The
ego-3 gene was identified in a screen for recessive enhancers of
glp-1(ts) (Qiao et al. 1995).
ego-3 mutations also enhance
lag-1(ts) (E. Maine, unpublished data) and cause a variety of germline and somatic defects resulting in Unc, early-adult sterile, and late-adult Mel phenotypes (Qiao et al. 1995).
ego-3 maps between the
stP6 polymorphism and
sdc-3 on LGV. To begin a molecular analysis, we attempted DNA-mediated transformation rescue of the
ego-3(
om40) Unc phenotype. The dominant
rol-6 allele contained on pRF4 was used as a marker. In previous meeting abstracts, we reported the incomplete rescue of
ego-3 by a set of overlapping cosmids (L. Qiao and E. Maine). However, these results are in doubt because it is now clear that pRF4 rescues the Unc phenotype in a high proportion of
ego-3(
om40) animals and weakly rescues the Mel phenotype in these animals as well. In a second attempt at transformation rescue of
ego-3, I am using rescue of
dpy-20(
e1282ts) as a marker. No alterations of the Ego-3 mutant phenotype are observed in
dpy-20(
e1282);
ego-3(
om40) double mutants and the Dpy-20 Ego-3 phenotype is clearly distinguishable from Ego-3 alone. Thus,
dpy-20 appears to be a good choice for this study. However, one inconvenience with this approach is the high proportion of somatic (non-heritable)
dpy-20(+) transformants in the F1 progeny of injected animals (as previously reported by other worm researchers). Despite dropping the concentration of the
dpy-20(+) plasmid, pMH86, to 10 ug/ml (out of a total of ~200 ug/ml of cosmid, pMH86, and Bluescript DNA), I only recover stable lines at an overall rate of ~3%. As a result, I am narrowing down the
ego-3 region by testing several cosmid pools rather than starting with (16) individual cosmids. The two cosmid gaps are bridged by YACs that will be used for transformation only in the event that no cosmid rescue is obtained. To test for rescue of
ego-3, lines are generated that contain both omEx and
ego-3 in a
dpy-20(
e1282ts) background. These
dpy-20(
e1282ts);
ego-3/+; omEx lines are monitored for rescue of the
ego-3(
om40) Unc and germline phenotypes by examining non-Dpy animals. If only the Unc phenotype is rescued, then non-Dpy animals with Ego-3 germ lines will be present. If the germline phenotype is rescued as well, then non-Dpy Ego-3 animals will be absent altogether. Generation of transgenic lines is well underway and tests for rescue of
ego-3 are in progress. Results of these studies will be reported at the meeting.