|dc.description.abstract||Cyclin-dependent kinases (CDKs) are crucial regulators of the cell cyele. CDKs
themselves are subject to control by both cyelins and CDK inhibitors. Among the
inhibitors, p 16 is very prominent, since it has been found to be mutated or lost in a
variety óf tumours, We are interested in mutations involved in the progression of
leukemia from the chronic to the acute phase. The p 16 gene has been implicated in
this progression, therefore we needed an assay for p 16 status that could be applied to
screen patients in chronic phase regularly.
Traditional mutation screening makes use of physical methods such as Single
Stranded Conformational Polymorphism (SSCP) analysis. These methods are
generally labour intensive and are not always informative. If tests for the actual
function for the gene products could be devised, it could be used to screen tumour
samples for the status of these genes.
We have decided to develop a yeast-based test that would directly assay for activity
rather than just nucleotide changes. The assay is based on the yeast two-hybrid
system, where protein-protein contact is reflected in colony colour. We have
designed a primer set to amplify the p16 reading frame by RT-PCR from small
amounts of leukocyte mRNA. This cDNA is then cotransformed with a gapped
plasmid containing terminal p 16 overlaps, allowing homologous recombination to
splice the reading frame into the plasmid. The host strain also contains a CDK4-
expressing plasmid and if the amplified p16 can still bind to CDK4, the colonies
would turn blue.
We have successfully constructed and tested the system and found it to be very
sensitive, being able to assay p 16 from as little as 300 microliters of whole blood.||en_ZA