Genetics (2)

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• Recombinant DNA
• Patterns of
inheritance
• Biochemical genetics
• Population studies
• Human genome
project
• Cloning

The Beginning
• Gregor Mendel – Pisum sativum
– pairs of contrasting characteristics in the
garden pea.
– Tall or dwarf, yellow or green seeds, violet or
white flowers etc.

Early Genetics
• Carvings in stone – 6000 years old
• Haemophillia - 1500 years ago
• Regnier de Graaf -
– male and the female parent transmitted
genetic characteristics to the off spring.
• Pierre Louis Moreau de Maupertuis
-1700s
– hereditary particles
– one from each parent

TT tt
F1 Tt
F2 TT Tt Tt tt
F3 All Same as All
Dominant F2 Recessive
Mendel’s Experiments

Mendel’s Laws
• Mendel’s 1st law, or the Law of
Segregation
• 2 factors for a specific character
• parent transmits only one to offspring
• matter of chance as to which unite

• Punnet’s Square
Mendel’s Laws
Male Gametes
FemaleGametes
T t
T
t
TT Tt
Tt tt

• The law of Unit Inheritance – Before
Mendel’s time it was believed that the
characteristics of parents blended into the
offspring. (Darwin)
• Blending did not occur.
• Characteristics of one parent may not
appear in one generation (F1) but may
reappear in the next generation (F2).
Mendel’s Laws

• Law of Independent assortment –
Members of different gene pairs assort to
the gametes (sex cells) independently of
one another.
Tt
T t
• Random recombination
Mendel’s Laws

• Work of Mendel was not widely noticed –
• Darwin’s theory – based on inheritance
But mechanism not known
• Rediscovered –
– Vries - Holland,
– Correns - Germany and
– Tschermak - Austria
Mendel’s Laws

• Homozygous – an individual who has the same factors
for a particular characteristic. (eg-TT or yy)
• Heterozygous – individual with different factors (Tt or Yy)
– character that is manifested - dominant,
– and the other - recessive .
• The term gene - Danish botanist Johannsen, represents
the hereditary factors.
• The genes responsible for contrasting characters are
called alleles.
Terminology

Human Genetics
• Family Studies – pedigree
• Albinism, Polydactyly (early 1700s) and
Hemophilia (early 1800s).
Consanguineous marriages.
• Effects of Nature and Nurture 1800s by
Galton
– Hereditary improvement of men and animals
by selective breeding – eugenics.

• 1900s Sir Garrod
• Alkaptonuria - dark urine
• Children were usually normal, but the disorder
could reappear later in the descendents.
• Mendelean recessive type of inheritance
• Excretion of homogentisic acid which is usually
metabolized in normal individuals.
Human Genetics

Human Genetics
• Connection between gene and enzyme
• This was the first time that the idea that
genes control the synthesis of enzymes
arose.
• Landsteinter  ABO blood groups
Blood group genetics

Chromosome Theory of Inheritance
• Thread like structures in
nucleus – the filaments of life.
• Chromosomes
– Chroma=colour
• Walter Sutton
• Boveri
• 1903

Chromosome Theory of Inheritance
• Interaction between these
chromosomes that lead to the
phenomenon of inheritance.
• They behaved just like
Mendel’s factors – occur in
pairs and come one from
each parent.

Nucleic Acid
• First isolated as early as 1869
by a Swiss doctor named
Meicher.
• Rich in phosphorous
• Nuclein
• 1892 – postulated that it is
the hereditary material.

Nucleic Acid
• Protein was the basic
substance of life
• Importance in protein
formation was not
appreciated until the work of
Griffith and later Avery,
Macleod and McCarthy (on
pneumococci) and Hershey
and Chase (using
bacteriophages).

Nucleic Acid - Structure
• long chains of molecules
called nucleotides
• Each nucleotide is composed
of :-
– A nitrogenous base
– A sugar molecule, and
– A phosphate molecule

• The nitrogenous bases are of
2 types – purines &
pyrimidines.
• The purines include –
adenine and guanine
• The pyrimidines include –
cytosine, thymine and uracil.

• Nucleic acids – 2 types (according
to sugar)
• Ribose  Ribonucleic acid or RNA
- nucleolus and cytoplasm
• Deoxyribose  Deoxyribonucleic
acid or DNA – nucleus

Structure of DNA
Requirements –
1. Versatile.
2. Produce identical replica is
formed at each cell division.

