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Studies on structure and function of molecules involved in gamete interactions

参与配子相互作用的分子的结构和功能研究

基本信息

批准号：
11680716
负责人：
URUSHIHARA Hideko
金额：
$ 2.37万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

This project intended to reveal the genetic control of gamete interactions using the macrocyst formation of the cellular slime mold, Dtctyostelium discoideum as a model system. For this purpose, immunochemical, forward genetic, and reverse genetic approaches have been taken.1. Results of immunochemical approach : GP138 multigene family, which had been shown to encode gp138, a target molecule for gamete-fusion blocking antibodies, was analyzed. Two new members, GP138C and GP138D, have been isolated as well as two truncated pseudogenes Analysis of total tetra knockout mutants, however, indicated that proteins encoded by GP138 multigenes are distinct from gP138. The possibility that GP138 multigenes are involved in asexual development has been suggested.2. Results of forward genetics : The effective method of insertional mutagenesis called REMI (Restriction Enzyme Mediated In tegration) was used to isolate sexually defective mutants and relevant genes. One of the obtained mutants, MCF1 is unable to undergo sexual cell fusion. Analysis of genome structure around insertion sites revealed an ORF of 6.3 Kb long including two short introns. The relevant gene was named macA. The actual function of macA in cell fusion is being analyzed.3. Results of reverse genetics : Random analysis of genes expressed in the gametes was attempted. First, oligo-(dT) primed directional cDNA library (FC) was constructed and randomly chosen 1,000 clones were sequenced to obtain gene expression profile in the gamete. Since the genes contained in the FC library were predominantly of housekeeping functions, a gamete-specific subtraction library (FC-IC) was constructed. All 901 clones in the FC-IC library were sequenced and characterized. Expression analysis of randomly chosen 100 clones indicated that more than 60 % of the clones are actually gamete specific and FC-IC library is an useful source for analysis of genes involved in gamete interactions.

本项目旨在揭示配子相互作用的遗传控制的细胞黏菌，Dtctyosteelium discoideum作为一个模型系统的大孢囊形成。为此，免疫化学、正向遗传学和反向遗传学方法已被采用。免疫化学方法的结果：分析了GP 138多基因家族，该家族已被证明编码gp 138，其是配子融合阻断抗体的靶分子。两个新的成员，GP 138 C和GP 138 D，已经被分离出来，以及两个截短的假基因。然而，对全四基因敲除突变体的分析表明，由GP 138多基因编码的蛋白质与GP 138不同。GP 138多基因参与无性发育的可能性已被证实.正向遗传学的结果：利用插入突变的有效方法REMI（Restriction Enzyme Mediated In Integration）分离性缺陷突变体及其相关基因。获得的突变体之一，MCF 1不能进行性细胞融合。对插入位点周围的基因组结构分析显示，ORF长6.3Kb，包括两个短内含子。相关基因被命名为macA。分析macA在细胞融合中的实际功能.反向遗传学的结果：尝试随机分析配子中表达的基因。首先构建寡聚脱氧胸苷（oligo-（dT））引物定向cDNA文库（FC），随机选取1,000个克隆进行测序，获得配子中基因表达谱。由于FC文库中含有的基因主要是管家功能，因此构建了配子特异性消减文库（FC-IC）。对FC-IC文库中的所有901个克隆进行测序和表征。对随机挑选的100个克隆的表达分析表明，超过60%的克隆实际上是配子特异性的，并且FC-IC文库是分析涉及配子相互作用的基因的有用来源。