Deletion mutants within the Py DNA region between the replication origin and the beginning of late protein coding sequences have been constructed and analysed for viability, early gene expression and viral DNA replication. Assay of replicative competence was facilitated by the use of Py transformed mouse cells (COP lines) which express functional large T-protein but contain no free viral DNA. Viable mutants defined three new nonessential regions of the genome. Certain deletions spanning the PvuII site at nt 5130 (67.4 mu) were unable to express early genes and had a cis-acting defect in DNA replication. Other mutants had intermediate phenotypes. Relevance of these results to eucaryotic "enhancer" elements is discussed.

Volatile anesthetics like halothane and enflurane are of interest to clinicians and neuroscientists because of their ability to preferentially disrupt higher functions that make up the conscious state. All volatiles were once thought to act identically; if so, they should be affected equally by genetic variants. However, mutations in two distinct genes, one in Caenorhabditis and one in Drosophila, have been reported to produce much larger effects on the response to halothane than enflurane [1, 2]. To see whether this anesthesia signature is adventitious or fundamental, we have identified orthologs of each gene and determined the mutant phenotype within each species. The fly gene, narrow abdomen (na), encodes a putative ion channel whose sequence places it in a unique family; the nematode gene, unc-79, is identified here as encoding a large cytosolic protein that lacks obvious motifs. In Caenorhabditis, mutations that inactivate both of the na orthologs produce an Unc-79 phenotype; in Drosophila, mutations that inactivate the unc-79 ortholog produce an na phenotype. In each organism, studies of double mutants place the genes in the same pathway, and biochemical studies show that proteins of the UNC-79 family control NA protein levels by a posttranscriptional mechanism. Thus, the anesthetic signature reflects an evolutionarily conserved role for the na orthologs, implying its intimate involvement in drug action.

Mutations in LRRK2 are thus far the most frequent known cause of autosomal dominant and idiopathic Parkinson’s disease (PD) with prevalent mutations being found within the GTPase (R1441C/G) and kinase (G2019S) domains. Previous in vitro studies have revealed that R1441C and G2019S mutations are associated with increased kinase activity. To better understand LRRK2-linked PD pathogenesis in vivo, we have generated transgenic C. elegans overexpressing human LRRK2 wild type, R1441C and G2019S in dopaminergic (DA) neurons. Overexpression of these LRRK2 proteins causes age-dependent DA neurodegeneration, behavioral deficits, and locomotor dysfunction that are accompanied by a reduction of dopamine levels in vivo. In comparison, R1441C and G2019S mutants cause more severe phenotypes than the wild type protein. Interestingly, treatment with exogenous dopamine rescues the LRRK2-induced behavioral and locomotor phenotypes. In contrast, expression of the GTP binding defective mutant, K1347A, or knockout of the C. elegans LRRK2 homolog, LRK-1, prevents the LRRK2-induced neurodegeneration and behavioral abnormalities. Hence, our transgenic LRRK2 C. elegans models recapitulate key features of PD including progressive neurodegeneration, impairment of dopamine-dependent behavior and locomotor function, and reduction in dopamine levels. Furthermore, our findings provide strong support for the critical role of GTPase/kinase activity in LRRK2-linked pathologies. These invertebrate models will be useful for studying pathogenesis of PD and for development of potential therapeutics for the disease.

Synaptojanin is a lipid phosphatase required to degrade phosphatidylinositol 4,5 bisphosphate (PIP(2)) at cell membranes during synaptic vesicle recycling. Synaptojanin mutants in C. elegans are severely uncoordinated and are depleted of synaptic vesicles, possibly because of accumulation of PIP(2). To identify proteins that act downstream of PIP(2) during endocytosis, we screened for suppressors of synaptojanin mutants in the nematode C. elegans. A class of uncoordinated mutants called "fainters" partially suppress the locomotory, vesicle depletion, and electrophysiological defects in synaptojanin mutants. These suppressor loci include the genes for the NCA ion channels, which are homologs of the vertebrate cation leak channel NALCN, and a novel gene called unc-80. We demonstrate that unc-80 encodes a novel, but highly conserved, neuronal protein required for the proper localization of the NCA-1 and NCA-2 ion channel subunits. These data suggest that activation of the NCA ion channel in synaptojanin mutants leads to defects in recycling of synaptic vesicles.