We have been analyzing mutations that affect speratogenisis in C. these were isolated in our laboratory by selecting sterile mutants that produced large number 9 of oocytes that could be fertilized by wild type male sperm; others were sent to us from other labs. All mutations were assigned to linkage groups and tested for complementation to all other known linked spermatogenesis- defective mutations. Many of the genes identified have been mapped. At this time, there are 50 mutations which we have organized into 32 complementation groups; seven complementation groups have more than one allele. For mutations in 9/10 genes tested, the phenotype over a deletion is identical to the homozygous mutant phenotype 80 the sperm defect seems to be the null phenotype. Most of our recent work on this project has been limited to linkage group I where we have identified 28 mutations in 12-13 complementation groups. We have recovered many of these mutants as cis doubles with
dpy-5. This has facilitated the linkage assignment, balancing of nonconditional mutants and identification of sterile worms for phenotypic analysis (see below). Depending on their position on the map, nonconditional steriles have been balanced to sDp2 or a complementing LG I deficiency. Five linkage group r complementation groups contain ore than one allele. Our present understanding of the genetic organization of these LG I spermatogenesis-defective mutants appears in Figure 1. We have analyzed the light microscopic phenotypes of most of our LG I spermatogenesis-defective mutants and many on the other five linkage groups (see summary Table and Fig. 2). The top row of cartoons in Fig. 2 shows the pathway of normal spermatogenesis. Just below this pathway fer and spe genes that arrest development at an apparently normal step in the pathway are shown at the step they arrest. Aberrant phenotypes that accumulate in some mutants are shown below the pathway. For example,
spe-4 mutations accumulate cells that look like spermatocytes but have 4 haploid nuclei;
fer-2, s accumulate aberrant looking spermatids which fail to activate to spermatozoa. Mutations in five genes (
fer-7, cumulate normal looking spermatozoa that are motile in vitro, but these fail to fertilize oocyt of them are swept out of the hermaphrodite spermatheca by the passage of oocytes, but others are retained. Previously, we thought sperm-defective sterile hermaphrodites that laid large numbers of oocytes were likely to be defective in the postmeiotic stages of spermatogenesis where the spermatid is transformed into a motile spermatozoa. While this is frequently the case, we have also recovered mutants defective in the earlier stages of spermatogenesis (see Fig. 2). We have decided to name all new sperm-defective genes spe, irrespective of their sperm-defective phenotypes. We will abandon the further use of the gene name fer which was intended to distinguish that subset of sperm-defective mutants that accumulate haploid gametes. There are now a number of intermediate phenotypes that make this distinction less useful. We are interested in cloning the genes that have been identified by our collection of spe and fer mutating. Unfortunately, it has not proven possible to isolate mutants out of 'high hopper' lines because there is a very high background of oocyte laying. For instance, we used Mike Finney's cleaned up 'high hopper' line (originally created in Phil Anderson's lab) in a precomplementation screen to
fer-1(hclts),
dpy-5(
e61)r; 15ts),
unc-4 (
e120)II and recovered oocyte layers at a frequency of 1 out of 30. We tried to recover a nonconditional sterile from 17 of these lines and were not successful. Since this did not look like a particularly promising approach, we have sought other methods. Fortunately, it appears that the
spe-4 gene has already been cloned. Genetic analysis indicates that
spe-4 complements sDf5 but fails to complement sDf6. This places this gene very close to
unc-15 which is in a contig identified by Allan Coulson and John Sulston. This same contig also contains a lambda phage that Dan Burke had identified as containing a sequence that hybridized specifically to male RNA in a differential hybridization assay. We are trying to determine the relationship (if any) of this lambda phage and the
spe-4 gene; even if there is not a relationship it seems likely that this contig contains the
spe-4 gene. We are also trying to clone the
fer-15 gene, which seems to be blocked in the spermatid- spermatozoon transition. This gene has been positioned near
emb-27 on LG II; it complements mnDf 92 but fails to complement mnDf91. Phil Carter has been kind enough to send us his 100 kb cosmid walk from the mnDf 103 breakpoint towards
fer-15. We hope to walk and jump through this region to clone the interval between the mnDf 91 and 92 breakpoints that define the
fer-15 gene. Differential hybridization assays and/or microinjection might then permit identification of the
fer-15 gene. {Figures 1, 2}