1. Introduction to Cytogenetics

7/29/2019 1. Introduction to Cytogenetics

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Advanced Genetics

Lecturer:

Dr. Winston Elibox

Instructor: Ms. Gabrielle Holder
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Course Assessment

Final Theory Paper = 50%

In-course = 50%

Three in-course tests = 30%

Tutorials = 10%

Labs = 10%


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LECTURE-1

PART- I CYTOGENETICS
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Chromosome theory of inheritance

States that chromosomes are the

unit of inheritance

Proposed by Walter Sutton and

Theodor Boveri (1907)

Behavior of chromosomes duringmeiosis paralleled that of Mendels

particles
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Meiosis: Segregation and

Independent assortment


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ChromosomesMendels principles


Pairs of homologous

chromosomes

Homologous

chromosomes separate

and go into gametes at

equal frequency

Different pairs ofchromosomes segregate

independently

Paired particles

Paired particles segregate

into gametes at equal

frequency

Different particles assort

independently


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Chromsome theory of inheritance

Chromosomes became the centre of

interest following the Chromosometheory of inheritance

Chromosomes are bodies that take up stain during

cell division.


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What is Cytogenetics ?

The study of the genetic constitutionof cells through the visualisation andanalysis of chromosomes.

Primarily concerned with genome and chromosome

characterization so that any changes to the

organization or structure can be detected andcorrelated with behavioural changes and

evolutionary leaps.


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What is Cytogenetics ?

Cytogenetics is the study of

Nuclear organisation of chromosomes

Macromutations that affect chromosome

structure or number

Effect of macromutations on

chromosomal behavior during meiosis

Effect of macromutations on phenotye

and evolution


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Chromosome organization

Chromosome number

Total number of chromosomes in asomatic cell

Basic chromosome number (x)

Number of unique chromosomes in asomatic cell (Monoploid number)

Basic chromosome set

The unique chromosome set of asomatic cell

Human genome

X = 23

2x = 46


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Genome characterisation

A genome can be characterisedbased on:

(a) Basic chromosome set

(b) Ploidythe number of repetitionsof the basic chromosome set

- Euploidy-whole set repetitions

- Aneuploidy-repetitions that are notwhole number replicates of the basicchromosome set.


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Size: 0.5 - 400 M length (A - G groups)(http://www.accessexcellence.org/AE/AEPC/WWC/1993/karyoteype.php)

Banding pattern:Imparted by staining. Provides a means of uniquelyidentifying each chromosome

Shape: - Position of centromere (primaryconstriction; non-stainable) = telocentric,

acrocentric, mesocentric or metacentric

- Position of secondary constrictions

(satellites and nuclear organisers)


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Position of nucleolarorganizer:

Represents the regionof the chromosomethat has the rRNAgene cluster

Forms a secondaryconstriction

The region isassociated with thenucleolus which

stores the rRNA


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Satellites (satellite DNA): Secondary constrictions in eukaryotic DNA (5 to 200pb) thatconsists of short, tandem repeated non-coding sequences ofnucleotide pairs, often found near the region of the centromereand occupying the majority of the heterochromatin.

Secondary

constriction


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Genome characterisation :

Banding patterns

Quinacrine stain = Q bands

Impermanence fluorescent bands;

fades quickly

Giemsa staining = G bands

Dark permanent bands (area of most

coiling)

Reverse Giemsa

R bands- complimentary

to G bands

C bands (Giemsa fixed with alkali-

only heterochromatic region is seen)

Dark bands- heterochromatin

Light bands- euchromatin


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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 revealsonly 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.


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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.


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Chromosomes are nucleoproteins- have both DNA

and proteins. Proteins form the structural framework

for the DNA.


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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)


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The Nucleosome threadThe 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.


Primary structure

1 nm = 10Ao


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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 coilingduring mitosis and meiosis.


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chromatin -secondary

structure of

chromosome


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Chromatin:

Euchromatin vs heterochromatin

Nuclei consist of chromatinstrands as loosely packed

euchromatin or densely packed

heterochromatin

Heterochromatin

Euchromatin


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Chromatin:

Euchromatin vs heterochromatin

Heterochromatin

Heavily coiled functionally inactive regions of the chromosome

Constitutive heterochromatin

Associated with centromere, telomere and intercalary (betweencentromere and tip) 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 duringdifferentiation

Facultative heterochromatin

reflects regulatory devices designed to adjust the dosage ofcertain genes


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Position effects

Expression of genes in the euchromatic region can be affectedby adjacent heterochromatic regions.

The highly coiled regions of the heterochromatin affecttranscription machinery from accessing the genes fortranscription.

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 heterochromaticregion on genes in the euchromatin region is referred to as

position effect.


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Position Effects on Gene

Expression

Heterochromatin: condensed

Euchromatin: loose

Position effects

Yeast

Fruit Fly


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Karyotyping

Diagrammaticrepresentation ofchromosomes of asomatic cell at the

mitotic metaphasearranged inhomologous pairsofdecreasing size.

Indicates landmark

features that allowchromosomes to beuniquely identified.


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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.


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Uses of a Karyotype

1. Provides a means of identifying chromosomal aberrations fromthe type (normal) karyotype.

Identifies changes in chromosomes structure, size and

chromosome number (Down syndrome-trisomy 21).

2. Comparison of karyotypes of different species allowdetermination of taxonomic relationships.

Helps reform and correct taxonomic relationships.

3. Enables the understanding of evolution, where smallchromosomal changes accumulate over time in a linear fashion.Fewer changes imply recent divergence. Can help us construct

phylogenetic trees.


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Karyotype: Polytene chromosomes

The nuclei in the salivary glands of Dipteran insects (true flies e.g.Drosophila) show enlargement due to extra replication ofchromosomesendopolyploidy orpolyteny

The replicated chromosome do not separate but stay in stacks to formthick giant chromosomes called polytene chromosomes.

The heterochromatic regions which are visible to the naked eye inthese chromosomes.
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chromocenter
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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 calledthe chromocenter.

Found in larvae of the insects and promote faster growth anddevelopment than the diploid state.


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The active region (chromomeres- thickened,tightly coiled DNA, stains darker than therest of the DNA and associated withhighly expressive genes) form puffs,

while other regions are condensed(Drosophila: 5000-6000 puffs)

In Drosophila all the four chromosomes areconnected together at the chromocenter

Chromosomal aberrations can be seen asloops


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Karyotype: Lampbrush chromosomes

Found in the oocytes of some amphibiawith yolky eggs.

Occurs during the prolonged prophase

during the first meiotic division

The meiotic chromosomes reach 1000 umin thickness with long lateral loops

Each loop emerges from one chromomereby duplication (puff)

Some loops are pinched off as balbianirings, which can also independentlyexpress gene products.


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Karyotype: Lampbrush chromosomes


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A model for the structure of a lampbrush

chromosome

Karyotype:

Lampbrush

chromosomes


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Notes

60% of DNA in higher eukaryotes is junk DNA.

Important for pairing of chromosomes and crossovers inconstitutive heterochromatin

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


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Summary

Chromosome organization


Chromosome organisation

Basic chromosome set

Ploidy

Karyotyping allow characterisation of the basic chromosome set

Length, shape, special features (satellites, nucleolar organiser)

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

Documents

1. Introduction to Cytogenetics