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At least 559 records · Page 31Linked to original sources
PubMed1991

Physical and biochemical characterization of recombination-dependent synthesis of linear plasmid multimers in Bacillus subtilis.

The synthesis and structure of linear multimeric plasmid molecules (hmw DNA) in Bacillus subtilis were investigated. The replication of covalently-closed-circular supercoiled (form I) DNA requires the rate-limiting plasmid-encoded replication initiation protein. Unlike form I, hmw DNA synthesis is partially resistant to inhibition of cellular transcription or translation and requires the host DnaB protein. In addition, hmw DNA synthesis involves host recombination and repair functions (RecE and Poll). Analysis of hmw DNA by electron microscopy displayed linear DNA molecules up to 100 kb in size, which were either single-stranded, double-stranded or double-stranded with single-stranded ends. Structural features of hmw DNA molecules were mapped by means of heteroduplex studies using defined strand-specific probes. The results suggest that a recombination intermediate, but not plasmid-encoded replication, is involved in the initiation of hmw DNA synthesis.

Bacillus subtilis↗
PubMed1984

Nucleotide sequence of the Galleria mellonella nuclear polyhedrosis virus origin of DNA replication.

The initiation sites of the Galleria mellonella L. nuclear polyhedrosis virus (G.m. NPV) DNA replication were revealed. For this purpose SCLd 135 cells permitting the G.m. NPV productive reproduction were transformed by the recombinant plasmids containing the viral genome individual fragments in pRSF 2124 and pBR 322 vectors. It was revealed that 2 of the 32 recombinant plasmids can autonomously replicate in the eucaryotic cells. According to the Maxam-Gilbert method the DNA G.m. NPV fragment (1300 bp) primary structure of pHBR plasmid was determined. The structure analysis revealed the typical regulator signals as in the replicons. The possible regulation mechanism of the DNA G.m. NPV synthesis initiation was supposed.

Base Sequence↗
PubMed1997

[Laser Raman spectral analysis of superhelical spatial conformation on plasmid DNA].

The pBR322 recombinant plasmids DNA products, which purified by phenol-extract and equilibrium centrifugation in CsCl-ethidium bromide gradients, have been shown as mainly spatial conformation of covalently closed circular DNA (i.e. cccDNA) by agarose gel electrophoresis for homogeneity detection. Laser Raman spectra in the region 450-1750 cm-1 have been obtained for the circular double-stranded plasmids DNA molecule in an aqueous solution, indicating not only contain those marker peaks of secondary structure as in conventional B-form of DNA, but also present both 854 and 1083 cm-1 diagnostic bands of reflecting the vibration state of deoxyribosyl phosphodiester backbone. The present analysis have demonstrated relationship between the superhelical state for cccDNA and the two conformational marker bands that can be considered as the tertiary structure marker on plasmids DNA. Because of markedly Raman hypochromicity of charecter band 1378cm-1 of dT in contrast to liear DNA molecule, as well as the carbonyl double bond vibration line of dT have shifted to higher wave number position, the base stacking analysis represent occurrence of both increase in staking reaction activity of dT, and injury of a number of the Hoogsteen hydrogen bond between dA and dT with exist supercoil structure in cccDNA.

DNA, Circular↗
PubMed1985

Minichromosome assembly of non-integrated plasmid DNA transfected into mammalian cells.

The nucleoprotein structures formed on various plasmid expression vectors transfected into mammalian cells by both the calcium phosphate and DEAE-dextran methods have been studied. We demonstrate by a variety of means that mammalian cells are capable of rapidly assembling non-integrated circular plasmids (both replicating and non-replicating) into typical "minichromosomes" containing nucleosomes with a 190 bp repetitive spacing. Treatment of recipient cells with sodium butyrate for a short period of time (12-16 h) immediately following transfection markedly increased the DNase I digestion sensitivity of the newly assembled plasmid chromatin. Furthermore, minichromosomes isolated from such butyrate-treated cells are depleted in histone H1 and contain highly acetylated forms of histone H4. These findings are entirely consistent with our earlier speculation (Gorman et al., Nucleic Acids Res. 11, 1044; 1983) that appropriate butyrate treatment might stimulate transient expression of newly transfected genes by facilitating their assembly into an "active" type of chromatin structure.

