Population genetics khaireni for agriculture

Population genetics khaireni for agriculture

2. The Nature of Gene
Presented by: Anup Adhikari

3. Gene
•Gene is defined as the molecular hereditary unit of all living
organisms.
•It is a segment of DNA that is capable of autonomous division
and contains genetic information in the form of genetic codes.
•It carries the valuable information to build and maintain cell
and pass those genetic information to the generations.

4. Gene theory
TH Morgan proposed the gene theory which
states that:
•Chromosomes are the bearers of hereditary
units and each chromosome carries hundreds
or thousands of genes.
•The genes are arranged on the chromosomes
in the linear order and on the special regions
or locus.

5. Terminologies
•Cistron:
A segment of DNA specifying a single polypeptide chain.
•Recon:
It is the smallest unit of DNA capable of undergoing
crossing over and recombination.
•Muton:
The smallest unit of genetic material which can undergo
mutation.

7. One gene one Polypeptide Hypothesis
•George Beadle and Edward Tatum were awarded with
Nobel prize for their work in One gene One Enzyme
Hypothesis.
•They worked on Neurospora crassa during 1940s for
this hypothesis.
•Tatum a research associate in Beadle’s laboratory,
learned that although bacteria are alike in their basic
biochemistry, they differ in their growth-factor
requirements.
•If these differences are genetic in origin, Beadle
reflected, it should be possible to induce mutations
producing them.

8. •Because there was then no genetics of bacteria, Beadle
chose another microorganism, the bread mould
Neurospora, to test his idea.
•The beautifully simple genetic machinery of Neurospora
was well understood, having been worked out largely by
B.O. Dodge at the New York Botanical Garden.
•Dodge was a close friend of T.H. Morgan and convinced him
to take some cultures with him in 1928 when he left
Columbia University to found the Biology Division at
Caltech.
•Dodge assured Morgan that Neurospora would be an
important organism for genetics some day.

9. Beadle and Tatum Experiment
•The experiment consisted of X-raying one parent of a cross and
culturing the progeny (haploid ascospores, isolated singly and
numbered) on a “complete” medium-i.e., one designed to
satisfy the maximal number of nutritional needs, known and
unknown.
•This medium was made by adding yeast and malt extracts to a
simple “minimal” medium consisting of salts, sugar, and biotin,
the only growth-factor required by wild-type Neurospora.
•Once progeny cultures were obtained, they were tested for
their ability to grow on minimal medium.
•Those that did not grow were further tested to identify their
growth requirement

10. Here, wild type of
Neurospora can grow in
minimal media(MM) but
mutants cannot grow.

11. •The chance for success in this venture seemed so low at
the time that Beadle and Tatum agreed at the outset to
test 5,000 spores before giving up.
•Success actually came with spore number 299, which
grew on minimal medium supplemented with
pyridoxine.
•Crossing to wild type showed that the requirement was
inherited, as if caused by mutation of a single gene.
•The phenotypic ratio of 1:1 was obtained which
indicated the inheritance of nutritional requirement in
mutant types.
•Other mutations soon followed number 299, resulting in
requirements for various vitamins, amino acids, and
nucleic acid bases.

13. •Beadle and Tatum wanted to identify which arg mutants
affected particular steps in the arginine synthesis pathway.
•They hypothesized that a defective gene would produce a
defective enzyme.
•A defective enzyme in one of the steps of the pathway would
mean that the intermediate compound it produced would not
be synthesized.
•The arg mutants were grown in media supplemented with
intermediates of the arginine synthesis pathway.
•Using this method, Beadle and Tatum isolated mutant strains
that were defective at a specific step.

16. Conclusion of Beadle and Tatum
•Beadle and Tatum concluded that one gene codes for
one enzyme.
•Genes control biochemical reactions by controlling
the production of enzymes.
•Each enzyme serves as catalyst of the enzymatic
pathway that leads to a certain metabolic activity
leading to a formation of final product.

17. •Since some enzymes are made up of two or more
different polypeptides and these polypeptides are
synthesized by two or more gene regions, this
relationship was updated to the one-gene/one-
polypeptide hypothesis,
•Example: Tryptophan synthetase enzyme is made up
of two polypeptides, α-polypeptide and β-
polypeptide which are synthetized by two different
genes trp A and trp B.
•Example: Haemoglobin is a protein that is made up
of four molecules of two different kind of
polypeptides.

18. Enzymatic explanation of
Genetic ratios
Presented by: Anup Adhikari

19. •There is relationship between enzyme and phenotype of an
organism.
•Enzyme controls the biochemical reaction and the reaction
produces biochemical product which is responsible to final
phenotype expression.
•So phenotypic ratio is determined by the enzyme and these
enzymes are the product of genes.

Gene A Gene B Gene C

Enzyme1 Enzyme2 Enzyme 3

Precursor Product 1 Product2 Phenotype

20. •In other words a gene is transcribed into RNA and
RNA is translated ta a kind of polypeptide.
•The polypeptide changes to enzyme or the
polypeptide behaves like the enzyme.
•The enzyme catalyzes the biochemical reaction to
produce a final product.
•The product of such reaction is responsible for the
expression of the phenotype and the phenotype
expression produces the phenotypic ratio.

21. •In Mendelian phenotypic ratio, one gene was
involved for the expression of a single phenotype.
•But there are cases of gene interaction in which
two or more genes are involved to determine a
single trait.
•In gene interaction the phenotypic ratio deviates
from the expected Mendelian ratio.
•There are a variety of phenotypic ratios which are
the result of a variety of gene interaction.

22. Example: Novel phenotype (9:3:3:1)
Eg: Comb shape in poultry.
Gene P Gene R

Enz P Enz R

Precursor Product Phenotype
(Single) (Pea) (Walnut)
Parents: Rose X Pea
RRpp rrPP
F1: PpRr (Walnut)
Gametes: RP Rp rP rp (Selfing)

F2: 9P-R-: Walnut
3ppR-: Rose
3P-rr: Pea
1pprr: Single

23. •The proportion of walnut, pea, rose and single
shaped comb in poultry in F2 generations is 9:3:3:1.
•In segregating population, P gene alone(rrPP)
produces pea shaped comb in poultry and R gene
alone (RRpp) pro bduces rose shaped comb.
•When both dominant genes P and R are present in
the genotype(P-R-) they produce walnut shaped
comb.
•recessive homozygous genotype pprr produces
single type of comb.

24. Example: Complementary action(9:7)
Eg: Flower color in pea.
Gene C Gene P

Enz C Enz P

Precursor Product 1 Phenotype
(White) (White) (Purple)

Parents: Purple X White
CCPP ccpp

F1: CcPp (Purple)
Gametes: CP Cp cP cp (Selfing)

F2: 9 C_P_: Purple
3 ccP_:
3 C_pp: white
1 ccpp:

25. •The proportion of purple and white flowers of pea
plants in F2 segregating population is 9:7.
•In segregating population, C gene alone(CCpp) P
gene alone (ccPP) don't produce any pigments for
flower color.
•When both dominant genes C and P are present in
the genotype(C-P-) they produce purple colored
pigments for production of purple flowers in pea
plants.
•recessive homozygous genotype ccpp produces
white flowers.

26. Example: Modifying action(9:3:4) or Recessive epistasis or
Supplementary action
Eg: Aleurone color in maize
Gene R Gene P

Enz R Enz P

Precursor Product1 Phenotype
(Colorless) (Red) (Purple)

Parents: Purple X White
PPRR pprr
F1: PpRr (purple)
Gametes: PR Pr pR pr (Selfing)

F2: 9P-R-: purple
3ppR-: red
3P-rr: white
1pprr: white

27. Fine structure of gene
There are two concepts of gene structure:
Old concept: Bead theory
•Classical concept of gene was introduced by Sutton
(1902) and was elaborated by Morgan (1913)
Modern concept : Molecular concept
•Fine structure or molecular structure of gene was
given by S. Benzer(1957).

28. Bead theory
•According to this theory a gene is the fundamental
unit of structure, change and function.
•As the beads are arranged in a necklace, genes are
arranged linearly in a chromosome.
•So chromosome is a linear array of genes according
to bead theory.
•This theory was later discarded due to its
shortcomings.

29. Gene is the fundamental unit of:
•Structure: Gene is the fundamental unit of structure
indivisible by crossing over. Crossing over occurs only
between genes.
•Change: the whole gene must change from one
allelic form to another, there are no smaller
components within gene that can change by
mutation.
•Function: the gene functions as a unit, parts of a
gene cannot function on their own.

30. Modern concept of fine structure of gene
•The word gene is derived from Greek word
‘genesis’ which means ‘to be born’.
•Mendel used the term hereditary
determiners, and later it was termed gene by
W. Johansen in 1909.
•Seymour Benzer coined different terms for
different nature of gene and genetic material
in relation to the chromosome.

31. •Gene as a cistron:
A segment of DNA specifying a single polypeptide
chain(100-30,000 nucleotide pairs). It transmits
characters from one generation to other as a unit of
transmission.
•Genes as a unit of recombination or Recon:
It is the smallest unit of DNA capable of undergoing
crossing over and recombination. It consists of no more
than 2 pairs of nucleotides.
•Gene as a unit of mutation or Muton:
The smallest unit of genetic material which when
changed or mutated produce a phenotypic trait. Thus
muton can be a single nucleotide.

