BBT: 511 Genetics

Dr. Bipinraj
Why to Study genetics?
Genetics has an important position in biology
Genetics is central to numerous human
interactions, such as behavior, character,
diseases, evolution etc.
It helps us to develop many advanced varieties
of plants and animal stock
It helps us to understand the pattern of
inheritance of many diseases
It helps us to control many infections

Important topics in genetics
Gene organization : molecular structure
Gene regulation : heredity and variation
Gene to proteins
Gene expression to environment
Evolution
How to study genetics ?
Observations (natural and experimental) . What ?
Experimental design . How ?
Molecular studies

Largely depends on statistics

Significance of Genetics in Evolution
and Breeding
Genetics : Study of heredity and variation
(Classical genetics)
: Study of gene and gene interaction
(Molecular genetics)
: Study of evolution of character/ species
(Evolutionary genetics)
Genetics helps us to understand heredity , variation
and speciation thus mechanism of evolution.
Breeding is the application of genetic principles of
to get better variety
Evolution: Is the genetic changes in successive
generations.
Evolution give rise to diversity and causes
speciation
Evolution occurs when there is a variation in
the allelic frequency in the population
Variation in allelic frequency can happen due
to natural selection, genetic drift and
mutation.

Industrial Melanism
Applications of Evolution:
Artificial selection or selective breeding
to get desired variety
Medicine to control genetic disorder

Plant breeding: Artificial breeding and selecting
procedure to
Increase quality and yield
Increase resistance against
Draught, salinity, temperature
Virus, fungus and bacteria
Increase tolerance against pesticide and herbicide
Classical breeding : relies on homologous
recombination chromosomes to introduce desired
variety
Modern breeding : use molecular techniques to select
or introduce desired traits

Steps involved are
Creation of variation, Selection and Evaluation
controlled pollination, genetic engineering
Marker assisted selection
Release
Multiplication
Distribution of the new variety


CHROMOSOME
Mendel Proposed Mechanisms of Heredity (1866)
Walther Flemming Describes Chromosomes (1878)
Theodor Boveri Links Chromosomes and Heredity
(study on round worm) (1888)
Walter Sutton Finds Evidence for Mendel's
Principles, chromosome separates during gametes
formation (study on grasshopper) (1902)
Thomas Hunt Morgan Experimentally
Demonstrates Chromosome Theory (red and
white eye fly). (1933)
Fern (Ophioglossum reticulatum) 1200
Drosophila melanogaster (fruit fly) 8
Dove (family Columbidae) 78
Mouse (Mus sp.) 40
Rat (Rattus sp.) 40
Nematode worm ( Caenorhabditis
elegans)
6
Zebrafish (Danio rerio) 50
Tobacco (Nicotiana tabacum) 48
Arabidopsis thaliana 10
Chromosome is the orderly arrangement of
DNA.
In eukaryote, DNA double helix is bound to an
octamer protein (HISTONE) to form a bead-
like structure called as (NUCLEOSOME )
Nucleosomes are connected by linker DNA
strand
Nucleosomes are further coiled to form
solenoid tubes
Solinoid tube
Solinoid tubes now spirally arranged on a non-
histone protein scaffold and this scaffold
further coils to form condensed chromosome.
On condensed chromatin the region at which
spindle fiber attaches is called as centromere
On chromosome arm there are more
condensed region called heterochomatin and
less condensed region called as euchromatin
Centromere
Arms
p (shorter)
q (longer)
Telomere
Types of Chromosomes
based on centromere
Telocentric : centromere at the end
Acrocentric : centromere at off center
Metacentric : Centromere in the middle
Holocentric : Whole chromosome act as centromere
Sex chromosome
Autosomes


CHROMOSOME STAINING

Chromosome staining
Staining: Used for visualizing the chromosomes
aceto-orcein, acetocarmine, gentian violet,
haematoxylin etc.
Giemsa, Wright and Leishman
Applications:
Chromosome analysis
Number
Morphology
Length
Position of centromere etc.
To study chromosome breakage

Giemsa staining:
It is made up of methylene blue, eosin, and Azure B.
Specifically binds the phosphate groups of DNA, and
attaches itself to regions of DNA where there are high
amounts of adenine-thymine bonding.
Procedure
Place slides in a staining dish.
Stain for 7 minutes.
Rinse slides in two changes of distilled water.
Air dry slides; mount them with a cover slip if desired.
Chromosome banding:
Differential intensity pattern along the length
of chromosome when stained are called as
chromosome banding.
This difference is due to the differences in the
base component of DNA
Based on the stain used, chromosome
produce different banding pattern
Uses of banding
Chromosome identification
Identifying abnormalities of chromosome number
Identifying translocations
Identifying deletions, inversions or amplifications
Comparisons of chromosome banding patterns
can confirm evolutionary relationships between
species

Standard banding methods
Q-Banding: pattern created by treating fluorochrome quinacrine. Forms
dark bands at AT rich regions (heterochomatid
G-Banding: pattern created by treating Giemsa stain. Forms dark bands at
AT rich regions (heterochomatid) producing
R-Banding : Reverse banding, produced by staining GC rich region by dyes
such as chromomycin A3, olivomycin and mithramycin.
C-Banding : Banding of non-coding constitutive heterochomotin by Giemsa
staining. C-Banding is produced by treatment of chromatin with acidic and
then basic solutions followed by staining with Giemsa.
NOR-banding: Involves silver staining (silver nitrate solution) of the
"nucleolar organizing region", which contains rRNA genes.
T-Banding: Involves the staining of telomeric regions of chromosomes
using either Giemsa or acridine orange after controlled thermal
denaturation. T bands apparently represent a subset of the R bands
because they are smaller that the corresponding R bands and are more
strictly telomeric.



High resolution banding
To produce more detailed analysis of chromosome
Hy-banding can be produced for entire chromosome or
a specific region
Methods:
Banding prophase or early metaphase chromosome By
decreasing the degree of condensation by treating with
chemicals such as Ethidium bromide, acridine orange
and actinomycin D etc.

