The
fog-1 and
fog-3 genes are required in both sexes to specify that germ cells differentiate as sperm rather than as oocytes. However, mutations in either
fog-1 or
fog-3 have no effect on somatic fates. Two observations suggest that these genes might directly control sexual fate for germ cells. First, mutations in either gene are epistatic to alleles of other known sex determination genes. Second, sequence analysis suggests that
fog-3 is regulated by TRA-1A, which acts near the end of the sex-determination pathway. To elucidate how
fog-1 controls germ cell fates, we are cloning the gene. Genetic and physical mapping indicate that
fog-1 lies in a large region of the YAC Y54E10 that has not been cloned in cosmids. Transformation rescue data confirm that Y54E10 carries a wild-type copy of
fog-1. In collaboration with the WUSTL Genome Sequencing Center, we are using the PCR and DNA sequence analysis to assemble DNA fragments from the gap under Y54E10 into contigs. These contigs are being tested for ability to rescue
fog-1(
q253ts) mutants, and for detection of rearrangements in
fog-1 mutant DNA. To identify genes whose products regulate or interact with FOG-1, we are screening for suppressors of
fog-1(
q253ts). Hermaphrodites that are homozygous for
q253 produce both sperm and oocytes at 15oC, but only oocytes at 25oC, preventing self-fertilization. We have therefore raised
fog-1(
q253) animals at 15oC, treated them with EMS, and shifted their F2 progeny to 25oC, to select for self-fertile individuals. From a screen of approximately 40,000 haploid genomes, we isolated 18 suppressor mutations. Most of the suppressors are recessive, and none are completely penetrant. The majority of these suppressors fall into two complementation groups. The alleles
v7,
v8,
v9,
v17,
v18,
v20,
v23 , and
v24 fail to complement each other, and map near
sma-3 on LGIII. These mutations appear to define a new locus,
sof-1, (suppressor of
fog-1). In our screen, these alleles of
sof-1 arose at a rate of approximately 1/5000 haploid genomes. Although most alleles of
sof-1 are good suppressors of
fog-1(
q253ts), they seem to have little effect on either fertility or viability in a wild-type background. To understand how
sof-1 influences germ cell fate, we are carrying out three experiments. First, to determine the null phenotype, we are screening for new alleles by non-complementation. Second, in order to clone the gene, we are mapping it with respect to several Tc1 insertions near
sma-3, so as to position
sof-1 more precisely on both the physical and genetic maps. Third, we are testing these mutations for the ability to suppress other alleles of
fog-1. The suppressor alleles
v3,
v15,
v16 and
v21 fail to complement each other, and map to the left arm of LGII. We do not yet know if they define a new locus. Finally, the alleles
v4,
q475, and
v11 complement both the suppressor on II and the
sof-1 on III, and so might identify additional genes. We are just beginning analysis of the remaining suppressor mutations.