32. Benzer’s Experiment
•The r mutants of T
4 Phage of E.coli are the rapid lysing mutants
of the phage and make large, sharp plaques on E. coli strain B.
•The wild type allele r
+
makes small, fuzzy plaques.
•There are several independent r loci (rI, rII, rIII) and the rII locus
was analyzed by Benzer.

•A strain of E.coli callled strain K, reacts differently to the rII
mutant, i.e. no plaques are formed by rII mutant on strain K
culture.
•The phage multiplies inside the bacterial cell and it kills the
bacterium but no lysis occurs with the result that no phage is
released.
•The wild type allele makes normal plaques on B-strain as well as
K strain.

35. •Benzer infected K-strains with rII
+
and rII and K cells
were lysed (rII
+
was responsible for this).
•Since rII cells do multiply inside the K-strain, this
time they were released by lysis cased by rII
+
phage.
•Benzer then infected the K-cells with two different
genetically separable mutants, i.e. rII
x
and rII
y
.
•Benzer could observe lysis in some cases which was
unexpected since rII mutant is unable to lyse the K-
cells.
•Various combinations of two different rII mutants
were plated together on K-strain to see the plaque
formation.

36. •Benzer on the basis of his experiments, grouped
the rII mutants into two types, type A and B.
•He had a conclusion that the mixture of rII
mutant from group A and another mutant rII
from group A formed no plaques.
•Similarly, the mixture of rII mutant from group B
and another rII mutant from group B formed no
plaques.
•Instead a mixture of mutants of group A and B
resulted in plaque formation.

Complementation test

37. Mutant1: Gene A Gene b

Enzyme A
Precursor Intermediate Product
Enzyme B

Mutant 2 Gene a Gene B
Production of wild type phenotype when two
different mutations are combined in a diploid.

38. •Benzer concluded that the mutants of group A and B
involve a separate mutable site within the rII locus or they
are the two functional units or cistrons in the rII region.
•Each governs the synthesis of a specific polypeptide
necessary for the growth in K strain.
•Mixture of two A-mutants or two B-mutants does not
cause this lysis because both the mutants involve the
same site and same functional deficiency.
•Whereas mixture of A and B types i.e. A and B are plated
together, they make up for each other’s functional
deficiency.
•The two mutants complement each other resulting in a
wild type and this gives rise to plaques on strain K
cultures.

39. Benzer’s complementation test

40. Recombination
•Benzer further plated a mixture of two independent
mutations rII
1
and rII
2
from group A-mutants on E.
coli strain B.
•After lysis he obtained the lysate which was mixture
of both the phages, i.e. rII
1
and rII
2
.
•He plated the lysate on strain K which resulted in
plaque formation which was unexpected as the
mutants of group-A alone cannot lyse the cells.
•According to benzer rII
1
was genetically dissimilar to
rII
2
and a recombination occurred between the two
resulting in wild type genome which lysed the cells.

41. •Same results were obtained from plating of mutants
of group-B.
•Recombination within the gene was the explanation
as the mutation was ruled out by control
experiments.
•This shows that within a gene, there are recons that
can be recombined.

43. Benzer’s definition of gene
•The rII locus as described consists of two cistrons, the two
functional units.
•Each cistron has a large mutable sites, each resulting in rII
mutant.
•Such unit of mutations (each nucleotide) are called mutons.
•The smallest distance within a gene between which two
mutons can recombine was named as recon by Benzer.
•Cistron therefore is a genetic unit of function and is
synonymous with term gene and muton and recon are its
subdivisions.
•The gene locus is a complex site having different units of
mutation, recombination and function in it.

44. Enzymatic explanation of
Genetic ratios
Presented by: Anup Adhikari

45. •There is relationship between enzyme and phenotype of an
organism.
•Enzyme controls the biochemical reaction and the reaction
produces biochemical product which is responsible to final
phenotype expression.
•So phenotypic ratio is determined by the enzyme and these
enzymes are the product of genes.

Gene A Gene B Gene C

Enzyme1 Enzyme2 Enzyme 3

Precursor Product 1 Product2 Phenotype

46. •In other words a gene is transcribed into RNA and
RNA is translated ta a kind of polypeptide.
•The polypeptide changes to enzyme or the
polypeptide behaves like the enzyme.
•The enzyme catalyzes the biochemical reaction to
produce a final product.
•The product of such reaction is responsible for the
expression of the phenotype and the phenotype
expression produces the phenotypic ratio.

47. •In Mendelian phenotypic ratio, one gene was
involved for the expression of a single phenotype.
•But there are cases of gene interaction in which
two or more genes are involved to determine a
single trait.
•In gene interaction the phenotypic ratio deviates
from the expected Mendelian ratio.
•There are a variety of phenotypic ratios which are
the result of a variety of gene interaction.

48. Example: Novel phenotype (9:3:3:1)
Eg: Comb shape in poultry.
Gene P Gene R

Enz P Enz R

Precursor Product Phenotype
(Single) (Pea) (Walnut)
Parents: Rose X Pea
RRpp rrPP
F1: PpRr (Walnut)
Gametes: RP Rp rP rp (Selfing)

F2: 9P-R-: Walnut
3ppR-: Rose
3P-rr: Pea
1pprr: Single

49. •The proportion of walnut, pea, rose and single
shaped comb in poultry in F2 generations is 9:3:3:1.
•In segregating population, P gene alone(rrPP)
produces pea shaped comb in poultry and R gene
alone (RRpp) pro bduces rose shaped comb.
•When both dominant genes P and R are present in
the genotype(P-R-) they produce walnut shaped
comb.
•recessive homozygous genotype pprr produces
single type of comb.

50. Example: Complementary action(9:7)
Eg: Flower color in pea.
Gene C Gene P

Enz C Enz P

Precursor Product 1 Phenotype
(White) (White) (Purple)

Parents: Purple X White
CCPP ccpp

F1: CcPp (Purple)
Gametes: CP Cp cP cp (Selfing)

F2: 9 C_P_: Purple
3 ccP_:
3 C_pp: white
1 ccpp:

51. •The proportion of purple and white flowers of pea
plants in F2 segregating population is 9:7.
•In segregating population, C gene alone(CCpp) P
gene alone (ccPP) don't produce any pigments for
flower color.
•When both dominant genes C and P are present in
the genotype(C-P-) they produce purple colored
pigments for production of purple flowers in pea
plants.
•recessive homozygous genotype ccpp produces
white flowers.

52. Example: Modifying action(9:3:4) or Recessive epistasis or
Supplementary action
Eg: Aleurone color in maize
Gene R Gene P

Enz R Enz P

Precursor Product1 Phenotype
(Colorless) (Red) (Purple)

Parents: Purple X White
PPRR pprr
F1: PpRr (purple)
Gametes: PR Pr pR pr (Selfing)

F2: 9P-R-: purple
3ppR-: red
3P-rr: white
1pprr: white

53. Fine structure of gene
There are two concepts of gene structure:
Old concept: Bead theory
•Classical concept of gene was introduced by Sutton
(1902) and was elaborated by Morgan (1913)
Modern concept : Molecular concept
•Fine structure or molecular structure of gene was
given by S. Benzer(1957).

54. Bead theory
•According to this theory a gene is the fundamental
unit of structure, change and function.
•As the beads are arranged in a necklace, genes are
arranged linearly in a chromosome.
•So chromosome is a linear array of genes according
to bead theory.
•This theory was later discarded due to its
shortcomings.

55. Gene is the fundamental unit of:
•Structure: Gene is the fundamental unit of structure
indivisible by crossing over. Crossing over occurs only
between genes.
•Change: the whole gene must change from one
allelic form to another, there are no smaller
components within gene that can change by
mutation.
•Function: the gene functions as a unit, parts of a
gene cannot function on their own.

56. Modern concept of fine structure of gene
•The word gene is derived from Greek word
‘genesis’ which means ‘to be born’.
•Mendel used the term hereditary
determiners, and later it was termed gene by
W. Johansen in 1909.
•Seymour Benzer coined different terms for
different nature of gene and genetic material
in relation to the chromosome.

57. •Gene as a cistron:
A segment of DNA specifying a single polypeptide
chain(100-30,000 nucleotide pairs). It transmits
characters from one generation to other as a unit of
transmission.
•Genes as a unit of recombination or Recon:
It is the smallest unit of DNA capable of undergoing
crossing over and recombination. It consists of no more
than 2 pairs of nucleotides.
•Gene as a unit of mutation or Muton:
The smallest unit of genetic material which when
changed or mutated produce a phenotypic trait. Thus
muton can be a single nucleotide.

58. Benzer’s Experiment
•The r mutants of T
4 Phage of E.coli are the rapid lysing mutants
of the phage and make large, sharp plaques on E. coli strain B.
•The wild type allele r
+
makes small, fuzzy plaques.
•There are several independent r loci (rI, rII, rIII) and the rII locus
was analyzed by Benzer.

•A strain of E.coli callled strain K, reacts differently to the rII
mutant, i.e. no plaques are formed by rII mutant on strain K
culture.
•The phage multiplies inside the bacterial cell and it kills the
bacterium but no lysis occurs with the result that no phage is
released.
•The wild type allele makes normal plaques on B-strain as well as
K strain.

