| Co-Investigator(Kenkyū-buntansha) |
OKUBO Kousaku Molecular and Cellular Biology, Osaka University, Assistant Professor, 細胞生体工学センター, 助教授 (40233069)
NISHIDA Kohji Medicine, Ophthalmology, Instructor, 医学部, 助手 (40244610)
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| Budget Amount *help |
¥12,800,000 (Direct Cost: ¥12,800,000)
Fiscal Year 1999: ¥6,100,000 (Direct Cost: ¥6,100,000)
Fiscal Year 1998: ¥6,700,000 (Direct Cost: ¥6,700,000)
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| Research Abstract |
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
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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
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