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PubMed1991

[Localization of camphor degradative plasmids on the chromosome of Pseudomonas putida strains PaW].

Camphor degradative plasmids (CAM, pRK1) are preferentially situated on chromosomes of Pseudomonas putida strains PaW. After having been transferred into Cam+ strains, the TOL plasmid pWWO dissociates into the cryptic plasmid pWWO-8 and chromosome-borne transposon Tn4651. The opposite situation, i.e. reconstruction of the TOL plasmid pWWO from the cryptic plasmid pWWO-8 and chromosome-borne catabolic operons of the pWWO plasmid has been described. Cam- derivatives of the CAM plasmid were obtained in vivo which contain the TOL plasmid transposons Tn4651 or Tn4652 as obligatory structural elements. These plasmids as well as pWWO-8 determine conjugational mobilization of chromosome-located cam operons followed by their integration into the chromosome of recipient.

Camphor↗
PubMed1996

Structural organization of pRAM4, a cryptic plasmid from Prevotella ruminicola.

A total of 530 strains of rumen bacteria were screened for the presence of plasmid DNA. The percentage of plasmid-bearing strains was found to be the highest among the Bacteroides/ Prevotella group (9.9%), while it was less than 1% in the Butyrivibrio (0.2%) and Clostridium (0.6%) genera. A small cryptic plasmid pRAM4 from Prevotella ruminicola T31 was subcloned in Escherichia coli and completely sequenced. Two open reading frames, encoding potential polypeptides of M(r) 32,322 (ORF1) and 32,122 (ORF2) with limited sequence similarity to replication initiation and mobilization proteins, respectively, could be identified within the sequence. The region upstream from ORF1 had an AT-rich (75%) region followed by four 22-bp direct repeats, a structure characteristic of replication origins. The plasmid hybridized at high stringency with plasmids from Bacteroides/Prevotella and Butyrivibrio, and with pBR322, suggesting that at least regions of the plasmid are widespread.

Amino Acid Sequence↗
PubMed1980

Membrane phospholipid synthesis in Escherichia coli. Cloning of a structural gene (plsB) of the sn-glycerol-3-phosphate acyl/transferase.

Si+ hybrid ColE1 plasmids of the Clarke-Carbon collection (Clarke, C., and Carbon, J. (1976) Cell 9, 91-99) which eliminate the sn-glycerol 3-phosphate growth requirement of a mutant of Escherichia coli with a Km defect in sn-glycerol-3-phosphate acyltransferase (plsB) were identified. Marked overproduction of a plasmid-encoded sn-glycerol-3-phosphate acyltransferase with a wild type Km in a host plsB- background indicates that the hybrid plasmids carry a structural gene for this enzyme. In addition, all of these plasmids suppress the phenotype of a mutation in a second locus involved in phospholipid biosynthesis, dgk (diglyceride kinase), and one of them also bears the dnaB structural gene. Diglyceride kinase activity is also overproduced in these strains. The linkage of plsB, dgk and dnaB loci was confirmed by transduction analysis which demonstrated the clockwise gene order malB, dnaB, dgk, plsB, and uvrA near Minute 91 on the E. coli linkage map. This is in contrast to the previously reported co-transduction of plsB with dctA near Minute 78 (Cronan, J. E., Jr., and Bell, R. M. (1974) J. Bacteriol., 120, 227-233). Recloning of restriction endonuclease fragments and in vitro mutagenesis have localized the dgk, and plsB loci to a 2.2-megadalton DNA segment, and have demonstrated that diglyceride kinase and sn-glycerol-3-phosphate acyltransferase activities reside in separate polypeptides. Availability of these clones and mutationally altered derivatives has allowed the identification of a single polypeptide (Mr = 83,000) corresponding to the sn-glycerol-3-phosphate acyltransferase and purification of this membrane-bound enzyme to near homogeneity (Larson, T. J., Lightner, V. A., Green, P. R., Modrich, P., and Bell, R. M. (1980) J. Biol. Chem. 255, 9421-9426). The size of the plsB polypeptide indicates that a major fraction of the DNA segment to which this gene has been localized is involved in coding for the sn-glycerol-3-phosphate acyltransferase.

