A yeast-based assay for detection of mutations in the human p16 gene

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.