A novel vector for positive selection of inserts harboring an open reading frame by translational coupling.
(17/196)
We have developed a novel vector pTCS, as a tool for efficient selection of open reading frame (ORF)-containing inserts. In pTCS clones containing an insert with an ORF a downstream marker gene (immE3, conferring resistance to colicin) is activated via translational coupling with the insert, and transformed cells can then be selected by exposure to colicin E3. Our method differs from previous methods in that the marker gene is activated without protein fusion, and that selection occurs irrespective of the reading frame of the insert. (+info)
Evolution of the iss gene in Escherichia coli.
(18/196)
Identification of a plasmid-coded protein required for initiation of ColE2 DNA replication.
(19/196)
The product of the rep gene of ColE2 is required for initiation of ColE2 DNA replication. The rep gene was placed under the control of the promoters, PL and PR, and the heat-labile cl857 repressor of bacteriophage lambda. The Rep protein was identified as a 35 Kd protein by the maxicell method in combination with heat-induced expression. The protein was efficiently expressed from these promoters in unirradiated cells and accumulated up to a few per cent of the total cellular proteins. It was partially purified (about 80% pure) and its properties examined. The amino acid sequence of the amino terminal portion of the partially purified protein agreed well with that predicted from the nucleotide sequence of the rep gene. One of the characteristic features of the rep gene is frequent usage of rare codons, especially those for arginine. The protein specifically stimulated replication of ColE2 DNA but not that of ColE3 DNA in crude cell extracts of Escherichia coli. Specific binding of the protein to plasmid DNA containing the origin region of ColE2 was demonstrated by the filter binding method. Neither endonuclease activity nor topoisomerase activity was detected by using ColE2 DNA. (+info)
Transcription and initiation of ColE1 DNA replication in Escherichia coli K-12.
(20/196)
By S1 nuclease protection mapping, we characterized RNA transcripts and nascent ColE1 DNA synthesized in wild-type Escherichia coli cells after infection with lambda-mini-ColE1 hybrid bacteriophages. Transcription of the RNA II region of ColE1 DNA in vivo starts mostly from the RNA II promoter, which was identified by in vitro experiments, and ends at or near the ori site. Synthesis of the leading strand of ColE1 DNA was found to start at the ori site. Nevertheless, the molar ratio of the nascent DNA to the synthesized transcripts ending at the ori site was less than 0.05. In bacterial rnh mutants whose RNase H activities were less than 0.06% of that of the wild type, transcription patterns, as well as nascent DNA synthesis, were still similar to those in rnh+ cells. However, in bacteria whose rnh gene was interrupted by insertion of a drug resistance gene, the number of transcripts ending at the ori site was much reduced and that of transcripts reading through the ori site was definitely increased relative to that observed in wild-type bacteria. These results suggested that cleavage of the RNA transcript at the ori site in vivo is dependent on RNase H activity, as demonstrated in the in vitro system, but most of the cleaved RNA is unable to prime initiation of ColE1 DNA synthesis efficiently. (+info)
Lysophospholipase L1 from Escherichia coli K-12 overproducer.
(21/196)
After screening 900 E. coli strains of the Clarke and Carbon collection for by lysophospholipase L1 activities, we isolated a clone bearing the plasmid pLC6-34, which showed an increased level of lysophospholipase L1 activity. Strains bearing the plasmid pC124, a subclone of pLC6-34 in plasmid vector pUC8, showed approximately 11.4 times higher lysophospholipase L1 activity than that of the parental strain. Starting from those overproducing strains, the lysophospholipase L1 was purified to near homogeneity by sequential use of ammonium sulfate fractionation, Sephacryl S-300, DEAE-cellulose, hydroxyapatite and Sephacryl S-200 column chromatographies. The apparent molecular weight of the purified lysophospholipase L1 was estimated to be 20,500-22,000 both by SDS-polyacrylamide gel electrophoresis and by gel permeation chromatography. The specific activity of the homogeneous lysophospholipase L1 was 10,400 nmol/min/mg protein when 1-acyl-sn-glycero-3-phosphoethanolamine was used as the substrate. The amino acid sequence of the amino-terminal portion of purified lysophospholipase L1 was determined and was different from that of lysophospholipase L2, which had previously been purified from the envelope fraction of E. coli strains bearing its cloned structural gene, pldB [Karasawa, K., Kudo, I., Kobayashi, T., Sa-eki, T., Inoue, K., & Nojima, S. (1985) J. Biochem, 98, 1117-1125]. The gene responsible for overproduction of lysophospholipase L1 activity was designated as pldC (phospholipid degradation C). Its restriction enzyme map was also different from that of cloned pldB. These results further confirmed that, in E. coli, there are two lysophospholipases with distinct characteristics. (+info)
Analysis of ColE1 MbeC unveils an extended ribbon-helix-helix family of nicking accessory proteins.
(22/196)
Diverse Antibacterial activity of Pectobacterium carotovorum subsp.carotovorum isolated in Korea.
(23/196)
Fifty-four Pectobacterium carotovorum subsp. carotovorum strains isolated in Korea were characterized by a spectrum of antibacterial activities against 7 indicator strains chosen to represent various regions and host plants. All P. carotovorum subsp. carotovorum isolates tested could be grouped into 4 classes depending on the pattern of antibacterial substance production. All tested strains had DNA fragment(s) homologous to the genes encoding carotovoricin and 21 of them had genes homologous to DNA invertase. Sixteen strains had genes homologous to the genes encoding carocin S1. Several isolates produced antibacterial substances active against strains in Brenneria, Pantoea, and Pectobacterium genera that belonged formerly to the genus Erwinia. Strains in Pseudomonas or Xanthomonas sp. were not sensitive to the antibacterial substances produced by P. carotovorum subsp. carotovorum, except for X. albilineans that was sensitive to antibacterial substances produced by most strains in P. carotovorum subsp. carotovorum and P. betavasculorum KACC10056. These results demonstrated the diverse patterns of antibacterial substance production and the possibility of the existence of new antibacterial substance(s) produced by P. carotovorum subsp. carotovorum isolated in Korea. (+info)
Evolution of colicin BM plasmids: the loss of the colicin B activity gene.
(24/196)