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.