to express themselves. Normally, it is covered by a repressor. Operator gene of lac operon is small, made of 27 base pairs.

Promoter gene (P) : It is recognition centre / initiation point for RNA polymerase of the operon.

Regulator gene (i Gene) : It produces a repressor that binds to operator gene for keeping it nonfunctional (preventing RNA polymerase to pass from promoter to structural genes).

Repressor : It is a small protein formed by regulator gene. Which binds to operator gene and blocks passage of RNA polymerase towards structural enzymes. Repressor has two allosteric sites, one for attaching to operator gene and second for binding to inducer. Repressor of lac operon has a molecular weight of 160,000 and 4 subunit of 40,000 each.

Inducer : It is a chemical which attaches to repressor, changes the shape of operator binding site so that repressor no more remain attached to operator.

Table : 4-8 Differences between induction and repression

Induction	Repression
It turns the operon on.	It turns the operon off.
It starts transcription and translation.	It stops transcription and translation.
It is caused by a new metabolite which needs enzymes to get metabolised.	It is caused by an excess of existing metabolite
It operates in a catabolic pathway.	It operates in an anabolic pathway.
Repressor is prevented by the inducer from joining the operator gene.	Aporepressor is enabled by a corepressor to join the operator gene

Lactose/galactoside is inducer of lac operon. As soon as the operator gene becomes free, RNA polymerase is recognised by promoter gene. cAMP is required, RNA polymerase passes over the operator gene and then reaches the area of structural genes. Here it catalyses transcription of mRNAs.

(2) Repressible operon system/tryptophan operon system : A repressible operon system is that regulated genetic material, which normally remains active/operational and enzymes formed by its structural genes present in the cell till the operon is switched off when concentration of an end product crosses a threshold value. Repressible operon system usually occurs in anabolic pathways, e.g., tryptophan operon, argnine operon. Each has the following parts.

Structural genes : They are genes, which take part in synthesis of polypeptides/proteins/enzymes through the formation of specific mRNAs. Tryptophan operon has five structural genes – E, D, C, B and A.

Operator gene : It provides passage to RNA polymerase moving from promoter to structural genes. Operator gene of repressible operon is normally kept switched on as aporepressor formad by regulator gene is unable to block the gene.

Promoter gene : It is initiation/recognition point for RNA polymerase.

Regulator gene : The gene produces an aporepressor.

Aporepressor : It is a proteinaceous substance formed through the activity of regulator gene. It is able to block operator gene only when a corepressor is also available.

Corepressor : The nonproteinaceous component of repressor, which can be end product (feed back inhibition/repression) of the reaction mediated through enzymes synthesized by structural genes. Corepressor of tryptophan operon is tryptophan. It combines with aporepressor, form repressor which then blocks the operator gene to switch off the operon.

Gene expression in eukaryotes

In regulation of gene expression in eukaryotes the chromosomal proteins play important role. The chromosomal proteins are of two types. They are histones and non-histones. The regulation of gene expression involves an interaction between histones and non-histones. Histones inhibit protein synthesis and non-histones induce RNA synthesis. There are four main steps in the expression of genes. Hence regulation is brought about by the regulation and modification of one or more of these steps. They are :

Regulation of replication : Differential gene expression is achieved by gene amplification.

Regulation of transcription : The regulation of the expression of gene is mainly done at transcription. Hybridization experiments clearly show that production of specialised protein is due to differential gene transcription.

Regulation of the processing level : Some of the RNA synthesized in the nucleus are destroyed without leaving the nucleus. 80% of the nuclear RNA has no equivalent in the cytoplasm and only 20% if the nuclear RNA is identical in the cytoplasm. All the genes in a cell are transcribed into mRNA at all times, but the mRNA produced by some genes is destroyed rapidly. But the mRNA modeled on other genes are stabilized and only these mRNAs are passed into the cytoplasm.

Regulation of translation : The control of mRNA-translation is a fundamental phenomenon. In sea-urchin eggs fertilisation is followed by a tremendous increase in protein synthesis; but in the unfertilised egg, there is no protein synthesis. Still the unfertilised egg has complete machinery (i.e., amino acids, ribosomes, mRNA) for protein synthesis. There are two model for regulation in eukaryotes.

(a) Frenster’s model : According to 1965, The histones act as repressor’s during protein synthesis.

(b) Britten Davidson model : This is also called gene battery model or operon-operator model. It was proposed by Britten and Davidson in 1969. They have been proposed four type of genes namely integrator sensor, producer and receptor.

Chromosomes

The chromosomes are capable of self-reproduction and maintaining morphological and physiological properties through successive generations. They are capable of transmitting the contained hereditary material to the next generation. Hence these are known as ‘hereditary vehicles’. The eukaryotic chromosomes occurs in the nucleus and in certain other organelles, and are respectively called nuclear and extranuclear chromosomes.

Discovery of chromosomes

Hofmeister (1848) : First observed chromosomes in microsporocytes (microspore mother cells) of Tradescantia.

Flemming (1879) : Observed splitting of chromosomes during cell division and coined the term, ‘chromatin’.

