Worm Breeder's Gazette 12(3): 41 (June 15, 1992)
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.
Mutations in the spe-11 gene (for spermatogenesis-defective) result in a paternal-effect embryonic-lethal phenotype. Fertilization of wild-type oocytes by sperm from mutant animals leads to abnormal early embryonic development. This and other studies suggest that the sperm must contain or supply the spe-11 gene product for normal early development to occur (Hill et al. Dev. Biol. 136, 154-166).
In order to molecularly characterize the gene, we are cloning spe-11 by transformation rescue using YACs, cosmids and phage. spe-11 maps .1-.2 m.u. Ieft of dpy-5 .We have co-injected a number of the cosmids and YACs from this region (provided by Alan Coulson and John Sulston) with rol-6 plasmid into + spe-11 dpy-51 unc-73 + + heterozygotes and tested homozygous spe-11 dpy-5 progeny for rescue. The YAC Y51G8 rescues both spe-11 and dpy-5 mutant phenotypes. This results in roller animals of wild-type length, which produce Dpy and wt length but no Unc animals. When these wt length animals are crossed to N2 males, all the Fl progeny produce some spe-11 dpy-5 animals. Thus, the genotype of these animals is spe-11 dpy-5 / spe-11 dpy-5 ,with the YAC rescuing both spe-11 and dpy-5 .
We hybridized Y51G8 to a Southern blot of some of the cosmids in the region [See Figure 1] and injected those cosmids to which it hybridized. The cosmid B0342 rescues dpy-5 .However, none of the cosmids we tested appear to rescue spe-11 .This could be due to deletions in the cosmid DNA we isolated. We are suspicious that cosmids from this region are prone to rearrangements. In some cases the vector bands are not the expected size, overlap with other cosmids is not as extensive as expected, and different isolates of the same cosmid show different restriction patterns. Consequently, rather than requesting additional cosmids from this region, we chose to screen an unamplified lambda DASH II C. elegans genomic library with the rescuing YAC and Y38H4 .Since by Southern analysis Y38H4 ends in B0342 ,we further characterized 22 phage which hybridize to Y51G8 but not Y38H4 .
We established the order of the phage by hybridization to cosmids which hybridize to Y51G8 and began injecting phage from the most suspicious areas. Lo and behold, one such phage with an insert of approximately 18kb rescues spe-11 .This phage lies on the very left edge of Y51G8 (the YAC hybridizes to only part of the phage) between C5 OD8and F55A12 . F55G5 should cover this region; however, our isolate does not We are currently determining which cosmids overlap with the phage. In addition, we are injecting restriction fragments from the phage to zero in on the gene. (The library and phage are available upon request.).