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[transforming activity of plasmid r6k dna on serratia marcescens strain 20-10. the behavior of the plasmid in the transformants].the authors described transformation of s. marcescens, strain 20-10, of the isolated r6k plasmide dna. as demonstrated by centrifugation in cesium chloride gradient and electrophoresis in agarose, the plasmide was present in the transformants in the form identical to r6k in e. coli k12. analysis of the transforming activity of r6k plasmide from serratia and e. coli k12 strains with a complete and defective restriction system showed s. marsescens, strain 20-10, to possess specific system of restr ...1978371301
equilibrium, kinetic, and footprinting studies of the tus-ter protein-dna interaction.arrest of dna replication in the terminus region of the escherichia coli chromosome is mediated by protein-dna complexes composed of the tus protein and 23 base pair sequences generically called ter sites. we have characterized the in vitro binding of purified tus protein to a 37-base pair oligodeoxyribonucleotide containing the terb sequence. the measured equilibrium binding constant (kd) for the chromosomal terb site in kg buffer (50 mm tris-cl, 150 mm potassium glutamate, 25 degrees c, ph 7.5 ...19921313800
construction of conjugative shuttle and suicide vectors for pasteurella haemolytica and p. multocida.a shuttle cloning vector, paka16, and suicide derivatives paka19 and paka22 have been developed for gene transfer to pasteurella haemolytica and p. multocida. paka16 was constructed by insertion of the lacz alpha-peptide-encoding region and a multiple cloning site into a plasmid which was originally isolated from p. haemolytica serotype a1. the vector encodes ampicillin resistance, and contains at least 14 unique restriction sites and the property of phenotypic identification of recombinant clon ...19948045428
investigations of pi initiator protein-mediated interaction between replication origins alpha and gamma of the plasmid r6k.a typical plasmid replicon of escherichia coli, such as ori gamma of r6k, contains tandem iterons (iterated initiator protein binding sites), an at-rich region that melts upon initiator-iteron interaction, two binding sites for the bacterial initiator protein dnaa, and a binding site for the dna-bending protein ihf. r6k also contains two structurally atypical origins called alpha and beta that are located on either side of gamma and contain a single and a half-iteron, respectively. individually, ...201020029091
the dnak-dnaj-grpe chaperone system activates inert wild type pi initiator protein of r6k into a form active in replication initiation.the plasmid r6k is an interesting model system for investigating initiation of dna replication, not only near the primary binding sites of the initiator protein pi but also at a distance, caused by pi -mediated dna looping. an important milestone in the mechanistic analysis of this replicon was the development of a reconstituted replication system consisting of 22 different highly purified proteins (abhyankar, m. a., zzaman, s., and bastia, d. (2003) j. biol. chem. 278, 45476-45484). although th ...200415485812
a cryptic plasmid of yersinia enterocolitica encodes a conjugative transfer system related to the regions of clodf13 mob and incx pil.yersinia enterocolitica 29930 (biotype 1a; o : 7,8), the producing strain of the phage-tail-like bacteriocin enterocoliticin, possesses a plasmid-encoded conjugative type iv transfer system. the genes of the conjugative system were found by screening of a cosmid library constructed from total dna of strain 29930. the cosmid cos100 consists of the vector supercos1 and an insert dna of 40 303 bp derived from a cryptic plasmid of strain 29930. the conjugative transfer system consists of genes encod ...200314523116
monomer/dimer ratios of replication protein modulate the dna strand-opening in a replication origin.dna opening is an essential step in the initiation of replication via the cairns mode of replication. the opening reaction was investigated in a gamma ori system by using hyperactive variants of plasmid r6k-encoded initiator protein, pi. reactivity to kmno4 (indicative of opening) within gamma ori dna occurred in both strands of a superhelical template upon the combined addition of wt pi, dnaa and integration host factor (ihf), each protein known to specifically bind gamma ori. ihf, examined sin ...200111237610
