The objective of this research is to study mutagenesis in mammals at the DNA level using both nuclear and mitochondrial DNA. Studies are to be based upon variation among individual copies of a particular sequence rather than variation between averages for the same sequence. Special emphasis is to be placed on the differential sensitivity to mutagenesis of gametogenic stages and in comparison with the response in the various somatic tissues. A major problem for detection of genetic damage directly in mammalian DNA is that most genes occur in one, or few copies. Our approach will utilize well characterized DNA sequences. The basic requirements for direct analysis of specific DNA sequencs are that the sequences: (1) are already amplified in the cell, (2) can be isolated from the mammalian genome, and (3) can be amplified in vitro. Two genetic entities met these requirements. The first is the use of mitochondrial (mt) DNA. Cloned mouse mtDNA has been used for restriction analysis of sperm mtDNA isolated from a single mouse; after additional technical improvements, the mtDNA from treated mice will be examined for mutations. The second is the use of viral DNA transformed into mammalian DNA. Double stranded DNA from PhiXDNA from the transformed mammalian cells. Conditions for purification of PhiX from the mammalian genome have been developed and conditions for measuring reverse mutations of am3 and cs70 have been established. Attempts are being made to create a mouse strain with PhiXDNA in the genome for the study of mutation induction in any part of the animal tissue.
