Selective detection of inactivating mutations of the p53 tumour suppressor gene: Development of a new functional assay in yeast
Abstract
Alterations of the p53 tumour suppressor gene in human cancer are mainly missense mutations. We have previously described a functional assay in Saccharomyces cerevisiae based on a HIS3 reporter system, which allows the selective detection of mutations which have inactivated the transcriptional activity of p53. We have now developed a simpler reporter system in which yeast change color according to their p53 status. We used an ade2-1 strain which contains an intergrated copy of the ADE2 open reading frame controlled by a p53-sensitive promoter. In the absence of wild-type p53, the strain is red due to the accumulation of an intermediate in adenine metabolism. As in the previous assay, we transform PCR-amplified p53 cDNA directly into the strain and clone them into a p53 expression vector by homologous recombination in vivo. Colonies that contain wild-type p53 overcome the block in adenine synthesis and become white. After two days, the white/red system allows to distinguish very easily wild-type and mutant p53 alleles. This new assay is a simple and rapid method to detect inactivating p53 mutations in clinical samples.
- Authors:
-
- CHU de Rouen (France)
- ISREC, Epalinges (Switzerland)
- Publication Date:
- OSTI Identifier:
- 133397
- Report Number(s):
- CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-0125
- Resource Type:
- Journal Article
- Journal Name:
- American Journal of Human Genetics
- Additional Journal Information:
- Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 55 BIOLOGY AND MEDICINE, BASIC STUDIES; GENES; TRANSCRIPTION; GENE MUTATIONS; GENE RECOMBINATION; PATIENTS; NEOPLASMS; DETECTION; YEASTS; ABSORPTION SPECTROSCOPY
Citation Formats
Frebourg, T, Flaman, J M, and Iggo, R. Selective detection of inactivating mutations of the p53 tumour suppressor gene: Development of a new functional assay in yeast. United States: N. p., 1994.
Web.
Frebourg, T, Flaman, J M, & Iggo, R. Selective detection of inactivating mutations of the p53 tumour suppressor gene: Development of a new functional assay in yeast. United States.
Frebourg, T, Flaman, J M, and Iggo, R. 1994.
"Selective detection of inactivating mutations of the p53 tumour suppressor gene: Development of a new functional assay in yeast". United States.
@article{osti_133397,
title = {Selective detection of inactivating mutations of the p53 tumour suppressor gene: Development of a new functional assay in yeast},
author = {Frebourg, T and Flaman, J M and Iggo, R},
abstractNote = {Alterations of the p53 tumour suppressor gene in human cancer are mainly missense mutations. We have previously described a functional assay in Saccharomyces cerevisiae based on a HIS3 reporter system, which allows the selective detection of mutations which have inactivated the transcriptional activity of p53. We have now developed a simpler reporter system in which yeast change color according to their p53 status. We used an ade2-1 strain which contains an intergrated copy of the ADE2 open reading frame controlled by a p53-sensitive promoter. In the absence of wild-type p53, the strain is red due to the accumulation of an intermediate in adenine metabolism. As in the previous assay, we transform PCR-amplified p53 cDNA directly into the strain and clone them into a p53 expression vector by homologous recombination in vivo. Colonies that contain wild-type p53 overcome the block in adenine synthesis and become white. After two days, the white/red system allows to distinguish very easily wild-type and mutant p53 alleles. This new assay is a simple and rapid method to detect inactivating p53 mutations in clinical samples.},
doi = {},
url = {https://www.osti.gov/biblio/133397},
journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = {Thu Sep 01 00:00:00 EDT 1994},
month = {Thu Sep 01 00:00:00 EDT 1994}
}