The 5'' region of the p53 gene: evolutionary conservation and evidence for a negative regulatory element.

The 5' regions of the mouse, rat and human functional p53 genes were isolated and analysed. All three genes possess a non-coding exon, comprising exclusively 5' untranslated sequences. This exon contains extensive diad symmetry near the 5' end of p53 mRNA, possibly allowing for the formation of a stable hairpin structure in this mRNA. The nucleotide sequence within this hairpin element is highly conserved among the species. A DNA stretch of 225 bp preceding the p53 mRNA cap site possesses distinct promoter activity when assayed in the CAT system. However, this activity is practically abolished when further upstream p53 sequences (approximately 120 bp) are included in front of the CAT gene. This suggests that the control of p53 gene expression is complex and involves a negative regulatory element.     

Characterization of the 5' and 3' untranslated regions in murine mdm2 mRNAs

The murine double minute 2 (mdm2) gene is essential for embryogenesis in mice that express the p53 tumor suppressor protein. Mdm2 levels must be regulated tightly because overexpression of mdm2 contributes to tumorigenesis. We investigated whether the 5' and 3' untranslated regions (UTRs) of murine mdm2 affect the expression of MDM2 proteins. Induction of mdm2 expression by p53 results in synthesis of an mdm2 mRNA with a short 5' UTR. The long 5' UTR increases internal initiation of translation of a minor MDM2 protein, p76(MDM2), without affecting the efficiency of translation of the full-length p90(MDM2). We discovered two alternative 3' untranslated regions in murine mdm2 mRNA expressed in the testis. The longer 3' UTR contains a consensus instability element, but mdm2 mRNAs containing the long and short 3' UTRs have comparable half-lives. The 3' UTRs do not affect either initiation codon use or translation efficiency. Thus, the murine 5' UTR, but not the 3'UTR, influences the ratio of the two MDM2 proteins but neither UTR affects MDM2 abundance significantly.     

Lack of expression of the protein p53 gene in a human T-cell leukemia line

Expression of the gene encoding the nuclear phosphoprotein p53 (a proto-oncogene classified in the same functional family as c-myc and E1a adenovirus gene) was examined in a human T-cell leukemia (KE-37R cell line). No p53 (or a modified product) could be detected by immunoprecipitation with monoclonal antibodies P Ab 421 and P Ab 122 in KE-37R cell extracts, and no p53-specific RNA was characterized by Northern blot analysis. Southern blot using a murine p53 cDNA clone as a probe, did not reveal any gross rearrangement in the structure of the gene. However, this molecular probe was not suited for investigating the 5' end of the gene which contains the promoter and the non coding exon 1. It is interesting to notice that in KE-37R cells, c-myc has been activated by a t(8; 14) (q24; q11) translocation, suggesting that the c-myc product might substitute to some functions normally requiring p53.     

The detection of mutations induced in vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay.

We have analysed five mutation hotspots within the p53 gene (codons 175, 213, 248, 249, and 282) for mutations induced by hydrogen peroxide (H(2)O(2)), employing the restriction site mutation (RSM) assay. In addition, four other restriction sites covering non-hotspot codons of exons 5-9 of the p53 gene (codons 126, 153/54, 189 and the 3' splice site of exon 9) were analysed by the RSM assay for H(2)O(2)-induced mutations. Two cell types were concurrently analysed in this study, i.e. primary fibroblast cells and a gastric cancer cell line. Using the RSM assay, H(2)O(2)-induced mutations were only detected in exon 7 of the p53 gene. This was true for both cell types. These mutations were mainly induced in the Msp I restriction site (codon 247/248) and were predominantly GC to AT transitions (71%). Hence these GC to AT mutations were presumably due to H(2)O(2) exposure, possibly implicating the 5OHdC adduct, which is known to induce C to T mutations upon misreplication. Importantly, this study demonstrates that the RSM methodology is capable of detecting rare oxidative mutations within the hotspot codons of the p53 tumour suppressor gene. Hence, this methodology may allow the detection of early p53 mutations in pre-malignant tissues.     

Identification of polypeptides encoded in open reading frame 1b of the putative polymerase gene of the murine coronavirus mouse hepatitis virus A59.

The polypeptides encoded in open reading frame (ORF) 1b of the mouse hepatitis virus A59 putative polymerase gene of RNA 1 were identified in the products of in vitro translation of genome RNA. Two antisera directed against fusion proteins containing sequences encoded in portions of the 3'-terminal 2.0 kb of ORF 1b were used to immunoprecipitate p90, p74, p53, p44, and p32 polypeptides. These polypeptides were clearly different in electrophoretic mobility, antiserum reactivity, and partial protease digestion pattern from viral structural proteins and from polypeptides encoded in the 5' end of ORF 1a, previously identified by in vitro translation. The largest of these polypeptides had partial protease digestion patterns similar to those of polypeptides generated by in vitro translation of a synthetic mRNA derived from the 3' end of ORF 1b. The polypeptides encoded in ORF 1b accumulated more slowly during in vitro translation than polypeptides encoded in ORF 1a. This is consistent with the hypothesis that translation of gene A initiates at the 5' end of ORF 1a and that translation of ORF 1b occurs following a frameshift at the ORF 1a-ORF 1b junction. The use of in vitro translation of genome RNA and immunoprecipitation with antisera directed against various regions of the polypeptides encoded in gene A should make it possible to study synthesis and processing of the putative coronavirus polymerase.     

Vectorettes for long chromosome walking in genomic DNA of the human p53 gene

Chromosome walking in mammalian DNA by vectorette PCR is not always very specific, and the walks have been limited to distances <1 kb. To improve the method, we have designed new vectorettes, which unlike the currently used ones have very little repetitive sequences or homology with known DNA sequences of various origins in the data banks. We have tested these new vectorettes for chromosome walking in human p53 tumor suppressor gene, human tissue-type plasminogen activator gene, and mouse stanniocalcin gene with good success. In chromosome walking of the human p53 gene, we isolated gene-specific fragments of 2.4. kb, and by walking in a bacterial artificial chromosome (BAC) clone carrying the mouse stanniocalcin gene, we isolated fragments up to about 7 kb in size. We further sequenced the 5' region of the p53 gene and found that the nucleotides upstream of -1009 are transcribed in antisense orientation into a messenger RNA (mRNA) (flj10385) encoding a putative serine/threonine kinase.