FUNCTION & EVOLUTION OF A CANDIDATE EGG-BINDING PROTEIN

功能

• 批准号: 2202687
• 负责人: LEE M SILVER
• 金额: $ 15.45万
• 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/2202687
• 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

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进行的 最终导致单个精子细胞与排卵的精子细胞融合 蛋。这个过程的第一步是结合自由游动的精子 通过某种机制到达细胞外蛋壳或透明带 一般来说，具有高度的物种特异性。这个的特殊性 相互作用暗示特定互补分子的存在 作用于卵子和精子，分别称为精子受体和 卵结合蛋白。小鼠卵子上的主要精子受体是 鉴定为单一透明带糖蛋白-ZP3。相比之下，一些 分子和酶已被提议作为精子的候选者 表面卵结合蛋白。已在两份报告中确定了一名此类候选人 测定 - 通过亲和层析并通过与 ZP3 交联 - 作为 Mr 在 SDS 凝胶上观察到 56,000 个多肽。克隆和表征 已经针对小鼠和人类基因的同源物（称为 根据功能分析，Tcte1) 似乎编码该多肽 以及进化方面的考虑。需要资金来执行 一系列遗传和进化研究，旨在了解 该基因产物在受精过程中的功能。两大 目标是回答以下问题：（1）TCTE1多肽是否必要 以及 (2) TCTE1 多肽是否足以受精？ 物种特异性受精的开始。定向诱变将 用于产生不表达Tcte-1基因的小鼠，以及转基因小鼠 将产生表达来自异源物种的 TCTE1 的小鼠，例如 如仓鼠、大鼠或豚鼠。生育力和卵子结合的研究 这些动物将为人们提出的问题提供明确的答案 被摆出姿势。第三个目标是确定 Tcte1 基因是否受到影响。 正达尔文选择，如果 TCTE1 发挥了作用，就会预测到 在建立物种间交配后生殖障碍方面的作用。 这个问题将通过比较序列分析来解决， 这也可以提供对交互本质的洞察 TCTE1 和鸡蛋表面之间。积极达尔文主义的体现 选择将为物种特异性作用提供确凿的证据 配子识别中的 TCTE1。