Graduation date: 2001

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

Life history within a single species can vary significantly. Many of these differences are associated with varying environmental conditions. Understanding what environmental conditions cue alternate life histories within a single species has been researched extensively. In salmonid fishes, more than almost any other group, varying environmental conditions give rise to individuals within species that take markedly different life history trajectories. Oncorhynchus mykissis a species of salmonid native to the Pacific Northwest region of North America. This species has two life history forms, anadromous and resident. The anadromous form spends a portion of its life in ocean while the resident life history form completes its entire life history in freshwater. Until the decision to migrate and morphological changes associated with smoltification occur, the two life history variants of this species are indistinguishable from each other. This ambiguity in juvenile O. mykiss morphology presents challenges for conservation managers charged with protecting and increasing threatened O. mykiss populations around the Pacific Northwest because conservation efforts cannot be evaluated until juvenile fish make the decision to migrate. Microarray gene expression analysis was used to profile gene expression in juvenile populations of wild and hatchery O. mykiss to identify gene expression variation associated with alternate life history variants. This analysis identified 8 DNA sequences present in both brain and gill tissues that differ in expression in rainbow trout and steelhead hatchery stocks. Differential expression as quantified by microarrays was validated with quantitative real-time PCR. Lastly, the expression of these putative life history markers was preliminarily evaluated in a wild population of O. mykiss at sample locations in the South Fork John Day River Basin, Oregon with known ratios of juvenile anadromous and resident fish.