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Biomedical subjects

J M Nigro

Publications and source records attributed to J M Nigro.

9 recordsLinked to original sources
PubMed1992

Human p53 and CDC2Hs genes combine to inhibit the proliferation of Saccharomyces cerevisiae.

Human wild-type and mutant p53 genes were expressed under the control of a galactose-inducible promoter in Saccharomyces cerevisiae. The growth rate of the yeast was reduced in cells expressing wild-type p53, whereas cells transformed with mutant p53 genes derived from human tumors were less affected. Coexpression of the normal p53 protein with the human cell cycle-regulated protein kinase CDC2Hs resulted in much more pronounced growth inhibition that for p53 alone. Cells expressing p53 and CDC2Hs were partially arrested in G1, as determined by morphological analysis and flow cytometry. p53 was phosphorylated when expressed in the yeast, but differences in phosphorylation did not explain the growth inhibition attributable to coexpression of p53 and CDC2Hs. These results suggest that wild-type p53 has a growth-inhibitory activity in S. cerevisiae similar to that observed in mammalian cells and suggests that this yeast may provide a useful model for defining the pathways through which p53 acts.

Base Sequence↗
PubMed1991

Scrambled exons.

Using a sensitive assay for RNA expression, we identified several abnormally spliced transcripts in which exons from a candidate tumor suppressor gene (DCC) were scrambled during the splicing process in vivo. Cloning and sequencing of PCR-amplified segments of the abnormally spliced transcripts showed that exons were joined accurately at consensus splice sites, but in an order different from that present in the primary transcript. Four scrambled transcripts were identified, each involving a different pair of exons. The scrambled transcripts were found at relatively low levels in a variety of normal and neoplastic cells of rodent and human origin, primarily in the nonpolyadenylated component of cytoplasmic RNA. These results demonstrate that the splicing process does not always pair sequential exons in the order predicted from their positions in genomic DNA, thus creating a novel type of RNA product.

Amino Acid Sequence↗
PubMed1991

Mutant p53 proteins bind DNA abnormally in vitro.

The p53 gene encodes a phosphoprotein which binds DNA. Many types of tumors contain mutant p53 genes, but the effects of these mutations on the intrinsic properties of p53 are largely unknown. In the present study, we tested the effect of p53 mutations on DNA-binding. Each of 15 different mutant p53 gene products derived from human tumors or mouse transformants bound calf thymus DNA more weakly than did wild-type products. A significant subset of mutant proteins were also found to be underphosphorylated compared to the wild-type protein when produced in a reticulocyte lysate system, but this did not appear to explain the pattern of alterations of DNA-binding. The tested mutations were dispersed over several regions of the p53 gene and included representatives of all four of the evolutionarily conserved domains that are the known 'hot spots' for p53 mutation. The results suggest common pathways by which these various mutations affect the normal function of p53.

DNA↗
PubMed1990

Identification of a chromosome 18q gene that is altered in colorectal cancers.

Allelic deletions involving chromosome 18q occur in more than 70 percent of colorectal cancers. Such deletions are thought to signal the existence of a tumor suppressor gene in the affected region, but until now a candidate suppressor gene on this chromosomal arm had not been identified. A contiguous stretch of DNA comprising 370 kilobase pairs (kb) has now been cloned from a region of chromosome 18q suspected to reside near this gene. Potential exons in the 370-kb region were defined by human-rodent sequence identities, and the expression of potential exons was assessed by an "exon-connection" strategy based on the polymerase chain reaction. Expressed exons were used as probes for cDNA screening to obtain clones that encoded a portion of a gene termed DCC; this cDNA was encoded by at least eight exons within the 370-kb genomic region. The predicted amino acid sequence of the cDNA specified a protein with sequence similarity to neural cell adhesion molecules and other related cell surface glycoproteins. While the DCC gene was expressed in most normal tissues, including colonic mucosa, its expression was greatly reduced or absent in most colorectal carcinomas tested. Somatic mutations within the DCC gene observed in colorectal cancers included a homozygous deletion of the 5' end of the gene, a point mutation within one of the introns, and ten examples of DNA insertions within a 0.17-kb fragment immediately downstream of one of the exons. The DCC gene may play a role in the pathogenesis of human colorectal neoplasia, perhaps through alteration of the normal cell-cell interactions controlling growth.

