A simple p53 functional assay for screening cell lines, blood, and tumors.

Flaman, Jean–Michel; Frébourg, Thierry; Moreau, Viviane; Charbonnier, Françoise; Martin, Cosette; Chappuis, Pierre O.; Sappino, André‐Pascal; Limacher, I M; Bron, Luc; Benhattar, Jean

doi:10.1073/pnas.92.9.3963

Cited by 409 publications

(481 citation statements)

References 25 publications

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“…RNA was isolated from fresh blood as described above in the DFCI laboratory and analysis of p53 transcriptional activity was performed by OncorMed, Inc. (Gaithersburg, MD, USA) according to Flaman et al (1995). …”

Section: Yeast P53 Functional Assaymentioning

confidence: 99%

Novel p53 splice site mutations in three families with Li-Fraumeni syndrome

et al. 2000

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Germline mutations in the p53 tumor suppressor gene predispose to a variety of cancers in families with LiFraumeni syndrome. Most germline p53 mutations observed to date cause amino acid substitutions in the protein's central sequence-speci®c DNA binding domain. Outside this conserved core region, however, we found novel alterations in sequences that regulate precursor mRNA splicing in three Li-Fraumeni syndrome families. Two splice site mutations a ected the consensus sequence at the splice donor sites of introns 1 and 9, and produced unstable variant transcripts in normal cells. A third mutation at the splice acceptor site of intron 9 generated splicing at a cryptic acceptor site in intron 9. These splice site alterations emphasize the need to examine both noncoding and untranslated regions of the p53 gene for germline mutations in Li-Fraumeni syndrome families. Oncogene (2000) 19, 4230 ± 4235.

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Section: Yeast P53 Functional Assaymentioning

confidence: 99%

Novel p53 splice site mutations in three families with Li-Fraumeni syndrome

et al. 2000

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“…The yeast reporter strain yIG397 (Flaman et al, 1995) was used throughout this study. The genotype is MATaade 2-1 leu2-3, 112trp1-1his3-11, 15can1-100ura3-1 URA3 36RGC::pCYC1::ADE2.…”

Section: Yeast P53 Functional Assaymentioning

confidence: 99%

“…Although these methods are practically sensitive and widely used, they cannot distinguish among polymorphisms, functionally silent mutations, and inactivating mutations. Recently, a di erent approach based on a biological assay for p53 function has been made, which uses yeast as a living test tube (Ishioka et al, 1993;Flaman et al, 1995). This method takes advantage that gapped plasmid is repaired by homologous recombination of the linear plasmid and DNA fragment in yeast (Orr-Weaver et al, 1983).…”

Section: Introductionmentioning

confidence: 99%

“…This method takes advantage that gapped plasmid is repaired by homologous recombination of the linear plasmid and DNA fragment in yeast (Orr-Weaver et al, 1983). The p53 functional assay in which yeast change color according to the p53 status raises the very real possibility of simple and large-scale screening for p53 functional mutations (Flaman et al, 1995) in clinical samples.…”

Section: Introductionmentioning

confidence: 99%

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High frequency of p53 mutations in human oral epithelial dysplasia and primary squamous cell carcinoma detected by yeast functional assay

et al. 1997

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To determine the timing and actual incidence of p53 mutations in oral epithelial lesions, we examined 33 primary squamous cell carcinomas (SCCs), 14 dysplasias and six hyperplasias from Japanese patients by a combination of yeast functional assay and DNA sequencing. The assay detects mutations of p53 mRNA between codons 67 and 347 on the basis of the DNAbinding activity of the protein. Twenty-six SCCs (79%) and ®ve dysplasias (36%) were positive for p53 mutation, while all six hyperplasias were negative for the mutation. Human papillomavirus type 16 E6 mRNA was detected in one of seven p53 mutation-negative SCCs by reverse transcription polymerase chain reaction (RT ± PCR). We further examined p53 mutations in 17 Sri Lankan oral SCCs using the yeast functional assay and the singlestrand conformation polymorphism analysis of PCRampli®ed DNA fragments (PCR ± SSCP) of exon 5 ± 8. The mutations were con®rmed by DNA sequencing and the detection sensitivity was compared between the two methods. Six samples (35%) were positive for p53 mutation in PCR ± SSCP analysis, while nine samples (53%) were positive in yeast functional assay. This suggests that the incidence of p53 mutations has been considerably underestimated in the conventional SSCP analysis. The present data indicate that p53 mutations are extremely frequent in oral cancers in the Japanese, and suggest that the timing and signi®cance of p53 mutation in oral tumor progression vary in di erent ethnic populations and areas.

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“…Initially the assay, based on the inability of the majority of TP53 mutants to transactivate target genes, was established in human cells (Frebourg et al, 1992). However with the demonstration that mammalian TP53 can function in yeast cells (Scharer and Iggo, 1992), a simple functional assay was described (Ishioka et al, 1993;Flaman et al, 1995). This assay will now provide a mechanism by which mutations can be assessed for their transactivation properties.…”

mentioning

confidence: 99%