Cloning of the syrian hamster p53 gene: Structural and functional characterization of the upstream promoter region

Abstract

We isolated the p53 gene from Syrian hamster embryo cells by cosmid cloning procedures. The organization of the hamster p53 gene was similar to that of other mammalian p53 genes; it had 11 exons, a noncoding exon 1, and a long intron 1 (about 6.5 kb). The upstream p53 promoter was isolated, and the nucleotide sequence of a region encompassing 694 bp upstream from the exon/intron 1 boundary plus the first 6 nt of intron 1 was determined. This genomic region was highly homologous to those of mice and humans but contained a repetitive element not present in either species. Sequence comparisons with the murine and human promoters revealed the presence of similar transcription-factor binding motifs mapping within a 431-bp SacI-PstI fragment. Transient transfection assays of primary Syrian hamster embryo cells and neoplastic cell lines with recombinant constructs in which the SacI-PstI fragment was placed upstream of a bacterial chloramphenicol acetyltransferase (CAT) gene revealed the efficient expression of CAT activity. Primer extension analyses identified several putative transcription initiation sites within the p53 upstream promoter, the strongest of which was located 315 bp upstream from the exon/intron 1 junction and about 30 bp upstream from the region encompassing the regulatory motifs.