identification of the central regulatory segment of plasmid r6k complexed with the membranes of escherichia coli.understanding the mechanisms which control replication of chromosomes with multiple origins of replication have been the subject of many investigations. the plasmid, r6k, has three origins of replication, alpha, beta, and gamma. in vivo, alpha and beta are utilized with equal frequencies while the gamma origin is rarely used in the parental plasmid, or remains silent in several miniplasmids. hence, in the present study, the multiple origin plasmid, r6k, was utilized as a model system to investig ...19957731391
rapid purification of a cloned gene product by genetic fusion and site-specific proteolysis.we have developed a rapid and general technique for purification of a protein encoded by a cistron contained in a recombinant dna clone. the technique consists of fusing the target cistron dna in the correct reading frame to a marker cistron via a piece of dna that codes for a linker peptide. the target cistron in the example presented here is the replication initiator cistron of the plasmid r6k. the linker is a dna fragment encoding 60 amino acids from the triple helical region of chicken pro a ...19846087342
positive and negative roles of an initiator protein at an origin of replication.the properties of mutants in the pir gene of plasmid r6k have suggested that the pi protein plays a dual role; it is required for replication to occur and also plays a role in the negative control of the plasmid copy number. in our present study, we have found that the pi level in cell extracts of escherichia coli strains containing r6k derivatives is surprisingly high (approximately equal to 10(4) dimers per cell) and that this level is not altered in cells carrying high copy number pir mutants ...19863540947
bacteriophage x-2: a filamentous phage lysing incx-plasmid-harbouring bacterial strains.phage x-2, a filamentous rod about 950 nm in length, was isolated from sewage as plating on strains of escherichia coli, salmonella typhimurium or serratia marcescens carrying either the incx plasmid r6k, or the unique plasmid r775. phage x-2 differs morphologically from a previously described very broad host range filamentous phage x which also lyses plasmid r6k-carrying strains and the phages differ in their resistance to inactivation by diethyl ether. phage x-2 is serologically unrelated to p ...19883076188
a host-encoded dna-binding protein promotes termination of plasmid replication at a sequence-specific replication terminus.we have purified approximately 6600-fold an approximately 40-kda protein (ter protein) encoded by escherichia coli that specifically binds to two sites at the 216-base-pair replication terminus (tau) of the plasmid r6k. chemical footprinting experiments have shown that the ter protein binds to two 14- to 16-base-pair sequences that exist as inverted repeats in the tau fragment. site-directed mutagenesis of one of the terminus sequences (tau r) resulted in a mutant tau r that failed to bind to th ...19892654932
a replication initiator protein enhances the rate of hybrid formation between a silencer rna and an activator rna.the replication origin gamma of plasmid r6k in certain miniplasmids is kept silent by a silencer rna. we have identified a major and three minor transcripts that are synthesized in a direction antiparallel and complementary to the silencer rna. the major rna, called the activator, is essential for replication from ori gamma. the complementary nature of the activator and silencer rnas strongly suggests that the former is a target of the latter. we have also discovered that the initiator protein i ...19872444344
translational options for the pir gene of plasmid r6k: multiple forms of the replication initiator protein pi.the autogenously controlled pir gene of plasmid r6k was believed to encode a single polypeptide that plays multiple roles in the plasmid's biology. we have isolated an opal (op) mutant at the 18th codon of the pir coding frame which does not totally abolish translation of pir mrna. in extracts of cells containing this mutation two translational products (35 kda and 30.2 kda) have been detected. we propose that the 35-kda polypeptide produced by the pir18 op mutation contains trp substituted for ...19921628846
[characteristics of the deletion mutant of plasmid r6k].deletion plasmide r6kdelta with the mol wt of 17.2.10(6) dalton isolated from the e. coli chi 925 (r6k) is described. this plasmide expresses no resistance to streptomycin, is replicated in the e. coli k12 under relaxed control and is resistant to the treatment with the eliminating agents. analysis of plasmide dna with the aid of electrophoresis in agarose gel demonstrated that r6k delta has one site attacked by restriction endonucleases eco. ri and bam hi. these data were confirmed by the deter ...1977339611
