Analysis of ras gene mutations in rainbow trout tumors Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/z029p8464

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  • For ras gene mutation analysis in the rainbow trout (Oncorhynchus mykiss) model system, a partial trout ras sequence was identified using the polymerase chain reaction (PCR). Two synthetic oligonucleotides based on rat K-ras gene sequence were used as primers for the PCR procedure. A 90 base pair (bp) sequence, referred to as the trout K-ras, was amplified from trout genomic DNA and cDNA. Cloned 90 by PCR products from several normal liver tissues were sequenced resulting in the same sequence. Large-sized PCR products, 111 and 237 bp, were also cloned and sequenced indicating that these fragments included the 90 by sequence information expressed in mRNA. This 5'-terminal partial trout K-ras nucleotide sequence was 88% homologous to that of the goldfish ras gene, and less homologous to those of mammalian ras genes. Based on the partial sequence information of two trout ras genes, K-ras and H-ras, DNA from trout tumors induced by chemical carcinogens, aflatoxin B1 (AFB1) and N-methyl-N'-nitro-N-nitrosoguanidene (MNNG), were analyzed for the presence of point mutations. Using the PCR and oligonucleotide hybridization methods, a high proportion (10/14) of the AFB1-initiated liver tumor DNA indicated evidence for ras point mutations. Of the 10 mutant ras genotypes, seven were probed as G to T transversions at the second position of codon 12, two were G to T transversions at the second position of codon 13, and one was a G to A transition at the first position of codon 12. Nucleotide sequence analysis of cloned PCR products from four of these tumor DNAs provided definitive mutation evidence in each case, which seemed to occur in only a fraction of the neoplastic cells. However, no mutations were detected in exon 1 of the trout K-ras gene, nor in DNA from trout normal livers. Results indicated that the hepatocarcinogen AFB1 induced similar ras gene mutations in trout as in rat liver tumors. By comparison, the mutation specificity of MNNG in trout liver tumors was for G to A transitions, but no ras mutations were detected in trout kidney tumors. This investigation was the initial study of experimentally induced ras gene point mutations in a lower vertebrate fish model.
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