A SIMPLE P53 FUNCTIONAL ASSAY FOR SCREENING CELL-LINES, BLOOD, AND TUMORS

Mutations in the p53 gene are implicated in the pathogenesis of half o f all human tumors, We have developed a simple functional assay for p5 3 mutation in which human p53 expressed in Saccharomyces cerevisiae ac tivates transcription of the ADE2 gene. Consequently, yeast colonies c ontaining wild-type p53 are white and colonies containing mutant p53 a re red, Since this assay tests the critical biological function of p53 , it can distinguish inactivating mutations from functionally silent m utations, By combining this approach with gap repair techniques in whi ch unpurified p53 reverse transcription-PCR products are cloned by hom ologous recombination in vivo it is possible to screen large numbers o f samples and multiple clones per sample for biologically important mu tations, This means that mutations can be detected in tumor specimens contaminated with large amounts of normal tissue. In addition, the ass ay detects temperature-sensitive mutants, which give pink colonies. We show here that this form of p53 functional assay can be used rapidly to detect germline mutations in blood samples, somatic mutations in tu mors, and mutations in cell lines.