In the absence of the
cog-2(connection-of-gonad-defective-2)/egl-13 gene, fusion between the anchor cell and uterine seam cell does not occur due to abnormalities in the uterine seam cell. This fusion event is required for functional egg-laying activity. The
cog-2 gene encodes a SOX domain transcription factor that is expressed in the uterine seam cell. To gain insight into how this gene affects uterine seam cell function and anchor cell - uterine seam cell fusion, we are investigating factors that interact with the
cog-2 gene or its product. To date, we have isolated two suppressors of a
cog-2(
ku207) mutation; the
cog-2(
ku207) allele is a missense mutation in the region encoding the SOX domain. The first of these suppressors is
him-8, which is located on chromosome IV, but has not yet been cloned (Phil Meneeley, personal correspondence). Suppression by
him-8 appears to be
cog-2-allele-specific (i.e., only the
ku207 mutant allele can be suppressed; putative null mutant alleles are not affected). Suppression by
him-8 is also semi-dominant, with suppression levels (in terms of the percentage of animals laying eggs) at approximately 25% in animals homozygous for
him-8 and 20% in heterozygous animals. Less than 3% of non-suppressed
cog-2(
ku207) mutants lay eggs, so these levels of suppression are clearly significant. We have mapped the other suppressor,
ku376, to the fourth chromosome, and are currently working to pinpoint its location and clone the corresponding gene. Like
him-8,
ku376 is also semi-dominant, showing approximately 35% suppression when it is homozygous and 15% when heterozygous. However, the
ku376 suppressor does not display a Him phenotype, suggesting that it is distinct from
him-8. To confirm this, complementation testing is now in progress. More phenotypic analysis is underway for both suppressors and we plan to present these studies at the time of the international conference. In addition, once we clone these suppressors, we will further characterize those genes and their products in order to determine their mechanisms of action as related to the proper formation of the egg-laying apparatus. In biochemical studies, we are using co-immunoprecipitation and yeast two-hybrid techniques to identify proteins that interact with the COG-2 protein. To prepare for co-IP studies, we are currently generating transgenic animals that will express a FLAG-tagged form of COG-2. In our yeast two-hybrid work, we have encountered the problem of self-activation by COG-2 constructs, so we are currently testing derivatives to find a suitable candidate for a yeast two-hybrid screen.