Mutation detection by a two-hybrid assay.

Yeast-based assays have been developed to detect inactivating mutations in human genes, but these assays generally rely on the human protein having a biological function in yeast. We describe a simple method to detect mutations by virtue of their ability to abolish a protein-protein interaction in the yeast two-hybrid assay. By the use of direct recombinational cloning in yeast of a reverse transcription-PCR product followed by a simple growth selection this method distinguished both homozygous and heterozygous mutations in the p53 tumor suppressor gene. This approach should be applicable to many human genes whose encoded proteins have suitable partners in the two-hybrid assay.

[1]  K. Isselbacher,et al.  Detection of heterozygous truncating mutations in the BRCA1 and APC genes by using a rapid screening assay in yeast. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[3]  S. Fields,et al.  Two cellular proteins that bind to wild-type but not mutant p53. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Fields,et al.  Analyzing protein-protein interactions using two-hybrid system. , 1995, Methods in enzymology.

[5]  S. Fields,et al.  Distinct residues of human p53 implicated in binding to DNA, simian virus 40 large T antigen, 53BP1, and 53BP2 , 1994, Molecular and cellular biology.

[6]  Anne M. Bowcock,et al.  Identification of a RING protein that can interact in vivo with the BRCA1 gene product , 1996, Nature Genetics.

[7]  F. Winston,et al.  A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. , 1987, Gene.

[8]  P. Chappuis,et al.  A simple p53 functional assay for screening cell lines, blood, and tumors. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[9]  P. Bartel,et al.  RAD51 Interacts with the Evolutionarily Conserved BRC Motifs in the Human Breast Cancer Susceptibility Gene brca2 * , 1997, The Journal of Biological Chemistry.

[10]  S. Elledge,et al.  The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. , 1993, Genes & development.

[11]  S. Friend,et al.  Screening patients for heterozygous p53 mutations using a functional assay in yeast , 1993, Nature genetics.

[12]  S. Powers,et al.  RAS in yeast: complementation assays for test of function. , 1995, Methods in enzymology.

[13]  A. Reymond,et al.  p16 proteins from melanoma-prone families are deficient in binding to Cdk4. , 1995, Oncogene.

[14]  G. Fink,et al.  Methods in yeast genetics , 1979 .

[15]  S. P. Fodor,et al.  Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two–colour fluorescence analysis , 1996, Nature Genetics.