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