61. •Benzer infected K-strains with rII
+
and rII and K cells
were lysed (rII
+
was responsible for this).
•Since rII cells do multiply inside the K-strain, this
time they were released by lysis cased by rII
+
phage.
•Benzer then infected the K-cells with two different
genetically separable mutants, i.e. rII
x
and rII
y
.
•Benzer could observe lysis in some cases which was
unexpected since rII mutant is unable to lyse the K-
cells.
•Various combinations of two different rII mutants
were plated together on K-strain to see the plaque
formation.

62. •Benzer on the basis of his experiments, grouped
the rII mutants into two types, type A and B.
•He had a conclusion that the mixture of rII
mutant from group A and another mutant rII
from group A formed no plaques.
•Similarly, the mixture of rII mutant from group B
and another rII mutant from group B formed no
plaques.
•Instead a mixture of mutants of group A and B
resulted in plaque formation.

Complementation test

63. Mutant1: Gene A Gene b

Enzyme A
Precursor Intermediate Product
Enzyme B

Mutant 2 Gene a Gene B
Production of wild type phenotype when two
different mutations are combined in a diploid.

64. •Benzer concluded that the mutants of group A and B
involve a separate mutable site within the rII locus or they
are the two functional units or cistrons in the rII region.
•Each governs the synthesis of a specific polypeptide
necessary for the growth in K strain.
•Mixture of two A-mutants or two B-mutants does not
cause this lysis because both the mutants involve the
same site and same functional deficiency.
•Whereas mixture of A and B types i.e. A and B are plated
together, they make up for each other’s functional
deficiency.
•The two mutants complement each other resulting in a
wild type and this gives rise to plaques on strain K
cultures.

65. Benzer’s complementation test

66. Recombination
•Benzer further plated a mixture of two independent
mutations rII
1
and rII
2
from group A-mutants on E.
coli strain B.
•After lysis he obtained the lysate which was mixture
of both the phages, i.e. rII
1
and rII
2
.
•He plated the lysate on strain K which resulted in
plaque formation which was unexpected as the
mutants of group-A alone cannot lyse the cells.
•According to benzer rII
1
was genetically dissimilar to
rII
2
and a recombination occurred between the two
resulting in wild type genome which lysed the cells.

67. •Same results were obtained from plating of mutants
of group-B.
•Recombination within the gene was the explanation
as the mutation was ruled out by control
experiments.
•This shows that within a gene, there are recons that
can be recombined.

69. Benzer’s definition of gene
•The rII locus as described consists of two cistrons, the two
functional units.
•Each cistron has a large mutable sites, each resulting in rII
mutant.
•Such unit of mutations (each nucleotide) are called mutons.
•The smallest distance within a gene between which two
mutons can recombine was named as recon by Benzer.
•Cistron therefore is a genetic unit of function and is
synonymous with term gene and muton and recon are its
subdivisions.
•The gene locus is a complex site having different units of
mutation, recombination and function in it.

70. DNA and DNA Manipulation
Presented by: Anup Adhikari

71. DNA
•It is a double stranded molecule that stores genetic
material of all living things, except RNA viruses.
•It is a double chain of linked nucleotides, the
fundamental substance of which genes are
composed.
•The backbone is made up of 5 carbon sugar and
phosphate.
•DNA contains four different subunits (two purine
nucleotides and two pyrimidine nucleotides).
•Correct structure of DNA was first deduced or
discovered by J.D. Watson and F.H.C. Crick in 1953.

72. Structure of DNA

73. Location of DNA

74. Gene Structure
•Genes consist of short coding sequences or exons which are
interrupted by a longer non-coding sequence or introns.
•A few genes in human genome have no introns.
•Genes also contain the promoter sites for recognition of
signals by enzymes for transcription.
Fig: Gene structure of eukaryotes
Fig: Gene structure of prokaryotes

75. Organelle genome: Mitochondrial DNA
(mtDNA)
•Mitochondrial DNA was discovered by Margit M. K.
Nass and Sylvan Nass by electron microscopy as
DNAase- sensitive thread inside mitochondria.
•The size of mt DNA range from 15k to 300kn in some
of the plants.
•They contain many genes and non-coding regions as
well but these non-coding regions are not found in
case of prokaryotes.
•The number of mtDNA varies according to the
number of mitochondria in a cell and the genome
size per mitochondria.

78. Chloroplast DNA (cpDNA)
•Structurally similar DNA to that of mtDNA but is several times
larger and contains genes involved in photosynthetic pathway.
•It is independent of nuclear DNA and is maternally inherited.
•In higher plants cpDNA ranges from 120-160 kb in size and in
algae from 85-292 kb.
•The number of cpDNA in a cell depends upon the number of
chloroplast and the number of cpDNA per chloroplast.
•All cpDNA carry the same set of genes but in different species
of plants these genes are arranged in a different way.
•Most cpDNAs have a pair of large inverted repeats that contain
the gene for ribosomal RNAs.

81. Isolation and Purification of DNA
•Isolation of DNA is a routine procedure to collect DNA for
subsequent molecular analysis.
•Isolating DNA from plant tissues can be very challenging as
the biochemistry between divergent plant species can be
extreme.
•Polysaccharides and polyphenols are two classes of plant
biomolecules that vary widely between species and are very
problematic when isolating DNA.
•Contaminating polysaccharides and polyphenols can interfere
with manipulations of DNA following isolation.
•The use of CTAB (cetyl trimethylammonium bromide), a
cationic detergent, facilitates the separation of
polysaccharides during purification while additives, such as
polyvinylpyrrolidone, can aid in removing polyphenols.

82. Materials Required
•Liquid Nitrogen
•CTAB buffer: 2% cetyl trimethylammonium bromide, 1%
polyvinyl pyrrolidone, 100 mM Tris-HCl, 1.4 M NaCl, 20 mM
EDTA, or CTAB Extraction Buffer
•RNase A Solution
•Isopropanol
•70% Ethanol
•2 ml centrifuge tubes
•SpeedVac
•Water bath, 65°C and water bath, 37°C
•TE Buffer (10 mM Tris-HCL, pH 8, 1 mM
Ethylenediaminetetraacetic acid (EDTA))

84. Procedure of DNA Extraction
•Process leaf tissue by freezing with liquid nitrogen and
grinding into a fine powder using a micro centrifuge tube
pestle or a mortar and pestle.
•Add 40mg of this leaf powder to a 2ml micro centrifuge tube.
•Add 600µl of Nuclei Lysis Solution and incubate at 65°C for 15
minutes.
•Add 3µl of RNase Solution to the cell lysate, and mix the
sample by inverting the tube 2–5 times. Incubate the mixture
at 37°C for 15 minutes.
•Allow the sample to cool to room temperature for 5 minutes
before proceeding.
•Proceed to Protein Precipitation and DNA Rehydration.

85. Protein Precipitation and DNA Rehydration
•Centrifuge at 13,000–16,000 × g for 3 minutes.
•Transfer supernatant to clean tube containing 600µl room
temperature isopropanol.
•Mix by inversion and centrifuge at 13,000–16,000 for 1 minute.
•Carefully remove the supernatant containing the DNA (leaving
the protein pellet behind) and transfer it to a clean 1.5ml micro-
centrifuge tube containing 600µl of room temperature
isopropanol.
•Gently mix the solution by inversion until thread-like strands of
DNA form a visible mass and centrifuge at 13,000–16,000 × g for
1 minute.

86. •Carefully decant the supernatant. Add 600µl of room
temperature 70% ethanol and gently invert the tube
several times to wash the DNA.
•Centrifuge at 13,000–16,000 × g for 1 minute at room
temperature.
•Carefully aspirate the ethanol using either a drawn
Pasteur pipette or a sequencing pipette tip.
•The DNA pellet is very loose at this point and care
must be used to avoid aspirating the pellet into the
pipette.

87. •Invert the tube onto clean absorbent paper and air-
dry the pellet for 15 minutes.
•Add 100µl of DNA Rehydration Solution and
rehydrate the DNA by incubating at 65°C for 1 hour.
•Periodically mix the solution by gently tapping the
tube.
•Alternatively, rehydrate the DNA by incubating the
solution overnight at room temperature or at 4°C.
14.
•Store the DNA at 2–8°C.

90. Genetic Engineering

91. Genetic Engineering
•Genetic engineering is a method in which genotype of the
plant cell is altered by the introduction of a foreign gene(s)
into the genome of host, other that by sexual crossing.
•The joining together of DNA segments derived from
biologically different source is the technology referred to as
recombinant DNA (r-DNA) technology or genetic engineering.
•A recombinant DNA molecule is produced by joining together
two or more DNA segments usually originating from different
organism.
•Plants obtained through genetic engineering contain a
gene/genes usually from an unrelated organism; such genes
are called as transgenes and the plants containing such genes
are known as transgenic plants.

92. General approach of Genetic Engineering
in Plants
Identification and isolation of desired gene(s).
Transfer of gene to the targeted recipient.
Stable integration of the genes into the
recipient genome.
Expression of the introduced genes.
Transmission of the introduced genome to the
progeny.

93. Components of genetic engineering
•Gene of interest or Foreign gene
•Gene transfer system or Vector
•Restriction Enzyme
•Ligase
•Host
•Marker Gene
•Tissue Culture for Regeneration

94. Identification Isolation Introduction Integration
(Gene) (Gene) (Host) (Genome)



Transmission Regeneration Selection
(Progeny) (Culture) & Expression
(Gene)


Steps of Genetic Engineering

96. Recombinant DNA (r DNA)
•A recombinant DNA molecule is a vector into which a desired
DNA fragment has been inserted to enable its cloning in a
appropriate host.
•It is achieved through use of specific enzymes that are able to
cut DNA into suitable fragments known as restriction enzymes.
•Enzyme ligase is used to join the appropriate fragments for
producing r-DNA molecules.
•r-DNA are produced for following objectives:
To obtain large number of copies of specific DNA fragments.
To recover large quantities of the protein produced by the
concerned gene,
To integrate the gene into the chromosome of a organism where
it expresses itself.

