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genes for the hook-basal body proteins of the flagellar apparatus in escherichia coli.of the more than 30 genes required for flagellar function, 6 are located between pyrc and ptsg on the escherichia coli genetic man. this cluster of genes is called flagellar region i. four-point transductional crosses were used to establish the position and order of the region i flagellar genes with respect to the outside markers ptsg and pyrc. bacteriophage lambda-e. coli hybrids that contained most of the genes necessary for flagellar formation were constructed. the properties of specific hybr ...1978350831
proteins synthesized in minicells containing plasmid cole1 and its mutants.minicells containing the cole1 plasmid and derivatives of it that contain a tn3 insertion and deletions of the plasmid were shown to synthesize a variety of polypeptides: (i) a 56,000-dalton polypeptide was found that is colicin e1. (ii) a 15,000-dalton polypeptide is involved in plasmid dna relaxation. (iii) a 14,000-dalton polypeptide may be the colicin immunity protein. (iv) a correlation between the location of various tn3 insertions and the proteins synthesized from the plasmid dna was foun ...1978346572
construction and characterization of new cloning vehicles. ii. a multipurpose cloning system.in vitro recombination techniques were used to construct a new cloning vehicle, pbr322. this plasmid, derived from pbr313, is a relaxed replicating plasmid, does not produce and is sensitive to colicin e1, and carries resistance genes to the antibiotics ampicillin (ap) and tetracycline (tc). the antibiotic-resistant genes on pbr322 are not transposable. the vector pbr322 was constructed in order to have a plasmid with a single psti site, located in the ampicillin-resistant gene (apr), in additio ...1977344137
cole1 plasmid mutants affecting growth of an escherichia coli recb recc sbcb mutant.three mutant derivatives of the plasmid cole1 were found to reduce the formation of plasmidless cells in a recb recc sbcb cell population. an active plasmid role in the plasmid-host interaction is suggested.1978338596
the transposon tn1 as a probe for studying cole1 structure and function.insertion of the transposable genetic element tn1 into different sites of plasmid cole1 results in a number of mutnat phenotypes. whereas all plasmid examined were present in normal amount, all showed reduced immunity to killing by colicin e1. of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome. the other is conjugally deficient, produces colicin normally and maps close to two ...1977327263
aminoglycoside-modifying enzyme of an antibiotic-producing bacterium acts as a determinant of antibiotic resistance in escherichia coli.bacillus circulans nrrl b-3312, a nonpathogenic bacterium that produces the aminoglycoside antibiotic butirosin, is known to contain an aminoglycoside phosphotransferase that is similar to the neomycin phosphotransferases of clinically isolated antibiotic-resistant bacteria. purified dnas from b. circulans and the plasmid cole1-apr were digested with ecori endonuclease and the resulting fragments covalently joined with polynucleotide ligase. the recombined dna was used to transform e. coli and a ...1977322154
deletions affecting the transposition of an antibiotic resistance gene.the structural gene for plasmid-mediated ampicillin resistance resides upon a 3.2 x 10(6) dalton transposable sequence (tna) flanked by short inverted repeated sequences that accompany its insertion. tna was transposed to pmb8, a 1.8 x 10(6) dalton derivative of the colicingenic plasmid cole1. random deletions were introduced in the resultant 5 x 10(6) dalton recombinant plasmid by a combination of nuclease treatments in vitro. from this set of deletions a subset was isolated that contained dele ...1977322140
functional expression of cloned yeast dna in escherichia coli.a collection of hybrid circular dnas was constructed in vitro using the poly(da-dt) "connector" method: each hybrid circle contained one molecule of poly(dt)-tailed dna of plasmid cole1 (made linear by digestion with ecori endonuclease) annealed to a poly(da)-tailed fragment of yeast (saccharomyces cerevisiae) dna, produced originally by shearing total yeast dna to an average size of 8 x 10(6) daltons. this dna preparation was used to transform e. coli cells, selecting colicin-e1-resistant clone ...1977322128
mitomycin c-induced expression of trpa of salmonella typhimurium inserted into the plasmid cole1.ecori endonuclease digestion of the deoxyribonucleic acid of a phi80 transducing phage carrying the entire tryptophan (trp) operon of salmonella typhimurium (phi80 s.t.trpe-a) yielded a 4.3 x 10(6)-dalton fragment containing intact trpe, trpd, and trpc and a 3.35 x 10(6)-dalton fragment containing intact trpa. the trpa fragment inserted into ecori-cleaved plasmids cole1 and cr1 was expressed regardless of its orientation of insertion. mitomycin c, a compound that induces colicin e1 production in ...1977318646
construction of a novel plasmid-phage hybrid: use of the hybrid to demonstrate cole1 dna replication in vivo in the absence of a cole1-specified protein.a hybrid bacteriophage, p420, was constructed in vitro; it contains part of bacteriophage p4 and a 3.6-kilobase derivative of plasmid cole1. in escherichia coli, the plasmid-phage hybrid can exist as a stable plasmid or can be packaged into infective bacteriophage particles. replication of p420, directed by the cole1 replicon, was found to occur after p420 phage infection of e. coli cells that had been incubated in the presence of chloramphenicol. replication began without a lag period and resul ...1978276860
modification of dna by the benzo[a]pyrene metabolite diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.the structural modification of double-stranded circular dna of simian virus 40 and plasmid cole1 by in vitro binding of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied. stepwise hydrolysis with endonuclease s1 and dnase followed by dna base analysis by thin-layer chromatography provided evidence that binding to adenine caused the local denaturation of dna, whereas the more than 10-fold greater binding to guanine did not create such local denaturation. of the two synth ...1978272658
a complementation analysis of mobilization deficient mutants of the plasmid cole1.hydroxylamine was used to induce mutants of the cole1 derived plasmid pml2 that are inefficiently mobilized (mob-) during conjugation by an hfr donor. the ability of those mutants to be complemented by deletion mutants and tn3 insertion mutants of cole1 was examined. three complementation groups were identified and localized on the cole1 genetic map (mob1, mob2, and mob3). one hydroxylamine mutant was not complemented by any mobilization deficient mutant but was complemented by mobilizable cole1 ...1979225640
initiation of replication of plasmid cole1 dna by rna polymerase, ribonuclease h, and dna polymerase i. 1979225109