a comparison of the origin of replication of psa with r6k.the plasmid pori3 is a derivative of the origin of replication of psa. replication is defective as a result of a truncated repa gene, the product of which is required for plasmid replication. the defective replication is complemented by the presence of the intact repa gene of psa, or by the presence of the plasmid r6k. the basis of this complementation has been examined by comparing the nucleotide sequence of the origin of psa with that of r6k. a 13 base pair sequence present twice in the origin ...19836358799
[streptomycin-3"-phosphotransferases from streptomycin-resistant cells of escherichia coli strains].streptomycin-3"-phosphotransferases were isolated and purified from e. coli cells containing plasmids 836, pbs52 or r6k, which determine the microorganisms resistance towards streptomycin and dihydrostreptomycin. phosphorylation of the 3"-hydroxylic group of dihydrostreptomycin was demonstrated by [13c]-nmr spectrometry. it was shown that streptomycin-3"-phosphotransferase, whose synthesis is determined by plasmid 836 (as well as by plasmid r6k), differs from the analogous enzyme, whose synthesi ...19806166328
strand and site specificity of the relaxation event for the relaxation complex of the antibiotic resistance plasmid r6k. 19744616719
activation in vivo of the minimal replication origin beta of plasmid r6k requires a small target sequence essential for dna looping.the plasmid r6k contains three distinct origins of replication: alpha, beta, and gamma. the gamma sequence is essential in cis and acts as an enhancer that activates the distant alpha and beta origins. r6k therefore represents a favorable procaryotic model system with which to unravel the biochemical mechanisms underlying selective origin activation, particularly activation involving distant sites on the same chromosome. we have discovered that plasmids containing the origins alpha and gamma req ...19921515418
mechanism of origin activation by monomers of r6k-encoded pi protein.one recurring theme in plasmid duplication is the recognition of the origin of replication (ori) by specific rep proteins that bind to dna sequences called iterons. for plasmid r6k, this process involves a complex interplay between monomers and dimers of the rep protein, pi, with seven tandem iterons of gamma ori. remarkably, both pi monomers and pi dimers can bind to iterons, a new paradigm in replication control. dimers, the predominant form in the cell, inhibit replication, while monomers fac ...200717383678
binding of dnaa protein to a replication enhancer counteracts the inhibition of plasmid r6k gamma origin replication mediated by elevated levels of r6k pi protein.replication of the gamma origin of escherichia coli plasmid r6k requires pi protein, encoded by the r6k pir gene, and many host factors, including dnaa protein. pi has dual roles, activating replication at low levels and inhibiting replication at high levels. the inhibitory function of pi is counteracted by integration host factor and a specific sequence of the origin called the enhancer. this 106-bp dna segment contains a binding site for dnaa protein (dnaa box 1). in this study, we mutated thi ...19947961437
the integration host factor of escherichia coli binds to multiple sites at plasmid r6k gamma origin and is essential for replication.examination of the effect of the hima and himd mutants of e. coli on the maintenance of plasmid r6k has revealed that the gamma origin-containing replicons cannot be established in any of the mutants deficient in the production of e. coli integration host factor (ihf). contrary, the r6k derivatives containing other origins of the plasmid (alpha and/or beta) replicate in a host lacking functional ihf protein. we show that ihf protein binds specifically to a segment of the replication region which ...19882967465
use of autonomously replicating mini-r6k plasmids in the analysis of the replication regions of the r plasmid r6k. 1979383375
molecular cloning of replication and incompatibility regions from the r-plasmid r6k. 1978361972
escherichia coli replication terminator protein impedes simian virus 40 (sv40) dna replication fork movement and sv40 large tumor antigen helicase activity in vitro at a prokaryotic terminus sequence.we have discovered that the escherichia coli terminator protein (ter) impedes replication fork movement, initiated in vitro from the simian virus 40 replication origin by the large tumor antigen (tag), at the terminator site (tau r) of the prokaryotic plasmid r6k preferentially when tau r is present in one orientation with respect to the origin. we also have discovered that ter impedes helicase activity of tag at the tau r site, when tau r is in this same orientation. in contrast with ter, a mut ...19911849268
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