Mobilization of a Drosophila transposon in the Caenorhabditis elegans germ line.

Transposons have been enormously useful for genetic analysis in both Drosophila and bacteria. Mutagenic insertions constitute molecular tags that are used to rapidly clone the mutated gene. Such techniques would be especially advantageous in the nematode Caenorhabditis elegans, as the entire sequence of the genome has been determined. Several different types of endogenous transposons are present in C. elegans, and these can be mobilized in mutator strains (reviewed in ref. 1). Unfortunately, use of these native transposons for regulated transposition in C. elegans is limited. First, all strains contain multiple copies of these transposons and thus new insertions do not provide unique tags. Second, mutator strains tend to activate the transposition of several classes of transposons, so that the type of transposon associated with a particular mutation is not known. Here we demonstrate that the Drosophila mariner element Mos1 can be mobilized in C. elegans. First, efficient mobilization of Mos1 is possible in somatic cells. Second, heritable insertions of the transposon can be generated in the germ line. Third, genes that have been mutated by insertion can be rapidly identified using inverse polymerase chain reaction. Fourth, these insertions can subsequently be remobilized to generate deletion and frameshift mutations by imperfect excision.

Studies of synaptic vesicle endocytosis in the nematode C. elegans.

After synaptic vesicle exocytosis, synaptic vesicle proteins must be retrieved from the plasma membrane, sorted away from other membrane proteins, and reconstituted into a functional synaptic vesicle. The nematode Caenorhabditis elegans is an organism well suited for a genetic analysis of this process. In particular, three types of genetic studies have contributed to our understanding of synaptic vesicle endocytosis. First, screens for mutants defective in synaptic vesicle recycling have identified new proteins that function specifically in neurons. Second, RNA interference has been used to quickly confirm the roles of known proteins in endocytosis. Third, gene targeting techniques have elucidated the roles of genes thought to play modulatory or subtle roles in synaptic vesicle recycling. We describe a molecular model for synaptic vesicle recycling and discuss how protein disruption experiments in C. elegans have contributed to this model.

Identification and characterization of the vesicular GABA transporter.

Synaptic transmission involves the regulated exocytosis of vesicles filled with neurotransmitter. Classical transmitters are synthesized in the cytoplasm, and so must be transported into synaptic vesicles. Although the vesicular transporters for monoamines and acetylcholine have been identified, the proteins responsible for packaging the primary inhibitory and excitatory transmitters, gamma-aminobutyric acid (GABA) and glutamate remain unknown. Studies in the nematode Caenorhabditis elegans have implicated the gene unc-47 in the release of GABA. Here we show that the sequence of unc-47 predicts a protein with ten transmembrane domains, that the gene is expressed by GABA neurons, and that the protein colocalizes with synaptic vesicles. Further, a rat homologue of unc-47 is expressed by central GABA neurons and confers vesicular GABA transport in transfected cells with kinetics and substrate specificity similar to those previously reported for synaptic vesicles from the brain. Comparison of this vesicular GABA transporter (VGAT) with a vesicular transporter for monoamines shows that there are differences in the bioenergetic dependence of transport, and these presumably account for the differences in structure. Thus VGAT is the first of a new family of neurotransmitter transporters.

Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans.

Synaptotagmin, an integral membrane protein of the synaptic vesicle, binds calcium and interacts with proteins of the plasma membrane. These observations suggest several possible functions for synaptotagmin in synaptic vesicle dynamics: it could facilitate exocytosis by promoting calcium-dependent fusion, inhibit exocytosis by preventing fusion, or facilitate endocytosis of synaptic vesicles from the plasma membrane by acting as a receptor for the endocytotic proteins of the clathrin AP2 complex. Here we show that synaptic vesicles are depleted at synaptic terminals in synaptotagmin mutants of the nematode Caenorhabditis elegans. This depletion is not caused by a defect in transport or by increased synaptic vesicle release, but rather by a defect in retrieval or synaptic vesicles from the plasma membrane. Thus we propose that, as well as being involved in exocytosis, synaptotagmin functions in vesicular recycling.

patched overexpression causes loss of wingless expression in Drosophila embryos.

The patched (ptc) segment polarity gene of Drosophila encodes a transmembrane protein involved in cell signaling that establishes pattern within the segment. In the posterior half of the parasegment Patched protein represses transcription of the wingless (wg) gene by an unknown mechanism. In the most posterior row of cells in each parasegment this repression is neutralized by a signal possibly carried by the product of the hedgehog gene, allowing wg expression. High levels of Patched expression might therefore overcome the repression and repress wg in all cells. Here we use a heat shock-inducible promoter to transiently express high levels of Patched in all cells. A single pulse of Patched transgene expression has little or no effect on the segmental pattern, as has been previously reported. Repeated pulses of Patched production drastically alter the segment pattern to mimic embryos lacking one of the wg class of segment polarity genes. We observe repression of wg and gooseberry (a wg class gene) transcription in the germband ectoderm but not in the head. Expression of two other segment polarity genes, engrailed and cubitus interruptus, is unaffected. Thus excess Patched is capable of overcoming the neutralizing signal.