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Chapter 1 Genetic Basis of Inheritance
 Definition = Genetics is the study of Heredity and Variation
Sibling = offspring of same parents but at different birth ex. Brother and Sister.
Discoverers and Discoveries Related to Genetics
Father of Genetics Gregor John Mendel
Father of Modern Genetics - Bateson
Father of Drosophila Genetics - Thomas Haunt Morgan
Father of Indian Genetics M.S. Swami Nathan
Father of Human Genetics Garrod
Father of Radiation Genetics - H. J. Muller
Branches of Genetics
(1) Eugenics = Improvement of human race by applying law of heredity.
(2) Euthenics = Improvement of human race by providing better nutrition or better environment.
(3) Euphenics = Improvement of human race by using genetic engineering.
Mendelism
Life-History
Full Name - Gregar Johann Mendel
22 July, 1822 - Birth at Silsian village of Heinzendorf of Austria.
- Belongs to a poor farmer family
- He was an Austrian Monk.
1884 - Death of Mendel
 Due to defected Kidney
Important Dates Related to Mendel
1856-64 - Duration of experiment on pea
1866 - Published his results & ideas in proceeding natural history society of Brunn
- Name of the paper - experiment on plant Hybridisation
1900 - Rediscovery of Mendel's work by 3 scientist named as
(i)Hugo de vries- Holland
(ii)Korl Correns - Germany
(iii) Tschermack- Austria
1901 - De vries got paper of mendel published in "Flora".
1983 - Celebrated as century year of Mendelism in International Genetics Congress, New Delhi
(12th Dec 1983)
More About Mendel's Work
Experimental material = Pea (Pisum sativum)
Later he also selected cow pea (or Bean) and Hawk weed (or Heracium) but did not succeed.
Reason for Success of Mendel
Mendel selected pea plants that is world wide in distribution
He studies the inheritance of one character at a time so, the complex problems became easier
He maintained statistical records of the results and analysed his results using Algebric formulae and probabilities.
He verified his results by further experiments such as F3 and test cross etc.
Terminology of Genetics
Gene : A hereditary unit. A Piece of DNA that can synthesize one molecule of protein. Mendel used 'FACTOR' term for genes
Homozygous : (TT or tt) also called Pure.
When both genes in a pair are similar
Heterozygous : (Tt or Rr) Also called Impure
When both genes of the Pair are dissimilar
(Genes are found in pairs because, the chromosomes remain in pair)
Hemizygous : (T or t) When one gene of the pair is lost e.g. - Gametes
Alleles or Allelomorphs : The genes occupying same locus (position) in homologous chromosomes are called alleles. Usually allelic genes interact each other.
Wild allele : The original allele that is dominant and widely spread is called wild allele
Mutant allele : A wild allele that undergoes mutation, recessive in expression and less common in population is called Mutant allele.
Pure Line : A race of genetically homozygous and true breeeding organisms resulting from continued selfing is called a pure line.
Genotype : Genetic constitution of an organisms. For example
(i) Pure Tall = TT
(ii) Hybrid Tall = Tt
Phenotype : External appearance of the organism is called phenotype. For example
A dwarf pea plant = tt
A tall pea plant will has 2 condition.
(i) Pure tall = TT
(ii) Hybrid tall = Tt
Genome : Haploid number of chromosome for an Individual is called genome. For example. In case of man 2n = 46, n = 23
* Genome of man has 23 chromosome
Gene Pool : Sum total of genes of a population in any area at a time constitute a gene pool.
* Back Cross : If member of F1 progeny is crossed with either of its parent for Increasing the traits of parents.
* Test Cross : If member of F1 progeny is crossed with its recessive parent for knowing whether individual is momogyzous or heterogyzous.
If F1 plants is crossed with the dominant parent, this is called Out cross.
* Punnet Square = Gametic checker board.
It is checker board used to show result of a cross between two organisms.
* Pure Line
Term by Johannsen
A strain of genetically pure true breeding individual which have been derived from identical homozygous ancestor.
Experiments & Laws of Mendel
A. Monobybrid cross: Mendel selected two Pea, Plants, a tall and a dwarf. The gene for tallness was supposed as TT and for dwarfness tt. He obtained all F1 plants tall.
Now, he self crossed two F1 plants and find the following results in F2 generation
So, Phenotypic ratio = 3 : 1 (3 tall and 1 dwarf) and, genotypic ratio = 1 : 2 : 1. On the basis of the above results one law was formulated
* Law of Segregation (Law of Purity of Gametes)
Genes remain together without containing each other and, both genes segregated in the next generation. So, the gametes are always pure.
Deviations from Mendelian ratios:
A. Incomplete dominance:
e.g. Flower colour of Mirabilis Jalapa (Expt. by Correns) and in Antirrhinum (also called snapdragon or Dog flower), short horned cattles etc.
B. Co-dominace : When both alleles of a pair are fully expressed in heterozygous conditions, the phenomena is called Co-dominance.
Here both products are similar in respect of function but different in exact amino acid sequence e.g. Skin colour of cattle.
Examples (i) ABO blood group (ii) MNS blood group (iii) Sickle cell haemoglobin etc.
C. Lethal genes : Discovered by L. Cuenot
Any gene combination (Gene) that is fatal to the individual is called lethal gene. e.g.,
Inheritance of sickle cell Anaemia
The genes for this disease show co-dominance in heterozygous condition but, in homozygous recessive condition this is lethal. The heterozygotes are called Sickle cell Trans (SCT). On this basis three types of individuals can be found
(i) HbN HbN  100% normal RBC - alive
(ii) 
(iii) HbS HbS 100% Sickle cell RBC - Dies
So, the phenotypic (or genotypic) ratio = 1 : 2.
* Most of the african people are SCT so, are resistant to malarial infections
In Sickle cell anaemia, the RBCs get elongated & curved due to polymerization of haemoglobin. This occurs due to substitution of valine for glutamic acid at the 6th. position the  -chain.
D. Multiple alleles : Discovered by H. Nilsson Ehle
Some times more than two different forms of a given gene exists in a species. They are called multiple alleles.
e.g.- Skin colour of rodents, Eye colour in Drosophila, self sterilty in Tobacco, ABO blood groups in human beings, Hemolytic jaundice etc.
Inheritance of ABO blood groups:
The human blood groups depend basically upon the type of antigen in them. The genes for antigen formation are present on a chromosome, I (I = isoagglutinogen). Two types of antigen are found A and B. on, this basis.
Genotypes Phenotype
IAIA Blood group A
IAI0 (i) Blood group A
IBIB Blood group B
IBI0 (i) Blood group B
IAIB Blood group A B
I0I0 (ii) Blood group O
So, No. of Alleles = 3 (A, B & O)
No. of Phenotypes = 4 (A, B, AB & O)
No. of genotypes = 6 (IAIA, IAiA, IAIB, IBIB, IBi, ii)
In such a condition if n is the no. of alleles of a gene, the no. of different genotypes possible is
Dihybrid Cross
Cross between two individuals that are different in two characters is called dihybrid cross.
So, phenotypic ratio = 9 : 3 : 3 : 1 (4 classes) and, Genotypic ratio = 1 : 2 : 2 : 4 : 1 : 2 : 1 : 2 : 1 (9 classes)
We may write down such results in the following table also for our convenience in further topics.
