Worm Breeder's Gazette 15(3): 39 (June 1, 1998)

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.

che-2 Encodes a Novel Protein with the WD40 Repeats

Manabi Fujiwara, Takeshi Ishihara, Isao Katsura

Graduate University for Advanced Studies & National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411-8540, Japan

   We are studying on che-2 mutants, which fail in chemotaxis, osmotic
avoidance, dye-filling and male mating behavior (J.A.Lewis and
J.A.Hodgkin  1977,  T.A Starich et al 1995). One allele of these mutants
has been analyzed already by electron microscopy, and shown to have
morphological defects in sensory cilia. The cilia of this mutant lack
the tip portions, and its microtubules assemble ectopically, creating
posterior projections (J.A.Lewis and J.A.Hodgkin  1977,  L.A.Perkins et
al 1986).

   By transformation rescue, we identified a 6.7kb EcoT14I fragment in
cosmid F38G1 which can rescue the defects of chemotaxis, osmotic
avoidance, dye-filling and male mating behavior. According to the genome
project, this fragment seemed to contain one predicted gene. We analyzed
the gene structure by RT-PCR and 3'-RACE, and found che-2 mRNA (2.7kb)
encodes a 760a.a. protein. This che-2 product  is a novel protein with
WD40 repeats in the N-terminal half region. The WD40 repeat is a motif
probably involved in  assembling multiprotein complexes. The C-terminal
half region shows no homology. We found one allele (mn395) has a
missense mutation in the WD40 repeats, while three alleles (e1033,
sa133, m127) have nonsense mutations in the C-terminal region.

    We studied the expression patterns using transgenic strains carrying
che-2::GFP fusion constructs (containing 3kb or 650bp of 5' upstream
region and almost all the coding region). The che-2::GFP is expressed in
the almost all ciliated sensory neurons. To know the subcellular
localization, we made a che-2::tagged GFP construct which can rescue the
defect of dye-filling of che-2 mutant. When this construct was injected
into N2, most of the GFP is localized at cilia.

    To confirm che-2 acts cell-autonomously, by using the sra-6 promoter
(E.R.Troemel et al 1995) che-2 cDNA was expressed only in ASH and ASI
amphid sensory neurons on the che-2 mutant background. This strain
showed dye-filling only in ASH and ASI neurons, normal osmotic avoidance
(mediated by ASH neurons), but showed still low response to the
chemoattractant benzaldehyde (mediated by  AWC neurons). So we concluded
che-2 acts cell-autonomously.

     Next, we studied when the expression of che-2 is necessary for
cilia formation during the development. We generated heat shock
promoter::che-2 cDNA fusion gene (pPD49.78 kindly given by A.Fire et al
), and injected into che-2 mutant . After heat shock induction at
embryo, the defect of dye-filling was rescued. Surprisingly, even if
this strain was heat-shocked at adult, they extended their deformed
cilia correctly and became almost normal in dye-filling.

     Now we are trying to express che-2 in mammalian culture cell lines,
and examine whether the che-2 protein is colocalized with the
cytoskeletons such as microtubules or actin filaments. We hope to
elucidate che-2 functions by such experiments.