Lens epithelial cell mRNA. I. Cloning and sequencing of a messenger RNA with a basic motif/leucine-rich domain specifically expressed in rat lens epithelial cells.
By methods of subtraction-hybridization of lambda ZAP cDNA libraries, prepared from 4-week-old rat lens epithelial cells (capsule) and lens fiber cells (decapsulated lens), we have isolated a specific cDNA clone whose target mRNA is about 600 b long. Northern blot hybridization analysis data showed that the target mRNA was preferentially expressed in the lens epithelial cells; it was not found in the retina or in non-ocular tissues. The complete sequence of the mRNA was obtained both by the 5' and 3' rapid amplification of cDNA ends (5'-RACE, 3'-RACE), and by sequencing of a clone containing the full-length cDNA insert. It contains 599 b. with an open reading frame at nucleotide (nt) 30 (ORF30). The base sequence appears to represent a complete sequence of the target mRNA, with a poly(A) signal at nt 563-569, and a poly(A) tail at nt 582 (GenBank accession #U15149). In situ hybridization showed that the target mRNA was localized in the anterior epithelial cells, and in the elongating cells in the bow region. The encoded protein of 57 amino acids (aa) contains a basic and an acidic domain. It is rich in leucine, which is located at the basic region of the ORF at every fourth aa, and at the acidic region at eight aa intervals. Therefore, there is a periodicity in the appearance of leucine residues in the deduced primary sequence, as has been seen in DNA binding proteins. Search of GenBank, EMBL and Swiss-Prot did not yield significant homology to any of the entered sequences in those databanks. Our data demonstrate that we have isolated a cDNA clone containing novel sequences specifically expressed in lens epithelial cells.
Wen
Y
Y
Laboratory of Lens Molecular Biology, Doheny Eye Institute, Los Angeles, CA, USA.
Li
G W
GW
Chen
P
P
Bekhor
I
I
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D018076:DNA, Complementary / Q000032:analysis; D004848:Epithelium / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D017403:In Situ Hybridization; D007908:Lens, Crystalline / Q000378:metabolism*; D007930:Leucine; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley
D018076:DNA, Complementary; D012333:RNA, Messenger; D007930:Leucine