the fate of phage lambda dna in lambda-infected minicells.the fate of phage lambda dna in lambda-infected escherichia coli minicells harboring the plasmid cole1, and in plasmid-free minicells, were studied. binding of lambda dna to the minicell membrane, and formation of the supercoiled covalently-closed circular structure has been demonstrated. phage infection abolishes plasmid dna synthesis. only a very slight, non-replicative lambda dna synthesis occurs, soon after infection. this synthesis is associated with fragments of lambda dna arising during, ...1979161175
structure and function of plasmid cole1 and related plasmids.analysis of plasmid cole1, its naturally occurring relatives colk and clodf13, and a wide range of cole1 derivatives containing either insertions or deletions of genetic material has allowed localization on the cole1 genome of dna sequences responsible for colicin e1 synthesis, immunity to colicin killing, conjugal mobility and incompatibility. we have examined incompatibility between pairs of cole1 derivatives ranging in size from 2.6 to 13.8 md. though all the plasmids tested exerted cole1 inc ...1979394924
nucleotide sequence of the region required for maintenance of colicin e1 plasmid.plasmids carrying various portions of colicin e1 plasmid (cole1) dna have been isolated in an attempt to determine the regions of cole1 dna which are required for maintenance of the plasmid in bacteria. to construct the plasmids, the dna of a cole1 derivative that contains a gene which controls ampicillin resistance was cleaved by the restriction endonuclease haeii. the digestion products were joined by t4 dna ligase and then used to transform bacteria to ampicillin resistance. the plasmid deriv ...1979393952
hypersensitivity to hg2+ and hyperbinding activity associated with cloned fragments of the mercurial resistance operon of plasmid nr1.the region of plasmid nr1 concerned with resistance to hg2+ and organomercurials consists of sequences found on restriction endonuclease fragments ecori-h and ecori-i. when both fragments were cloned together into a derivative of plasmid cole1, the hybrid plasmid conferred properties indistinguishable from those of the parental plasmid, nr1: resistance to hg2+ and to the organomercurials merbromin and fluoresceinmercuric acetate and the inducible synthesis of the enzyme mercuric reductase. when ...1979387720
studies of colicin induction with an imm- col+ mutant of the plasmid colicin e1.a mutant of a derivative of the colicin e1 plasmid has been isolated that does not confer immunity to colicin e1 on its host (imm-) although it is still capable of producing colicin (col+). cells carrying the col+, imm- plasmid are capable of forming colonies and grow best in liquid culture in the presence of trypsin. the induction of colicin synthesis by ultraviolet light has been analysed using this mutant plasmid. the results suggest that a) the expression of the col+ gene may be delayed for ...1979384154
regulation of the l-arabinose operon in strains of escherichia coli containing cole1-ara hybrid plasmids.hybrid plasmids were constructed from fragments of f'ara episomes formed by the restriction endonuclease ecori and a linear form of the plasmid cole1 created by cleavage with ecori. hybrid plasmids were constructed containing the entire ara region or the ara region with various parts deleted. e. coli k12 host strains were constructed which contained different deletions of the ara region. the hybrid plasmids were transferred to those strains whose ara deletion complemented that of the plasmid. th ...1979384153
the structure of a transcriptional unit on colicin e1 plasmid.in an rna-synthesizing system in vitro, a low-molecular-weight rna consisting of about 110 residues (rna-i) was efficiently synthesized on dna of colicin e 1 plasmid (cole1) and its deletion derivatives. the promoter site for rna-i was analysed by testing the rna polymerase-binding ability and template activity of restriction fragments; it was mapped in the region between the replication initiation site and the colicin immunity gene of cole1. the direction of transcription was determined by hybr ...1979380993
cole1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility.deletion mutants of plasmid cole1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. it was observed that (i) a region of cole1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of cole1 dna between base pairs -185 and -572 is essential for the expression of ...1979378980
isolation and characterization of replication-deficient mutants of cole1 plasmids.replication-defective mutants of plasmid cole1 were isolated from a chimeric plasmid formed by ligating a temperature-sensitive replication derivative of psc101, phsg1, with a cole1-tn3-containing plasmid. the replication-defective cole1 mutants isolated were all spontaneous deletion mutants that had lost the cole1 replication origin and regions adjacent to it. the extent of a deletion was determined by analyzing restriction endonuclease-generated deoxyribonucleic acid fragments of the cole1 pla ...1979378979
a relationship between dna helix stability and recognition sites for rna polymerase.the rna polymerase binding sites on the dna of (i) the aroe-trka-spc segment of the escherichia coli genome, (ii) transposon tn3, (iii) plasmid cole1, and (iv) coliphage lambda were mapped by electron microscopy, with the use of the bac technique; these maps were compared with the maps of the early-melting regions for the same genomes. the results indicate that in all these cases the binding sites for the e. coli rna polymerase lie preferentially in the early melting regions of dna. these data i ...1979377494
total synthesis of a tyrosine suppressor trna gene. xviii. biological activity and transcription, in vitro, of the cloned gene.the chemically synthesized gene for escherichia coli tyrosine suppressor trna has been joined to both plasmid (cole1 ampr) and bacteriophage (charon 3a) vector chromosomes after the latter had been digested with the restriction endonuclease ecori. suppression of both bacterial (trpa, his, lacz) and bacteriophage lambda amber mutations (aam32, bam1) has been demonstrated after transformation of e. coli with the recombinant dna molecules carrying the synthetic suppressor trna gene. the cloned synt ...1979376520
altered dna-protein relaxation complex in a replication mutant of plasmid cole1.approximately 200,000 clones of escherichia coli carrying mutagen-treated colicinogenic plasmid e1 (cole1) were examined for irreversible loss of the plasmid at 43 degrees. thirty of these clones that appeared to be most defective in plasmid dna replication at the non-permissive temperature were selected for the study of: (a) the kinetics of plasmid and chromosomal dna replication during a temperature shift in either the presence or absence of chloramphenicol; (b) the temperature stability of th ...1978368585
mobilization of the escherichia coli plasmid cole1 (colicin e1) and cole1 vectors used in recombinant dna experiments.the escherichia coli co1e1 plasmid, which codes for production of colicin e1, is inherently nontransferable (nonconjugative) by bacterial mating. co1e1 can be transmitted at mating by a process called mobilization if co1e1 is coresident with a transfer plasmid. mobilization is governed in part by a co1e1 gene called mob. co1e1 is mob+. several co1e1 derivatives employed in recombinant dna experiments, notably pbr313 and pbr322, are mob-. these cloning vehicles are mobilized at markedly reduced f ...1978351085