Acyltransferases↗
PubMed1992

Genetic organization and structural features of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa.

The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-1 codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasmids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a lysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.

Amino Acid Sequence↗
PubMed1978

Construction and identification by positive hybridization-translation of a bacterial plasmid containing a rat growth hormone structural gene sequence.

The construction, identification, and use of a recombinant DNA clone containing a growth hormone structural gene sequence is described. A cDNA copy of partially purified pregrowth hormone mRNA from cultured rat pituitary tumor (GC) cells was employed in the construction of a hybrid plasmid, designated pBR322-GH1. The cloned DNA sequence was positively identified by a hybridization-translation procedure which should be applicable to any cloned structural gene sequence. This procedure involved hybridization of cytoplasmic poly(A)-containing RNA from GC cells to the cloned DNA immobilized on nitrocellulose filters, followed by elution of the hybridized RNA and translation in a mRNA-depleted rabbit reticulocyte lysate system. Physical and immunological criteria were employed to show that the translation products were enriched for pregrowth hormone. Hybridization to excess plasmid DNA of [3H]uridine-labeled, size fractionated GC cell cytoplasmic RNA was used to show that all growth hormone-specific RNA sequences are the same size as functional pregrowth hormone mRNA.

Cell Line↗
PubMed1987

Plasmid-mediated resistance to phagocytosis in Yersinia enterocolitica.

Results of our previous studies have shown that the chemiluminescence response of human neutrophils (polymorphonuclear leukocytes [PMNs]) is inhibited by plasmid-mediated cell surface components from Yersinia enterocolitica. In this study we examined the susceptibility to phagocytosis of Y. enterocolitica cells with or without plasmid-mediated surface structure and the effect of isolated outer membrane fragments on phagocytosis of Escherichia coli by PMNs in vitro. Y. enterocolitica cells with expressed plasmid-mediated surface structure were much less sensitive to ingestion by PMNs than those without it, and the resistance to phagocytosis was readily eliminated in a dose-dependent fashion by pronase treatment of whole cells, which was shown to remove plasmid-encoded outer membrane proteins. Ingestion and intracellular killing of E. coli were inhibited significantly in the presence of isolated outer membrane fragments derived from plasmid-bearing Y. enterocolitica cells. To assess the interaction of Y. enterocolitica with phagocytic cells in vivo, two isogenic strains of Y. enterocolitica, differing only in the presence or absence of the virulence plasmid, were inoculated intradermally into the backs of rabbits; and tissue sections obtained at 12 h postinoculation were examined by light and electron microscopy. The plasmidless strain was found almost entirely in PMNs or mononuclear cells. In contrast, the plasmid-bearing strain was found to be surrounded by, or interspersed with, PMNs and mononuclear cells; but most bacteria were extracellular, with little evidence of phagocytosis. These results suggest that plasmid-mediated cell surface components of Y. enterocolitica act as antiphagocytic factors, thus facilitating the survival and proliferation of the organism in the host tissue.

Animals↗
PubMed2003

A track structure model for simulation of strand breaks in plasmid DNA after heavy ion irradiation.

We present a track structure model based on the local dose deposited around heavy ion tracks to explain the cross sections for single-strand and double-strand break induction in plasmid DNA in different aqueous buffers. The model is based only on measurable quantities, namely the effect distribution for inducing strand breaks after x-ray irradiation as a function of dose, and the radial dose distribution of the heavy ion track. The effect of indirect DNA damage mediated by free radicals produced in the water surrounding the DNA is accounted for by allowing the radial dose distribution to be smeared in space by an effective target size corresponding to the squared sum of the geometrical extension of the plasmid molecule and the mean free drift path of the radicals in the buffer solution. Our calculations reproduce well the measured cross sections for single-strand and double-strand break induction in SV40 plasmid DNA in various buffer solutions both as a function of the LET and of the specific energy of the heavy ion.

Buffers↗
PubMed2000

Immune responses to hepatitis C virus structural and nonstructural proteins induced by plasmid DNA immunizations.

