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 蛋这个过程的第一步是自由游动的精子的结合 通过一种机制， 一般来说，这具有高度的物种特异性。这件事的特殊性 相互作用暗示了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 卵结合蛋白小鼠卵子上的初级精子受体 被鉴定为单一的糖蛋白-ZP 3。相反，一些 分子和酶被认为是精子的候选者 表面卵结合蛋白其中一名候选人已在两个 测定-通过亲和色谱法和通过与ZP 3交联-作为Mr 在SDS凝胶上观察到56，000个多肽。克隆和鉴定 已经完成了小鼠和人类同源基因（称为 Tcte 1），其基于功能测定似乎编码该多肽 以及进化的考虑。要求提供资金， 一系列的基因和进化研究旨在了解 这种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）TCTE 1多肽是必需的吗 为了受精？和（2）TCTE 1多肽是否足以用于 物种特异性受精的开始。靶向诱变将是 用于产生不表达Tcte-1基因的小鼠， 将产生表达来自异源物种的TCTE 1的小鼠 仓鼠、大鼠或豚鼠。研究生育力和卵子结合与 这些动物将为那些 被摆了姿势第三个目标是确定Tcte 1基因是否在 积极的达尔文选择，这将是预测，如果TCTE 1发挥作用， 在建立物种间交配后生殖障碍中的作用。 这个问题将通过比较序列分析来解决， 这也可以让我们深入了解 在TCTE 1和卵表面之间。积极达尔文主义的证明 选择将提供一个物种特异性的作用， TCTE 1在配子识别中的作用