| replication of plasmid r6k in dna polymerase deficient mutants of escherichia coli. | the plasmid r6k has been introduced into a number of escherichia coli polymerase deficient (pol) mutants. in polcts mutants transferred to the nonpermissive temperature to inactivate polymerase iii, r6k replicates but the replication products have a density in dye-cscl gradients intermediate between supercoiled and linear forms. this aberrant replication requires normal cellular levels of polymerase i since it does not occur in pola polcts mutants. normal r6k replication and maintenance occur in ... | 1978 | 86279 |
| [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 ... | 1977 | 339611 |
| [organization of plasmid r6k genome]. | | 1978 | 344018 |
| replication of antibiotic resistance plasmid r6k dna in vitro. | a soluble extract prepared from cells of an escherichia coli strain carrying the antibiotic resistance plasmid r6k is capable of carrying out the complete process of r6k dna replication. dna synthesis in vitro is dependent on the four deoxyribo- and ribonucleotide triphosphates and is sensitive to rifampin and streptolydigin, inhibitors of dna-dependent rna polymerase. the incorporation of deoxyribonucleotides into r6k dna also is sensitive to actinomycin d, novobiocin, arabinofuranosyl-ctp, and ... | 1978 | 354690 |
| partial amino acid sequence of penicillinase coded by escherichia coli plasmid r6k. | direct studies of the amino acid sequence of an escherichia coli plasmid-coded penicillinase (penicillin amido-beta-lactamhydrolase, ec 3.5.2.6) are in complete agreement with results derived from the translation of the dna sequence of a related plasmid, apart from a single amino acid substitution. this penicillinase from a gram-negative bacterium shows 30-35% identity with functionally similar enzymes from gram-positive bacteria. this paper should be read in conjunction with the report of the d ... | 1978 | 358199 |
| interaction between the plasmid r6k and escherichia coli with defective dna polymerase i. | | 1978 | 361497 |
| activity of the replication terminus of plasmid r6k in hybrid replicons in escherichia coli. | | 1978 | 361971 |
| molecular cloning of replication and incompatibility regions from the r-plasmid r6k. | | 1978 | 361972 |
| [genetic heteroduplex analysis of the r6k plasmid]. | the results of genetic studies of r6k tra1- and r6kdelta[sm1] mutants of r6k plasmid and those of heteroduplex analysis of dnas have shown that dna of this drug-resistant factor contains three loops flanked by the inverted repeats. the latter are designated as ir1, ir2 and ir3 and are of 50, 100 and 120 nucleotides in size respectively. ir1 is inserted into the loop flanked by ir2. loops with these two repeats are located in major ecor1 fragment, ir3 having been found in minor ecori fragment of ... | 1978 | 363508 |
| requirement of a plasmid-encoded protein for replication in vitro of plasmid r6k. | conditions are described for the replication of exogeneous r6k dna in an in vitro system prepared from escherichia coli cells. replication of plasmid dna in this system is semiconservative and sensitive to actinomycin d, novobiocin, arabinofuranosyl-ctp,n-ethylmaleimide, and inhibitors of dna-dependent rna polymerase. an ammonium sulfate fraction prepared from cells carrying the r6k plasmid is required for replication. a direct role in replication for a plasmid-encoded protein, designated pi, in ... | 1978 | 364478 |
| transposition of a duplicate antibiotic resistance gene and generation of deletions in plasmid r6k. | transformation experiments showed that spontaneous deletions which result in loss of streptomycin resistance and an increase in conjugal transfer efficiency are present at a frequency of about 10(-4) in plasmid molecules of r6k. similar deletions were thus readily selected by conjugal transfer of r6k, and their appearance was dependent upon reca+ activity in either donor or recipient host. the deoxyribonucleic acid segment deleted in four mutants examined was concluded to extend from the same te ... | 1979 | 370107 |
| [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 ... | 1978 | 371301 |
| construction of plasmid r6k derivatives in vitro: characterization of the r6k replication region. | | 1978 | 372982 |
| nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid r6k. | a 2.1-kilobase segment of the antibiotic resistance plasmid r6k carries sufficient information to replicate as a plasmid in escherichia coli. this segment contains a functional origin of replication and a structural gene for a protein, designated pi, that is required for the initiation of r6k replication. the nucleotide sequence of a 520-base-pair portion of this 2.1-kilobase segment that includes the functional origin of replication and the region adjacent to the start of the pi structural gene ... | 1979 | 375227 |
| use of autonomously replicating mini-r6k plasmids in the analysis of the replication regions of the r plasmid r6k. | | 1979 | 383375 |
| trans-complementation-dependent replication of a low molecular weight origin fragment from plasmid r6k. | a non-self-replicating segment (1370 base pairs) of plasmid r6k was cloned in e. coli and shown to trans-complement temperature-sensitive replication mutants of this plasmid. this segment contains the gene which codes for a protein required for initiation of replication of the plasmid, and was used as a helper in a functional assay for an origin of replication in r6k derivatives. a 420 bp fragment, derived from r6k dna, was shown to carry a functional origin since it was capable of replicating a ... | 1978 | 728998 |