Roux (1883) : He believed the chromosomes take part in inheritance.

W.Waldeyer (1888) : He coined the term ‘chromosome’.

Benden and Boveri (1887) : They found a fixed number of chromosomes in each species.

Chromosomal theory of inheritance

It was proposed independently by Sutton and Boveri in 1902. The chromosome theory of inheritance proposes that chromosomes are vehicles of hereditary information and expression as Mendelian factors or genes.

Kinds of chromosomes

Viral chromosomes : In viruses and bacteriophages a single molecule of DNA or RNA represents the viral chromosome.

Prokaryotic / Bacterial chromosomes : In bacteria and cyanobacteria, the hereditary matter is organized into a single large, circular molecule of double stranded DNA, which is loosely packed in the nuclear zone. It is known as bacterial chromosome or nucleoid.

Eukaryotic chromosomes : Chromosomes of eukaryotic cells are specific individualized bodies, formed of deoxyribonucleo proteins (DNA + Proteins).

Number of chromosomes

The number of chromosomes varies from two, the least number an organism can have, to a few hundred in different species. The least number of chromosomes are found in Ascaris megalocephala i.e., 2 (n = 2 in Mucor hiemalis in plants) while in a radiolarian protist (Aulocantha) has maximum number of chromosomes is 1600 (Ophioglossum reticulatum, 2n = 1262 in plants). The male of some roundworms and insects have one chromosome less than the females.

Table : 4-9 Diploid number of chromosomes in some organisms

Common name	Scientific name	Chromosomes
Amoeba	Amoeba proteus	500
Man	Homo sapiens	46
Gorilla	Maccaca mulatta	48
Pig	Sas scrofa	40
Sheep	Ovis aries	54
Cat	Felis maniculata	38
Dog	Canis familiaris	78
Rat	Rattus rattus	42
Rabbit	Oryctolagus cuniculus	44
Honey bee	Apis mellifera	32, 16
Mosquito	Culex sp	6
Grasshopper	Gryllus	23(M), 24(F)
Pink bread meld	Neurospora crassa	14
Baker’s yeast	Saccharomyces cerevisiae	34
Broad bean	Vicia faba	12
Garden pea	Pisum sativum	14
Onion	Allium cepa	16
Maize	Zea mays	20
Potato	Solanum tuberosum	48
Cabbage	Brassica oleracea	18
Radish	Raphanus sativum	18
Compositae	Haplopappus gracilis	4
Adder’s tongue fern	Ophioglossum reticulatum	1262
Jimson weed	Datura stramonium	24
Evening primrose	Oenothera biennis	14
Bread wheat	Triticum aestivum	42
Emmer wheat	Triticum turgidum	28
Tomato	Lycopersicon esculentum	24
Giant sequoia	Sequoia sempervirens	22

Structure of chromosome

Different regions or structure recognized in chromosomes are as under

Pellicle : It is the outer thin but doubtful covering or sheath of the chromosome.

Matrix : Matrix or ground substance of the chromosome is made up of proteins, small quantities of RNA and lipid. It has one or two chromonemata (singular-chromonema) depending upon the state of chromosome.

Chromonemata : They are coiled threads which form the bulk of chromosomes. A chromosome may have one (anaphase) or two (prophase and metaphase) chromonemata. The coiled filament was called chromonema by Vejdovsky in 1912. The coils may be of the following 2 types :

(1) Paranemic coils : When the chromonemal threads are easily separable from their coils then such coils are known as paranemic coils.

(2) Plectonemic coils : When the chromosomal threads remain inter-twined so intimately that they cannot be separated easily are known as plectonemic coils.

Primary constriction : A part of the chromosome is marked by a constriction. It is comparatively narrow than the remaining chromosome. It is known as primary constriction or centromere.

The microtubules of the chromosomal spindle fibres are attached to the centromere. Therefore, centromere is associated with the chromosomal movement during cell division. Kinetochore lies in the region of primary constriction. Kinetochore is the outermost covering of centromere.

Secondary constriction or nucleolar organizer : Sometimes one or both the arms of a chromosome are marked by a constriction other than the primary constriction. In certain chromosomes, the secondary constriction is (In human beings 13, 14, 15, 20 and 21 chromosome are nucleolar organizer) intimately associated with the nucleolus during interphase. It contains genes coding for 18S and 28S ribosomal RNA and is responsible for the formation of nucleolus. Therefore, it is known as nucleolar organizer region (NOR).

Chromomeres : Chromomeres are linearly arranged bead-like and compact segments described by J. Bellings. They are identified by their characteristic size and linear arrangement along a chromosome.

Telomeres : The tips of the chromosomes are rounded, sealed and are called telomeres which play role in Biological clock. The terminal part of a chromosome beyond secondary constriction is called satellite. The chromosome with satellite is known as sat chromosome, which have repeated base sequence.

Chromatids : At metaphase stage a chromosome consists of two chromatids joined at the common centromere. In the beginning of anaphase when centromere divides, the two chromatids acquire independent centromere and each one changes into a chromosome.

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