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.
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.