This project seeks to apply genetic manipulations possible in the trout system to examine genetic mechanisms of carcinogenesis. The study should provide basic insights into the carcinogenesis process using this non-mammalian vertebrate model. The first specific aim is to examine tumor incidence in diploid, triploid and clonal rainbow trout. In fish, unlike mammals, triploid individuals can be readily induced and are viable. In our studies to date, induced tumors were much more common in diploid than in triploid rainbow trout in three of four target organs tested. This might be due to the involvement of tumor suppressor genes in carcinogenesis in those tissues; triploids might be more tumor- resistant because it would be more difficult to inactivate or alter all three copies of a gene than just two. These interesting results need to be extended over a wider dose-response range for the three carcinogens studied to date (AFB1, DMBA, MNNG). Substantial differences in tumor susceptibility may also exist among clonal lines and analysis of these differences may provide insights into underlying genetic mechanism of carcinogenesis. The second specific aim is to test a large number of RFLP markers in tumor tissues from isogenic, hybrid trout for loss of heterozygosity. Screening for the loss of heterozygosity (LOH) with RFLP markers has been a primary tool for identifying candidate tumor suppressor genes in humans. The same approach should be useful in trout as well. RFLPs detectable using DNA fingerprint polymorphisms, trout cDNA probes and known human tumor suppressor gene probes, where feasible, will be screened for LOH. The third specific aim is to initiate development of a genetic map of DNA markers for a rainbow trout. It is important to document the proportion of the genome being scanned in our LOH studies. We will seek to map the DNA markers being used in our LOH studies by generating recombinant clonal lines from the two hybrid lines used in the LOH studies and by backcrossing the lines to one of their homozygous parents. This should also provide insights into the distribution of repetitive elements in the salmonid genome and chromosomal evolution in salmonids in light of their tetraploid ancestry.
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