hybrid plasmids complement a putp mutation in escherichia coli k12.clarke and carbon have prepared a colony bank of 2000 escherichia coli strains each containing a random segment of the escherichia coli chromosome inserted into the ecor1 restriction site of the plasmid cole1. we have screened the colony bank by conjugation and have identified three strains bearing hybrid plasmids that complement a defective putp gene. each of these strains shows increased l-proline uptake activity in comparison with the unmodified host or with the host bearing noncomplementing ...1979396012
bidirectional replication of the mini-cole1 plasmid pvh51.replicating molecules of the min-cole1 plasmid pvh51 have been examined by electron microscopy after cleavage with the restriction endonuclease ecori. replication apparently starts at a unique site indistinguishable from the origin of replication used by the parental plasmid cole1. in contrast to cole1, the structure of the majority of the replication intermediates was consistent with a bidirectional mode of replication. a minor portion of the molecules appeared to replicate unidirectionally in ...1979438132
characterization of a mini-colc1 plasmid.an in vitro constructed plasmid, pvh15, consisting of the entire genome of the plasmid cole1, the tryptophan operon of escherichia coli, and regions of the bacteriophage phi80pt190, spontaneously gave rise in e. coli to a mini-cole1 plasmid consisting of approximately one-half of the cole1 genome and a small segment of phi80pt190 dna. this mini-cole1 plasmid, designated pvh51, has a molecular weight of approximately 2.1 x 10(6) and possesses a single ecori restriction site. heteroduplex analyses ...1976770430
on the mechanism of genetic recombination: electron microscopic observation of recombination intermediates.this paper deals with the nature of recombination intermediates. using the electron microscope to study the dna of the plasmid colicin e1, we have observed more than 800 molecules that appear to represent intermediates in the process of recombination. specifically, after isolating colicin dna and linearizing it with the restriction enzyme ecori, we find crossed molecules with twice the normal colicin dna content. these forms consist of two genome-length elements held together at a region of dna ...1976787981
the generation of a cole1-apr cloning vehicle which allows detection of inserted dna.a 3.2 mdal sequence of dna, tna, which contains the ampicillin (ap) resistance determinant has been translocated from an r plasmid to the plasmid cole1. a total of 12 isolates were studied. there are at least 8 sites in cole1 at which tna has inserted. insertion at five of these has resulted in a col-phenotype. one cole1-apr plasmid, rsf2124, was examined further and its replication properties are found to be similar to that of the parent plasmid. rsf2124 appears to be a useful plasmid vehicle f ...19751045010
relaxation complexes of plasmid dna and protein. ii. characterization of the proteins associated with the unrelaxed and relaxed complexes of plasmid cole1.the proteins of the dna-protein relaxation complex of plasmid cole1 were labeled with [3h]leucine by growth of cole1 containing escherichia coli cells in the presence of this radioactive labeled amino acid. three major [3h]leucine-labeled proteins are found associated with the supercoiled dna in the cole1 relaxation complex. the molecular weights of these proteins, determined by sodium dodecyl sulfate-acrylamide gel electrophoresis, are 60,000, 16,000, and 11,000, respectively. induction of rela ...19751102544
relaxation complexes of poasmid dna and protein. iii. association of protein with the 5' terminus of the broken dna strand in the relaxed complex of plasmid cole1.the location of the protein in the open circular dna form of the cole1 dna-protein relaxation complex, induced by treatment with sodium dodecyl sulfate, has been studied using several enzymes of dna metabolism. escherichia coli exonucleases i and iii are able to degrade extensively the nicked strand of the relaxed complex from the 3' end. dna polymerase i can initiate synthesis using the relaxed complex as template-primer and specifically extends the 3' end of the nicked strand. the 5' end of th ...19751102545
structure of the cole1 dna molecule before segregation to daughter molecules.the segregation of daughter dna molecules at the end stage of replication of plasmid cole1 was examined. when circular cole1 dna replicates in a cell extract at a high kcl concentration (140 mm), a unique class of molecules accumulates. when the molecule is cleaved by a restriction enzyme that cuts the cole1 dna at a single site, an x-shaped molecule in which two linear components are held together around the origin of dna replication is made. for a large fraction of these molecules, the 5' end ...19921438203
the tram protein of the conjugative plasmid f binds to the origin of transfer of the f and cole1 plasmids.the gene encoding the tram protein of the conjugative plasmid f was cloned, overexpressed and the gene product was purified. the tram protein was found in the cytoplasm of cells carrying the f plasmid with a smaller amount in the inner membrane. dnase i footprinting experiments showed that the purified protein protects three regions in the f orit locus with different affinity for the upper and lower strands of dna. a 15-nucleotide motif was identified within the protected regions that represente ...19921479887
quantitation of cole1-encoded replication elements.replication of the escherichia coli plasmid cole1 initiates from an rna primer. this primer is formed by a cole1 rna ii molecule that remains hybridized to its dna template in the origin region after transcription. continued hybridization is inhibited by prior binding to rna ii of another cole1 transcript, rna i; and this interaction is regulated by the plasmid-encoded rom protein. to understand the quantitative aspects of regulation of cole1 synthesis, we have measured the levels of rna i, rna ...19911703297
complexes formed by complementary rna stem-loops. their formations, structures and interaction with cole1 rom protein.regulation of replication of plasmid cole1 involves interaction of two plasmid-specified rna transcripts. one of these rnas (rna ii) serves as a primer for dna synthesis, and the other (rna i) is complementary to part of rna ii. the complementary regions of rna i and rna ii form several stem-loop structures. binding of these rnas that regulates dna replication begins by interaction at the loop regions. plasmid-coded rom protein stabilizes the product of the interaction. in this paper, the mechan ...19911715406
site-specific recombination promoted by a short dna segment of plasmid r1 and by a homologous segment in the terminus region of the escherichia coli chromosome.a short dna segment located in the kanamycin resistance region of plasmid r1 promotes site-specific recombination and plasmid maintenance. this segment has been reduced to 100 bp and subsequently to 44 bp without losing these properties. it can recombine with a similar segment located in the terminus region of the escherichia coli chromosome. it is proposed that this recombination is responsible for the plasmid maintenance properties of the r1 segment. the chromosomal site has been isolated; it ...19911931823
