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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

为了研究小鼠col 4a 6基因的结构，我们筛选了小鼠129基因组文库，并分离到一个大小约为18.3kb的TSg 6片段。对基因组片段的精细分析以及与cDNA结构的比较表明，在小鼠转录时，在人类基因中观察到的替代转录物不存在。为了建立col 4a 6基因敲除小鼠模型，我们将NcoR基因插入col 4a 6基因的第2外显子。转染129 v小鼠R1-Es细胞，G418筛选阳性ES细胞。将阳性ES细胞植入囊胚内，移植假孕小鼠子宫。嵌合体小鼠，杂合子和纯合子现在正在检查，如果任何表型被注意到。当用a6特异性单克隆抗体对纯合子小鼠进行染色时，在任何组织中都没有得到阳性信号，这表明a6多肽在小鼠的骨髓中不表达。 ...更多信息虽然由α 1和α 2链组成的胶原IV分子广泛分布，但包含其它四条链α 3-α 6的组合的分子是特化基底膜的重要组分。由α 3-α 6链组装的三螺旋分子的精确链组成尚不完全清楚，但假设α 3-α 5链以及α 5和α 6链形成异源三聚体分子。有证据表明，α 3/α 4/α 5和α 5/α 6分子是基底膜网络的组成部分，我们鉴定了日本病例的DL/AS缺失，并确定了断点序列。结果表明，COL 4A 5的第一个编码外显子和COL 4A 6的前两个外显子缺失。他们还表明，断裂点共享相同的序列，这反过来又与拓扑异构酶I和II的共有序列密切同源。额外的DNA证据表明，男性患者是突变的体细胞嵌合体。使用α链特异性单克隆抗体的免疫组织化学分析证实了这一结论，因为它们揭示了在大多数但不是所有平滑肌细胞肿瘤的基底膜中缺乏α 5（IV）和α 6（IV）胶原链。本研究对于深入了解DL的发病机制和病因学具有重要意义。少