Construction of human corneal endothelial cDNA library and identification of novel active genes.
OBJECTIVE
To describe genes expressed in human corneal endothelial cells and identify novel genes.


METHODS
Sixteen human donor corneas that had no history of corneal disease, infection, or intraocular surgery were used within 7 days of death. Total RNA was extracted from corneal endothelial cells with attached Descemet membranes. A 3'-directed cDNA library was constructed from mRNA by using a pUC19-based primer. These sequences were compared with each other to determine their frequency and were searched against GenBank for identification. To identify novel specific and abundant transcript genes in corneal endothelial cells, the novel genes were compared with an expressed sequence tag database, the expected sequence extended, and 5' rapid amplification of cDNA ends-polymerase chain reaction cloning performed.


RESULTS
The human corneal endothelial cDNA library showed that the most abundant transcript was prostaglandin D2 synthase. The remaining transcript genes that were present in abundance consisted of lactate dehydrogenase-A, gene signature (GS) 3582, which is a novel gene without a known function, and matrix Gla protein. The full-length sequence of GS3582 showed similarity to genes obtained in ovary and TESTIS.


CONCLUSIONS
A human corneal endothelial cDNA library was constructed. An expression profile of corneal endothelium provides probes to monitor physiologic and pathologic conditions of this tissue in terms of gene expression.
Sakai
Rieko
R
Department of Ophthalmology, Jichi Medical School, Kawachi-gun, Tochigi, Japan.
Kinouchi
Tadatoshi
T
Kawamoto
Shoko
S
Dana
M Reza
MR
Hamamoto
Toshirou
T
Tsuru
Tadahiko
T
Okubo
Kousaku
K
Yamagami
Satoru
S
D000368:Aged; D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000378:metabolism*; D004728:Endothelium, Corneal / Q000378:metabolism*; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D015723:Gene Library; D006801:Humans; D008875:Middle Aged; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D012313:RNA / Q000302:isolation & purification; D014019:Tissue Donors
D018076:DNA, Complementary; D005136:Eye Proteins; D012313:RNA