escherichia coli xerc recombinase is required for chromosomal segregation at cell division.xerc is a site-specific recombinase of the bacteriophage lambda integrase family that is encoded by xerc at 3700 kbp on the genetic map of escherichia coli. the protein was originally identified through its role in converting multimers of plasmid cole1 to monomers; only monomers are stably inherited. here we demonstrate that xerc also has a role in the segregation of replicated chromosomes at cell division. xerc mutants form filaments with aberrant nucleotides that appear unable to partition cor ...19911931824
differential sequence dynamics of homopolymeric and alternating at tracts in a small plasmid dna.the location of oso4 bispyridine hyper- and hyporeactivity in a small deletion derivative of plasmid cole1 (ptc12, 1727 bp) has been determined for approximately 70% of the molecule. thymine bases in homopolymeric (da)n.(dt)n tracts (n greater than or equal to 4) were always found to be resistant toward oso4 modification. dna supercoiling did not destabilize these tracts. the extent of oso4 bispyridine reactivity of homopolymeric (da)n.(dt)n tracts, where n = 3, was found to be dependent on the ...19912001354
effect of altering gatc sequences in the plasmid cole1 primer promoter.plasmid cole1 has three recognition sites for the escherichia coli dna adenine methylase in the immediate upstream region of the primer promoter. two of these sites are conserved among all plasmid relatives of cole1 and constitute parts of an inverted repeat that can conceivably form a cruciform structure. recent experiments have indicated that hemimethylated cole1-type plasmids are inefficiently replicated after transformation (d. w. russell and n. zinder, cell 50:1071-1079, 1987). by mutating ...19902156802
site-specific transposition of insertion sequence is630.is630 is a 1.15-kilobase sequence in shigella sonnei that, unlike many mobile elements, seems not to mediate cointegration between different replicons. to assess its transposition, we constructed composite elements containing inverted copies of is630 flanking a drug resistance gene. we found that these composite elements transposed to plasmid cole1 in escherichia coli. dna sequencing showed that transposition was, in all cases, to the dinucleotide sequence 5'-ta-3'. there were two preferred inse ...19902163390
recombination at cole1 cer requires the escherichia coli xerc gene product, a member of the lambda integrase family of site-specific recombinases.site-specific recombination at the plasmid cole1 cer site requires the escherichia coli chromosomal gene xerc. the xerc gene has been localized to the 85-min region of the e. coli chromosome, between cya and uvrd. the nucleotide sequences of the xerc gene and flanking regions have been determined. the xerc gene encodes a protein with a calculated molecular mass of 33.8 kda. this protein has substantial sequence similarity to the lambda integrase family of site-specific recombinases and is probab ...19902254268
isolation and characterization of mutants affecting functional domains of cole1 rnai.the control of dna replication initiation in the plasmid cole1 is mediated by rnai, a 108 nucleotide plasmid-encoded rna that is entirely complementary to the 5'-terminal region of the replication primer rna. rnai acts in trans to inhibit primer maturation. previously, we constructed a plasmid in which the cole1 rnai was separated from the primer and placed under transcriptional control of the serratia marcesens tryptophan promoter. this plasmid provides rnai in trans in vivo and mediates cole1- ...19852416940
factor-independent termination of transcription in a stretch of deoxyadenosine residues in the template dna.the primer transcript of plasmid cole1 extends beyond the replication origin in either of two different modes. it does or does not form a hybrid with the template dna. when a stretch of 20 deoxyadenosine residues is inserted into the template strand downstream of the origin, more than 90% of hybridized transcripts and about 10% of unhybridized transcripts terminate at the insert. when the number of inserted residues is reduced to ten, the corresponding values are decreased considerably, while th ...19872445490
multiple mechanisms for initiation of cole1 dna replication: dna synthesis in the presence and absence of ribonuclease h.a transcript (rna ii) of plasmid cole1 that hybridizes with the template dna is cleaved by rnaase h and used as a primer by dna polymerase i. however, the plasmid can replicate in bacteria lacking both enzymes, apparently using a different mechanism of initiation of replication. here we report in vivo and in vitro studies on initiation of dna replication in the presence or absence of either or both enzymes. hybridization of rna ii with the template dna is always required for initiation. hybridiz ...19872446774
transcriptional activation of cole1 dna synthesis by displacement of the nontranscribed strand.plasmid cole1 can replicate using rnaase h and dna polymerase i. however, it can also replicate in the absence of these enzymes. in this case, formation of a persistent hybrid between a transcript (rna ii) and the dna indirectly activates subsequent dna synthesis, instead of providing a primer as it does in the presence of these enzymes. to activate dna synthesis, a certain length is required for the hybridized region and the region of minimum length cannot include a palindrome. these results sh ...19872446775
suppression of cole1 rna-rna mismatch mutations in vivo by the cole1 rop protein.in the bacterial plasmid cole1 the control of initiation of dna replication is mediated by the interaction of two complementary rna molecules, the replication primer and rna1. the rate of interaction between rna1 and the primer rna in vitro can be increased by the product of the cole1 rop gene, a 63-amino-acid polypeptide. we have investigated the role of the rop protein in suppressing the incompatibility defects of 13 rna1-mutant alleles. these rna1 mutants are defective due to single nucleotid ...19872447599
conditional high copy number cole1 mutants: resistance to rna1 inhibition in vivo and in vitro.we describe three independently isolated copy number mutants of a plasmid cole1 derivative which undergo temperature- and growth-phase-dependent dna amplification in escherichia coli. these mutants have single base-pair alterations in a highly localized region of the plasmid genome encoding the replication primer rna. the mutations map immediately upstream of the rna1 transcript, altering the sequence between conserved elements of the rna1 promoter. these mutants have 2- to 4-fold increased copy ...19882460340
rom transcript of plasmid cole1.the cole1 rom protein contributes to copy number control by affecting the rate of formation of a complex between rna ii, the precursor of the primer for dna replication, and rna i, a small rna complementary to the 5' end of rna ii. interaction of rna i with rna ii can affect plasmid copy number by preventing primer formation. although the rna i and rna ii transcripts have been well characterized, the rom mrna has not previously been detected. we have now identified the rom mrna, and determined i ...19892472606