| mode of replication of the conjugative r-plasmid rsf1040 in escherichia coli. | replicating deoxyribonucleic acid (dna) molecules of plasmid rsf1040, a deletion mutant of the conjugative r plasmid r6k, appear in the electron microscope as partially supercoiled structures with two open circular branches of equal size, although open structures with three branches, two branching points and no supercoiled regions (theta structures) were also found at a lower frequency. the partially supercoiled molecules sediment more rapidly than native covalently closed circular dna in neutra ... | 1976 | 770431 |
| plasmid dna replication. | recent studies have provided some insight to the overall characteristics of plasmid replication in bacteria. the cole 1 and r6k plasmids replicate via a covalently-closed circular intermediate. replication is initiated at a fixed origin and is unidirectional in the case of cole 1 and bidirectional for r6k. in the case of the plasmid r6k. the bidirectional replication is asymmetric and sequential, proceeding from a fixed origin to a fixed terminus located approximately 20% of the r6k genome from ... | 1976 | 776703 |
| bidirectional replication of plasmid r6k dna in escherichia coli; correspondence between origin of replication and position of single-strand break in relaxed complex. | replicating molecules of plasmid r6k dna have been purified as covalently closed circular dna forms and analyzed in the electron microscopy after cleavage with the ecori restriction endonuclease. it has been determined that in most cases replication proceeds bidirectionally from an origin whose position is indistinguishable from the site of the single-strand break (nick) in the open circular dna form of the relaxation complex of r6k dna and protein. evidence is presented for the existence of a u ... | 1975 | 1103126 |
| 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 ... | 1992 | 1313800 |
| terf, the sixth identified replication arrest site in escherichia coli, is located within the rcsc gene. | we report the existence of a sixth replication arrest site, terf, that is located within the coding sequences of the rcsc gene, a negative regulator of capsule biosynthesis. the terf site is oriented to allow transcription of the rcsc gene but prevent dna replication in the terminus-to-origin direction. our results demonstrate that the terf site is functional in both chromosomal and plasmid environments and that the stability of the tus-terf protein-dna complex more closely resembles the plasmid ... | 1992 | 1447156 |
| two alternative structures can be formed by ihf protein binding to the plasmid r6k gamma origin. | escherichia coli integration host factor (ihf) contributes to the regulation of r6k plasmid copy number by counteracting the inhibitory activity of the plasmid-encoded replication protein pi. two ihf-binding sites (ihf1 and ihf2) flank seven iterons in the origin which bind pi protein. as previously shown by electron microscopy, ihf can compact a large segment of the r6k gamma origin dna, encompassing site ihf1, an at-rich domain containing ihf1, and some of the seven iterons located downstream ... | 1992 | 1447190 |
| an improved suicide vector for construction of chromosomal insertion mutations in bacteria. | we have constructed an r6k-based suicide vector (pjp5603) that requires a trans supply of the pir-encoded pi protein of plasmid r6k for replication. therefore, efficient plasmid suicide results upon transfer to bacteria not harbouring pir. the 3.1-kb vector encodes kanamycin resistance and is mobilizable. when used in conjunction with a jm109 strain carrying pir, it has nine unique restriction sites available for alpha-complementation cloning. vector functionality was demonstrated in rhodobacter ... | 1992 | 1511879 |
| 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 ... | 1992 | 1515418 |
| activation of distant replication origins in vivo by dna looping as revealed by a novel mutant form of an initiator protein defective in cooperativity at a distance. | we have isolated mutants of the pi initiator protein of the plasmid r6k that are defective in dna looping in vitro but retain their normal dna binding affinity for the primary binding sites (iterons) at the gamma origin/enhancer. one such looping defective mutant called r6 was determined to be a proline to leucine change at position 46 near the n terminus of the pi protein. using a set of genetic assays that discriminate between the activation of the gamma origin/enhancer from those of the dista ... | 1992 | 1547780 |
| structural and functional analysis of a replication enhancer: separation of the enhancer activity from origin function by mutational dissection of the replication origin gamma of plasmid r6k. | the plasmid r6k possesses three distinct origins of replication: alpha, beta, and gamma. the replication origin gamma of plasmid r6k performs a dual function: (i) as an origin itself and (ii) as an enhancer element required in cis for the activation at a distance of the other two replication origins alpha and beta. we have dissected the gamma origin/enhancer by site-directed mutagenesis and have reached the following conclusions. the origin function can be specifically inactivated without impair ... | 1992 | 1594615 |
| identification of a novel promoter in the replication control region of plasmid r6k. | a novel source of transcription has been detected in the replication region of plasmid r6k by using fusions involving the galk reporter gene. the -35 and -10 consensus rna polymerase binding sites were identified in the region overlapping the binding sites for the r6k-encoded replication protein pi. transcription from this promoter, designated p2, is repressed in vivo by pi-protein levels that are inhibitory for replication. promoter-down mutations in p2 induced in vitro by bisulfite mutagenesis ... | 1992 | 1624464 |
