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PubMed2001

Progressive alteration of telomeric sequences at one end of a yeast linear plasmid and its possible association with reduced plasmid stability.

After selection for migration into the nucleus, a cytoplasmic yeast linear plasmid bearing an inverted terminal repeat (ITRs) at each end replicates in Saccharomyces cerevisiae in a linear form, called pTLU, which carries host telomeric repeats (TG(1-3))(n) of about 300-350 bp added to the ITR ends. We previously showed that the nucleotide composition of the added telomeric sequences varied among individual pTLU isolates, while those on the two ends of any given pTLU were always identical. The telomeric sequences of pTLU remained unchanged over numbers of cell generations when cells were selected for expression of the plasmid-borne nuclear marker. We report here that progressive alterations in telomeric sequences can be detected in cells which are grown under non-selective conditions. Surprisingly, in any given molecule, the telomeric alterations occur exclusively on one side, either the left or the right end, while the sequence at the opposite end remained identical to the original, suggesting a difference in the mode of DNA replication between the plasmid ends. These alterations occur over a broad area extending from the termini of telomeres to nucleotides near the junction between the telomeric sequences and the pTLU-ITR, implying that the plasmid ends undergo successive rounds of extension and contraction. Clonal analysis under non-selective conditions indicated that the alterations in telomeric sequences are generally associated with extreme instability of the pTLU plasmid.

Base Sequence↗
PubMed1986

Regulation of IncFII plasmid DNA replication. A quantitative model for control of plasmid NR1 replication in the bacterial cell division cycle.

A quantitative model for the regulation of replication of the low copy number IncFII plasmid NR1 in the Escherichia coli cell division cycle has been developed. The initiation of NR1 replication requires a cis-acting initiator protein whose synthesis is regulated by several mechanisms. The NR1 regulatory processes include co-operative protein-protein interactions in the formation of an active transcription repressor, the interaction of repressor with a rightward operator site in the control of transcription of the initiator gene, and the interaction of an inhibitor RNA transcript with the initiator mRNA in the control of translation of the initiation protein. A statistical thermodynamic model was used to predict probable configurations of the regulatory processes in a single growing cell. These probabilities were coupled by a kinetic model to the events of the cell cycle, such as initiation of mRNA transcription and protein translation, and the initiation of plasmid DNA replication. Parameter values were chosen so that the simulated values for plasmid copy number and the intracellular concentrations of repressor protein and mRNA agreed with experimentally determined estimates. A number of different copy number mutants that have altered one or another of the regulatory processes were simulated by the model. The contributions of each of the regulatory processes toward the overall stability of inheritance of plasmid NR1 in a population of cells in culture were examined. These simulations predict a very stable pattern of inheritance for plasmid NR1 despite its low copy number, in agreement with experimental observation.

Cell Cycle↗
PubMed1979

Role of plasmid-coded RNA and ribonuclease III in plasmid DNA replication.

An in vitro replication system has been used to study the control of DNA replication of the relaxed plasmids Col E1 and RSF1030. An RNA transcript approximately 100 nucleotides long is synthesized during the in vitro DNA replication reaction. This RNA is synthesized approximately 450 bp away from the origin of replication. A small insertion in the coding sequence for the RNA made from Col E1 DNA leads to a larger RNA species and simultaneously to an increase in plasmid copy number. Revertants missing the specific insertion show shorter RNA transcripts and wild-type copy number. Although plasmids Col E1 and RSF1030 have no extensive sequence homology, the RNA synthesized during RSF1030 replication has almost the same mobility as the Col E1 RNA on polyacrylamide gels and hybridizes to the Col E1 origin region. Extracts prepared from mutants of Escherichia coli deficient in ribonuclease III do not replicate RSF1030 or Col E1 plasmids in vitro. When supplemented with homogeneous RNAase III, such extracts do support DNA replication on these templates, indicating that RNAase III is required for DNA replication. We propose that the 100 nucleotide RNA species is involved in regulating the initiation of DNA replication of these plasmids, and that RNAase III may be involved in processing this RNA.