Structure of DNA

• Purine ↔ Pyrimidine
• Guanine ↔ cytosine
• Adenine ↔thymine
Structure of DNA

Replication
• Semi-conservative
method.
• Complementary chain
is formed
• Daughter cell
– one parent strand
– one new strand

Genetic Code
• Genes code the sequence of the amino
acids
• 4 bases  20 amino acids
• 3 bases are essential for coding the amino
acids.
• Triplet code = codon

Genetic Code

Transcription
• Information from DNA
 messenger –
RNA
• Complementary
bases are found in
the RNA.
– Cytosine with guanine,
– thymine with adenine,
and
– adenine with uracil

Translation
• mRNA associates
with ribosomes
• Proteins formed
around mRNA
template
• Amino acids collected
from cytoplasm
(AA + ATP) ↔ tRNA 
Ribosome

“Central dogma”

A Closer Look at Genes
• But about 80% of human
DNA does not code for
proteins.
• The coding part of the
DNA is known as exons,
and the intervening non-
coding sequences are
called introns.

• These genes have a regulatory effect.
• In prokaryots (without nucleus)
– Operon = operator gene and structural gene
– Remote regulator gene
• Produces repressor.
– Repressor can be inactivated by inducer.

• In humans – certain flanking regions
referred to as enhancers and promoters.
• Structure of Globin gene -
– CAT box = CCAAT
– TATA box = TATA
– End flanking region = AATAAA

• Exon-intron pattern of a particular gene appears
to be conserved during evolution
• Two introns at precisely the same locations
since their appearance 500 million years ago
• Alterations in exons are slow, and mutations are
rarely retained
• Changes in the introns occur much more rapidly

• There are several meters of DNA in a
human body, and the total length of the
chromosomes is less than a millimeter.
• Finch and Klug suggested the “Solenoid
model”
Structure of Chromosomes

• Each DNA duplex is coiled around itself – primary coiling
• This is couled around histone ‘beads’ – secondary coiling
– called nucleosomes
• Nucleosomes are coiled to form chromatin fibres, around
a protein matrix or scaffold – tertiary coiling
• Chromatin fibres are coiled to form loops – quaternary
coiling
• The loops are further wound in a tight helix to form the
chromosome – that can be seen under a microscope.

Human Chromosomes
• 46 chromosomes in the normal human – 23 pairs.
– 22 pairs are alike in males and females – known as autosomes
– 1 pair differs – the sex chromosomes.
• Pair of chromosomes are microscopically
indisdtinguishable, except the x and y chromosomes.
• Y is smaller than x, but the 2 are thought to have a
homologous short segment.

Studying Chromosomes
• cells to be studied must be able to grow
and divide rapidly.
• WBC cultures - usually short lived
• skin cultures – for biochemical and
histochemical studies

Cells
Culture
phytohemagglutinin
(mitogenic agent)
colchicine
Metaphase
hypotonic solution
Staining
Cells rapidly dividing

Chromosome Spread

•1960 - Denver
classification
•7 chromosome
groups (A through
G) based on length
and centromere
position.

Paris Classification

• The location of the centromere can be
used to classify the chromosomes –
– Metacentric – central centromere
– Submetacentric - off –centre
– Acrocentric – towards one end
– Telocentric – terminal centromere (does not
occur in man)

• Acrocentric - small masses of chromatin known
as satellites attached to their short arms by
narrow stalks (secondary constrictions)
• Stalks contain the genes for 18S and 28S
ribosomal RNA (rRNA).
• The rRNA transcribed from these areas, along
with 5S rRNA (from another location), is utilized
in the nucleolus to synthesize ribosomes.

• Autoradiography
• radioactive thymidine
• cell divisions are stopped
• not all chromosomes replicate at the same
time. But this process is laborious and
time consuming, and is rarely used.

• After 1970
• Q banding - quinacrine mustard or related
compounds
– fluroscence microscopy.
• G banding – widely used
– tripsin to denature the protein
– Giemsa stain
– dark bands correspond to the bright Q bands
Studying Chromosomes-
Staining

• R banding
– less widely used
– heat treated
– then stained with Giemsa
– results are the REVERSE of G banding
• C banding – centromere
– regions of the chromosome containing constrictive
heterochromatin
– secondary constrictions of chromosomes 1, 9, 16
Staining

• NOR staining –
– ammoniacal silver to stain the nucleolus
• High resolution banding -– used for
staining cells in prophase – shows much
more bands than the metaphase staining.
Staining

• Clinical diagnosis – congenital
malformations, mental retardation
disorders of sexual development etc.
• Linkage and Mapping – Assignment of
specific human genes to their
chromosomal positions.
Studying Chromosomes- Medical
applications

• Polymorphisms – Minor heritable
differences in chromosomes are
common, especially in chromosomes 1,
9, and 16 and the Y chromosome.
• Highly specific  genetic marker
applications

