Worm Breeder's Gazette 15(1): 63 (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.

Role of Cdc25 Cell Cycle Regulators in C. elegans

Neville Ashcroft, Mary Kosinski, Andy Golden

ABL-Basic Research Program, FCRDC, Frederick, MD 21702

Cancers develop from cells that inappropriately divide.  Understanding
the control mechanisms that regulate cell divisions would provide
insight into how tumors develop and possible ways of treatment.
Although a lot is already known about the biochemistry of cell cycle
regulation, little is known about how the regulatory genes function in
multicellular organisms.

Simply put, the dual specificity phosphatase, cdc25, activates cyclin
dependent kinases (such as cdc2) which in turn drives the cell cycle
machinery.  Searching the C. elegans' genome sequence database,
numerous cell cycle regulator homologues can be found including cdks
and cyclins. From the cdc25 family, four homologues have been
identified which we have named after the cosmid they were sequenced
from: ZK637, R05H5, F16B4, K06A5.  Why are there so many cdc25s in the
worm while only 3 can be found in mammals, 2 in Drosophila, and only 1
in yeast?  Perhaps the C. elegans' cdc25s act in different cells, or at
different developmental stages, or perhaps some may regulate meiosis
while others regulate mitosis?

The protein sequences of all the C. elegans' CDC25s all share
remarkable conservation of the catalytic domain.  However, this
homology diverges outside the phosphatase domain, the predicted
intron/exon structures are distinct and all map to separate
chromosomes.  We subcloned the cdc25 genes and reintroduced them back
into the worm as multicopy extrachromosomal arrays with no noticeable
effect on the animals' development. Characterization of the expression
patterns of all these genes is continuing.  Our data suggest that only
three of the four cdc25s are expressed genes: RT-PCR, RNA in situ
experiments, Northern blot analysis, and GFP and LacZ fusion constructs
all show that the R05H5 cdc25 is either expressed weakly or not at all.
One (ZK637 cdc25 mRNA pattern) has been reported in an earlier report
[WBG 14(2): 76].  An interesting expression pattern has also been
characterized for the K06A5 cdc25 by indirect immunohistochemistry.  We
first saw antibody staining in oocyte nuclei.  After fertilization,
staining in the polar bodies was absent despite still being present in
both pronuclei.  After the first mitosis, we also saw staining in the
embryonic nuclei as well as plasma membranes.  This pattern persisted
at least until gastrulation.

To perturb expression of each of the cdc25 genes, we injected antisense
RNAs corresponding to all four cdc25s.  While R05H5 cdc25 and ZK637
cdc25 antisense RNA injections, singly or in combination, did not
create a noticeable effect, both K06A5 and F16B4 cdc25s produced lethal
phenotypes.  Antisense K06A5 cdc25 RNA generated aneuploidy in the
early embryo resulting from meiotic defects.  Using videomicroscopy of
live embryos treated with K06A5 cdc25 antisense RNA, enlarged polar
bodies, multiple pronuclei and unstable cleavage furrows were often
observed.  Antisense F16B4 cdc25 RNA caused L1 larval lethality.
Interestingly, a few embryos that were laid soon after the antisense
K06A5 and F16B4 cdc25 treatment matured into sterile adults.
Additional experiments demonstrated redundancy between the genes since
antisense RNA injections of ZK637 and F16B4 cdc25 together caused a
late embryonic lethality.  Furthermore, antisense RNA injections of
ZK637, K06A5 and F16B4 cdc25 caused embryos to arrest development at
the single cell stage.  This was a phenotype similar to that of cdc2
RNA antisense treatment (van den Heuvel, personal communication and our

Candidate lethal mutants for the K06A5 and F16B4 cdc25 are now being
evaluated.  Weak rescue was obtained for a K06A5 cdc25 candidate,
mei-2, after injection of a cocktail of neighboring cosmids including
K06A5 (Srayko & Mains, 1997 International Worm Meeting abstract 565).
However, sequencing the cdc25 gene from homozygous mei-2 (ct102)
animals showed that K06A5 cdc25 is not mei-2.  We are preparing to
screen for deletion alleles of each of these cdc25s.

Research sponsored by the National Cancer Institute, DHHS, under
contract with ABL.