FISH
Fluorescence In Situ Hybridization: it is a
technique used to detect the presence or
absence of a specific DNA sequence or a gene
in an organism.
Applications of FISH
FISH to Identify the Positions of
Genes
for identifying the location of a cloned
DNA sequence on metaphase
chromosomes.
Diagnosing Chromosomal
Abnormalities
to Paint Entire Chromosomes
Analyze Interphase Chromosomes

Karyotyping
Karyotyping is the process of pairing and orderly arranging all
the chromosomes of an organism, thus providing a genome-
wide snapshot of an individual's chromosomes.
Karyotypes are prepared using standardized staining
procedures that reveal characteristic structural features for
each chromosome.
Applications:
To detect gross genetic changesanomalies involving several
megabases or more of DNA.
Karyotypes can reveal changes in chromosome number
Careful analysis of karyotypes can also reveal more subtle
structural changes, such as chromosomal deletion, duplications,
translocations, or inversions.
Karyotypes are becoming a source of diagnostic information for
specific birth defects, genetic disorders, and even cancers.
Blood is drawn and coagulation prevented by addition of heparin.
Mononuclear cells (lymphocytes and monocytes) are purified by density
gradient centrifugation
Mononuclear cells are cultured for 3-4 days in a medium containing a
mitogen such as phytohemagglutinin. (Phytohemagglutinin stimulates lymphocytes to
proliferate.)
After the culture period when a large population of dividing cells is present,
colcemid is added. (Colcemid disrupts the mitotic spindles and prevents completion of mitosis, thereby
assembling cells arrested in metaphase.)
Lymphocytes are harvested and treated briefly with a hypotonic solution.
The hyportonic solution causes the nuclei to swell through osmosis
facilitating the spreading of the chromosomes of the metaphase
preparation.
The swollen cells are fixed, dropped onto a microscope slide and dried.
Slides are stained.
Stained slides are scanned and good preparations are then photographed.
Karyograms are prepared from developed photos. Chromosomes are cut
out and arranged and pasted orderly (from large to small in their different
groups) on a prepared form.
Karyogram of Klinefelter's syndrome
Karyogram of down syndrome male
Nomenclature:
Standard nomenclature of chromosome is done
by the size of the chromosome,
So in human, all non-sex chromosome arranged
according to the size, and 22
nd
, the sex
chromosome arranged at the end.
They are again sub divided into 7 groups (A-G)
based on the size and position of centromere.

Group Chromosom
e no.
Size and position of centromere
Group A 1-3 Largest , Median centromere
Group B 4-5 Large, Submedian centromere
Group C 6-12 Medium, Submedian centromere
Group D 13-15 Medium, Acrocentric centromere
Group E 16-18 Short, Median or submedian
centromere
Group F 19-20 Short, Median centromere
Group G 21-22 Very short, Acrocentric centromere
In 1881 a French embryologist E G Balbiani observed chromosome as a cylindrical cord
that repeatedly unraveled and filled the Nucleus of larval salivary glands, Malpighian
tubules, intestine, hypoderm and muscles a midge.

He called this structure a permanent spireme.

In 19331934, three groups of researchers (Painter, 1933; Heitz and Bauer, 1933; King and
Beams, 1934), using squashed preparations, showed that the spireme is not continuous
but consists of separate elements whose number is close to the haploid number of the
mitotic chromosomes.

Each element is formed as a result of the tight synapsis (joining together) of homologous
chromosomes, Painter, 1933

Many scientists studied this structure and later it was termed as Polytene chromosome.


Peculiarities of Polyteny
The formation of polytene chromosomes is associated with the
elimination of the entire mechanism of mitosis after DNA synthesis as a
result of which the cell cycle consists of just two periods, synthetic (S)
and intersynthetic (G).
at the end of each replication period,DNAstrands do not segregate;
rather, they remain paired to each other to different degrees
The polytene chromosomes formed are incapable of being involved in
mitosis.
the nuclear membrane and nucleolus remain intact during consecutive
DNA replication cycles
Polyteny arises and attains high levels in tissues, organs and at
developmental stages when there is need for the rapid development
of an organ at an unaltered high level of function.
Morphology of Polytene Chromosomes
Polytene chromosomes develop from the
chromosomes of diploid nuclei by successive
duplication of each chromatid.
Based on the conjugation of homologous
chromosomes there are three types of
polyteny
classic polytene chromosomes
cryptic polyteny
pompon-like chromosome
In polyteney nonhomologous chromosomes
are joined by their centromeric regions to form
a common chromocentre
Banding pattern of polytene chromosmes are
unique for a species
A series of swollen regions were also seen on
the light banded regions called as Puffs.
Polyteny is found in a wide range of organisms,
appearing at many stages of evolution
The experimental plants must necessarily:
Possess constant differentiating characteristics
The hybrids of such plants must, during the
flowering period, be protected from the influence
of all foreign pollen, or be easily capable of such
protection.
The hybrids and their offspring should suffer no
marked disturbance in their fertility in the
successive generations.

Leguminosae has been selected on account of
their peculiar floral structure.

Distinct characters which are constant, and easily and certainly
recognizable, and when their hybrids are mutually crossed they
yield perfectly fertile progeny.
Disturbance through foreign pollen cannot easily occur, since
the fertilizing organs are closely packed inside the keel and the
anthers burst within the bud, so that the stigma becomes
covered with pollen even before the flower opens.
easy culture of these plants in the open ground and in pots, and
also their relatively short period of growth
Artificial fertilization was possible by opening the bud before it
is perfectly developed, the keel is removed, and each stamen
carefully extracted by means of forceps, after which the stigma
can at once be dusted over with the foreign pollen
Characters selected
1. The difference in the form of the ripe seeds
Round and wrinkle
2. Difference in the color of the seed albumen
Either pale yellow, bright yellow and orange colored, or it
possesses a more or less intense green tint.
3. Difference in the color of the seedcoat
White and violet
4. Difference in the form of the ripe pods.
Inflated or contricted
5. difference in the color of the unripe pods
Green or yellow
6. Differencein the position of the flowers
Terminal or axial
7. Difference in the length of the stem
Tall 6 -7 ft or short ft. - 1 ft.