| roles of a 106-bp origin enhancer and escherichia coli dnaa protein in replication of plasmid r6k. | a dnaa 'null' strain could not support replication of intact plasmid r6k or derivatives containing combinations of its three replication origins (alpha, gamma, beta). dnaa binds in vitro to sites in two functionally distinct segments of the central gamma origin. the 277-bp core segment is common to all three origins and contains dnaa box 2, which cannot be deleted without preventing replication. immediately to the left of the core lies the 106-bp origin enhancer, which contains dnaa box 1. when ... | 1992 | 1627205 |
| 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 ... | 1992 | 1628846 |
| conformational changes induced by integration host factor at origin gamma of r6k and copy number control. | we have investigated the role of integration host factor (ihf) in the replication of plasmid r6k by studying the maintainance of the plasmid in a strain of escherichia coli that lacks both subunits of ihf and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of ihf; however, loss of ihf reduced the copy number of those replicons initiating solely from ori gamma by 5-fold. concomitant loss of direct repeats within the origin t ... | 1991 | 1651928 |
| replication of plasmid r6k origin gamma in vitro. dependence on dual initiator proteins and inhibition by transcription. | we have developed a more efficient in vitro replication system for the plasmid r6k with the objective of dissecting the mechanism of activation of replication origins at a distance. using this in vitro system we have shown that the activation of replication origin gamma of r6k is absolutely dependent on two exogenously added initiator proteins: namely the host-encoded dnaa and the plasmid-encoded pi proteins. replication was inhibited by novobiocin, suggesting a requirement for dna gyrase. surpr ... | 1991 | 1651932 |
| replication of the r6k plasmid during the escherichia coli cell cycle. | the cell-cycle replication pattern of the r6k plasmid has been investigated by using the membrane-elution technique to produce cells labelled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid dna. the high-copy plasmid r6k replicates exponentially in a cell-cycle-independent manner. a mini-r6k plasmid deleted for the ori alpha origin of replication also replicates, exponentially in a cell-cycle-independent manner. | 1992 | 1732198 |
| a compact nucleoprotein structure is produced by binding of escherichia coli integration host factor (ihf) to the replication origin of plasmid r6k. | understanding the role of escherichia coli histone-like protein integration host factor (ihf) in replication of r6k plasmid (dellis, s., and filutowicz, m. (1991) j. bacteriol. 173, 1279-1286) requires detailed analyses of the interaction of ihf protein with the plasmid's replication origin (gamma ori). we describe an electron microscopic analysis which shows that a compact structure can be formed in the presence of ihf, in which, on average, a 102-base pair (bp) ori segment is involved. ihf.gam ... | 1991 | 1836213 |
| 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 ... | 1991 | 1849268 |
| dna-protein interaction at the replication termini of plasmid r6k. | understanding the molecular mechanism of specific and polarized termination of dna replication at a sequence-specific replication terminus requires detailed analyses of the interaction of terminator protein (ter) with specific dna sequences (tau), constituting the replication terminus. such analyses should provide the structural basis of the functional polarity of replication inhibition observed in vivo and in vitro at tau sites. with this objective in mind, we have purified the replication term ... | 1991 | 1989907 |
| integration host factor of escherichia coli reverses the inhibition of r6k plasmid replication by pi initiator protein. | integration host factor (ihf) protein is the only host-encoded protein known to bind and to affect replication of the gamma origin of escherichia coli plasmid r6k. we examined the ability of r6k origins to replicate in cells lacking either of the two subunits of ihf. as shown previously, the gamma origin cannot replicate in ihf-deficient cells. however, this inability to replicate was relieved under the following conditions: underproduction of the wild-type pi replication protein of r6k or produ ... | 1991 | 1991721 |
| alpha and beta replication origins of plasmid r6k show similar distortions of the dna helix in vivo. | plasmid r6k contains inverted repeats of an approximately 100-base-pair sequence separated by 5.5 kilobases. these long inverted repeats (lirs) occur within the alpha and beta origins of replication and are essential for origin function. in this study, primer-extension analysis of dna modified in vivo by dimethyl sulfate or kmno4 revealed that both alpha and beta lirs acquire similar structural distortions of the dna helix in a functional r6k replicon. these distortions were not seen in plasmids ... | 1990 | 2251253 |
| n-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid r6k replication. | the replication initiation protein pi of the escherichia coli plasmid r6k is a dual regulator in the control of plasmid copy number, functioning both as a specific initiator and inhibitor of replication. while the biochemical basis of these activities is not known, initiator activity requires binding of the protein to the seven 22 bp direct repeats within the gamma-origin region. by deleting c-terminal segments of the pi coding region, we have found that the n-terminal polypeptides of pi that ar ... | 1990 | 2277631 |
| elimination of multicopy plasmid r6k by bleomycin. | bleomycin eliminated multicopy plasmid r6k from growing cells of escherichia coli ab1157 but failed to cure either of the low-copy plasmids r1 or r46. measurements of r6k-encoded beta-lactamase and of covalently closed plasmid dna indicated that the drug causes a progressive reduction in plasmid copy number. | 1985 | 2411218 |
| 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 ... | 1987 | 2444344 |
