Introduction To Cytogenetics

BIOL2165
Genetics II
Lecturer:
Dr. Winston Elibox
Course Assessment
• Final Theory Paper = 50%

• In-course = 50%
– Three in-course tests = 25%
– Tutorials = 10%
– Labs = 15%
LECTURE-1
PART- I CYTOGENETICS
Chromosome theory of inheritance
• States that chromosomes are the

unit of inheritance
• Proposed by Walter Sutton and

Theodor Boveri (1907)
• Behavior of chromosomes during

meiosis (segregation/
independent assortment)
paralleled that of Mendel’s
particles
Chromsome theory of inheritance
Chromosomes became the centre of

interest following the ‘Chromosome
theory of inheritance’
Chromosomes are nucleoproteins that take

up stain during cell division.
What is Cytogenetics ?
The study of the genetic constitution of cells through
the visualisation and analysis of chromosomes.
It involves the study of:

• nuclear organization of chromosomes.
• macromutations
Primarily concernedthat
withaffect chromosome
genome structure or number.
and chromosome
characterization so that any changes to the organization or
structure can be
• the effects of detected and correlated
macromutations with behavioural
on chromosomal behavior
changes
duringand evolutionary leaps.
meiosis.
• the effects of macromutations on phenotype and evolution.

Chromosome organization
• Chromosome number
Total number of chromosomes in a
somatic cell
• Basic chromosome number (x)

Number of unique chromosomes in a
somatic cell (Monoploid number)
Human genome • Basic chromosome set

X = 23 The unique chromosome set of a
2x = 46
somatic cell
Genome characterisation
A genome can be characterized

based on:
(a) Basic chromosome set
(b) Ploidy – the number of repetitions

of the basic chromosome set:
- Euploidy-whole set repetitions
- Aneuploidy-repetitions that are not

whole number replicates of the basic
chromosome set.
Shape: - Position of centromere (primary

constriction; non-stainable) = telocentric,
acrocentric, mesocentric or metacentric
- Position of secondary constrictions

(satellites and nuclear organisers)
Size: 0.5 - 400 μM length (A - G groups)

(http://www.accessexcellence.org/AE/AEPC/WWC/1993/karyoteype.php)
Banding pattern: Imparted by staining. Provides a means of uniquely

identifying each chromosome
Human chromosomes are divided into 7 groups & sex chromosomes:
• A: 1-3 - Large metacentric
• B: 4,5 - Large submetacentric
• C: 6-12, X - Medium sized, submetacentric
• D: 13-15 - medium-sized acrocentric plus

satellites
• E: 16-18 - short metacentric 16 or

submetacentric 17,18
• F: 19-20 - Short metacentrics
• G: 21,22,Y - Short acrocentrics with satellites. Y has no satellites.

Position of nucleolar
organizer:
– Represents the region
of the chromosome
that has the rRNA
gene cluster
– Forms a secondary
constriction
– The region is
associated with the
nucleolus which
stores the rRNA
Genome characterization
Satellites (satellite DNA):
• Secondary constrictions in eukaryotic DNA (5 to 200pb) that consists of
short, tandem repeated non-coding sequences of nucleotide pairs, often
found near the region of the centromere and occupying the majority of
the heterochromatin.
Secondary
constriction
Genome characterisation: Banding patterns
Dark bands- heterochromatin; Light bands- euchromatin
Quinacrine stain = Q bands Giemsa staining = G bands

Impermanence fluorescent bands; Dark permanent bands (area of most coiling)
fades quickly
Reverse Giemsa C bands (Giemsa fixed with alkali-

R bands- complimentary to G bands only heterochromatic region is seen)
Genome characterization :
Giemsa staining
Giemsa stain = G bands; most popular staining method (permanent).
Reveals areas of most coiling- dark bands associated with heterochromatin.
Lighter bands are the euchromatin.
Also reveals regions called chromomeres- heavily stained regions associated with
genes (associated with the euchromatic regions).
If Giemsa stain is fixed with alkali, a different banding pattern called C bands
reveals only the heterochromatic regions.
If the chromosomes are heated in a phosphate buffer, then treated with Giemsa
stain, an R banding patterns occurs- that is the reverse of that produced in G-
banding.
Cytogeneticists use stains to differentiate between chromosomes.

