Publications
| Title | Abstract | Year Filter | PMID(sorted descending) Filter |
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| entry exclusion and orit of a conjugative system encoded by the cryptic plasmid p29930 of yersinia enterocolitica. | the conjugative transfer system of yersinia enterocolitica 29930 present on the cryptic plasmid p29930 comprises a mating pore formation system (mpf) related to that of the incx plasmid r6k and a dna transfer and replication system (dtr) with close relationship to the mob region of the mobilizable plasmid clodf13. two regions of the transfer system were selected for more detailed analyses of basic functions of conjugative transfer. the putative open reading frame orf22 located in the mpf region ... | 2010 | 20470820 |
| 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, ... | 2010 | 20029091 |
| replication initiation at a distance: determination of the cis- and trans-acting elements of replication origin alpha of plasmid r6k. | plasmid r6k, which contains 3 replication origins called alpha, gamma, and beta, is a favorable system to investigate the molecular mechanism(s) of action at a distance, i.e. replication initiation at a considerable distance from the primary initiator protein binding sites (iterons). the centrally located gamma origin contains 7 iterons that bind to the plasmid-encoded initiator protein, pi. ori alpha, located at a distance of approximately 4 kb from gamma, contains a single iteron that does not ... | 2010 | 20018882 |
| nucleotide sequence of pola52: a conjugative incx1 plasmid from escherichia coli which enables biofilm formation and multidrug efflux. | the large conjugative multidrug resistance (mdr) plasmid pola52 was sequenced and annotated. the plasmid encodes two phenotypes normally associated with the chromosomes of opportunistic pathogens, namely mdr via a resistance-nodulation-division (rnd)-type efflux-pump (oqxab), and the formation of type 3 fimbriae (mrkabcdf). the plasmid was found to be 51,602 bp long with 68 putative genes. about half of the plasmid constituted a conserved incx1-type backbone with predicted regions for conjugatio ... | 2008 | 18440636 |
| cooperative binding mode of the inhibitors of r6k replication, pi dimers. | the replication initiator protein, pi, plays an essential role in the initiation of plasmid r6k replication. both monomers and dimers of pi bind to iterons in the gamma origin of plasmid r6k, yet monomers facilitate open complex formation, while dimers, the predominant form in the cell, do not. consequently, pi monomers activate replication, while pi dimers inhibit replication. recently, it was shown that the monomeric form of pi binds multiple tandem iterons in a strongly cooperative fashion, w ... | 2008 | 18295232 |
| conjugal transfer of plasmid r6k gamma ori minireplicon derivatives from escherichia coli to various genera of pathogenic bacteria. | three r6k-derived gamma ori minireplicons were successfully transferred by conjugation from escherichia coli to several species of pathogenic bacteria. the pfl129 replicon encodes the wild-type initiation replication protein pi, while plasmids pfl130 and pag101 encode mutant forms of the pi protein conferring the plasmid copy-up phenotype. plasmids could be transferred to all recipient species tested, although high efficiency conjugal transfer was only obtained with genera of the enterobacteriac ... | 2007 | 17909888 |
| structure-based functional analysis of the replication protein of plasmid r6k: key amino acids at the pi/dna interface. | in previous work, we characterized the bases in an iteron of plasmid r6k that are important for the binding of pi protein monomers and dimers. here we investigate the following six amino acids of pi, encoded by pir, hypothesized to be important for dna contact: ser71, try74, gly131, gly211, arg225, and arg254. | 2007 | 17449630 |
| 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 ... | 2007 | 17383678 |
| crystal structure of pi initiator protein-iteron complex of plasmid r6k: implications for initiation of plasmid dna replication. | we have determined the crystal structure of a monomeric biologically active form of the pi initiator protein of plasmid r6k as a complex with a single copy of its cognate dna-binding site (iteron) at 3.1-a resolution. the initiator belongs to the family of winged helix type of proteins. the structure reveals that the protein contacts the iteron dna at two primary recognition helices, namely the c-terminal alpha4' and the n-terminal alpha4 helices, that recognize the 5' half and the 3' half of th ... | 2006 | 17124167 |
