TRANSGENIC REPORTERS OF ENVIRONMENTAL GENOTOXICANTS

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.