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Biological Function of Type VIII Collagen

VIII 型胶原蛋白的生物学功能

基本信息

批准号：
04044124
负责人：
NINOMIYA Yoshifumi
金额：
$ 1.66万
依托单位：
Okayama University Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

Since several amino-terminal amino acid sequences determined from peptides isolated from Descemet's membranes were found within deduced amino acid sequence from the cDNA clones we isolated, we designated the polypeptide chain as alpha1(VIII) chain. We have isolated genomic DNA encoding the rabbit alpha1(VIII) chain. Intriguingly, the gene contains three small and one large exons. The largest exon contains a short NC2 domain, the entire COL and NC1 domains and 3' UTR. The human gene, COL8A1, has been assigned to chromosome 3q11.1-q13.2. The question here was whether the molecule contains another alpha chains. A set of PCR primers was designed and the PCR products were screened by various probes. The amino acid sequence deduced from a candidate clone contained several collagenous amino acid sequences obtained from Descemet's membrane that were not found in the alpha1(VIII) chain, therefore we identified the collagenous polypeptide as the second chain, alpha2(VIII), that joins the type VIII collagen molecule. The gene coding for the alpha2(VIII) chain was assigned to chromosome 1p32.3-1p34.3. Mouse cDNA and genomic DNA clones were isolated and the expression in various tissues and cells were demonstrated by Northern-blot analyzes and in situ hybridization. The results showed that the alpha1(VIII) mRNA is predominantly expressed in keratinocytes, corneal endothelial and epithelial cells, lens epithelial cells as well as mesenchymal cells surrounding cartilage and calvarial bone and in the meninges surrounding the brain.In order to know the biological function of type VIII collagen molecule, we are now trying to create transgenic animals. The first experiment we have done so far was to use presumptive regulatory region of type VIII collagen gene joined to Lac Z gene. Several lines of transgenic mice we obtained did not show typical expression features of the Lac Z gene. Upstream region of type VIII collagen gene needs to characterized further.

由于从Descemet膜中分离的多肽在我们所分离的克隆的推导氨基酸序列中发现了几个氨基末端氨基酸序列，我们将该多肽链命名为α1(Viii)链。我们已经分离了编码兔α1(Viii)链的基因组DNA。有趣的是，该基因包含三个小外显子和一个大外显子。最大的外显子包含一个短的NC2结构域、完整的COL和NC1域以及3‘非编码区。人类基因COL8A1已被分配到染色体3q11.1-q13.2。这里的问题是，这个分子是否含有另一个阿尔法链。设计了一套聚合酶链式反应引物，用不同的探针对扩增产物进行筛选。从一个候选克隆推导的氨基酸序列包含了从Descemet膜获得的几个胶原氨基酸序列，这些氨基酸序列在α1(Viii)链中没有发现，因此我们将该胶原多肽鉴定为连接VIII型胶原分子的第二链α2(Viii)。编码α2(Viii)链的基因位于染色体1p32.3-1p34.3。通过Northern-Blot分析和原位杂交分析，证实了其在不同组织和细胞中的表达。结果表明，在角质形成细胞、角膜内皮和上皮细胞、晶状体上皮细胞以及软骨和颅骨周围的间充质细胞以及脑周围的脑膜中主要表达α1(VIII)mRNA。为了了解VIII型胶原分子的生物学功能，我们正在尝试建立转基因动物。到目前为止，我们所做的第一个实验是将VIII型胶原基因的假定调节区连接到Lac Z基因上。我们获得的几株转基因小鼠没有表现出Lac Z基因的典型表达特征。VIII型胶原基因的上游区域需要进一步鉴定。