| small deletion variants of the replication protein, pi, and their potential for over-replication-based antimicrobial activity. | the emergence of multiply antibiotic-resistant microorganisms in the environment has become a serious public health threat. to address this, our lab has devised a methodology in which antimicrobial agents are transferred into unwanted cells using the process of bacterial conjugation. in the work described here, we pursued proteins that cause plasmid over-replication as potential antimicrobial agents. our focus was on the pir-encoded pi protein of plasmid r6k that possesses both positive and nega ... | 2006 | 16907728 |
| characterization of broad host range cryptic plasmid pcr1 from corynebacterium renale. | plasmid pcr1 is a cryptic plasmid harboured by corynebacterium renale. it is the smallest corynebacterial plasmid known to date. although its natural host is animal corynebacteria, it can replicate in several strains of soil corynebacteria. it can also replicate in escherichia coli, in which it is stably maintained. the copy number of pcr1 in this host is higher than that of puc19, with which it shows unidirectional incompatibility. it is also incompatible with pbk2, a plasmid bearing the common ... | 2006 | 16545871 |
| transformation of escherichia coli mediated by natural phospholipids. | transformation system for escherichia coli based upon introduction of plasmid dna by natural phospholipids has been developed. transformants are easily obtained by treatment with natural phospholipids such as phosphatidylethanolamine, phosphatidylcoline, and phosphatidylserine, where the presence of mgcl2 or cacl2 is essential. this method of transformation is applicable not only for small plasmid phsg399 (2.3 kb) but also for giant plasmid r6k (100 kb). | 2005 | 15665495 |
| 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 ... | 2004 | 15485812 |
| binding modes of the initiator and inhibitor forms of the replication protein pi to the gamma ori iteron of plasmid r6k. | discerning the interactions between initiator protein and the origin of replication should provide insights into the mechanism of dna replication initiation. in the gamma origin of plasmid r6k, the rep protein, pi, is distinctive in that it can bind the seven 22-bp iterons in two forms; pi monomers activate replication, whereas pi dimers act as inhibitors. in this work, we used wild type and variants of the pi protein with altered monomer/dimer ratios to study iteron/pi interactions. high resolu ... | 2004 | 15247259 |
| isomerization and apparent dna bending by pi, the replication protein of plasmid r6k. | plasmid r6k-encoded pi protein has multiple regulatory functions in replication and transcription. these functions rely, in part, on a complex set of interactions between monomers and dimers of the protein and distinct dna targets, the direct and inverted repeats (drs, irs). in the work described here, we examine the isomerization and dna bending properties of pi using electrophoretic mobility shift assays and circular permutation assays. our data suggest that pi dimers can bend irs, and dimer s ... | 2004 | 14706617 |
| biochemical investigations of control of replication initiation of plasmid r6k. | the mechanistic basis of control of replication initiation of plasmid r6k was investigated by addressing the following questions. what are the biochemical attributes of mutations in the pi initiator protein that caused loss of negative control of initiation? did the primary control involve only initiator protein-ori dna interaction or did it also involve protein-protein interactions between pi and several host-encoded proteins? mutations at two different regions of the pi-encoding sequence indiv ... | 2004 | 14665626 |
| pi protein- and atp-dependent transitions from 'closed' to 'open' complexes at the gamma ori of plasmid r6k. | r6k-encoded pi protein can bind to the seven, 22 bp tandem iterons of the gamma origin. in this work, we use a variant of pi, his-pi.f107s, that is hyperactive in replication. in vitro, his-pi.f107s-dependent local dna melting (open complex formation) occurs in the absence of host proteins (ihf/hu or dnaa) and it is positioned in the a + t-rich region adjacent to iterons. experiments described here examine the effects of atp, mg2+ and temperature on the opening reaction. we show that the opening ... | 2003 | 14530447 |
| 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 ... | 2003 | 14523116 |