Chromosome Mapping↗
PubMed1980

Partitioning of bacterial plasmids during cell division: a cis-acting locus that accomplishes stable plasmid inheritance.

We have identified and characterized a genetic function (designated par, for partition) that is required for stable maintenance of plasmids within exponentially growing cell populations. This function, which accomplishes the active distribution of plasmid DNA molecules to daughter cells, has been localized within the pSC101 plasmid to a 270 bp segment adjacent to the replication origin. The par locus, which appears to be functionally equivalent to the centromere of eucaryotic cells, is able to rescue unstable pSC101-derived replicons or an unrelated par- P15A-derived multicopy replicon in the cis, but not the trans, configuration. It is independent of copy number control and dose not specify plasmid incompatibility. Furthermore, it is not associated directly with plasmid replication functions.

Cell Division↗
PubMed1983

Partition mechanism of F plasmid: two plasmid gene-encoded products and a cis-acting region are involved in partition.

Plasmids that replicate using the replication origin (oriC) of the E. coli chromosome are not stably inherited through cell division, but can be stabilized by joining with a particular segment of F plasmid that presumably provides the partition function. The segment necessary for stabilization has been located within a 3.0 kb segment outside of the region essential for autonomous replication of the F plasmid. This segment contains three functionally distinct regions: two of them (designated sopA and sopB) specify gene products that act in trans, whereas the third region (sopC) acts in cis. All three functions seem to be essential for normal partition of the plasmid into daughter cells during cell division. The cis-acting region also specifies plasmid incompatibility.

Bacterial Proteins↗
PubMed1980

Physical mapping of plasmid pDB101: a potential vector plasmid for molecular cloning in streptococci.

A physical map of the streptococcal macrolides, lincomycin, and streptogramin B (MLS) resistance plasmid pDB101 was constructed using six different restriction endonucleases. Ten recognition sites were found for HindIII, seven for HindII, eight for HaeII, and one each for EcoRI, HpaII, and KpnI. The localization of the restriction cleavage sites was determined by double and triple digestions of the plasmid DNA or sequential digestions of partial cleavage products and isolated restriction fragments, and all sites were aligned with a single EcoRI reference site. Plasmid pDB101 meets all requirements essential for a potential molecular cloning vehicle in streptococci; i.e., single restriction sites, a MLS selection marker, and a multiple plasmid copy number. The vector plasmid described here makes it possible to clone selectively any fragment of DNA cleaved with EcoRI, HpaII, or KpnI, or since the sites are close to each other in map position, any combination of two of these restriction enzymes.

Cloning, Molecular↗
PubMed1983

DNA homology between the arsenate resistance plasmid pSX267 from Staphylococcus xylosus and the penicillinase plasmid pI258 from Staphylococcus aureus.

A 29.5-kb plasmid, pSX267, from Staphylococcus xylosus DSM 20267 was found to code for arsenate, arsenite, and antimony (III) resistance. The isolated plasmid was transformed into S. aureus, where the same resistances were expressed. It was of special interest to see whether pSX267 showed any DNA sequence homology with the well-studied penicillinase plasmid from S. aureus pI258, also conferring arsenate, arsenite, and antimony III resistance. By the use of the Southern blotting technique, it was found that DNA sequence homology exists in the region of arsenate, arsenite, and antimony resistance, in addition to the region where the origin of replication, the incompatibility, and the replication A function were mapped on pI258. This finding was confirmed by electron microscope heteroduplex analysis, which allowed a correlation between the genetic and physical maps of pI258 and pSX267. Duplex DNA was formed at the arsenate operon of pI258, with a length of 2.6 kb, and at the incompatibility and replication A region, comprising a length of 2.5 kb. Adjacent to this latter region, two small regions of DNA homology were present, with lengths of 0.2 and 0.27 kb. Both plasmids share approximately 20% DNA sequence homology. The DNA homology of the arsenate, arsenite, and antimony III resistance coding regions between pI258 and pSX267 indicate that these plasmid-determined resistance markers are highly conserved and distributed among different staphylococcal species.