• Chromosomes and Neoplasia –
Philadelphia chromosome
• Reproductive problems
• Prenatal Diagnosis – Amniocentesis
– Useful in older pregnant women, and families
with a history of chromosomal abnormalities.
applications

• Nuclear material is conserved in the
daughter cell
• Cytoplasm seem to split
• Nuclear division – 4 stages
– Prophase
– Metaphase
– Anaphase
– Telophase
Mitosis

Mitosis
• Interphase -
chromosome divides
longitudinally into 2
daughter chromosomes,
or chromatids, which
remain attached to each
other at the centromere.
Interphase
G1
S
G2
M
G1
S
G2 M

Mitosis
• Prophase
– chromosomes can be seen
and easily discerned
– chromatids can be seen
– centriole,
– each one migrates to the
opposite pole of the cell
– nuclear membrane
disappears and the
nucleus begins to loose its
identity.

Mitosis
• Metaphase
– chromosomes are
maximally contracted and
deeply staining
– 2 dimensional metaphase
plate
– spindle is now formed
– (microtubles of protein)
– spindle fibres centrioles to
kinetochores (sites of
attachment at the
centromere)

Mitosis
• Anaphase
– centromeres divide
– spindles contract
– actin-myosin
interactions?
• Telophase
– daughter
chromosomes arrive at
the poles
– Cytokinesis
– chromosomes unwind
– nuclear membrane

• 2 phases –
– Meiosis I – the
reduction division
– Meiosis II – an
ordinary mitosis,
without DNA
replication
Meiosis

• Leptotene
– chromosomes begin
to condense
– consist of alternating
thick and thin
regions –
(chromomeres), -
characteristic for
each chromosome.
Meiosis I - Prophase

• Zygotene
– pairing (synapsis)
– bivalents
• Pachytene
– chromomeres become
more prominent
– bivalent
– actually a tetrad
– crossing over occurs

• Diplotene –
– Two components of the
bivalent begin to
separate
– Centromere of each
chromosome remains
intact
– Chromatids seem to be
contact at several
places, called chiasmata
• Diakinesis – more
condensation

Meiosis I
• Metaphase I –
– the nuclear membrane
disappears, and the
chromosomes move to
the equatorial plane.
• Anaphase I
– 2 members of the
bivalent disjoin, and
one member goes to
each pole

Meiosis II
• Resembles mitosis,
but without DNA
replication
• Without an interphase
• Centromeres divide,
and the sister
chromatids disjoin,
passing to opposite
poles and produce 2
daughter cells.

Crossing Over
• Reorganization of genes among the
chromosomes  hence increases genetic
variability.
• Chiasmata  sites of cross over
• 2 chromatids take part in any crossover. But all
4 chromatids of the bivalent may be
simultaneously involved in crossovers at
different sites.

Crossing Over
• Crossing over in
mitosis is much less
common
• Important effect in
case of heterozygous
cells

Crossing Over
• Sometimes crossing over can also occur
between the sister chromatids.
• Bloom Syndrome
– growth retardation, prenatally and postnatally,
and a butterfly rash is seen on the face
• But the correlation of these findings to the
sister chromatid exchange is unknown.

Recombinant DNA Technology
• Genetic Engineering
• Portion of DNA cut  introduced into another host
 cultured
• Discovery on restriction endonucleases (Smith in
1970).
• A restriction endonulease is an enzyme that cuts
DNA at specific sequences, called restriction
sites. Over 200 such enzymes are known.

• Principles of the technique
– Use of the restriction enzyme to cut away a DNA
fragment which includes certain gene/genes.
– Incorporation of these fragments into a carrier or
vector.
– Transformation of a host organism eg-E. coli by the
vector.
– Culturing of the host organism in a suitable medium.
– Selection of the bacteria containing the relavent DNA
fragment.

• Cleavage of the DNA
is done with the
restriction
endonucleases.

• DNA fragment  vector.
– Phage, or a plasmid
(circular mass of DNA in
bacteria, which replicate
independent of the main
bacterial chromosomes).
• The vector DNA is usually
cleaved with the same
enzyme as the DNA
fragment, and the
complementary base
pairs that result are
combined together.

