FUNCTION & EVOLUTION OF A CANDIDATE EGG-BINDING PROTEIN

功能

• 批准号: 2025450
• 负责人: LEE M SILVER
• 金额: $ 16.71万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2025450
• 关键词: Mus musculus; animal genetic material tag; animal population genetics; binding proteins; biochemical evolution; egg /ovum; embryonic stem cell; fertilization; gene expression; genetic polymorphism; genetically modified animals; glycoproteins; guinea pigs; hamsters; heterozygote; human genetic material tag; laboratory mouse; laboratory rat; nucleic acid sequence; polymerase chain reaction; protein structure function; species difference; sperm; zona pellucida glycoproteins

Fertilization in mammals occurs through a series of highly ordered steps that lead ultimately to the fusion of a single sperm cell with an ovulated egg. The first step in this process is the binding of free-swimming sperm to the extracellular egg coat, or zona pellucida, through a mechanism which, in general, is highly species-specific. The specificity of this interaction implicates the existence of specific complementary molecules on egg and sperm, referred to respectively as the sperm receptor and the egg binding protein. The primary sperm receptor on mouse eggs has been identified as a single zona glycoprotein - ZP3. In contrast, a number of molecules and enzymes have been proposed as candidates for the sperm surface egg-binding protein. One such candidate has been identified in two assays - by affinity chromatography and by crosslinking to ZP3 - as a Mr 56,000 polypeptide observed on SDS gels. Cloning and characterization has been accomplished for the mouse and human homologs of a gene (called Tcte1) that appears to encode this polypeptide based on functional assays as well as evolutionary considerations. Funds are requested to carry out a series of genetic and evolutionary studies aimed at understanding the function of this gene product in the process of fertilization. Two major goals are to answer the questions: (1) Is the TCTE1 polypeptide necessary for fertilization?, and (2) Is the TCTE1 polypeptide sufficient for the initiation of species-specific fertilization. Targeted mutagenesis will be used to produce mice that do not express the Tcte-1 gene, and transgenic mice will be generated that express TCTE1 from a heterologous species such as hamster, rat, or guinea pig. Studies of fertility and egg binding with these animals will provide definitive answers to the questions that have been posed. A third goal is to determine whether the Tcte1 gene is under positive Darwinian selection, which would be predicted if TCTE1 played a role in establishing post-mating reproductive barriers between species. This question will be approached through comparative sequence analysis, which could also provide insight into the nature of the interaction between TCTE1 and the egg surface. The demonstration of positive Darwinian selection would provide confirming evidence for a species-specific role of TCTE1 in gamete recognition.

哺乳动物的受精是通过一系列高度有序的步骤发生的 最终导致单个精子细胞与排卵的融合 蛋。此过程的第一步是结合自由迅速的精子 通过机制到达细胞外鸡蛋或Zona pellucida 通常，这是高度物种特异的。这个特异性 相互作用暗示了特定互补分子的存在 在鸡蛋和精子上，分别称为精子受体和 卵结合蛋白。小鼠卵上的主要精子受体已经 被识别为单个Zona糖蛋白-ZP3。相反，许多 已经提出了分子和酶作为精子的候选者 表面结合蛋白。一个这样的候选人已经在两个中确定 测定 - 通过亲和力色谱法，并通过交联ZP3-作为MR 在SDS凝胶上观察到56,000多肽。克隆和表征具有 是针对基因的小鼠和人类同源物完成的（称为 TCTE1）似乎根据功能测定法编码此多肽 以及进化考虑。要求资金进行 一系列旨在理解的遗传和进化研究 该基因产物在受精过程中的功能。两个主要 目标是回答问题：（1）是必需的TCTE1多肽 施肥？和（2）TCTE1多肽是否足以使 物种特异性施肥的启动。目标诱变将是 用于产生不表达TCTE-1基因的小鼠和转基因 将产生从异源物种表达TCTE1的小鼠 作为仓鼠，大鼠或豚鼠。与生育和卵结合的研究 这些动物将为有问题提供明确的答案 被摆姿势。第三个目标是确定TCTE1基因是否处于 达尔文积极的选择，如果TCTE1扮演 在建立物种之间建立后的生殖屏障中的作用。 这个问题将通过比较序列分析来解决 这也可以提供对互动本质的见解 在TCTE1和鸡蛋表面之间。积极的达尔文人的演示 选择将为某些特定于物种的作用提供证据 TCTE1在配子识别中。