97. Restriction enzymes
•Nucleases are the enzymes that degrade
nucleic acids, i.e they hydrolyses or break
down the poly nucleotide chain into its
component nucleotides.
•The poly nucleotide chain is held together by
3’ and 5’ ends of phosphodiester linkages.
•Nucleases are of two types:
•Exonucleases and
•Endonucleases

98. a. Exonucleases
•They degrade nucleic acids at one or both the ends
of polynucleotide chain.
•They hydrolyze the phosphodiester bonds of the
terminal nucleotide.
•The exonuclease will cut either the 3’-OH end or the
free 5’-P end and digest the polynucleotide in 5’-3’
end direction.
•In both cases the enzymes travel along the chain
liberating single nucleotides and degrade the entire
polymer.

99. Endonuclease or
Restriction Endonuclease
•Endonucleases are the enzymes that cleave nucleic acids at
internal sites.
•A class of endonucleases which cleave DNA only within or
near the sites which have specific base sequence are known
as restriction endonucleases.
•The term Restriction endonuclease was coined by Hederberg
and Meselson in 1968.
•The sites recognized by them are called Restriction sequences
or restriction sites or recognition sites.
•These are prokaryotic enzymes which were discovered by
1970 by S.Smith and D. Nathans from Haemophilus influenza.
•These are popularly called molecular knives/molecular
scissor/molecular scalpels/biological scissors.

100. Nomenclature of Restriction Enzymes
•A system based on the proposals of Smith and Nathans has
been followed:
•The first letter of this code is derived from first letter of genus
name.
•The second and third letters are from the species name.
•Eg: Eco from E. coli , Hin from Haemophilus influenzae.
•This is followed by the strain molecules. Eg: EcoR
•If a particular strain has more than one restriction enzyme,
these will be identified by sequential roman numbers I, II and
III etc.
•Eg: EcoR I for the first enzyme of Escherichia coli strain R.
H. influenzae strain d are named Hind II, Hind III etc.

101. Classes of Restriction enzymes
•Based on type of sequence recognized and the nature of cut
made in DNA, these enzymes have been classified into three
different types.

102. Recognition sequences or Restriction site
•The recognition sequences of type II endonuclease form
palindromes with rotational symmetry.
•In a palindrome the nucleotide base sequences in half
of the DNA strand is the mirror image of the sequence
in its other half. Eg: GTAATG
•The inverted repeat palindromes is also a sequence
that reads the same forward and backwards, but the
forward and backward sequences are found in
complementary DNA strands (eg: GTATAC is
complementary to CATATG).
•Inverted repeat palindromes are more common and
have greater biological importance than mirror like
palindromes.

103. The restriction enzymes cut DNA molecule by cleavage
which occurs in two types:
i. Blunt end style
•Certain restriction enzymes eg: Alu I (Arthrobacter
luteus ) make cuts across both strands of DNA at the
same position.
•The resulting termini or ends have blunt end in which
the two strands end at the same point.
ii. Sticky or cohesive ends/staggered cuts/palindromes
•in this style the restriction endonuclease such as EcoR I,
Hind I, Hind III make single strand cuts that produce
sticky ends.
•The two strands of DNA are cleaved at different
locations generating fragments with protruding ends.

105. Gene Cloning vectors
•Gene cloning or DNA cloning is defined as insertion
of fragment of DNA representing a gene into a
cloning vector and subsequent propagation of DNA
molecule in a host organism.
•Gene cloning is isolation and amplification of an
individual gene sequence by immersion of that
sequence into a bacterium where it can be
replicated.
•A vector is a DNA molecule that has the ability to
replicate in an appropriate host cell.
•Vector must have a origin of DNA replication (ori)
that functions efficiently in the concerned host cell.

106. •The vector is a vehicle or carrier which is used
for cloning foreign DNA in bacteria.
•Any extra chromosomal small genomes eg:
plasmid, phage and viruses may be used as a
vector.
•Vector carries the foreign DNA integrated with
vector DNA molecule and is used for cloning
the foreign DNA in bacteria.

107. Properties of a good vector
(i)They should independently (autonomously) replicate themselves
and the foreign DNA segments they carry.
(ii)It should be easy to isolate and purify.
(iii)It should be easy to introduced into the host cells.
(iv)The vector should carry a selectable marker to distinguish host
cells that carry vectors from host cells that do not contain a
vector. Eg: genes for Ampicillin and Tetracycline resistance.
(v)A vector should contain unique target sites for as many
restriction enzyme as possible.
(vi)When expression of the DNA insert is desired, the vector should
contain regulatory sites like promoter, operator, ribosome
binding sites etc.

108. Cloning and Expression vectors
•The vectors used for propagation of DNA inserts
in a suitable host are called cloning vectors.
•When a vector is designed for the expression of
foreign gene, i.e. production of the protein
specified by the DNA insert is known as
expression vector.
•The expression vectors contain the regulatory
sites.
•The genes carried by the expression vector is
efficiently transcribed and translated by the host
cell.

109. Gene cloning vectors

•Plasmids
*pBR 322, Ti Plasmid, Ri Plasmid
•Cosmids
•Phages: Bacteriophage(lambda phase, M 13
phage)
•Yeast artificial chromosomes (YAC):
developed by David Burke et al. (1987)
•Bacterial artificial chromosomes (BAC)

110. Plasmid Vectors
•Plasmids are small circular DNA molecules other than bacterial
chromosomal DNA that are capable of autonomous replication
and transmission.
•Plasmids multiply at the same rate as that of bacterial cell and
this rate is independent of that of host cell.
•Use of plasmids as vector provide even up to 1000 copies per
cell.
•Length of DNA segment that plasmids can accommodate is 0.1 to
15 kb base pairs.
pBR-322 (Ideal Plasmid vector)
•The name pBR denotes ‘p’ as plasmids and BR is from Boliver
Rodriguez (1977) the two initials of scientists who developed
pBR-322 which is the most widely used plasmids.

112. Ti Plasmid
•A. tumefaciens has the Ti plasmid (200kb) while A. rhizogenes
have the Ri plasmid which are similar in their general features.
•A. tumefaciens is a plant pathogenic gram negative bacteria
that cause crown gall in dicot plants (A rhizogenes casuse hairy
root diseases).
•These plasmids naturally transfer a part of their DNA, the T-
DNA into the host plant genome, which makes Agrobacterium a
natural genetic engineer.
•The T-region (23kb) is transferred into host cells and is
integrated into their genomes.
•They have another region called ‘vir’ region which produces an
endonuclease essential for the transfer of T-region into plant
cells.
•They can accommodate up to 10 kb long foreign DNA segment.

114. Bacteriophase vectors
•Bacteriophage or phase are the viruses that infect
bacteria.
•They are made up of two components: capsid or
protein coat and nucleic acid genome enclosed inside
capsid.
•The phase genome can be double stranded DNA (T2,
T4 and T6) or single stranded (X174 and M-13) and
RNA virus-MS2.
•These bacteriophages are used as cloning vectors, most
common ones are: lambda phase and M-13 phases.
•They are more efficient for cloning large DNA fragments
as they can accommodate foreign segments up to 25
kb.

115. Lambda vectors
•The λ- genome contains an origin of replication and
genes for head and tail, proteins and enzymes of DNA
replication, lysis and lysogeny and single stranded
protruding cohesive ends of 12 bases at its 5’ ends.
•These two cohesive ends are referred as cos sites
(sticky ends).
•These ends enable DNA to form a circular molecule
when it is injected into E. coli cells.
•The central part of λ-chromosome may be axcised with
restriction enzymes and replaced with foreign DNA.
•They can accommodate large size DNA fragment upto
25 kb.

117. Cosmid vectors
•Cosmids are the plasmids that contain a minimum of
250 bp of λ-DNA including cos site and restriction sites.
•Packaged cosmids infect the host cells like λ- particles
but inside the host they replicate and propagate like
plasmids.
•Cosmids can be used to clone DNA inserts upto 45 kb.
•They can be packaged into λ- particles which infect host
cells, which is many fold more efficient than plasmid
transformation.
•Selection of recombinant DNA is based on the
procedure applicable to the plasmid making up the
cosmid.

118. Transformation
•Introduction of r-DNA into a host is called
transformation.
•The process of transfer, integration and
expression of transgene in the host cell is known
as genetic transformation.
•It is required for stable introduction of genetic
material into cells and its subsequent expression.
•Possibility of specific introduction of traits of
economic importance.

119. Methods of transformation

1. Indirect method:
Agrobacterium tumefaciens mediated:
2. Direct methods:
Particle gun
Electroporation
Polyethylene glycol
Micro injection
Fibre mediated transformation
Liposome mediated DNA transfer.