Multiple allele
M is a multiple allelic gene, having four allele
(M, M1, M2 & M3). How many alleles would
be present in an individual with heterozygous
genotype?
How many alleles would be present in an
individual with homozygous genotype?
C
+
C
+
, C
+
c
ch
, Cc
h
,
Cc
c
ch
c
ch
, c
h
c
h
, c
h
c Cc
However a new phenotype (Light Gray) emerged during the cross of chincilla with Himalayan
or albino c
ch
c , c
ch
c
h

Feather pattern in duck
M,produces the wild-type mallard pattern.
MR , produces pattern called restricted
md , produces a pattern termed dusky.
In this allelic series, restricted is dominant
over mallard and dusky, and mallard is
dominant over dusky: MR < M < md .
Heredity
Genetics is based on hereditary variation
Continuous variation
Discontinuous variation
Inheritance
Autosomal inheritance
Sex-linked inheritance
Cytoplasmic inheritance

Patterns of inheritance
Autosomal dominant inheritance :
One mutated copy of the gene in each cell is sufficient
for a person to be affected by an autosomal dominant
disorder.

Eg: Huntington disease
Progressive brain disorder that
causes uncontrolled movements,
emotional problems, and loss of
thinking ability
Mutation in HTT gene : important in
neuron function
CAG repeats 10 35 in normal
36 120 in abnormal
Autosomal Recessive
Two mutated copies of the gene are present in each
cell when a person has an autosomal recessive
disorder.
Eg: Cystic fibrosis
Inherited disease of the mucus
glands include progressive damage
to the respiratory system and
chronic digestive system problems
Mutations in CFTR gene
Impaired function of the chloride
channels, preventing them from
regulating the flow of chloride ions
and water across cell membranes
X-linked dominant
Caused by dominant mutations in genes on the X
chromosome
Eg: Fragile X syndrome
This disease is the most common
form of inherited mental
retardation
It is manifested cytologically by a
fragile site in the X chromosome
that results in breaks in vitro
X-linked recessive
Caused by recessive mutations in genes on the X
chromosome
Eg: Hemophilia
Due to the absence or malformation
of factor VIII
Codominant
In codominant inheritance, two different versions (alleles) of
a gene can be expressed, and each version makes a slightly
different protein. Both alleles influence the genetic trait or
determine the characteristics of the genetic condition.
Eg: ABO blood group
Mitochondrial
Eg: Optic neuropathy
GENE INTERACTION
Influence of two or more non-allelic pairs of
gene over a character
Epistasis
Epistasis:
One gene override the expression of other gene
Expressed gene is called as epistatic
Overridden gene is called as hypostatic
Dominant
Recessive
Complementary
Duplicate


9 C_P_ : 3 C_pp :3 ccP_ : 1 ccpp
purple white
Epistatic Gene Interaction
Complementary gene action
Enzyme C and enzyme P cooperate to make a
product, therefore they complement one
another

Enzyme C Enzyme P
Purple
pigment
Colorless
intermediate
Colorless
precursor
Colorless
precursor
Colorless
intermediate
Purple
pigment
Enzyme C Enzyme P
genotype cc
genotype pp
Colorless
precursor
Colorless
intermediate
Purple
pigment
Enzyme C
Enzyme P

Recessive Epistasis
Epistasis due to recessive genes is called recessive
epistasis


White coat produced by a recessive gene aa
Gene B dominant state produces grey coat
colour called agouti, and when recessive (bb)
leads to black coat colour.
The recessive gene for albinism (aa) is found
to be epistatic to the gene for agouti (BB and
Bb), and also to its recessive, homozygous
allele (bb) for black.
Dominant epistasis

Cytoplasmic Inheritance
In 1909, cytoplasmic inheritance was recognized
by Carl Correns as one of the first exceptions to
Mendels principles.
Uniparental inheritance: progeny inherit
organelle genes exclusively from one parent.
Maternal inheritance. Reason?
Heteroplasmons : Normal as well as mutated
organelle is present in the same zygote
Cytoplasmic segregation : segregation of wild
and mutated organelle to different daughter
cells are called as cytoplamic segregation.


In a dihybrid cross between Purple long (PPLL)
and red round (ppll). F1 produced Purple
flower with long pollen (PpLl).
In F2 generation it produced total 2134 seeds.
What would be the number of seeds with
Purple long, Purple round, Red Long and red
round genotype?
Bateson, Saunders and Punnett (1905, 1906)
Linkage and recombination
Genetic Maps
Gene Mapping with Recombination Frequencies
Crossingover occurs more or less at randomly
Two genes that lie far apart are more likely to undergo a
crossover than are two genes that lie close together.
Thus recombination frequencies could provide a convenient way
to determine the order of genes along a chromosome and
would give estimates of the relative distances between the
genes.
Chromosome maps calculated by using recombination
frequencies are called genetic maps.
Distances on genetic maps are measured in map units ( m.u.);
one map unit equals 1% recombination.
Map units are also called centimorgans (cM), in honor of
Thomas Hunt Morgan; one morgan equals 100 m.u.
Recombination frequency:


Normal leaves, tall X Molted leaves, dwarf (recessive
(MD) (F1) Parent) (md)
Progenies :
55MD, 8 Md, 7 mD and 53 md
What is the recombination frequency?

1
Constructing a Genetic Map
with Two-Point Testcrosses
Genetic maps can be constructed by
conducting a series of testcrosses between
pairs of genes and examining the
recombination frequencies between them.


Gene Mapping with the
Three-Point Testcross
Linkage and Recombination
Between Three Genes
Normal eye v
+
/ vermillion eye v
Cross vein cv
+
/ cross vein less
Normal wing ct
+
/ cut wing


Test cross :
Variation
Discontinuous
Continuous
Nilsson-Ehle Experiment
Deep dark red medium light very white
Red red red red light red
1 6 15 20 15 6 1

deep red
white
Variation
Discontinuous
Individuals can be grouped in distinct class
Controlled by one or few genes
Not affected by environmental factors
Eg. Blood groups
Continuous
Difficult to group in distinct group
Controlled by many genes
Mostly affected by environment
When plotted as a histogram, these data show a typical bell-
shaped normal distribution curve, with the mean, mode and
median all being the same.
Eg. Height, colour,

Nilsson-Ehle's experiment on wheat
kernel color
Changes in genetic material
Variation
Chromosomal aberrations
Mutation
Numerical Structural
Numerical Aberration
Euploidy : Addition or removal of a complete
set of chromosome
Aneuploidy : Addition or removal of individual
chromosome

