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.