Week 2 - Cytogenetics Activity 1

Bone marrow aspirate/trephine Peripheral blood Lymph nodes Solid tumours

• Start off with dividing cells • Eventually analyse metaphase chromosomes (because that’s when we have all 46 chromosomes which are condensed for optimal analysis) • Mitotic arrest at metaphase • Block (synchronise) at S phase (interphase where DNA is replicated) to increase number of metaphase chromosomes

Acts as an antagonist to thymidylate synthetase interrupting the purine pathway (A&G) in the ‘S’ phase (holds cells in S phase to synchronise the culture) After time, all cells will be held in the ‘S’ phase

Thymidine unblocks the synchronised cells in the ‘S’ phase (that have been blocked by FdU) This allows cells to continue through the cell cycle

Colchicine is added at the metaphase step of the cell cycle which holds all cells at metaphase It inactivates the spindle formation These metaphase chromosomes are then put onto a slide for analysis

Treat sample with hypotonic solution of potassium chloride to swell the cells Fixed with methanol and acetic acid Done in a heat chamber as the humidity and temperature of the room affects the spreading of the metpahse chromosome and the banding quality

Technique to visualise bands/regions within metaphase chromosomes Trypsin partially digests the chromosome Dark bands: A-T rich, late replicating, heterochromatic regions (v few active genes) Light bands: C-G rich, early replicating, euchromatic regions (active genes)

Pericentric - involves the centromere Paracentric - do not involve the centromere (occur within a single arm of a chromosome)

Mutation - indels, point - e.g. deletions: if 1 copy deleted it leads to expression/change of function Amplification - repeated copying of DNA in replication leads to expansion in copy number, increases oncogene expression - e.g. double minutes - small pseudo chromosomes (dmins), homogenously staining regions (hsr’s) Recurrent chromosomal rearrangements - e.g. translocations and inversions - juxtaposition of gene next to IGH gene, falls under its control and is dysregulated OR - juxtaposition of gene next to another gene, other than IGH gene, forms novel hybrid fusion gene which produces chimeric protein with altered activity/function and transforming ability

BCL on chromosome 11 translocates to chromosome 14 which house the IGH gene, and due to the BCL gene being close to the IGH gene, it enhances cell proliferation