kinetics of complementary rna-rna interaction involved in plasmid cole1 copy number control.binding of a small antisense rna (rna i) to the primer transcript (rna ii) of plasmid cole1 inhibits formation of primer for dna polymerase i-mediated plasmid replication. it is thought that rna i and rna ii transiently interact via their single-stranded loop regions to form an unstable complex that subsequently converts into a more stable complex by hybridization. rom (or rop) protein enhances the inhibitory effect of rna i on replication by enhancing the binding of the two rnas. in this paper, ...19892475639
characterization of the cole1 primer-rna1 complex: analysis of a domain of cole1 rna1 necessary for its interaction with primer rna.rna1 is a small, plasmid-encoded transcript involved in the replication control of the plasmid cole1. rna1 blocks replication by preventing processing of the primer rna necessary for the initiation of replication. it has been proposed that inhibition by rna1 involves a direct interaction between rna1 and primer rna. here we describe an in vitro system that allows the detection and characterization of the rna1-primer complex. using this system, we have demonstrated that the association between co ...19852581244
[structuro-functional organization of segments of co1a plasmid dna, determining its stable inheritance].two par regions were localized within the structure of a small colicinogenic plasmid cola. one of them functions at the expense of plasmid multimere resolution. analysis of the nucleotide sequence of the region revealed the existence of essential homology with the par locus of plasmid cole1. as compared to e. coli c600, the function of multimere forms' resolution of plasmid dna in e. coli c is reduced or absent due to par regions of the cole1 type. par regions of various degrees of homology with ...19892586498
preferential transposition of an is630-associated composite transposon to ta in the 5'-ctag-3' sequence.a composite transposon, tn4731, associated with is630 has been shown to transpose preferentially to 5'-ta-3' sequences that are located at two sites in a rho-dependent transcription terminator in plasmid cole1 in escherichia coli (t. tenzen, s. matsutani, and e. ohtsubo, j. bacteriol. 172:3830-3836, 1990). here we demonstrated that tn4731 preferentially transposes to ta sequences at four sites in plasmid puc118 and its derivatives: the ta sequence (hot spot i) in the intergenic region of phage m ...19911655702
dif, a reca-independent recombination site in the terminus region of the chromosome of escherichia coli.dif (deletion induced filamentation) is a newly identified locus that lies within the terminus region of the escherichia coli chromosome. the dif phenotype was characterized by a subpopulation of filamentous cells with abnormal nucleoids and induction of the sos repair system. interactions between dif-carrying plasmids as well as between such plasmids and the bacterial chromosome demonstrated that dif is a cis-acting, reca-independent recombination site. filamentation continued in dif mutants in ...19911657123
mutations affecting primer rna interaction with the replication repressor rna i in plasmid coie1: potential rna folding pathway mutants.the control of plasmid cole1 copy number is mediated by the kinetics of interaction of two complementary plasmid-encoded rnas. one rna is the primer precursor and the other is a small counter-transcript called rna i. the interaction of these highly structured rnas results in inhibition of formation of mature primer rna necessary for replication initiation. we have studied several plasmid copy number mutants which have single base changes in the primer which render the primer resistant to inhibit ...19901688532
control of cole1 plasmid replication. intermediates in the binding of rna i and rna ii.replication of plasmid cole1 is regulated by a plasmid-specified small rna (rna i). rna i binds to the precursor (rna ii) of the primer for dna synthesis and inhibits primer formation. the process of binding of rna i to rna ii that results in formation of a stably bound complex consists of a series of reactions forming complexes differing in the stability. formation of a very unstable early intermediate that was previously inferred from the inhibition of stable binding caused by a second rna i s ...19901691790
size and base composition of rna segments in complementary strands of supercoiled plasmid cole1 dna. 19826177012
nucleotide sequence and gene organization of cole1 dna.the primary structure of the plasmid cole1 dna has been determined. the plasmid dna consists of 6646 base pairs (molecular mass of 4.43 mda) and is 48.46% in gc content. the phi 80 trp insert of the composite plasmid of cole1, pvh51, has also been determined. the determination of the nucleotide sequence of cole1 dna provides the basis for examining the relationships between the dna sequence and the gene organization of the plasmid. the focus of this paper is to use this sequence data coupled wit ...19852991225
the nucleotide sequences of the replication origins of plasmids cola and cold.the nucleotide sequences of the replication origin regions of plasmids cola-ca31 and cold-ca23 were obtained. analysis of the nucleotide sequences showed a high degree of homology of these regions with the plasmid cole1 region responsible for its autonomous replication. in the cola and cold regions involved in the regulation of replication, sites have been revealed identical to those participating in the transcription initiation of the cole1 plasmid rna i and primer rna. the presumed rna polymer ...19853006099
restriction endonuclease ecori alters the enantiomeric preference of chiral metallointercalators for dna: an illustration of a protein-induced dna conformational change.a conformational change in the dna plasmid cole1 appears to occur upon specific binding of the restriction endonuclease ecori. enzyme association alters the chiral discrimination found in binding metallointercalators to dna sites. the complexes tris(1,10-phenanthroline)ruthenium(ii), ru(phen)3(2+), tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(ii), ru(dip)3(2+), and tris(4,7-diphenyl-1,10-phenanthroline)cobalt(iii), co(dip)3(3+), in general, bind stereoselectively to dna helices, with enantiom ...19863011080
polar mobilization of the escherichia coli chromosome by the cole1 transfer origin.mobilization of the plasmid cole1 from cells containing a conjugative plasmid (such as f) requires the synthesis of cole1 mob proteins, and the presence, in cis, of bom (basis of mobility), a region of cole1 containing the origin of transfer (orit). the process of cole1 transfer is thought to resemble that of the conjugative plasmid f, although the plasmids share little sequence homology. in f, conjugation is preceded by a strand-specific nicking event at orit. the nicked strand is then conducte ...19863018432
