• Carmine is a dye extracted from the cochineal insect (Coccus cacti).
• Haematoxylin is a dye extracted from the heartwood of a tropical tree etc. Haematoxylin campechianum both are stain the chromosome and nucleus.

  Linkage.                                                                                                                                                                  

Introduction : "When genes are closely present link together in a group and transmitted as a single unit, the is phenomenon is called linkage".

(1)Theories of linkage

1. Sutton's hypothesis of linkage (1903) : The number of groups of genes are equivalent to the number of chromosomes.
2. 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.
3. 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 F₁ 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

F₁ 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.                                         A                                    a                       A                                    a

In repulsion phase the normal       B                                    b                       b                                    B

alleles as well as mutant alleles lie in

opposite   chromosomes   of    the

Fig : CIS – Arrangement of genes                         Fig : TRANS – Arrangement of genes

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).

4. 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.
5. 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.

1. Complete linkage : 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.

2. 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.
   3. 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.

4. 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.
5. Factor affected to linkage : Linkage is affected by the following factors.
   1. Distance : Closely located genes show strong linkage while genes widely located show weak linkage.
   2. Age : With increasing age the strength of linkage decreases.
   3. Temperature : Increasing temperature decreases the strength of linkage.
   4. X-rays : X-rays treatment reduces the strength of linkage.

(6)Significance of linkage

1. Due to linkage new recombinants are formed.
2. It helps in maintaining the valuable traits of a newly developed variety.
3. It helps locating genes on chromosome.
4. It disallows the breeders to combine all the desirable traits in a single variety.

           Important Tips                                                                                                                                                                                                

• Cinderella of genetics is Drosophila melanogaster.
• H. Lamprecht (1961) demonstrated that seven genes used by Mendel belonged to only four linkage groups (not seven as thought earlier).
• Blue green algae and bacteria contain one linkage group.
• Two dominant nonallelic genes are 50map unit apart then the linkage will be absent.
• Linkage decrease frequency of hybridization.
• In order to remain linked the distance between two genes should not increase beyond 40 map units.
• Linkage was first studied in Lathyrus-odoratus.
• Drosophilla was first animal for which a linkage map was constructed.
• Law of linkage is an exception to Mendel's law.

 Crossing over.                                                                                                                                                      

Introduction : 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.

(1)Kinds of crossing over

1. Somatic crossing over : It is found in somatic cells e.g., Curt stern in Drosophila and Potnecorvo in

Aspergillus nidulans shown somatic crossing over i.e. mitotic crossing over.

2. Germinal crossing over : It is found in germinal cells during gametogenesis. This is also known as meiotic crossing over.
3. Single cross over : It takes place at one point only on the non-sister chromatids, only 2 chromatids are involved.
4. Double cross over : It is the formation of 2 chiasmata in the same chromosome independent of each other. In a double cross over the genes lying outside the crossed regions will retain their original association.
5. Multiple cross over : It is formed when more than 2 chiasmata are formed. It is very rare.
6. Two-stranded crossing over : It takes place before splitting of homologous chromosomes so all the four resultants are recombinants.
7. Four stranded crossing over : It takes place after splitting of homologous chromosomes only 2 non- sister chromatids take part in crossing over resulting in 2 parental and 2 recombinant types.
2. Crossing over and chiasma : There are two views extended to explain the relationship between crossing over and chiasma formation. They are summarised here under.
   1. 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.
   2. 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.
3. Mechanism of crossing over : There are different views put forward to explain the mechanism of crossing over.

1. 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.

2. 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.

In fact, the crossing over is the event which, precisely at molecular level, results in the formation of a hybrid DNA molecule. Such models have been proposed by White house, 1963 as also by Holliday, 1964. These models mainly elaborate the mechanism of breakage and reunion of DNA helicase.

4. 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.
5. Coincidence : Coincidence or coefficient of coincidence is inverse measure of interface and is expressed as the ratio between the actual number of double cross over and the expected number of such double cross. That is:

Coincidence = Actual number of double cross over Expected number of double cross over

6. 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:

1. Temperature : High and low temperature increase the frequency of crossing over.
2. X-ray : Muller has discovered that exposure to X-ray and other radiations increases the frequency of crossing over.
3. Age : The frequency of crossing over decreases with increasing age in female Drosophila.
4. 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.
5. 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.
6. Chiasmata formation : Chiasmata formation at one point discourages chiasmata formation and

crossing over in the vicinity. This phenomenon is known as interference.

7. Inversions : Inversions of chromosome segments suppresses crossing over.
8. 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.

a

b

c

A                     B                     C                      D

Fig :       Diagram showing possibilities of crossing over between genes at different distances

9. Cytoplasm : Factor for crossing over is present in cytoplasm and is inherited to the offspring.

10. Nutritional effect : Crossing over frequencies are affected by concentration of metallic ions, such as calcium and magnesium.
11. Genotypic effect : Crossing over frequencies between the same two loci in different strains of the same species show variation because of numerous gene differences.
12. Chromosome structure effect : Changes in the order of genes on a chromosome produced by chromosomal aberrations usually act as cross over suppressors.
13. Centromere effect : Genes present close to the centromere region show reduced crossing over.
14. 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.
    7. Significance of crossing over : This phenomenon is of great biological significance, which are as under:

1. It gives evidence that the genes are linearly arranged on a chromosome. Thus, it throws light on the nature and working of the genes.
2. 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.
3. The crossing over is helpful in the chromosomal mapping. The percentage of crossing over is proportional to the distance between two genes.
4. 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.
5. 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.

