The G2 to M phase progression of the cell cycle is controlled by the universal regulator, CDC2. The WEE1 kinase family negatively regulates CDC2 by phosphorylation of amino terminal threonine and tyrosine residues. These phosphorylations prevent the progression of the cell cycle from G2 to M phase. As reported earlier, three WEE1 homologs were identified in the C. elegans genome database. We showed previously that one of the three homologs,
wee-1.3 , produced a novel phenotype when its expression was perturbed by the reverse genetic technique, RNA-mediated interference (RNAi). When expression is perturbed, the injected animal becomes sterile. This sterility is caused by the most proximal oocytes exiting from diakinesis prematurely and thus clogging up the gonad arm. This data, along with epistatic experiments with
cdc25 and
cdc2 RNAi, has allowed us to construct a model in which WEE-1.3 is required to keep CDC2 inactive in ooyctes until fertilization occurs. Once CDC2 is activated by CDC25, upon fertilization, meiosis resumes and normal development occurs. In addition to the above RNAi phenotype seen in injected animals, a few of the F1 progeny laid by the injected animal before it went sterile also have a sterile phenotype. Sterility in the F1 progeny is caused by the absences of a germline. To further characterize this phenotype we would like to obtain a mutant of
wee-1.3 . To accomplish this goal we are performing both a reverse and a forward genetic screen. Our first approach consists of a PCR based deletion screen in which a library of mutagenized animals is screened for a deletion in the
wee-1.3 gene. The second approach is a linked sterile screen. The
wee-1.3 gene is located 0.1 map units from
unc-4 , (uncoordinated movement Unc). This close proximity allows us to use the
unc-4 gene as a linked marker, thus we screen for sterile mutants linked to
unc-4 . To then further narrow down the candidates to a
wee-1.3 mutant we can cross them into several deficiencies that map to this region of chromosome II and score for a sterile phenotype. Once a mutant has been scored under the smallest deficiency, we can then determine whether it is a
wee-1.3 mutant by performing cosmid rescue and sequencing the
wee-1.3 gene from the sterile mutants. This research was sponsored by the National Cancer Institute, DHHS, under contract with ABL.