GENETIC ANALYSIS OF SMALL WING PLC-ALPHA IN DROSOPHILA

果蝇小翼PLC-α的遗传分析

• 批准号: 2024432
• 负责人: JUSTIN R THACKERAY
• 金额: $ 11.03万
• 依托单位: CLARK UNIVERSITY (WORCESTER, MA)
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2024432
• 关键词: Drosophilidae; alleles; biological signal transduction; enzyme activity; gene deletion mutation; gene expression; gene interaction; gene mutation; genetic enhancer element; genetic mapping; genetic markers; genetic polymorphism; growth factor; phospholipase C; polymerase chain reaction; suppressor mutations; transposon /insertion element; visual photoreceptor

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.

描述：长期目标是理解 果蝇小翅编码的磷脂酶C-γ(PCL-g) (SL)基因，通过研究与RAS介导的信号通路的相互作用 在果蝇眼睛里。Sl等位基因是目前仅有的plc-g突变。 在任何系统中都存在，这种酶是一种重要的胞内酶 它是响应生长因子刺激而激活的。 该提议有两个具体目标，第一，生成一个真正的空值 S1的等位基因，第二个是寻找参与 SL-PLC-g在信号通路中的表达。隔离的两种替代策略 对s1零突变进行了详细的分析。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，纯合子 它们本身可能没有明显的表型。因此，他的主要战略是 使用屏幕检测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)和 PI将寻找P元素重新插入到SL中或接近SL的位置。 对可能的P元件插入的检测将依赖于聚合酶链式反应 在约sl的18kb区域内产生的产品。曾经是一个新的 获得接近SL的插入，则第二轮动员可以 通过不精确地删除P元素来生成sl的删除。空的 将检查等位基因以确定去除sl-PLC-g对 无七(SEV)途径中的信号转导。新的sl等位基因的双突变 和SEV途径的其他成员将被检查在PLC-g中的作用(S 路径。萨克雷博士建议，如果P元素突变筛查失败 利用EMS诱变sl.评分的主要方法 将通过检查机翼的翼脉缺陷变短， 钝化的翅膀。然后将对突变体进行遗传检查，以确保它们 映射到sl，然后用逆转录聚合酶链式反应方法识别单个 链构象多态将被用来筛选整个sl ORF。 第二个目的是筛选sl表型的增强和抑制因子。 从而识别相互作用的基因座。一般的方法是 对已经纯合的果蝇进行s1突变的诱变并筛选 没有表现出典型的短而钝的翅膀的突变表型的苍蝇， 异位的翼脉和略微粗糙的眼睛。十字架已经设计好了 在X，2，Ar，3染色体上发现增强子/抑制子突变体。 然后，带有多个标记的染色体将被用来绘制 新诱导的突变体。地图位置将通过测试进一步细化 每个地区的不足之处，如果它们存在的话。以前的突变体 然后，将未鉴定的基因座作为克隆和测序的目标 授权期结束。