Euploidy: In euploidy an organism acquires an
additional set of chromosomes over and
above the diploid complement
Triploid (3n), tetraploid (4n); pentaploid (5n),
and hexaploid (6n)
MONOPLOIDS: Male bees, wasps, and ants are
monoploid. In the normal life cycles of these
insects, males develop by parthenogenesis (the
development of a specialized type of unfertilized
egg into an embryo without the need for
fertilization).
POLYPLOIDS Polyploidy is very common in plants
but rarer in animals. Indeed, an increase in the
number of chromosome sets has been an
important factor in the origin of new plant
species.
Autopolyploids : If source of extra chromosome
are same species
Allopolyploids : If source of extra chromosome are
Different species
Autotriploid : Mostly sterile
They arise spontaneously in nature, but they can be
constructed by geneticists from the cross of a 4n
(tetraploid) and a 2n (diploid). The 2n and the n
gametes produced by the tetraploid and the diploid,
respectively, unite to form a 3n triploid.
Triploids are characteristically sterile.
Once formed, the triploid plants are healthy and robust
and are propagated through asexual cuttings
Eg. seedless watermelons, European pears and apples

Autotetraploids; either fertile or only partially
sterile
Autotetraploids arise by the doubling of a 2n
complement to 4n. This doubling can occur
spontaneously, but it can also be induced
artificially by applying chemical agents that
disrupt microtubule polymerization.
Eg. Groundnut, potatoes and coffee
The use of colchicine to generate a tetraploid from a diploid.
Colchicine added to mitotic cells during metaphase and anaphase
disrupts spindle-fiber formation, preventing the migration of
chromatids after the centromere is split. A single cell is created that
contains pairs of identical chromosomes that are homozygous at all
loci.
Allopolyploidy: An allopolyploid is a plant that is a
hybrid of two or more species, containing two or
more copies of each of the input genomes.
Eg. The hybrid produced between radish and
cabbage






hexaploid Triticum (2n + 44) X diploid Secale (2n =
14) to produce : Octaploid Triticale


Polyploidy in agriculture:
Wheat : Einkorn, Emmer and Vulgare
Einkorn (diploid 2n = 14). Not used for consumption
Emmer (Tetraploid). Hybrid of Einkorn and Goat
grass.
T. durum, T. dicoccum
Vulgare (Hexaploid)
T. aestivum (bread wheat) (AABBDD; 2n =
42)
Originated through hybridisation between T.
dicoccum (AABB) and a different species of
goat grass followed by chromosome doubling

Aneuploidy
Aneuploidy can arise in several ways.
First, a chromosome may be lost in the course of mitosis or meiosis
if, for example, its centromere is deleted. Loss of the centromere
prevents the spindle fibers from attaching; so the chromosome fails
to move to the spindle pole and does not become incorporated into
a nucleus after cell division.
Second, the small chromosome generated by a Robertsonian
translocation may be lost in mitosis or meiosis.
Third, aneuploids may arise through nondisjunction, the failure of
homologous chromosomes or sister chromatids to separate in
meiosis or mitosis. Nondisjunction leads to some gametes or cells
that contain an extra chromosome and others that are missing a
chromosome
2n+1 : Trisomy : 48 chromosome
2n+2 : Tetrasomy : 47 chromosome
2n- 1 : Monosomy: 45 chromosome
2n-2 : Nullisomy : 44 chromosome
Klinefelter syndrome XXY (Sterile male with low
IQ),
Turner Syndrome XO (sterile female)
XYY Normal male
Triplo-X trisomics (XXX) are phenotypically
normal and fertile females
Down syndrome 21
st
trisomy
Edward syndrome 18
th
trisomy
Pataus syndrome 13
th
trisomy

Structural aberrations
DELETION : Deletion of a small portion of a
chromosome resulting in loss of one or more genes.
It originates from breakage occurring at random in
both chromatids of a chromosome (called
chromosome break), or only in one chromatid
(chromatid break).
Terminal : Single break at the end of the chromosome
Interstitial : Two breaks occur in a middle portion of the
chromosome.
Multigenic deletion : several genes are mission due to
deletion. More lethal than Interstitial
Restitution: If the two raw ends of the same
chromosome unite and restore the structure
Exchange union: if two single breaks occur in
two different chromosomes in a cell, the
deleted segment of one chromosome may
unite with the raw broken end on the other
chromosome; this is called exchange union
Fate of the deleted segment:
Deletion loop:
Pseudodominance :
Eg: Cri du chat
heterozygous deletion of 5p15.2 and 5p15.3, of
chromosome 5.
Williams syndrome.
An autosomal dominant syndrome characterized by
unusual development of the nervous system and
certain external features.
Frequency 1 in 10,000 people.
Patients often have pronounced musical or singing
ability.
Caused by a 1.5 Mb deletion on one homolog of
chromosome 7.
Duplications
A chromosome duplication is a mutation in
which part of the chromosome has been
doubled
Tandem
Reverse tandom
Displacement duplication
Displaced on same arm
Displaced on different arm


Origin: Unequal crossing over
Eg: Bar eye mutant in Drosophila
The Bar mutant results from a small duplication
on the X chromosome, which is inherited as an
incompletely dominant, X-linked trait

779




358




68




45
Inversion
Inversions result when there are two breaks in a
chromosome and the detached segment
becomes reinserted in the reversed order
Paracentric: both breaks occur in one arm of the
chromosome it leads to a paracentric inversion
Pericentric : when a break occurs in each of the two
arms, the centromere is included in the detached
segment and leads to a pericentric inversion
Translocations
A translocation entails the movement of
genetic material between nonhomologous
chromosomes
Nonreciprocal translocations: genetic
material moves from one chromosome to
another without any reciprocal exchange
Reciprocal translocation : A two-way
exchange of segments between the
chromosomes
Effect of Phenotype:
May create new linkage relations that affect
gene expression (a position effect)
Chromosomal break can disrupt gene function
Eg. Neurofibromatosis
Robertsonian translocation