120. Agrobacterium mediated
transformation
•It is a gram negative rod shaped bacteria which causes tumours
(crown galls) in dicot plants.
•Ti plasmids of A. tumefaciens are the most widely used vectors
for indirect gene transfer in plants.
•Ti plasmids have a T-region and a vir (virulence) region.
•The T-DNA is flanked on both sides by a border (B) of direct
repeat sequences each of 25 bp responsible for the T-DNA
transfer to infected plant cell.
•T-DNA has 4 genes:
Tms1, tms2 – Synthesis of IAA
Tmr- Production of cytokinin
os- Opine synthesis

121. •tms1., tms2 and tmr are called onco genes and are to be
removed from the T-DNA of Ti plasmid.
•Removal of three tumour producing genes from T-DNA is called
disarming and the resulting Ti plasmid is referred as disramed.
•After deletion of these genes, this region is replaced by marker
genes or desirable genes.
•Opine serves as carbon, nitrogen and energy source for bacteria.
•The vir region is essential for t-DNA transfer though it is not
transferred to plant self itself.
•The vir region contains 6-8 operons depending on the type of Ti
plasmid.
•Vir D operon codes for an endonuclease responsible for excision
of a single stranded copy of T-DNA from the Ti Plasmid and then
guiding this DNA into the plant cell nucleus.

122. Fig: Indirect method or A. tumefaciens mediated gene transfer

123. Direct methods of gene transfer
1.Gene gun/Particle bombardment/microprojectile

124. 2. Electroporation
•Induction of DNA into cell by exposing them for a
very brief period to high voltage electrical pulses.

125. 3.Chemical/Polytheylene glycol

126. 4. Microinjection
The DNA
solution is
directly injected
inside the cell
using capillary
glass micro-
pipette with the
help of
micromanipulat
ors of a
microinjection
assembly.

127. 5. Fibre mediated transformation
•The DNA is delivered into the cell cytoplasm and nucleus by
silicon carbide fibres of 0.6 μm diameter and 10-80μm length.
•The method was successful with maize nad tobacco
suspension cell culture.

128. 6. Lypofection
•Introduction of DNA into cells via liposomes.
•Liposomes are small lipid artificial vesicles.
•The DNA enclosed in the lipid vesicles when
mixed with protoplast under appropriate
condition penetrates into the protoplast where
lipase activity of the protoplast dissolves the
lipid vesicles and DNA gets released for
integration into the host genome.

129. Some examples of genetically modified crops

130. Molecular genetics

131. Gel Electrophoresis
•Electrophoresis is the standard method for analyzing,
identifying, and purifying fragments of DNA or RNA that differ
in size, charge, or conformation.
•When charged molecules are placed in an electric field, they
migrate toward the positive (anode, red) or negative
(cathode, black) pole according to their charge.
•Nucleic acids have a consistent negative change due to their
phosphate backbone, and hence they migrate toward the
anode.
•Most commonly, the gel is cast in the shape of a thin slab,
with wells for loading the sample.
•The gels used for electrophoresis are composed either of
agarose or polyacrylamide.

132. •Agarose gels are used in a horizontal gel apparatus, while
polyacrylamide gels are used in a vertical gel apparatus.
•Agarose gels are used for the analysis and preparation of
fragments between 100 and 50,000 bp in size with moderate
resolution, and polyacrylamide gels are used for the analysis
and preparation of small molecules with single nucleotide
resolution.
•Maker DNA fragments of known size are run in a separate
lane which permits accurate determination of size of
unknown DNA.
•The gels are stained with dye ethidium bromide which gives
visible fluorescence on illumination of the gel with UV light
giving the bands of the DNA fragments.

136. Southern Hybridization (Blotting)
•A sample of DNA fragments of different sizes is subjected to
electrophoresis using either polyacrylamide or agarose gel.
•By the electrophoresis specific DNA molecule from a mixture is
detected and isolated.
•The restriction fragments of DNA presents in agarose gel are
denatured into single stranded form by alkali treatments.
•They are then transferred into a nitrocellulose filter membrane
which is done by placing some dry filter papers on top of
membrane.
•The buffer moves due to capillary action from bottom filter paper
to that on top through gel carrying with it denatured DNA present
in gel, DNA become trapped in nitrocellulose membrane as buffer
passes through.
•This process is known as blotting and takes several hours to
complete.

137. •The nitrocellulose membrane is now removed from blotting stack
and DNA is permanently immobilized on membrane by baking it
at 80 °c in vacuum.
•Single stranded DNA has high affinity for nitrocellulose filter.
•So, the baked membrane is treated with solution containing 0.2
% each of Ficoll ( an artificial polymer of sucrose),
polyvinylpyrrolidone and bovine serum albumin.
•The pretreated membrane is placed in a solution of radioactive,
SS DNA or an oligo-deoxynucleotide called probe.
•This signifies that DNA molecule is used to detect and identify
that DNA segments in gel/ membrane which is complementary
to probe. This step is known as hybridization.
•The membrane is washed to remove unbound probes.
•The membrane is placed in close contact with an X-ray film and
incubated for desired period for visualization.

139. Polymerase chain reaction (PCR)
•The polymerase chain reaction (PCR) is the one of the most
powerful techniques that has been developed recently in the
area of recombinant DNA research.
•It is a fast and inexpensive technique used to "amplify" - copy -
small segments of DNA.
•There are three major reactions in PCR i.e. Denaturing,
Annealing and Primer extension.
•The entire cycling process of PCR is automated and can be
completed in just a few hours.
•It is directed by a machine called a thermocycler, which is
programmed to alter the temperature of the reaction every few
minutes to allow DNA denaturing and synthesis.
•Polymerase chain reaction (PCR) was invented by Mullis in 1983
and patented in 1985.

140. Fig: Procedure of PCR

141. Steps of PCR
1.Denaturation: In the first step, the target sequence of DNA is
heated (95°c for 30 sec) to denature the template strands
and render the DNA single-stranded.
2.Annealing: The DNA is then cooled (55°c for 30 sec) to allow
the primers to anneal, that is, to bind the appropriate
complementary strand. Primers are generally DNA
oligonucleotides of approximately 20 bases each.
3.Primer extension: In the presence of Mg++, DNA
polymerase extends the primers on both strands from 5′ to
3′ by its polymerase activity.
•Primer extension (72°c for 1.5 min) is performed at a
temperature optimal for the particular polymerase that is
used. Currently, the most popular enzyme for this step is Taq
polymerase, the DNA polymerase from the thermophilic
(heat-loving) bacteria Thermus aquaticus.

142. Applications of PCR
•To amplify the DNA segment required for scientific
studies or gene cloning.
•Study of patterns of gene expression.
•Assist in DNA sequencing.
•Phylogenetic study of DNA from ancient sources.
•Study of patterns of genetic mapping.
•DNA fingerprinting and parental testing, forensic
sciences etc.
•RT-PCR for amplification of cDNA made by reverse
transcription of viral single stranded RNA genome.

143. Polymorphism
•Polymorphism is the term used to describe the variation.
Consequently, a genetic variant should appear in at least 1% of the
population to be declared polymorphic.
•In terms of markers, polymorphism may be defined as the
simultaneous occurrence of more than one allele or genetic
marker at the same locus.
•The most common type of polymorphism derives from variation at
a single base pair and is called a single nucleotide polymorphism
(SNP).
•Depending upon the mutational event, the polymorphism may be
due to variation in length of the DNA fragment (fragment length
polymorphism).
•Because molecular markers are usually located in non-coding
regions of DNA, they are selectively neutral and unaffected by
environment and stage of development.

144. Genetic Markers
•Genetic markers are simply landmarks on chromosomes that
serve as reference points to the location of other genes of
interest when a genetic map is constructed.
•Breeders are interested in knowing the association (linkage) of
markers to genes controlling traits they are trying to manipulate.
•Genetic markers can be detected at both the morphological level
and the molecular or cellular level.
•Morphological markers are phenotypes, the products of the
interaction of genes and the environment (e.g., seed shape,
flower color, and growth habit).
•On the other hand, molecular markers are detected at the
subcellular level and can be assayed before the adult stage in the
lifecycle of the organism.

145. Morphological markers
•Morphological markers generally correspond to the
qualitative traits that can be scored visually.
•They can be either dominant or recessive.
•Morphological markers cause large effects on
phenotype and are undesirable in breeding
programs.
•They mask the effects of linked minor genes.
•The are more influenced by environments.
•Examples: Seed shape, flower color, plant height,
maturity period etc.

146. Biochemical Markers
•Biochemical markers are the proteins produced by
gene expression.
•These proteins can be isolated and identified by
electrophoresis and staining.
•Isozymes are the different molecular forms of the
same enzyme that catalyzes the same reaction.
•They are generally co-dominant and are revealed on
electrophoregrams through a colored reaction
associated with enzymatic activity.
•They are products of the various alleles of one or
several genes.

147. Molecular markers
•They are landmarks within a chromosomal DNA which help
to locate genes or regions of genes.
•A DNA marker is a small region of DNA showing sequence of
polymorphism in different individuals within a species or
group of individuals.
•They are the powerful tools in the field of molecular genetics
that are used to identify either presence or absence of any
character in an individual.
•Molecular markers have been proven the fast and precise
indirect tools of selection in breeding programs.
•Detection of quantitative trait locus (QTL) and marker
assisted selection (MAS) are the important applications of
molecular markers for crop improvements.

