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ego-6 (J. Spoerke, M. Klein, and E. Maine, unpublished), map between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ego-1 mutations (S. Stacey and E. Maine, unpublished data).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.