Molecular cloning of the bovine MYOC and induction of its expression in trabecular meshwork cells.
OBJECTIVE
Myocilin gene (MYOC) was identified as one of the disease-causing genes of primary open-angle glaucoma. This study was conducted to establish a system for the investigation of the biological role of MYOC in vitro by using bovine eyes, which are easy to obtain and have been widely used to examine the aqueous outflow system. The cDNA sequence of the bovine MYOC was determined and its expression in bovine eyes was examined with a quantitative polymerase chain reaction (PCR) assay.


METHODS
Bovine MYOC cDNA was obtained from cultured bovine trabecular meshwork cells, and part of its sequence was determined using a primer pair designed based on the known sequence of the human MYOC gene. The 3' and 5' ends of this sequence were determined using the method of 3' and 5' rapid amplification of cDNA ends. The induction of the MYOC gene in cultured bovine trabecular meshwork cells after exposure to dexamethasone was quantitatively examined with real-time quantitative PCR using a probe designed according to the sequence of the determined bovine MYOC gene.


RESULTS
Bovine MYOC protein was composed of 490 amino acids, which was 81.6% identical with that of human MYOC protein. Most of the amino acid residues of which mutation was reported to cause glaucoma were conserved in the bovine MYOC protein. After 2 weeks of treatment with 500 nM dexamethasone, expression of bovine MYOC mRNA was amplified 14-fold (14.1+/-5.1-fold, mean +/- SEM) measured by real-time quantitative PCR.


CONCLUSIONS
The cDNA sequence of the bovine MYOC gene had a high degree of similarity to that of the human MYOC gene. Investigation of the function of bovine MYOC may contribute to identifying the role of MYOC protein in the aqueous outflow system.
Taniguchi
F
F
Eye Clinic, Ohmiya Red Cross Hosptial, Saitama, Japan. dzs02553@nifty.ne.jp
Suzuki
Y
Y
Kurihara
H
H
Kurihara
Y
Y
Kasai
H
H
Shirato
S
S
Araie
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D017931:DNA Primers / Q000737:chemistry; D015342:DNA Probes / Q000737:chemistry; D003907:Dexamethasone / Q000494:pharmacology; D005136:Eye Proteins / Q000096:biosynthesis* / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014129:Trabecular Meshwork / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D017931:DNA Primers; D015342:DNA Probes; D003907:Dexamethasone; D005136:Eye Proteins; D005938:Glucocorticoids; D006023:Glycoproteins; D012333:RNA, Messenger; D000099179:Myocilin