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

[摘要] Cyclin-dependent kinases (CDKs) are crucial regulators of the cell cyele. CDKsthemselves are subject to control by both cyelins and CDK inhibitors. Among theinhibitors, p 16 is very prominent, since it has been found to be mutated or lost in avariety óf tumours, We are interested in mutations involved in the progression ofleukemia from the chronic to the acute phase. The p 16 gene has been implicated inthis progression, therefore we needed an assay for p 16 status that could be applied toscreen patients in chronic phase regularly.Traditional mutation screening makes use of physical methods such as SingleStranded Conformational Polymorphism (SSCP) analysis. These methods aregenerally labour intensive and are not always informative. If tests for the actualfunction for the gene products could be devised, it could be used to screen tumoursamples for the status of these genes.We have decided to develop a yeast-based test that would directly assay for activityrather than just nucleotide changes. The assay is based on the yeast two-hybridsystem, where protein-protein contact is reflected in colony colour. We havedesigned a primer set to amplify the p16 reading frame by RT-PCR from smallamounts of leukocyte mRNA. This cDNA is then cotransformed with a gappedplasmid containing terminal p 16 overlaps, allowing homologous recombination tosplice 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 colonieswould turn blue.We have successfully constructed and tested the system and found it to be verysensitive, being able to assay p 16 from as little as 300 microliters of whole blood.

[发布机构] University of the Free State