• Bactriophages  larger fragments of DNA,
• cosmids – which are a combination of
plasmids and phages  Even larger
fragments

• Vector is incorporated
into a host bacteria
(usually E. coli).
• Bacteria is cultured

• Southern Blot
Technique

Recombinant DNA Technology -
Applications
• Analysis of gene structure
• Restriction fragment length polymorphisms (RFLPs)
– small changes in the nucleotide sequences without phenotypic
effects (small deletions or insertions)
• length of the fragments generated by a particular
restriction enzyme will be different,
• inherited as Mendelian characters
– As genetic markers – which can be used to study genetic
structure – similar to blood groups and serum proteins.
– In the prenatal diagnosis of any genetic disorder which is linked
to an RFLP, or to identify carriers of the disorder.

• In detecting the products of various
genes
• In producing probes to diagnose viral
diseases like Hepatitis B or HIV
Applications

• Biosynthesis of gene products
– no antigenic properties, and free from contamination
by HIV etc.
• Gene therapy
– The missing gene is created and introduced into the
host
– single gene disorders
• most genetic problems are multifactorial, this
seems to have only limited application.
Therapeutic Applications

Genetics – Part II
Dr. Punit Thawani

Mutations
• Muller – Mutation of fruit flies by X-rays
• 3 types
– Substitution
– Deletion
– Insertion

Mutations
• Single Base Substitutions
– alter the triplet codon
– one amino acid to be replaced
• Deletions and insersions
– if a single base pair is deleted or inserted, the
entire frame of the DNA strand gets shifted
• Single base substitutions – proteins
produced
• Frame shift mutations – no proteins

Mutations
• Chain termination mutations –
– Termination codons can be added
prematurely or be deleted
• Splice Mutations –
– These interfere with the way
introns are removed from the
messenger RNA.
• Mutations in regulatory
sequences
– These affect the TATA box and the
CAT box regions of the gene.

Inheritance
• Diseases in families – The family history
• Environmental factors play a role –
Multifactorial disorders

• Single Gene disorders [1 in 2000 or less]
• Chromosome disorders [7 in 1000]
• Multifactorial disorders
Inheritance

• Indications that a condition has a genetic
etiology (Neel and Schull 1954)
1. Occurrence of a disease in definite
proportions in families when environmental
factors can be ruled out.
2. Absence of disease in unrelated lines
3. Characteristic age of onset, absence of
precipitating factors
Inheritance

4. More in monozygotic than dizygotic twins.
5. Demonstration of characteristic phenotype and
chromosomal abnormality, with or without family
history.
Inheritance

Dominant
Autosomal
Recessive
Dominant
Sex-linked
(Gonosomal) Recessive
Inheritance – Single gene
disorders

• Autosomal dominant
– Rare
– Patient usually heterozygous
– Parent affected
– New mutation
– Achondroplasia, Osteogenesis imperfecta
– Porphyria variegata – one couple (1688)
– ½ the children affected – irrespective of sex
disorders

• Experssivity – polydactyly
• Non – penetrance
• Sex influence
– Gout
– Presenile baldness
– “Eunuchs neither get gout nor grow bald”
Hippocrates
• Viral etiology? – Alzheimer’s disease.
disorders

• Autosomal Recessive inheritance
– Both sexes – homozygous
– Extremely rare – heterozygous are normal
disorders
A a
A
a
AA Aa
Aa aa

• Consanguineous marriages
– Chance that cousins will carry the same
genes is 1 in 8
– The rarer the disease  more probability of
consanguineous marriage of parents.
–Eg – Alkaptonuria
• Most common autosomal recessive
disorder – Cystic fibrosis – 1 in 22 is a
carrier.
disorders

• Intermediate inheritance
– Sickle cell trait – recessive/dominant?
• Codominance
– Blood group AB
disorders

• Sex-Linked inheritance
– X Linked
– Y Linked
• Female must be homozygous
• Male is hemizygous (since only 1 X chr.)
• Eg – Haemophillia.
disorders

• Females with hemophillia
– XO (Turner’s syndrome)
– Homozygous
– Mutation during gametogenesis
– Manifesting heterozygote.
disorders

• X linked dominant
– Similar to autosomal dominant
– Male transmits disease to all daughters but
none of his sons – Vit. D resistant Rickets
disorders
X Y
X
X
XX XY
XX XY
Xh Y
X
X
XXh XY
XXh XY

• Y linked inheritance
– Hairy pinna
– Transmitted from father to all his sons
– Females not affected
disorders

• Establishing modes on inheritance
• Autosomal dominant – Verical pattern
• Autosomal recessive – Horizontal
• Sex linked – Oblique if male does not
produce offspring
disorders

• Multiple alleles
– Some characters may have more than one
allele
– Blood group genes
• A1 A2 B O
– Any one may be transmitted to the offspring.
disorders

Chromosomal Abnormalities
Autosomes Numerical
Sex Chromosomes Structural
Inheritance – Chromosomal
Abnormalities