1. - TTRR - Tall plant and round seeds
2. - TT Rr - DO -9
2. - Tt RR - DO
4. - TtRr - DO
1. - TT rr - Tall plant wrinkled seeds-3
2. - Tt rr - DO
1. - tt RR - Dwarf plant and & round seeds
2. - ttRr - Dwarf plant and & round seeds
1. - ttrr - Dwarf plant & wrinkled seeds -1
* Formula to find out types of gametes produced = 2n
where n = the number of heterozygous gene pairs
* Formula to find out phenotypic classes
= 2n, where n = the number of heterozygous gene pairs
* Formula to find out genotypic classes = 3n
where n = the number of heterozygous gene pairs
* Formula to find out fraction of a gemete = 1 / 2n,
where n = the number of heterozygous gene pairs
* Formula to find out possibilities for fertilization
= 2n1 × 2n2,
where n1 = number of heterozygous gene pairs of one parent and
n2 = number of heterozygous gene pairs of another parent
Q. What will be dihybrid phenotypic ratio if both gene pairs show incomplete dominance.
A. Ans. 1 : 2 : 2 : 4 : 1 : 2 : 1 : 2 : 1
Q. What will be the dihybrid genotypic ratio if both gene pairs show in-complete dominance.
A. 1 : 2 : 2 : 4 : 1 : 2 : 1 : 2 : 1
Q. What will be the dihybrid phenotypic ratio if one gene pair is lethal in homozygous recessive state (rr)
A. Ans. 9 : 3
Some More Useful Informations
Atavism - Occasional reappearance of a remote ancestral trait in some individuals is called atavism. This occurs due to activity of some Junk genes. ex. a short tail in some babies.
Penetrance - The percentage of individuals that show a particular phenotype among those capable of showing it. e.g. All individuals with geotype AA or Aa may not express the dominant phenotype due to presence of modifiers, suppressors epistatic genes, environment etc. If only 80% individuals with AA or Aa express dominant phenotype so, this may be described as 80% penetrance of gene A.
Expressivity - The degree to which a given genotype is espressed phenotypically in a particular individual is called expressivity. So, degree of expression of any penetrant gene is called expressivity. ex. Red eye colour in Drosophila flies may show different shades of colour suggesting variable expression of wild allele W or WW or Ww.
Selfish Gene - The genes which propagate themselves, despite being detrimental to the organisms that carry them.
Heterosis - The superiority of a heterozygote over other homozygote for a given trait is called heterosis or Gigas effect-discovered by Shull in Capsella.
Homoetic Mutation - A mutation that causes a body part to develop in an inappropriate position in an organism eg. In Drosophila sometimes legs develop at the place of antennae in head.
Electroporation - A process where the cell membranes are made permeable to DNA by applying an intense electric current.
Gene Interactions
A. Simple interactions - 9 : 3 : 3 : 1
B. Complementary genes - 9 : 7 (eg. - sweet Pea)
C. Recessive epistasis - 9 : 3 : 4 (e.g. skin colour of Mice)
(or supplementary genes)
D. Dominant epistasis - 12 : 3 : 1 (e.g. Fruit colour of summersquash)
E. Duplicate genes - 15 : 1 (eg - capsella bursa pastoris shephard in purse)
F. Inhibitory genes - 13 : 3 (e.g. Plumage colour in Poultries)
G. Novel phenotypes - 9 : 6 : 1 (e.g. fruit shape of summer squash)
or Additive - 1 : 2 [snapdragon, mouse coat colour)
H. Polygenic inheritance - 1 : 4 : 6 : 4 : 1 (e.g. kernal colour of wheat)
1 : 6 : 1 : 15 : 20 : 15 : 6 : 1 (Human skin colour)
Pedigree analysis - Pedigree is a tool most widely used for study representation of inheritance of human traits
Chapter 2 Gene & Chromosomes
Chromosomes : "These are self duplicating basophilic structures"
"Chromosomes are the vehicles, drived by centromere, carrying passengers (genes) from station to station (generation to generation)".
Chromosomes were first observed by -Hofmeister (in Tradescantia) (Pollen mother cell)
First observed by - Holf meischer.
Discovered by - Strasburger
Named by - Waldayer because of their affinities for basic dye.
Chromatin - Term - Flemming
Sutton & Boveri - Proposed chromosomal theory of inheritance - Morgan confirmed it.
Chemical compostion : Chromosomes are Nucleoproteinaceous thus, are made up of
1. DNA
2.
 
* DNA - 40%
Histone + other proteins = 50%
Lipids - traces
RNA - 1.5%
NHC - 8.5% - Ca2+, Mg2+ and Fe2+ are present
- The relation of DNA with chromosome can be visualised as -
––– 
Karyotype
The chromosome complement of a cell or an individual is called karyotype and is used to refer to the arrrangement of chromosomes in Metaphase according to their length and position of centromere. Karyotype of any cell is represented diagramatically by IDIOGRAM.
Classification of human chromosomes:
In a human diploid cell 23 pairs of chromosome are found and have been divided into 7 groups according to their lengthwise arrangement, ABCDEF & G. Longest chromosome are present near the poles and are respectively more and more smaller towards the centre. The complete picture looks like -
Chromosome 'X' is supposed to be of group 'C' & chromosome 'Y' of the group 'G'
Euchromatins and Heterochromatins
Terms-Emil Heitz If a chromosome is stained, mostly parts get deeply stained while, a few parts get lightly stained. The former is called Heterochromatins and the later Euchromatins. Heterochromatic regions are deeply stained due to high amount of DNA but, are genetically almost inert and only euchromatic regions are active. Actually, due to high amount DNA, strands are unable to separate for transcription.
Mutation
Term-by Hugo Devries. Discovered by Hugo De Vries in Oenothera lamarkiana .
He wrote a book "The Mutation Theory"
Any qualitative or quantitative change in the genetic material or chromosomes is called Mutation.
Spontaneous Mutation :
Mutation that occurs automaticaly in nature.
Induced Mutation :
Mutation is done artifically is called induced mutation.
Forward mutation :
Mutation that occurs continuously from generation to generation.
Backward mutation :
Mutation occurs generation to generation and returns back to its original gene in the same way.
Somatic Mutation :
Mutation in somatic cells. This is not inherited in the next generation.
Silent Mutation :
Mutations that do not appear phenotypically.
Biochemical Mutation
We may distinguish mutations into 2 main parts
A. Chromosomal Mutations B : Gene Mutations
A. Chromosomal mutations -
Two types
(i) By change in number and position of gene on chromosome
(called chromosomal aberrations)
1. By deletion:
If any segment of a chromosome is detached from either end, this is called deletion.
If the segment is detached from intermediate portions, this is called deficiency.
2. By Duplication : If a segment of a chromosome gets attached with its homologous chromosome, this is called duplication.
3. By Inversion :
If a segment of a chromosome is detached and reattached at the same place but, in reverse order, this is called inversion. 
4. By Translocation :
This occurs between two non-homologous chromosomes. This may be of two sub-types.
(a) Simple translocation :
Segment of a chromosome is attached to some other non-homologous chromosome in this case.
