The objective of this research is to study mutagenesis at the DNA level in mammals and teleost fish. A major problem in mutagenesis is that the level and specificity of response are very different between indicator organisms; predictions about induced mutagenesis may not be relevant. Significant variation is due to the diversity of target 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. In addition transgenic fish will be used for mutagenesis studies on ova and during differentiation. The well characterized am3 mutation of phiX174 is used to evaluate substitutions at the A:T base pair. An inbred line of C57B1/6 mice was established that is homozygous for 100 copies of phiX174 in a tandem array and free of any detrimental effect from the insertion. Reversions via one transition and two transversions are detected by selection among progeny phage recovered from the transgenic animals. The vector can be recovered in a range of 1-10 million PFU per microgram restriction digested DNA from various mouse tissue and fish. Both the recovery and the putative mutation frequencies are independent of any endogenous variation in CpG methylation among host animals or tissues. Spontaneous mutation frequencies are in the range of 2-4 per 10 million for liver, spleen, brain, kidney and testis. In adult animals treated with 200mg/kg N-methyl-N-nitrosourea (ENU) there are significant increases in mutation frequencies in the spleen and liver, but not the kidney or brain. This follows the pattern of tumor induction for ENU in adult animals. The accomplishments for the fish studies are: (1) produced numerous transgenic founders of Fundulus and Medaka containing phiX174 as a single gene marker and (2) demonstrated that the transgenic vector can be recovered from the chromosomal DNA of fish in the range of 10 million PFU per microgram genomic DNA. The spontaneous mutation frequency for Fundulus is less than 1 per 50 million, probably in the same range as found in mammals. Efforts are underway to produce additional transgenic animals containing other specific markers for anaylsis of CpG-dependent and CpG-independent mutagenesis at the single gene copy level. The use of well-defined phage sequences with unbiased recovery and mutation detection may allow us to compare spontaneous and induced intragenic events.