Alleles↗
PubMed1989

Mutations in the p53 gene occur in diverse human tumour types.

The p53 gene has been a constant source of fascination since its discovery nearly a decade ago. Originally considered to be an oncogene, several convergent lines of research have indicated that the wild-type gene product actually functions as a tumour suppressor gene. For example, expression of the neoplastic phenotype is inhibited, rather than promoted, when rat cells are transfected with the murine wild-type p53 gene together with mutant p53 genes and/or other oncogenes. Moreover, in human tumours, the short arm of chromosome 17 is often deleted. In colorectal cancers, the smallest common region of deletion is centred at 17p13.1; this region harbours the p53 gene, and in two tumours examined in detail, the remaining (non-deleted) p53 alleles were found to contain mutations. This result was provocative because allelic deletion coupled with mutation of the remaining allele is a theoretical hallmark of tumour-suppressor genes. In the present report, we have attempted to determine the generality of this observation; that is, whether tumours with allelic deletions of chromosome 17p contain mutant p53 genes in the allele that is retained. Our results suggest that (1) most tumours with such allelic deletions contain p53 point mutations resulting in amino-acid substitutions, (2) such mutations are not confined to tumours with allelic deletion, but also occur in at least some tumours that have retained both parental 17p alleles, and (3) p53 gene mutations are clustered in four 'hot-spots' which exactly coincide with the four most highly conserved regions of the gene. These results suggest that p53 mutations play a role in the development of many common human malignancies.

Alleles↗
PubMed1989

Chromosome 17 deletions and p53 gene mutations in colorectal carcinomas.

Previous studies have demonstrated that allelic deletions of the short arm of chromosome 17 occur in over 75% of colorectal carcinomas. Twenty chromosome 17p markers were used to localize the common region of deletion in these tumors to a region contained within bands 17p12 to 17p13.3. This region contains the gene for the transformation-associated protein p53. Southern and Northern blot hybridization experiments provided no evidence for gross alterations of the p53 gene or surrounding sequences. As a more rigorous test of the possibility that p53 was a target of the deletions, the p53 coding regions from two tumors were analyzed; these two tumors, like most colorectal carcinomas, had allelic deletions of chromosome 17p and expressed considerable amounts of p53 messenger RNA from the remaining allele. The remaining p53 allele was mutated in both tumors, with an alanine substituted for valine at codon 143 of one tumor and a histidine substituted for arginine at codon 175 of the second tumor. Both mutations occurred in a highly conserved region of the p53 gene that was previously found to be mutated in murine p53 oncogenes. The data suggest that p53 gene mutations may be involved in colorectal neoplasia, perhaps through inactivation of a tumor suppressor function of the wild-type p53 gene.

Alleles↗
PubMed1987

cDNA cloning and mapping of the human creatine kinase M gene to 19q13.

We describe the first isolation of a human creatine kinase M cDNA clone and its mapping of the gene to human chromosome 19. A human creatine kinase M cDNA clone, pJN2CK-M, harboring a 1,160-bp insert, was isolated by colony hybridization with a previously sequenced chicken creatine kinase M cDNA probe. The human cDNA was used as a probe in Southern transfers of TaqI-digested genomic DNA from mouse/human somatic-cell hybrids to localize the human creatine kinase-M gene to chromosome 19. In situ hybridization of the tritiated cDNA probe to metaphase chromosomes of peripheral blood lymphocytes from normal males revealed significant labeling to chromosome 19. These two independent methodologies assign the human creatine kinase-M gene to chromosome 19. Since greater than 69% of the grains of chromosome 19 label band q13, the human creatine kinase-M gene has been mapped to 19q13. On the basis of high-resolution G-banding, the predominant labeling site was 19q13.2-q13.3.

Animals↗