| characterization of his-tagged, r6k-encoded pi protein variants. | the pi protein of plasmid r6k is a multifunctional replication (rep) protein, its different activities attributable, in part, to different oligomeric states: monomers and dimers. we have previously shown that his-tagged variants of the protein can exhibit alterations in dimer stability. herein, we examined the functional properties of selected his-tagged derivatives of pi (his-pi x wt and three hyperactive replication variants) to determine if the functionality of these proteins in replication, ... | 2003 | 12826061 |
| genetic analysis of pigment biosynthesis in xanthobacter autotrophicus py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. | a highly efficient method of transposon mutagenesis was developed for genetic analysis of xanthobacter autotrophicus py2. the method makes use of a transposon delivery vector that encodes a hyperactive tn 5 transposase that is 1,000-fold more active than the wild-type transposase. in this construct, the transposase is expressed from the promoter of the teta gene of plasmid rp4, which is functional in a wide variety of organisms. the transposon itself contains a kanamycin resistance gene as a sel ... | 2002 | 12189420 |
| simple and efficient vectors for retrofitting bacs and pacs with mammalian neor and egfp marker genes. | bacterial artificial chromosomes (bacs) and p1 artificial chromosomes (pacs) are widely used to investigate the functions of genes and genomes in mammalian cells in vitro and in vivo. we have developed a series of vectors which can simply and efficiently be retrofitted onto bacs or pacs. these vectors carry a neor gene for selection in cells in tissue culture, including es cells, and also an egfp gene driven by the strong cag promoter for quick detection of the dna in cells. all the plasmids are ... | 2001 | 11290429 |
| 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 ... | 2001 | 11237610 |
| dimers of pi protein bind the a+t-rich region of the r6k gamma origin near the leading-strand synthesis start sites: regulatory implications. | the replication of gamma origin, a minimal replicon derived from plasmid r6k, is controlled by the rep protein pi. at low intracellular concentrations, pi activates the gamma origin, while it inhibits replication at elevated concentrations. additionally, pi acts as a transcription factor (auto)repressing its own synthesis. these varied regulatory functions depend on pi binding to reiterated dna sequences bearing a tgagng motif. however, pi also binds to a "non-iteron" site (i.e., not tgagng) tha ... | 2000 | 10762246 |
| hexahistidine (his6)-tag dependent protein dimerization: a cautionary tale. | nickel nitrilotriacetic acid (ni2+-nta) immobilization of hexahistidine (his6) tagged proteins has become one of the most commonly used methods of affinity chromatography. perhaps the greatest utility of this protein purification method stems from the general belief that his-tagged proteins (comprised of his6) are little affected in their activities or efficiencies, while alterations in specificity are unexpected. although this is certainly true in many instances, we present a case in which the ... | 1999 | 10698267 |
| regulatory implications of protein assemblies at the gamma origin of plasmid r6k - a review. | recognition of the replication origin (ori) by initiator protein is a recurring theme for the regulated initiation of dna replication in diverse biological systems. the objective of the work reviewed here is to understand the initiation process focusing specifically on the gamma-ori of the antibiotic-resistance plasmid r6k. the control of gamma-ori copy number is determined by both plasmid-encoded and host-encoded factors. the two central regulatory elements of the plasmid are a multifunctional ... | 1998 | 9858731 |
| assemblies of replication initiator protein on symmetric and asymmetric dna sequences depend on multiple protein oligomerization surfaces. | the pi35.0 protein of plasmid r6k regulates transcription and replication by binding a dna sequence motif (tgagr) arranged either asymmetrically into 22 bp direct repeats (drs) in the gamma origin, or symmetrically into inverted half-repeats (irs) in the operator of its own gene, pir. the binding patterns of the two natural forms of the pi protein and their heterodimers revealed that the predominant species, pi35.0 (35.0 kda), can bind to a single copy of the dr as either a monomer or a dimer wh ... | 1998 | 9784371 |
