Worm Breeder's Gazette 15(1): 76 (October 1, 1997)
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.
|1||Dept. Biology, University of Michigan, Ann Arbor MI 48109|
|2||HHMI and University of Wisconsin, Madison, WI 53706|
Previous studies showed that fog-3 is required to specify that germ cells develop as sperm rather than oocytes. Furthermore, tests of epistasis indicate that fog-3 functions at the very end of the sex determination pathway in the germ line. As a first step towards learning how FOG-3 functions, we have cloned the gene. By using a variety of cosmids for transformation rescue, we defined a small region at the right end of K07A5 that is likely to contain fog-3. Extrachromosomal arrays containing a 3.9 kb subclone from this region restore self-fertility to more than 80% of fog-3(q504) hermaphrodites. Using Northern analysis, we have identified a single transcript of approximately 1200 nucleotides from this region. We used RACE and RT-PCR to clone cDNA copies of this transcript. Injection of adult hermaphrodites with 'anti-sense' RNA to the 5' half of the transcript causes many of the progeny to develop as females, supporting the claim that the transcript encodes FOG-3. The genomic and cDNA sequences indicate that this transcript encodes a single protein of 263 amino acids. Although much of the protein appears novel, the first 116 amino acids show 28% identity and 48% similarity to mammalian Tob proteins (Matsuda et al. 1996). The Tob proteins are known to bind the erbB-2 receptor tyrosine kinase; furthermore, over-expression of these proteins can inhibit cell proliferation (Matsuda et al. 1996). It is interesting to note that genes of the ras pathway are active in the C. elegans germline and control whether or not developing germ cells remain arrested in the pachytene stage of meiosis (Church et al. 1994). One experiment hints at a role for fog-3 in this process - whereas gld-1(q93Mog) animals produce only sperm, germ cells in gld-1(q93Mog) fog-3 animals arrest permanently at the pachytene stage of meiosis. We are now sequencing the 11 fog-3 mutations to determine if any affect regions of homology with the Tob proteins. Extrachromosomal arrays that lack the 3' end of the fog-3 gene cause all germ cells to differentiate as oocytes. This dominant negative phenotype suggests that FOG-3 might bind another protein to specify spermatogenesis, and that truncated FOG-3 can still bind, but cannot cause spermatogenesis. To identify proteins that interact with FOG-3 we are preparing to screen a yeast two hybrid library using FOG-3 as bait. The promotor region on our smallest subclone is less than 1400 base pairs in extent. It contains three perfect TRA-1A binding sites, as predicted by Zarkower and Hodgkin. The sequence of each site is TTTCnnnnTGGGTGGTC. Only four additional sites with this sequence occur in the 63 Mb of sequenced C. elegans DNA. We propose that TRA-1A binds to these sites to repress transcription of fog-3. When the three FEM proteins are active, they prevent TRA-1A from functioning, allowing transcription of fog-3. However, because mutations in the fem genes are epistatic to mutations in tra-1, the FEM proteins must also directly activate FOG-3 or FOG-1. There are many potential serine and threonine phosphorylation sites in FOG-3; one possibility is that some of these are targets of the FEM-2 phosphatase. Church, D. L., K.-L. Guan and E. J. Lambie 1995. Three genes of the MAP kinase cascade, mek!2, mpk-1/sur-1, and let-60 ras, are required for meiotic cell cycle progression in Caenorhabditis elegans. Development 121: 2525-2535. Matsuda S. et al. 1996. Tob, a novel protein that interacts with p185erbB2, is associated with anti-proliferative activity. Oncogene 12(4):705-713.