The aim of this study was to examine the claim that a single, mutant oncogene can transform NIH 3T3 mouse fibroblasts into transformed, tumorigenic cells, acting in a genetically dominant fashion. A c.Ha-ras 1 oncogene, cloned from the EJ human bladder carcinoma cell line, was inserted into a shuttle vector carrying the selectable marker gene gpt, which encodes the enzyme XPRT (xanthine-guanine phosphoribosyl transferase). This construct, pSV2gptEJ, was transfected into NIH 3T3 cells by the calcium phosphate precipitation method and cells which had incorporated the plasmid were selected by growth in mycophenolic acidcontaining medium, to which gpt confers resistance. A number of clonal lines were established and their tumorigenicity tested. Tumour cell lines derived from these transfectants were back-selected using 2-thioxanthine, a cytotoxic analogue of the xanthine-guanine phosphoribosyl transferase substrate, to isolate clones which no longer contained functional pSV2gptEJ sequences. Six sub-clones which did not express detectable levels of the ras oncogene product, p21^H.ras , were obtained. All were judged to be less transformed than the transfected parent cells: they appeared morphologically normal, were more serum-sensitive, showed clear saturation densities and were more anchorage-dependent. Three of these sub-clones were found to be tumorigenic at all sites tested. Cytological examination of the NIH 3T3 transfectants revealed that significant perturbation of their chromosome complement accompanied transfection. The transfection process, in the absence of DNA or with pSV2gpt alone, was found to be capable of transforming NIH 3T3 cells. Finally, a brief investigation of the effect of a "functional EJ-ras gene upon the differentiated phenotypes of these cell lines was attempted by comparing their ability to produce an extracellular matrix.

No description available.

Characterization of the 5' flanking region of rainbow trout ki-ras gene was begun with the cloning and sequencing of this region by the inverse PCR technique and dideoxynucleotide chain termination method. In total, a nucleotide sequence of 1080 bp upstream from the first coding ATG was sequenced. Although this region showed certain promoter elements, it does not share common features with other mammalian ras promoters, which lack the TATA and contain multiple GC boxes with Spl binding activities. In contrast, this region in trout ras contains typical TATA and CCAAT boxes. This structural difference of the trout ki-ras promoter from that of other mammalian ras genes may suggest that different transcriptional regulation mechanisms of the ras ger.e are used at various levels in evolution. The chromatin structure of the trout ki-ras gene was studied by probing invivo for DNase I hypersensitive sites. To overcome the difficulties of using the traditional indirect end labeling method for a single-copy gene, the technique of ligation-mediated PCR was applied. No hypersensitive sites were observed at or near the codon 12 region of the gene, either in normal (protooncogene) or tumor (oncogene) tissue from the liver. This result suggests that the local chromatin structure of trout ki-ras gene may not be an important factor for codon 12 mutations induced by genotoxins, and that changes of chromatin structure are unlikely to be promoted after tumor formation. Studies by micrococcal nuclease demonstrate that this ras gene, in the region around 12, lacks ordered nucleosome positioning or may be even free of nucleosomes. Such an irregular organization of ras oncogenic chromatin would resemble that of many other "normal", highly active eukaryotic genes. The intrinsic affinity of trout ki-ras gene for aflatoxin B₁ was determined by in vitro alkylation experiments. Exon 1 of the gene was synthesized and labeled at the 5'end of the coding strand by the PCR technique. Taking advantage of the selective cleavage of AFB1-DNA adducts by piperidine under alkali conditions, the frequency of AFB 1 attack to each guanyl site was determined by densitometric scans after the cleaved fragments were electrophoresed on sequencing gels. The results demonstrated that two guanyl sites of codon 12 had differential affinity to AFBl, the more 5' G was relatively inaccessible but the more 3' G was accessible, indicating that the sequence selectivity of AFB I may contribute to the preference of the initial adduction in vivo. / Graduation date: 1993

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Thesis (Ph.D.)--Ohio University, August, 2008. / Title from PDF t.p. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains iv, 171 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 252-253). Also available in print.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.