basic genetics and methods of karyotyping.

1. Dr. R.A. Siddique
M.V by Dr D N MISHRA Asst Prof
Anatomy SCB Medical College
Cuttack..Sc PhD Scholar
National Dairy Research Institute
Karnal, (Haryana) 132001
India

2. HISTORY
• Chromosomes were first described by
Strausberger in 1875.
• The term “Chromosome”, however was
first used by Waldeyer in 1888.
• They were given the name chromosome
(Chromo = colour; Soma = body) due to
their marked affinity for basic dyes.
• Their number can be counted easily only
during mitotic metaphase.

3. Chromosomes
• Normal cells of humans contain 46chromosomes;
• occurring in pairs and numbered from
1 (the largest) to 22 (the smallest);
• The chromosomes may be divided into two major
types: the 44 autosomes and the 2 sex
chromosomes.
• Autosomes are indistinguishable in males and
females.
• Males have an X and a Y chromosome, and females
have two X chromosomes.

4. What are chromosomes?
Chromosomes are the
rod-shaped, filamentous
bodies present in the
nucleus, which become
visible during cell
division.
They are the carriers of
the gene or unit of
heredity.
Chromosome are not
visible in active nucleus
due to their high water
content,but are clearly
seen during cell Division.

5. Contd…..
• Chromosomes are the structures that contain the
genetic material
– They are complexes of DNA and proteins
• The genome comprises all the genetic material that an
organism possesses
• The main function of the genetic material is to store
information required to produce an organism
• DNA sequences are necessary for
– 1. Synthesis of RNA and cellular proteins
– 2. Proper segregation of chromosomes
– 3. Replication of chromosomes
– 4. Compaction of chromosomes
• So they can fit within living cells

6. Number of chromosomes
Normally, all the individuals of a species
have the same number of
chromosomes. Presence of a whole sets
of chromosomes is called euploidy.
It includes haploids(23), diploids(46),
triploids(69), tetraploids(92) etc.
Gametes normally contain only one set of
chromosome is called - n: Haploid
Somatic cells usually contain two sets of
chromosome - 2n : Diploid

7. Organism No. chromosomes
Human 46
Chimpanzee 48
Dog 78
Horse 64
Chicken 78
Goldfish 94
Fruit fly 8
Mosquito 6
Nematode 11(m), 12(f)
Horsetail 216
Sequoia 22
Round worm 2
On the extreme, round worm shows only two
chromosomes, while the other extreme is
represented by Protozoa having 300 or more
chromosomes. However, most organisms have
numbers between 12 to 50.

8. Chromosome Size
• The size of chromosomes shows a remarkable
variation depending upon the stages of cell division.
• Interphase: chromosome are longest & thinnest
• Prophase: there is a progressive decrease in their
length accompanied with an increase in thickness
• Anaphase: chromosomes are smallest.
• Metaphase: Chromosomes are the most easily
observed and studied during metaphase when they
are very thick, quite short and well spread in the cell.
Therefore, chromosomes measurements are
generally taken during mitotic metaphase.

9. • In a cell which is not
about to divide, the
• structures in the
nucleus are not distinct

10. • Just before cell division,
thread-like
• structures appear in
the nucleus

11. • These structures are
called chromosomes
• They get shorter and
thicker and take up
• stains very strongly

12. • The shortening and
thickening continues.
• Now the chromosomes
are seen to be in pairs
• The chromosomes are
always in pairs because
one of them is derived
from the male parent
and the other from the
female parent

13. • Because the chromosomes are in
pairs, the genes they carry are also
in pair. Each member of a pair of
genes comes from either the male
or the female parent just as the
chromosomes do
• The individual genes of a pair,
control the same characteristic, e.g.
B and b could control eye colour; G
and g could control hair colour
A
B
C
D
E
F
G
H
I
a
b
c
d
e
f
g
h
I

14. Chromosomes/ chromatids/
chromatin
• Chromosomes are made of 2 chromatids. This
is because you can only see them during the
stage of the cell cycle called Mitosis, when the
nucleus splits.
• The chromatids are made of a substance
called chromatin.
• This is a single, very long strand of DNA.

