Analysis of expressed sequence tags of retinal pigment epithelium: cystatin C is an abundant transcript

L Paraoan, I Grierson, B E Maden

Research output: Contribution to journalArticle (journal)peer-review

37 Citations (Scopus)

Abstract

In order to identify genes that are expressed in the retinal pigment epithelium (RPE), randomly chosen clones of a cDNA library of cultured human foetal RPE cells were analyzed by sequencing. Of 164 informative expressed sequence tags (ESTs), 88 matched the sequences of 74 genes for proteins of known or presumed function. Approximately a third of these represented genes with involvement in gene/protein expression, with a major subcategory concerned with protein turnover. In particular, the gene coding for precursor cystatin C was represented by 3 independent ESTs, and plaque hybridization estimated the frequency of cystatin C clones in the library to be 1.3%. Cystatin C mRNA in cultured RPE cells was confirmed by Northern blotting and by reverse transcription polymerase chain reaction (RT-PCR) with identification of the cystatin C sequence as the product of the reaction. The survey also revealed 25 novel human sequences representing genes that are active in RPE. One of these was localized near a recently identified, new autosomal recessive retinitis pigmentosa locus. In conclusion, the findings specifically demonstrate the unexpected presence of cystatin C mRNA at fairly high abundance in cultured human RPE cells, and, more generally, serve as a model study establishing the usefulness of the EST approach for further characterizing the molecular basis of the activities of the RPE.

Original languageEnglish
Pages (from-to)417-26
Number of pages10
JournalInternational Journal of Biochemistry and Cell Biology
Volume32
Issue number4
DOIs
Publication statusPublished - 1 Apr 2000
Externally publishedYes

Keywords

  • Cells, Cultured
  • Cystatin C
  • Cystatins/genetics
  • Cysteine Proteinase Inhibitors/genetics
  • DNA, Complementary/genetics
  • Expressed Sequence Tags
  • Gene Library
  • Humans
  • Pigment Epithelium of Eye/cytology
  • RNA/analysis
  • Reverse Transcriptase Polymerase Chain Reaction

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