| replication of r6k gamma origin in vitro: discrete start sites for dna synthesis dependent on pi and its copy-up variants. | the regulation of the plasmid r6k gamma origin (gamma ori) is accomplished through the ability of the pi protein to act as an initiator and inhibitor of replication. hyperactive variants of this protein, called copy-up pi, allow four to tenfold increases of gamma ori plasmid dna in vivo. the higher activity of copy-up pi variants could be explained by an increase in the initiator function, a decrease in the inhibitor activity, or a derepression of a more efficient mechanism of replication that c ... | 1998 | 9743626 |
| cre/loxp-mediated excision and amplification of large segments of the escherichia coli genome. | the isolation and amplification of large, predetermined segments of a genome from its host have been explored. the prototype of our approach was the excisional replication of some viruses such as the lambda-lysogen. similar machinery was used to excise and amplify large genomic segments of escherichia coli in its host. two loxp sequences for a site-specific recombinase cre, together with a conditional replication origin (pi-dependent gamma-ori), were inserted into the genome by homologous recomb ... | 1998 | 9526700 |
| two forms of replication initiator protein: positive and negative controls. | the pir gene of plasmid r6k encodes the protein, pi, a replication and transcription factor. two translational options for the pir gene give rise to two forms of pi protein: a 35.0-kda form (pi35.0) and a shortened 30.5-kda form (pi30.5). although both proteins bind to a series of 22-bp direct repeats essential for plasmid r6k replication, only pi35.0 can bind to a site in the (a.t)-rich segment of its gamma ori and activate the gamma ori in vivo and in vitro. however, unlike pi35.0, pi30.5can i ... | 1997 | 9391136 |
| replication of the r6k gamma origin in vitro: dependence on wt pi and hyperactive pis87n protein variant. | the pi protein of plasmid r6k is involved in control of replication. the aim of this study was to use an in vitro replication system dependent on an r6k-derived gamma origin of replication (gamma ori) to compare replication characteristics of wt pi and a hyperactive variant of pi protein (pis87n; filutowicz et al., 1994b. cooperative binding of initiator protein to replication origin conferred by single amino acid substitution. nucleic acids res. 22, 4211-4215). the characteristics of in vitro r ... | 1997 | 9249072 |
| genes involved in conjugative dna processing of plasmid r6k. | the conjugative transfer region of the incx plasmid r6k (tra(x)) was analysed by transposon mutagenesis and dna sequencing. tn5tac1 insertional mutations localized tra(x) to a 14.8 kb segment containing the alpha origin of transfer (orit alpha), genes involved in conjugative dna-processing (dtr(x)) and genes involved in pilus synthesis and assembly (mpf(x)). a second functional orit, oritbeta, was located at a distance of 5.3 kb from orit alpha and was outside tra(x). mpf(x) occupied a segment o ... | 1997 | 9218765 |
| a broad-host-range in vivo pop-out and amplification system for generating large quantities of 50- to 100-kb genomic fragments for direct dna sequencing. | a prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic dna fragments in adequate quantity. previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the escherichia coli genome. this system, which employed the yeast flp/frt elements for excision and the plasmid r6k-based replication machinery for dna amplification, permits one to bypass conventional cloning [pósfai et al. (1994) nucleic acids res. 22, 2392-2398]. to exte ... | 1996 | 8955645 |
| three novel plasmid r6k proteins act in concert to distort dna within the alpha and beta origins of dna replication. | three novel r6k genes which are responsible for expression of dna distortion polypeptides (ddp) were identified. the ddps act in vivo in concert to induce similar stepwise dna helix distortions within two long inverted repeats (alpha lir and beta lir), which are essential elements for the two distally located r6k alpha and beta dna replication origins. ddp1 and ddp2 are encoded by two tandem genes located at the 5' end of alpha lir, whereas a gene coding for ddp3 is located at the 3' end of beta ... | 1996 | 8830279 |
| preponderance of fis-binding sites in the r6k gamma origin and the curious effect of the penicillin resistance marker on replication of this origin in the absence of fis. | fis protein is shown here to bind to 10 sites in the gamma origin of plasmid r6k. the fis-binding sites overlap all the previously identified binding sites in the gamma origin for the plasmid-encoded pi initiator protein and three host-encoded proteins, dnaa, integration host factor, and rna polymerase. however, the requirement of fis for r6k replication depends on the use of copy-up pi-protein variants and, oddly, the antibiotic resistance marker on the plasmid. in fis-deficient cells, copy-up ... | 1996 | 8759862 |
