Worm Breeder's Gazette 11(4): 22
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.
Mutation of the spe-4 gene disrupts C. elegans sperm meiosis so cells that appear by Nomarski optics to be spermatocytes actually contain four haploid nuclei. This phenotype is not changed when either of two EMS induced alleles of spe-4 are placed in trans to the noncomplementing chromosome I deletion sDf6, suggesting that this gene might have a sperm-limited function (S. L'Hernault et al., 1988 Genetics 120: 435). We have undertaken a molecular analysis of the spe-4 gene in order to better understand its role in spermatogenesis. Initially, our strategy was to map both breakpoints of sDf6, which we knew contained at least part of spe-4, and sDf5, which was outside spe-4 but included unc-13, a gene that is very close to spe-4. We obtained a number of cosmids between unc-15 and lin-10 and probed Southern blots of genomic DNA's prepared from all three spe-4 alleles ( balanced to sDf5), veral different balancers) and wild type. We have not found any novel restriction fragments associated with sDf5 or the right breakpoint of sDf6 but have confirmed the apparent location of the left sDf6 breakpoint as under C05B12 (an unc-15 cosmid), as first discovered by I. Maruyama. However, the cosmid C44E1 identified an allele specific deletion of ~200 b.p. associated with the spontaneous spe-4 allele q347 (provided by Tim Schedl); this mutation arose on a Bristol chromosome I. We have two-factor mapped spe-4(q347) relative to unc-13 to obtain recombinants for molecular characterization. We analyzed 2,951 F2 from spe-4(q347) 091)/++ F1 animals and only one nonSpe Unc recombinant was obtained. This recombinant was picked and allowed to propagate, and it did not contain the ~200 b.p. deletion in its DNA, further suggesting the association between this deletion and spe-4.The cosmid C44E1 has been extensively characterized by I. Maruyama because it contains the unc-13 gene; this gene encodes a 6 kb RNA. We have probed Southern blots with oligo labeled restriction fragments from this cosmid and have localized the above-mentioned 200 b.p. deletion to a 2.1 kb HindIII fragment. This fragment has been cloned and its insert only hybridizes to an abundant 1.3 kb male specific RNA on total RNA male vs female Northerns (male=RNA from Mog fem-3 gf; female= RNA from fem-1 lf). We will shortly begin trangenic rescue experiments with C44E1 and various subclones in order to determine if spe-4 is on this cosmid.