Worm Breeder's Gazette 14(2): 77 (February 1, 1996)
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, Frederick Cancer Research and Development Center, Frederick, MD 21702
We have initiated a project to examine the regulation of the cell cycle during C. elegans development (please see accompanying abstract by Wilson et al.). Biochemical and genetic experiments from other systems have revealed that the tyrosine kinase WEE1 is a negative regulator of cell cycle progression. WEE1 phosphorylates the serine/threonine kinase, CDC2, on a single tyrosine residue to maintain it as an inactive kinase during much of the cell cycle. A search of the available sequences from the Genome Consortium revealed a candidate wee1 gene (cosmid F35H8). The WEE1 candidate, as predicted by GENEFINDER, has all of the distinguishing features that define it as a member of the WEE1 tyrosine kinase family. Using SL1, SL2, and 3¹ UTR specific primers and RT-PCR, we identified a SL1-trans-spliced cDNA. Interestingly, the SL1 is trans-spliced to the second exon predicted by GENEFINDER. We have yet to detect a message that incorporates the predicted exon 1. We have used this cDNA for RNA in situ studies in embryos. The wee1 message appears not to be maternal. The first stage at which we observe mRNA expression is the 12-cell stage, in a single prometaphase nucleus. By co-staining with an anti-P-granule antibody (for orientation), we have determined that this single nucleus is that of the E blastomere. wee1 expression is then seen in 5-8 distinct prometaphase nuclei in the 16-cell embryo. We believe these 5-8 nuclei are those of the 8 AB-derived cells. Interestingly, the message is only observed in cells with condensed chromatin (as seen with DAPI staining). This expression is observed at a time in the cell cycle when nuclear envelope breakdown is occurring. The wee1 message does not appear to be cytoplasmic at all. We interpret this distribution as a burst of embryonic wee1 transcription (in these 5-8 prometaphase cells), followed by rapid mRNA turnover. Anaphase, telophase, and interphase cells appear devoid of wee1 message. We are currently attempting to determine whether this burst of embryonic transcription is truly cell cycle stage-specific and lineage-restricted. We have yet to observe any mRNA expression in the MS, C, D, or P3 (or P4) blastomeres. Also, no wee1 message has been observed beyond the 16-cell stage of embryonic development. We are currently testing WEE1 antibodies to determine the protein expression patterns of this cell cycle regulator during embryogenesis. We are also performing whole animal RNA and protein in situs to determine the expression patterns of this gene during the proliferation and development of the germline in the gonad. We would like to acknowledge and thank the C. elegans Genome Consortium for sharing all of their sequence information with the scientific community and for making this project possible. This research was sponsored by the National Cancer Institute, DHHS, under contract with ABL.