| plasmid r6k contains two functional orits which can assemble simultaneously in relaxosomes in vivo. | plasmid r6k contains two functional origins or transfer (orit), in contrast to previously characterized conjugative plasmids. the orits are formed by 98 bp palindromic sequences invertedly orientated with respect to each other and located in the immediate vicinity of the alpha and beta origins of replication. the gene for r6k orit-nickase, taxc, was identified by transposon mutagenesis and sequenced, revealing that taxc belongs to the vird2 nickase family. the protein was overproduced and purifi ... | 1996 | 8757282 |
| characterization of bacterial cell membrane attachment sites of plasmid r6k. | in vitro binding studies revealed that plasmid r6k could attach to both inner and outer membrane fractions of its host cell, escherichia coli. derivatives of r6k carrying one or two of its three origins of replication could not bind stably to the same membrane fractions in the presence of salt. however, the derivative, prk35, carrying the intact three origins of replication could bind stably to membrane fractions from its host in the presence or absence of salt. these observations suggest that t ... | 1996 | 8660333 |
| initiator protein pi can bind independently to two domains of the gamma origin core of plasmid r6k: the direct repeats and the a+t-rich segment. | the pi protein of plasmid r6k functions in both replication and transcription. pi autoregulates its own synthesis and is required for replication of the risk gamma origin. pi performs these functions by binding to specific dna sites arranged as pairs of 6-10 bp inverted repeats (irs) or as a cluster of seven tandem 22 bp direct repeats (drs) which lack symmetry. the sites share the tgagrg nucleotide motif (where r is a or g). the drs and irs flank the central a+t-rich segment of the gamma origin ... | 1996 | 8657577 |
| replication of plasmid r6k gamma origin in vivo and in vitro: dependence on ihf binding to the ihf1 site. | the gamma origin of plasmid r6k requires the specific initiator protein pi for initiation of replication. however, increased pi concentrations inhibit replication. the host-encoded integration host factor (ihf) protein permits gamma origin replication at otherwise inhibitory pi levels. ihf is thought to mediate this positive effect by directly binding to the gamma origin. in this study we demonstrate that ihf binding to one ihf site in the gama origin, ihf1, but not to the other side, ihf2, is n ... | 1996 | 8648623 |
| the replication initiator protein pi of the plasmid r6k specifically interacts with the host-encoded helicase dnab. | the replication initiator protein pi of plasmid r6k is known to interact with the seven iterons of the gamma origin/enhancer and activate distant replication origins alpha and beta (ori alpha and ori beta) by pi-mediated dna looping. here we show that pi protein specifically interacts in vitro with the host-encoded helicase dnab. the site of interaction of pi on dnab has been localized to a 37-aa-long region located between amino acids 151 and 189 of dnab. the surface of pi that interacts with d ... | 1996 | 8643608 |
| new suicide vector for gene replacement in yersiniae and other gram-negative bacteria. | a suicide vector named puk4134 was constructed to enlarge the repertoire of vectors available for allelic exchange of mutated sequences in gram-negative bacteria. this plasmid combines the properties of two previously described plasmid vectors, pjm703.1 and prtp1. puk4134 is a suicide vector, carrying the origin of replication of the plasmid r6k and thus requiring the product of the pir gene for its stable maintenance. the rpsl gene encoding escherichia coli ribosomal protein s12 confers strepto ... | 1993 | 8469722 |
| autoregulation-deficient mutant of the plasmid r6k-encoded pi protein distinguishes between palindromic and nonpalindromic binding sites. | the autogenously regulated gene pir of escherichia coli plasmid r6k encodes the replication protein pi. this protein binds to two sites in the operator region of the pir gene: a 22-base pair nonpalindromic sequence and a pair of palindromic 9-base pair sequences. these pi-binding sites are similar, suggesting that pi uses a single dna-binding domain in recognizing them. we devised a plasmid system permitting isolation of mutants of the pi protein which are altered in autoregulation. a ser87 to a ... | 1993 | 8408040 |
| 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 ... | 1994 | 8045428 |