Important Tips

• Separation of a chromosome segment and its union to non-homologous chromosomes is called illegitimate crossing over.
• Two genes situated very close on the chromosome show hardly any crossing over.
• The most acceptable theory to explain crossing over is of Muller.
• Genes of Antibiotic resistance on bacteria are located on plasmid.
• Barr and Bertram (1949) discovered barr body in nerve cell of female cats. Later found in cells of human females
• Study of phenotype to DNA sequence in gene come under forward genetics.
• First chromosomal map of a plant was of maize, it was prepared by Emerson.
• Plotting of specific genes of the chromosomes is chromosomes map, linkage map, genetic map.
• The most important use in producing transgenic plants and animals is of Reverse genetics.
• tt ´ tt ® Tt , This type of inheritance is an example of de-novo mutation.
• Hugo de Vries worked on evening primrose in preparation of mutation theory.
• E. coil is an important material for genetic experiment because it is haploid in nature and also easilly cultured.
• The percentage of individuals with a given genotype exhibiting, the phenotype associated genotype is known as penetrance.

 Chromosomal maps.                                                                                                                                          

On the basis of the following information, chromosomal maps have been prepared.

1. The genes are linearly arranged on a chromosome and therefore, the gene order should be known.
2. The percentage of crossing over between two genes is directly proportional to their distance. It is infact the index of 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 %).
3. 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:

C                                 A             B

9                       4

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.

A            B                 C

4               5

(4)Uses of chromosomal map

1. Finding exact location of gene on chromosomes.
2. Knowing recombination of various genes in a linkage group of chromosomes.
3. 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 by Friedrich Miescher in 1868 from the nuclei of pus cells and was named nuclein. The term nuclein was given by Altman.

DNA (Deoxyribonucleic Acid)

Introduction : 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.

1. Chemical composition : The chemical analysis has shown that DNA is composed of three different types of compound.
   1. Sugar molecule : Represented by a pentose sugar the deoxyribose or 2-deoxyribose which derived from ribose due to the deletion of oxygen from the second carbon.
   2. Phosphoric acid : H₃PO₄ that makes DNA acidic in nature.
   3. Nitrogeneous base : These are nitrogen containing ring compound. Which classified into two groups:

1. Purines : Two ring compound namely as Adenine and Guanine.
2. Pyramidine : 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.

Fig  :  Diagrammatic  representation  of  Watson's

and Crick's modal of DNA

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.

Composition of nucleoside and nucleotides of DNA and RNA

(D= Deoxyribose sugar, R = Ribose sugar, P = Phosphoric acid)

2. Watson and Crick’s model of DNA : In 1953 Watson and Crick suggested that in a DNA molecule there are two such polynucleotide chains arranged antiparallal or is 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.

(i)Important features of Watson and Crick double helical model of DNA

There are important features of DNA double helix.

1. The double helix comprises of two polynucleotide chains.
2. The two strands (polynucleotide chains) of double helix are anti-parallel due to phosphodiester bond.
3. Each polynucleotide chain has a sugar-phosphate ‘backbone’ with nitrogeneous bases directed inside the helix.
4. 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.
5. 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.
6. Erwin Chargaff (1950) made quantitative analysis of DNA and proposed “base equivalence rule” starting that molar

concentration of A = T & G º C or

 A + G =1& A + T

which is constant

Fig :       Helical     structure     of     DNA     as

suggested by Watson and Crick

for a species.

C + T

G + C

7. Ten base pairs occur per turn of helix (abbreviated 10bp). The spacing between adjacent base pairs is 10Å. The helix is 20Å in diameter and DNA molecule found 360^o in a clockwise.
   3. 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:
      1. The numbers of base pairs, present in each turn of DNA helix.
      2. The pitch or angle between each base pair.
      3. The helical diameter of DNA molecule.

4. The handedness of double helix. Which is mentioned in table.

Comparison of different types of DNA

4. Characteristics of DNA
   1. Denaturation or melting : The phenomenon of separating of two strand of DNA molecule by breaking of hydrogen bond at the temp. 90⁰C.
   2. 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.

5. Evidences of DNA as the genetic material : The following experiments conducted by the molecular bioloigists provide direct evidences of DNA being the genetic material bacterial transformation, bacterial recombination and bacteriophage infection.
   1. 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.
   2. 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.

1. 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.

2. 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.

3. 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 ³⁵S and ³²P in the following steps.

1. 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 ³⁵ S formed the proteins of phage.
2. 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 bacter