The
mut-2 mutator plays multiple regulatory roles in the C. elegans germline. In addition to regulating activity of at least four distinct transposon families it is implicated in chromosome segregation, gametogenesis, germline gene silencing and DNA double-strand break repair. Elucidating the function of
mut-2 will enhance the utility of transposons for functional genomic studies in this organism and shed light on mechanisms that maintain structural and functional integrity of eukaryotic genomes. Using the Him phenotype conferred by
mut-2(
r459), we mapped the gene between
dpy-14 and
sem-4 on LGI. However, efforts to identify a molecular clone of the gene were hampered because the available phenotypes were unsuitable for standard transformation rescue approaches. This roadblock led us to examine the temperature-sensitive (ts) behavior of
mut-2: the original
mut-2 isolates TR674 and TR679 are inviable at 25 o C. Analysis of ancestors and recombinants derived from these isolates demonstrated that the ts phenotype cosegregates with
mut-2 and is unrelated to the Bergerac ancestry of these strains. Closer inspection (with assistance from Eric Lambie) revealed that sterility results from a defect in gametogenesis. Gonads of
mut-2 animals raised at 25 o C appear normal but have a dearth of sperm which exhibit variable morphology. Oocytes show no gross abnormalities but may be affected as well. DAPI staining reveals no obvious chromosomal defects. We are testing if wild-type sperm can rescue sterility and determining the temperature-critical stage of sperm (and oocyte?) development. The ts sterile phenotype is rescued by sDp2, a large free duplication that covers the
dpy-14 sem-4 interval. This suggests it may be a suitable phenotype for identifying a molecular clone of
mut-2. Two smaller free duplications that bisect this interval, hDp62 and hDp65, were tested; hDp65 rescues, hDp62 does not. This positions
mut-2 between +1.57 and +1.66, a region covered by three cosmids, containing ~20 ORFs. Work is in progress to clone
mut-2 by rescuing sterility. Recent evidence suggests
mut-2 strains may be resistant to RNAi (1). We are exploring the use of this phenotype in our cloning efforts as well. 1) Tabara, H and Mello, C. 1999. (this meeting)