148. Mostly used molecular markers
•Restriction fragment length polymorphism
(RFLP)
•Random amplified polymorphic DNAs (RAPD)
•Amplified fragment length polymorphism
(AFLP)
•Microsatellite or simple sequence repeat (SSR)
•Single nucleotide polymorphism (SNP)
•Sequence characterized amplified region
markers (SCAR)

149. Non-PCR based molecular markers
Restriction fragment length polymorphism (RFLP)
•It is the first generation of DNA markers and one of
the best for plant genome mapping.
•The simplest type of RFLP is 2-allele system involving
the presence or absence of a recognition site for a
single restriction enzyme.
•They are co-dominant markers i.e. help to distinguish
between dominant, recessive and heterozygotes.
•The main disadvantage is they require relatively large
amount of DNA.

150. RFLP method of analysis

151. PCR based markers
Random Amplified Polymorphic DNA (RAPD)
•It is a PCR-based marker system in which the total genomic DNA is
amplified using a single short (about 10 bps) random primer
•It differs from traditional PCR analysis in that it does not require
specific knowledge of the DNA sequence of the target organism T
•The primer will or will not amplify a segment of DNA, depending
on whether the positions are complementary to the primer’s
sequence.
•RAPD markers are mostly dominant markers (impossible to
distinguish between DNA amplified from a heterozygous locus or
homozygous locus).
•When using RAPD markers, using only the reproducible major
bands for identification may minimize its shortcomings. Further,
parental genomes may be included where available to help
determine bands of genetic origin.

152. Amplified Fragment Length Polymorphism
(AFLP)
•It is the combination of RFLP and RAPD techniques and is a
dominant marker.
•The technique uses primers that are 17–21 nucleotides in
length and are capable of annealing perfectly to their target
sequences.
•Another advantage of the technology is that it does not require
sequence information or probe collections prior to generating
the fingerprints.
•Some of the applications of AFLP markers include biodiversity
studies, analysis of germplasm collections, genotyping of
individuals, identification of closely linked DNA markers,
construction of genetic DNA marker maps, construction of
physical maps, gene mapping, and transcript profiling.

153. Microsatellite or Simple Sequence
Repeats (SSRs)
•Microsatellites (or simple sequence repeats (SSRs), sequence-
tagged microsatellite sites (STMS), simple sequence repeat
polymorphisms (SSRP) are repetitive DNA sequences.
•The repeats may be di-, tri or tetranucleotides (e.g., GT, GAC,
GACA; or generally (CA) n repeat where n varies among alleles) in
the nuclear genome.
•Microsatellites were the first most successful and widely
exploited PCR-based markers.
•In addition to being highly polymorphic, SSRs occur frequently
and randomly distributed throughout eukaryotic genomes.
•Their high variability is due to the fact that they have a higher
mutation rate as compared to the neutral parts of the DNA.

156. Applications of DNA markers
•Construction of high density genetic maps of crops for
genetic studies.
•Comparative gene mapping in different species to
determine their similarity in gene order and
evolutionary relationships.
•Marker assisted selection of breeding lines for speed
up of breeding program.
•Mapping of polygenes or QTL.
•Germplasm characterization and evaluation.
•Estimation of genetic diversity.
•Genomic selection of crops.

157. FISH (Fluorescent in situ Hybridization)
•It is a process which vividly paints chromosomes or portions of
chromosomes with fluorescent molecules.
•It identifies chromosomal abnormalities.
•Aids in gene mapping, toxicological studies, analysis of
chromosome structural aberrations, and ploidy determination.
•Used to identify the presence and location of a region of DNA
or RNA within morphologically preserved chromosome
preparations, fixed cells or tissue sections.
•This means we can view a segment or entire chromosome with
our own eyes.
•It was often used during M phase but is now used on I phase
chromosomes as well.

158. Procedure of FISH
•Denature the chromosomes
•Denature the probe
•Hybridization
•Fluorescence staining
•Examine slides or store in the dark

160. LOD Score
•Sample of offspring is the powerful determinant for the
construction of linkage map.
•Raising a second sample from the same population of the
progenies will generally yield a different recombination
estimate.
•The estimate will be more reliable if the sample size is large.
•The precise recombination is reflected by the variance or the
standard error.
•For example: in a sample of offspring sample size N if we
observe k recombinants and N-k non-recombinants than
estimate of r can be given by:
r= k/N and its variance is given by Var (r) = r(1-r)/N

161. •Higher sample size give more confidence in the
estimate of r.
•In linkage analysis, an alternative measure of
confidence is used known as LOD score ( Logarithm
of odds).
LOD = N[r log r+(1-r(log(1-r) + log(2)]
•LOD value give the idea about proof of linkage or
evidence of linkage.
•Higher the value of LOD score, higher will be the
confidence about linkage.
•The score of LOD lower than 3 shows poor reliability
about linkage of the loci.

162. Example:
•Let k=20 and N= 100 ( there are 20 recombinants in a
population size of offspring of 100.
•We have recombination estimate (r) = 20/100=0.20

LOD= N[r log r+(10r(log(1-r) + log(2)]
Or LOD= 100[0.2 log 0.2+(1-0.2(log(1-0.2) + log(2)]
Or LOD= 8.37

This means that it is 10
8.37
times as likely to obtain the
sample when r=0.2 than to obtain the sample whebr=0.5
(no linkage). It is a very large number and we are very
confident that the loci are linked.

163. Genetic control mechanism in
Eukaryotes

164. Gene Regulation
•Genes code specific proteins or enzymes, which ultimately
produce specific phenotypes or control specific activities.
•There is precise control on the type of proteins or enzymes
produced by these genes and it is known as gene regulation.
•In prokaryotes cells produce only those enzymes which are
required in a given time.
•In eukaryotes cells of different organs produce different
proteins or enzymes needed for their function.
•Gene regulation occurs in two ways:
–By controlling the synthesis of the enzymes
–By regulating the activity of enzyme molecule already
present in the cells.

165. The operon model of gene regulation in prokaryotes
•Transcription is the first step of gene expression and controlling
of transcription helps to regulate the expression of gene.
•An operon is the functional unit of genetic expression
consisting of a group of structural genes whose transcription is
regulated by the coordinated action of regulator gene and an
operator sequence. Eg: Lac operon, trp (tryptophan ) operon in
E. coli.
a.Structural genes: these genes produce one long polycistronic
mRNA that code for more than one polypeptide.
b. Operator gene: it is the DNA segment to which repressor
binds in order to prevent transcription
c.Promoter gene: the promoter region consist of DNA sequence
that provide signal to RNA polymerase for binding to DNA and
initiation of transcription.

166. Fig: Operon model in Prokaryotes

167. Eukaryotic gene regulation is regulated at six levels
•Transcription
•RNA processing
•mRNA transport
•mRNA translation
•mRNA degradation
•Protein degradation

168. Gene control in Eukaryotes
•Some genes have to respond to changes in physiological
condition.
•Many others are part of developmentally triggered genetic
circuits that organize cells into tissues and tissues into an entire
organ (except unicellular eukaryotes).
•In these cases, the signals controlling gene expression are the
products of developmental regulatory genes, rather than
signals from the external environment.
•There are three components of gene control in eukaryotes.
- Signals
- Levels
- Mechanisms (molecular control)

169. Signals for gene control in Eukaryotes:
1. Hormones
•Both small molecules and polypeptide can cause
different gene expression: In multicellular
Eukaryotes, one type of cell can signal another by
secreting a hormone.
•Hormones circulate through the body, make contact
with the target cells and then initiate a series of
events that regulate the expression of particular
genes. In animal there are two classes of hormones.
•Steroid hormones: are small lipid soluble molecule
derived from cholesterol.
•Peptide hormones: linear chain of amino-acids.

170. 1.The steroid hormones
enter its target cell
and combines with a
receptor protein.
2.The hormone-
receptor complex
binds to a hormone
response elements in
the DNA.
3.The bound complex
stimulates
transcription.
4.The transcript is
processed and
transported to the
cytoplasm.
5.The mRNA is
translated into
proteins.
Regulation of gene expression by steroid hormones

171. Regulation of gene expression by peptide hormones:

172. 2. Cell to cell contacts can act as signals to control genes:
•This signal is not well studied but hoped that cell to cell contacts
can act as signals to control gene for e.g., contact between the
mesenchymal cells (derived from mesoderm) and either
endodermal or ectodermals.
•The nature of the information passed in such cell to cell is
unknown, but it may be that a surface signal mediated by the
contact between cell surface proteins leads to gene control.
3. Environmental and nutritional signals:
•Eukaryotic cells like yeast and mold responds to such signals as
nutritional stress.
•For e.g., yeast cells increase the level of enzymes for the synthesis
of tryptophan, histidine, isoleucine and valine by a factor of 2-10
when the cells are starved for these aminoacids.

173. Post-Replicative Modification of DNA
•DNA methylation is the addition or removal of a
methyl group predominantly where cytosine base
occur, most often in CG dinucleotide.
•DNA methylation plays an important role for gene
regulation in epigenetic development and disease.
•DNA methylation leads to heterochromatin
formation.
•Histone Acetylation is the modification that leads to
euchromatin formation.

174. Mechanisms of transcriptional control in
Eukaryotes
•Initiation of DNA transcription by RNA polymerase II is activated
by cis-and trans-acting elements cooperating with each other.
•As the name suggests, cis-acting elements are DNA sequences
that can influence transcription of a gene.
•They can be functionally subdivided into promoter and
enhancer elements, both of which are necessary for the
specific and activated expression of a gene.
•Trans-acting elements are proteins (transcriptional activators)
that bind to specific cis-acting elements to activate
transcription.
•There are also negative cis-acting elements that bind
transcriptional repressors to turn down expression of genes.