• Numerical abnormalities
– Aneuploidy
– Polyploidy
• Monosomy
– Lethal – one infant with monosomy 21
• Trisomy – Non-disjunction
– Lejeune (1959) – Down’s syndrome
– Patau’s syndrome – trisomy 13
– Edward’s syndrome – trisomy 18
Abnormalities

• Structural abnormalities –
– Translocations – exchange of segments
– Deletion – loss of a segment.
• Translocation
Abnormalities

• Gametes –
(14/21) 14 21
Abnormalities
14/21 14 21
14/21 21
14/21
14 21
14
Down’s syndrome
Carrier
Normal
Monosomy - death

• Reciprocal translocation
• Person is normal
• Gametes produced are abnormal
Abnormalities

• Deletions
– Results in partial monosomy
• Lejeune – ‘cri du chat’
• Deletion of short arm of chromosome 5
• Formation of ring chromosomes
Abnormalities

• Sex chromosome abnormalities
– Kleinfelter’s syndrome – XXY
– Turner’s syndrome – XO
– Multiple X
– XYY males
• Structural abnormalities
– Isochromosome – long X
Abnormalities

• More than one gene involved + environment
• Familial tendency
– Incidence in family more than in general population
– Less common than unifactorial disorders
• Normal traits – intelligence, skin colour, blood
pressure, etc.
• Abnormal traits – schizophrenia, diabetes, peptic
ulcer, ischemic heart disease, ankylosing
spondylitis etc.
Inheritance – Multifactorial

• Relatives generally have higher incidence
than general population

• The incidence is greater among relatives
of individuals with more severe form of the
disease
– Pt. with bilateral cleft lip – 6%
– Pt. with unilateral cleft lip – 2.5%
• Similarly, subsequent children have more
chance of being affected.

• Heritability – proportion of the total
variation of a character which can be
attributed to genetic factors.
– Greater the heritability, greater the genetic
component.

Homeobox Genes
• Development of a
head-tail axis.
• According to their
location – cells
differentiate – under
the regulatory effect
of homeotic or
homeobox genes.

Homeobox Genes
• These genes contain a specific 180 base
pair region called the homeobox.
• Produces proteins – transcription factors –
bind to DNA and regulate its expression.
• First experiments on Drosophilla
melanogaster later found in vertebrates.

Homeobox Genes
• Hox genes form “Hox
codes” which specify
the position of cells.
• Anterior to posterior
arrangement

Homeobox Genes

Homeobox Genes
• In mice – disruption of Hox a-2
– Lack of derivatives of 2nd
branchial arch
– Some 2nd
arch structures changed to 1st
arch
structures
• Stapes missing – 2 malleus’
• Ectopic expression of Hox d-4
– Occipital bones to vertebrae
• Deletion of Hox a-3 and Hox d-3
– Atlas is deleted

Homeobox Genes
• Each branchial arch exhibits a specific
combination of Hox gene expression
• No Hox genes been detected in the brain

Homeobox Genes
• Msx -1  development of secondary
palate and tooth.
– familial tooth agenesis –
missing 2nd
premolar and 3rd
molar
• Studies in Finnish families
(Nieminen et al 1995).
• Msx- 2  Craniosynostosis

Cloning
• Cloning is the process of making a
genetically identical organism through
nonsexual means.
• First animal cloned in 1997, at the Roslin
Institute in Scotland

Cloning

Cloning
• Proposed benefits of cloning
– Use of clones as donors – cloning only organs
– Saving endangered species –Noah the gaur
– Cloning of stem cells
• Claims of first cloned baby born on 26 Dec
2002.

Human Genome Project
• Begun formally in 1990,
the U.S. Human Genome
Project is a 13-year effort
coordinated by the U.S.
Department of Energy
and the National
Institutes of Health
• Expected completion date
–sometime in 2003

• Identify all human
genes
• Determine the entire
base pair sequence
• Store the information
in databases

• Study of various non
human organisms –
E.Coli, fruit fly, mice

• Molecular Medicine
– Knowledge of which
genes cause which
disorders
– Based on the causes,
specific treatments
can be devised.

• Microbial Genomics
– Use of bacteria useful in
energy production (like
photosynthesis), toxic
waste reduction, and
industrial processing.
– Better understanding of
micro-organisms and
hence development of new
drugs

• Risk Assessment
– Genetic basis to
variable response
to toxins, x-rays,
cigarette smoke,
and susceptibility to
cancer.

• Anthropology and
Evolution
– By studying mutations
and variations in
genes
• DNA forensics
• Agriculture, animal
breeding etc.

Genetics (2)

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