A simple p53 functional assay for screening cell lines, blood, and tumors.
Mutations in the p53 gene are implicated in the pathogenesis of half of all human tumors. We have developed a simple functional assay for p53 mutation in which human p53 expressed in Saccharomyces cerevisiae activates transcription of the ADE2 gene. Consequently, yeast colonies containing wild-type p53 are white and colonies containing mutant p53 are red. Since this assay tests the critical biological function of p53, it can distinguish inactivating mutations from functionally silent mutations. By combining this approach with gap repair techniques in which unpurified p53 reverse transcription-PCR products are cloned by homologous recombination in vivo it is possible to screen large numbers of samples and multiple clones per sample for biologically important mutations. This means that mutations can be detected in tumor specimens contaminated with large amounts of normal tissue. In addition, the assay 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 tumors, and mutations in cell lines.
Flaman
J M
JM
Unité de Génétique Moléculaire, Centre Hospitalier, Universitaire de Rouen, France.
Frebourg
T
T
Moreau
V
V
Charbonnier
F
F
Martin
C
C
Chappuis
P
P
Sappino
A P
AP
Limacher
I M
IM
Bron
L
L
Benhattar
J
J
D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D002262:Carboxy-Lyases / Q000096:biosynthesis; D002460:Cell Line; D003001:Cloning, Molecular; D003062:Codon; D017931:DNA Primers; D005260:Female; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D016158:Genes, p53; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis; D012016:Reference Values; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis*
D003062:Codon; D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D002262:Carboxy-Lyases; C022378:phosphoribosylaminoimidazole carboxylase