Worm Breeder's Gazette 13(4): 68 (October 1, 1994)
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.
The Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, Downing Street, Cambridge UK.
The intracellular second messenger inositol 1,4,5 trisphosphate (InsP) binds to a specific receptor (InsPR) that releases Ca[2+] from intracellular storage sites, thereby modulating various Ca[2+]-associated proteins in cells (Mikoshiba et al 1993 Ann. NY Acad Sci 707, 178-197). As part of a project to study inositol 1,4,5 trisphosphate (InsP) signalling using C. elegans we have been attempting to clone the nematode InsP receptor. Using degenerate primers to two sequences conserved amongst InsP receptors in mammals, Xenopus and Drosophila we were able to amplify a 240bp (IP240) fragment of the coding sequence, either from genomic DNA or from random primed 1st strand cDNA. In addition, a search of the NCBI EST database with the Drosophila sequence revealed that one of Yuji Kohara's (WBG 13 20-21) cDNAs ( yk33 g8 )encoded 445 bp covering the end of the gene and part or all of the 3' untranslated region. This clone was clearly different from cDNA clones encoding the known ryanodine receptor (eg yk4 h3 ).The amino acid sequences encoded by the two sequences are clearly much closer to InsP receptors than to the ryanodine receptors. Thus the evidence to date suggests that this is the InsP receptor. An alignment of one part of the C. elegans sequence and InsP receptor sequences from other organisms (figure 1) shows that some of the residues which are identical in all the other sequences are not conserved in C. elegans. Thus the sequence of this molecule may provide insights into structure and function of the protein. We have recently obtained a PCR product covering the 3kb between the two fragments and are currently working towards obtaining a full length cDNA (expected to be in the region of 8-9kb) and a genomic clone. From here we plan to study the function of the receptor using C. elegans genetics and in vitro expression and electrophysiology. We are indebted to Drs Katsuhiko Mikoshiba, Teiichi Furuichi, Yuji Kohara and John Fleming for their assistance during this study. IP240 2056 (Drosophila amino acid numbering) DROS. NpdmQNlLRnQNNKTNnNLVsETLmFLDciCGSTTGGLGLLGLYINEhNlaLinQTLEaL RAT 1 NrdLQNFLRcQNNKTNyNLVcETLqFLDciCGSTTGGLGLLGLYINEkNvaLinQTLEsL RAT 2 NreLQNFLRnQNNKTNyNLVcETLqFLDciCGSTTGGLGLLGLYINEkNvaLvnQTLEsL XENOPUS NrdLQNFLRcQNNKTNyNLVcETLqFLDimCGSTTGGLGLLGLYINEdNvgLviQTLEtL RAT 3 NrdLQNFLRcQNNKsNyNLVcETLqFLDciCGSTTGGLGLLGLYINEkNvaLinQTLEsL HUMAN 3 NrdLQNFLRcQNNKTNyNLVcETLqFLDimCGSTTGGLGLLGLYINEdNvgLviQTLEtL MOUSE 1 NrdLQNFLRcQNNKTNyNLVcETLqFLDciCGSTTGGLGLLGLYINEkNvaLinQTLEsL IP240 NslLQNFLRkQsdrTNhNLVsETLsFLDtvCGSTkGsLGvfGe.IgEhNfsLitQTLatL Consensus N--LQNFLR-QNNKTN-NLV-ETL-FLD--CGSTTGGLGLLGLYINE-N--L--QTLE-L Figure 1. An alignment of the amino acid sequence encoded by IP240 to InsP receptors from other species. Positions which are identical tothe consensus are shown in upper case. The consensus is where 7 (of the 8) sequences are identical.