Worm Breeder's Gazette 12(3): 71 (June 15, 1992)

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.

Homologs of Mammalian Synaptic Vesicle Protein Genes are Expressed in the C. elegans Nervous System

Michael L. Nonet, Barbara J. Meyer

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720

We are beginning to examine synaptogenesis and synaptic function in C. elegans utilizing both molecular and genetic approaches. Utilizing PCR, we have isolated three genes that probably encode components of synaptic vesicles in C. elegans. We have acquired complete cDNA sequence for each gene, and we have positioned the genes on the physical and genetic maps. We are interested in determining the functions of these genes in C. elegans either by identifying previously existing mutations that disrupt these genes, or by isolating mutants de novo that affect their gene function. Additionally, we wish to use antibodies directed against these components to identify and characterize mutants with developmental defects in synaptogenesis.

First, we have characterized a homolog of a gene encoding VAMP (also called synaptobrevin), an integral membrane protein found in synaptic vesicles. No clear function in neurotransmitter release has been assigned to VAMP (1). The C. elegans gene shows 67% amino acid identity with human VAMP- 1. Physically, the C. elegans VAMP gene was localized to the T10H9 cosmid on chromosome V. Genetically, we have positioned the VAMP gene to the 0.25 cM deficiency interval spanning unc-70 ,between the right end of nDf18 and the right end of nDf32 .We are now determining if unc-70 or one of the four let genes in the interval encode VAMP. Antibodies directed against human VAMP stain the C. elegans nervous system in situ and also cross react with a C. elegans VAMP fusion protein. The ventral cord, dorsal cord and nerve ring all stain clearly. The staining intensity is not strong enough to determine if individual axons or commisures stain. We are raising antibodies against the C. elegans protein that should permit us to further characterize the localization of VAMP in C. elegans.

Second, we have isolated and sequenced a homolog of a gene encoding p65 (also called synaptotagmin), another integral membrane protein localized to synaptic vesicles. The presence of two motifs homologous to calcium-dependent protein kinase C (PKC) regulatory domains suggested this molecule may be involved in calcium-regulated fusion of synaptic vesicles at synapses (1). C. elegans p65 shows 51% amino acid identity (71% in the PKC-like regulatory domains) with the human sequence. Physically, this gene has been positioned on cosmid K1 OB7,indicating that the gene maps in the interval between tra-2 and dpy-10 on chromosome II. Antibodies directed against mammalian p65 stain the C. elegans nervous system in situ and cross react with a C. elegans p65 fusion protein. The staining is also competed away by the C. elegans p65 protein fusion. The nerve ring stains very brightly and has a punctate appearance, different from the VAMP staining. The ventral and dorsal cord stain also, but much less intensely. From the intensity of the ventral cord staining, we would not expect to be able to determine if individual commisures stain. We anticipate that antibodies we are making against the C. elegans protein, will allow us to characterize the staining in more detail.

Third, we have also characterized a homolog of the rab3 Agene (69% amino acid identity with human rab3 A),which encodes a synaptic vesicle associated ras-like GTP binding protein proposed to be involved in the regulation of synaptic vesicle fusion at synapses (1). Physically, this gene has been positioned to cosmid C02C12 ,indicating the gene maps somewhere between hlh-1 and lin-4 on chromosome II. We must await C. elegans rab3 Aantibodies to localize the rab3 Aprotein, since the mammalian rab3 Aantibodies do not stain C. elegans.

Finally, we have isolated new recessive lethal mutants that have similar phenotypes to well-characterized mutants with known synaptic defects (subject of a future gazette article). Using antibodies directed against synaptic components, we are proceeding to characterize these new mutants, and other unc mutants likely to have synaptic defects.

Literature Cited:

Sudhof and Jahn. Neuron 6: 665-677. 1991