MOLECULAR BASIS OF INCOMPATIBILITY IN DROSOPHILA

果蝇不相容性的分子基础

• 批准号: 2185387
• 负责人: Timothy Lawrence Karr
• 金额: $ 9.3万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185387
• 关键词: Drosophilidae; Rickettsia; bacterial genetics; cytogenetics; cytoplasm; gene expression; genetic strain; genetic transcription; intracellular transport; lethal genes; microinjections; molecular biology; nucleic acid sequence; sex linked trait; sperm; transfection

DESCRIPTION: In this amended proposal Dr. Karr proposes to continue his studies to understand the cellular and molecular basis for cytoplasmic incompatibility (CI) in insects. In CI, crosses of strains of the same species fail to yield many progeny.In such crosses the sperm is able to enter the egg, but it is unable to contribute its genetic material to the developing embryo. The syndrome is associated with a rickettsial endosymbiont (a Wolbachia species). CI is inherited maternally, but acts by affecting the sperm of males carrying the incompatibility factor. Crosses between CI males and uninfected females are unsuccessful, while all other combinations are successful. In preliminary studies Dr. Karr has made a number of valuable findings. He has shown a bidirectional incompatibility system in Drosophila simulans, he has developed a technique for monitoring the endosymbiont via DNA staining in early embryos, he has isolated Wolbachia specific monoclonal antibodies, he has shown an association between Wolbachia and the centrosome of the host in the early embryo, and he has developed a partial purification scheme for the endosymbiont. Perhaps his most important preliminary result is the successful transfer of the endosymbiont from Drosophila simulans to tetracycline-cured D. simulans and to Drosophila melanogaster. Furthermore, via a selection scheme he has achieved a high level of CI in D. melanogaster. The proposed research is aimed to a large extent toward placing many of the assumptions about CI on a more rigorous experimental basis, and on developing tools that will be essential for the long term success of the research program. In the first set of proposed experiments, Dr. Karr will test the hypothesis that the presence of Wolbachia in pole cells is sufficient for vertical transmission of CI via pole cell transplantation experiments. He also proposes to obtain definitive evidence that Wolbachia is the cause of CI by microinjection of purified bacteria, and by showing that infected lines can be produced which display CI. He will also quantify CI and Wolbachia levels during the reestablishment of the syndrome after antibiotic treatment. In further experiments he will examine the intracellular localization of Wolbachia during gametogenesis. In another line of research Dr. Karr will attempt to determine the basis for the bidirectional incompatibility found in D. simulans. In these experiments bacteria from one type of strain will be introduced by transplantation into cured strains of the other crossing type. He will also introduce each type of bacteria into D. melanogaster and then determine if bidirectional CI is displayed by the resulting stocks. In preliminary results, Dr. Karr has identified a protein found in sperm from CI carrying males, but not uninfected males. He will purify enough of this protein for microsequencing. He will then use the sequence information to synthesize oligonucleotides that will allow him to clone the gene that encodes the protein. He will then raise antibodies to the protein and determine its cytological location and behavior during CI.

描述：在此修订的建议中，卡尔博士建议继续他的 了解细胞质的细胞和分子基础 昆虫中的不兼容（CI）。 在CI中，相同的菌株交叉 物种无法产生许多后代。在这样的杂交中，精子能够 输入鸡蛋，但无法将其遗传物质贡献给 开发胚胎。 该综合征与立克相关 内共生体（沃尔巴氏菌种）。 CI是母亲继承的，但是 通过影响携带不兼容的雄性精子来行动 因素。 CI男性和未感染的女性之间的十字架是 不成功，而所有其他组合都是成功的。 在 初步研究Karr博士做出了许多宝贵的发现。 他在果蝇中显示了一个双向不兼容系统 Simulans，他开发了一种用于监测内共生体的技术 通过早期胚胎中的DNA染色，他具有孤立的Wolbachia特异性 单克隆抗体，他显示了沃尔巴基亚和 宿主在早期胚胎中的中心体，他已经开发了一个 内共生体的部分纯化方案。 也许是他最大的 重要的初步结果是成功转移 从果蝇simulans到四环素固化的D. simulans的内共生体 并到果蝇Melanogaster。 此外，通过选择方案 在D. melanogaster中获得了高水平的CI。 拟议的研究在很大程度上旨在放置许多 关于CI的假设在更严格的实验基础上以及 开发对长期成功至关重要的工具 研究计划。 在第一组提议的实验中，Karr博士 将检验以下假设： 足以通过极细胞垂直CI垂直传输 移植实验。 他还建议获得确定的 沃尔巴基亚是通过对纯化的微注射而引起CI原因的证据 细菌，通过表明可以产生受感染的线 显示CI。 他还将量化CI和Wolbachia的水平 抗生素治疗后综合征的重建。 进一步 实验他将检查Wolbachia的细胞内定位 在配子发生期间。 在另一项研究中，卡尔博士将尝试 确定在中的双向不相容性的基础 D. Simulans。 在这些实验中，一种类型的菌株细菌将 通过将移植到另一个的固化菌株中引入 交叉类型。 他还将将每种类型的细菌引入D。 melanogaster，然后确定双向CI是否由 由此产生的股票。 在初步结果中，Karr博士确定了精子中发现的蛋白质 来自CI携带男性，但没有未感染的雄性。 他将足够净化 该蛋白用于微粘结法。 然后他将使用序列 信息综合寡核苷酸，使他可以克隆 编码蛋白质的基因。 然后，他将提出抗体 蛋白质并确定其在CI期间的细胞学位置和行为。