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Supramolecular Assembly and Function of New Basement Membrane Collagen Molecules

新型基底膜胶原分子的超分子组装及其功能

基本信息

批准号：
08457154
负责人：
NINOMIYA Yoshifumi
金额：
$ 5.63万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

To characterize the structure of mouse col4a6 gene, we screened mouse 129 genomic library and isolated a fragment of TSg6, approximately 18.3kb in size. Fine analysis of the genomic fragment and comparison of the structure to that of the cDNA revealed that alternative transcripts that are observed in the human gene do not exist when the mouse is transcribed. In order to create null mouse for col4a6 gene, we manipulated genomic fragment and inserted NcoR gene into the exon 2 of col4a6 gene. The vector was transfected into the R1-Es cells derived from 129v mouse and positive ES cells screened by G418. The positive ES cells were inserted into blastocysts, then the blastocysts were moved uterus of pseudopregnant mice. Chimera mice, heterozygotes and homozygotes are now being checked if any phenotypes are noticed. When homozygote mice were stained by a6 specific monoclonal antibodies, no positive signals was obtained from any tissues, indicating that a6 polypeptide is not expressed in the m … More ice.While collagen IV molecules composed of alpha1 and alpha2 chains are broadly distributed, molecules comprising combinations of the other four chains, alpha3-alpha6, are important components of specialized basement membranes. The precise chain composition of triple-helical molecules assembled from the alpha3-alpha6 chains is not entirely clear, but it is hypothesized that alpha3-alpha5 chains and alpha5 and alpha6 chains form heterotrimeric molecules. Several pieces of evidence indicate that alpha3/alpha4/alpha5 molecules and alpha5/alpha6 molecules are components of the basement membrane network.We identified an DL/AS deletion and determined the breakpoint wequences of the Japanese case. The results demonstrated that a deletion clininates the first coding exon of COL4A5 and the first two of COL4A6. They also showed that the breakpoints share the same sequence, which is in turn closely homologous to the consensuses of topoisomerases I and II.Additional DNA evidence suggestetd that the male patient is a somatic mosaic for the mutation. Immunohistochemical analysis using alpha chain-specific monoclonal antibodies supportedthis conclusion in that they revealed the absence of the alpha5 (IV) and alpha6 (IV) collagen chains in most but not all of basement membranes of the smooth muscle cell tumor. This study is greatly relevant to the understanding of DL pathogenesis and its etiology. Less

为了表征小鼠col4a6基因的结构，我们筛选了小鼠129基因组文库并分离了TSg6的片段，大小约为18.3kb。对基因组片段的精细分析以及与 cDNA 结构的比较表明，当小鼠转录时，在人类基因中观察到的替代转录物并不存在。为了创建col4a6基因的空鼠，我们操纵基因组片段并将NcoR基因插入到col4a6基因的外显子2中。将载体转染至129v小鼠来源的R1-Es细胞和G418筛选的阳性ES细胞中。将阳性ES细胞植入囊胚中，然后将囊胚移至假孕小鼠子宫中。现在正在检查嵌合体小鼠、杂合子和纯合子是否注意到任何表型。当纯合子小鼠用a6特异性单克隆抗体染色时，没有从任何组织获得阳性信号，表明a6多肽在m… Moreice中不表达。虽然由α1和α2链组成的IV型胶原蛋白分子分布广泛，但由其他四种链α3-α6组合而成的分子很重要特殊基底膜的组成部分。由α3-α6链组装而成的三螺旋分子的精确链组成尚不完全清楚，但假设α3-α5链以及α5和α6链形成异三聚体分子。一些证据表明α3/α4/α5分子和α5/α6分子是基底膜网络的组成部分。我们鉴定了DL/AS缺失并确定了日本病例的断点序列。结果表明，COL4A5 的第一个编码外显子和 COL4A6 的前两个编码外显子被删除。他们还表明，断点具有相同的序列，这又与拓扑异构酶 I 和 II 的共有序列密切同源。额外的 DNA 证据表明，该男性患者是该突变的体细胞嵌合体。使用α链特异性单克隆抗体进行的免疫组织化学分析支持了这一结论，因为它们揭示了平滑肌细胞肿瘤的大多数（但不是全部）基底膜中不存在α5（IV）和α6（IV）胶原链。这项研究对于理解 DL 发病机制及其病因学具有重要意义。较少的