Worm Breeder's Gazette 15(3): 31 (June 1, 1998)
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.
Dept. of Biology, Syracuse University, Syracuse, NY 13244
We are characterizing a number of ego (enhancer of glp-1) genes at the molecular level. Two genes, ego-1 (Qiao et al. 1995) and ego-6 (J. Spoerke, M. Klein, and E. Maine, unpublished), map between goa-1 and gld-1 (most likely to the right of lrp-1) and show intergenic non-complementation (i.e., ego-6 +/ + ego-1 animals are mutant). The only genetic evidence to suggest that ego-6 and ego-1 are different genes is deficiency mapping data; two independently derived chromosomal deficiencies, nDf25 and mnDf111, uncover ego-1 alleles (om18, om71) but not ego-6 alleles (om54, om58, om84, om96, om97, om119). ego-6 mutations cause a variety of germline defects including enhancement of glp-1(ts) and lag-1(ts), premature onset of meiosis, slow progression through the early meiotic "transition zone", and various gametogenesis defects. ego-1 mutations cause a similar, but not identical, phenotype. ego-6 +/ + ego-1 transheterozygotes resemble ego-6 mutants. To determine whether ego-6 and ego-1 indeed are different genes and to better understand their role(s) in germline development, we have begun molecular studies. Using cosmids spanning the goa-1 to gld-1 region, we looked for DNA rearrangements associated with ego-6 alleles. Using cosmid F26A3 as a probe, we detected a deletion of ~300 bp associated with the UV-induced ego-6(om84) allele. It maps to the vicinity of the predicted transcription unit, "F26A3.3". No other RFLPs were detected with any other ego-6 allele or cosmid probe. Earlier studies using cosmids spanning the lrp-1 to gld-1 region failed to detect any polymorphisms associated with ego-1 mutations (S. Stacey and E. Maine, unpublished data). We isolated cDNAs spanning the F26A3.3 region and carried out RNA blots with several subfragments of F26A3 as probes. The GeneFinder prediction for this region was fairly accurate, but incorrectly assigned two separate genes to one transcription unit. Interestingly, the two genes are structurally related and encode "novel" proteins that are ~58% identical at the amino acid level (as predicted from cDNA sequences). Based on our studies, the om84 deletion interrupts the coding region of the upstream gene; we presume that this gene is ego-6. In support of this conclusion, ego-6 can be phenocopied by injection of RNA made from a partial cDNA corresponding to the upstream gene. C. elegans appears to contain at least one other relative based on GeneFinder predictions (on cosmid F10B5 on LG II); a loosely related gene is predicted by the S. pombe genome project. In-progess experiments aim to (1) determine determine whether any existing ego-6 allele is a null; (2) investigate whether or not ego-1 and ego-6 are in fact the same gene; (3) examine the tissue-specificity of ego-6 expression; (4) examine the function of the downstream, ego-6-related gene. To examine (1), we are amplifying and sequencing the ego-6 gene from ego-6 mutant strains. In particular, we are interested in knowing whether the deletion in om84 shifts the open reading frame. To investigate (2), we are amplifying the ego-6 gene from ego-1 mutant strains to determine if it contains mutations associated with the ego-1 alleles. To investigate (3), we are examining glp-4(bn2ts) mutants (raised at 25!C) for the presence of ego-6 RNA. Based on its mutant phenotype, we suspect that ego-6 might be expressed specifically in the germ line. To examine (4), we are carrying out RNA interference experiments. Also, we are amplifying and sequencing this gene from ego-1 mutant strains to determine whether it might in fact correspond to ego-1.