Abstract
Deletions of the promoter region for the tetracycline-resistance (Tcr) gene(s) of pBR322 were constructed in order to generate new promoter-probe plasmid cloning vectors. The deletions were constructed in vitro by exonuclease digestion at the HindIII site and blunt-end ligation of the digestion products. Plasmids which lost the HindIII site but retained the EcoRI site carried deletions ranging from 5 to 60 bp. Some of the plasmids lacked the nucleotide sequences required for initiation of transcription from the Tcr promoter and "anti-Tcr" promoter. Three of the promoter-deletion plasmids (containing deletions of 5-29 bp) formed tight-binding complexes with RNA polymerase in vitro, despite their tetracycline sensitive phenotype. One deletion plasmid, pPV33, retained three out-of-phase stop codons located between the promoter-cloning site (EcoRI) and the translational start codon for the tetracycline resistance gene. These features give pPV33 several advantages over previously described promoter-cloning vehicles.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 291-304 |
| Number of pages | 14 |
| Journal | Gene |
| Volume | 20 |
| Issue number | 2 |
| DOIs | |
| State | Published - 1982 |
Fingerprint
Keywords
- Antibiotic resistance
- gene cartridge
- insertional activation
- overlapping promoters
- pPV33
- RNA polymerase binding
ASJC Scopus subject areas
- Genetics
Cite this
Construction and characterization of E. coli promoter-probe plasmid vectors III. pBR322 derivatives with deletions in the tetracycline resistance promoter region. / West, Robert W.; Rodriguez, R. L.
In: Gene, Vol. 20, No. 2, 1982, p. 291-304.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Construction and characterization of E. coli promoter-probe plasmid vectors III. pBR322 derivatives with deletions in the tetracycline resistance promoter region
AU - West, Robert W.
AU - Rodriguez, R. L.
PY - 1982
Y1 - 1982
N2 - Deletions of the promoter region for the tetracycline-resistance (Tcr) gene(s) of pBR322 were constructed in order to generate new promoter-probe plasmid cloning vectors. The deletions were constructed in vitro by exonuclease digestion at the HindIII site and blunt-end ligation of the digestion products. Plasmids which lost the HindIII site but retained the EcoRI site carried deletions ranging from 5 to 60 bp. Some of the plasmids lacked the nucleotide sequences required for initiation of transcription from the Tcr promoter and "anti-Tcr" promoter. Three of the promoter-deletion plasmids (containing deletions of 5-29 bp) formed tight-binding complexes with RNA polymerase in vitro, despite their tetracycline sensitive phenotype. One deletion plasmid, pPV33, retained three out-of-phase stop codons located between the promoter-cloning site (EcoRI) and the translational start codon for the tetracycline resistance gene. These features give pPV33 several advantages over previously described promoter-cloning vehicles.
AB - Deletions of the promoter region for the tetracycline-resistance (Tcr) gene(s) of pBR322 were constructed in order to generate new promoter-probe plasmid cloning vectors. The deletions were constructed in vitro by exonuclease digestion at the HindIII site and blunt-end ligation of the digestion products. Plasmids which lost the HindIII site but retained the EcoRI site carried deletions ranging from 5 to 60 bp. Some of the plasmids lacked the nucleotide sequences required for initiation of transcription from the Tcr promoter and "anti-Tcr" promoter. Three of the promoter-deletion plasmids (containing deletions of 5-29 bp) formed tight-binding complexes with RNA polymerase in vitro, despite their tetracycline sensitive phenotype. One deletion plasmid, pPV33, retained three out-of-phase stop codons located between the promoter-cloning site (EcoRI) and the translational start codon for the tetracycline resistance gene. These features give pPV33 several advantages over previously described promoter-cloning vehicles.
KW - Antibiotic resistance
KW - gene cartridge
KW - insertional activation
KW - overlapping promoters
KW - pPV33
KW - RNA polymerase binding
UR - http://www.scopus.com/inward/record.url?scp=0020352609&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0020352609&partnerID=8YFLogxK
U2 - 10.1016/0378-1119(82)90047-6
DO - 10.1016/0378-1119(82)90047-6
M3 - Article
C2 - 6299894
AN - SCOPUS:0020352609
VL - 20
SP - 291
EP - 304
JO - Gene
JF - Gene
SN - 0378-1119
IS - 2
ER -