Mutation
A mutation is defined as an inherited change in
genetic information
Mutation is the source of all genetic variation
Mutation is the cause of evolution
Mutation helps organisms to survive in a particular
conditions
Mutation can be the reason for disease and disorder
Mutations serve as important tools of genetic analysis
Mutations are also useful for probing fundamental
biological processes
Types of Mutations
Somatic mutations
Germ-line mutations
Mutation can be spontaneous or induced
Point mutations
Alterations of single base pairs of DNA or of a
small number of adjacent base
Types:
Base substitutions : Alternation of a single
nucleotide in the DNA
Insertions and deletions (Indel): The addition or
the removal, respectively, of one or more
nucleotide pairs
Base substitution: Transition or Transversion
Indel Mutations: Insertions and deletions
Insertions and deletions within sequences that
encode proteins may lead to frameshift
mutations, changes in the reading frame of the
gene

Expanding trinucleotide repeats:
Fragile X syndrome; the most common hereditary
cause of mental retardation
The FMR 1 (Fragile X Mental retardation) gene
mutation has 60 or fewer copies of this
trinucleotide CGG, but in persons with fragile-X
syndrome, the allele may harbor hundreds or even
thousands of copies
Forward mutation: A mutation that alters the
wild-type phenotype,
Reverse mutation : A reversion that changes a
mutant phenotype back into the wild type.
Suppressor mutations:
Is a genetic change that hides or suppresses the
effect of an other mutation
A suppressor mutation occurs at a site that is
distinct from the site of the original mutation;
thus, an individual organism with a suppressor
mutation is a double mutant, possessing both the
original mutation and the suppressor mutation
but exhibiting the phenotype of an unmutated
wild type
Intragenic suppression Mutation: happens on the same gene but different
nucleotide or codon

Intergenic suppression Mutation: Happens on different gene
Lethal Mutation: mutation that causes death
of the organism
Recessive lethal
Dominant lethal
Semi lethal
Hemophilia

http://www.nature.com/scitable/topicpage
/mendelian-ratios-and-lethal-genes-557
Causes of mutation
Spontaneous Replication Errors
Tautomeric shifts, in which the positions of protons in
the DNA bases change. Purine and pyrimidine bases
exist in different chemical forms called tautomers
The two tautomeric forms of each base are in dynamic
equilibrium, although one form is more common than
the other.
The standard Watson and Crick base pairings
adenine with thymine, and cytosine with guanine
are between the common forms of the bases, but, if
the bases are in their rare tautomeric forms, other
base pairings are possible.
Mechanism of point mutation: Base
replacement

Tautomerization due to
ionization
Base analogues
The mutagen 5-bromouracil (5-BU) is an analogue
of thymine and binds to adenine under normal
conditions
5-BU frequently change to either the enol form or
an ionized form that can bind to guanine
Mechanism of point mutation: Base alteration
Some mutagens causes specific mispairing by
adding alkylating agents ;
eg Ethylmethanesulfonate (EMS) Adds ethyl group
Nitrosoguanidine (NG) adds methyl group

Transposable Elements
Transposable elements are mobile DNA sequences that
insert into many locations within a genome and often
cause mutations and DNA rearrangements.
They make up at least 50% of human DNA
Transposition is the movement of a transposable
element from one location to another.
Short, flanking direct repeats of 3 to 12 base pairs are
present on both sides of most transposable elements
Some have terminal inverted repeats, which are
sequences from 9 to 40 bp in length
Structure
In bacteria:
Insertion sequences (IS): Simple transposable
elements
Size 700 to 2000bp
Contains one or two genes that code for
transposase
Flanking direct repeats
Trasposones (Tn):
Larger than IS
May carry genes other than transposase between
the inverted sequences
Some are with Tra gene helps in transfer to other
bacteria during conjugation (conjugative
transposons)


Integrones:
Integrones are transposons that can capture
and express genes from other sources
They are highly selective in insertion
It contains , integrase, transposase, a gene
cassette
Eukaryotic TE
Ty elements in yeast
These elements are retrotransposons
Ac element in maize
Ac elements are about 4500 bp long, including
terminal inverted repeats of 11 bp, and the
flanking direct repeats that they generate are 8 bp
in length
Each Ac element contains a single gene that
encodes a transposase enzyme
Ds element in maize
Ds elements are Ac elements with one or more
deletions that have inactivated the transposase
gene
Ds elements can transpose in the presence of Ac
elements
Mechanism of trasposition
Conservative:



Replicative:

Transposition
through an RNA
intermediate
(Retrotransposition)
SEX DETERMINATION
The mechanism by which sex is established is termed sex
determination
In some species, both sexes are present in the same
individual, a condition termed hermaphroditism
Organisms that bear both male and female reproductive
structures are said to be monoecious (meaning one
house).
Species in which an individual has either male or female
reproductive structures are said to be dioecious (meaning
two houses).
Humans are dioecious.
Among dioecious species, the sex of an individual may be
determined chromosomally, genetically, or
environmentally.
Chromosomal Sex-Determining Systems
XX/XY sex-determination system
Most familiar, as it is found in humans.
Females have two of the same kind of sex
chromosome (XX, Homogametic)
Males have two distinct sex chromosomes (XY,
heterogametic).
Y chromosome carries factors responsible for
triggering male development
XX-XO sex determination
In the grasshoppers
In this system, females have two X chromosomes
(XX),
Males possess a single X chromosome (XO).
O means no chromosome
ZZ-ZW sex determination
W chromosome carries factors responsible for
female development
Male are homogametic (ZZ)
Female are heterogametic (ZW)
Eg Birds


Environmental Sex Determination
Marine mollusk Crepidula fornicata (slipper
snail )
This type of sexual development is called sequential
hermaphroditism; each individual animal can be both male and
female, although not at the same time. In Crepidula fornicata, sex
is determined environmentally by the limpets position in the
stack.
American alligator
Red-eared slider turtle
Alligator snapping turtle
Other mechanisms
Other insects, including honey bees and ants,
use a haplodiploid sex-determination system.
In this case diploid individuals are generally
female, and haploid individuals (which
develop from unfertilized eggs) are male.
This sex-determination system results in highly
biased sex ratios, as the sex of offspring is
determined by fertilization rather than the
assortment of chromosomes during meiosis
Sex determination in Drosophila
Total 8 chromosomes
3 pairs of autosomes 1 pair of sex chromosome
6A XX in Female and 6A XY in Male.
Here presence of the Y chromosome does not
determine maleness in Drosophila;
Flys sex is determined by a balance between
genes on the autosomes and genes on the X
chromosome.
This type of sex determination is called the genic
balance system.
Here number of genes seem to influence sexual
development.
The X chromosome contains genes with female
producing effects, whereas the autosomes
contain genes with male-producing effects.
Consequently, a flys sex is determined by the X:A
ratio, the number of X chromosomes divided by
the number of haploid sets of autosomal
chromosomes.
An X:A ratio of 1.0 produces a female fly; an X:A
ratio of 0.5 produces a male.
Genic system of sex determination is seen in the
case of many plants.
Here there are no sex chromosomes, and the sex
is determined by genome of one or more loci.
Even in dioecious organism although there are
sex chromosomes, the sex charecters are
determined by few genes in the sex
chromosomes.
Eg. SRY genes in mammalian Y chromosomes
determine male phenotype.