(b) Reciprocal translocation :
Any segment of a chromosome is when detached and, attached to any non-homologous chromosome and vice-versa. (illegimate crossing over)
Robertsonian translocation:
This may arise from aberrations that lead to the fusion of two formerly separate non-homologous chromosomes into a single chromosome, or to the separation of formely joined sections of a single chromosome into two chromosomes.
(ii) By change in chromosome number
I. Euploidy :
Exact duplication in the number of chromosomes of any cell is called euploidy. 3 sub-type.
(a) Autopolyploidy :
Duplication in the original set of chromosomes in any cell. e.g. Autotriploids - Banana, Ananas, Cynadon etc. Autotetraploids - Grapes, Tagetes, Maize, Potato, secale (Rye), Barseem. Autopentaploids - Rosa Autohexaploids - Some grasses
(b) Allopolyploidy :
Duplication of non-homologous chromosomes is called allopolyploidy. Actually the two set of chromosomes come from two different species and get duplicated.
Ex. 1. Rahphanobrassica
- (Prepared by G. D. Karpenchinko ) ( c ) Autoallopolyploidy : e.g., AAAABB - Helianthus tuberosus.
Ex. 2.
Triticale is allohexaploid (6n), 1st. man made cereal, formed from cross between Triticum & Rye.
Ex. 3. Triticum aestivum
- This is a hexallopolyploid
* Gigas effect
= Increase in characters due to increase in frequency of dominant allele is called Gigas effect . In broad sense, this is also called Heterosis and the example is called Hybrid vigour . * Non sense Mutation = A mutation which stops polypeptide synthesis due to formation of terminating or nonsense codon. Ex. ATA
UAA. * Mis sense Mutation = A mutation which involves changes in a codon that produce different amino acid at specific site in a polypeptide chain e.g., Sickle cell Anaemia GAA
GUA. * Same sense mutation = A silent mutation in which codon is changed but not after the amino acid specificity. GCA GCT.
B. Gene Mutation :
First gene mutation was recorded by Sethwrights in Ancon sheep.
All genes can mutate
Mostly mutation are recessive
1. By Transition : A purine is replaced by another purine base or a pyrimidine base is replaced by another pyrimidine base.
2. By Transversion
: A Purine base is replaced by a pyrimidine base or vice versa.
3. By base analogous
-5-Bu (5-bromouracil), 5-chlorouracil (5-Cu), 5-iodouracil etc. are analogous to Thymine. 2-Aminopurine (2-A-P) is analogous to Adenine 5-Bu has affinity with Guanine So, it may replace T but, pairs always with G. So, ultimately A = T is converted into G 
C.
4. By deamination
: HNO 2 deaminates Adenine to Hypoxanthine (H) cytosine to uracil (U) and Guanine to xanthine (x). H pairs C, U with A and x with C. So, A = T is converted into G
C. ( b ) Gibberish mutation or Frame shift Mutation (Gross mutation) : may be of 2 types 
When due to mutation, the whole gene is changed, this is called Gross mutation.
Induced Mutation :
Mutation may be produced artificially by chemicals or radiations etc. * Muller was 1st scientist to produce induced mutation in Drosophila by x-rays and got noble prize of 1946.
Chemical Mutagens
1. HNO2 -
It is a common industrial chemical, used as a meat preservative HNO2 changes ( i ) Cytosine
Uracil [C - U) ( ii ) Guanine
Xanthein [G - X) ( iii ) Adenine
Hypoxanthein (A - H)
2. Alkylating Agents =
Produce methylation or ethylation of N2 bases.
EES =
Ethyl ethane sulphonate.
NTG
= Nitrosoguanidine.
EMS =
Ethyl methane sulphonate
DES =
Die ethyl sulphonate
NM =
Nitrogen mustard
EDB =
Ethyl Die bromide - Food preservative
GF =
Griseofuvin - Antifungal
3. Base anulogue (T-G)
Bu - 5 Bromouracil Iu - 5 Idouracil Cu - 5 Chlorouracil
4. Acridines =
Dyes as proflavin, Acriflavin etc. They get interculated in between base pair produce insertion or deletion. They give rise to Frame shift or Gibbish mutation addition or loss of some nucleotords from DNA give rise to laterally or forward shifting of base sequence hence mutation . ( i ) By lowering pH - Some bases become positively charged and so, strand separation occurs. This may be lethal sometimes. Sometimes at low pH, A&G are completely removed. ( ii ) At High temperature - Strands get separated and sometimes this may cause depurination. In Rice low temperature causes mutation. ( iii ) By radiations - Ionising radiations usually cause mutation ex. x -rays, g-rays, a-rays, cosmic rays. laser rays, protons, neutrons etc.
By x -rays mutation is assured in prokaryotes.
1st - Gamma - Garden in India was established in Bose's Research Institute, Calcutta.
UV-rays are non-ionising but, may cause mutation.
Linkage
Discovered by Bateson & Punnet in Lathyrus odoratus .
Boveri stated that "Genes are linearly present on chromosomes". The tendency of genes present on the same chromosome to stay together in hereditary transmission is called linkage. Usually, the linked genes do not show independent assortment.
Linkage is the exception of law of independent assortment.
Mendel could not discovered linkage selected because the genes for characters were either present on different chromosomes or were (swee pea) syntenic.
Degree of Linkage
( a ) Complete linkage : This occurs between two very closely located genes as they have no chance of separating by crossing over. e.g ., Grey body, normal wings × black body & vestigeal wings. F 2 ratio comes out to be 3 : 1 and Test cross ratio = 1 : 1. ( b ) Incomplete linkage : This occurs in genes distantly located on the same chromosome so, crossing over may occur between them (genes). e.g., Flower colour and size of pollens. (In Maize reported by Hutchinson).
Chromosomal theory of linkage
was Postulated by Morgan & Castle
Linkage Groups All the genes on one chromosome which remain linked together during hereditary transmission are said to form a linkage group. The number of linkage groups in an organism is equal to the number of chromosome pairs or haploid number of chromosomes of the organism e.g. pea has 7 linkage groups & maize has 10.
Coupling and Repulsion
A. Coupling :
Either dominant or recessive genes when come from the same parent (PPLL or ppll). They tend to remain together in the next generation. This is called coupling.
Repulsion
Either dominant or recessive genes when come from different parents (PPll or ppLL), they tend to remain separated in the next generation. This is called repulsion.
Cis genes and Trans genes :
If the dominant alleles A & B of two linked genes are present on the same chromosome and their recessive alleles are present on its homologous chromosomes, the arrangement of genes is called cis-arrangement.
If one dominant gene and another recessive gene are present on one chromosome (A and b) and their alleles type (a and B) on the homologus chromosome, this type of arrangement is called Trans arrangement.
Crossing Over
Discovered by Morgan This is exchange of corresponding parts of the non-sister chromatids of homologus chromosomes during pachytene of prophase-I of Meiosis. This results in recombination of genes present on the same chromosome and, the resultants are called recombinants.
Recombinants
= Discovered by Morgan = Product of crossing over = The non sister chromatids which take part in crossing over are called recombinants.
Types of Crossing Over
1. Single crossing over
: Exchange of genetic mateerial take place at one point so 2 recombinant and 2-parental types are produced.
2. Double crossing over
Crossing over is found at 2 points in between the any two genes in homologous chromosomes.
Inteference and Coincidence
Usually, crossing over in one region interferes with the crossing over in the adjacent region. This is called interference and, is applied in double cross-over cases. If double cross-over are absent altogether, we would say that the interference is 100% (or 1).
