Isolation and functional analysis of genes uniquely expressed in human corneal epithelium and conjunctival epithelium

人角膜上皮和结膜上皮独特表达基因的分离和功能分析

基本信息

批准号：
10470365
负责人：
KINOSHITA Shigeru
金额：
$ 8.19万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10470365/
关键词：
corneal epithelium conjunctival epithelium human cDNA library specific gene cathepsin V uroplakin Ib Cl channel cDNA解析

项目摘要

We constructed the mRNA expression profile from human corneal epithelium and analyzed the genes expressed abundantly or specifically in corneal epithelium. From these data, a novel cathepsin (cathepsin V), uroplakin Ib and Cl channel were isolated and analyzed as to their functions and chromosomal localizations. The expression profile of human conjunctival epithelium was also constructed and compared to that of human corneal epithelium.Cathepsin V was a novel cathepsin, which contained ORF of 77% identical homology to human cathepsin L. A recombinant cathepsin V protein produced using a baculovirus expression system has proteolytic activity as a cysteine proteinase. By RT-PCR, only in cornea, the expression level of cathepsin V was higher than that of cathepsin L. Cathepsin V may play an important role in corneal physiology. By the FISH method, cathepsin V genes was mapped to chromosomal region 9q22.2, 15cM from the cathepsin L gene. This suggests that cathepsin L and V evolved more re … More cently by gene duplication from an ancestral gene. Uroplakin Ib protein had four transmembrane domains. This clone was an isoform of uroplakin Ib, which had already published, because a 3'-UTR of this clone was differed from published uroplakin Ib. By RT-PCR, uroplakin Ib mRNA was detected not only in transitional epithelium, but also on the ocular surface. By immunohistochemistry using antiserum against uroplakin Ib peptide, uroplakin Ib protein was found in cell membranes of corneal, limbal and conjunctival epithelium, especially in the superficial half of the corneal epithelial layer. A novel C1 channel coded 943 amino acids and mapped to chromosomal region 1p32. C1 channel mRNA was 100 times more plentiful than other C1 channels, implying an important rule of corneal transparency.The expression profile of normal conjunctival epithelium was so differed greatly from those of corneal epithelium. In this expression profile, keratin 13, beta- 2 microglobulin and lipocortin were highly expressed. Among the abundant transcripts appearing in three or more clones, two unknown conjunctival specific genes were included. Less

我们从人角膜上皮构建了mRNA表达谱，并分析了在角膜上皮中表达的基因。从这些数据中，将新型的组织蛋白酶（组织蛋白酶V），乌罗翼蛋白IB和CL通道分离出来，并分析其功能和染色体局部化。还构建了人类结膜上皮的表达曲线，并将其与人角膜上皮的表达曲线进行了比较。Cathepsinv是一种新型的组织蛋白酶，它包含与人类组织蛋白酶L的77％相同同源性的ORF。一种使用细菌性表达系统产生的重组callosinant v蛋白具有蛋白酶蛋白酶的蛋白酶。通过RT-PCR，仅在角膜中，组织蛋白酶V的表达水平高于组织蛋白酶L. calterpsin v的表达水平，在角膜生理中可能起重要作用。通过鱼方法，将组织蛋白酶V基因映射到距组织蛋白酶L基因15厘米的染色体区域9q22.2。这表明组织蛋白酶L和V进化了更多……通过祖先基因重复的基因重复。乌洛飞金IB蛋白具有四个跨膜结构域。这个克隆是乌洛普拉金IB的同工型，它已经出版了，因为该克隆的3'-UTR与已出版的Uroplakin IB不同。通过RT-PCR，不仅在过渡上皮，而且在眼表面上检测到尿素粉Ib mRNA。通过使用抗乌洛飞蛋白IB肽的抗血清的免疫组织化学，在角膜，边缘和结膜上皮的细胞膜中发现了尿藻蛋白IB蛋白，尤其是在角膜上皮层的表层一半中。一个新颖的C1通道编码943个氨基酸，并映射到1p32染色体区域。 C1通道mRNA比其他C1通道多100倍，这表明正常结膜上皮的表达曲线与角膜上皮的表达曲线差异很大。在此表达曲线中，角蛋白13，β-2微球蛋白和脂皮质素被高度表达。在三个或三个克隆中出现的丰富转录本中，包括两个未知的结膜特异性基因。较少的