Lecture 7 (ch8&9)

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Chromosome Analysis and Karyotyping

Introduction to Karyotyping

Karyotyping is a laboratory technique used to visualize and analyze the number and structure of chromosomes in a cell. It is essential for diagnosing genetic diseases and certain birth defects.

Karyotype: The complete set of chromosomes in a cell, displayed in a standard format.
Applications: Detection of chromosomal abnormalities such as aneuploidies, deletions, duplications, and translocations.

Steps in Karyotyping

Collect a cell from an individual (e.g., blood, amniotic fluid).
Induce the cell to divide in culture.
Arrest cell division at metaphase when chromosomes are most visible.
Stain the chromosomes to enhance visibility.
View and photograph the chromosomes under a microscope for analysis.

Chromatin Structure: Euchromatin vs. Heterochromatin

Definitions and Characteristics

Chromatin is the complex of DNA and proteins that forms chromosomes. It exists in two main forms, each with distinct properties and functions.

Heterochromatin:
- Highly condensed and transcriptionally inactive.
- Gene-poor, often contains repetitive sequences.
- Stains darkly with certain dyes (e.g., Giemsa).
- Located at centromeres, telomeres, and other specific regions.
- Replicates late in S phase.
Euchromatin:
- Less condensed and transcriptionally active.
- Gene-rich, with unique sequences.
- Stains lightly.
- Found on chromosome arms.
- Replicates throughout S phase.

Banding Patterns and Chromosome Identification

Banding Techniques

Chromosome banding patterns are used to identify individual chromosomes and detect structural abnormalities.

G-banding: Uses Giemsa stain to produce a series of dark and light bands unique to each chromosome.
R-banding: Reverse of G-banding, highlights GC-rich regions.
C-banding: Stains constitutive heterochromatin, especially at centromeres.
NOR-banding: Identifies nucleolar organizer regions (NORs), which contain rRNA gene repeats.

Banding patterns help in diagnosing chromosomal aberrations such as deletions, duplications, and inversions.

Chromosome Mutations: Variations in Number

Aneuploidy and Polyploidy

Chromosome mutations can involve changes in chromosome number (aneuploidy, polyploidy) or structure (deletions, duplications, inversions, translocations).

Aneuploidy: The presence of an abnormal number of chromosomes (not a multiple of the haploid set).
Polyploidy: The presence of more than two complete sets of chromosomes.

Types of Aneuploidy

Type	Description	Human Example
Nullisomy	Loss of both members of a homologous pair (2n-2)	Not viable in humans
Monosomy	Loss of a single chromosome (2n-1)	Turner syndrome (45,X)
Trisomy	Gain of a single chromosome (2n+1)	Down syndrome (Trisomy 21)
Tetrasomy	Gain of two homologous chromosomes (2n+2)	Rare, usually lethal

Causes of Aneuploidy

Nondisjunction: Failure of homologous chromosomes or sister chromatids to separate during meiosis or mitosis, leading to gametes with abnormal chromosome numbers.
Consequences: Monosomy of autosomes is usually lethal due to unmasking of lethal recessive alleles and haploinsufficiency. Trisomy of small chromosomes may be tolerated (e.g., Trisomy 21), but larger trisomies are often lethal.

Polyploidy

Autopolyploidy: Multiple chromosome sets from the same species.
Allopolyploidy: Chromosome sets from different species.
Mosaic/Endopolyploidy: Only certain cells are polyploid (e.g., human liver cells).
Common in plants and can lead to evolutionary success.

Chromosome Mutations: Structural Changes

Types of Structural Mutations

Duplication: A segment of the chromosome is duplicated, resulting in extra genetic material.
Deletion: Loss of a chromosome segment. Can be terminal (end) or intercalary/interstitial (internal).
Inversion: A chromosome segment is reversed end to end. Can be paracentric (not including centromere) or pericentric (including centromere).
Translocation: A segment from one chromosome is transferred to another. Includes reciprocal and Robertsonian translocations.
Isochromosome: Chromosome with identical arms due to abnormal division at the centromere.
Ring chromosome: Chromosome forms a ring due to fusion of ends after deletions.
Dicentric chromosome: Chromosome with two centromeres, often unstable.

Special Cases

Robertsonian Translocation: Fusion of two acrocentric chromosomes, leading to one large and one small (often lost) chromosome. Can cause familial Down syndrome.
Gene Fusion: Parts of two genes combine, potentially creating a chimeric gene with new functions.

Extranuclear (Cytoplasmic) Inheritance

Overview

Extranuclear inheritance refers to the transmission of genetic material through the cytoplasm, typically via organelles such as mitochondria and chloroplasts, rather than through nuclear DNA.

Organelle Heredity: Traits determined by DNA in mitochondria (mtDNA) or chloroplasts (cpDNA).
Infectious Heredity: Traits influenced by symbiotic or parasitic microorganisms.
Maternal Effect: Phenotype determined by substances (e.g., proteins, RNAs) present in the egg cytoplasm.

Mitochondrial and Chloroplast Genetics

Mitochondria and chloroplasts have their own circular DNA, similar to prokaryotes.
Endosymbiotic theory: These organelles originated from free-living bacteria engulfed by ancestral eukaryotic cells.
Most organelle proteins are encoded by nuclear genes and imported into the organelle.

Human mtDNA

Size: 16-18 kbp in mammals.
Encodes 13 polypeptides, 22 tRNAs, and 2 rRNAs.
Replication and transcription are independent of nuclear DNA.
Contains a D-loop region for replication and transcription control.
Exhibits differences from the universal genetic code (e.g., some codons code for different amino acids or stop signals).

Inheritance Patterns

Uniparental Inheritance: In animals, mitochondria are typically inherited maternally (from the egg).
Homoplasmy: All organelle DNA copies in a cell are identical.
Heteroplasmy: A mixture of normal and mutant organelle DNA within a cell, leading to variable expression of mitochondrial diseases.

Examples of Mitochondrial Disorders

MERRF (Myoclonic Epilepsy with Ragged Red Fibers): Caused by mutations in mitochondrial tRNA genes, leading to defects in ATP production. Severity depends on the proportion of mutant mtDNA (heteroplasmy).
Fragile X Syndrome: Although not mitochondrial, it is a genetic disorder caused by CGG repeat expansion on the X chromosome, leading to intellectual disability and developmental anomalies.

Chloroplast Inheritance

Chloroplasts also exhibit cytoplasmic inheritance, usually maternal but sometimes biparental.
Chloroplast DNA (cpDNA) is similar to bacterial DNA in structure and gene content.
Inheritance patterns can be observed in variegated plants, where leaf color depends on the source of the chloroplasts.

Source of Pollen	White Branch	Green Branch	Variegated Branch
White Branch	White	Green	White, green, or variegated
Green Branch	White	Green	White, green, or variegated
Variegated Branch	White	Green	White, green, or variegated

Cooperation Between Nuclear and Organelle Genomes

Many proteins required for mitochondrial and chloroplast function are encoded by nuclear genes.
Example: Cytochrome oxidase complex in mitochondria has subunits encoded by both nuclear and mitochondrial genomes.
Genetic material can be exchanged between the nucleus, mitochondria, and chloroplasts ("promiscuous DNA").