Chromosomal Mapping :
Morgan and strutevant's hypothesis states that frequency of crossing over between two genes present on the same chromosome is directly proportional to the distance between genes. If two genes are quite apart a crossing over will most probably occur resulting in nearly 50% recombination. The probability of crossing over and the recombination will go on decreasing, as the distance between the genes decreases. Thus recombination frequency can indicate the relative distance between genes.
First chromosomal map was prepared by Sturtevante (1911) for x- chromosome of Drosophila.
First chromosomal map among plants was prepared by Barbara MC Clintock in Maize.
Complete gene mapping was 1st. done by Barbara McClintock in Maize, then by Morgan in Drosophila. Distance 
Sxe Determination Study of factors which are responsible for making any Individual male or female, known as Sxe Determining factor and this phenomenon is called Sxe determinator . It is of four types:
1. Environmental
Sxe Determination: In case of marine mollusc crepidula, marine - worm bonellia crocodiles and some lizards environment determinance type of Sxe.
2. Genic Determination of
Sxe 
In case of bacteria, chlamydomonas and male infloresence in maize monogenic gene controlled six determination . In Jula diagenic determination of Sxeis found.
3. Hormonal control of
Sxe
Sometimes Sxe is determined by hormones released by Sxe organs. For Ex. In case of
1. Bonellia -
A marrine worm
2. Free Martin -
In case of cattles, when twins are produced, one of being male and other female.
Here female is sterile and male is normal.
This is known as free martin after the name of worker.
The condition is present only when there is vascular connection between them.
4. Chromosomal Determination of
Sxe
Chromosomal theory of Sxe determination was given by Wilson and Stevens.
Term x chromosome and y chromosome was given by Wilson and Stevens
Henking discovered X chromosome in reproductive cells of fire fly.
Stevens discovered Y chromosome
Some additional points regarding
Sxe -chromosomes.
Sxe chromosomes were discovered by Mc clung.
Sxe-chromosomes are mostly heterochromatic and, Y is more heterochromatic than X.
In human beings Y chromosome has 4 - 6 functional genes only, like TDF, SRY, genes fro hypertrichosis etc.
The genes found on Y chromosome are known as HOLANDRIC GENES .
In drosophila the Y-chromosome looks inverted J-like and is almost 100% inert.
Chromosomal determination of
Sxe may be of following types
XX - XY Type (or Lygeous Type)
Found in mostly animals & plants
Males are heterogametic (XY) and females are homogametic (XX) e.g. like in human beings
Total number of chromosomes = 46 (23 pairs)
1 Pair is of Sxe chromosomes XX in females & XY in males.
Male gametes are of two types ( i ) Androsperms = 22 + Y (Heterogametric) ( ii ) Gynosperms = 22 + X When sperm having X chromosome fuses with the ovum, the foetus will be a female and, when the sperm with Y chromosome fuses with ovum the foetus will be male.
XX-XO Types (Frotanor Type)
Found in round worms and insects like, grasshoppers. etc (except - Drosophila).
Females have 2 Sxe chromosomes while male have only one Sxe chromosome. so female have 1 chromosome less than the males.
Genic Balance theory of
Sxe determination
A/c to the theory: ( i ) In insects X determines femaleness which maleness is determined by some of the autosomes. ( ii ) One X is heavier than 1 autosome but, lighter than 2 autosomes. ( iii ) There is a balance remains constant between X-chromosome & autosomes from beginning to the last of life span.
Theory was given by Calvin Bridges.
According to this theory, ratio between number of x chromosomes and one or more number of autosomes will determine the Sxe.
Bridge sugested that in insects X determines femalenes while some of the autosomes determine maleness
One X is heavier than one autosome but lighter than two autosomes.
Number of X-chromosomes and autosomes participating in Sxe determination remains balanced throughout the whole life span.
If ratio between x and A is 1.0, it will be a female individual.
When this ratio is more than one (or - about 1.5), it will be metafemale.
* Hormonal control of Sxe :
Sometimes Sxe is determined by hormones released by Sxe organs. For Ex. In case of 1. Bonellia - A marrine worm 2. Free martin - In case of cattle, then twins are produced, one of being ale and other female.
*
Here female is sterile and male is normal.
*
This is known as free martin after the name of worker.
*
The condition is present only when there is vascular connection between them.
ZW - ZZ Type
Found in birds and some reptiles
Females are heterogametic
Males are homogametic
ZO - ZZ Type
Found in some butterflies and moth.
Females are heterogametic. Sxe Linked Inheritence
Genes present on Sxe chromosomes are known as Sxe linked genes.
Inheritence of such type of genes is called Sxe linked inheritence.
Morgan reported that all Sxelinked characters show Ctis cross inheritence.
Two important Sxe linked disease are haemophilia and colourblindness, both the disease perform criss-cross inheritence.
Criss Cross Inheritence
A type of Sxe linked inheritence where a parent passes that traits to the grand child of same Sxe through offspring of opposite Sxe.
Hence father is Diagynic , and mother is Diandric. Sxe Linkage
The Sxe chromosome (x+y) besides carrying the Sxe determining genes, also have genes for several other character which are inherited together from one generation to the next and, are called Sxe linked genes. In heritance of there Sxe linked genes or traits is called Sxe linkage .
Ex. 1.
Haemopbhilia
Discovered by John Otto.
Also known as Bleeder's disease or princess diasease or Royal Family disease.
Patient lacks Antihaemophilic Globulin.
So natural phenomena of blood clotting is not found.
Disease is due to presence of a recessive gene h on x chromosome.
Inheritence
Genotypes :
(1) Carrier female = XXh
(2) Haemophilic female dies at birth = XhXh
(3) Haemophilic male = Xhy
Ex. 2.
Colour Blindness
A. Red Green colour blindness
Also known as Daltonism.
B. Protanopia
Red blindness
C. Deuteronopia
Green blindness
Defination
- A recessive Sxe linked trait where eyes fails to distinguish between red and green colour so name is red green colour blindness.
Sxe limited traits
Traits which are expressed in particular Sxe but genes are present in both of the Sxe.
The genes for such characters are found on autosomes but, such genes express only in particular Sxe.
Ex.
( i ) Milk secretion in mamalian females. ( ii ) Beard and moustache etc.
Sxe influenced traits
These are not due to particulars genes but are activated by production of particular hormones.
The genes for these traits are present in autosomes but influenced by particular Sxe hormone.s
Ex. (i)
Low pitched voice
(ii)
Baldness in human
Gene for baldness behaves as an autosomal dominant in males and autosomal recessive in females.
Other Sxe influenced traits are
1.
Forinstance stuttering.
2.
Cleft palate.
3.
Hare lip
Cytoplasmic Inheritence
Dr. Ruth Sanger reported that some self prepetuating hereditary particles are made up of DNA are found in certain cell organelles.
These particles are known as extranuclear genes and their inheritence is called cytoplasmic or extra nuclear inheritence.
The genetic material found in cytoplasm is also known as plasmon and these genes are known as plamagenes.
Plasmagenes are found in cytoplasm. We known that cytoplasm of mother get transferred to next progeny (via egg), so this type of inheritence is also called as maternal inheritence.