multicopy plasmid stability in escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination.the heritable stability of the multicopy plasmid cole1 and its natural relatives, requires the presence in the plasmid of a site (cer in cole1) that acts as a substrate for site-specific recombination, thereby maintaining plasmids in the monomeric state. multimerization, promoted by homologous recombination, leads to plasmid loss. here we show that the escherichia coli chromosome encodes at least two unlinked functions that act on cer and its analogous sites, to promote stabilizing site-specific ...19883067082
the arginine repressor is essential for plasmid-stabilizing site-specific recombination at the cole1 cer locus.the heritable stability in escherichia coli of the multicopy plasmid cole1 and its natural relatives requires that the plasmids be maintained in the monomeric state. plasmid multimers, that arise through reca-dependent homologous recombination, are normally converted to monomers by a site-specific recombination system that acts at a specific plasmid site (cer in cole1). no plasmid functions that act at this site have been identified. in contrast, two unlinked e.coli genes that encode functions r ...19883149585
resolution of cole1 dimers requires a dna sequence implicated in the three-dimensional organization of the cer site.plasmid cole1 specifies a recombination site (cer) which participates in the conversion of plasmid dimers to monomers. the uncontrolled accumulation of dimers (and higher oligomeric forms) would otherwise lead to plasmid instability. exonuclease iii-generated deletions have been used to define the left-hand boundary of the cer site. deletions which have lost up to 60 bp adjacent to the boundary no longer mediate the conversion of plasmid dimers to monomers, but still recombine with a wild-type s ...19883294000
searching for potential z-dna in genomic escherichia coli dna.the clarke-carbon library with escherichia coli dna cloned into plasmid cole1 was partially screened for z-dna with the monoclonal antibody z-d11 using the retardation of the covalently closed circular dna-protein complex by nitrocellulose filters. about 85% of the plasmids tested at "natural" supercoil density bound to the filter. together with binding studies of the iodinated antibody, one z-dna segment per about 18,000 base-pairs of e. coli dna is observed. one clone containing the region aro ...19873295260
regulation of gene expression in plasmid cole1: delayed expression of the kil gene.cea, imm, and kil are a cluster of three functionally related genes of the plasmid cole1. the cea and kil genes are in the same inducible operon, with transcription being initiated from a promoter adjacent to the cea gene. the imm gene is located between the cea and kil genes, but it is transcribed in the opposite direction. complementary interaction between the imm mrna and the anti-imm sequences in the middle of the cea-kil transcript causes a pronounced delay in expression of the kil gene whe ...19883142845
mitomycin c-induced bidirectional transcription from the colicin e1 promoter region in plasmid cole1.treatment of a colicinogenic culture with mitomycin c induces convergent transcription from two adjacent promoters at the beginning of the colicin e1 gene. s1-mapping and primer extension assays indicate that the mitomycin c-inducible transcripts correspond to colicin e1 mrna (cea mrna) and to a transcript, designated rna-c, that may code for an entry exclusion function. nucleotide sequences that strongly resemble a consensus sequence for lexa protein binding sites span the transcription start p ...19863092859
nucleotide sequence of the partition function of escherichia coli plasmid cole1.the dna nucleotide sequence of a 382-bp hpa ii fragment containing cer (cole1 resolution) function responsible for cole1 plasmid stability in dividing escherichia coli was determined. the partition (par) region of psc101 and the cer region have similar biological functions, as they both maintain plasmid stability through plasmid monomerization. both regions contain 40- to 70-bp hairpin-loop structures that resemble bidirectional transcription terminators and share sequence homology with each oth ...19853908032
minicircular cole1-related dna in strains of klebsiella aerogenes selected for fast growth on xylitol.we have previously described a large family of mutants of klebsiella aerogene which were selected by continuous on xylitol and which superproduce ribitol dehydrogenase. one of these strains was found to harbour a high copy number 2.1 x 10(6) dalton plasmid. this plasmid is a deletion derivative of a low copy number 3.5 x 10(6) dalton plasmid present in the ancestral strain of k. aerogenes. however, since these plasmids do not contain the genes required for pentitol catabolism and some enzyme-sup ...19806999122
structural and functional organization of the colicin e1 operon.we analyzed the structural and functional relationships among independently cloned segments of the plasmid cole1 region that regulates and codes for colicin e1 (cea), immunity (imm), and the mitomycin c-induced lethality function (lys). on the basis of physiological properties, restriction endonuclease mapping, and dna sequence analysis, the following recombinant plasmids were determined to represent three major functional classes: pnp12 (cea+, imm+, lys+), pnp4 (cea+, imm+, lys-), and pnp6 (cea ...19853936034
effect of inhibitors of ribonucleic acid and protein synthesis on the cyclic adenosine monophosphate stimulation of plasmid cole1 replication.addition of cyclic adenosine 3'-5'-monophosphate (c-amp) to growing escherichia coli cells, colicinogenic for the plasmid cole1, results in a fourfold stimulation in the rate of synthesis of the plasmid deoxyribonucleic acid (dna). the stimulation is transient (30 min) and is succeeded by a brief period (30 min) of cessation of plasmid dna replication. the stimulation of cole1 dna replication also occurs in chloramphenicol-treated cells. rifampin inhibits cole1 dna replication in the presence or ...19744368626
functional relationship between parts of the replication region of plasmid cole1.the inhibition of plasmid cole1 replication caused by a deletion of the cole1 plasmid replication origin has been previously reported (t. hashimoto-gotoh and j. inselburg, j. bacteriol. 139:597-619). evidence is presented showing that restoration of the deleted nucleotide sequence in the precise relationship it normally has to the rest of the replication region is essential for restoration of cole1 replication capability to the deletion mutant.19807009549
unidirectional replication of plasmid cole1 dna. 19744610399
plasmid colel as a molecular vehicle for cloning and amplification of dna.dna fragments obtained from ecori endonuclease digestion of bacteriophage varphi80pt190 (trp(+)) and the plasmid cole1 were covalently joined with polynucleotide ligase. transformation of escherichia coli trp(-) strains to tryptophan independence with the recombined dna selected for reconstituted cole1 plasmids containing the tryptophan operon and the varphi80 immunity region. similarly, an ecori endonuclease generated fragment of plasmid psc105 dna containing the genetic determinant of kanamyci ...19744610576