| evidence of a ter specific binding protein essential for the termination reaction of dna replication in escherichia coli. | activity binding specifically to the 22 bp of the dna replication terminus (ter) sequence on plasmid r6k and the escherichia coli genome was detected in the crude extract of e. coli cells. this activity was inactivated by heat or by protease but not by rnase treatments. overproduction of the ter binding activity was observed when the extract was prepared from the cell carrying a plasmid with a chromosomal-derived 5.0 kb ecori fragment, on which one of the four terc sites, terc2, was also located ... | 1989 | 2551684 |
| 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 ... | 1989 | 2654932 |
| [biological characteristics of plasmid carrying a repeated deoxyribonucleic acid sequence]. | it was found that a plasmid which had a foreign deoxyribonucleic acid (dna) between two repeated sequences did not multiply in e. coli recbcsbcb, even if it multiplied in wild-type e. coli, e. coli recbc or e. coli recbcsbcbrecf when the insert was longer than 351 base pair. the multiplication of these plasmids were, however, inhibited when a plasmid expressing recf gene was introduced into e. coli recbcsbcbrecf. the inviability of the plasmid carrying the repeated sequence in e. coli recbcsbcb ... | 1989 | 2681680 |
| negative control of plasmid r6k replication: possible role of intermolecular coupling of replication origins. | the gamma origin binding sites of the replication initiator pi protein, composed of seven 22-base-pair (bp) direct repeats and previously shown to be essential for replication of plasmid r6k, can also act as an inhibitor of r6k replication in escherichia coli cells if provided in trans. inhibition is dependent upon the ability of these repeats to bind the r6k-encoded pi protein but is not overcome by increasing the intracellular pi level. the insertion of a second repeat cluster in close proximi ... | 1989 | 2682632 |
| convenience of plasmid r6k higher-copy-number deletion derivative for cloning of larger dna fragments. | vector properties of plasmid pnh602, a higher-copy-number deletion mutant of plasmid r6k, were tested by cloning the 6.5 mg/mol bamhi psa fragment carrying determinants of resistance to four antibiotics in the unique bamhi site of pnh602. the resulting in vitro constructed recombinant plasmid pnh606 was found to be stable, conjugative, multicopy (20 copies of pnh606 per e. coli chromosome were estimated) and to ensure the increased expression of different genes responsible for the antibiotic res ... | 1987 | 2822553 |
| identification of the dna sequence from the e. coli terminus region that halts replication forks. | the terminus region of the e. coli chromosome contains two loci, t1 and t2, that inhibit the progress of replication forks and require the trans-acting factor tus. we have identified a 23 bp terminator signal at t1 and t2 that is within 100 bp of the sites of replication arrest. when an oligodeoxyribonucleotide containing the terminator signal was inserted into a plasmid, replication was halted only in a tus+ strain and when the terminator signal was oriented properly. we also found this termina ... | 1988 | 2846183 |
| a family of cosmid vectors with the multi-copy r6k replication origin. | a family of cosmid vectors was constructed which contain replication origins (ori) derived from the multicopy plasmid r6k, a kanamycin resistance gene and two cos sites, permitting efficient library construction. additional features of later constructs are (i) the presence of noti sites flanking the site of insertion to allow intact excision of inserts, (ii) the facility for selective cloning of the ends of inserts for rapid chromosome walking, and (iii) the use of a mutated r6k ori leading to a ... | 1987 | 2961651 |
| 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 ... | 1988 | 2967465 |
| role of the pi initiation protein and direct nucleotide sequence repeats in the regulation of plasmid r6k replication. | | 1985 | 2990404 |
| new replication mutant pnh602 and its relationship to plasmid pas3, another deletion derivative of plasmid r6k. | a stable deletion derivative pnh602 was obtained when the recently described higher-copy-number point mutant pnh601 of plasmid r6k was introduced to a minicells-producing strain of escherichia coli. the size of plasmid pnh602 is 18.8 mg/mol as determined by electron microscopy. the 7.2 mg/mol fragment of r6k genome missing in pnh602 carries the smr-determinant and the region fino and, according to the results of restriction analysis, it includes one ecori site. with its radioisotopically determi ... | 1985 | 2997007 |
| conformational changes in a replication origin induced by an initiator protein. | the replication initiator protein of the plasmid r6k binds to seven contiguous 22 bp direct repeats that form an indispensable part of the three replication origins alpha, beta, and gamma. binding of the initiator to the direct repeats induced a marked bending of the region of gamma replication origin. binding of the initiator also promoted unwinding of the origin dna by at least two turns. distamycin appeared to antagonize the binding of the initiator to the seven 22 bp direct repeats. at the a ... | 1985 | 3000600 |
| a single amino acid alteration in the initiation protein is responsible for the dna overproduction phenotype of copy number mutants of plasmid r6k. | a novel type of high copy-number (cop) mutants of a mini-r6k plasmid were isolated. the mutations were mapped in the pir gene which encodes the pi initiation protein for plasmid r6k dna replication. they resulted in an alteration by substitution of a single amino acid: threonine to isoleucine at the 108th position for the cop41, and proline to serine at the 113th position for the cop50, of the 305 amino acid pi protein. the cop41 mutation in the pi protein was found to be trans-dominant over the ... | 1985 | 3000771 |
