Screening patients for heterozygous p53 mutations using a functional assay in yeast.
Inherited mutations of the p53 gene significantly increase the risk of developing diverse malignancies, and germline p53 mutations can be detected by assaying the transcriptional activity of the p53 protein in mammalian cells. Here we describe a method starting with lymphocytes that allows detection of germline p53 mutations by 'functional' analysis of p53 protein expressed in Saccharomyces cerevisiae. The p53 PCR products are directly cloned into yeast expression vectors in vivo and subsequently tested for transcriptional activity in a simple growth assay. This technique, functional analysis of separated alleles in yeast (FASAY), requires only a few steps, can be automated readily and should permit screening for germline or somatic heterozygous mutations in any gene whose function can be monitored in yeast.
Ishioka
C
C
Massachusetts General Hospital Cancer Center, Charlestown 02129.
Frebourg
T
T
Yan
Y X
YX
Vidal
M
M
Friend
S H
SH
Schmidt
S
S
Iggo
R
R
D000818:Animals; D001483:Base Sequence; D004247:DNA; D005820:Genetic Testing; D005822:Genetic Vectors; D006579:Heterozygote; D008214:Lymphocytes; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013698:Templates, Genetic; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D016159:Tumor Suppressor Protein p53; D004247:DNA