15. Chromatids
• Before cell division, it
can be seen that the
chromosomes have
replicated; that is each
chromosome has made
a copy of itself
(including its DNA).
These copies and the
originals are now
called chromatids

16. centromere
• The chromatids tend
to separate but are
held together by a
special region called
the centromere

17. CHROMOSOME PARTS
• A typical mitotic
chromosome at
metaphase

18. CHROMOSOME PARTS
• Heterochromatin: Deep Stain
More condensed
– Silenced genes (methylated)
– Gene poor (high AT content)
– Stains darker
• Euchromatin: Light stain
– Less condensed
– Gene expressing
– Gene rich (higher GC content)
– Stains lighter

19. CHROMOSOME PARTS
• Telomeres – chromosome tips
– Repeats
– Act as sort of biological clock
– Being whittled down at each
Mitosis
• Centromeres – middle
– Highly condensed
– Also repetitive sequence
– Region where spindle fibers attach
– Pulling chromatids apart during
Mitosis

20. Telomeres, our biological clocks
that can reveal our lifespan
• When we are born, our telomeres are longer.
As you get older, they shorten and this is
progressive in time.
When the telomeres run out, the cell becomes
inactive or dies, which leads to disease and
later to death..
• Scientists are still working on this, which
seems to be the key of the eternal life, but its
still too soon to have results.

21. CHROMOSOME PARTS
• p arm – the smaller of the two
arms
– p stands for petite
• q arm – the longer of the two
arms
• Bands are numbered from
centromere outward

22. CHROMOSOME PARTS
• Homologous chromosomes are not identical
– Can have different alleles of genes
• Sister chromatids are identical
– Form as cells go through S phase (replication)
– Attached to each other by centromere
– Until Anaphase of Mitosis
– Once separated each is again referred
to as a chromosome

24. Sister chromatids
Typically a chromosome is
made of two chromatids, a
centromere and a secondary
constriction.
Sister chromatids are two
identical copies of the
chromosome connected by a
centromere. The two
chromatids of one
homologous chromosome
with respect to those of the
other homologue are called
Nonsister chromatids.

25. CHROMOSOME CONTAINS
• Non histone protins and
• Histone protins
• Non histone protins are
• DNA and RNA polymerase,
• Gene regulatory proteins and
• HMG (high mobility Gr proteins)

26. Histone protins
• Histones are 5 proteins according
to concentration
• arginine and lysine residue
• H1, H2A,H2B,H3 and H4 combine
to form spherical octamer known
as NUCLEOSOME CORE

27. Histone
 Histone proteins are basic and bind with the
phosphates along the DNA backbone
 They contain many positively-charged amino acids

Lysine and arginine
 There are five types of histones
H2A, H2B, H3 and H4 are the core histones
Two of each make up the octamer
H1 is the linker histone

Binds to linker DNA

Also binds to nucleosomes

28. 2 nm
 If stretched end to end, a single
set of human chromosomes will
be over 1 meter long!
 The cell’s nucleus is only 2 to 4
µm in diameter
 Therefore, the DNA must be
tightly compacted to fit

29. secondary coiling. 11 nm
• Each nucleosome is
rapped twice by
146 base pairs of
helical DNA to form
Chromatin fibre 11
nm diameter
secondary coiling.

30. Nucleosome 30 nm

It is composed of double-stranded
DNA wrapped around an octamer of
histone proteins
 An octamer is composed two
copies each of four different
histones
 146 bp of DNA make 1.65 negative
superhelical turns around the
octamer

31. nucleosomes resembles
“beads on a string”
This structure shortens the DNA length about seven-fold

32. Nucleosomes Join to Form
a 30 nm Fiber

33. Nucleosomes
 Nucleosomes associate with each other to
form a more compact structure termed the
30 nm fiber
 Histone H1 plays a role in this compaction
 At moderate salt concentrations, H1 is
removed

The result is the classic beads-on-a-string
morphology
 At low salt concentrations, H1 remains bound

Beads associate together into a more compact
morphology

34. Tertiary coiling 30nm

35. Solenoid model
 The 30 nm fiber shortens the total length of DNA
another seven-fold

Two models have been proposed

Solenoid model

Three-dimensional zigzag
model

36. Solenoid model

37. SCAFOLD Protein / nuclear matrix
quaternary coiling
 The two events we have discussed so far have
shortened the DNA about 50-fold
 A third level of compaction involves interaction
between the 30 nm fiber and the nuclear matrix
 The nuclear matrix is composed of two parts
 Nuclear lamina
 Internal matrix proteins
 10 nm fiber and associated proteins

