FUNCTION & EVOLUTION OF A CANDIDATE EGG-BINDING PROTEIN

功能

• 批准号: 2403305
• 负责人: LEE M SILVER
• 金额: $ 17.38万
• 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 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2403305
• 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进行的 最终导致单个精子细胞与一个排卵的 蛋。这一过程的第一步是结合自由游动的精子。 细胞外卵壳，或透明带，通过一种机制 一般来说，这是高度物种特异性的。这一点的特殊性 相互作用意味着特定互补分子的存在 在卵子和精子上，分别称为精子受体和 卵子结合蛋白。小鼠卵子上的主要精子受体是 鉴定为单一的带状糖蛋白-ZP3。相比之下，一些 分子和酶被认为是精子的候选者。 表面卵子结合蛋白。已经确定了两个候选人中的一个 用亲和层析法和ZP3交联法进行MR分析 在十二烷基硫酸钠凝胶上观察到56,000个多肽。克隆和鉴定具有 已经完成了小鼠和人类的基因同源物(称为 Tcte1)，根据功能分析，似乎编码该多肽 以及进化方面的考虑。需要资金来进行 一系列的遗传和进化研究，旨在了解 该基因产物在受精过程中的作用。两大 目的是回答以下问题：(1)TCTE1多肽是必需的吗 用于受精？以及(2)TCTE1多肽是否足以用于 启动物种特定受精。定向突变将是 用于生产不表达Tcte-1基因的小鼠，并转基因 将产生表达来自异源物种的TCTE1的小鼠 如仓鼠、老鼠或豚鼠。受精性和卵子结合的研究 这些动物将为以下问题提供明确的答案 已经摆好姿势了。第三个目标是确定Tcte1基因是否处于 积极的达尔文选择，这是可以预测的，如果TCTE1发挥 在物种间建立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， 它还可以提供对交互的性质的洞察 在TCTE1和鸡蛋表面之间。达尔文正定论的证明 选择将为物种特有的作用提供确凿的证据 配子识别中的TCTE1。