Worm Breeder's Gazette 15(1): 62 (October 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
ABL-Basic Research Program, NCI-FCRDC, Frederick, MD 21702
The universal regulator of entry into mitosis is the serine/threonine kinase CDC2. This kinase is regulated by its phosphorylation state. Amino terminal threonine and tyrosine residues are phosphorylated by members of the WEE1 kinase family to keep CDC2 inactive during interphase of the cell cycle. For example, MYT1 is a dual-specificity kinase that can phosphorylate both threonine and tyrosine residues of CDC2 in Xenopus and mammalian cells. MYT1 is a unique member of the WEE1 family in that it has a hydrophobic domain and is membrane-associated. We performed BLAST searches with the WEE1, MYT1, and MIK1 (another member of this family found in S. pombe) using the Genome Database and, as of Sept. 1, 1997, there were three highly related sequences that were predicted by Genefinder to encode homologs. We are calling these predicted kinases WEE1A, B, and C for now. All three genes map to LG II. We have previously described the cDNA cloning and the mRNA expression patterns of wee1A [F35H7.7; see WBG 14(2): 77 & WM 97: 243]. We are currently characterizing the expression patterns of the other two wee1 genes. WEE1C is represented by at least four Kohara ESTs and we are currently characterizing and sequencing these four. WEE1C looks most like the MYT1 proteins as it also has a hydrophobic domain outside the kinase domain. In order to perturb expression of these three related sequences, we have injected antisense RNAs corresponding to various exons of each of these genes. Our hypothesis was that perturbation of these negative regulators of cell cycle progression would lead to excessive proliferation of specific lineages, and thus lead to visible phenotypes, or would disrupt germline development. For wee1A and B antisense RNAs, we have not observed any obvious phenotypes. Furthermore, co-injection of both antisense RNAs also did not perturb development. We are currently re-examining these genes using an improved procedure for dsRNA inhibition developed by S. Xu and A. Fire. Antisense RNA injections of wee1C sequences resulted in the injected animals becoming sterile. These injected animals laid less than 20 embryos before going sterile. DAPI staining of the injected animals revealed that the oocytes most proximal to the spermatheca were no longer diakinetic, but appeared to have decondensed chromatin. Oocytes in the uterus appear to have an Emo phenotype as well. We are currently examining such animals further to determine whether these oocytes have progressed through prophase of meiosis I prematurely. Our observations suggest that WEE1C may be required for the arrest of oocytes in diakinesis of prophase I of the meiotic cell cycle. In addition to the above observations, we have also seen that some of the F1 progeny (that were born before the injected animals went sterile) were also sterile. DAPI staining has revealed that these animals have no germ cells. We are currently examining these sterile F1 animals to determine whether this lack of germline proliferation (Glp phenotype) is due to defects in generating Z2, Z3, and/or the DTCs, or truely represents a signalling defect in the germline. We intend to pursue our studies of this gene by generating a deletion allele via chemical mutagensis and PCR screening. This research was sponsored by the National Cancer Institute, DHHS, under contract with ABL.