38. Scaffold/matrix-attachment regions
Radial Loops (SARs/MAR) 300nm
Matrix-attachment
regions
MARs are anchored
to the nuclear
matrix, thus creating
radial loops

39. Compaction of Radial Loops

42. Heterochromatin vs
Euchromatin
 The compaction level of interphase
chromosomes is not completely uniform
 Euchromatin

Less condensed regions of chromosomes

Transcriptionally active

Regions where 30 nm fiber forms radial loop domains
 Heterochromatin

Tightly compacted regions of chromosomes

Transcriptionally inactive (in general)

Radial loop domains compacted even further

43.  There are two types of heterochromatin
 Constitutive heterochromatin

Regions that are always heterochromatic

Permanently inactive with regard to transcription
 Facultative heterochromatin

Regions that can interconvert between euchromatin and
heterochromatin

Example: Barr body

44. Constitutive heterochromatin
It is fixed and irreversible in form and
function.
It does not reverse to the euchromatic
stage.
Chromosomes 1, 9, 16 and the
Y chromosome contain regions of
constitutive heterochromatin

45. Facultative heterochromatin
• It has the faculty to return to the
normal euchromatic state.
• Consists of euchromatin that takes
on the staining and compactness
characteristics of heterochromatin
during some phase of development.
• The inactive X chromosome is made
up of facultative heterochromatin.

46. SMC proteins
 Structural maintenance of chromosomes
 Two multiprotein complexes help to form and
organize metaphase chromosomes.
 Condensin

Plays a critical role in chromosome
condensation
 Cohesin

Plays a critical role in sister chromatid
alignment
 Both contain are called SMC proteins

47. Cohesin at
centromer is
degraded

48. Constitutive heterochromatin
• Constitutive heterochromatin is found more commonly in the
periphery of the nucleus attached to the nuclear membrane.
This concentrates the euchromatic DNA in the center of the
nucleus where it can be actively transcribed.
• During mitosis it is believed that constitutive heterochromatin
is necessary for proper segregation of sister chromatids and
centromere function.
• The repeat sequences found at the peri-centromeres are not
conserved throughout ,while telomeres show more
conserved sequences.

49. Chromosome like twisted ladder : primary
coiling

50. STRUCTURE
• Each bead is separated by length of 50
base pairs of DNA intercepted with a
Linker Histone H1
• This form of extended chromatin with
beads of nucleosomes is known as
Euchromatin when DNA is biologically
active - transcribed to form RNA

51. SOLENOIDS (tertiary coiling)
• Then helical segregation of six nucleosomes
per turn forms SOLENOIDS (tertiary coiling)
fibre thickness upto 30 nm make the
appearance of hetero chromatin with
biologically inactive DNA
• Condenced chromatin is further folded into
giant super coiled loops to form chromosomes
quaternary coiling.

52. SAT chromosome
• A satellite
chromosome or SAT
chromosome has a
chromosome segment that
is separated from the main
body of the chromosome
by such a secondary
constriction
• the chromosomes
number 13, 14, 15, 21 and
22 are examples of SAT
chromosomes

53. Nucleolar Organizing Regions( NOR)
• Some parts of these constrictions
indicate sites of nucleolus
formation and are called
"Nucleolar Organizing Regions.
(NOR)"
• The formations of nucleolus takes
place around the NOR region.
• The secondary constriction also
contains the genes for r RNA
synthesis (18 S, 5.8 S, 28 S)

54. • Mammalian cells have 2 mitochondrial (12S
and 16S) rRNA molecules and 4 types of
cytoplasmic rRNA (the 28S, 5.8S, 18S, and 5S
subunits). The 28S, 5.8S, and 18SrRNAs are
encoded by a single transcription unit (45S)
separated by 2 internally transcribed spacers.