Sex linked inheritance

X-Linked Colour Blindness in Humans
Ability to detect colour lies in cone cells
Each cone cell contains one of three pigments
capable of absorbing light of a particular
wavelength; one absorbs blue light, a second
absorbs red light, and a third absorbs green light
Each of the three pigments is encoded by a
separate locus; the locus for the blue pigment is
found on chromosome 7, and those for green
and red pigments lie close together on the X
chromosome

Z-Linked Characteristics
In Indian blue peafowl, wild-type plumage is a
glossy, metallic blue (Z
Ca+
)
Cameo plumage, which produces brown
feathers, results from a Z-linked allele (Z
ca
)
that is recessive to the wild-type blue allele
(Z
Ca+
).

Dosage Compensation
In XX/XY system, female has two X chromosome
and male has one X. As a result X will over
express causes problem in development.
In animals there are different mechanism to
reduce the effect of extra X chromosome, and
are called as dosage compensation.
By dosage compensation animals equalizes the
amount of protein produced by X-linked genes in
the two sexes.
Dosage compensation in Humans
Lyon hypothesis (Mary Lyon, 1961): within
each female cell, one of the two X
chromosomes randomly becomes inactive;
If a cell contains more than two X
chromosomes, all but one of them is
inactivated by methylation.
X inactive-specific transcript or XIST gene is
active in future inactivated X (Xi).
X inactivation takes place relatively early in
development
In humans, within the first few weeks of
development.
Once an X chromosome becomes inactive in a
cell, it remains inactivated and is inactive in all
somatic cells that descend from the cell.
Thus, neighboring cells tend to have the same X
chromosome inactivated, producing a patchy
pattern (mosaic) for the expression of an X-
linked characteristic in heterozygous females.

Although many genes contribute to
coat color and pattern in domestic
cats, a single X-linked locus
determines the presence of orange
color.
There are possible two alleles at this
locus:
X+ : nonorange (usually black) fur,
Xo : which produces orange fur.
Males are hemizygous and thus may
be black (X+Y) or orange (Xo Y) but
not black and orange.
(Rare tortoiseshell males can arise
from the presence of two X
chromosomes, X+ Xo Y.)
Females may be black (X+X+), orange
(Xo Xo ), or tortoiseshell (X+Xo ), the
tortoiseshell pattern arising from a
patchy mixture of black and orange
fur.
Each orange patch is a clone of cells
derived from an original cell with the
black allele inactivated, and each
black patch is a clone of cells derived
from an original cell with the orange
allele inactivated.
Population Genetics
Population genetics is concerned with the
genetic composition or allele frequency
distribution of a population and how it
changes with time under the influence of
several factors



The genetic composition of a population is the
collection of frequencies of different
genotypes in the population.
These frequencies are the consequence of
processes that act at the level of individual
organisms to increase or decrease the number
of organisms of each genotype.
Population genetics is both an experimental and a
theoretical science
On the experimental side, it provides descriptions
of the actual patterns of genetic variation among
individuals in populations and estimates the rates
of the processes
On the theoretical side, it makes predictions of
how the genetic composition of populations can
be expected to change as a consequence of the
various forces operating on them
Variation and its modulation
Observations of variation
Protein polymorphisms
IMMUNOLOGIC POLYMORPHISM
AMINO ACID SEQUENCE POLYMORPHISM
DNA structure and sequence polymorphism
CHROMOSOMAL POLYMORPHISM
RESTRICTION-SITE VARIATION
TANDEM REPEATS
COMPLETE SEQUENCE VARIATION

Calculation of Genotypic Frequencies
A frequency is simply a proportion or a
percentage, usually expressed as a decimal
fraction.

The sum of all the genotypic frequencies always equals 1.
Calculation of Allelic Frequencies
The gene pool of a population can also be
described in terms of the allelic frequencies.

The primary goal of population genetics is to understand the
processes that shape a populations gene pool.
Allelic frequency is the result of selection of a particular allele and
its segregation according to various laws of genetics
How various laws affects the allelic frequency is explained with the
help of Hardy Weinberg law
For an autosomal locus with two alleles, the Hardy- Weinberg law
can be stated as follows:
AssumptionsIf a population is large, randomly mating, and not
affected by mutation, migration, or natural selection, then:
Prediction 1 the allelic frequencies of a population do not change;
Prediction 2 the genotypic frequencies stabilize (will not change)
after one generation in the proportions p2 (the frequency of AA), 2pq
(the frequency of Aa), and q2 (the frequency of aa), where p equals
the frequency of allele A and q equals the frequency of allele a.



The Hardy-Weinberg law indicates that, when
the assumptions are met, reproduction alone
does not alter allelic or genotypic frequencies
and the allelic frequencies determine the
frequencies of genotypes.
When genotypes are in the expected
proportions of p2 , 2pq, and q2 , the
population is said to be in Hardy-Weinberg
equilibrium.


