Worm Breeder's Gazette 12(5): 56 (February 1, 1993)
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.
C. elegans spermatids discard much of their cytoplasm during their formation from spermatocytes. Retention of essential components occurs, in part, because they reside within specific organelles, the fibrous body-membranous organelle (FB-MO) complexes, and these organelles segregate to spermatids during formation of the cell (Wolf et al., J. Ultrastruc. Res. 63: 155-169. 1978; Ward et al., J. Cell Biol 91: 26-44, 1981). We are interested in spermatogenesis defective (spe) genes that affect morphogenesis and function of the FB-MO complexes in order to better understand their role during spermatogenesis. We have discovered that the previously described spe-5 gene is in this phenotypic class (L'Hernault et al., Genetics 120: 435-452, 1988).
Ultrastructural analyses of spe-5 mutants reveals structurally abnormal FB-MO complexes, but in a way that differs from the disruption of these organelles previously observed in other spe mutants, such as, for instance, spe-4 (L'Hernault and Arduengo, J. Cell Biol. 119.55-68, 1992) or spe-6 (Varkey et al., Genetics 133: 79-86, 1993). spe-5 is presently defined by four alleles, all of which are slightly self-fertile. Most commonly, spermatogenesis arrests prior to spermatid formation, but spe-5 mutants produce some free spermatids, and a few must be capable of differentiating into functional spermatozoa because progeny are produced. Further genetic characterization reveals that spe-5 mutants show an unusual cold sensitivity: more oocytes are produced at 25° than at 15°. This observation is true for all four spe-5 alleles, and contrasts with wild type animals that produce more progeny and oocytes at 15°.
We are interested in cloning and sequencing spe-5 to gain insight into how it alters spermatogenesis. To that aim, three factor crosses were performed to map spe-5 in relation to other known genes in its region of chromosome I. The F1 progeny of + spe-5 +/ unc-63 + dpy-5 hermaphrodites were screened and 1/4 of the Unc non-Dpy, and 1/8 of the Dpy non-Unc recombinants picked up spe-5 .This places spe-5 between unc-63 and dpy-5 .Furthermore, from the F1 progeny of + spe-5 dpy-5 / mes-3 + +, 25 Dpy fertile recombinants were recovered, all of which were + spe-5 dpy-5 / mes-3 + dpy-5 .This implies a gene order of mes-3 spe-5 dpy-5 .Previous work had mapped unc-63 to the right of mes-3 (Caprowski et al. Genetics 129 1061-1072, 1991), which if combined with our 3 point data, gives a gene order of: mes-3 unc-63 spe-5 dpy-5 .
mes-3 was placed on the physical map by J. Paulsen and S. Strome, when the mes-3 phenotype was rescued by a B0231 cosmid/ rol-6 ( su1006 )transgene. They generously provided the transgene containing this cosmid, and it was crossed into an unc-63 spe-5 background. Four non-Unc fertile rolling lines were recovered and their genotype was confirmed as unc-63 spe-5 /B0231.The brood size of these transgenic lines was 213.1 + 44.2 (n=10), with ~58% of the progeny segregating as rollers at 20°. This is statistically indistinguishable from wild type, which has a brood size of 199 + 35 (n=14), and indicates that B0231 covers both spe-5 and unc-63 .
Presently we are screening genomic Southerns containing spe-5 mutant DNA's and several gamma-lay induced unc-63 mutant DNA's (provided by Jim Lewis) to locate RFLPs in either gene, which will help us localize spe-5 more specifically within the B0231 cosmid. In parallel, we are constructing a restriction map of B0231 ,creating transgenic animals with subclones of B0231 and screening Northerns containing polyA+ RNA from fem-3 gf( q23 ), him-5 ( e1490 )and fem-3 lf( hc17 )with different fragments of B0231 to identify male specific messages. This latter strategy is based on the fact that B0231 identifies two male specific messages (J. Paulsen and S. Strome personal communication), and, hopefully, one is spe-5 .