Acetylation↗
PubMed1993

[Nucleotide sequence and properties of an inverted repeating element of a Bordetella pertussis chromosome].

The repeated sequence from Bordetella pertussis chromosome was cloned using the method described by Ohtsubo. The sequences of the characterized B. pertussis chromosome are homologous to the previously described sequences and analogous in the structure to the already known IS elements. The parental recombinant plasmids containing the RS were unstable and segregated to the plasmids of different structure. The segregants' structure is characterized in this paper. It is shown in our study that the RS element is able to stimulate intragenomic rearrangements, such as deletions. It is shown that at least one deletion begins precisely after 3' end of RS and terminates with the sequence which is completely homologous to ten terminal nucleotides of this one. Probably, RSs stimulate at high frequency the formation of deletions, which appear to be the result of recA-independent site-specific recombination between short direct repeats.

Base Sequence↗
PubMed1998

Structural organization of virulence-associated plasmids of Yersinia pestis.

The complete nucleotide sequence and gene organization of the three virulence plasmids from Yersinia pestis KIM5 were determined. Plasmid pPCP1 (9,610 bp) has a GC content of 45.3% and encodes two previously known virulence factors, an associated protein, and a single copy of IS100. Plasmid pCD1 (70,504 bp) has a GC content of 44.8%. It is known to encode a number of essential virulence determinants, regulatory functions, and a multiprotein secretory system comprising the low-calcium response stimulation that is shared with the other two Yersinia species pathogenic for humans (Y. pseudotuberculosis and Y. enterocolitica). A new pseudogene, which occurs as an intact gene in the Y. enterocolitica and Y. pseudotuberculosis-derived analogues, was found in pCD1. It corresponds to that encoding the lipoprotein YlpA. Several intact and partial insertion sequences and/or transposons were also found in pCD1, as well as six putative structural genes with high homology to proteins of unknown function in other yersiniae. The sequences of the genes involved in the replication of pCD1 are highly homologous to those of the cognate plasmids in Y. pseudotuberculosis and Y. enterocolitica, but their localization within the plasmid differs markedly from those of the latter. Plasmid pMT1 (100,984 bp) has a GC content of 50.2%. It possesses two copies of IS100, which are located 25 kb apart and in opposite orientations. Adjacent to one of these IS100 inserts is a partial copy of IS285. A single copy of an IS200-like element (recently named IS1541) was also located in pMT1. In addition to 5 previously described genes, such as murine toxin, capsule antigen, capsule anchoring protein, etc., 30 homologues to genes of several bacterial species were found in this plasmid, and another 44 open reading frames without homology to any known or hypothetical protein in the databases were predicted.

Base Composition↗
PubMed1994

An analysis of the complete nucleotide sequence of the Haemophilus ducreyi broad-host-range plasmid pLS88.

We present an analysis of the complete nucleotide sequence of pLS88, a naturally occurring, 4.8-kb broad-host-range plasmid isolated from Haemophilus ducreyi and encoding resistance to sulfonamides, streptomycin, and kanamycin. Sequence analysis of the genes encoding sulfonamide and streptomycin resistance revealed homology to the RSF1010 sulII and strA genes. The sulII-strA intergenic region of pLS88 has a 38-bp deletion identical to that of the RSF1010-like plasmid pHD8.1, isolated from Actinobacillus pleuropneumoniae. The kanamycin resistance gene shows strong homology to Tn903, but lacks the inverted repeats of the transposon. No other genes have been identified. The region downstream of the kanamycin resistance gene shows homology to the RSF1010 oriV region; however, this region is not essential to plasmid replication. The ori of pLS88 is contained within a 1060-bp region and does not appear to contain structures typical of plasmid origins. This region is flanked by DNA showing strong homology to regions both upstream and downstream of the Haemophilus influenzae ROB-1 beta-lactamase gene. Because of the small size of the origin, pLS88 appears to resemble the structure of narrow-host-range plasmids, but replicates, via an as yet unidentified mechanism, as a broad-host-range plasmid.

