OVARIAN RING CANALS IN DROSOPHILA

描述：林恩库利博士的这份修订申请概述了 实验研究蛋白质参与的结构和功能， 一种特殊的细胞内桥，称为环形通道， 雌性果蝇的卵室 环形管道状结构是 也存在于包括人类在内的其他生物的生殖细胞中。 在 果蝇卵子发生时，环管是重要的流动管道 从滋养细胞到卵母细胞的细胞质物质。 他们的 形成涉及至少四种蛋白质：磷酸酪氨酸蛋白， 丝状肌动蛋白、胡里太少（hts）蛋白和kelch 蛋白 据认为，肌动蛋白丝提供了途径， 通过环形运河的运动。 博士库利假设，这些细丝是稳定的周围， 通过HTS蛋白质的运河的边缘，并且它们被分组为 bundles束by the kelch Kelch Kelch蛋白. 此外，她认为， 另一个基因cheerio参与了环管的形成。 库利博士计划的第一部分是研究kelch蛋白质。 一 目的是使用生殖系转化构建体来测试 对kelch基因的两个ORF的产物的要求。 这些orf 被一个单一的终止密码子分开， 翻译抑制 因此，kelch基因使两个 多肽，一个对应于第一ORF，另一个对应于第二ORF。 两个ORF的组合;两种多肽都存在于环管中。 博士库利将研究这些多肽的功能， 制备其中第二ORF缺失的转化构建体，或 其中ORF之间的终止密码子突变或缺失。 这些 构建体将由来自卵巢肿瘤的启动子驱动， (otu)或山雀基因，一旦插入基因组， 测试了它们拯救不育Kelch突变的能力。 完全或部分获救动物的抗体染色将用于 研究转基因kelch蛋白的细胞定位并 评估它们的环形运河的完整性 相似变换 将进行实验以分析kelch内的域。 proteins. 这些实验将辅以体外试验， 确定Kelch蛋白的片段是否可以结合，捆绑，帽化， 使肌动蛋白丝成核。 这些测试的蛋白质将是 在E.大肠杆菌中或感染杆状病毒的昆虫细胞中 表达结构 库利博士还将使用抗原决定簇标记 测定Kelch蛋白在体内是否二聚化的方法。 DNA序列 EMS诱导的kelch突变和细胞定位的分析 用针对较大Kelch的ORF 2部分的抗体进行的实验 多肽也被计划。 库利博士项目的第二部分是研究hts蛋白。 该蛋白含有一个与脊椎动物同源的N端结构域， 内收蛋白 C-末端结构域是新的，并且似乎在 只在环形运河里。 库利博士假设它是由 通过蛋白水解切割由蛋白酶产生的前体蛋白， 交替剪接的卵巢特异性RNA 种系转化 将使用实验来确定卵巢特异性RNA是否， 事实上，产生60 kD HTS环管蛋白。 然后各种 结构将调查的功能意义， HTS基因的最后三个外显子。 主要的问题是 hts蛋白的C-末端部分可以拯救hts突变体 表型 此外，纯化的HTS蛋白将用于肌动蛋白 结合、捆绑、加帽和成核测定，如已经描述的 对于Kelch蛋白质。 库利博士计划的第三部分是克隆和描述 干杯基因。 初步研究表明，cheerio位于 III号染色体上89 F和90 A之间的间隔（66 A中没有， 以前认为）。 库利博士会用转座子跳跃法 将89 E11 -12或90 B3 -4中的P元件插入跳跃到cheerio中， 基因座 将通过筛选一种 雌性不育表型，当带有跳跃元件的染色体 一个已知的啦啦队等位基因 标准程序将用于 分离并克隆该基因。

DESCRIPTION: This revised application from Dr. Lynn Cooley outlines experiments to study proteins involved in the structure and function of specialized intracellular bridges called ring canals, which are found in the egg chambers of Drosophila females. Ring canal-like structures are also found in the germlines of other organisms, including humans. In Drosophila oogenesis, the ring canals are important conduits for the flow of cytoplasmic materials from the nurse cells to the oocyte. Their formation involves at least four proteins: a phosphotyrosine protein, filamentous actin, the hu-li tai shao (hts) protein, and the kelch protein. It is thought that actin filaments provide the pathway for movement through the ring canals. Dr. Cooley hypothesizes that these filaments are stabilized around the rim of the canal by the hts protein and that they are grouped into bundles by the kelch protein. In addition, she believes that the product of another gene, cheerio, is involved in ring canal formation. The first part of Dr. Cooley's plan is to study the kelch protein. One aim is to use germline transformation constructs to test the requirements for products of the two ORFs of the kelch gene. These ORFs are separated by a single stop codon which is evidentally subject to translational suppression. The kelch gene therefore makes two polypeptides, one corresponding to the first ORF, the other to a combination of the two ORFs; both polypeptides are found in ring canals. Dr. Cooley will investigate the functions of these polypeptides by making transformation constructs in which the second ORF is deleted, or in which the stop codon between the ORFs is mutated or deleted. These constructs will be driven by a promoter from either the ovarian tumor (otu) or chickadee genes, and once inserted into the genome, will be tested for their ability to rescue sterilizing kelch mutations. Antibody staining of fully or partially rescued animals will be used to study the cellular localization of the transgenic kelch proteins and to assess the integrity of their ring canals. Similar transformation experiments will be carried out to analyze domains within the kelch proteins. These experiments will be supplemented with in vitro tests to determine if fragments of the kelch proteins can bind, bundle, cap and nucleate actin filaments. The proteins for these tests will be synthesized in E. coli or in insect cells infected with a baculovirus expression construct. Dr. Cooley will also use an epitope tagging procedure to determine if kelch proteins dimerize in vivo. DNA sequence analysis of EMS-induced kelch mutations and cellular localization experiments with antibodies to the ORF2 portion of the larger kelch polypeptide are also planned. The second part of Dr. Cooley's project is to study the hts protein. This protein contains an N-terminal domain homologous to vertebrate adducin. The C-terminal domain is novel and appears to be found exclusively in ring canals. Dr. Cooley hypothesizes that it is produced by proteolytic cleavage of a precursor protein that is made from an alternately spliced, ovary-specific RNA. Germline transformation experiments will be used to determine if the ovary-specific RNA does, in fact, produce the 60 kd hts ring canal protein. Then various constructs will be made to investigate the functional significance of the last three exons of the hts gene. The main question is whether or not the C-terminal part of the hts protein can rescue the hts mutant phenotype. In addition, purified hts protein will be used in actin binding, bundling, capping and nucleating assays, as already described for the kelch proteins. The third part of Dr. Cooley's plan is to clone and characterize the cheerio gene. Preliminary work has shown that cheerio lies in the interval between 89F and 90A on chromosome III (not in 66A, as previously thought). Dr. Cooley will use a transposon hopping scheme to jump P element insertions in 89E11-12 or 90B3-4 into the cheerio locus. Jumps into this gene will be identified by screening for a female sterile phenotype when a chromosome with a jumped element is put over a known cheerio allele. Standard procedures will then be used to isolate and clone the gene.