| Summary: | 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. === Graduation date: 1991
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