| dna segments of the incx plasmid r485 determining replication and incompatibility with plasmid r6k. | the expression of incompatibility properties between the incx plasmids r6k and r485 of escherichia coli was examined. for small autonomously replicating derivatives of both plasmid elements, the requirements for incompatibility expression include a functional r485 replicon and an active r6k beta-origin region. functional r6k alpha and gamma origins are not directly involved in incompatibility expression between r6k and r485. a trans-acting replication system was constructed for plasmid r485. it ... | 1985 | 3006104 |
| binding of purified wild-type and mutant pi initiation proteins to a replication origin region of plasmid r6k. | the three replication origins of the antibiotic resistance plasmid r6k require for their activity in escherichia coli a dna segment containing seven 22 base-pair direct repeats and a plasmid-encoded initiation protein (pi). the pi protein functions in the negative control of r6k replication, in addition to its requirement for the initiation of replication. construction of a plasmid containing the pi structural gene (pir) downstream from the inducible pr promoter of bacteriophage lambda provided ... | 1986 | 3009827 |
| replication from one of the three origins of the plasmid r6k requires coupled expression of two plasmid-encoded proteins. | the minimal beta-replicon of plasmid r6k contains an open reading frame for a 151-amino acid protein in addition to the seven 22-base pair direct nucleotide sequence repeats, the structural gene (pir) for the pi initiation protein, and the beta-origin sequence that have been shown to be required for replication activity. in this work, a site-specific mutation by linker insertion in this putative coding sequence, designated bis, resulted in a nonfunctional beta-replicon. the nonfunctional beta-re ... | 1986 | 3013894 |
| 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 ... | 1988 | 3076188 |
| an initiator protein for plasmid r6k dna replication. mutations affecting the copy-number control. | two kinds of mutations affecting the copy-number control of plasmid r6k were isolated and identified in an initiator pi protein by dna sequencing. firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. this cop21 ... | 1988 | 3277861 |
| rna polymerase binding sites on a plasmid r6k derivative with increased copy number. | the specific binding of escherichia coli rna polymerase molecules to the dna of plasmid pnh602, a deletion derivative of r6k having an increased copy number, was detected by electron microscopy. seven strong rnapol binding sites were found on pnh602 dna linearized with bamhi or ecori restriction endonuclease. all of these specific sites occur in genetically defined regions of the pnh602 molecule. two of them correspond with the recently reported transcription initiation sites within a region ess ... | 1987 | 3298616 |
| dna and protein interactions in the regulation of plasmid replication. | as for bacterial and animal viruses that employ different mechanisms for their duplication in a host cell, plasmids have evolved different strategies to assure their hereditary stability or maintenance at a specific copy number during cell growth and division. a characteristic feature of plasmid replication control, however, is an involvement of one or more negatively controlling elements. furthermore, a majority of the bacterial plasmids examined to date contain direct nucleotide sequence repea ... | 1987 | 3332651 |
| enhancer-origin interaction in plasmid r6k involves a dna loop mediated by initiator protein. | initiation of dna replication from ori beta of plasmid r6k requires the presence of the ori gamma sequence in cis. we demonstrate that binding of initiator protein to the seven strong, tandem binding sites in gamma increases binding of the protein at the very weak binding site present in ori beta by cooperativity at a distance. the gamma-beta interaction via the initiator results in a dna loop, as revealed by the novel technique of cyclization enhancement and as confirmed by exonuclease iii prot ... | 1988 | 3345564 |
| detection of dna looping due to simultaneous interaction of a dna-binding protein with two spatially separated binding sites on dna. | we describe different and relatively rapid biochemical techniques to detect protein-mediated dna looping. these techniques, based on enhancement of dna knotting and that of ligase-catalyzed cyclization, were used to show that the replication initiator protein of plasmid r6k can bring together two intramolecular gamma origin of replication sequences located as far apart as 2 kilobases. the site-site interaction causes looping out of the intervening dna sequence as visualized by electron microscop ... | 1988 | 3413096 |
| 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 ... | 1986 | 3540947 |
| replication initiator protein of plasmid r6k autoregulates its own synthesis at the transcriptional step. | the replication initiator protein of plasmid r6k preferentially repressed transcription initiated in vitro from the promoter of the initiator protein cistron. dnase i protection experiments revealed that the sequences in the region of the promoter recognized by the initiator protein partially overlapped the sequences of the same promoter recognized by rna polymerase of escherichia coli. competitive dnase i protection experiments revealed that the initiator not only prevented the rna polymerase f ... | 1985 | 3857600 |
| plasmid-encoded initiation protein is required for activity at all three origins of plasmid r6k dna replication in vitro. | dna replication of plasmid r6k initiates at three unique sites, ori alpha, ori beta, and ori gamma. replicating dna molecules of a deletion derivative of r6k were synthesized in an in vitro system containing pi protein fraction from cells carrying a mini-r6k derivative that produced only this initiation protein as an r6k-encoded protein and analyzed by electron miscroscopy. requirement of pi protein for the activity of all these three replication origins in vitro was verified. frequencies of ini ... | 1985 | 3882456 |
| mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid r6k. | plasmid pmm3 is a pbr322 derivative carrying the gamma origin of replication of the naturally occurring plasmid r6k. we have produced a gamma-origin mutant bank of this plasmid using the single-strand-specific mutagen sodium bisulfite. members of this bank contain single or multiple mutations in the seven direct repeats and the flanking sequences in the gamma origin. three mutants with defective gamma origins have been isolated from this mutant bank. two of these direct repeat mutants, gamma 117 ... | 1985 | 3883361 |
| transcription signals in a region essential for replication of plasmid r6k. | a dna fragment encoding for the enzyme chloramphenicol acetyltransferase (cat) but lacking the cat promoter sequence was used to probe transcription signals in a dna fragment (640 base pairs) from plasmid r6k that plays a central role in r6k dna replication. the r6k dna fragment analyzed contains the seven 22-bp direct repeats which express r6k incompatibility and are required in cis for activity of all three of the r6k origins of replication. in addition to the previously identified promoter se ... | 1985 | 3887441 |
| autorepressor properties of the pi-initiation protein encoded by plasmid r6k. | a dna fusion containing the promoter of the pir gene of plasmid r6k that encodes for the pi-initiation protein and the beta-galactosidase gene of escherichia coli (lacz) is described. the synthesis of beta-galactosidase promoted by this pir-lac fusion was almost completely inhibited when an r6k sequence containing the pir gene was provided in trans in e. coli. transcription in vitro from the pir promoter but not the trp promoter of e. coli, was inhibited by purified pi protein indicating that th ... | 1985 | 3923429 |
| strand and site specificity of the relaxation event for the relaxation complex of the antibiotic resistance plasmid r6k. | | 1974 | 4616719 |
| 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 ... | 1984 | 6087342 |
| phage x: a plasmid-dependent, broad host range, filamentous bacterial virus. | phage x was isolated from sewage as plating on escherichia coli or salmonella typhimurium strains harbouring the incompatibility group x plasmid r6k. it also plated on a strain of serratia marcescens carrying this plasmid. it failed to form plaques on proteus mirabilis, p. morganii or providencia alcalifaciens harbouring r6k, but did multiply on them. no phage increase occurred with homologous r- strains. phage x also plated or registered an increase in titre on e. coli or s. typhimurium strains ... | 1981 | 6121839 |
| [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 ... | 1980 | 6166328 |
| interaction of the plasmid r6k-encoded replication initiator protein with its binding sites on dna. | initiation of dna replication in plasmid r6k is potentiated by the plasmid-encoded 35 kd replication initiator protein. we had previously reported that the initiator bound to two regions of r6k dna called site i and site ii. using dnaase i footprinting technique we have demonstrated that the initiator bound to seven tandem repeats of a 22 bp long sequence in site i. in site ii, the initiator bound to a single repeat having the same consensus sequence and to two partial repeats that most likely o ... | 1983 | 6224568 |
| control of plasmid r6k copy numbers in isogenic rep+ and rep escherichia coli strains. | | 1980 | 6246401 |
| intramolecular transposition and inversion in plasmid r6k. | selection was made in escherichia coli k-12 reca hosts carrying plasmid r6k for ampicillin hyperresistance. twenty-two selected strains were found to carry mutant plasmids, which, from electron microscopy and restriction enzyme analysis, were concluded to arise by a duplication of transposon tn2660, which confers ampicillin resistance, in all cases the duplicate transposon being in an inverted orientation with respect to the resident tn2660. a mutant of r6k, psjc301, which was temperature sensit ... | 1980 | 6247328 |
| location of two relaxation nick sites in r6k and single sites in psc101 and rsf1010 close to origins of vegetative replication: implication for conjugal transfer of plasmid deoxyribonucleic acid. | a nick-labeling method has been used to localize the relaxation complex nick sites in three plasmids (psc101, rsf1010, and r6k) that differ markedly in their host range, deoxyribonucleic acid replication, and conjugal transfer properties. single specific relaxation sites were located in psc101 and rsf1010, but surprisingly two distinct sites could be identified in the bi-origin plasmid r6k. in all cases, relaxation nick sites, which are thought to be origins of plasmid conjugal transfer, were sh ... | 1980 | 6254952 |
| three origins of replication are active in vivo in the r plasmid rsf1040. | replicating dna molecules of rsf1040, a deletion derivative of the conjugative r plasmid r6k, are cleaved at a single site by the eco ri restriction endonuclease. microscopic analysis of eco ri-cleaved rsf1040 replicative intermediates synthesized in vivo indicates that initiation of replication occurs at three unique sites, ori alpha, ori beta, and ori gamma. the relative frequencies of initiations at these three origins are different from those found in vitro. | 1980 | 6254957 |
| [genetic control of plasmid r6k conjugativity]. | the regions determining conjugation ability of plasmid r6k were localized by means of deletion mutants obtained in vitro and in vivo and tra- mutants induced by integration of transposons tn5, tn7 and tn9 into different dna sites of the conjugative deletion mutant pas3. at least 13 genes were found to be involved in the genetic control of r6k conjugativity, on the basis of genetic, restriction and heteroduplex studies. they were mapped within the two dna regions having the total molecular weight ... | 1981 | 6271623 |
