Isolation and characterization of cell-specific cDNA clones from a subtractive library of the ocular ciliary body of a single normal human donor: transcription and synthesis of plasma proteins.
A subtractive cDNA library was developed for the purpose of identifying cell-specific genes expressed within the human ocular ciliary body, a tissue responsible for regulating aqueous humor secretion and intraocular pressure. Partial DNA sequence of a large number of cDNA clones and homology searches of nucleic acid and protein databases revealed significant homologies to at least 90 independently known genes. A group of biologically significant genes that were previously not known to have transcriptional expression in the ciliary body, complement component C4; alpha 2 macroglobulin; selenoprotein-P; and apolipoprotein D, were further demonstrated by Northern hybridization. Antibodies to these and other proteins (i.e., tyrosinase-related protein and pigment epithelium-derived factor) confirmed their cell-restricted expression in ciliary epithelial cells (pigmented, nonpigmented), or vascular endothelial cells. We provide evidence that two human plasma proteins, complement component C4 and alpha 2-macroglobulin are metabolically labeled with [35S]methionine in ciliary processes explants, suggesting that the ciliary body is an organ of synthesis and secretion of plasma proteins present in aqueous humor. These results challenge the notion that plasma proteins in aqueous humor are imported from outside of the eye. The subtractive cDNA library reported in this work should be very useful for identifying potential candidate genes in ocular abnormalities affecting the ciliary body, or involved in the regulation of intraocular pressure.
Escribano
J
J
Department of Ophthalmology and Visual Science, Yale University School of Medicine, New Haven, CT 06520, USA.
Ortego
J
J
Coca-Prados
M
M
D000328:Adult; D001483:Base Sequence; D001798:Blood Proteins / Q000096:biosynthesis / Q000235:genetics*; D002924:Ciliary Body / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000302:isolation & purification*; D005260:Female; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D012016:Reference Values; D014019:Tissue Donors; D014158:Transcription, Genetic
D001798:Blood Proteins; D018076:DNA, Complementary