Molecular analyses of synaptic vesicle proteins/genetic and molecular studies of Drosophila learning mutants

I have attempted to define specific roles for individual synaptic vesicle proteins. The cloning and sequencing of one synaptic vesicle protein, SV2, revealed that the protein displays significant topological and sequence similarity to various transmembrane transporters.; Expression of SV2, synaptotagmin and synaptophysin CHO fibroblasts also allowed me to examine the targeting information contained by each protein. All three proteins entered different cellular compartments. Synaptotagmin was found on the plasma membrane. Both SV2 and synaptophysin were sorted to small intracellular vesicles, but synaptophysin colocalized with early endosomal markers, while SV2 did not. I also created cell lines expressing both SV2 and synaptotagmin, both synaptotagmin and synaptophysin, and lines expressing all three synaptic vesicle proteins. In all cases, the proteins maintained their distinct compartmentalizations, were not found in the same organelle, and did not create synaptic vesicle-like structures.; In the second part of my thesis I describe molecular, genetic and behavioral studies of normal and mutant Drosophila melanogaster. Fruit flies can learn a number of simple tasks, and single gene mutations that fail to learn or remember have been isolated. Two of these mutations, dunce and rutabaga, have been extensively characterized and encode cAMP phosphodiesterase and adenylate cyclase, respectively. I found that, unlike the original rutabaga allele {dollar}(rutsp{lcub}it 1{rcub}),{dollar} the {dollar}rutsp{lcub}it 2{rcub}{dollar} mutation could partially rescue the dunce learning defect. This result suggests that the dunce and rutubaga mutations do influence the same pool of intracellular cAMP.; I used a second site suppressor screen to isolate new Drosophila learning and memory mutants. Since rutabaga alleles can suppress both the female specific sterility and behavioral phenotype of dunce flies, it seemed likely that new learning mutants could be isolated as suppressors of dunce female sterility. Only progeny of rare fertile females are candidate mutants, and isolation of these flies requires significantly less time and effort than the traditional approach of creating multiple isogenic mutagenized stocks and testing the behavior of each individually. A dysgenic mutagenesis was used to recover P element induced X-chromosomal mutations that suppress dunce sterility. One of the mutants recovered was in the previously characterized amnesiac locus. Molecular cloning of the locus revealed that amnesiac encodes a novel neuropeptide gene. (Abstract shortened by UMI.)...