Worm Breeder's Gazette 11(1): 33
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.
As an approach to analyze neuronal signal transduction in C. elegans, we attempted to characterize a gene for guanine nucleotide- binding regulatory protein (G protein). Rat cDNA probes for Gsalpha and Goalpha, described by Itoh et al., Proc. Natl. Acad. Sci. USA. 83, 3776-3780 (1986), revealed a few bands on a Southern blot of C. elegans N2 total DNA digested with HindIII, EcoRI or XbaI. A single clone was isolated from N2 genomic library in lambdaEMBL-4 from Dr. C. Link, and another from a library in lambda2001 from Dr. A. Coulson, by screening with the 1.1kb EcoRI fragment corresponding to the C- terminal 2/3 of rat Gsalpha (pGI11L). The two clones are similar and probably overlapping. The former clone (lambdaEGLI-9) produces hybridizing fragments on a Southern blot comparable to those from total DNA, and it was further characterized. A 0.8kb XbaI fragment, which hybridizes strongly with the 11L probe, was sequenced. It carries a sequence potentially coding for 48 amino acid sequence which is highly homologous to that found at the C-terminus of the rat Gsalpha (see below). Fourty-four amino acids out of 48 are identical between the two sequences. The sequence 5' to this region and within the XbaI fragment does not show significant homology with the probe, and seems to be an intron. Another fragment, which hybridizes to the rat Gsalpha cDNA fragment corresponding to the N-terminal 1/3 (pGI11S), resides 5' to the sequenced XbaI fragment. Therefore, this clone may carry the entire gene coding for a Gsalpha subunit. We are further analyzing this clone, and also trying to clone a corresponding cDNA from the library of Dr. B. Barstead. Screening the lambda2001 genomic library with the N-terminal cDNA probe produced seven positive clones. A few of these were partially sequenced, but none revealed a putative coding region for Gsalpha. Synthetic oligonucleotide probes for the conserved GTPase or guanine-binding regions revealed several bands on a Southern blot of C. elegans total DNA. We are also screening genomic clones with these oligonucleotide probes. [See Figure 1]