Worm Breeder's Gazette 15(3): 26 (June 1, 1998)
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.
University of Wisconsin, 425 Henry Mall Rm. 5350, Madison, WI 53706
We are characterizing the temperature-sensitive mutant stu-4 (e2406) (originally isolated by David Livingstone) which maps to -4.42 on the left arm of LGI. When gravid adults are shifted to the non-permissive temperature 100% of the embryos die due to maternal effect lethality. We examined the cause of this lethality using 4-D time-lapse (Nomarski) microscopy. The polar bodies fail to be extruded resulting in several ooctye !pronuclei! migrating toward the posteriorly located sperm pronucleus where they generally meet at the center of the embryo. Spindle formation, and reformation of the nuclei following chromosome segregation appears to be normal, but at no time point is there any sign of furrowing at the cortex. This results in a multi-nucleated single celled embryo. When L2 or L3 larval stage e2406 animals are shifted to the non permissive temperature 100% of them become sterile and exhibit an uncoordinated phenotype to a lesser extent. These defects are presumably due to failures in cell divisions in the post-embryonic lineages giving rise to the gonad and ventral nerve cord respectively. At an intermediate temperature of 20 degrees only 7 per cent of the homozygotes shifted as L2 or L3 larvae were fertile. We were able to rescue the post-embryonic defects with a small group of cosmids containing K03E5, W05F2 and W07C11. Up to 60% of the rolling homozygotes shifted to 20 degrees as L2 or L3 larvae becoming fertile adults. Of these three cosmids only K03E5 had been sequenced by the genome consortium, and contains 5 predicted genes. One of them K03E5.cand3 codes for a protein with strong homology to calponins which are actin-binding proteins that regulate acto-myosin contraction in a calcium-dependent manner in smooth muscles. When this gene!s function was perturbed in N2 animals using double-stranded RNA mediated interference we obtained early eggs that showed a strikingly similar phenotype to e2406 embryos at the non-permissive temperature. It is possible that this gene may be a non-muscle isoform that plays an important role in either the specification or regulation of the acto-myosin contractile apparatus involved in cytokinesis. To confirm that this gene corresponds to stu-4 we would like to sequence e2406, and further alleles which we are currently in the process of recovering. In a non-complementation screen singled F1 males from EMS-mutagenized him-8 hermaphrodites were crossed to stu-4(e2406) dyp-5(e61)/szT1 hermaphrodites and we looked for the presence of non dpy sterile progeny. From 740 haploid genomes screened we have obtained one additional allele which we are currently in the process of backcrossing.