DNA-based immunizations have been used to elicit cellular immunity to hepatitis C virus (HCV) proteins in mice. Mice were immunized by intramuscular or intradermal injections of plasmid DNA derived from a near-full-length HCV genotype 1b genomic clone (pRC/B2) or individual genomic clones. These immunizations induced cytotoxic T lymphocytes (CTLs), as revealed in standard chromium-release assays that used syngeneic peptide-pulsed or transfected target cells. These assays identified four CTL epitopes within the capsid, E1, and E2 regions of the polyprotein sequence of HCV genotype 1a that were cross-reactive with HCV genotype 1b. Additionally, CTLs derived from mice immunized with either NS3 or NS5 specifically lysed target cells sensitized to either the genotype 1a or 1b gene products. Nucleic acid immunizations also generated humoral immunity to HCV proteins, as detected by anti-HCV reactivity to NS3 and capsid in ELISAs and immunoblot assays.

Animals↗
PubMed1998

Structural analysis of a new cryptic plasmid pAR67 isolated from Ruminococcus albus AR67.

The complete nucleotide sequence of a new cryptic plasmid pAR67 isolated from a rumen bacterium Ruminococcus albus AR67 has been determined. The plasmid pAR67 was 3419 bp in size with a 45% GC content. Two open reading frames, ORF1 and ORF2, encoding potential polypeptides of 285 and 165 amino acids, with limited sequence similarity to replication and mobilization proteins, respectively, were identified within the sequence. The region upstream of ORF1 had an AT-rich (80%) segment followed by four 19-bp direct repeats, which is similar to the structural organization characteristic of replication origins of some bacterial plasmids.

Amino Acid Sequence↗
PubMed1997

The use of sonication for the efficient delivery of plasmid DNA into cells.

PURPOSE: Ultrasonic methods have considerable potential for the introduction of macromolecules into cells. In this paper we demonstrate that, under controlled conditions, application of 20 kHz ultrasound to a suspension of yeast cells facilitates the delivery of plasmid DNA into these cells. METHODS: Aliquots of growing yeast cells (Saccharomyces cerevisae, strain AH22) were suspended in buffer and exposed to 20 kHz ultrasound from a laboratory (probe-type) sonicator in the presence of microgram quantities of plasmid DNA. Efficiency of DNA delivery was scored as the number of cells transformed. RESULTS: Cell transformation was optimal at 30 seconds sonication using an output of 2.0 watts and resulted in a 20 fold enhancement over control values. At extended sonication times, fewer cells showed evidence of transformation because of reduced cell viability. The increased DNA uptake and the decreased cell viability were both attributable to acoustic cavitation events during sonication. The extent of acoustic cavitation was measured and it was found that there was an increase in cavitation events with increased sonication time. Cell viability was shown to be directly related to the number of cavitation events. The effects of sonication on plasmid DNA were investigated and indicated that the structural integrity of plasmid DNA was unaffected by the sonication conditions employed. CONCLUSIONS: Under controlled conditions, ultrasound is an effective means of delivering plasmid DNA into cells. The subsequent expression of DNA molecules in cells depends upon a balance between transient cell damage and cell death.

DNA Damage↗
PubMed1998

Rolling-circle plasmids from Bacillus subtilis: complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from gram-positive bacteria.

Most small plasmids of Gram-positive bacteria use the rolling-circle mechanism of replication and several of these have been studied in considerable detail at the DNA level and for the function of their genes. Although most of the common laboratory Bacillus subtilis 168 strains do not contain plasmids, several industrial strains and natural soil isolates do contain rolling-circle replicating (RCR) plasmids. So far, knowledge about these plasmids was mainly limited to: (i) a classification into seven groups, based on size and restriction patterns; and (ii) DNA sequences of the replication region of a limited number of them. To increase the knowledge, also with respect to other functions specified by these plasmids, we have determined the complete DNA sequence of four plasmids, representing different groups, and performed computer-assisted and experimental analyses on the possible function of their genes. The plasmids analyzed are pTA1015 (5.8 kbp), pTA1040 (7.8 kbp), pTA1050 (8.4 kbp), and pTA1060 (8.7 kbp). These plasmids have a structural organization similar to most other known RCR plasmids. They contain highly related replication functions, both for leading and lagging strand synthesis. pTA1015 and pTA1060 contain a mobilization gene enabling their conjugative transfer. Strikingly, in addition to the conserved replication modules, these plasmids contain unique module(s) with genes which are not present on known RCR plasmids of other Gram-positive bacteria. Examples are genes encoding a type I signal peptidase and genes encoding proteins belonging to the family of response regulator aspartate phosphatases. The latter are likely to be involved in the regulation of post-exponential phase processes. The presence of these modules on plasmids may reflect an adaptation to the special conditions to which the host cells were exposed.

