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.