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Titolo:
MOLECULAR-CLONING AND EXPRESSION OF RPE65, A NOVEL RETINAL-PIGMENT EPITHELIUM-SPECIFIC MICROSOMAL PROTEIN THAT IS POSTTRANSCRIPTIONALLY REGULATED IN-VITRO
Autore:
HAMEL CP; TSILOU E; PFEFFER BA; HOOKS JJ; DETRICK B; REDMOND TM;
Indirizzi:
NEI,RETINAL CELL & MOLEC BIOL LAB,BLDG 6,RM 339,9000 ROCKVILLE PIKE BETHESDA MD 20892 NEI,RETINAL CELL & MOLEC BIOL LAB,BLDG 6,RM 339,9000 ROCKVILLE PIKE BETHESDA MD 20892 NEI,IMMUNOL & VIROL LAB BETHESDA MD 20892 GEORGE WASHINGTON UNIV,MED CTR,DEPT PATHOL WASHINGTON DC 20037
Titolo Testata:
The Journal of biological chemistry
fascicolo: 21, volume: 268, anno: 1993,
pagine: 15751 - 15757
SICI:
0021-9258(1993)268:21<15751:MAEORA>2.0.ZU;2-G
Fonte:
ISI
Lingua:
ENG
Soggetto:
BINDING PROTEIN; SEQUENCE-ANALYSIS; BOVINE RETINA; CELLS; IDENTIFICATION; DIFFERENTIATION; 11-CIS-RETINYL; NITROCELLULOSE; PURIFICATION; TRANSLATION;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
49
Recensione:
Indirizzi per estratti:
Citazione:
C.P. Hamel et al., "MOLECULAR-CLONING AND EXPRESSION OF RPE65, A NOVEL RETINAL-PIGMENT EPITHELIUM-SPECIFIC MICROSOMAL PROTEIN THAT IS POSTTRANSCRIPTIONALLY REGULATED IN-VITRO", The Journal of biological chemistry, 268(21), 1993, pp. 15751-15757

Abstract

Studies reported previously from this laboratory have shown that microsomal membranes of the vertebrate retinal pigment epithelium (RPE) contain an RPE-specific 65-kDa protein, RPE65, which bears the determinant recognized by the strictly tissue-specific monoclonal antibody RPE9, and which is developmentally regulated (Hamel, C. P., Tsilou, E., Harris, E., Pfeffer, B. A., Hooks, J. J., Detrick, B., and Redmond, T. M. (1993) J. Neurosci. Res. 34, 414-425). Microsequencing of 17 trypticand chymotryptic peptides obtained from the in situ digestion of the RPE65 blotted on nitrocellulose yielded primary sequences that were used to generate oligonucleotide probes. An 84-nucleotide guessmer was used to isolate two clones from a bovine RPE lambdaZap II cDNA library. Rapid amplification of cDNA ends was used to complete the 5' and 3' ends, resulting in a 3,115-base pair composite cDNA. The open reading frame encodes a novel protein of 533 amino acid residues with a computed molecular weight of 60,940. This protein does not match any other sequence in the data bases. The 231 amino acids obtained from peptide sequencing match 43% of the amino acid sequence deduced from the cDNA. The protein has a calculated pI of 6.41 and is not predicted to have any transmembrane segments. The open reading frame expressed in Escherichia coli has an apparent molecular weight identical to that of the native protein and is recognized by the monoclonal antibody RPE9, furthercorroborating its validity. Northern blot analysis detected a major mRNA species of 3.15 kilobases for RPE65, as well as shorter species, only in RPE and not in other tissues (including other ocular tissues). Cultured RPE cells (7 weeks in primary culture) contained RPE65 mRNA in amounts equivalent to fresh RPE. Such cells, however, contained no immunodetectable RPE65. The possible structure of this RPE-specific protein and hypotheses for the absence of translation in vitro are discussed.

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Documento generato il 05/12/20 alle ore 23:58:32