Bacillus subtilis↗
PubMed1987

Intermolecular homologous recombination between transfected sequences in mammalian cells is primarily nonconservative.

Intermolecular recombination in mammalian cells was studied by coinfecting African green monkey cells in culture with two shuttle vector plasmids, each carrying an incomplete but overlapping portion of the gene for neomycin resistance. The region of homology between the two plasmids was about 0.6 kilobases. Recombination between the homology regions could reconstruct the neomycin resistance gene, which was monitored by analysis of progeny plasmids in bacteria. The individual plasmids carried additional markers which, in combination with restriction analysis, allowed the determination of the frequency of formation of the heterodimeric plasmid which would be formed in a conservative recombination reaction between the homologous sequences. Reconstruction of the neomycin resistance gene was readily observed, but only 1 to 2% of the neomycin resistance plasmids had the structure of the conservative heterodimer. Treatment of the plasmids which enhanced the frequency of the neomycin resistance gene reconstruction reaction did not significantly increase the relative frequency of conservative product plasmids. The results support nonconservative models for recombination of these sequences.

Animals↗
PubMed2005

Preparation of poly(ethylene glycol)-introduced cationized gelatin as a non-viral gene carrier.

The objective of this study was to prepare cationized gelatins grafted with poly(ethylene glycol) (PEG) (PEG-cationized gelatin) and evaluate the in vivo efficiency as a non-viral gene carrier. Cationized gelatin was prepared by chemical introduction of ethylenediamine to the carboxyl groups of gelatin. PEG with one terminal of active ester group was coupled to the amino groups of cationized gelatin to prepare PEG-cationized gelatins. Electrophoretic experiments revealed that the PEG-cationized gelatin with low PEGylation degrees was complexed with a plasmid DNA of luciferase, in remarked contrast to that with high PEGylation degrees. When the plasmid DNA complexed with the cationized gelatin or PEG-cationized gelatin was mixed with deoxyribonuclease I (DNase I) in solution to evaluate the resistance to enzymatic degradation, stronger protection effect of the PEG-cationized gelatin was observed than that of the cationized gelatin. The complex of plasmid DNA and PEG-cationized gelatin had an apparent molecular size of about 300 nm and almost zero surface charge. These findings indicate that the PEG-cationized gelatin-plasmid DNA complex has a nano-order structure where the plasmid DNA is covered with PEG molecules. When the PEG-cationized gelatin-plasmid DNA complex was intramuscularly injected, the level of gene expression was significantly increased compared with the injection of plasmid DNA solution. It is concluded that the PEG-cationized gelatin was a promising non-viral gene carrier to enhance gene expression in vivo.

Animals↗
PubMed1988

Drug-resistant plasmids from fish pathogens.

The epidemiological surveillance of drug-resistant strains of the fish pathogenic bacteria Vibrio anguillarum and Pasteurella piscicida carrying transferable R plasmids in fish farms is described. The DNA structure of R plasmids, and the drug-resistant determinants of R plasmids from the fish pathogens Aeromonas hydrophila,aeromonas salmonicida, Edwardsiella tarda, V. anguillarum, and P. piscicida are discussed.

Animals↗
PubMed2004

Structure and function of the shufflon in plasmid R64.

