Publications

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
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
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
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
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
[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
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
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 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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
use of autonomously replicating mini-r6k plasmids in the analysis of the replication regions of the r plasmid r6k.		1979	383375
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
construction of plasmid r6k derivatives in vitro: characterization of the r6k replication region.		1978	372982
[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
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
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
[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
molecular cloning of replication and incompatibility regions from the r-plasmid r6k.		1978	361972
activity of the replication terminus of plasmid r6k in hybrid replicons in escherichia coli.		1978	361971
interaction between the plasmid r6k and escherichia coli with defective dna polymerase i.		1978	361497
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
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
[organization of plasmid r6k genome].		1978	344018
[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
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