Antimony↗
PubMed1984

T-DNA fragments of hairy root plasmid pRi8196 are distantly related to octopine and nopaline Ti plasmid T-DNA.

Agrobacterium Ti (tumor-inducing) and Ri (root-inducing) plasmids transform dicot plant cells by insertion of a specific plasmid sector called T-DNA (transferred DNA) into host plant nuclear DNA. The mannopine -type Ri plasmid pRi8196 contains four BamHI fragments that encompass core T-DNA. We report Southern hybridization studies that show that these four fragments have no strong homology to octopine-, nopaline-, or agropine -type Ti plasmids. We detected and mapped very weak homology regions, most of which are assignable to opine synthase or opine catabolic functions on the Ti plasmid. We found no homology between Ri T-DNA and the region of Ti T-DNA that encodes tumor morphology functions.

Arginine↗
PubMed1984

Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid R1 system.

Plasmids are autonomously replicating DNA molecules that are present in defined copy numbers in bacteria. This number may for some plasmids be very low (2-5 per average cell). In order to be stably inherited, replication and partitioning of the plasmid have to be strictly controlled. Plasmids carry genetic information for both processes. In the present paper we summarize what is known about the replication control system of one low-copy-number plasmid, R1, belonging to the FII incompatibility group. We do so because the FII group seems to be one of the best understood examples with respect to genetics, molecular biology, and physiology of the replication control system. The paper is not a classical review, but rather an essay in which we discuss the aspects of replication control that we regard as being important.

Bacterial Proteins↗
PubMed1986

Effect of the deletion of a fragment dispensable for the autonomous maintenance of plasmid pT181 on the competition between incompatible plasmids.

The deletion of the 560-bp HindIII C fragment from pT181 derivatives does not change the stability or copy number of the plasmid but affects its ability to compete with undeleted, incompatible plasmids for maintenance in the host cell. The disadvantage of the deleted plasmids seems to be manifested at the level of replication. It results that for plasmid pT181 a sequence dispensable for autonomous maintenance and replication control could affect the outcome of the competition between autonomous, incompatible plasmids.

Base Sequence↗
PubMed1987

Incompatibility between a Rhizobium Sym plasmid and a Ri plasmid of Agrobacterium.

The symbiotic plasmid of Rhizobium trifolii G1008 was mobilized to other Rhizobium strains and to Agrobacterium using Tn5-Mob, a transposon that confers on a host replicon the ability to be mobilized in trans by RP4. Incompatibility was observed between pSymG1008 and the hairy-root-inducing plasmid pRi1855. Agarose gel electrophoresis revealed that pRi1855 was eliminated as an autonomous element in the presence of pSymG1008 and its absence was correlated with loss of the ability to induce hairy root disease. This indicates a close ancestral relationship between a Rhizobium symbiotic plasmid and a plant pathogenic plasmid of Agrobacterium. pSymG1008 and pRi1855 can be assigned to the IncRh-3 incompatibility group. Furthermore, pSymG1008 was mobilized at low frequency to R. phaseoli 51E and the transconjugants isolated had lost the indigenous Sym plasmid and the ability to nodulate beans.

Escherichia coli↗
PubMed1991

The large Bacillus plasmid pTB19 contains two integrated rolling-circle plasmids carrying mobilization functions.

Plasmid pTB19 is a 27-kb plasmid originating from a thermophilic Bacillus species. It was shown previously that pTB19 contains an integrated copy of the rolling-circle type plasmid pTB913. Here we describe the analysis of a 4324-bp region of pTB19 conferring resistance to tetracycline. The nucleotide sequence of this region revealed all the characteristics of a second plasmid replicating via the rolling-circle mechanism. This sequence contained (i) the tetracycline resistance marker of pTB19, which is highly similar to other tetL-genes of gram-positive bacteria; (ii) a hybrid mob gene, which bears relatedness to both the mob-genes of pUB110 and pTB913; (iii) a palU type minus origin identical to those of pUB110 and pTB913; and (iv) a plus origin of replication similar to that of pTB913. A repB-type replication initiation gene sequence identical to that of pTB913 was present, which lacked the middle part (492 bp), thus preventing autonomous replication of this region. The hybrid mob gene was functional in conjugative mobilization of plasmids between strains of Bacillus subtilis.

