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 .