Following are the examples which explain cytoplasmic and maternal inheritence.
( i ) Cytoplasmic male sterlity in maize
Rhoades first of all discovered first cytoplasmic male male sterility of plants in maize
Factor responsible for this cytoplasmic male sterility are located in mitochondrial DNA ( ii ) Maternal Influence on shell coiling in Limnae
The snail exhibit two types of coiling of their shells.
1.
Right directional - Dextral
2.
Left directional - Sinistral ( iii ) Plastid Inheritance in 4 'O' clock plant
( iv ) Sigma factor in Drosophila
( v )Kappa particles Inheritance in paramecium ( vi ) Poky in Neurospora
Chapter 3 Expression of gene DNA (Deoxy ribonucleic Acid)
In vitro synthesis of RNA was first done by Ochoa.
The DNA in centrosome is non-genetic.
It vitro synthesis of DNA has been carried out by korenberg. Altman gave the name - nucleic acid (discovered mitochondria)
Discovered by -F. Miescher (1569) - in pus cells and named nuclein.
Presence of Pentose sugar (both ribose and Deoxyribose) was demonstrated by - Levene.
Size - Length not constant, Diameter is 20 
(approximately)
No.
- Not countable
DNA is the largest macromolecule. * DNAs are measured in weight. Unit of measurement is picogram (1pg = 10 -12 gram) 1 pg DNA is about 31 cms. - long. In each human cell about 5.6 pg (about 174 cms - long) DNA is found.
Chemical composition -
Made up of three components.
1. Pentose sugar :
Deoxyribose type (oxygen is removed from 2nd - carbon atom of sugar)
2. Phosphoric acid :
(H 3 PO 4 ) attached with 3rd and 5th carbon atom of sugar.
3. Nitrogenous bases :
Two types
Purines
Adenine (A) & Guanine (G)- double ring compounds.
9 membered double ringed, with N at 1, 3, 7 and 9 position.
Pyrimidines 6 membered single ringed, No is at 1, 3 Positions.
The structure of these sugars looks like -
A molecule of water is removed during the formation of each phosphodiester bond and also of Glycosidic bond is they are hydrophobic / water releasing bonds while in the formation of H bonds water is wither absorbed nor released. Antill nature of DNA strands occurs due to phosphodiester bonds. * Mononucleotides ex . AMP, ADP, ATP, TDP, CMP, CTP, GMP, GDP etc. * Dinucleotides ex. FAD & NAD. * Polynucleotides ex. DNA & RNA.
One mole of sugar + one base + phosphorus together constitute a nucleotide. A nucleotide without phosphororus is called a nucleoside. DNA is a polynucleotide chain.
Phosphoric acid are linked with 3rd and 5th - carbon atoms of the sugar. Now only 1st. carbon-atom of sugar is free to which any one base is attached. Purines are attached with sugar with its 9th position while pyrimidine with its 3rd position.
Double Helix Model of DNA -
Proposed by Watson & Crick - 1953.
Nobel Prize to-Watson, crick and wilkins-1962.
DNA is double stranded and both the strands are antiparallel i.e., one strand runs in 5 1 3 1 direction while the another strand runs in 5 1 3 1 direction.
The strands remain coiled about the same axis in such a way that they can separate only by uncoiling so lateral separation is not possible.
Strands are coiled together plectonemically so, separation is more easier. Paranemical coiling rarely is seen near the site of transcription (only).
The bases are set in a plane at 90 o to the long axis.
The strands complete one turn at every 34
. There are 10 base pairs (steps) present in each turn so, the distance between two base pairs is 3.4
.
The DNA is right handed and a major groove and a minor groove are alternately found.
A pairs with T by two H-bonds while C pairs with G by three H-bonds so, a purine base pairs with a pyrimidine base and vice versa.
The bases are arranged in zig-zag fashion and placed at 36 o to each other.
Angle of each turn (major to major grooves and minor to minor grooves) is of 360 o so, the angle between a major and a minor groove is 180 o . Walkin studied DNA using (X ray crystallography technique)
* A purine pairs with a pyrimidine & vice-versa because, The space between the two strands is constant and this allows only pairing between a purine and a pyrimidine base. * Chargaff's rule : Amount of purines in a DNA is always equialent to the amount of pyrimidines. According to chargaff the ratio of is constant for a species.
The flow of information from gene (DNA molecule) to regulatory proteins in a outline manner looks like -
The melting temperature of DNA is 80 o C. This is called denaturation. After cooling the DNA is again formed. This is called Renaturation. This was studied by J. Murmur.
DNA is stained with basic fuschin (Also called Fuelgen stain or Parasanalin).
*
RNA is stained in Methyl green, chemically known as Pyronin.
*
The denaturing temp. for RNA is 100-120°C since it is difficult to break the phosphodiester bonds than hydrogen bonds.
*
Althrough either of the 3 DNA polymerases may polymrize complimentary nucleotides but polymerization is generally done by side polymerase III.
DNA Replication
Replication means formation of carbon copies.
Eukaryotic DNA is multirepliconic since, this may start from more than one place while, Prokaryotic DNA is monorepliconic. (Only one replicating point is present in Prokaryotes DNA).
Non-histonic proteins separate DNA and histones to start replication in DNA.
DNA replication is usually bidirectional Three models were proposed for DNA-replication.
C. Semi Conservative Model
: Proposed by CRICK
According to this model in the daughter DNA, one strand is newer while, the another remains older. This was proved to be true. The difference between conservative, semiconservative and dispersive modes can be visualised as -
Semiconservative model of DNA replication was proved by Messelson and Stahl by using radioactive nitrogen (N 15 ) in E. coli. Also proved by Taylor in Vicia faba ( Broad Bean ) by using radioactive thymidine, that in chromosomes semiconservative type of replication is found.
During replication in Eukaryotes Replication fork is formed while in prokaryotes Replication eye (or replication q) is formed. The same DNA-Polymerase-III Catalyses the formation of complementary newer strands along both older strands simultaneously.
In prokaryotes replication always starts from a specific point, called REPLICON . So, point of replication and termination is always the same.
DNA replication occur during S phase of cell cycle.
Some Important Enzymes For replication
A. DNA-Polymerase
: 1st obtained by Korenberg so, also often called Korenberg enzyme . These are of three types in prokaryotes, DNA polymerase-I, II and III.
In Eukaryotes 5 types of DNA polymerase are found to be reported 1. DNA-polymerase 
(cytoplasmic or large polymerase) In nucleus chain. 2. DNA-polymerase
(Nuclear polymerase) found only in vertebrates. 3. DNA -polymerase 
- Mitochondrial polymerase-encoded in nucleus. 4. DNA-polymerase 
- In mammalian cells it is PCNA (Proliferating cell nuclear antigen). 5. DNA-polymerase 
- In mammalian Hela cells & budding yeasts.
C. Topoisomerase -
The DNA molecule is double helical but, it may even twist further & becomes supercoiled. Due to supercoiling of DNA around a histone core, nucleosome is formed. It (loosening of super coilings)is brought about by the enzyme Topoisomerase.
All types of DNA mols. - exhibit a considerable conformational flexibility.
A and B forms are found almost in all DNA mols. In RL model the B-DNA is left-handed only.
