Myosin V, an unconventional myosin, seems to be common to most organisms but takes part in rather diverse processes, including localization of mRNA, recycling plasma membrane receptors, and positioning melanosomes and synaptic vesicles. Myosin V is expressed in mammalian nervous systems, and mutations in human myosin V are associated with at least one neurological disease. Accordingly, we are interested in whether HUM-2, the C. elegans homologue of myosin V (Baker and Titus, 1997), is expressed in neurons. HUM-2 is predicted to have several alternatively spliced variants, the longest of which is HUM-2b. We performed 5' RACE for the
hum-2b transcript and found that the actual start codon does not correspond to the site predicted by genefinder. We used the PCR-based method of Hobert et al (2002) to fuse the upstream promoter region and first exon of
hum-2b to GFP. This product, along with pBX, was injected into Pha-1 worms, and seven GFP-expressing transgenics were obtained. Most of the transgenic lines express GFP in six to eight head neurons with cell bodies in the anterior and lateral ganglia. At least four of these neurons send processes to the nose as well as into the nerve ring and occasionally into the ventral nerve cord. Those worms showing the strongest expression also have one or two GFP-positive cell bodies in the pre-anal ganglion, as well as the anal sphincter muscle. In addition, all lines express the promoter driven GFP in the pharyngeal-intestinal valve, and several lines weakly express GFP in the excretory system. The C. elegans knockout consortium has isolated a 2.1 kb deletion in the
hum-2 gene (OK596) that begins in the region corresponding to the motor domain of the predicted protein. This deletion is expected to result in a truncated, null protein. The outcrossed
hum-2 mutants do not have an obvious phenotype although they do not exhibit the typical slowing response upon entering food, suggesting that there is either a mechano- or chemosensory defect. We are in the process of identifying the relevant neurons, but the GFP expression pattern and the modulation defect in the mutant suggets that the dopaminergic neurons are involved. Finally, we are determining the expression pattern of the predicted splice variants of HUM-2, including HUM-2c, which is a truncated version of HUM-2b lacking the motor domain.