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基因组库，并隔离了TSG6的片段，大小约为18.3kb。对基因组片段和结构与cDNA的比较进行了很好的分析表明，在小鼠转录时，在人基因中观察到的替代转录本不存在。为了为COL4A6基因创建零小鼠，我们操纵基因组片段并将NCOR基因插入Col4a6基因的外显子2中。将载体转化为源自129V小鼠的R1-ES细胞，并通过G418筛选的阳性ES细胞。将阳性ES细胞插入胚泡中，然后将胚泡的子宫移动为假小鼠的子宫。是否注意到任何表型，现在正在检查嵌合体小鼠，杂合子和纯合子。当纯合小鼠用A6特异性单克隆抗体染色时，未从任何组织中获得正信号，表明在M…中未表达A6多肽。在M…更多的冰中，collagen IV分子由Alpha1和Alpha2链组成，由Alpha1和Alpha2链组成，分布宽广，分布在其他重要的Chains中。专业地下膜的组成部分。从alpha3-Alpha6链中组装的三螺旋分子的精确链组成并不完全清楚，但可以假设α3-Alpha5链和α5和α6链形成杂素分子。几个证据表明，alpha3/alpha4/alpha5分子和alpha5/alpha6分子是基底膜网络的组成部分。我们将DL/确定为删除，并确定了日本案例的断点威胁。结果表明，删除是COL4A5的第一个编码外显子和Col4a6的前两个。他们还表明，断点共享相同的序列，这反过来与拓扑异构酶I和II的共识紧密同源。使用α链特异性单克隆抗体的免疫组织化学分析支持了这一结论，因为它们揭示了大多数但不是所有平滑肌细胞肿瘤的基底膜中α5（IV）和α6（IV）胶原链的缺失。这项研究与对DL发病机理及其病因的理解非常相关。较少的