Genetic equilibrium
The frequencies of alleles will remain constant in the
absence of selection, mutation, migration and genetic
drift.
Factors affecting the equilibrium or allelic frequency
mating patterns : random, inbreeding or assortive
mating
Migration
Mutation
Recombination
natural selection
Variation in the allelic frequency due
to mutation
The rate of change in gene frequency from the
mutation process is very low because spontaneous
mutation rates are low
The mutation rate is defined as the probability that a
copy of an allele changes to some other allelic form in
one generation.


is mutation rate
P s frequency of wild allele
Pt is frequency of wild allele after t time

Variation due to recombination
Recombinations brings new gene combinations
AB and ab
Ab and aB
The creation of genetic variation by
recombination can be a much faster process than
its creation by mutation.
Since recombination is a random process it brings
out an equilibrium between the allele on the
same chromosome called as linkage equilibrium

Variation due to migration
Migration causes changes in the allelic frequency
of a population by introducing alleles from other
populations
The magnitude of change due to migration
depends on both the extent of migration and the
difference in allelic frequencies between the
source and the recipient populations
Migration decreases genetic differences between
populations and increases genetic variation
within populations.
Selection
Selection is the differential rates of survival and
reproduction of an allele
Selection alters the frequencies of the various
genotypes in the population
Selection depends on the reproductive fitness of an
allele
Fitness is a consequence of the relation between the
phenotype of the organism and the environment in
which the organism lives
As a result of selection, the allele with the higher mean
fitness relative to the mean fitnesses of other alleles
increases in frequency in the population.
Random events
Genetic drift
If a population consists of a finite number of
individuals (as all real populations do) and if a
given pair of parents has only a small number of
offspring, then even in the absence of all
selective forces, the frequency of a gene will not
be the same in the next generation.
Just by chance, the composition of this sample
may deviate from that of the parental gene pool,
and this deviation may cause allelic frequencies
to change
Evolution
Evolution is the change in the inherited
characteristics of biological populations over
successive generations.
Theory of evolution by natural selection,
which was formulated by Charles Darwin.
Population growth would lead to a "struggle for
existence" where favourable variations could
prevail as others perished. It is based three simple
facts:
1. Heritable variation exists within populations of
organisms.
2. Organisms produce more progeny than can survive.
3. These offspring vary in their ability to survive and
reproduce
However, he could not explain origin of variation
and how it is transmitted to next generation
He tried to explain heritability by Pangenesis
Inheritance of tiny heredity particles he called
gemmules that could be transmitted from parent
to offspring

Modern evolutionary synthesis
It is a union of ideas from several biological specialties
which provides a widely accepted account of evolution
All evolutionary phenomena can be explained with known
genetic mechanisms and the observational evidence of
naturalists.
the genetic diversity existing in natural populations is a key
factor in evolution
Evolution is gradual: small genetic changes regulated by
natural selection accumulate over long periods.
Natural selection is by far the main mechanism of change;
even slight advantages are important when continued. The
object of selection is the phenotype in its surrounding
environment.

Genome evolution or molecular evolution
Microevolution : Changes in allele frequencies
that occur over time within a population
Macroevolution : Evolution on a scale of
separated gene pools.

Macroevolutionary
studies focus on change that occurs at or
above the level of species.
How evolution leads to speciation?
Mutation leads to variation
Recombination leads to formation of new
combination
New combination leads to adaptation
Adaptation leads to selection
Selection leads to evolution
High level of adaptation in a specific environment
leads to reproductive isolation and speciation.

Speciation
A group of organisms that exchanges genes
within the group but cannot do so with other
groups is what is meant by a species
All the species now existing are related to
each other, having had a common ancestor at
some time in the evolutionary past.
Then how did they separate?
Allopatric speciation,
A population splits into two geographically
isolated populations by habitat fragmentation or
migration
The isolated populations then undergo genotypic
and/or phenotypic divergence as:
(a) They become subjected to dissimilar selective
pressures;
(b) They independently undergo genetic drift
(c) Different mutations arise in the two populations.
Peripatric speciation
In peripatric speciation a small population
moves away from parent population and
undergoes high level of inbreeding and
became distinct from the parental population
Parapatric speciation:
There is only partial separation of the zones of
two diverging populations afforded by geography;
individuals of each species may come in contact or
cross habitats from time to time, but reduced
fitness of the heterozygote leads to selection for
behaviours or mechanisms that prevent their
inter-breeding.
Sympatric speciation
It refers to the formation of two or more
descendant species from a single ancestral species
all occupying the same geographic location.
Divergence and speciation in Lake Malawi cichlids
Biological reasons of isolation
Prezygotic isolation occurs when there is failure to form
zygotes.
The cause of this failure may be that
the different species mate at different seasons or in different habitats.
the species are not sexually attractive to each other
their genitalia do not match or male gametes are physiologically
incompatable with the female.
Postzygotic isolation results from the failure of fertilized
zygotes to contribute gametes to future generations.
Hybrids may fail to develop or have a lower probability of
survival than that of the parental species or the hybrids may be
partly or completely sterile.
Origin of new genes
Genetic level evolution consists of formation
of new allele, formation new functions or
genes, increasing the expression of genes,
modification of metabolic activities etc. All
these functions involves formation of new
DNA.
Where does the DNA for new genes come
from?



Mutation
Polyploidy
Duplications
Imported DNA
CELLULAR ORGANELLES
HORIZONTAL TRANSFER

FISH
Infective particles

Model Organisms
A model organism is a non-human species
that is extensively studied to understand
particular biological phenomena
Its genetic mechanisms are common either to
all species or to a large group of related
organisms.
We need a variety of model organisms to
understand differences in general features of
inheritance and development.

VIRUSES : Bacteriophage
PROKARYOTES : Escherichia coli
EUKARYOTES :
Yeast: Saccharomyces cerevisiae
Filamentous fungi : Neurospora
Multicellular organisms : Arabidopsis thaliana,
Drosophila melanogaster, Caenorhabditis elegans,
Mus musculus



Selection of Model organism
Different basic biological properties,
Size of individuals
Short generation time
The ease with which they can be grown and
mated under simple controlled conditions.
Eg. For the study of vertebrate genetics, mice are
to be preferred to elephants.

Three types of model organisms
Genetic model organisms:
These are species that are amenable to genetic
analysis, i.e. they breed in large numbers and have
a short generation time so large-scale crosses can
be set up and followed over several generations.
Many different mutants are generally available
and highly detailed genetic maps can be created
Eg. Saccharomyces cerevisiae, Drosophila
melanogaster and Caenorhabditis elegans

Experimental model organisms:
These species may not necessarily be genetically
amenable (i.e. they may have long generation
intervals and poor genetic maps) but they have other
experimental advantages.
For example, the chicken and the African clawed frog
Xenopus laevis have many disadvantages in terms of
genetics but they produce robust embryos that can be
studied and manipulated with ease. These species are
widely used model organisms in developmental
biology.

