Worm Breeder's Gazette 14(3): 42 (June 1, 1996)
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.
|1||The Babraham Institute Laboratory of Molecular Signalling, Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, UK|
|2||Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan|
|3||Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan and Molecular Neurobiology Laboratory, The Institute of Physical and Chemical Research (RIKEN), Tsukuba Life Science Centre, Koyodai 3-1-1, Tsukuba, Ibaraki 305, Japan|
The intracellular second messenger inositol 1,4,5-trisphosphate (InsP3) binds to a specific receptor (InsP3R)1,2 that releases Ca2+ from intracellular stores, thereby modulating various Ca2+-associated processes in cells1. Using degenerate oligonucleotide primers and PCR cDNA clones for a putative InsP3R have been isolated from C. elegans. The same gene is also represented by ESTs. The gene, itr-1, is located on chromosome 4 between dif-1 and col-4. PCR and Southern blot experiments indicate that this is the only gene of this type in C. elegans. The full sequence of the cDNA (8.9kb) has been determined and encodes a putative InsP3R of 2849 amino acids (the longest yet identified). The predicted amino acid sequence is approximately 40% identical to all other known InsP3Rs. High levels of homology are observed in particular regions of the protein for example within the InsP3 binding domain, in the putative channel pore and in transmembrane domain 6. Analysis of cDNAs has revealed that the mRNA encoding the receptor is transpliced to SL1 and that there are at least 3 alternative splicing events in the production of the mRNA. One such event (at site A) alters the most 5' exon of the gene and is predicted to result in proteins with alternative N-termini. Events B and C occur in the InsP3 binding region and the modulatory regions of the protein respectively, in positions either very close to (B), or identical to (C) the SI and SII alternative splice sites in the mouse type 1 receptor. The conservation of these sites (although not the sequences) suggests they are important in receptor function. The genomic sequence of the region of chromosome 4 encoding itr-1 is now being determined by the C. elegans genome project. Thus we now know that the gene consists of 35 exons spread over 20kb. The exons encoding the two versions of the N-terminal are 3.5kb apart and hence the two forms may result from alternative promoters. An antibody against a peptide representing the C-terminal of the ITR-1 protein (Ab022) has been generated. We have shown that this antibody and another (#263) directed against part of the mouse type 1 receptor InsP3 binding domain recognise a protein of Mr 210kDa in C. elegans membrane preparations. Immunoprecipitation of solubilized C. elegans membrane proteins with antibody #263 results in the precipitation of a 210kDA protein recognised by Ab022. Using antibody #263, we have succesfully performed in situ staining of fixed, whole C. elegans. The highest levels of staining are observed in the nerve ring and the ventral cord indicating an important role for ITR-1 in neuronal intracellular signalling. To analyse further the function of the receptor in C. elegans we are generating knock-out animals using transposon, Tc1, mutagenesis. A screen for insertions throughout the gene identified two lines carrying Tc1 inserts in the region encoding the modulatory region of the protein. Cultures in which excision of Tc1 has produced deletions in itr-1 have now been identified and single worms carrying these deletions are being isolated. 1 Berridge M J (1993) Nature 361, 315-325. 2 Furuichi T, Kohda K, Miyawaki A and Mikoshiba K (1994) Curr. Op. in Neurobiol. 4, 294-303.