GENETIC ANALYSIS OF SMALL WING PLC-ALPHA IN DROSOPHILA

描述：长期目标是了解 磷脂酶C-γ（PCL-g）是由果蝇小翅膀编码的 (sl)基因，通过研究与Ras介导的信号通路的相互作用 在果蝇的眼睛里 sl等位基因是目前唯一的PLC-g突变 发现于任何系统中，并且该酶是重要的细胞内酶 其响应于生长因子刺激而被激活。 这个提议有两个具体的目的，首先是生成一个真正的null sl的等位基因，其次寻找其他参与 sl-PLC-g在信号通路中的作用。 隔离的两种替代策略 Sl无效突变的详细描述。 萨克雷博士认为，现有的 等位基因可能产生难以解释的表型， 它们本身可能没有可见的表型。 因此，他的主要战略是， 用筛选器检测sl基因座的分子变化。 P[ry+]元素 (eas p372）将从附近的易休克基因（EAS）动员， PI将寻找P元件在SL中或接近SL的重新插入。 可能的感兴趣的P元件插入的检测将依赖于PCR 从大约S1内的18 kb区域产生的产物。 一旦新的 获得接近S1的插入，第二轮活动可 通过P元件的不精确切除产生s1的缺失。 零 将检查等位基因以确定去除s1-PLC-g对 在Sevenless（Sev）信号通路中。 新sl等位基因的双突变体 将检查Sev途径的其他成员在PLC-g中的作用 通路 如果P元素突变筛选失败，萨克雷博士建议 用EMS诱导sl. 评分的主要方法 将通过检查发现的翅脉缺损较短的翅， 钝的翅膀 然后将对突变体进行基因检查，以确保它们 映射到sl，然后逆转录PCR方案来鉴定单个 链构象多态性将用于筛选整个S1 ORF。 第二个目的是筛选SL表型的增强子和抑制子 从而鉴定相互作用的基因座。 一般的做法是 诱变已经对SL突变纯合的果蝇，并筛选 没有表现出短而钝的翅膀的典型突变表型的苍蝇， 异位翼静脉和轻微粗糙的眼睛 十字架的设计 在X、2和3染色体上发现增强子/抑制子突变体。 然后，将使用具有多个标记的染色体来绘制 新诱导的突变体 地图位置将通过测试进一步完善 每个地区都有不足之处，如果有的话。 以前的突变体 然后将未表征的基因座靶向用于克隆和测序， 补助期结束时。

DESCRIPTION: 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 is 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.