Worm Breeder's Gazette 11(4): 21
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Wild type spermatids can be treated in vitro with a number of agents that cause the sessile spermatid to extend a single pseudopod that permits crawling locomotion. (S. Ward et al. 1983, Dev. Biol 98: 70). Mutation of the fer-15 gene allows worms to make spermatids but disrupts this transition of non-motile spermatids to motile spermatozoa. The fer-15 gene has been genetically mapped within a narrow interval defined by chromosome II deletions (S. L'Hernault and S. Ward, unpublished observations), and this has permitted us to identify the region that encodes this gene. We prepared heterozygous DNA from 10 chromosome II deletions ( including mnDf92, 93 and 101) and one recessive lethal putative point mutant (as control) for Southern blot analysis. We probed these genomic DNA's with a number of cosmids spanning the region between zyg- 11 and let-237 and found that the cosmids C41C4 and C30D12 detected several deletion breakpoints that define the genetic location of fer- 15. All our subsequent analysis has been with cosmid C30D12, which hybridizes to novel restriction fragments associated with mnDf92, 93 and 101.We have more precisely localized the above-mentioned deletion breakpoints by probing blots with restriction fragments derived from C30D12. A 17kb BamHI restriction fragment hybridizes with novel bands derived from both mnDf92 and mnDf93. This BamHI fragment hybridizes to a single, ~5 kb band on total RNA Northern blots; this RNA does not appear to be sex-specific. We are currently cloning the DNA within this 17 kb interval and screening cDNA libraries. The cosmid C30D12 has been microinjected into fer-15(hc89ts) animals in order to confirm its association with this gene; this fer-15 allele causes complete sterility in hermaphrodites raised at 25 C. Permissively raised adult hermaphrodites were injected with cosmid DNA and then allowed to produce a F1 under restrictive conditions. To date, 18/78 injected worms gave rise to F2's (range, 10-48 progeny). Decendants of 4 of the 18 successfully injected worms gave rise to F3's (range, 5-30 progeny) and, in one case, F4 worms (8 progeny) were obtained. We have not yet obtained stable rescue of the fer-15 sterile phenotype. [See Figure 1]