| the dimerization domain of r6k plasmid replication initiator protein pi revealed by analysis of a truncated protein. | replication of plasmid r6k is controlled by the homodimeric initiator protein pi, which binds to seven 22-bp direct repeats (iterons) in the gamma-origin. one of the genetically engineered pi variants (delta c164 pi) contains only the 164 n-terminal amino acids (aa) of the 305-aa pi molecule. this truncated pi polypeptide retains the ability to function as a specific inhibitor of r6k replication in vivo, though it neither drives replication, nor binds to iterons [greener et al., mol. gen. genet. ... | 1994 | 8045425 |
| in vivo excision and amplification of large segments of the escherichia coli genome. | in vivo excision and amplification of large segments of a genome offer an alternative to heterologous dna cloning. by obtaining predetermined fragments of the chromosome directly from the original organism, the problems of clone stability and clone identification are alleviated. this approach involves the insertion of two recognition sequences for a site-specific recombinase into the genome at predetermined sites, 50-100 kb apart. the integration of these sequences, together with a conditional r ... | 1994 | 8036169 |
| 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 ... | 1994 | 7961437 |
| cooperative binding of initiator protein to replication origin conferred by single amino acid substitution. | the replication initiator protein pi of plasmid r6k binds seven 22 bp direct repeats (dr) in the gamma origin. the pi protein also binds to an inverted repeat (ir) in the operator of its own gene, pir, which lies outside the gamma origin sequences. a genetic system was devised to select for pi protein mutants which discriminate between ir and dr (york et al., gene (amst.) 116, 7-12, 1992; york and filutowicz, j. biol. chem. 268, 21854-21861, 1993). from this selection the mutant pi s87n protein ... | 1994 | 7937147 |
| 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 ... | 1995 | 7731391 |
| altered (copy-up) forms of initiator protein pi suppress the point mutations inactivating the gamma origin of plasmid r6k. | the r6k gamma origin core contains the p2 promoter, whose -10 and -35 hexamers overlap two of the seven binding sites for the r6k-encoded pi protein. two mutations, p2-201 and p2-203, which lie within the -35 region of p2, are shown to confer a promoter-down phenotype. we demonstrate here that these mutations prevent replication of a gamma origin core plasmid. to determine whether or not the reduced promoter activity caused by these mutations is responsible for their effect on replication, we ge ... | 1995 | 7592461 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| [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 |
| [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 |
| 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 |
| plasmid r6k dna replication. iii. regulatory properties of the pi initiation protein. | 1982 | 6818353 | |
| plasmid r6k dna replication. i. complete nucleotide sequence of an autonomously replicating segment. | 1982 | 6759660 | |
| 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 |
| 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 |
| [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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| plasmid r6k dna replication. ii. direct nucleotide sequence repeats are required for an active gamma-origin. | 1982 | 6296394 | |
| 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 |
| 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 |
| [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 |
| [organization of the genetic system of plasmid r6k conjugativity]. | 1982 | 6277584 | |
| [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 |
| 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 |
| 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 |
| 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 |
| control of plasmid r6k copy numbers in isogenic rep+ and rep escherichia coli strains. | 1980 | 6246401 | |
| 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 |
| [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 |
| 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 |
| 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 |
| strand and site specificity of the relaxation event for the relaxation complex of the antibiotic resistance plasmid r6k. | 1974 | 4616719 | |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| role of the pi initiation protein and direct nucleotide sequence repeats in the regulation of plasmid r6k replication. | 1985 | 2990404 |