175. Cis-acting regulatory elements
•Promoter region: It is located immediately upstream of the
transcription start site and serves as the binding site of RNA-
polymerase II complex.
•It is similar to that of prokaryote and consist of TATA box.
•Enhancer region: They are the distance independent site that
activate the expression of genes upon specific stimuli.
•They increase the rate of transcription. They are composed of a
number of specific sequences that can bind different
transcriptional activators.
•Insulators: The sequence that have the ability to protect genes
from inappropriate signals coming out from their surrounding
genome.

176. Fig: Eukaryotic transcription complex

177. Trans-control of transcription:
•A large number of trans-acting regulatory proteins have now
been identified in eukaryotic cells.
•Like their counterparts in prokaryotes, these regulatory
proteins act by binding to specific target DNA sequences.
•Regulatory proteins that bind the core-promoter and promoter-
proximal elements help RNA-polymerase, they form an
initiation complex.
•Several transcription factor complexes (TFII complexes)
interact with RNA-polymerase II for initiation of transcription.
•For example, the TFIID complex consists of a TATA-box-binding
protein (TBP) and more than eight additional subunits (TAFs).
•This transcript mRNA is then processed in the nucleus and
release in cytoplasm through ribosome then it undergoes to
translation to give coded protein.

178. Post-transcriptional modification/eukaryotic mRNA
processing
1. Modification of 5’ end of preRNA by a formation of a cap
•A 7-methylguanosine is added to the 5’ end. Protects 5’ from
enzymatic degradation in the nucleus and assists in export to
cytosol
2. RNA splicing
•Removal of large non coding sequences (introns) from the primary
RNA transcript followed by rejoining of the non adjacent coding
sequences (exons) to produce the functional mRNA.
3.Enzymatic addition of poly A tail to the 3’ end of the molecule
•Addition of approximately 200 nucleotide-long sequences of
adenylate nucleotides (“Poly A tails”). Protect mRNA from
degradation of the coding sequence at the 3’ end by exonuclease.

180. Alternate splicing or Exon shuffling
•Different combinations of exons from different mRNA
resulting in multiple proteins from the same gene.
•Humans have 30,000 genes are capable of producing 100,000
proteins.

181. Translational control in Eukaryotes
•There are regions on the beginning of mRNA which
don’t code for proteins known as leaders.
•Proteins and other molecules can bind to the leader
which can enhance or restrict ribosome binding and
hence translation.
•mRNA molecules in cytoplasm may be degraded and
recycled to make more RNA.
•This varies the amount of gene product that is
produced (degraded mRNA cannot express much
protein).

183. Structures of Chromosomes

184. Chromosome
•The word chromosome was derived from two Greek
words Chroma(color) and soma (body).
•The name is given due to their property of being
stained very strongly by some dyes during mitosis.
•During interphase two types of chromatin are
distinguished according to intensity of DNA
condensation, staining and transcription.
•Euchromatin is the area of chromatin with active
genes which is lightly stained .
•Heterochromatin is the dark stained region of
chromatin that contains mostly inactive DNA.

185. Early model of chromosome
•Accoring to this model the basic component of chromosome
structure is chromonema (pl - chromonemata), which is
composed of chromatin and contains genes.
•The variation in chromosome length and thickness is proposed to
be due to coiling and uncoiling of chromonemata.
•Each chromatid of a chromosome may contain two or more
chromonemata, which run across through the centromere.
•In the case of metaphase chromosome, chromonemata are
surrounded by an amorphous matrix the outer side of which is
enclosed in the membrane called pellicle.
•However, electron micrographs of metaphase chromosomes do
not show any evidence for the existence of either matrix or
pellicle. So, this model of chromosome structure is inadequate
and of historical interest only.

186. Recent Models of Chromosome
structure
•Multistranded model
•Coiled DNA model
•Folded fiber model of chromosome
•Nucleosome solenoid model of chromatin
fibre
•Special chromosomes
–Lampbrush chromosomes
–Giant/Polytene chromosomes

187. I. Multistranded model:
•According to to this model, each chromatin fiber is on an average
100 Å in diameter.
•Each chromatin fiber is composed of two strands, each strand
being 35- 40 Å in diameter.
•Each strand consists of single DNA double helix (20Å in diameter)
and the associated histone and non-histone proteins; thus a
chromatin fibre contains two DNA double helices (separated
from each other by a space of about 25Å) and associated protein.
•Four chromatin fibers (each composed of two DNA double
helices) coil around each other to form a quarter chromatid,
which is smallest subunit of the chromosome visible under light
microscope in many organisms.

188. Fig: Multistranded model of chromosome

189. •Association of two quarter chromatids (each of 400 Å
diameters) gives rise to one half chromatids of 800 Å
diameters, which is composed of 16 DNA double helices.
•Finally, two half chromatids coil around each other to produce
one chromatid which is 1600Å in diameter, and is made up of
32 DNA double helices.
•Thus, a metaphase chromosome has 64 DNA double helices
i.e., strands and would be about 3200 Å in diameter. The
variation in chromosome thickness would be due to the
differences in coiling of chromatids.
•The numbers of DNA double helices in quarter, half and full
chromatids are proposed to vary from 8 to 64 depending
upon the species. Evidence in favour of this model studies on
Drosophila.

190. II. Coiled DNA model
•According to this model, the single DNA molecule of a
chromatin fiber is coiled in a manner similar to the wire in a
spring.
•The coils are held together by histone bridges produced by
binding of histone molecules in a large groove of DNA
molecules.
•Such a coiled structure would be stabilized as a single histone
molecule that would bind to several coils of DNA.
•This coiled structure is further coated with chromosomal
proteins to yield the basic structure of chromatin fibers.
•This fiber (type A) may undergo supercoiling to produce the
another type of fiber (type B).

192. III. Folded-fiber model
•This model was proposed by DuPraw in 1965 is the most
widely accepted model of chromosome.
•According to this model chromosomes are made up of
chromatin fibers of about 230 Å diameters.
•Each chromatin fiber contains only one DNA double helix,
which is in coiled state, this DNA coil is coated with histone
and non-histone proteins.
•Thus, 230 Å chromatin fiber is produced by coiling of a single
DNA double helix; the coils of DNA molecule are stabilized by
proteins divalent cations (Ca++ and Mg++).
•Each chromatid contains a single enormously long chromatid
fiber.

193. •The DNA of this chromatin fiber replicates during interphase
producing two sister chromatin fibers.
•It remains unreplicated in the centromeric region. So that the
two sister chromatin fibers remained joined in this region.
•During division subsequent replication in the centromeric
region to separate chromatin fibers.
•During cell division, the two sister chromatin fibers after
replication undergo extensive folding separately in an irregular
manner to give rise to two sister chromatids.
•This cause reduction in length and increase in their thickness
and stainability. Then, this structure further undergos
supercoiling which further reduces the length and increase
thickness of chromosomes.

195. IV. Nucleosome- solenoid model
•This model was proposed by Kornberg and Thomas in 1974 and
is universally accepted.
•According to this model chromatin is composed of a repeating
unit called nucleosome.
•One complete nucleosome consists of: 1. Nucleosome core 2.
Linker DNA 3. An average of one molecule of H1 histone 4. The
other associated chromosomal proteins
•Nucleosome core: It consist of a histone octamer composed of
two molecules each of histones H2a, H2b, H3, and H4.
•Histone octamer is surrounded by DNA known as core DNA of
length 146 bp (base pair) 2) Linker DNA: The size of linker DNA
varies from 8bp to 114 bp.

197. •A complete nucleosome contains two full turns of DNA
super-helix (a 166 nucleotide pair of DNA) on surface of
histone octamer and stabilization of this structure by binding
of one molecule of histone H1
•Nucleosome are in close contact along the chain and the
close-packed chain is called nucleofilament (10nm) in which
packing of DNA is about five-to- seven fold.
•Nucleofilament is coiled to form supercoil or solenoidal
structure of pitch about 300A
0 which further coiled into
super-solenoid or chromonema of 2000-4000A
0
•Chromonema is folded in chromosomal matrix to produce a
chromatid or chromosome

199. V. Lampbrush chromosomes:
•They are found in oocytes of many invertebrates and all
vertebrates, except mammals.
•They have also been reported in human and rodent oocytes,
but most extensively studied in amphibian oocytes.
•During diplotene, the homologous chromosomes begin to
separate from each other, remaining in contact only at
several points along their length.
•From each of a majority of the chromosomes generally a pair
of lateral loops extends in the opposite directions
perpendicular to the main axis of chromosome.

200. Fig: Lampbrush Chromosome

201. •As the oocytes progresses from diplotene to
metaphase I, the loops are slowly withdrawn and
reassembled into chromosomes.
•Each loop represents one chromatid of a
chromosome and is composed of one DNA double
helix.
•One end of each loop is markedly thinner (thin end)
and other is thicker (thick end).
•Loops represent the site of gene action
(transcription), and the function of lampbrush
chromosome is to produce large numbers and
quantities of proteins and RNAs stored in eggs.