Base Sequence↗
PubMed1991

Molecular and genetic analysis of a region of plasmid pCF10 containing positive control genes and structural genes encoding surface proteins involved in pheromone-inducible conjugation in Enterococcus faecalis.

Exposure of Enterococcus faecalis cells carrying the tetracycline resistance plasmid pCF10 to the heptapeptide pheromone cCF10 results in an increase in conjugal transfer frequency by as much as 10(6)-fold. Pheromone-induced donor cells also express at least two plasmid-encoded surface proteins, the 130-kDa Sec 10 protein, which is involved in surface exclusion, and the 150-kDa Asc10 protein, which has been associated with the formation of mating aggregates. Previous subcloning and transposon mutagenesis studies indicated that the adjacent EcoRI c (7.5 kb) and e (4.5 kb) fragments of pCF10 encode the structural genes for these proteins and that the EcoRI c fragment also encodes at least two regulatory genes involved in activation of the expression of the genes encoding Asc10 and Sec10. In this paper, the results of physical and genetic analysis of this region of pCF10, along with the complete DNA sequences of the EcoRI c and e fragments, are reported. The results of the genetic studies indicate the location of the structural genes for the surface proteins and reveal important features of their transcription. In addition, we provide evidence here and in the accompanying paper (S. B. Olmsted, S.-M. Kao, L. J. van Putte, J. C. Gallo, and G. M. Dunny, J. Bacteriol. 173:7665-7672, 1991) for a role of Asc10 in mating aggregate formation. The data also reveal a complex positive control system that acts at distances of at least 3 to 6 kb to activate expression of Asc10. DNA sequence analysis presented here reveals the positions of a number of specific genes, termed prg (pheromone-responsive genes) in this region of pCF10. The genes mapped include prgA (encoding Sec10) and prgB (encoding Asc10), as well as four putative regulatory genes, prgX, -R, -S, and -T. Although the predicted amino acid sequences of Sec10 and Asc10 have some structural features in common with a number of surface proteins of gram-positive cocci, and the Asc10 sequence is highly similar to that of a similar protein encoded by the pheromone-inducible plasmid pAD1 (D. Galli, F. Lottspeich, and R. Wirth, Mol. Microbiol. 4:895-904, 1990), the regulatory genes show relatively little resemblance to any previously sequenced genes from either procaryotes or eucaryotes.

Amino Acid Sequence↗
PubMed1986

Structural stability of heterologous genes cloned in Streptomyces plasmid pIJ702.

A recombinant plasmid pWCL1 containing Streptomyces plasmid pIJ702, E. coli plasmid pUC12, and hepatitis B viral surface antigen (HBsAg) gene was stably maintained in E. coli, but exhibited structural instability in S. lividans 1326. The deletions were found ranging from 2.75 to 5.65 kilobases (kb) and most of them occurred within the melanin (mel) gene of pIJ702, resulting in the loss of part of the mel gene sequence plus the insert. The removal of the pUC12 sequence from pWCL1 eliminated the instability. However, pUC12 alone inserted in either orientation on pIJ702 also caused the deletion in S. lividans 1326. The results indicated that the structural instability of hybrid plasmid of pIJ702 depended on the interaction between the mel sequence and the inserted sequence.

Chromosome Deletion↗
PubMed2000

Stabilized plasmid-lipid particles for systemic gene therapy.