Conservative site-specific recombination plays key roles in creating biological diversity in prokaryotes. Most site-specific inversion systems consist of two recombination sites and a recombinase gene. In contrast, the shufflon multiple inversion system of plasmid R64 consists of seven sfx recombination sites, which separate four invertible DNA segments, and the rci gene encoding a site-specific recombinase of the integrase family. The rci product mediates recombination between any two inverted sfx sites, resulting in the inversion of four DNA segments independently or in groups. Random shufflon inversions construct seven pilV genes encoding constant N-terminal segment with different C-terminal segments. The pilV products are tip-located adhesins of the type IV pilus, called the thin pilus, of R64 and recognize lipopolysaccharides of recipient bacterial cells during R64 liquid matings. Thus, the shufflon determines the recipient specificity of liquid matings. Rci protein of R64 was overexpressed, purified, and used for in vitro recombination reactions. The cleavage and rejoining of DNA strands in shufflon recombinations were found to take place in the form of a 5' protruding 7-bp staggered cut within sfx sequences. Thus, the sfx sequence is asymmetric: only the 7-bp spacer sequence and the right arm sequence are conserved among various R64 sfxs, whereas the sfx left arm sequences are not conserved. Rci protein was shown to bind to entire sfx sequences, suggesting that it binds to the right arms of the sfx sequences in a sequence-specific manner and to their left arms in a non-sequence-specific manner. The sfx left arm sequences greatly affected the shufflon inversion frequency. The artificial symmetric sfx sequence, in which the sfx left arm was changed to the inverted repeat sequence of the right arm, exhibited the highest inversion frequency. Rci-dependent deletion of a DNA segment flanked by two symmetric sfx sequences in direct orientation was observed, suggesting that the asymmetry of sfx sequences may prevent recombination between sfx sequences in direct orientation in the R64 shufflon. The Rci C-terminal domain was not required for recombination using the symmetric sfx sequence. A model, where the C-terminal domain of Rci protein plays a key role in the sequence-specific and non-specific binding of Rci to asymmetric sfx sites, was proposed. Site-specific recombination in the temperate phage Mx8 of M. xanthus was also described. The Mx8 attP site is located within the coding sequence of the Mx8 intP gene. Therefore, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the intP gene into a new gene, intR. As a result of this conversion, the 112-amino-acid C-terminal sequence of the intP product is replaced with a 13-amino acid sequence of the intR product. The C-terminal domain of Mx8 IntP recombinase is only required for integration and not for excision.

Bacteria↗
PubMed1994

Structure-activity relationships in the induction of single-strand breakage in plasmid pBR322 DNA by amino sugars and derivatives.

Structure-activity relationships in the induction of strand breakage in plasmid pBR322 DNA by amino sugars and their derivatives were investigated using agarose gel electrophoresis. The coexistence of a potential free aldehyde group at the C-1 position and a free amino group at the C-2 position in the molecules was indispensable for the display of DNA strand-breaking activity in both mono- and oligo-aminosaccharides. The activity was increased by the introduction of an acidic group, especially a phosphate group, at the C-6 position. The activity was also increased by the addition of Cu2+. The order of activity of the amino monosaccharides tested was D-isoglucosamine > D-mannosamine > D-galactosamine > D-glucosamine, and it is suggested that this order is correlated with the portion of acyclic (aldehydo) form in the solution of each sugar. The possible chemical basis for DNA strand breakage by amino sugars is discussed.

Carbohydrate Conformation↗
PubMed1980

IS-like element IS8 in RP4 plasmid and its involvement in cointegration.

The structure of the cointegrate plasmids formed by fusion of RP4 and the tumour-inducing plasmid (pTi) of Agrobacterium tumefaciens was analyzed. In all of the nine independently isolated pTi::RP4 cointegrates, the integration occurred at the same site on the RP4 genome. Moreover, a 1.2 Md (1750 bp) RP4 sequence (IS8) was directly repeated at both junction sites of the two replicons. The insertion of RP4 generated deletions, starting from the IS8 sequence and extending into the Ti part of the cointegrate. Dissociation of the cointegrates resulted in wild-type RP4 and Ti-plasmids with the IS8 sequence inserted at the original RP4 insertion site. The processes of integration and dissociation and the genetic properties of the cointegrates indicate that the IS8 sequence has unique characteristics defining a new insertion sequence.

Base Sequence↗