xerb, an escherichia coli gene required for plasmid cole1 site-specific recombination, is identical to pepa, encoding aminopeptidase a, a protein with substantial similarity to bovine lens leucine aminopeptidase.the heritable stability of cole1 is dependent on a site-specific recombination system which acts to resolve plasmid multimers into monomers. this plasmid stabilizing recombination system requires the presence in cis of the cole1 cer region, plus at least two trans-acting factors encoded by the xera and xerb genes of escherichia coli. the xerb gene has been cloned and sequenced and found to encode a polypeptide with a calculated mol. wt of 55.3 kd. the predicted amino acid sequence of this protei ...19892670557
[analysis of functioning of cole1 plasmid par-regions during various conditions of culturing].the functioning of the par-regions of the small multi-copied colicinogenic plasmids cole1 and cola has been analysed under the different conditions of plasmid-containing cells cultivation. the efficient cointegrate resolution of the multimeric forms of the plasmid cole1 has been demonstrated in escherichia coli k12 cells under the different conditions of cell cultivation. it took place at 30 degrees-42 degrees c on the minimal as well as rich medium, at the different stages of cell cycle in the ...19892697803
the effect of dnaa protein and n' sites on the replication of plasmid cole1.the role of the dna a protein in the replication of plasmid cole1 and its derivatives was examined. wild-type and mutant cole1 plasmids were compared as to their ability to replicate in an in vitro replication system supplemented with ammonium sulfate fractionated extracts from a dnaa-overproducing strain. synthesis on plasmid templates containing the wild-type origin of replication was stimulated 1.3-fold by addition of the dnaa-overproducing extract. a larger effect was observed after deletion ...19882844794
formation of an rna primer for initiation of replication of cole1 dna by ribonuclease h.a plasmid that consists of an 812-base-pair segment containing the replication origin of plasmid cole1 and of a 1240-base-pair segment containing a beta-lactamase gene has been constructed. the plasmid dna has three principal sites where transcription is initiated in vitro. one is located in the cole1 segment 555 nucleotides upstream from the origin. most transcription from this site extends past the origin; some of the transcripts form hybrids spontaneously with the template at their 3' portion ...19806156450
differential scanning calorimetric and theoretical studies of cole1 dna.the differential scanning calorimetry (dsc) of plasmid cole1 dna was carried out. the dsc curve under the solvent condition of 1.0 x ssc buffer gave eleven clear peaks over the temperature range of 83 to 98 degrees c. the dsc curves obtained here were essentially in good agreement with the optical melting curves of cole1 dna reported previously. the theoretical melting profiles of cole1 dna calculated from its entire nucleotide sequence showed a good agreement with the dsc curves. the theoretica ...19863550711
synthesis and repair of rna-containing supercoiled plasmid cole1 dna in escherichia coli. 19826177011
rna1 is sufficient to mediate plasmid cole1 incompatibility in vivo.the multicopy plasmid cole1 specifies a small rna designated rna1 that has been implicated in copy number control and incompatibility. we have inserted a 148 base-pair cole1 dna fragment containing a promoter-less rna1 gene into a plasmid vector downstream from the tryptophan promoter of serratia marcesens . the cole1 rna1 produced by this plasmid is not functional in vivo due to the presence of 49 nucleotides appended to the 5'-terminus of the wild-type rna1 sequence. deletions of these sequenc ...19846202877
aminoglycoside-modifying enzymes of staphylococcus aureus; expression in escherichia coli.staphylococcus aureus plasmids psh2, rn1956 and pwa1 code for an aminoglycoside phosphotransferase; plasmid pwa1 also encodes an aminoglycoside-aminocyclitol adenylyltransferase. s. aureus plasmid pwa2 confers resistance to erythromycin and sulfonamide. using plasmid cole1-apr (rsf2124) as a vehicle, we have transferred the genes determining aminoglycoside phosphotransferase and aminoglycoside-aminocyclitol adenylyltransferase activities from s. aureus to escherichia coli. the new plasmids obtai ...19806248429
insertion element is102 resides in plasmid psc101.in vivo recombination was found to occur between plasmid phs1, a temperature-sensitive replication mutant of psc101 carrying tetracycline resistance, and plasmid cole1 after selection for tetracycline resistance at the restrictive temperature, 42 degrees c. extensive analysis of the physical structures of three of these recombinant plasmids, using restriction endonucleases and the electron microscope heteroduplex method, revealed that the plasmid phs1 was integrated into different sites on cole1 ...19806252187
equilibrium melting of plasmid cole1 dna: electron-microscopic visualization.the fine structure of the melting curve for the linear cole1 dna has been obtained. to find the cole1 dna regions corresponding to peaks in the melting curve's fine structure, we fixed the melted dna regions with glyoxal /12/. electron-microscopic denaturation maps were obtained for nine temperature points within the melting range. thereby the whole process of cole1 dna melting was reconstructed in detail. spectrophotometric and electron microscopic data were used for mapping the distribution of ...19806253910
isolation of recombinant plasmids and phage carrying the lexa gene of escherichia coli k-12.the lexa gene of escherichia coli k-12 was cloned from the plasmid plc44-14 into pbr322. plasmids carrying lexa+ were selected by their ability to complement a recessive tsl mutation, which is believed to be a mutation in lexa. the smallest lexa+ recombinant plasmid, pjl21, contained an ecori-psti fragment 2.9 kilobases (kb) in length; two larger plasmids also contained this fragment, and genetic material to one or both sides of the ecori-psti fragment. plasmids homologous to pjl21, but carrying ...19806254842
peptidase activity of escherichia coli aminopeptidase a is not required for its role in xer site-specific recombination.xer site-specific recombination is required for the stable inheritance of multicopy plasmids and the normal segregation of the bacterial chromosome in escherichia coli. two related recombinases and two accessory proteins are essential for xer-mediated recombination at cer, a recombination site in the plasmid cole1. the accessory proteins, argr and pepa, function in ensuring that the xer recombination reaction acts exclusively intramolecularly, converting plasmid dimers into monomers and not vice ...19948057849
isolation and characterization of the yeast 3-phosphoglycerokinase gene (pgk) by an immunological screening technique.an immunological screening technique has been used for the detection of a specific antigen-producing clone in a bank of bacterial colonies containing hybrid plasmids. this technique involves covalent attachment of antiserum to cyanogen bromide-activated paper discs, contact of this paper with lysed colonies on agar plates, and finally detection of the bound antigen with 125i-labeled antibody. using this method, we have identified an escherichia coli colony, containing a yeast dna insert in plasm ...19806254992