The structure of 'stabilized plasmid-lipid particles' (SPLP) and their properties as systemic gene therapy vectors has been investigated. We show that SPLP can be visualized employing cryo-electron microscopy to be homogeneous particles of diameter 72 +/- 5 nm consisting of a lipid bilayer surrounding a core of plasmid DNA. It is also shown that SPLP exhibit long circulation lifetimes (circulation half-life >6 h) following intravenous (i.v.) injection in a murine tumor model resulting in accumulation of up to 3% of the total injected dose and concomitant reporter gene expression at a distal (hind flank) tumor site. In contrast, i v. injection of naked plasmid DNA or plasmid DNA-cationic liposome complexes did not result in significant plasmid delivery to the tumor site or gene expression at that site. Furthermore, it is shown that high doses of SPLP corresponding to 175 microg plasmid per mouse are nontoxic as assayed by monitoring serum enzyme levels, whereas i.v. injection of complexes give rise to significant toxicity at dose levels above 20 microg plasmid per mouse. It is concluded that SPLP exhibit properties consistent with potential utility as a nontoxic systemic gene therapy vector.

Animals↗
PubMed2003

ParG, a protein required for active partition of bacterial plasmids, has a dimeric ribbon-helix-helix structure.

The ParG protein (8.6 kDa) is an essential component of the DNA partition complex of multidrug resistance plasmid TP228. ParG is a dimer in solution, interacts with DNA sequences upstream of the parFG genes and also with the ParF partition protein both in the absence and presence of target DNA. Here, the solution nuclear magnetic resonance structure of ParG is reported. The ParG dimer is composed of a folded domain formed by two closely intertwined C-terminal parts (residues 33-76), and two highly mobile tails consisting of N-terminal regions (residues 1-32). The folded part of ParG has the ribbon-helix-helix (RHH) architecture similar to that of the Arc/MetJ superfamily of DNA-binding transcriptional repressors, although the primary sequence similarity is very low. ParG interacts with DNA predominantly via its folded domain; this interaction is coupled with ParG oligomerization. The dimeric RHH structure of ParG suggests that it binds to DNA by inserting the double-stranded beta-sheet into the major groove of DNA, in a manner similar to transcriptional repressors from the Arc/MetJ superfamily, and that ParG can function as a transcriptional repressor itself. A new classification of proteins belonging to the Arc/MetJ superfamily and ParG homologues is proposed, based on the location of a conserved positively charged residue at either the beginning or at the end of the beta-strand which forms part of the DNA recognition motif.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence and structural relationships of a chloramphenicol acetyltransferase encoded by the plasmid pSCS6 from Staphylococcus aureus.

The 4.6 kb chloramphenicol resistance (Cm) plasmid, pSCS6, isolated from a naturally occurring Staphylococcus aureus biotype C encoded an inducible chloramphenicol acetyltransferase (CAT). The respective cat gene and its regulatory region were cloned. Sequence analyses revealed two open reading frames: one for a 9-amino acid leader peptide and the other for the 215-amino acid CAT monomer. Comparisons of the predicted CAT amino acid sequences revealed a high degree of similarity between CAT from pSCS6 and the CAT variants encoded by Cm plasmids of the pC221 family. These close structural relationships suggested an intraspecific exchange of Cm-determinants between Staph. aureus of human and bovine biotype.

Amino Acid Sequence↗
PubMed1987

Molecular analysis of regulatory and structural xyl genes of the TOL plasmid pWW53-4.

pWW53-4 is a cointegrate between RP4 and the catabolic plasmid pWW53 from Pseudomonas putida MT53, which contains 36 kbp of pWW53 DNA inserted close to the oriV gene of RP4; it encodes the ability to grow on toluene and the xylenes, characteristic of pWW53, as well as resistance to tetracycline, kanamycin and carbenicillin, characteristic of RP4. A physical map of the 36 kbp insert of pWW53 DNA for 11 restriction enzymes is presented, showing that the relative positions of the two xyl operons are different from those on the archetypal TOL plasmid pWW0. The location of the genes for 4-oxalocrotonate decarboxylase (xylI) and 4-oxalocrotonate tautomerase (xylH) were shown by subcloning and enzyme assay to lie at the distal end of the meta pathway operon. Although 2-oxopent-4-enoate hydratase (xylJ) and 4-hydroxy-2-oxovalerate aldolase (xylK) could be detected on a large cloned HindIII fragment, they could not be accurately located on smaller subcloned DNA, but the only credible position for them is between xylF and xylI. The gene order in the meta pathway operon is therefore xylDLEGF(J,K)IH. The regulatory genes xylS and xylR were located close to and downstream of the meta pathway operon, and the restriction map of the DNA in this region, as has previously been shown for the two operons carrying the structural genes, shows similarities with the corresponding region on pWW0. Evidence is also presented for the existence of two promoters, termed P3 and P4, internal to the meta pathway operon which support low constitutive expression of the structural genes downstream in Pseudomonas hosts but not in E. coli.