Genomic model organisms
Regardless of their genetic or experimental advantages
and disadvantages, certain species are chosen as model
organisms because they occupy a pivotal position in the
evolutionary tree or because some quality of their
genome makes them ideal to study.
Eg. Mouse for studying immune system.
The mouse genome is similarly organised to the human
genome and large blocks of genes are even arranged in the
same order.
Mice have been extensively used to establish disease
models by mimicking the gene defects seen in humans,
and these models can be used to test the efficacy of new
drugs.
Escherichia coli
Escherichia coli was discovered by German pediatrician
and bacteriologist, Theodor Escherich in 1800s.
Due to its rapid growth rate (20 Min.)
Simple nutritional requirements
Well established genetics and completed genomic
sequence
Mostly used to study:
Bacterial genetics
Biochemistry
Bacterial physiology
Recombinant DNA technology
Saccharomyces cerevisiae
The yeast genome is just over 12 million base pairs in length and
contains about 6000 genes.
About 20 per cent of human disease genes have counterparts in
yeast
This suggests that such diseases result from the disruption of very
basic cellular processes, such as DNA repair, cell division or the
control of gene expression
It also means that yeast can be exploited to look at functional
relationships involving these genes, and to test new drugs.
A yeast mutant that has lost the functional equivalent of a human
disease gene can be screened with thousands of potential drugs in
order to identify compounds that restore normal function to the
yeast cell. These compounds, or molecules like them, might also be
useful in humans.
Mouse
The mouse is the most closely related model organism
The mouse and human genomes are approximately the same size,
contain the same number of genes and show extensive synteny
(conserved gene order).
Most human genes have mouse counterparts and the functions of
these genes are closely related.
Mutations that cause diseases in humans often cause similar
diseases in mice.
Importantly, mice have genes that are not represented in other
animal models including the genes of the immune system
Mice are small, easy to maintain in the laboratory and (compared
to most mammals) have a short breeding cycle (about 2-3 months).
They can produce 10-15 offspring per litter and approximately one
litter every month. This makes them suitable for genetic analysis.
Many mutants are available and new mutations can be introduced
easily by irradiation, feeding with chemical mutagens or inserting
DNA fragments into the genome to interrupt genes.
Inter species hybrids can be produced in mice, which are
heterozygous in nature.
They can be used for accurate mapping of genes
Advanced breeding strategies can be used to make
specialised strains such as congenics, which are genetically
identical with the exception of polymorphism for one
specific gene.
By gene transfer technology transgenic mice can be created
carrying any foreign gene of interest.
Also, the mouse is the only vertebrate species in which pre-
selected genes can be deliberately mutated in a precise
manner (knockout mice)
Another technological wonder is human-mouse hybrid
Drosophila melanogaster
The fruit fly Drosophila melanogaster has the longest
history of any model organism and has been widely used
to study genetics and developmental biology.
The fruit fly is small and has a simple diet. Therefore, large
numbers of flies can be maintained inexpensively in the
laboratory.
The life cycle is also very short, taking about two weeks, so
large-scale crosses can be set up and followed through
several generations in a matter of months.
Fruit flies also have large polytene chromosomes, whose
barcode patterns of light and dark bands allow genes to be
mapped accurately.
Mutants are available for a large number of
genes and new mutations can be induced very
easily by exposing flies to radiation or adding
mutagenic chemicals to their food.
This ability to recover mutants means that
flies can be used to investigate the genetic
basis of any conceivable biological process.

The nematode worm Caenorhabditis elegans
The nematode worm Caenorhabditis elegans is a very simple
animal that can be handled like a microbe but it shares many genes
and molecular pathways with humans
Caenorhabditis elegans is a soil-dwelling nematode worm about 1
mm in length that feeds on bacteria.
It was chosen because it is perhaps the simplest multicellular
organism in existence, containing fewer than 1000 somatic cells.
Despite this simplicity, C. elegans has a multitude of cell types and a
fully functioning nervous system.
Large numbers of worms can be maintained inexpensively on lawns
of bacteria growing on standard agar plates, but viable cultures can
be stored as frozen stocks and then revived when required.
C. elegans does not have male and female sexes.
Instead, there are males and hermaphrodites.
A single hermaphrodite can self-fertilise and
produce over 1000 eggs per day.
If crosses need to be carried out, hermaphrodites
can be mated with males.
The life cycle of C. elegans is two weeks long,
similar to that of the fruit fly, so it is very
amenable to genetic analysis.
C. elegans also has an impressive list of experimental
advantages.
The worm is transparent throughout its life. Therefore, the
behaviour of individual cells can be followed through
development and gene expression patterns can be
monitored very easily.
A unique feature of this animal is that development is
stereotypical, i.e. cells divide and specialise in a totally
characteristic way so that every normal worm has the same
number and type of cells arranged in the same manner
A complete wiring map of the 302 neurons that make up
the nervous system is also available
The C. elegans genome is 97 million base pairs in
length and contains about 20 000 genes
Many of these genes appear to have functional
counterparts in humans, and whole pathways are
often conserved.
This makes C. elegans a useful model for human
diseases.
For example, the insulin signalling pathway is fully
conserved between humans and nematodes so
mutant worms impaired for insulin signalling are
useful models of type II diabetes.

C. elegans mutants provide models of many
other diseases including neurological
disorders, congenital heart disease and
kidney disease.
Other important phenomenon studied using C
elegans is cell death and ageing.
Arabidopsis
Arabidopsis thaliana is a small flowering plant
that is widely used as a model organism in
plant biology especially used for studies of
the cellular and molecular biology of
flowering plants.
Approximately 115 Mb of the 125 Mb
genome has been sequenced and annotated
Extensive genetic and physical maps of all 5
chromosomes are available.
The life cycle is short--about 6 weeks from
germination to seed maturation.
Seed production is prolific and the plant is
easily cultivated in restricted space.
Transformation is efficient utilizing
Agrobacterium tumefaciens.
A large number of mutant lines and genomic
resources is available.