GENETICS I

Dr. Monali Sonawane

Associate Professor
Department of Anatomy
ZMCH,Dahod
Learning Objectives,

At the end of lecture students should able to,

• Describe the structure of chromosomes with classification

• Describe technique of karyotyping with its applications

• Describe the Lyon's hypothesis

In resting stage cells’s nuclear material (chromatin) has a homogeneous appearance

When a cell divides, nuclear material (chromatin) condenses to appear as a number of rod –
shaped organelles - chromosome

Individual chromosome are visualized under microscope only during cell division
Chromosome act as a carrier unit of
inheritance in the form of gene of nuclear
DNA

Gene are borne by chromosome in linear

series as parts of specific DNA Molecule

Number of chromosome: in each cell is fixed

for a particular species
• Human beings: 46 (2n) arranged in 23 pairs
• In Spermatozoa / Ova: 23 (n)

One member of each chromosome is

derived from the mother & other from the
father
Types:
• 22 out of 23 pairs are identical and are known as autosomes

• Chromosome in the 23rd pair is known as sex chromosome

• In female both sex chromosome are identical and are called X- chromosome In Male 2 sex
chromosome are not identical and are called X & Y- chromome

• Female germ cell always has X chromosome where as Male germ cell may either have X or Y
chromosome
 Structure:
• Chromosome is made up of two rod shaped structure – chromatids, which are identical &
lie parallel to each
other.
• Two chromatids are united with each other at pale staining area : Centromere ( Primary
constriction)
• Centromere divides each chromatid into two arms
• Centromere is associated with formation of spindles and chromosomal movement during
cell division
• Free end of chromatids are known as Telomeres, which when intact do not permit fusion
with adjacent cromosomes
• In certain chromosome, secondary constriction exists near one end of
chromatid Part of chromatid beyond sencondary constriction looks like
satellites.
• Chromosomes with satellite bodies are known as SAT- chromosomes.
• Secondary constriction are concerned with formation of nucleoli, so
they are also known as Nucleolar Organizers
Chemical composition of chromosome:

Deoxyribonuclic acid (DNA): Most essential & stable molecular

constituent of chromosomes. It is made up of deoxyribose sugar
molecule and nucleotides. Each chromosome contains a single
continuous double –stranded DNA molecule

Ribose nuclic acid (RNA) : Single stranded structure having ribose

as a sugar molecule

Histones: are the protein rich in arginine & lysine. They are
aggregated along the DNA strand, Which is coiled around each
particle to form a complex body known as nucleosomes having 4
histones

Acidic proteins: are nonhistone proteins & form many enzymes

e.g. DNA polymerase & RNA Polymerase
 Classification of chrmosome:

On basis of :
 Position of centromere
 Numbr of centromere
 According to Denver system

 Depending on function:
Autosomes: Thre are 22 pair of autosomes-
responsible for determination of body parts &
their functions
Sex chromosomes: There is one pair of sex
chromosome in each sex. In male it is XX and
in female it is XY
 According to position of centromere:

1. Metacentric: Chromosome with a centromere

located in middle of chromosome ; Two arm are
almost equal

2. Submetacentric: Chromosome with a centromere

located slightly away form midpoint; Two arm are
unequal

3. Acrocentric: centromere occupying subterminal

position; one are is very long & other is short

4. Telocentric: centromere at its Terminal position;

Each chromatid therefore has only one arm
 According to number of centromere:
1. Monocentric: Having one centromere only, which
is usual and normal

2. Dicentric: Having two centromere, which is found

in some species of wheat

3. Polycentric: Having more than two centromere

seen in some forms of roundworm

4. A-centric: Represents only a fragment of

chromosome having no centromere. It is not
visible
 SEX CHROMATIN

 1949 Barr and Bertram- cat neurons

 Seen only in females not in males – sex chromatin or
Barr body
 Chromatin positive & Chromatin negative
 Used as a diagnostic tool to confirm the disorders of
sexual development
 No. of Barr bodies = no of X chromosomes -1
 46,XX - 1 barr body
 46XY - negative
 47XXX, barr bodies-2
 45XO - negative
Barr body represents one of two X chromosomes of a female
cell.
This remains condensed and is in inactive state throughout
interphase.
Its replication is also late as compared to its homologue.
 SEX CHROMATIN STUDY

 Scrapping from the inner side of the cheek

is taken on a slide and smeared evenly.
 Subsequently it is fixed in alcohol and
stained with thionine.
 It is then mounted in neutral medium and
observed under a microscope.
BARR BODY
KARYOTYPING
Karyotype: is a complete set of chromosome in terms of number of chromosomes &
morphology of chromosomes of an individual

Karyogram( karyotype/ idiogram): Photomicrograph of chromosomes of an individual arranged

in a standard manner/ Standard classification
Karyotyping: is the process of pairing & arranging all the chromosomes in a
standard manner of an individual & providing a hotomicrograph of an individual's
chromosomes

: is a process by which karyotype is obtained.