Ultrastructure of a chromosome
One chromosome = One molecule

of double helical DNA packaged in
a lattice work of histone proteins
Unineme model- each chromosome comprises 1 DNA double helix extending from one end
of the chromosome to the other.
Chromosomes are nucleoproteins- have both DNA and

proteins. Proteins form the structural framework for the
DNA.
Primary structure
• Nucleosome
- The unit of packaging of a chromosome
- Nucleosome = Histone core (2 H2A, 2 H2B, 2 H3 and 2 H4) +
two turns of double helical DNA (140 bp)
Primary structure
The Nucleosome thread
The primary structure of the chromosome (100 Ao) = 0.000001 cm. (1 Angstrom
= 10-8 cm).
The nucleosomes are linked together by linker DNA and stabilized by H1 protein
to form the nucleosome thread.
1 nm = 10Ao
Ultrastructure of a chromosome:
Secondary structure
• Chromatin
- The secondary structure of the chromosome
- The nucleosome thread is thrown into coils to form a solenoid
structure (200-300 Ao)
- approximately six nucleosomes per turn.
• Meiotic chromosomes
- Chromatin is thrown into tertiary and quaternary coiling
during mitosis and meiosis.
Chromatin:
Euchromatin vs heterochromatin
Nuclei consist of chromatin

strands as loosely packed
euchromatin or densely packed
heterochromatin Euchromatin
Heterochromatin
Chromatin:
Euchromatin vs. heterochromatin
• Heterochromatin
– Heavily coiled functionally inactive regions of the
chromosome
– Constitutive heterochromatin
Associated with centromere, telomere and intercalary (DNA
sequences between structural genes) regions = represent
highly repetitive non-coding regions
– Condensed heterochromatin
Distributed differently from tissue to tissue and appears during
cell maturation. Reflects permanent turning off of certain
genes during differentiation
– Facultative heterochromatin
Reflects regulatory devices designed to adjust the dosage of
certain genes
Position effects
• Expression of genes in the euchromatic region can be affected

by adjacent heterochromatic regions.
• The highly coiled regions of the heterochromatin affect

transcription machinery from accessing the genes for
transcription.
• The nearer a gene is to the heterochromatic region, the more its

expression is affected by the heterochromatic region.
• The spreading suppressing influence of the heterochromatic

region on genes in the euchromatin region is referred to as
position effect.
Position effects
Yeast
Position Effects on Gene

Expression
Heterochromatin: condensed
Euchromatin: loose
Fruit Fly
Karyotyping
• Diagrammatic representation of
chromosomes of a somatic cell at
the mitotic metaphase arranged in
homologous pairs of decreasing
size.
• Indicates landmark features that

allow chromosomes to be uniquely
identified.
How do you do a Karyotype?
1. Growing root tips squashed and stained
2. Identify mitotic metaphase in transverse

(cross sectional) view
3. Take microphotograph
4. Enlarge photograph
5. Cut and paste in descending order of size
6. Indicate landmark features.

Uses of a Karyotype
1. Provides a means of identifying chromosomal aberrations from

the type (normal) karyotype.
Identifies changes in chromosomes structure, size and
chromosome number (Down syndrome-trisomy 21).
2. Comparison of karyotypes of different species allow

determination of taxonomic relationships.
Helps reform and correct taxonomic relationships.
3. Enables the understanding of evolution, where small

chromosomal changes accumulate over time in a linear fashion.
Fewer changes imply recent divergence. Can help us construct
phylogenetic trees.
Karyotype: Polytene chromosomes
The nuclei in the salivary glands of Dipteran insects (true flies e.g.
Drosophila) show enlargement due to extra replication of chromosomes-
endopolyploidy or polyteny
The heterochromatic regions are visible to the naked eye in these

chromosomes during interphase.
chromocenter
Polytene chromosomes begin as normal chromosomes.
Undergo repeated rounds of DNA replication without cell division.
They become large, banded chromosomes.
Centromeric regions do not endoreplicate very well.
Centromeres of all the chromosomes bundle together in a mass called

the chromocenter.
Found in larvae and promote faster growth and development than the
diploid state.
The active region (chromomeres- thickened,

tightly coiled DNA, stains darker than the rest
of the DNA and associated with highly
expressive genes) form puffs, while other
regions are condensed
(Drosophila: 5000-6000 puffs)
In Drosophila all the four chromosomes are

connected together at the chromocenter
Chromosomal aberrations can be seen as loops

