Worm Breeder's Gazette 12(4): 33 (October 1, 1992)
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.
We have shown perviously that two C. elegans strains defective in muscle contraction, kra-1 and unc-68 ,are derived from mutations in closely linked loci. In brief, both unc-68 ( e540 , x14 and x24 )and kra-1 ( kh30 )animals exhibited normal muscle morphology but were resistant against either cholinergic agonists or ouabain (Lewis et al., 1980; Ando and Kagawa, 1991). The two loci were localized within the same 0.08 map unit on linkage group V, although they showed incomplete phenotypic complementations; this would probably be ascribed to gain-of-function nature of the mutations. We thus proposed that kra-1 and unc-68 genes are identical or form a complex locus, whose product(s) function downstream of cholinergic excitation and upstream of muscle filament assembly in the signaling pathway leading to muscle contraction (Ando and Kagawa, manuscript submitted).
In order to define the coding region of kra-1 / unc-68 ,we conducted poly(A)+ RNA blot analysis using 12 cosmids, spanning the breakpoints of nDf32 and sDf20 ,as probes. have classes of transcripts were detected but there were no size-differences between wild-type and mutant animals.
We then microinjected the cosmid DNAs into oocytes of x14 animals, and found a cosmid C46C8 reproducibly rescued the unc-68 mutant phenotype, i.e. a constant muscle relaxation. Among subclones of C46C8 ,a 2.5kb Hin dIII fragment was able to recover the mutant muscle contraction on regular media. A transgenic strain (pECT05R-2), which carried a 53kb Eco R1 transgene including the 2.5kb Hin dIII fragment and regained contractibility of the body wall muscle, was also obtained. Southern blot analysis indicated that the DNA fragment was present as multi-copies. pECT05 R-2animals recovered ouabaininduced muscle contraction in cut worm assays while x14 animals remained relaxed.
A 0.5kb transcript was found to be the only one coding unit in the 2.5kb Hin dIII fragment. By screening 150,000 plaques of lZAP cDNA library (R.J. Barsted and R.H. Waterston) using the 2.5kb fragment as a probe, three positively hybridized clones were isolated. One clone was sequenced and found to encode a 125kD protein. The predicted protein had a leucine-rich hydrophobic region on its NH2 terminus and Ser- and Thr- rich hydrophilic regions in the COOH-terminal half, where six potential phosphorylation sites were noted. In screening for possible relationships to other genes and proteins stored in the nucleic acid and protein data banks, neither identical nor significantly similar sequences were found.
Thus, we speculate that unc-68 codes for a novel protein participating in muscle contraction, which may serve as a substrate for certain kinases.
Ando H. and Kagawa H. (1991) J.Pharmacobio-Dyn. 14, s-82