Cloning the polB gene of Escherichia coli and identification of its product

H. Chen, S. K. Bryan, Robb Moses

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Using an in vivo mini-Mu cloning system, we have cloned the polB gene of Escherichia coli into the multicopy plasmid, pUC18. A chromosomal insert of 4.9 kilobases gave 30-40-fold overproduction of DNA polymerase II, and the cells containing the plasmid showed normal growth. The restriction pattern of the polB gene does not match that of either the polA gene or polC gene. Plasmid-directed protein synthesis demonstrates peptides of 99 and 82 kDa which are not expressed by derivative plasmids without DNA polymerase II activity. It appears from in situ gel assays and high performance liquid chromatography that 82- and 55-kDa proteins are derived from the 99-kDA protein by degradation, but all retain activity. DNA polymerase I or DNA polymerase III antibody does not inhibit the synthesis reaction of partially purified DNA polymerase II, but DNA polymerase II antibody does. By the criteria of restriction pattern of the polB gene, molecular weight of the protein, and antibody inhibition of reaction, DNA polymerase II can be demonstrated to be a distinct DNA polymerase.

Original languageEnglish (US)
Pages (from-to)20591-20595
Number of pages5
JournalJournal of Biological Chemistry
Volume264
Issue number34
StatePublished - 1989
Externally publishedYes

Fingerprint

DNA Polymerase II
Cloning
Escherichia coli
Organism Cloning
Genes
Plasmids
Antibodies
Proteins
DNA Polymerase III
DNA Polymerase I
High performance liquid chromatography
DNA-Directed DNA Polymerase
Proteolysis
Assays
Molecular Weight
Gels
Molecular weight
High Pressure Liquid Chromatography
Derivatives
Degradation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cloning the polB gene of Escherichia coli and identification of its product. / Chen, H.; Bryan, S. K.; Moses, Robb.

In: Journal of Biological Chemistry, Vol. 264, No. 34, 1989, p. 20591-20595.

Research output: Contribution to journalArticle

@article{390f4ed276ea47d9a02e31c636d831d2,
title = "Cloning the polB gene of Escherichia coli and identification of its product",
abstract = "Using an in vivo mini-Mu cloning system, we have cloned the polB gene of Escherichia coli into the multicopy plasmid, pUC18. A chromosomal insert of 4.9 kilobases gave 30-40-fold overproduction of DNA polymerase II, and the cells containing the plasmid showed normal growth. The restriction pattern of the polB gene does not match that of either the polA gene or polC gene. Plasmid-directed protein synthesis demonstrates peptides of 99 and 82 kDa which are not expressed by derivative plasmids without DNA polymerase II activity. It appears from in situ gel assays and high performance liquid chromatography that 82- and 55-kDa proteins are derived from the 99-kDA protein by degradation, but all retain activity. DNA polymerase I or DNA polymerase III antibody does not inhibit the synthesis reaction of partially purified DNA polymerase II, but DNA polymerase II antibody does. By the criteria of restriction pattern of the polB gene, molecular weight of the protein, and antibody inhibition of reaction, DNA polymerase II can be demonstrated to be a distinct DNA polymerase.",
author = "H. Chen and Bryan, {S. K.} and Robb Moses",
year = "1989",
language = "English (US)",
volume = "264",
pages = "20591--20595",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "34",

}

TY - JOUR

T1 - Cloning the polB gene of Escherichia coli and identification of its product

AU - Chen, H.

AU - Bryan, S. K.

AU - Moses, Robb

PY - 1989

Y1 - 1989

N2 - Using an in vivo mini-Mu cloning system, we have cloned the polB gene of Escherichia coli into the multicopy plasmid, pUC18. A chromosomal insert of 4.9 kilobases gave 30-40-fold overproduction of DNA polymerase II, and the cells containing the plasmid showed normal growth. The restriction pattern of the polB gene does not match that of either the polA gene or polC gene. Plasmid-directed protein synthesis demonstrates peptides of 99 and 82 kDa which are not expressed by derivative plasmids without DNA polymerase II activity. It appears from in situ gel assays and high performance liquid chromatography that 82- and 55-kDa proteins are derived from the 99-kDA protein by degradation, but all retain activity. DNA polymerase I or DNA polymerase III antibody does not inhibit the synthesis reaction of partially purified DNA polymerase II, but DNA polymerase II antibody does. By the criteria of restriction pattern of the polB gene, molecular weight of the protein, and antibody inhibition of reaction, DNA polymerase II can be demonstrated to be a distinct DNA polymerase.

AB - Using an in vivo mini-Mu cloning system, we have cloned the polB gene of Escherichia coli into the multicopy plasmid, pUC18. A chromosomal insert of 4.9 kilobases gave 30-40-fold overproduction of DNA polymerase II, and the cells containing the plasmid showed normal growth. The restriction pattern of the polB gene does not match that of either the polA gene or polC gene. Plasmid-directed protein synthesis demonstrates peptides of 99 and 82 kDa which are not expressed by derivative plasmids without DNA polymerase II activity. It appears from in situ gel assays and high performance liquid chromatography that 82- and 55-kDa proteins are derived from the 99-kDA protein by degradation, but all retain activity. DNA polymerase I or DNA polymerase III antibody does not inhibit the synthesis reaction of partially purified DNA polymerase II, but DNA polymerase II antibody does. By the criteria of restriction pattern of the polB gene, molecular weight of the protein, and antibody inhibition of reaction, DNA polymerase II can be demonstrated to be a distinct DNA polymerase.

UR - http://www.scopus.com/inward/record.url?scp=0024398715&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024398715&partnerID=8YFLogxK

M3 - Article

C2 - 2684981

AN - SCOPUS:0024398715

VL - 264

SP - 20591

EP - 20595

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 34

ER -