Worm Breeder's Gazette 15(3): e1 (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.
Sussex Centre for Neuroscience, University of Sussex, Falmer, Brighton, England. BN1 9QG
Soluble guanylate cyclase (sGC) is the main receptor of the gaseous signalling molecule, nitric oxide (NO), and catalyses the cyclization of GTP to the intracellular second messenger, cGMP. The NO-GC pathway is implicated in neuronal signalling throughout the animal kingdom and we are investigating its presence in C. elegans. sGC is a heterodimer composed of two subunits (alpha and beta) of around 70kDa. The C. elegans genomic sequencing project has revealed five putative subunits of sGC. One of these (C06B3.8) has been cloned and sequenced by us from cDNA and found to contain a splice site not predicted by the Genefinder software, and six point mutations compared to the genomic sequence. We are currently investigating the significance of this difference between the cDNA and genomic sequence. The full length cDNA was assembled from two overlapping fragments, cloned using a combination of nested PCR with a cDNA library as template, and RT-PCR on C. elegans mRNA. The fragments share a unique EaeI restriction site which was used for assembly of the full length clone. The cDNA encodes a 685 residue protein which shows a 28% homology to the rat sGC beta-2 subunit overall, and 51% homology in the catalytic domain. This has been subcloned into a mammalian expression vector and preliminary results will be presented regarding the cyclase activity of this enzyme in COS-7 cells. We have recently raised an antibody against this enzyme using the following peptide; (KY)TAMPLHDATRDLILLNQQRLSDV, taken from a predicted surface domain of C06B3.8. This was crosslinked to thyroglobulin using gluteraldehyde to generate the antigen. The antibody is currently being characterised and we will report on our preliminary findings using Western blotting and immunocytochemistry. In related experiments, a radioimmunoassay has been developed to detect cGMP generation in extracts of C. elegans tissue. Interestingly, worm cytosolic extract appears to be insensitive to NO stimulation. However, the amino acid sequence of the heme binding domain is atypical of NO-activated sGCs and may explain our result. The identity of the natural activator of this worm sGC remain unknown.