The objective of this research is to study mutagenesis at the DNA level in mammals and teleost fish. The fish will be used for research on ova and on differentiation. A major problem, in mutagenesis is that the level and specificity of response are very different between indicator organisms; predictions abut induced mutagenesis may not be relevant. Significant variation is due to the diversity of marker genes; a single sequence needs to be used among various organisms and tissues. The use of integrated transgenic vector can combine a theoretical study of mutations in several model organisms with assessment of mutagenic hazard and development of biomonitoring systems for aquatic biospheres. Our analyses detect reverse mutations among single copies of the phi-X174 virus as a shuttle vector in different species. The accomplishments are: 1) Transgenic inbred C57BI/6 mice containing the phi-X vector reached the 5th generation without any change in the number of incorporated phi-X genomes (100 per genome). 2) The methylation pattern was different in the founder animal and later generations. 3) The spontaneous and ENU induced mutation frequencies were determined 3 months after treatment. 4) Work is in progress to expand the type of detected genetic alterations using recombinant vectors of phi-Xl74. 5) NIH has filed a patent application based on this work. 6) A new E. coli strain was filed with the Amer. Type Col. for the patent application. 7) Methods were developed to produce transgenic fish with high efficiency. 8) PCR from fin clips was developed to identify the founders and follow the transgene through descendants. A single sequence can be exposed and analyzed as naked DNA, as a single stranded virus, double stranded in bacteria, and in the nuclear genome of mammalian cells, transgenic animals. Such an approach may allow us to examine in vivo mutagenesis and repair in somatic and gametogenic tissue during development, across species, and as a function of aging, and various conditions of environmental exposure.