Mechanism of DNA replication:
1. Activation of Deoxy Ribonucleotides.
1st of all AMP, GMP, CMP and TMP with the help of enzyme phosphorylase get activated by energy. So converted into ATP, GTP, CTP and TTp respectively.
2. Exposure of DNA strands
The two strands of DNA start to separate slowly from replication site (Replicon) by action of helicase (unwindase) . Topoisomerase start to open the coilings (twistings) in Eukaryotes and DNA Gyrase in prokaryotes. Replication starts near 5 1 end of the strands.
Just above the replication fork, the degree of coiling remains maximum - Supercoiling.
SSB (stabilizing protein) to stabilize the nurinding of DNA strands.
3. Formation of RNA primer
At 3 1 end of each strand a small RNA Primer is synthesized by the enzyme primase as a footstep at the 5 1 end (initiation) of the new strands.
4. Base Pairing
The nucleotides ATP, GTP, CTP and TTP get linked opposite to the complementary nucleotides of the template strands (older) between A and T two H-bonds and between C and G three H-bonds are formed and energy is released.
5. Chain Formation
Since, Replication takes place in 5 1 3 1 direction along both template strands this is formed continuously along one strand and interruptly along the another strand (zipper fashion). The formation is known as leading strand and the later is called lagging strand.
In this way DNA-polymerase-III proceeds continuously in 5 1 3 1 direction and along both template strands (crick) a new strand (watson) is formed.
6. Replication Reading
DNA polymerase shows polymerization always in 5'
3' direction and exonucleus activity always in 3'
5' direction.
Window
The main proof reader is DNA poly - I (3' 
5'). If this is absent poly. - III, acts as proof reader (in 3'
5' (direction). So, in proof reading the enzyme DNA polymerases function in 3'
5' direction.
In several prokaryotes the DNA poly - I Functions as proof reader in 5'
3' direction if the DNA segment is damaged by UV-rays or some other agents.
RNA
A. Genetic RNA : Found in RNA viruses only.
B. Non-genetic RNA : eg m-RNA, t-RNA and r-RNA.
RNAs are primitive than DNA.
RNAs are short lived due to failure of Chargaff's rule.
Synthesis of RNA from DNA is called Transcription.
Transcription
In each gene there is a initiation site for transcription found, called PROMOTOR SITE . All promotors are rich in A = T (TATA BOX) Contents.
Window
Only one strand of DNA participates in transcription and called SENSE or plus strand Chain. The another strand is called MISSENSE or Minus strand chain and this is meant for coiling with the sense chain. RNA - poly-III-for synthesis of t-RNA
(Rho) factor has ATP ase activity.
Still the m-RNA is not completed. Now, a small segment of 3 bases gets attached near 5 1 - end called Initiation codon. Initiation codon is AUG or rarely GUG. AUG codes for methionine in Eukaryotes and N-formyl methionine in prokaryotes. Though GUG codes for valine but, as initiation codon this codes methionine and N-formyl methionine respectively in Eukaryotes and prokaryotes. - The process in a nut shell looks like -
At the last, at 3 ' end a termination codon (non-sense codon) is attached. There are 3 termination codons, known, UAA (ocher), UAG (Amber) and UGA (opal).
Genetic Codes :
DNA sends message by giving codes on m-RNA for translation. These are called Genetic codes.
Characters :
1.
Genetic codes are non overlapping (i.e. the last base of one code and the first base at the next code are never similar).
2.
Genetic codes are universal (The same 64 types of genetic codes, including the termination codons are also found in all living oranism).
Wooble Hypothesis - By Crick
The t-RNA identifies genetic codes on the basis of the first two bases and, the 3rd - base of the code is not very important. So, wobbling allows economy of the no. of t-RNA mols
m - RNA
Synthesized maximally but found minimally.
These constitute 3-5% of the total cellular RNAs
Base pairings 10-15%
Life span
Half hour in prokaryotes and half hr. to 2 hrs, in eukaryotes.
Carries genetic informations from DNA to the site of translation, Ribosomes in the form of genetic codes.
t-RNA
Soluble in 1 mole NaCl solution. Also called s-RNA (Soluble RNA or Supernated RNA) due to its very small size (Adapter RNA is N-RNA)
Formed from nuclear DNA. These looks like as -
Base pairings 30-35% So, more stable than m-RNAs
At least 20 type of t-RNAs are essentially found in cells though, may be more.
The 3D model of t RNA looks like -
Made up of 70-85 nucleotides and diameter is 8-9
When t-RNA formation is completed a code of 3 bases - CCA is attached to the 3 ' end
Remain scattered in the cytoplasm.
Different sites on t RNA molecule looks like -
t-RNAs are specific in action so, a particular t-RNA carries a particular amino acid
r-RNA
Formed from nucleolar DNA. (Synthesized by nucleolar DNA)
Constitutes 80-85% of the total cellular RNAs
This is integral part of ribosomes and help in translating genetic codes of m-RNA
This is the most stable RNA, since this has 70-80% or more base pairings. But the pairing behaviour of RNA varies according to the temperature, pH, medium etc. rRNA looks like -
The base sequence of all r-RNAs in all organisms generally the same in all individuals of a species, so r-RNA is supposed to be the best raw material for the study of evolution.
Translation :
Following events occur
1. Activation of Aminoacids :
Aminoacids first get activated by consuming ATP. For activation of every aminoa cid a molecule of ATP is required. Now, the aminoacid is called activated.
2. Attachment of Aminoacids :
This activated aminoacid gets attached to the -CCA end of t-RNA to form amino acyl-t-RNA complex with the help of enzyme amino-acyl t-RNA synthetase. Now, the t-RNA is called charged t-RNA.
Formation of Polypeptide
In the larger subunit of ribosome a longitudinal tunnel is found through which this synthesized Polypeptide chain come out in the cytoplasm.
Regulation of Protein Synthesis
Out of sum 4000 genes in bacteria, only a few are expressed at a given time. The same is true for other organism, although the number of genes may vary significantly. Thus, there must to be some sort of genetic machinery to regulate the synthesis of certain proteins just for the following reasons: ( i ) Specific proteins are synthesized in those cells only, where they are needed, although their genes are present in all the cells of an organism. ( ii ) A particular protein in synthesized only at the time when it is needed by the organism. ( iii ) Only the required amount of a protein is synthesized. At a particular time certain set of genes is ' switched on' and other ' switched off '. When switched on, a gene is active for synthesizing proteins and when switched off, it is inactive.
The Operon
Jacob and Monod, 1961 proposed a scheme for induction and repression of enzyme synthesis popularly known as operon model . For their excellent work, they were awarded Nobel prize in 1965. An operon is a group of genes situated next to each other in the DNA that can be switched on or off in a unified manner. In other words, it is a unit of transcription. The lactose (lac) operon in E. coli has been studied in greater details.
Inducible system
: in E. coli the utilization of the disaccharide lactose is brought about by three enzymes namely
- galctosidase, lac permease and transcetylase . The enzyme
- galctosidase, hydrolyses lactose into glucose and galactose. When lactose is added in the medium, the production of the enzymes is enhanced by 100 times. This phenonenon is called induction . Such enzymes whose production is induced by adding the substrate are called inducible enzymes and the genetic system which controls their production as inducible system . The substrates whose addition induces the synthesis of enzyme are called inducers. In the absence of an inducer the genes for the synthesis of the enzyme do not function.
