Cloning and Analysis of a Gamete Fusion Gene

描述（由申请人提供）：莱茵衣藻（Chlamydomonas reinhardtii）的受精是一种确定配子融合所需蛋白质的新模型系统，可能有助于确定更复杂系统中精子-卵子融合的基本要求。我们的长期目标是了解配子融合的机制。哺乳动物的受精在融合之前需要很多的相互作用，因此很难确定哪些基因/蛋白质是精子和卵子膜融合所必需的。虽然只有少数参与配子融合的蛋白质被确定，但我们已经产生了衣藻的条件和非条件（插入）突变体，这些突变体在交配过程中进行早期识别、信号传导和粘附步骤，但在受精的最后阶段被阻断。此外，这是唯一可以独立检测配子粘附和融合的系统，因此突变可以被定义为影响质膜粘附或质膜融合。利用我们的插入突变体，TAIL-PCR（热不对称交错pcr）使我们能够识别衣藻基因组的两个区域，这些区域可能包含插入融合缺陷突变体中有缺陷的基因。将我们的融合缺陷突变体转化为含有野生型序列的细菌人工染色体（BACs），将使我们能够确定这些区域中的一个基因是否可以挽救融合活性。一旦我们确定了突变体中有缺陷的基因，我们将使用条件突变体来绘制鉴定蛋白质的功能域。荧光和电子显微镜以及Northern blotting和RT-PCR将用于研究该蛋白的调控、产生和定位，生物信息学和分子建模将帮助我们了解该蛋白的功能。未来的实验可能还包括对BAC序列的定点诱变，以进一步绘制蛋白质的功能域，以及实时RT-PCR，以了解更多关于基因表达的调控。我们的假设，由我们的遗传数据支持，是只有一个基因控制配子融合在衣藻。衣藻遗传学和分子生物学的强大力量、受精过程的简单以及该系统与多细胞生物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。在一个简单的模型系统中了解配子融合的分子基础可能会为治疗人类不孕症（哺乳动物的某些类型的不孕症可能是由于精子-卵子融合失败引起的）以及新型避孕药（破坏融合的分子可能表现出避孕活性）带来新的方法。

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).