Advanced Genetics

Lecturer: Dr. Winston Elibox Instructor: Ms. Gabrielle Holder

Course Assessment
Final Theory Paper = 50% In-course = 50%
Three in-course tests = 30% Tutorials = 10% Labs = 10%

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 paralleled that of Mendels particles

Chromosome theory of inheritance

Meiosis: Segregation and Independent assortment

Chromosome theory of inheritance

Mendels principles Paired particles Paired particles segregate into gametes at equal frequency Different particles assort independently Chromosomes Pairs of homologous chromosomes Homologous chromosomes separate and go into gametes at equal frequency Different pairs of chromosomes segregate independently

Chromsome theory of inheritance

Chromosomes became the centre of interest following the Chromosome theory of inheritance

Chromosomes are bodies 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.

Primarily concerned with genome and chromosome characterization so that any changes to the organization or structure can be detected and correlated with behavioural changes and evolutionary leaps.

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

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 X = 23 2x = 46

Basic chromosome set The unique chromosome set of a somatic cell

Genome characterisation
A genome can be characterised 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.

Genome characterisation
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 M 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

Genome characterisation
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 characterisation
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 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 alkalionly 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.

Ultrastructure of a chromosome

Chromosomes are nucleoproteins- have both DNA and proteins. Proteins form the structural framework for the DNA.

Ultrastructure of a chromosome 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)

Ultrastructure of a chromosome 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.

Ultrastructure of a chromosome

chromatin secondary structure of chromosome

Chromatin: Euchromatin vs heterochromatin

Nuclei consist of chromatin strands as loosely packed euchromatin or densely packed heterochromatin
Heterochromatin

Euchromatin

Chromatin: Euchromatin vs heterochromatin

Heterochromatin Heavily coiled functionally inactive regions of the chromosome
Constitutive heterochromatin

Associated with centromere, telomere and intercalary (between centromere and tip) regions = represent highly repetitive noncoding 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. 2. 3. 4. 5. 6.
Growing root tips squashed and stained Identify mitotic metaphase in transverse (cross sectional) view Take microphotograph Enlarge photograph Cut and paste in descending order of size 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 replicated chromosome do not separate but stay in stacks to form thick giant chromosomes called polytene chromosomes.
The heterochromatic regions which are visible to the naked eye in these chromosomes.

Karyotype: Polytene chromosomes

chromocenter

Karyotype: Polytene chromosomes

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 of the insects and promote faster growth and development than the diploid state.

Karyotype: Polytene chromosomes

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 amphibia with yolky eggs. Occurs during the prolonged prophase during the first meiotic division The meiotic chromosomes reach 1000 um 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

Karyotype: Lampbrush chromosomes

A model for the structure of a lampbrush chromosome

Notes
60% of DNA in higher eukaryotes 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 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