Repressible system :
The synthesis of amino acid tryptophan needs five enzymes in E. coli . If tryptophan is added in the medium, the synthesis of enzymes is reduced. This phenomenon is called repression . Such enzymes, whose production is checked by the addition of the end product are repressible enzymes and the governing system as repressible system. Such end products which bring about repression are called
co-repressors.
The cell possesses certain molecules called repressors which check the activity of genes. A repressore may be active or inactive. The active repressor becomes inactive by adding an inducer. The inactive repressor is called aporepressor . The aporepressor becomes active if a co-repressor is added.
Deciphering of Genetic Codes (By Neirenberg)
UUU - Phenylalanine AAA - Lysine CCC - Proline GGG - Glycine CUC - Sereine UCU - Leucine GUG - Valine UGU - Systeine AUG - Methionine UGG - Tryptophane
Recombinant DNA Technology
The phenomenon of alternation of genetic constitution of cells or individuals by directed & selective modification, insertion or deletion of an individual gene or genes for human welfare is known as genetic engineering. Mostly such manipulation in DNA or gene are done by the process known as Recombinant DNA Technology that is why mostly genetic engineering & Recombiant DNA Technology are used as synonyms. This research involves two main steps.
1. Gene Splicing
- It involves breakage of DNA molecule at two desired places to isolates a specific DNA segment & invitro insertion of this segment into another DNA molecule at desired position by the mediation of highly specific enzyme. DNA molecule thus obtained as known as. Recombiant DNA or chimeric DNA .
2. Gene Cloning -
Clone means genetically identical copies of individual or cell or DNA.
By Gene Cloning technique from the single copy of recombinant DNA, multiple copies of the same can be obtained. Clones of desired DNA fragments are obtained by inserting in into suitable vector ( Plasmid, Phagamid, Cosmids etc.) & then cloning these vectors into suitable host cell ( e.g. E. coli, yeast ) where they can faithfully replicate with host genome & many clones of inserted DNA fragments are obtained. Replication of Recombinant DNA Molecule in host cell is known as molecular cloning.
Tools & Techniques Involved in Genetic Engineering
1. Restriction Enzyme
These enzymes are the indispensable tool of recombinant DNA tech. They are also called as restriction endonucleases. They are isolated from bacteria.
W. Arber for the first time postulated the existence of this enzyme & it was firstly isolated by bacteria. Haemophilus influenzae by Smith & Nathans.
Arber Nathans & Smith were awarded the Nobel Prize in 1978 for this discoveries. Some examples of restriction enzyme are EcoRI, Eco B, Hpa I, BamH 1 .
Sticky or cohesive end style or staggered cutting
- Enzyme makes the cut across the strand of DNA at different position (few nucleotide apart) & generate the fragments with protruding ends these ends are said to be sticky or cohesive ends.
2. Cloning Vectors & Cloning of DNA
The cloning of a foreign fragment of DNA (or gene) in bacteria is made possible due to the ability of cloning 'vectors' or 'carries' to continue their life-style after additional sequences of DNA have been inserted into their genome. The insertion results in a hybrid or chimeric' or recombinant vector which consists in part of the additional 'foreign' fragment of DNA. These chimeric vector, when cloned in bacteria., replicate in exactly the same way as the original vector and so are obtained in large amounts. In this way, the inserted foreign DNA simultaneously replicates with the remaining part of chimeric vector and copies of the original foreign DNA then can be retrieved from the progeny. Most common vectors used for cloning DNA sequences in bacterial cell (specially E. coli ) are Plasmids bacteriophages & Cosmids.
(a) Plasmids - Plasmids are most widely used cloning vectors. They are extra chromosomal genetic elements found in bacteria that replicate autonomously with in the host cell. Their DNA is circular & Double stranded. Plasmid carries the sequences required for the replication of the plasmid known as origin denoted by ori. A Very common plasmid cloning vector i.e. E. coli plasmid cloning vector must have three features.
I.
An ori sequence which allows plasmid to replicate in E. coli.
II. A dominant selectable marker which enables E. coli cell carrying that plasmid to get easily distinguished from those cell lacking plasmids.
III. Unique cleavage site
for restriction enzyme. Mostly all these requirements are not found in naturally occurring plasmids so that are engineered invitro to obtain these feature & finally of facilitate gene cloning. Some of genetically engineered plasmids are pBR322, pBR327, pUC19 cloning vectors, cloning technique.
The plasmid vector is isolated from the bacterial cell and cleaved at one site by restriction endonuclease. The cleavage converts the circular plasmid into a linear molecule. Now the two ends of linear plasmid are joined to the ends of the foreign DNA (the gene) to be inserted with the help of enzyme DNA ligase. This regenerates a circular hybrid or chimeric plasmid. The chimeric plasmid is transferred to a bacterium wherein it replicates and perpetuates indefinitely.
(b) Phage Cloning Vectors
- Commonly used phage cloning vectors are derivatives of bacteriophage  it consists of linear DNA. Which have been engineered in the way that there lytic cycle is possible but lysogenic cycle is not possible. First vector is cut with the help of restriction enzyme. Desired foreign DNA fragment is inserted between the arms of vector DNA Inserted DNA is sealed with vector by ligase enzyme. Phage-particle can take up only DNA fragment ranging from 40-50 kb thus only 15 kb of foreign DNA can be inserted in phage vector.
(c) Phagemids Cloning Vector
- They are prepared artificially by combining the features of phages with plasmids (e.g. pBluescript 11 kb ). There are certain other vectors which are used in introducing recombinant DNA molecule into variety of prokaryotic & Eukaryotic organisms these are
(d) Shuttle Vector
- This cloning vector can replicate in two or more host organisms. Example - YEp24 .
(e) Yeast Artificial Chromosome Vector (YACS)
- They are artificial chromosome cloned in yeast cell.
3. DNA Ligases
DNA ligase enzymes are those that seal nicks between adjacent nucleotides in a double stranded DNA molecule. Ligases are present in both prokaryotes and eukaryotes.
4. Linkers
Linkers are short DNA double strands which contains site for the action of one or more restriction enzyme. Linker sequences are artificially jointed with naturally occurring plasmids to provide them with unique restriction site.
Among eukaryotes, DNA cloning has been done in yeast, mouse
and to some extent even in some higher plant species.
Selection & Screening of Recombinant Clones
- Cells having recombinant DNA are selected & screened from the population of cells. This is the final stage of gene cloning process. This process is achieved by Nucleic acid hybridization method in which radioactively labelled mRNA sequence complementary to recombinant DNA sequence is hybridized in situ & hybridized recombinant DNA : mRNA sequence is then screened.
Molecular Probes
Molecular probes are small DNA segments or RNA segments (often synthesized on DNA tempelate) that recognize complementary sequences in DNA or RNA molecules and thus allow identification and isolation of these specific DNA sequences from an organism. Antibodies are also occasionally use as probes to recognize specific protein sequences. These probes also frequently used for a variety of other purposes including diagnosis of infectious diseases, identification of food contaminants, variety of microbiological tests, forensic tests (e.g. DNA fingerprinting of nurderers or rapists ), etc. Probes can also be used to identify strains of an organism e.g. varieties of a crop species. Detection of homologous sequence after hybridization with the probe is very difficult task and to come over this difficulty probes are labelled by radioactive isotopes e.g. 32P (Phosphorus). After hybridixation with radioactively labelled probe, hybrids can be detected by auto radiography.