Amino Acid Sequence↗
PubMed1992

Characterization and properties of a novel plasmid vector for Bacillus thuringiensis displaying compatibility with host plasmids.

A novel plasmid vector, composed of a 1.7-kb Bacillus thuringiensis (B.t.) replicon, a multiple cloning site, and an erythromycin-resistance marker gene from Bacillus subtilis, was constructed for use in B.t. Unlike other vectors which have been reported to be acceptable for B.t., this new B.t. vector was stably maintained in the absence of Er and did not displace host plasmids, some of which carry crystal protein-encoding genes (cry genes). The compatibility of this B.t. vector with native plasmids is highly desirable when introducing new cry genes into a wild-type B.t. strain. When a cryIIIA gene of B.t. tenebrionis was cloned in this vector and introduced into B.t. kurstaki (kur) HD119, cryIIIA was highly expressed without affecting the level of expression of native cry genes. The stability of this vector and its compatibility with native B.t. plasmids were achieved by subcloning only nucleotide sequences required for the vector to replicate in B.t. The origin of replication was first cloned on a 9.6-kb Bg/II fragment from a 75-kb plasmid of B.t. kur HD73 and then localized to a 2.4-kb region within the 9.6-kb fragment. Sequencing of the 2.4-kb region revealed the presence of an open reading frame (ORF), encoding a putative 312-amino acid (aa) protein. The deduced aa sequence of the ORF showed no homology to any published aa sequences. Deletion analysis indicated that the B.t. vector required at least the ORF and up to 300 bp surrounding the ORF, in order to replicate.

Amino Acid Sequence↗
PubMed2005

Transcription of plasmid DNA: influence of plasmid DNA/polyethylenimine complex formation.

Polyethylenimine (PEI) is one of the most potent non-viral vectors. We have developed a lactosylated PEI (Lac-PEI) to enhance cell-specific transfection and have shown that Lac-PEI is more efficient than unsubstituted PEI for gene transfer into immortalized cystic fibrosis airway epithelial SigmaCFTE29o-cells. As both intact PEI/plasmid and Lac-PEI/plasmid complexes are found in the cell nucleus, we have investigated the transcription efficiency of the plasmid complexed with PEI or Lac-PEI, according to the polymer nitrogen/DNA phosphate (N/P) ratio (from 0 to 20). The initiation of transgene transcription was analyzed in an acellular nuclease S1 transcription assay. For both PEI and Lac-PEI complexes, transcription efficiency varied with the N/P ratio of the complexes. Transcription inhibition was observed when plasmid DNA was either loosely (N/P<5) or tightly condensed (N/P>15). For an N/P ratio of 5 and up to 15, transcription of the complexed plasmid was as efficient as that of the free plasmid. Similar results were observed when gene expression was studied after nuclear microinjection of the complexes into SigmaCFTE29o-cells. Our study shows that condensation of DNA influences the accessibility of the plasmid to the transcription machinery. Interestingly, the charge ratios that allow the most efficient transcription are those usually known to be the most efficient for gene transfer in vitro and in vivo.

Chemical Phenomena↗
PubMed2006

The cell-mediated immune response induced by plasmid encoding bovine herpesvirus 1 glycoprotein B is enhanced by plasmid encoding IL-12 when delivered intramuscularly or by gene gun, but not after intradermal injection.

