Worm Breeder's Gazette 13(4): 66 (October 1, 1994)

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.

Cloning of C. elegans 14-3-3 and GAP cDNAs By Functional Screening Using the Fission Yeast S. pombe.

Seiji Hayashizaki, Yuichi Iino, Masayuki Yamamoto

Department of Biophysics and Biochemistry, School of Science. University of Tokyo PO Hongo. Bunkyo-ku. Tokyo 113, Japan.

  Since M. Lee and P. Nurse cloned human cdc2 cDNA based on its ability to
rescue the fission yeast cdc2 mutant, complementation cloning by the use of yeast
systems is widely employed (e.g. WBG 13(3), p21 ).In order to apply this method to
cloning of C. elegans homologs of the S. pombe genes which we previously isolated in
our laboratory, we constructed a C. elegans cDNA library in a fission yeast expression
  We report here the progress of two lines of screening we performed using this
library. First, we used the sme2 -deletionmutant of fission yeast as a recipient of the
library. The sme2 gene encodes an RNA product expressed during meiosis in S.
pombe. The sme3062 mutant is defective in meiosis, arresting after premeiotic DNA
replication and before meiosis I. The RNA product of this gene has been shown to
work in association with an RNA binding protein, M ei2 ,which is a multifunctional
regulator of meiosis (Y. Watanabe and M. Yamamoto, Cell 78: 487-498, 1994). We
screened for C. elegans
  cDNA clones that can rescue the meiotic defect of the sme2 306mutant and
obtained one positive clone. Sequencing of this cDNA revealed that its translation
product has sequence homology to the 14-3-3 family of proteins. The cDNA
corresponds only to the C-terminal half of the family proteins, apparently lacking the
authentic N-terminus. When a frameshift mutation was introduced to this ORF, it
resulted in loss of the rescue activity, suggesting that the translation product is
responsible for the function. Within the region obtained, this cDNA had highest
similarity to D14 -3-3,a 14-3-3 homolog of Drosophila (76% amino acid identity). It
has 100% nucleotide sequence identity in the 66bp overlap with the EST sequence
registered to the EMBL database by W. R. McCombie et al. under the accession
number T02212 .It is different from the 14-3-3 cDNAs reported by W. Wang and D.
Shakes (WBG 13(2), p89 ).
  A variety of functions have been reported for the 14-3-3 family, including
activation of tyrosine hydroxylase and tryptophan hydroxylase, inhibition of PKC,
activation of ExoS ADP-ribosylase and so on. In S. pombe, two 14-3-3 homologs are
reported to be involved in the cell cycle checkpoint control. In order to clarify the in vivo
role of the 14-3-3 gene in C. elegans, we are going to attempt to isolate Tc1 insertion
mutants in collaboration with Y. Andachi and Y. Kohara.
  In the second line of screening. we used the gap1 -deletionmutant of S. pombe
as the recipient. The gap1 gene, which is homologous with mammalian ras-GAP and
NF1 and budding yeast IRA1 and IRA2 ,serves as a negative regulator of S. pombe
ras1 ,which is essential for the mating pheromone signaling. The gap1 306mutant is
hypersensitive to the mating pheromones: they elongate conjugation tubes extensively
and cannot mate efficiently (Y. Imai et al., Mol. Cell. Biol. I 1: 3088-3094, 1991). C.
elegans cDNA clones which can rescue the sterility of the gap1 306mutant was screened
for and two positive clones were isolated. One of them had a predicted amino acid
sequence that bears homology to GAPs (48% identity with human ras-GAP and 42%
identity with budding yeast BUD2 in the 67 amino acid core sequence of the
GAP-homology region). The other clone had no homology to known genes. We plan to
knock out the C. elegans GAP gene as well and see if it has any role in vulval
differentiation or other aspects of signal transduction in C. elegans.