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

doi:10.1016/S0161-5890(97)00075-8

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

Immunology

Research output: Contribution to journal › Article › peer-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 language	English (US)
Pages (from-to)	753-763
Number of pages	11
Journal	Molecular Immunology
Volume	34
Issue number	11
DOIs	https://doi.org/10.1016/S0161-5890(97)00075-8
State	Published - Aug 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

Access to Document

10.1016/S0161-5890(97)00075-8

Cite this

@article{4046f325ab4d4d65b8d0540e699b3189,

title = "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",

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.",

keywords = "Cell free transcription, Cell free translation, Database, Image analysis, Lymphocyte proteins, Proteinpaedia, Two-dimensional gel electrophoresis, cDNA library",

author = "Frey, {J. R.} and C. Coleclough and Kettman, {J. R.} and Hofmann, {J. P.} and L. Kuhn and I. Lefkovits",

note = "Funding Information: Acknowled#ements--We value the contribution of the program for assembling the data matrix provided by Roy Reese. We thank Jos6 Garcia-Sanz and Rolf Jessberger for critical reading of this manuscript. The Basel Institute for Immunology was founded and is supported by F. Hoffmann La-Roche, Basel, Switzerland. Copyright: Copyright 2007 Elsevier B.V., All rights reserved.",

year = "1997",

month = aug,

doi = "10.1016/S0161-5890(97)00075-8",

language = "English (US)",

volume = "34",

pages = "753--763",

journal = "Molecular Immunology",

issn = "0161-5890",

publisher = "Elsevier Limited",

number = "11",

}

TY - JOUR

T1 - 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

AU - Frey, J. R.

AU - Coleclough, C.

AU - Kettman, J. R.

AU - Hofmann, J. P.

AU - Kuhn, L.

AU - Lefkovits, I.

N1 - Funding Information: Acknowled#ements--We value the contribution of the program for assembling the data matrix provided by Roy Reese. We thank Jos6 Garcia-Sanz and Rolf Jessberger for critical reading of this manuscript. The Basel Institute for Immunology was founded and is supported by F. Hoffmann La-Roche, Basel, Switzerland. Copyright: Copyright 2007 Elsevier B.V., All rights reserved.

PY - 1997/8

Y1 - 1997/8

N2 - 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.

AB - 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.

KW - Cell free transcription

KW - Cell free translation

KW - Database

KW - Image analysis

KW - Lymphocyte proteins

KW - Proteinpaedia

KW - Two-dimensional gel electrophoresis

KW - cDNA library

UR - http://www.scopus.com/inward/record.url?scp=0031440138&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031440138&partnerID=8YFLogxK

U2 - 10.1016/S0161-5890(97)00075-8

DO - 10.1016/S0161-5890(97)00075-8

M3 - Article

C2 - 9444974

AN - SCOPUS:0031440138

SN - 0161-5890

VL - 34

SP - 753

EP - 763

JO - Molecular Immunology

JF - Molecular Immunology

IS - 11

ER -

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

Abstract

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this