selection and characterization of cole1 plasmid mutants that exhibit altered stability and replication.this report describes a method for isolating mutants of plasmid cole1 that exhibit unstable maintenance and altered replication characteristics. it also describes the initial characterization of four mutants isolated by that method. a chimeric plasmid, phsg124, containing a cole1 derivative and a temperature-sensitive replication derivative of psc101 was mutagenized in vitro, using hydroxylamine. by adjusting the growth conditions of transformants containing the mutagenized chimeric deoxyribonuc ...19816268615
tn292l, a transposon encoding fosfomycin resistance.the determinant of resistance to fosfomycin of the serratia marcescens plasmid pou900 was transposed into the plasmid cole1 and into the plasmid rp4 in the absence of the reca function of the host. in each case, the acquisition of fosfomycin resistance was correlated with the presence of a discrete piece of dna, uniform in size and in restriction pattern, this new, 7.5-megadalton transposable element encoding resistance to fosfomycin has been called tn2921. a preliminary map of the transposon is ...19826282810
regulatory regions of cole1 that are involved in determination of plasmid copy number.the copy number of plasmid cole1 has been known to increase when the hae ii-c segment downstream from the replication origin is deleted. the presence of the 306-base-pair (bp) hpa ii region in the segment is sufficient for reduction of the copy number. plasmids harboring the region express a trans-acting function that is responsible for the copy number reduction and synthesize a unique protein. a protein specified by the region is purified to near homogeneity and identified as the 63-amino-acid ...19836304700
plasmid-encoded regulation of colicin e1 gene expression.a plasmid-encoded factor that regulates the expression of the colicin e1 gene was found in molecular cloning experiments. the 2,294-base-pair avaii fragment of the colicin e1 plasmid (cole1) carrying the colicin e1 structural gene and the promoter-operator region had the same information with respect to the repressibility and inducibility of colicin e1 synthesis as the original cole1 plasmid. an operon fusion was constructed between the 204-bp fragment containing the colicin e1 promoter-operator ...19836313603
accumulation of cole1 early replicative intermediates catalyzed by extracts of escherichia coli dnag mutant strains.to investigate the events occurring at the replication forks during dna synthesis, we studied the replication of plasmid cole1 dna in vivo and in vitro, using strains of escherichia coli carrying either the dnag3(ts) or dnag308(ts) mutation. extracts of both mutant strains supported in vitro dna synthesis, but the amount of [3h]tmp incorporated into dna was always less for mutant extracts than for extracts of revertant strains, which were able to grow at 42 degrees c. sucrose gradient analysis, ...19836343345
the copy number of bacillus plasmid prbh1 is negatively controlled by repb protein.the replication control mechanism of bacillus plasmid prbh1 was analysed; prbh1 contains four promoters, p1 to p4, and a large inverted repeat (63 base pairs) upstream of the protein (repb) coding sequence. the stem and loop structure is surrounded by two promoters, p1 and p3, with different directions of transcription. one base substitution in the loop structure caused a change in copy number. since the p1 promoter is located upstream of the replication origin of prbh1, the transcript from the ...19863487022
precolicin e1, the major gene product of plasmid-cole1 deoxyribonucleic acid in vitro.coupled transcription and translation of plasmid-cole1 dna in vitro under optimized conditions gave one major product. this has an apparent weight of 71 000, the same n-terminal sequence as colicin e1 and was not digested by deoxyribonuclease or ribonuclease. it differed from colicin e1 in its c-terminal residue and amino acid composition. it had lower specific activities in cell killing and in the fluorescence-enhancement in vitro assay of phillips & cramer [(1973) biochemistry 12, 1170--1176] ...19806994710
nucleoprotein architecture and cole1 dimer resolution: a hypothesis.dimers of plasmid cole1 are converted to monomers by site-specific recombination, a process that requires 240 bp of dna (cer) and four host-encoded proteins (xerc, xerd, argr and pepa). here, we propose structures for nucleoprotein complexes involved in cer-xer recombination based upon existing knowledge of the structures of component proteins and computational analyses of protein structure and dna curvature. we propose that, in the nucleoprotein complex at a single cer site, a pepa hexamer acts ...19989720871
expression of a dna strand initiation sequence of cole1 plasmid in a single-stranded dna phage.in order to investigate initiation of h-strand (lagging strand) replication of the plasmid cole1, the origin region fragment (hae ii-e) of cole1 was inserted into the intergenic region of filamentous dna phage m13 and cloned. a site capable of promoting dna strand initiation on a single-stranded dna template has been detected on the l-strand (leading strand) of the cloned fragment. the site, named rri-1 rifampicin-resistant initiation), directs conversion of chimeric phage single-stranded dna to ...19807005899
sequence-specific recombination of plasmid cole1.the conjugal transmission of plasmid cole1 and its derivatives can lead to reca-independent recombination. the recombination is not observed during vegetative growth and takes place specifically between the sequences that are thought to represent the transfer origin of cole1. this provides genetic evidence for breakage and reunion events during the transmission of cole1.19807005900
replication of plasmids from staphylococcus aureus in escherichia coli.plasmid pbr322 derives from plasmid cole1 and does not replicate in escherichia coli strains lacking dna polymerase i. hybrids between pbr322 and a plasmid isolated from staphylococcus aureus, pc194, replicate in such e. coli strains, provided that the pc194 replication region is intact. inactivation of the pbr322 replication region does not interfere with the replication of hybrids in e. coli. hybrids between pbr322 and two other plasmids from s. aureus, pt127 and pub112, and replicate at the r ...19807012836
the synthesis of ribosomal 5 s rna in cultured hamster cells during the inhibition of protein synthesis.ribosomal 5 s rna synthesis in chinese hamster v79 cells treated with an inhibitory antibiotic of protein synthesis, cycloheximide, was quantitated by hybridization of rna preparations with plasmid cole1-psc101 dna carrying xenopus 5 s dna. in v79 cells, cycloheximide produced a notable decrease in the production of the higher molecular weight ribosomal rna (rrna) fraction, whereas the accumulation of both 5 s rna and polyadenylate-containing messenger rna was much less affected. when the cellul ...19817020763
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