Cloning and Analysis of a Gamete Fusion Gene

描述（由申请人提供）：莱茵衣藻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，可能有助于确定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使用的融合机制。哺乳动物受精在融合之前需要如此多的相互作用，因此很难确定精子和卵膜融合实际上需要哪些基因/蛋白质。虽然只鉴定了一些参与配子融合的蛋白质，但我们已经产生了衣藻的条件和非条件（插入）突变体，它们在交配过程中执行早期识别、信号传导和粘附步骤，但在受精的最后阶段被阻断。此外，这是唯一可以独立测定配子粘附和融合的可用系统，因此可以将突变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）使我们能够识别衣藻基因组的 2 个区域，这些区域可能包含我们的插入融合缺陷突变体中存在缺陷的基因。用包含这些基因组已识别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的基因是否可以挽救融合活性。一旦我们确定了突变体中存在缺陷的基因，我们将使用条件突变体来绘制已识别蛋白质的功能域图。荧光和电子显微镜以及Northern印迹和RT-PCR将用于研究该蛋白质的调控、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质的功能。未来的实验还可能涉及 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，以了解有关基因表达调控的更多信息。我们的假设（得到遗传数据的支持）是，衣藻中只有一个基因控制配子融合。衣藻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带来治疗人类不孕症的新方法（哺乳动物的某些类型的不孕症可能是由于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）。

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).