| [organization of the genetic system of plasmid r6k conjugativity]. | | 1982 | 6277584 |
| [location of the tras- and fino-type genes and of the orit region of plasmid r6k]. | the regions of b6k dna corresponding to orit, fino and tras are mapped using a number of hybrid plasmids and deletion mutants pas3::tn5, pas3::tn7 and pas3::tn9 obtained in vitro after treatment with restriction endonucleases ecori and bamhi. fino- and tras-like genes were mapped in the regions of 10 and 25,0-25,4 md, respectively, orit being situated in the region of 4,6-4,7 md. | 1982 | 6290313 |
| primary structure of the replication initiation protein of plasmid r6k. | the cistron of the replication initiation protein of plasmid r6k has been cloned into the single-strand dna vectors m13mp8 and m13mp9 and its complete nucleotide sequence has been determined. the amino acid sequence of the initiator protein as predicted from its nucleotide sequence shows that the protein is lysine rich and weakly basic and has a molecular weight of 35,000, which is in close agreement with that estimated from the mobility in nadodso4/acrylamide gels. the secondary structure of th ... | 1982 | 6291046 |
| the nucleotide sequence of the replication origin beta of the plasmid r6k. | we h ave identified by molecular cloning a region of 283 base pairs of the hindiii 2 fragment of r6k which corresponds to the region of the replication origin beta. this 283 base-pair dna fragment, when present contiguously with the structural gene for the replication initiation protein of r6k, encoded in the hindiii 9-15 and part of hindiii 2 restriction fragments, will support the replication of a plasmid chimera containing the pbr322 replicon in a pol ats host at the nonpermissive temperature ... | 1982 | 6292210 |
| plasmid r6k dna replication. ii. direct nucleotide sequence repeats are required for an active gamma-origin. | | 1982 | 6296394 |
| the replication initiator protein of plasmid r6k tagged with beta-galactosidase shows sequence-specific dna-binding. | we have tagged the replication initiator protein of the plasmid r6k near the c-terminal end by fusion, in the correct reading frame, with the 89 amino acid long n-terminal alpha-donor polypeptide of beta-galactosidase of e. coli. this fusion was carried out with recombinant dna methods. the protein chimera thus generated retained the activities of both initiation of dna replication in vivo at the replication origin gamma of r6k and hydrolysis of beta-galactopyranoside when complemented in vivo w ... | 1983 | 6297781 |
| structural properties of the beta origin of replication of plasmid r6k. | the beta origin of replication of plasmid r6k, one of three active r6k origins of replication, requires most or all of a 1962-base pair (bp) sequence for activity. the nucleotide sequence of a portion of this functional beta origin was determined in an earlier study (stalker, d., kolter, r., and helinski, d. (1982) j. mol. biol. 161, 33-43). in this work, the sequence of the remaining portion of this 1964-bp segment was obtained. in addition to its activity as an origin of replication, this sequ ... | 1983 | 6300075 |
| release of initiation control by a mutational alteration in the r6k pi protein required for plasmid dna replication. | plasmid prk419, a derivative of the naturally occurring antibiotic resistance plasmid r6k, contains the pir gene that codes for the pi initiation protein and the beta and gamma replication origins of r6k. a mutation in plasmid prk419, designated cos405, results in an elevated plasmid copy number in escherichia coli growing at 42 degrees c and an even greater increase in copy number when the cells are shifted to 30 degrees c. this mutation was assigned to the structural gene for the pi protein on ... | 1983 | 6310578 |
| use of gene fusions and protein-protein interaction in the isolation of a biologically active regulatory protein: the replication initiator protein of plasmid r6k. | the initiation of dna replication of plasmid r6k is triggered by a 35-kilodalton initiator protein. the initiator protein had been elusive because of its lability and the lack of a convenient assay procedure to aid its purification. using recombinant dna techniques, we have fused the cistron of the initiator near its cooh-terminal end, in the correct reading frame, to the lacz cistron of escherichia coli at the ninth codon from the nh2 terminus. the fused cistron yielded a protein that was not o ... | 1983 | 6316329 |
| two improved promoter sequences for the beta-lactamase expression arising from a single base-pair substitution. | a mutation in the transposon tn2660 derived from the plasmid r6k, and resulting in an approximate tenfold increase in penicillin resistance is shown to be a single gc to at substitution 177 base pairs 'upstream' of the initiation codon of the structural beta-lactamase (bla) gene. this substitution leads to the transcription of two new mrnas which can be ascribed to the creation of two new overlapping promoter sequences. all the sequences (450bp) examined in the wild-type tn2660 are identical to ... | 1984 | 6326054 |
| [effect of plasmid r6k on the radiation resistance of escherichia coli k-12 cells]. | in y; dp(1;3)scj4, y+m(3)i55pc2 ssak/mwh ssak stock, somatic y; mwh m+ clones were induced at different developmental stages by 60co gamma-irradiation (1000 rad; 12,2 rad/sec). expression of pc2 (the development of sex-combs on the 2nd and the third leg-pairs) in the non-m clones was similar to that in y; dp(1;3)scj4, y+m(3)i55 pc2ssak/mwh ssak flies, but significantly lower than in pc2 ssak/+ssak flies. such non-autonomous minute effect may be due to the early repression of the pc prior to the ... | 1983 | 6339323 |
