Brister JR et al. (1990) Worm Breeder's Gazette "MOLECULAR LOCALIZATION OF fer-"

L'Hernault SW, Brister JR

[Worm Breeder's Gazette, 1990]

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has been genetically mapped within a narrow interval defined by chromosome II deletions (S. L'Hernault and S. Ward, unpublished observations), and this has permitted us to identify the region that encodes this gene.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]

Tedesco PM et al. (1992) Worm Breeder's Gazette "Integrated Transgenic Lines Rescue Low Fertility in Age Strains"

Johnson TE, Tedesco PM

[Worm Breeder's Gazette, 1992]

Our main focus is on the positional cloning of age-1 .We based our initial cloning strategy on the cosegregation of Age and a reduced fertility (Fer) phenotype in backcrosses of age-1 to N2 ,making the assumption that the same mutational event gave rise to both traits. This assumption simplified the cloning because it is much easier to identify cosmids complementing the Fer trait than the Age trait. We showed that the fer-15 region was responsible for the reduced fertility by deficiency mapping and presented the corresponding physical map (WBG 11.4:14,15). Cosmid T13F9 detected end points of deficiencies immediately to the left and right of fer-15 ;thus T13F9 contains fer-15 ,while age-1 maps closer to unc-4 (WBG 11.4:74). As a final proof that the Age phenotype is separable from Fer we made a series of transgenic animals using the rol-6 coinjection strategy; we also included an actin-4 plasmid (pA4 Gal,Jocelyn Shaw) that was reported to increase the frequency of line formation (Mello, WBG 11.3:12). The injections were successful when we coinjected T13F9 and we saw complementation for fer-15 (b26)at 25 C, although the level of complementation was low. At 20 C little effect on fertility was observed (Table 1). We isolated two independent integrated lines starting with one of the cosmid/Rol-6 arrays and have found that one of these lines complements fertility quite well at 20 C and at 25 C. The other integrated line fails to complement at 20 C and has lower levels of complementation at 25 C. We were not able to get complementation using C26D10 which overlaps extensively with T13F9 and which was reported by Brister and L'Hernault (WBG 11.4:21) to contain fer-15 . These results show that integrated arrays work better for complementation than do extrachromosomal arrays. The quantitative approach we've employed is useful because, unlike qualitative analyses, quantitative analysis can provide better insight into the the level of function of integrated arrays positioned in different chromosomal milieus. We are planning life span assays of these integrated transgenics and hope that they will confirm the fact that the Age and Fer traits are genetically separable. [See Figure 1]