Graduate Thesis Or Dissertation
 

Characterization of a ras gene in rainbow trout

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  • The rainbow trout (Oncorhynchus mykiss) model of chemical carcinogenesis is becoming increasingly important as a supplement to rodent studies. However, much of the molecular biology of the carcinogenic response is still unknown in the trout model. The ras gene family has been implicated in the tumorigenesis of both spontaneous and chemically-induced tumors in mammals. This study is the first to characterize a ras proto-oncogene in rainbow trout. To accomplish this, the ras gene sequence was amplified in vitro by using polymerase chain reaction (PCR). Two synthetic and degenerative oligonucleotide sequences based on a consensus mammal/goldfish ras sequence were used as primers in the PCR procedure. An 800 base pair (bp) sequence was amplified from trout genomic DNA and hybridized with a human c-Ha-ras sequence. The initial amplifications of trout liver cDNA using the PCR procedure with the synthetic ras primers resulted in a single product of approximately 216 bps. However, this amplified "trout" 216 by product was subsequently shown to be an artifact of carryover from a human Ki-ras plasmid. Carryover is a common problem found in many laboratories involved with the PCR procedure, and extensive precautions were used to eliminate the problem in our laboratories. The 800 by PCR product was cloned and sequenced using Taq polymerase. RT-8, a clone containing the 800 bp insert, was shown to have 91% homology to the first two exons of mammalian c-Ha-ras gene and lesser homology to other ras genes. Amplification of trout liver cDNA using specific primers based on the RT-8 sequence resulted in the amplification of sequences identical to the sequence of the RT-8 insert without an intron, as well as unique sequences, which may represent additional trout ras genes. The PCR procedure was modified to identify sequence information immediately 3' of the known trout ras sequence. Partial sequences of at least two different trout ras genes are presented. With this new information, analysis of DNA sequence information from chemically initiated tumors may elucidate the role activation of ras genes plays in the trout model of carcinogenesis.
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