| isolation and characterization of a higher-copy-number mutant of plasmid r6k. | a stable copy-number mutant (pnh601) of plasmid r6k was isolated by selection for increased resistance to ampicillin determined by this plasmid. the size of the mutant plasmid was found to be unchanged (26 mg/mol) but it is present in 27 copies of pnh601 per e. coli k-12 chromosome which represents a two-fold increase of r6k copy number value. the following genetic properties of pnh601 are reported and compared with those of r6k: conjugative transfer, fertility inhibition of plasmids belonging t ... | 1983 | 6357969 |
| 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 ... | 1983 | 6358799 |
| plasmid r6k dna replication. i. complete nucleotide sequence of an autonomously replicating segment. | | 1982 | 6759660 |
| plasmid r6k dna replication. iii. regulatory properties of the pi initiation protein. | | 1982 | 6818353 |
| the nucleotide sequence surrounding the replication terminus of r6k. | the replication terminus of the plasmid r6k has been cloned into the single-stranded dna phage vector m13mp5 and also into the plasmid vectors pbr313 and pbr322. by using single-stranded dna templates prepared from the recombinant dna clones, the sequence of 215 base pairs of dna containing the replication terminus has been determined. the dna sequence of the region of the terminus does not contain any 2-fold rotational symmetry. therefore, folding of the dna at the region of the terminus is unl ... | 1981 | 6941271 |
| [effect of plasmid r6k on expression of the temperature-sensitive mutation in gene dnae of escherichia coli k-12]. | the multicopy, conjugative r6k plasmid is responsible for the increase in the number of temperature-resistant (tr) clones formed by escherichia coli k-12 e486 strain, carrying a ts mutation dnae486 in the gene coding for dna polymerase iii. the effect observed is not due to the mutator action of r6k or a mutation in the plasmid and requires the intactness of the host reca function. tr derivatives mg488 and mg492, isolated under non-permissive condition from the strain e486(r6k), still possess th ... | 1980 | 7007157 |
| [expression of plasmid r6k in a cell-free coupled transcription-translation system]. | the modified method for obtaining e. coli extract s 30 for the coupled transcription--translation system is described. the extract treated with immobilized dnaase and micrococcal nuclease had the minimal endogenous protein synthesis and high activity in respect to exogenous dna matrices. the synthesis of 9-12 proteins with molecular weights ranging from 14,000 to 178,000 daltons was shown to occur in this system in the presence of the dna of r6k plasmid. the use of the modification of novick's m ... | 1981 | 7018127 |
| a rapid and economic method for estimation of the number of plasmid copies in escherichia coli cells. | an economic method for a rapid estimation of the number of copies of plasmid r6k delta 1 in e. coli cells using lysis of the cells directly on the agarose gel and electrophoretic separation of radioactively labelled plasmid and chromosomal dna's is described. the method, particularly useful for screening purposes, permits detection of both the ccc and oc forms of plasmids of molar mass 2-150 mg/mol and it can be applied to other bacterial systems. | 1982 | 7044920 |
| molecular cloning of the replication terminus of the plasmid r6k. | analyses of the intermediates of dna replication of the r6k plasmid derivatives, rsf1040, rjhc12 and rjhc26 demonstrate the transient accumulation of open circular dna molecules with a discontinuity in either the plus or the minus strand of the dna. the location of this discontinuity is nonrandom and is near the terminus of dna replication. the discontinuity is not due to the activation of a relaxation complex since neither rjhc12 nor rjhc26 are relaxable. the replication terminus is functional ... | 1981 | 7262563 |
| new mini-tn5 derivatives for insertion mutagenesis and genetic engineering in gram-negative bacteria. | five mini-tn5 derivatives encoding resistance to km, cm, gm, tc, and sm, coupled with the polylinker of the pbluescriptii plasmid, were constructed. these derivatives are carried by an ampicillin-resistant plasmid that has a conditional origin of replication from plasmid r6k and origin of conjugal transfer from the broad host range plasmid rp4. the new vectors are smaller than those previously described and possess numerous unique restriction sites inside the minitransposons for gene cloning in ... | 1995 | 7497352 |
| buffer composition mediates a switch between cooperative and independent binding of an initiator protein to dna. | the regulation of many biological processes, including dna replication, is frequently achieved by protein-protein interactions, as well as protein-dna interactions. multiple protein-binding sites are often involved. for example, the replication of plasmid r6k involves binding of the initiator protein pi to seven 22-bp direct repeats (dr) in the gamma origin of replication (gamma ori). a mutant protein pi s87n has been isolated, that in tris.borate buffer (tb) binds cooperatively to seven dr, whe ... | 1995 | 7590295 |
| a dna segment conferring stable maintenance on r6k gamma-origin core replicons. | the plasmid r6k gamma origin consists of two adjacent modules, the enhancer and the core, and requires r6k initiator protein pi for replication. while the core alone can replicate at a low level of wild-type pi protein, we show here that host cells do not stably maintain core plasmids. the presence of the enhancer segment confers stable inheritance on core plasmids without a significant change in average plasmid copy number. deletions and site-directed mutagenesis indicated that the stability of ... | 1995 | 7592407 |