At the present time environmentally induced germ cell mutations cannot be detected in male mammals without testis biopsy or progeny testing. In humans both are impractical. I propose to develop a method through which environmentally induced mutations in the germ cells of male mammals will be detected by analysis of the sperm of the affected individuals. Mice will be used as a model with ready adaptability to human males. In this method undecamer deoxyribonucleotide probes will be synthesized which are either fully complementary to mouse sperm mitochondrial DNA (mtDNA) or which contain a single mismatched base. The fully complementary probe is expected to hybridize to mtDNA more tightly than one with a mismatched base. In mammalian sperm, mtDNA is sequestered into the sperm mid-pieces,while nuclear DNA is found in the sperm heads. The two can be easily separated. Because the mtDNA is much smaller than the nuclear DNA and is fully sequenced, we will be working with mtDNA. We expect the fully complementary probes to tightly hybridize with the vast majority of sperm mid-pieces, but the single mismatch probe would hybridize only with less than 1 x 10-5 sperm mid-pieces. Such tight hybridizations with the single base mismatch probe would be possible only if the mtDNA carried a newly arisen base-pair substitution mutation complementary to the single mismatch probe. The mutagen/carcinogen ethylnitrosourea will be used to see if induced mutations can be detected in sperm mtDNA. When fully developed and validated, the method may be used to detect base-pair substitution mutations in laboratory mammals and human males without a requirement for progeny testing.
| Rank, K; McManus, T P; Ginsberg, L C et al. (1991) Preparation of mouse-sperm DNA for PCR. Mutat Res 264:67-9 |
| Ficsor, G; Ginsberg, L C; Klepetka, J F et al. (1990) Detection of gene mutations in mouse sperm with polymerase chain reaction (PCR). Prog Clin Biol Res 340C:213-22 |