Bovine herpesvirus 1 (BHV-1) causes respiratory and genital infections in cattle. Previously we demonstrated that a DNA vaccine encoding a truncated, secreted form of BHV-1 glycoprotein B (tgB) induces cytotoxic T lymphocyte (CTL) responses in C3H mice. In this study we investigated the potential of interleukin 12 (IL-12) to further enhance the CTL response. C3H mice were immunized with a plasmid encoding tgB or with plasmids encoding tgB and murine IL-12. When the plasmid encoding tgB was delivered intramuscularly or epidermally by a gene gun, co-administration with IL-12 plasmid stimulated the synthesis of more IgG2a, the production of higher levels of IFN-gamma, and more effective killing by CTLs. In contrast, after intradermal delivery no effect of co-administration of IL-12 encoding plasmid was observed. Further investigation suggested that antigen and IL-12 need to be expressed in the draining lymph nodes, where IL-12 can have a direct effect on T cells.

Animals↗
PubMed2002

Prevotella intermedia native plasmid can be mobilized by an Escherichia coli conjugal IncP plasmid.

We have determined the nucleotide sequence of a small Prevotella intermedia cryptic plasmid, pYHBi1, which consisted of sequences that were highly homologous to the amino acid sequence of the replication and mobilization proteins found in related organisms. We have also demonstrated that chimeric plasmids derived from this P. intermedia native plasmid can be mobilized between Escherichia coli strains by using a broad-host-range E. coli conjugative plasmid, IncP plasmid RP4. The results suggest that pYHBi1 possesses gene(s) responsible for conjugal transfer.

Bacterial Proteins↗
PubMed2001

Plasmid DNA encoding caprine interferon gamma inhibits antibody response to caprine arthritis-encephalitis virus (CAEV) surface protein encoded by a co-administered plasmid expressing CAEV env and tat genes.

This study characterized immune responses of Saanen goats co-immunized with pUC18 based plasmids expressing caprine arthritis-encephalitis virus (CAEV) rev-env (pENV) or tat-rev-env (pTAT-ENV) and a second plasmid encoding caprine interferon gamma (IFNgamma). A previous study reported that immunization with pENV or pTAT-ENV induces IgG2 biased antibody responses to plasmid encoded CAEV surface envelope protein (SU). We show here that regional lymph nodes (LN) of pENV and pTAT-ENV immunized goats contain a dominant subset of SU activated IFNgamma+ Th1 lymphocytes. Co-immunization with pENV or pTAT-ENV and a caprine IFNgamma cDNA expression plasmid (pcIFNgamma) did not potentiate activation of SU responsive Th1 lymphocytes. However, the antibody response to SU encoded by pTAT-ENV was inhibited by co-immunization with pcIFNgamma. Results indicate that synergistic effects of CAEV Tat and IFNgamma suppress the primary adaptive B cell response to plasmid encoded SU.

Animals↗
PubMed2003

A LuxR-type regulator from Agrobacterium tumefaciens elevates Ti plasmid copy number by activating transcription of plasmid replication genes.

TraR, a LuxR-type quorum-sensing transcription factor in Agrobacterium tumefaciens, activates genes required for conjugal transfer of the Ti plasmid and also enhances the copy number of a nopaline-type Ti plasmid. Here, we show that TraR increases the copy number of an octopine-type Ti plasmid up to eightfold and that TraR activates transcription of the repABC operon up to 25-fold. The ability of TraR to increase copy number was strictly dependent on several TraR-activated promoters of this operon, indicating that TraR affects copy number solely at the level of transcription. Promoter resections and mRNA transcript analysis revealed the presence of three TraR-dependent promoters. Two TraR-dependent transcription start sites are located 45.5 and 65.5 nucleotides downstream of a site called tra box II, whereas the third start site lies 42.5 nucleotides downstream of a site called tra box III. Purified TraR bound to both tra boxes with comparable affinities, causing moderate DNA bending. TraR bound and bent these two sites independently rather than synergistically. Alteration of tra box III to match the consensus sequence dramatically increased TraR-dependent expression of repABC and plasmid copy number. TraR-dependent elevation of Ti plasmid copy number caused a three- to fourfold increase in plant tumorigenesis.

Agrobacterium tumefaciens↗