Karyotype: Lampbrush chromosomes
Found in the oocytes of some amphibians

with yolky eggs.
Occurs during the prolonged prophase

during the first meiotic division
The meiotic chromosomes reach 1000 µm

in thickness with long lateral loops
Each loop emerges from one chromomere

by duplication (puff)
Some loops are pinched off as balbiani

rings, which can also independently
express gene products.
Karyotype:
Lampbrush
chromosomes
A model for the structure of a lampbrush

chromosome
Notes
• 60% of DNA in higher eukaryotes on average is junk DNA.
• Important for pairing of chromosomes and crossovers in

constitutive heterochromatin
• Coding regions (genes) are not highly repetitive or may be unique.

Summary
Chromosome organization
• Chromosome theory of inheritance
• Chromosome organization
– Basic chromosome set
– Ploidy
• Karyotyping allow characterization of the basic chromosome set
– Length, shape, special features (satellites, nucleolar organizer)
– Various types of banding patterns by differential staining
• Ultrastructure of the chromosome
– Primary, secondary, tertiary and quaternary packaging of DNA
• Heterochromatin vs. euchromatin
• Position effects

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BIOL2165

• Final Theory Paper = 50%

• States that chromosomes are the

• Proposed by Walter Sutton and

• Behavior of chromosomes during

Chromosomes became the centre of

Chromosomes are nucleoproteins that take

It involves the study of:

• the effects of macromutations on phenotype and evolution.

• Basic chromosome number (x)

Human genome • Basic chromosome set

A genome can be characterized

(b) Ploidy – the number of repetitions

- Euploidy-whole set repetitions

- Aneuploidy-repetitions that are not

Shape: - Position of centromere (primary

- Position of secondary constrictions

Size: 0.5 - 400 μM length (A - G groups)

Banding pattern: Imparted by staining. Provides a means of uniquely

• B: 4,5 - Large submetacentric

• C: 6-12, X - Medium sized, submetacentric

• D: 13-15 - medium-sized acrocentric plus

• E: 16-18 - short metacentric 16 or

• F: 19-20 - Short metacentrics

• G: 21,22,Y - Short acrocentrics with satellites. Y has no satellites.

Quinacrine stain = Q bands Giemsa staining = G bands

Reverse Giemsa C bands (Giemsa fixed with alkali-

Reveals areas of most coiling- dark bands associated with heterochromatin.

Lighter bands are the euchromatin.

Cytogeneticists use stains to differentiate between chromosomes.

One chromosome = One molecule

Chromosomes are nucleoproteins- have both DNA and

Nuclei consist of chromatin

• Expression of genes in the euchromatic region can be affected

• The highly coiled regions of the heterochromatin affect

• The nearer a gene is to the heterochromatic region, the more its

• The spreading suppressing influence of the heterochromatic

Position Effects on Gene

• Indicates landmark features that

1. Growing root tips squashed and stained

2. Identify mitotic metaphase in transverse

5. Cut and paste in descending order of size

6. Indicate landmark features.

1. Provides a means of identifying chromosomal aberrations from

2. Comparison of karyotypes of different species allow

3. Enables the understanding of evolution, where small

The heterochromatic regions are visible to the naked eye in these

Polytene chromosomes begin as normal chromosomes.

Undergo repeated rounds of DNA replication without cell division.

They become large, banded chromosomes.

Centromeric regions do not endoreplicate very well.

Centromeres of all the chromosomes bundle together in a mass called

The active region (chromomeres- thickened,

(Drosophila: 5000-6000 puffs)

In Drosophila all the four chromosomes are

Chromosomal aberrations can be seen as loops

Found in the oocytes of some amphibians

Occurs during the prolonged prophase