55. p arm & q arm
• p arm – the smaller of
the two arms
– p stands for petite
• q arm – the longer of
the two arms
• Bands are numbered
from centromere
outward
•

56. Classification of Chromosomes
There are four types of chromosomes:
1. Telocentric
2. Acrocentric
3. Submetacentric
4. Metacentric
• Divided based on the position of the
centromere

57. Nomenclature of chromosomes

58. According to the Position of
centromere
1. Telocentric – no p arm; centromere is on
end
2. Acrocentric – very small p arm; centromere
is very near end
3. Submetacentric – p arm just a little smaller
than q arm; centromere in middle
4. Metacentric – p and q arms are exactly the
same length; centromere in exact middle of
chromosome

59. CLASSIFICATION according to
Denver-London system

60. according to length into 7
major groups :A,B,C,D,E,F&G

61. Group A
• Chromosomes 1: 3
• Chromosomes 1 & 2
have similar lengths
but….
• Chromosmes 1& 3 are
METACENTRIC

62. Group B
• Chromosomes 4 & 5.
• Chromosomes 4 & 5
are very similar!
• They are large & sub-
metacentric.

63. GROUP C
• Chromosomes 6
to 12 & (XX) or
only one X.
• middle-sized &
Metacentric or
Sub-metacentric

64. Group D
Chromosomes
13:15.
middle-sized &
Acrocentric.
They have Satellites.
Easy to be
distinguished!

65. Group E
Chromosomes 16:18.
Smaller than chrom. D
P-arm of chrom. 17 &18 is
shorter than chrom. 16.
16 Metacentric . 17&18
sub-metacentric

66. Group F
• Chromosomes 19&20.
• They are Metacentric.
• Chromosomes 19 and
20 can’t be
distinguished!

67. Group G
• Chromosomes 21,22 &
Y.
• Chrom. 21&22 are the
smallest pair. They have
Satellites.
• Chrom. Y is longer than
21&22. Y does not have
Satellites

68. Spectral Karyotype in cancer
cytogenetics.(SKY)
• Chromosomes painting using different probes.
• 24 colours are usually used .

73. Karyotype/ Idiotype
• Karyotype: is the general morphology of the
somatic chromosome. Generally, karyotypes
represent by arranging in the descending
order of size keeping their centromeres in a
straight line.
• Idiotype: the karyotype of a species may be
represented diagrammatically, showing all the
morphological features of the chromosome;
such a diagram is known as Idiotype.

74. Karyotype/ Idiotype

77. CYTOGENETICS
• Is the study of the structure and properties of
chromosomes, chromosomal behaviour during
mitosis and meiosis, chromosomal influence on
the phenotype and the factors that cause
chromosomal changes (Hare and Singh, 1979).

78. Nomenclature of chromosomes

80. PREPARATION OF CHROMOSOMES

82. METHODOLOGY
• Aseptic precautions
• Preparation of RPMI 1640 medium
• Collection of 10ml of blood with
heparin
• Setting of culture
8 ml of medium
0.1 ml of PHA-M
0.5 ml of blood/plasma
2 ml of plasma
• Incubate at 37°C for 72 hours

83. METHODOLOGY…
Harvesting of culture
• Spindle inhibitors – Colchicine/colcemed
(0.1µg/ml)
• Hypotonic treatment – 0.075M KCl
• Fixation (3:1 methanol : acetic acid)
• Preparation of slides
• Slides stained with 4% Giemsa for 20-25min
• Screening of slides to study the morphology of
chromosome
• Construction of karyotype

84. • CYTOGENETIC STUDY
• The following chemicals were used for the
present study:
• Anticoagulant
• Heparin solution of 200 units was taken in a
disposable syringe before bone marrow
aspiration and peripheral blood collection, to
prevent clotting.

85. Culture Media
• Culture Media
• RPMI 1640 culture media with L-glutamine
was used for sample collection. It contains all
essential amino acids and micronutrients
required for cell survival. Bovine calf serum
was used as growth factor.

86. Spindle Inhibitor
• Since chromosomes are analysed at
metaphase, colchicines was used as a spindle
inhibitor, which resulted in accumulation of
chromosomes in rapidly dividing ‘T’
lymphocytes. In order to achieve adequate
metaphase 0.005 gm of colchicine was added
in 10 ml of distilled water, from which 0.2 ml
was added to the culture and it was kept for
20 minutes

87. Hypotonic Solution
• Hypotonic solution having a lower salt
concentration than in the cytoplasm of normal
cell was taken.
• According to the rule of Osmosis there was a
movement of fluid from the hypotonic solution to
the cell through the cell membrane, leading to
the swelling of the cells, with subsequent
dispersion of the chromosomes.
• Potassium chloride was used to harvest
chromosomes in a concentration of (0.600 mg%)
for the above experiment

88. • Fixatives
• Fixatives were used to preserve, the form of
the cells and their contents as closely as
possible to their living state and to render
them resistant to further changes caused by
subsequent processing. The commonly used
fixative fluid was a mixture of glacial acetic
acid and absolute methyl alcohol in the ratio
of 1:3 proportions.

