Reporter gene regulation in Saccharomyces cerevisiae by the human p53 tumor suppressor protein.
This study evaluated the transcriptional regulation of four reporter genes in Saccharomyces cerevisiae by the human tumor suppressor protein p53. The S. cerevisiae ADE2, HIS3 and URA3 genes were used with nutritional selections and the E. coli LacZ gene was used to quantitate reporter gene activation. DNA elements containing binding sites for p53 were introduced upstream of several 5' truncated yeast promoters and used to express reporter genes. Human p53 cDNA was expressed at different levels by utilizing three different yeast promoters. All reporter genes were activated by p53, and in the case of nutritional selections, basal reporter gene expression could be detected in the absence of p53. A gap repair assay was evaluated and optimized for the purpose of determining whether p53 encoded in various cDNA sources was functional in transcriptional transactivation. The basal levels of reporter gene transcription in the absence of p53 could be decreased by integration of the reporter gene in the chromosome. For several expression systems, p53 appears to be limiting since higher levels of reporter gene expression were observed when the p53 cDNA was expressed from more efficient promoters. The gap repair assay can be used to determine the genotype (homozygous wild type, homozygous mutant or heterozygous) for cDNA generated from human cell lines or tissue samples. This assay can also be used to evaluate mutation rates associated with various conditions for in vitro PCR amplification of DNA.
Bitter
Grant A
GA
BitTech, Inc., Agoura Hills, CA 91376-1499, USA. gab@bittech.net
Schaeffer
Timothy N
TN
Ellison
Aaron R
AR
D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D006801:Humans; D007763:Lac Operon / Q000502:physiology; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012641:Selection, Genetic; D015533:Transcriptional Activation; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*
D029701:Saccharomyces cerevisiae Proteins; D016159:Tumor Suppressor Protein p53