202. VI. Giant chromosome/Polytenes
•Giant chromosomes are found in certain tissues e.g., salivary
glands of larvae, gut epithelium, Malphigian tubules and some
fat bodies of some diptera. E.g., Drosophila, Chironomous.
•These chromosomes are very long (upto 200 times size during
mitotic metaphase in case of Drosophila) and thick, hence they
are known as giant chromosomes.
•Homologous chromosomes pair to form giant chromosome
(somatic pairing) so that total number of giant chromosome in
a 2n nucleus in only n.
•In D. melanogaster salivary glands, the giant chromosomes
radiate as five long and one short arms from a deeply staining
and more or less amorphous structure called chromocenter.

203. •Each giant chromosome is composed of numerous
strands, each strands representing one chromatid.
•So, these are known as polytene chromosomes and
condition is referred as polyteny.
•The numerous strands of these chromosomes are
produced due to repeated replication of paired
chromosomes without any nuclear or cell division so
that the number of strands (chromatids) doubles
after every round of DNA replication.

204. Fig: Giant/Polytene Chromosome

205. Developmental genetics:

206. Variegation in biological tissue
•Variegation is the existence of difference looking
sectors of somatic tissue whatever may be the
cause.
•Geneticists gave another name mosaic useful in
relation to these phenomena.
•Thus mosaic is an individual composed of tissue of
two or more different genotype often recognizable
because of their phenotypes.
•It is also called as sectoring in biological tissue

207. Causes of variegation
1. Mitotic crossing over
•Besides meiotic crossing over, there occurs crossing over of
somatic chromosome which causes sectoring in somatic tissue.
•It may be defined as only mitotic process that generates diploid
daughter cells with a combination of gene different from diploid
parental cell in which mitosis occur.
2. Mitotic chromosome non-disjunction
•The failure in mitosis for the two members of a chromosome
pair to separate (to disjoin) normally, which causes both
chromosomes to go to one daughter cell while none go to the
other daughter cell.
•Which also leads to phenotypic segregation thereby causing
variegation.

208. 3. Position effect variegation
•A further causes of variegation is associated with
translocation because the expression of gene can be
affected by its position in genome and this will cause
variegation.
•Both translocation and inversion result in
rearrangement of gene in chromosome and hence
form new association
4. Fusion of different zygote
•By fusion of different zygote the cell line shows
peculiar mosaic.
•This could be seen in mouse, the blastoderm cell.
When fused in embryo form a mouse.

209. 5. Somatic mutation:
•Mutation in somatic cell is known as somatic mutation
which leads to a sector of identical mutant cell.
•There is little chance of translocation of such mutant to
progeny unless germinal cell is involved. E.g., bud
mutant.

6. Cytoplasmic mutation and segregation:
•There are genetic material present in plastids which
inherit certain genes.
•These are known as plastome and transmitted
generation after generation through cytoplasm carrying
by ovule.

210. Development and Pattern
•Fertilization gives rise to zygote which undergoes
different stages of division to develop into triploblastic
gastrula.
•Blastula is the hollow mass of undifferentiated cells of
embryo.
•Gastrulation is the rearrangement of embryonic blastula
into a three layered gastrula.
•Gastrula consist of the three germ layers which later
develop into various parts of plants or animals.
•These germ layers are:
–Ectoderm
–Mesoderm
–Endoderm

211. Fig: Development of gastrula

212. Fate of primary germ layers
•Ectoderm
Hair, nails, epidermis, brain, nerves
•Mesoderm
Dermis, blood vessels, heart, bones,
cartilage, muscle
•Endoderm
Internal lining of the gut and respiratory
pathways, liver, pancreas

213. Organogenesis
•Differentiation of primary germ layers into tissues
and organs.
•Differentiation is the process by which cells
become specialized and take on specific roles in
an organism.
•During human development, a single
cell(fertilized egg differentiates into every
different kind of specialized cell type found in the
body, including blood, muscle and nerve cells.
•The primary cells that are differentiated into
various cells are known as stem cells.

214. Stem cells
•Stem cells are those cells that can differentiate into many
different cell types.
•The daughter cells have the same DNA but different genes may
be turned on or off.
•The embryonic stem cells can be either totipotent, pluripotent
or multipotent.
•Stem cells that have the ability to differentiate into every type
of cell in the body are known as totipotent.
•Pluripotent cells can differentiate into most but not all type of
cells. Cells present in the blastocyst of human are pluripotent.
•Stem cells present in adult organism that can differentiate into
a limited number of cell types are called multipotent.

217. Effect of Environment on cell
differentiation in plants
•Environmental effects on gene expression are more
distinct in plants.
•Example: the environment helps to determine the
patterns of gene expression that lead to flowering.
•Short day plants will flower only when exposed to
extended period of darkness.
•Long day plants will flower only when exposed to
short periods of darkness.

218. Genetics of Cancer
•Cancer is the unwanted modification and growth
of certain cells and formation of uncontrolled
tumors.
•It is a genetic disease caused by the mutation in
the genes that control cell division.
•The factor that causes the cancer is known as
carcinogen. Eg: chemicals, radiations, virus,
bacteria, fungi, smoking etc.
•The study of tumor formation is called as
oncology.

219. Tumor
•It is the abnormal proliferation of cells that results
from uncontrolled, abnormal cell division.
•Benign: a non-cancerous tumor that remains within
a mass in a particular location.
•Malignant tumor: the cancerous tumor with
uncontrolled dividing cells that invade and destroy
healthy tissue elsewhere in the body.
•Metastasis: spread of cancer cells beyond their
original site.

221. Types of cancer
Carcinomas:
•Grow in skin and tissues that line the organs of the
body
•Eg: lungs and breast
Sarcomas:
•Grow in bone and muscle tissue
Lymphomas
•Solid tumors that grow in tissues that form blood cells
•Eg: leukemia

222. Mutation
•The basic mechanism in all cancer is mutation.
•Mutation is change in DNA sequence and this change can be
attributed to particular point or a segment of chromosome or
the number of chromosomes.
Carcinogenic agents are involved in causing mutations. The
mutation may be:
•Germline mutation: These mutation are present in every cell
of the body and are passed from parent to child.
•Sporadic cancer or somatic mutation: caused by tobacco, UV
radiation or other toxins and chemicals. They are not present
in every cell of the body and not passed from parent to child.

223. Genes & cancer
Four classes of normal regulatory genes are the
principle target of genetic damage.
•The growth promoting Proto-oncogenes
•The growth inhibiting tumor suppressor genes
•Genes that regulate programmed cell
death(Apoptosis)
•DNA repair genes

224. Proto-oncogene
•Have multiple roles, participating in cellular functions
related to growth & proliferation.
•Proteins encoded may function as growth factors or
their receptors, signal transducers, transcription
factors or cell cycle components.
•Mutations convert proto-oncogene into
constitutively active cellular oncogene that are
involved in tumor development.
•When a proto-oncogene becomes activated it is
called an oncogene.

226. Immuno-genetics
•The immune system is that system that defenses against the
antigens that result from the invasion of disease causing
organisms as bacteria, viruses and fungi.
•Immune defense mechanisms can be divided into two main
types:
•Innate immunity, which includes a number of nonspecific
systems that do not require or involve prior contact with the
infectious agent,
•And specific acquired or adaptive immunity, which involves a
tailor-made immune response that occurs after exposure to an
infectious agent.
•Both types can involve either humoral immunity, which
combats extracellular infections, or cell-mediated immunity,
which fights intracellular infections.

227. •The protein responsible for the immune response in
vertebrates is called the antibodies, which are
synthesized in response to antigens.
•Each antibody molecule is made up of four
immunoglobulin chains held together in a form of Y-
shaped.
•There are two identical heavy chains, of 330 or 440
amino acids, and two identical light chains, of 220
amino acids in each.
•Light chains are specified by variable(V), joining (J)
and constant (C) gene segments.
•Heavy chains are specified by V, diversity (D), J and C.

228. Fig: Antibody molecule

229. •Here, at the amino terminal ends of the four
polypeptide molecules, each antibody differs slightly
from every other antibody known as variables.
•This is also where the antigenic specificity of each
antibody exists.
•Based on amino acid sequence, it would appear that
each polypeptide chain of an antibody is constructed
of smaller subunit protein called domains.
•Each antibody is made up of four polypeptide chains
containing larger constant regions of amino acid
sequences and small variable regions of amino acid
sequences.

230. Theories of antibody diversity
a.Somatic recombination theory
•A functional antibody gene is made up in β- lymphocytes
(somatic cell derived from germ line) by somatically recombining
V and C segment into a functional antibody gene.
•Combining a few 100 V segments with 20 D segments and 4J
segments generates approximately 10,000 combinations.
•Heavy and light chain combination are included, there are over
10 million unique antibody molecules possible.
•Four different types of segments:
–Approximately 10 C segments coding for constant region of
both chains
–Several 100 V segments coding for variable portion of protein.
–Distinct 4 J segments used for joining V and C segments
–Different 20 D segments for diversity of heavy chain.

231. b. Clonal selection theory
•The β-lymphocytes originate from bone marrow cells. Each
cell produces antibodies against only specific antigens by the
process of somatic recombination.
•As this specific cell type proliferates, it produces a small clone
of identical cells, all producing the same antibodies.
•The antibodies remain bound to cell membrane.
•Whenever they bind to their specific antigen, the cell is
stimulated to proliferate rapidly to greatly increase the clone
and respond quickly to specific antigen.
•This is clonal selection and also explains how once immunity is
developed against a foreign antigen and it remains
throughout the life time of the individual.