89. Staining
• The conventional Giemsa staining was used in
this study. The Giemsa stain was prepared
from stock solution by adding 2 ml of stock
concentrate to 38 ml of phosphate buffer (pH-
7.2). The prepared stain was filtered using
Whatman filter paper. Air-dried slides were
treated with Giesma stain for 5 minutes. After
5 minutes, slides were rinsed twice in distilled
water and left for drying. Slides were then
screened under the microscope for better
metaphase

90. • G-Banding
• The chromosome of the slides was treated
with 0.50mg % of trypsin (1:250) solution. The
proteolytic enzyme (trypsin) denatures
chemically the A=T & G C bonds and≡
produces alternate dark & light bands on the
chromosomes when stained by Giemsa
solution. This procedure was known as
Giemsa banding.

93. Karyotype of bone marrow cell of male
AML patient 46XY t(11q+:17q-)

95. Karyotype of bone marrow cell of male
AML patient showing 46XY t(15:17)

97. Karyotype of bone marrow cell of female
ALL patient showing 70XXXX

99. Karyotype of bone marrow cell of female
ALL patient showing 46XY t(1:19)

101. Karyotype of bone marrow cell of female
ALL patient showing 69 XXX Triploidy

103. Karyotype of bone marrow cell of male ALL
patient showing 45XY t(8:14), -7

105. Karyotype of bone marrow cell of male ALL
patient showing 42XY. –3, -7

106. NORMAL
KARYOTYPE
OF CATTLE

108. TERMS AND DEFINITIONS OF VARIOUS
ABERRATIONS OF CHROMOSOMES
• Ring( r) Minute (min)
• Dicentric (d) Hyperdiploid (h)
• Chromosome gap (sg) Chromatid deletion (td)
• Fragment (f) Acentric fragment (af)
• Translocation (t)
• Complex rearrangement (cr)
• Polyploid (pp) or endoreduplication

112. G banding
• G banding is obtained with Giemsa
stain following digestion of
chromosomes with trypsin.
It yields a series of lightly and darkly
stained bands - The dark regions
tend to be heterochromatic, late-
replicating and A-T rich.
The light regions tend to be
euchromatic, early-replicating and G-
C rich.
• This method will normally produce
• 300–400 bands on chromosome

113. R - Banding
• It is the reverse of G-banding
(the R stands for "reverse").
• Treatmet of heat 65° C
denatures the DNA rich in A -T
• The dark regions are
euchromatic (guanine-cytosine
rich regions)
• the bright regions are
heterochromatic (thymine-
adenine rich regions).

118. COMPARISION G-R-C BANDING

119. COMPARISION G-R-Q BANDING

120. METHODOLOGY
G- Banding technique
• Ageing of good slides for 10 days
• Normal saline
• Treated with trypsin 0.25% solution 10-15 sec
• Immersed in 70% ethanol for few minutes
• Stained with 10% Giemsa for 6-10min
• Microphotograph good spreads
• Construction of G-banded karyotype

121. G-BANDED MITOTIC CHROMOSOMAL
SPREAD OF HUMAN

122. G-BANDED
KARYOTYPE
OF CATTLE

123. Inactive X chromosome
• A Barr body represents an inactive X chromosome in
a female’s somatic cell.
• Barr bodies are small, dark stained mass of inactive
X-chromosomes in females within the nucleus.
• Mc.Barr” and “Bertram were the two scientists to
observe the deeply stained chromatin body in the
nerve cell of female cat in the year 1943.
• Later they came to know that the chromatin body
was found absent in male cat.
• This chromatin body is called as a sex chromatin or
Barr body, which is named after the name of its
discoverer “Dr. Murray Llewellyn Barr”.

124. Lyon hypothesis
• Random inactivation of one X chromosome in
early foetal development the BARR body
• A Barr body can be defined as a small dark
stained mass of X chromosome, which is
inactive and are found only in the female cells.
• It is present in the nuclei of all cells except the
germ cells.
• It is also called as sex chromatin as it indicates
the presence of sex hormone.

126. X Inactivation
• The process of inactivation
occurs arround 15 – 16 days
of gestation approximately
5000 cells
• The process of X inactivation
takes place by mythylation
and is initiated by a locus
known as the X inactivation
centre