Karyotype

Question 1

What is karyotype?

Answer 1

A karyotype is the complete set of an organism’s chromosomes, often referring to a lab-produced image of these chromosomes, organized by size, shape, and number into homologous pairs.
Karyotyping is the process of pairing and ordering all the chromosomes of an organism.
Karyotypes are prepared using standardized staining procedures that reveal characteristic structural features for each chromosome.

Question 2

What is normal chromosome morphology?

Answer 2

p (petit) is the short arm of the chromosome
The centromere is the point of constriction
q is the long arm of the chromosome.
If chromosome not attached to mitotic spindle will be left out of daughter cell.

The telomeres are repeat, non-coding DNA at the end of the chromosomes.
The main functions of a telomere are to maintain chromosomal stability and prevent chromosomal degradation. Additionally, telomeres protect the ends of the chromosome from DNA end-joining to one another (short & long end), damage response to DNA, and accidental DNA recombination (especially during replication).

If the telomeres are lost during DNA replication, then the chromosome will be functional and no coding DNA will be lost.

When the telomeres are too short, cell enters senescence –> apoptosis.
The condensation of chromosomes in mitosis prevents transcription as transcription factors cannot bind to promoters.

Question 3

How are chromosomes sorted?

Answer 3

Chromosomes are categorised by size (length) and centromere position.

Metacentric chromosomes have their centromere located precisely in the middle, resulting in two arms of approximately equal length.

Submetacentric - the centromere is positioned slightly off-center, resulting in one shorter chromosome arm (the p arm) and one longer chromosome arm (the q arm). (Eg. Chromosomes 5,7 &18).

Acrocentric - centromere is not central and is instead located near the end of the chromosome. One arm is much shorter than the other. (No real short arms) Eg. chromosomes 13, 14, 15, 21, 22 and Y. These chromosomes can undergo certain types of structural abnormalities.

Question 4

How does banding differentiate chromosomes?

Answer 4

Each chromosome has a unique banding (staining) pattern.
Sub-bands (subdivision) denoted by decimal points.

Question 5

What are the functional elements of chromosomes?

Answer 5

Proper chromosome function requires:
1. Centromeric sequences - repetitive DNA elements that form the core of the centromere, the region on a chromosome where the kinetochore assembles and spindle fibers attach.

2. Autonomous replicating sequences (ARS, origin of replication.
3. Telomeric Sequences/Telomeres - structural integrity (prevents sticky ends); solves end-replication problem; assists with chromosome positioning.
4. Nucleolar organising regions (NORS - acrocentric) - specific chromosomal locations (satellite stalks) containing ribosomal DNA (rDNA) genes, which are crucial for ribosome biogenesis and the formation of the nucleolus (interphase). 10 ineach cell (2 per chromosome)

Question 6

Explain Centromeric DNA

Answer 6

Question 7

Explain Autonomously relpicating sequences

Answer 7

Question 8

Describe mitotic chromosomes

Answer 8

They are condensed; seen individually under the microscope; extreme packaging ensures genes are switched off.

Question 9

Describe Interphase chromosomes

Answer 9

They are long, extended; not seen individually.
Tangled mass (chromatin)
Allows gene expression.

Question 10

What are the proteins and their arrangement in the nuclesome core?

Answer 10

The nucleosome core has 8 histone (H) molecules in an octamer arrangement.

2 each of H2A; H2B; H3 & H4
There are 147bp of DNA wrapped around the core 1.67 times.

Each nucleosome separated from next 50-70bp of linker DNA.

The 5th type of histone (H1) attaches to linker DNA between 2 nucleosomes.

Question 11

Differentiate between Heterochromatin and Euchromatin

Answer 11

Question 12

Talk about the sex chromosomes

Answer 12

Question 13

What is Dosage Compensation

Answer 13

A mechanism which ensures that girls (two X chromosmes) do not have too much or boys (one X and one Y chromosome) too little expression of genes on the X chromosome. It allows standard structural genes on the X chromosome to be expressed at the same levels in females and males, regardless of the numbe of X chromosomes.

It occurs in females via inactivation of one X chromosome - done mainly by DNA methylation.

Question 14

How is X chromosome inactivation done

Answer 14

The Inactive X has the properties of heterochromatin (highly condensed, methylated cytosine residues).

This is seen as a Barr body (dark-staining chromatin) within the nucleus.

Inactivation must occur for at least on X chromosome or miscarriage.

In Trisomy X (48,XXX), 2/3 X usually inactivated. An extra X chromosome usually causes symptoms.

Question 15

Which gene controls X inactivation?

Answer 15

Question 16

What is the process of karyotyping?

Answer 16

Step 1: Obtain cells (assuming all cells affected - genetic defect constitutional) can take any cells.
Eg. Peripheral Nervous Blood, Solid Tumour cells, Amniotic fluid, chorionic villus biopsy and bone marrow biopsy.

2. Set up culture media with mitotic stimulants.
3. Incubate culture 37C. 5%-6%CO2
4. Harvest cells - mitotic arrest in metaphase (with colcemid). Hypotonic solution (usually KCl). Fixation (methanol and acetic acid).
5. Slide preparation -fixed cells placed on glass slide. Slides dried, aged and stained.
6. Microscopy - slides viewed with different methods. - G-banding, computer generated (image captured with digital camera, sent to computer , software pairs chromosomes to produce karyotype). The banding patterns for each different chromosome are characteristic and reproducible, resembling a barcode. Cytogeneticist compares banding patterns of homologous chromosomes to determine earrangements, deletions or duplications.

Question 17

Describe G-banding Microscopy

Answer 17

Question 18

What is Fluorescence in situ Hybridisation? (FISH)

Answer 18

Use of probes to identify regions of chromosome.
Probes may be manufactured to hybridise with : Centromere,
Telomeres,
Specific regions of interest,
Entire chromosomes (whole chromosome probes/whole chromosome paint).

Probe labelled with fluorophore to enable viualisation under fluorescence microscope.

Question 19

What are the applications of FISH?

Answer 19

Accurate delineation of reciprocal translocations

Detection of microdeletions

Detection of complex translocations

Detection of fusion genes in tumours (hybrid genes formed from two different, previously independent genes due to chromosomal rearrangements like translocations, inversions, or deletions that drive diseases or cause cancers).

Preimplantation Genetic diagnosis (PGD)