Chapter 4
Application of Recombinant DNA Technology
or
Genetic Engineering
1. Transgenic organisms
(i)
The modern techniques of gene transfer using recombinant DNA technology, however, allow transfer of genes from even bacteria and insects (or even artificially synthesized genes) to higher plants and animals, thus increasing manifold the possibilities of improving animals (e.g. livestock) and plants.
(ii)
Organisms developed by gene transfer method are known as transgenic organisms.
(iii) In animals DNA is injected into either the pronuclei of embryonic cell or the embryo stem cell . Techniques used in production of transgenic animals are In Vitro Fertilization (IVF) and Embryo Transfer (ET) .
(iv)
In plants technique of cell culture or protoplast culture and then regeneration of cultured cell or protoplast into whole plant is used.
(v)
Gene transfer or uptake of genes by cells of bacteria or microbes or higher plants is known as transformation.
(vi) Uptake of genes by animals cell on the other hand is known as transfection .
Transgenic Animals
- One of the first reports of transgenic animals published in December, 1982 involved transfer of growth hormone (GH) gene, fused to the promoter of metallothionine (MT) 1 gene. Since then transgenic animals have been produced in a variety of animals including mice, rabbits, sheep pigs, goats, cows, fish
Transgenic plants - Transgenic plants have been produced in more than 50 species by the end of the year 1992 and are being produced in a number of additional species every year. The progress has been so spectacular that by the turn of the century, we hope to be growing corps, which have been tailored to market specification by addition, substraction of moldification of genes. Transgenic plants have been produced, which are resistant to herbicides, insects, viruses and a variety of other biotic and abiotic stresses.
Agrobacterium tumifaciens
Agrobacterium
is gram negative soil bacteria infects over 330 genera of dicot plants. A tumifaciens causes crown gall disease . In dicot disease causing property of bacteria is because of its plasmid known as Ti-Plasmid. Small segment of Ti-plasmid is transferred and gets integrated with nuclear DNA of plant Cell. This segment is known as T-DNA. This bacterium is being highly utilized in gene transfer processes for higher plants. Recently A. Rhizogenes having Ri-Plasmid is alos being exploited in this field.
2. Hybridoma and Production of Monoclonal Antibodies (MAbs)
Monoclonal antibodies are derived from single lymphocyte cell, ( i.e., they are the clones of same lymphocyte cell)
3. Commercial Products
(i)
The gene of human insulin has been inserted into plasmid pPBR322 and cloned in E. coli where it is estimated to produce 100,000 insulin molecules per cell. Insulin produced in this manner is now available commercially.
(ii) Insulin from bacteria became first genetically engineered material to be licenced for use in human.
(iii)
Gene of human leucocyte interferon was prepared as cDNA form its mRNA this gene has been expressed and cloned in E. coli and such preparations of interferon are now available in the market.
(iv)
Tissue plasminogen activator (TPA) - used to prevent or reverse blood clots, therefore, preventing strokes, heart attacks, or pulmonary embolisms.
(v)
Human growth hormone - used to treat pituitary dwarfism.
(vi)
Human blood clotting factor VIII-to treat hemophilics
(vii)
DNAase for treating human patients with cystic fibrosis.
(viii)
Bovine growth hormone-to increase cattle and dairy yields.
(ix)
Recombinant vaccines-for treatment of human and animal viral diseases ( e.g ., hepatitis B in humans).
(x)
Genetically engineered bacteria and other microorganisms for improved production of, for example, industrial enzymes (e.g., amylases to break down starch to glucose), citric acid (flavouring), and ethanol.
(xi)
Genetically engineered bacteria that can accelerate the degradation of oil pollutants or of certain chemicals in toxic wastes ( e.g ., dioxin).
(xii)
Genetically engineered bacteria that, when sprayed on fields of crops such as strawberries or potatoes, lower by a degree or two the temperature at which the leaves will freeze, thus providing some protection against frost damage compared with untreated plants.
Animal Cloning and Genetic Engineering
1. Animal Cloning
Animal cloning is more difficult than plant cloning as animal cells lost their totipotency on reaching the gastrula stage of animal development. However, animal tissue cultures especially from tumours and embryonic tissue cells have been successfully since a long time and standard techniques are available for isolating animal cells and tissues from different systems. Some examples of animal cloning and genetic engineering are tissue culture somatic cell fusion, cell cloning and creating transgenics.
Examples of animal cloning and genetic engineering
( i ) Hela cells : The Hela cells lines are from the tumours of Henrietta Lacks who has been immortalised because of her cells growing in cell cultures. ( ii ) Hybridomas : These are the cells obtained from fusion of human cells and cells producing antibodies. ( iii ) Organ culture : There are reports that organs of pigs for organ transplantation in humans are being developed. A particular biotechnology company has created through genetic cloning a litter of five cloned piglets. Pigs breed easily and mature quickly and their organs are approximately the same size as those of humans. Techniques are being employed to have genetically enginered clones of pigs whose organs humans shall not reject upon transplantation. There is hope then, that gravely ill patients will not have to away donors. ( iv ) Transgenics : Any organism containing a foreign gene (segment of DNA) from a different species is a transgenic organism. Since genes can be transferred to the genome of another organism only through genetic engineering, transgenics can be defined as "genetically engineered organisms carrying gene/genes of another species in their genome". The gene for human growth hormone has been successfully introduced into mice. Transgenic mice are larger.
2. Gene Transfer in Animals
Gene transfer in animals is mostly through direct methods such as electroporation or microinjection or using particle gun. In creating 'Dolly' the cloned sheep, fertilised eggs of its mother was removed by microneedle and nucleus from an udder cell of a donor sheep was microinjected in the egg after removing egg nucleus. The egg developed into 'Dolly' with genes identical to its mother.
Animal Cloning
* ANDI was the first cloned monkey by embryo splitting derived its name from a backward abbreviation for "Inserted DNA" .
* Ian Wilmut
of Roslin Institute, Edinburgh, U. K. has produced a clone of adult lamb named "Dolly" (Feb. 1997). * Recent genetic analysis of Dolly's DNA has shown that she is a "chimera", not a perfect clone. * Dolly has two genetic mothers as confirmed by the analysis of her mitochondria by Eric Schon and Ian Wilmut in 1999. * The Scottish scientists who cloned Dolly have (July 1997) produced Molly and Polly , two lambs cloned with a human gene for blood clotting Factor IX. * The milk of "Molly" and "Polly" contains Factor IX that can be extracted for use in treating human haemophilia. * Steven Stice and James Robl (USA) have deeloped a technique for genetically customised calves that will be able to produce medicines for human in their milk. * The first cloned calves George and Charlie were born in January 1998.
* Brigitte Boisslier
, a 46-year old French chemist announced the creation of the world's first cloned human baby nicknamed "Eve" (December 2002).
Genomics
* The total genetic constitution of an organism. * The term "genomics" give by Thomas Roderick (1986). * 
Began in 1990.
Goals of this project are : * To develop ways of mapping the human genome at increasing fine level of precision. * To store this information in databases and develop tools for data analysis. * To address the ethical, legal and social issues that may arise from this project.
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