Cloning and Analysis of a Gamete Fusion Gene

配子融合基因的克隆与分析

• 批准号: 7842376
• 负责人: Charlene L. Forest
• 金额: $ 2.61万
• 依托单位: BROOKLYN COLLEGE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2009-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842376
• 关键词: Adhesions; Affect; Antibodies; Area; Bacterial Artificial Chromosomes; Bioinformatics; Biological Assay; Biological Models; Cell membrane; Cells; Chimeric Proteins; Chlamydomonas; Chlamydomonas reinhardtii; Cloning; Complement; Complex; Contraceptive Agents; Data; Electron Microscopy; Employee Strikes; Exhibits; Failure; Fertilization; Fluorescence; Fluorescence Microscopy; Future; Gene Expression Regulation; Gene Proteins; Genes; Genetic; Genome; Genomics; Germ Cells; Goals; Homologous Gene; Homology Modeling; Human; Infertility; Label; Lead; Learning; Mammals; Maps; Membrane; Membrane Fusion; Messenger RNA; Methods; Microscopic; Modeling; Molecular; Molecular Analysis; Molecular Biology; Molecular Models; Morus; Mutate; Mutation; Northern Blotting; Organism; Partner in relationship; Pathway interactions; Phenotype; Plasmids; Point Mutation; Process; Production; Proteins; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Sequence Analysis; Signal Transduction; Site; Site-Directed Mutagenesis; Staging; Structure; System; Tail; Temperature; Time; base; egg; follow-up; fusion gene; molecular modeling; mutant; novel; novel strategies; protein function; research study; sperm cell; tool

DESCRIPTION (provided by applicant): Fertilization in Chlamydomonas reinhardtii, a novel model system for determining the protein(s) required for gamete fusion, may help define the basic requirements for sperm- egg fusion in more complex systems. Our long term goal is to understand the mechanism of fusion used by gametes. Mammalian fertilization requires so many interactions prior to fusion, that it is difficult to determine which genes/proteins are actually required for fusion of the sperm and egg membranes. While only a few proteins involved in gamete fusion have been identified, we have produced conditional and non-conditional (insertional) mutants of Chlamydomonas that carry out the early recognition, signaling and adhesion steps in mating but are blocked in the final stage of fertilization. In addition, this is the only available system in which gamete adhesion and fusion can be assayed independently and where mutations can therefore be defined as affecting plasma membrane adhesion or plasma membrane fusion. Using our insertional mutants, TAIL-PCR (Thermal asymmetric interlaced-PCR) has allowed us to identify 2 regions of the Chlamydomonas genome that may contain the gene that is defective in our insertional fusion-defective mutants. Transforming our fusion-defective mutants with bacterial artificial chromosomes (BACs) containing the wild type sequences from these identified regions of the genome, will allow us to determine if a gene in one of these regions can rescue fusion activity. Once we have identified the gene that is defective in our mutants, we will use our conditional mutants to map the functional domains of the identified protein. Fluorescence and electron microscopy as well as Northern blotting and RT-PCR will be used to study the regulation, production and localization of this protein and bioinformatics and molecular modeling will help us understand this protein's function. Future experiments may also involve site directed mutagenesis of the BAC sequences to allow further mapping of the protein's functional domains as well as real time RT-PCR, to learn more about the regulation of gene expression. Our hypothesis, supported by our genetic data, is that there is only a single gene controlling gamete fusion in Chlamydomonas. The power of Chlamydomonas genetics and molecular biology, the simplicity of the process of fertilization and the striking similarity of this system to fertilization in multicellular organisms makes it an important model system for studying the mechanism of gamete fusion. Understanding the molecular basis of gamete fusion in a simple model system may lead to new approaches for the treatment of human infertility (some types of infertility in mammals may be caused by the failure of sperm-egg fusion) as well as new types of contraceptives (molecules that impair fusion can exhibit contraceptive activity).

描述（由申请方提供）：莱茵衣原体的受精是一种用于确定配子融合所需蛋白质的新型模型系统，可能有助于定义更复杂系统中精卵融合的基本要求。我们的长期目标是了解配子融合的机制。哺乳动物受精在融合之前需要如此多的相互作用，以至于难以确定精子和卵膜的融合实际上需要哪些基因/蛋白质。虽然只有少数参与配子融合的蛋白质已被确定，我们已经产生了条件和非条件（插入）衣原体的突变体进行早期识别，信号和粘附步骤在交配，但在受精的最后阶段被阻止。此外，这是唯一可用的系统，其中配子粘附和融合可以独立地进行测定，并且其中突变因此可以被定义为影响质膜粘附或质膜融合。使用我们的插入突变体，TAIL-PCR（热不对称交错PCR）使我们能够确定衣原体基因组的2个区域，这些区域可能包含在我们的插入融合缺陷突变体中有缺陷的基因。用含有来自这些鉴定的基因组区域的野生型序列的细菌人工染色体（BAC）转化我们的融合缺陷突变体，将使我们能够确定这些区域之一中的基因是否可以拯救融合活性。一旦我们确定了突变体中有缺陷的基因，我们将使用我们的条件突变体来定位所识别蛋白质的功能结构域。荧光显微镜、电子显微镜、北方印迹和RT-PCR等技术将用于研究该蛋白的调控、产生和定位，生物信息学和分子模拟将有助于我们了解该蛋白的功能。未来的实验还可能涉及BAC序列的定点诱变，以允许进一步绘制蛋白质的功能结构域以及真实的时间RT-PCR，以了解更多关于基因表达调控的信息。我们的假设，我们的遗传数据支持，是只有一个基因控制配子融合衣原体。衣原体遗传学和分子生物学的力量，受精过程的简单性以及该系统与多细胞生物中受精的惊人相似性使其成为研究配子融合机制的重要模型系统。在简单的模型系统中了解配子融合的分子基础可能会导致治疗人类不育症的新方法（哺乳动物中某些类型的不育症可能是由精卵融合失败引起的）以及新型避孕药（损害融合的分子可以表现出避孕活性）。