Analysis of a randomly assorted cDNA library from BW 5147 lymphoid cells. Frequency estimates based on computer aided concatenation matching of 2D gel polypeptide products

J. R. Frey, C. Coleclough, J. R. Kettman, J. P. Hofmann, L. Kuhn, I. Lefkovits

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

A cDNA library was divided into 291 sectors of low complexity, 800-1000 recombinant phage plaques per sector. Aliquots of DNA from sectors prepared from high titer phage were subjected to in vitro transcription using T7 polymerase and thereafter RNA was translated in a cell-free rabbit reticulocyte system in the presence of [35S] methionine. The resulting polypeptides were separated by 2D gel electrophoresis. Radiofluorographs of the gels prepared from 10 sectors were subjected to scanning and image analysis. A multilevel spot matching procedure was employed allowing us to recognise spot occurrence in the 10 sectors. The number of detected polypeptide spots per sector varied between 147 and 325. Overall, 1082 different spots were counted totalling 1983 spots considering multiple entries. The distribution of the spots and the frequency distribution of the RNA population among the 10 sectors are shown. The highest detected frequency is 10-2, while one half of the RNA molecular species are present at a frequency of 10-3 or lower.

Original languageEnglish (US)
Pages (from-to)753-763
Number of pages11
JournalMolecular Immunology
Volume34
Issue number11
DOIs
StatePublished - Aug 1 1997

Keywords

  • Cell free transcription
  • Cell free translation
  • Database
  • Image analysis
  • Lymphocyte proteins
  • Proteinpaedia
  • Two-dimensional gel electrophoresis
  • cDNA library

ASJC Scopus subject areas

  • Immunology
  • Molecular Biology

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