The long term goal is to understand the role of the Phospolipase C-gamma (PCL-g) which is encoded by the Drosophila small wing (sl) gene, by studying interactions with the Ras mediated signaling pathway in the Drosophila eye. The sl alleles are the only PLC-g mutations yet found in any system and the enzyme is an important intracellular enzyme which is activated in response to growth factor stimulation. There are two specific aims for this proposal, first to generate a true null allele of sl, and second to look for other genes that participate with sl-PLC-g in signaling pathways. Two alternative strategies to the isolation of a sl null mutation are detailed. Dr. Thackeray believes that existing allele may produce a phenotype difficult to interpret and the homozygotes themselves may have no visible phenotype. Thus, his primary strategy is to use a screen to detect molecular changes in the sl locus. A P[ry+] element (eas p372) will be mobilized from the nearby easily shocked gene (eas) and the PI will look for reinsertion of the P-element in or close to the sl. Detection of possible P-element insertions of interest will rely on PCR products generated from an 18kb region within around sl. Once a new insertion close to sl is obtained, a second round of mobilization could generate deletions of sl by imprecise excision of the P-element. The null allele will be examined to determine the effect of removing sl-PLC-g on signaling in the Sevenless (Sev) pathway. Double mutants of new sl alleles and other member of Sev pathway will be examined for role(s) in the PLC-g pathway. If the P-element mutagenesis screen fails, Dr. Thackeray proposes to use EMS to induce point mutants in sl. The primary method of scoring will be by examination of the wings for wing vein defects of shorter, blunted wings. The mutants will then be examined genetically to ensure they map to sl and then reverse transcriptase PCR protocol to identify single strand conformation polymorphisms will be used to screen the entire sl ORF.
The second aim i s to screen for enhances and suppressors of the sl phenotype and thereby identify interacting loci. The general approach is to mutagenize flies already homozygous for a sl mutation and to screen for flies that do not show the typical mutant phenotype of short, blunted wings, ectopic wing veins and a mildly rough eye. The crosses have been designed to pick up enhancer/ suppressor mutants in the X, 2nd ar 3rd chromosomes. Chromosomes with multiple markers will then be used to map the location the newly induced mutants. Map positions will be further refined by testing with deficiencies for each region, if they exist. Mutants of previously uncharacterized loci would then be targeted for cloning and sequencing at the end of the grant period.
| Mankidy, Rishikesh; Hastings, Jeremy; Thackeray, Justin R (2003) Distinct phospholipase C-gamma-dependent signaling pathways in the Drosophila eye and wing are revealed by a new small wing allele. Genetics 164:553-63 |
| Manning, Charlene M; Mathews, Wendy R; Fico, Leah P et al. (2003) Phospholipase C-gamma contains introns shared by src homology 2 domains in many unrelated proteins. Genetics 164:433-42 |
| Abbeyquaye, Tetteh; Riesgo-Escovar, Juan; Raabe, Thomas et al. (2003) Evolution of Gab family adaptor proteins. Gene 311:43-50 |