Chromosome Mapping↗
PubMed2000

Carbofuran degradation mediated by three related plasmid systems.

Two carbofuran-metabolizing Sphingomonas strains, TA and CD, were isolated from soils with differing histories of exposure to carbofuran. These strains were compared with a previously described strain, Sphingomonas sp. CFO6, with regard to growth rate, formation of metabolites, and plasmid content and structure. Extensive regions of similarity were observed between the three different plasmid systems as evidenced by cross hybridization. In addition, all three systems harbor IS1412, an insertion sequence (IS) element involved in heat-induced loss of carbofuran phenotype in CFO6, and heat-induced carbofuran deficient mutants of all three strains correlated with loss of IS1412. A carbofuran deficient mutant of TA generated by induction of IS elements was complemented by reintroduction of the wild-type plasmid, confirming the presence of genes required for carbofuran metabolism on this plasmid. Carbofuran metabolism in these three strains is clearly linked via plasmids of different numbers and sizes that share extensive common regions, and carbofuran-degrading genes may be associated with active IS elements.

Journal Article↗
PubMed1997

De novo and DNA primer-mediated initiation of cDNA synthesis by the mauriceville retroplasmid reverse transcriptase involve recognition of a 3' CCA sequence.

The Mauriceville mitochondrial retroplasmid of Neurospora encodes a novel reverse transcriptase that initiates cDNA synthesis at a 3' tRNA-like structure of the plasmid transcript, either de novo (i.e. without a primer) or by using the 3' OH group of a DNA primer. Both the de novo and primer-mediated initiations involve recognition of structural features at the 3' end of the retroplasmid transcript, which ends with a 3' CCACCA. Here, detailed biochemical characterization of the retroplasmid reverse transcriptase shows that the 3' CCA of the plasmid transcript is the major structural feature recognized by the reverse transcriptase for both the de novo and primer-mediated initiations. Complementarity between the DNA primer and RNA template is not required for the primer-mediated initiation, although short (1 to 3 nt) base-pairing interactions can influence both the efficiency and site of initiation near the 3' end of the transcript. Single nucleotide changes in the 3' CCA lead to less efficient initiation in the upstream CCA with an increased propensity to add extra "non-coded" nucleotides to the 5' end of the cDNA during de novo initiation or to the 3' end of the primer during primer-mediated initiation. Secondary structure features upstream of the 3' CCA also influence the efficiency of initiation, but are not stringently required in vitro. Finally, we find that the retroplasmid reverse transcriptase does not efficiently use DNA primers that are base-paired to internal positions in the RNA template, nor does it use analogs of natural substrates used by non-long terminal repeat retrotransposon or retroviral reverse transcriptases. Our results indicate that the retroplasmid reverse transcriptase is uniquely adapted to initiate cDNA synthesis by recognizing a 3' CCA sequence. The ability to recognize a specific template sequence is common for RNA polymerases, but unprecedented for a reverse transcriptase.

Base Composition↗
PubMed1984

Isolation and structure of the bacterial sex pheromone, cAD1, that induces plasmid transfer in Streptococcus faecalis.

The Streptococcus faecalis sex pheromone cAD1, which is involved in the conjugative transfer of the hemolysin plasmid pAD1, has been isolated and its structure determined. Its Mr is 818 and its amino acid sequence is H-Leu-Phe-Ser-Leu-Val-Leu-Ala-Gly-OH. A replicate of the pheromone synthesized by the liquid-phase method showed the same biological activity and chromatographic behavior as the isolated cAD1. Pheromone activity was detectable at a concentration of approximately 5 X 10(-11) M.

Amino Acid Sequence↗