Short term bacterial and mammalian cell mutagenesis tests have contributed much to our appreciation of the mutagenicity of many environmental agents. A major shortcoming of most is their inability to incorporate tissue differences in P450 activities into their design. Transgenic mouse technology has made it possible to precisely design reporter genes that have the potential to identify different types of mutational events in different tissues and organs of tester animals, and at different times of development. This program project grant describes five projects, all of which utilize transgenic mice, to assess the mutagenic behavior of environmental agents. The five projects are complementary and each addresses a different facet of the overall program. As common themes, they all address issues relating to mutations and/or tumor development and to mutational events induced by genotoxic agents.
The aims of individual projects are: Project 1) Dr. Stambrook will produce a set of transgenic mice each bearing a different mutant lacZ gene, encoding nonfunctional bacterial Beta-galactosidase. Each mutation will be a base-substitution within an inviolable amino acid so that only same-site reversion will reestablish B-galactosidase activity. As in projects 2 and 4, mutation is identified histochemically by cells that statin blue against an unstained background. Project 2) Dr. Sinden asks whether alternative DNA structures may serve as foci for spontaneous mutation in whole animals, and whether administration of mutagens/carcinogens increases the frequency of those classes of mutation. The strategy is to place DNA sequences that produce alternate DNA structures into the Beta-galactosidase gene, so as to interfere with function. Spontaneous or mutagen-induced resolution of the structure will result in resumption of enzymatic activity which can be detected histochemically in cultured cells or in tissues and organs of transgenic mice. Project 3) Dr. Doetschman will produce transgenic mice that are heterozygous for the tumor suppressor gene encoding p53. Thus, loss or mutation of the single functional p53 allele will predispose an affected cell to tumor formation. The single p53 allele will provide the mutagenic target and tumor production will serve as the indicator for the mutagenic event. Project 4) Dr. stringer has devised a reversion assay that will detect homologous recombination events and their potential enhancement by environmental agents. The constructs which allow detection of recombination events will also be targeted to a defined locus within the genome, so that reversion in embryoid bodies and whole animals can be assayed independent of position effects. Project 5) Dr. Dixon will utilize available transgenic mice that carry integrated lambda phage genomes bearing bacterial lacZ genes. These phage can be rescued, and mutant lacZ genes analyzed at the nucleotide sequence level. Dr. Dixon proposes to breed the target transgenes into mice with high affinity aryl hydroxylase (AH) receptors and into mice with low ;affinity AH receptors to help establish the roles that these receptors play in the extent and distribution of mutagenic events. This strategy will also be used in the other projects.
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