 Indications
1. Women with amenorrhoea & couples with Infertility or habitual abortion

2. Pregnancy in a elderly woman (fetal chromosome analysis)

3. Still births & Neonatal deaths

,
4. Problems in early growth- Developmental delay, Dysmorphic facies

5. First degree relatives of a known or suspected case of chromosome abnormality

6. Neoplasia
 Selection of tissue for Karyotyping:
Any cell capable of growth & rapid cell division in a tissue culture medium is suitable
for karyotyping.

A. T-lymphpcytes in peripheral blood- most commonly used

B. Skin Fibroblasts (from skin biopsy

C. Bone marrow cells

D. Fetal blood

E. Amniotic fluid cells

For Prenatal diagnosis
F. chorionic villi
 Procedure

1. Collection of peripheral venous blood in strictly aseptic condition in heparinized vaccute

2. Planting: should be done within 4-5 hours of collection Blood
sample is transferred to the culture tube containing:
a.Culture Media: RPMI( Roswell Park Memorial Institute), TC 199 etc.
b.Neonatal calf serum/ fetal bovine serum: to nourish the culture
cells
c.Phytoheamaglutinin : Mitotic aget – to increase the rate of mitosis
of cultured cells
d.Antibiotics: Penicilllin & streptomycin combination – to prevent
bacterial growth
3. Culture tube is Incubated at 37 degree C for 3 days – during this period lymphocytes
undergo mitosis

4. Harvasting: 69-72 hours after planting, Colchicin is added to culture tube to arrest
mitosis at metaphase by preventing the formation of spindle tubules.
5.After 2 hours cells are collected by Centrifugation (for separating the cells)

6.After discarding the supernatant fluid, Cell pellate are treated with Hypotonic solution
{sodium citrate /( 0.56% KCL)} and incubated for 20 min so that the cells swells &
chromosomes are dispersed

7.Hypotonic solution is discarded by centrifugation.

7. Cell pellate is fixed with methanol and acetic acid mixture and then the test tube is shaken
9. Cell suspension is dropped form a height on a clean slide, dried at room temperatue or spirit lamp flame &
then stained
10.Staining: For chromosome analysis various banding technique are vailabe:
G(giemsa)-banding, Q (quinacrine)-banding, R(reverse)-banding, C(centromeric heterochromatin) -banding &
NOR Banding
G- banding:
slides with chromosome prepration are first treated with solution of trypsin (Trypsin denatures the
chromosome protein) Slides are then treated with Giemsa solution
Chromosome shows dark and light band that can be observed under microscope
 Banding pattern helps to identify individual chromosomes
11. Micoscopy: Stained slide are examined under the oil immersion lens and Photomicrograph is taken
13. From the Enlarged photographs individual members of each homologous pair are matched and placed side
by side.
22 autosomes & a pair of sex chromosome are identified and arranged in pairs using
following parameters: Shape of chromosome
Length of chromosome

𝑆ℎ𝑜𝑟𝑡 𝑎 𝑟 𝑚
Centromeric Index =
𝑙𝑒𝑛𝑡ℎ
𝑇𝑜𝑡𝑎𝑙 𝑐ℎ𝑟𝑜𝑚𝑜𝑠𝑜𝑚𝑒
𝑙𝑒𝑛𝑔𝑡ℎ
G (giemsa) banding: Chromosomes are treated with trypsin( denatures their protein content) &
then Stained with a DNA-binding dye known as Giemsa, which gives each chromosome a
characteristic and reproducible pattern of light and dark bands.

Q (quinacrine) banding: Gives a banding pattern similar to that obtained with Giemsa, But
requires examination of chromosomes with an ultraviolet fluorescent microscope.

R (reverse) banding: Chromosomes are heat-denatured before staining with Giemsa, yielding
light & dark bands which are the reverse of those obtained using conventional G banding .

C (centromeric heterochromatin) banding: Chromosomes are pretreated with acid followed by

alkali prior to G banding, centromeres & other heterochromatic regions containing
highly repetitive DNA sequences are preferentially stained.
Symboles used in describing karyotype: {According to International system of Human cytogenetic Nomenclature (ISCN) 1985}
1. A-G: Chromosome groups
2. 1-22: Autosome numbers
3. X,Y: Sex Chromosome
4. p(= petite): Short arm of chromosome
5. Q ('g' = grande): Long arm of chromosome
6. mat: Maternal origin
7. pat: paternal origin
8. t: translocation
9. rob: robertsonian translocation
10. rep: reciprocal
11. r: ring chromomsome
12. i: Iso chromosoe
13. del: Deletion
14. dup: Duplication
15. inv: Inversion
16. fra: Fragile site
17. ter: terminal end of chromome
18. + or - : Befor a chromosome: indicate gain or loss of that chromosome. Eg:47, xx+21; means female with trisomy
21;Down’s syndrome
After a chromosome: indicate gain or loss of part of that chromosome , e.g. 46, XY, 5p-; means male with 46
chromosome but having deletion of short arm of chromosome 5 ( cri-du – chat syndrome)
 According to Denver System:

In 1960 at conference of genetics at Denver, human chromosome including sex

chromosome were arranged into 7 groups form I to VII depending upon size of
chromosome – Denver System

According to modern convention, chromosome are subdivided into Group A to Group G

depending upon position of centromere-patau’s modification of Denver method
 Long
Group A
metacentric
Chromosome Nos. 1,2,3
ChromosomeNo 2 is
submetacentric

Group B Fairly long

Chromosome Nos. 4,5 Submetacentric

Group C Medium Long

Chromosome Nos. 6 to Submetacentric
12 & X
Group D Medium Long
Chromosome Nos. Acrocentric ;
13,14,15
Sat body +

Group E Fairly Short

Chromosome Nos. Submetacentric
16,17,18

Group F Short metacentric

Chromosome Nos.
19,20

Group G Very Short acrocentric

Chromosome Nos.
21,22 Sat body +
# Group A & F are Metacentric
# Group D & G are Acrocentric
# Other groups are
Submetacentric
# 5 chromosomes ( 13,14, 15,
21 & 22) (D & G )posses
satellite bodies – sat
chromosomes – concerned
 APPLICATIONS OF KARYOTYPING

1.Clinical Diagnosis –
 Suspected
or clinically obvious chromosomal syndromes such as Down syndrome, Klinfelter
syndrome and Turner syndrome.
 Unexplained mental retardation with or without malformations.
 Proportionate short stature in a prepubertal female.
 Abnormal sexual development and differentiation
- Ambiguous genitalia
- Delayed or incomplete pubertal development in males and females.
- Oligospermia or Azoospermia
- Primary Amenorrhoea and Secondary Amenorrhoea
 Infertility
 Recurrent spontaneous abortions
 Unexplained still birth
2.Gene Mapping -
Proper localization of human genes to their specific position on chromosome.

3. Role in cancer –
Neoplastic conditions, particularly heamatological malignancies .
E.g – CML, Philadelphia chromosome
The abl (Abelson leukemia virus) proto-ancogen from chr 9 is juxtaposed to the bcr
(break point cluster region) on chr 22.

4. Prenatal diagnosis –
Pregnancy at risk of chromosomal abnormality.
 Parents of a child with a structural chromosomal anomaly.
Siblings and parents of a person with balanced structural rearrangement.
Positive triple or quadruple test.
 LYON’S HYPOTHESIS

 Dr Mary Lyon -1962 – Inactivation of X chromosome.

 At prophase, one X chromosome is late replicating and
heteropyknotic.
 Of the two X Chromosomes only one is active in cellular metabolism
while the other forms sex chromatin.
 In males there is only one X chromosome, which is active and hence
they do not show Barr body
Lyon’s hypothesis states that:
1.In female somatic cells, only one X chromosome is active. The
second is inactive, condensed and appears in the form of sex
chromatin in interphase.

2. Inactivation occurs early in embryonic life.

3.Inactivation is random but fixed.The inactive X can be maternal or

paternal in different cells of the same individual.
 Sex chromatin is detected in blastocyst at 9-12 days.
 First it is detectable in syncytiotrophoblast, then in chorionic
mesoderm followed by yolk sac.
 In embryo proper, it is detected after 18th day.
MECHANISM OF X INACTIVATION

 It involves DNA methylation

 Cytosine – 5 methyl cytosine at certain sites in
DNA.
 DNA methylation is responsible for inactivtion
of genes.
 Inactivation centre
 INACTIVATION CENTRE
 Proximal part of long arm of X chromosome around which barr body
condenses.
 An abnormal X lacking proximal part of Xq has not been observed to
form barr body.
 An abnormal X with dupliction of this part of X chromosome, forms
bipartite barr body.
GENETIC SIGNIFICANCE OF X INACTIVATION

 It is three fold
1. Dosage compensation
2.Variability of expression
3.Mosaicism
Dosage compensation
The X chromosome inactivation explains why the X-linked gene product is equivalent in both
sexes in spite of two X chromosome in female and only one in male.

Variability of Expression
 As inactivation is random, female heterozygotes for X-linked genes present a considerable
phenotypic variation.
 Variation in expression of X-linked disorders can be ranging from completely normal to full
expression of the defect.
 A carrier who exhibits an X linked trait is called manifesting heterozygote.
 e.g- colour blindness, heamophilia,Duchenne muscular dystrophy.
Mosaicism
 Females are mosaics in respect to X chromosome.
THANK YOU