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Results for “plasmid characterization”

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PubMed1985

Worldwide distribution of the conjugative Clostridium perfringens tetracycline resistance plasmid, pCW3.

The aim of this study was to test the hypothesis that all conjugative R-plasmids of Clostridium perfringens are closely related to the previously characterized tetracycline resistance plasmid, pCW3. Fourteen conjugative R-plasmids derived from 11 C. perfringens strains isolated in Australia, the United States, France, Belgium, and Japan were analyzed. Eleven of the plasmids encoded tetracycline resistance while three carried both tetracycline and chloramphenicol resistance. Each of these plasmids was compared, by restriction analysis, to the reference plasmid, pCW3. Seven of the tetracycline resistance plasmids had EcoRI, XbaI, and ClaI restriction profiles that were identical to those of the corresponding pCW3 digests. The seven remaining R-plasmids were different from pCW3. Comparison of partial restriction maps of these plasmids with a complete map of pCW3 indicated that they contained at least 17 kb of DNA that also was present in pCW3. Hybridization analysis confirmed that these plasmids shared substantial homology with pCW3. The three tetracycline and chloramphenicol resistance plasmids frequently lost a 6-kb chloramphenicol resistance segment during conjugation. Cloning experiments showed that the chloramphenicol resistance determinant was expressed in Escherichia coli and that the chloramphenicol resistance gene of one of these plasmids, pIP401, was contained within a 1.5-kb region of the 6-kb deletion segment. Hybridization analysis indicated that the deletion segment of pIP401 was related to those of the other two chloramphenicol resistance plasmids. During the course of this study, conjugative R-plasmids which appear to be identical to pCW3 or closely related to pCW3 were identified from C. perfringens strains from human, animal and environmental sources in five countries. It is concluded that C. perfringens strains in humans and animals throughout the world have overlapping gene pools and that all the conjugative C. perfringens R-plasmids examined probably evolved from a pCW3-like element.

Animal Diseases↗

PubMed2006

Electrophoretic behavior of plasmid DNA in the presence of various intercalating dyes.

In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.

3T3 Cells↗

PubMed1992

The relapsing fever agent Borrelia hermsii has multiple copies of its chromosome and linear plasmids.

Borrelia hermsii, a spirochete which causes relapsing fever in humans and other mammals, eludes the immune response by antigenic variation of the "Vmp" proteins. This occurs by replacement of an expressed vmp gene with a copy of a silent vmp gene. Silent and expressed vmp genes are located on separate linear plasmids. To further characterize vmp recombination, copy numbers were determined for two linear plasmids and for the 1-megabase chromosome by comparing hybridization of probes to native DNA with hybridization to recombinant plasmids containing borrelial DNA. Plasmid copy numbers were also estimated by ethidium bromide fluorescence. Total cellular DNA content was determined by spectrophotometry. For borrelias grown in mice, copy numbers and 95% confidence intervals were 14 (12-17) for an expression plasmid, 8 (7-9) for a silent plasmid, and 16 (13-18) for the chromosome. Borrelias grown in broth medium had one-fourth to one-half this number of plasmids and chromosomes. Staining of cells with 4',6-diamidino-2-phenylindole revealed DNA to be distributed throughout most of the spirochete's length. These findings indicate that borrelias organize their total cellular DNA into several complete genomes and that cells undergoing serotype switches do one or more of the following: (1) coexpress Vmps from switched and unswitched expression plasmids for at least three to five generations, (2) suppress transcription from some expression plasmid copies, or (3) partition expression plasmids nonrandomly. The lower copy number of the silent plasmid indicates that nonreciprocal Vmp gene recombination may result from loss of recombinant silent plasmids by segregation.

Bacterial Outer Membrane Proteins↗

PubMed1999

Nuclear targeting of plasmid DNA in human corneal cells.

PURPOSE: To characterize the mechanisms of plasmid DNA nuclear localization in primary cultures of human corneal epithelial cells and keratocytes. METHODS: Purified, supercoiled plasmid DNA was microinjected into the cytoplasm of human corneal epithelial cells and keratocytes that had been established from donor corneas two to three passages previously, and localized 8 hours later by in situ hybridization. To confirm the sequence-specificity of nuclear import observed in microinjected cells, liposome-mediated transient transfection experiments also were performed on human corneal epithelial cell and keratocyte cultures. RESULTS: Primary cultures of human corneal epithelial cells and keratocytes have the capacity to transport plasmid DNA from the cytoplasm to the nucleus in the absence of cell division. This transport activity is sequence-dependent requiring portions of the simian virus 40 (SV40) early promoter and enhancer. The majority of this nuclear transport activity resides within the enhancer domain of the SV40 DNA, a region rich in transcription factor binding sites. This DNA nuclear import sequence also manifested itself in liposome-mediated transfection experiments, causing a greater than 2-fold increase in reporter gene expression in human corneal cells in a beta-galactosidase-expressing vector and up to a 1000-fold increase in a luciferase-expressing vector when compared to similar expression plasmids lacking the sequence. CONCLUSION: These results demonstrate that primary, non-transformed human corneal epithelial cells and keratocytes display sequence-specific nuclear import of plasmid DNA in the absence of mitosis. The small sequence that mediates nuclear localization of plasmids is active both in microinjected and cationic liposome transfected cells, and leads to increased gene expression. Thus, inclusion of this DNA sequence into non-viral vectors should improve the efficiency of ocular gene transfer in vivo.

Base Sequence↗

PubMed1979

Insertion element analysis and mapping of the Pseudomonas plasmid alk regulon.

We characterized and mapped new mutations of the alk (alkane utilization) genes found on Pseudomonas plasmids of the Inc P-2 group. These mutations were isolated after (i) nitrosoguanidine mutagenesis, (ii) transposition of the Tn7 trimethoprim and streptomycin resistance determinant, and (iii) reversion of polarity effects of alk::Tn7 insertion mutations. Our results indicate the existence of two alk loci not previously described--alkD, whose product is required for synthesis of membrane alkane-oxidizing activities, and alkE, whose product is required for synthesis of inducible membrane alcohol dehydrogenase activity. Polarity of alk::Tn7 insertion mutations indicates the existence of an alkBAE operon. Mapping of alk loci by transduction in P. aeruginosa shows that there are at least three alk clusters in the CAM-OCT plasmid--alkRD, containing regulatory genes; alkBAE, containing genes for specific biochemical activities; and alkC, containing one or more genes needed for normal synthesis of membrane alcohol dehydrogenase. The alkRD and alkBAE clusters are linked but separated by about 42 kilobases. The alkC cluster is not linked to either of the other two alk regions. Altogether, these results indicate a complex genetic control of the alkane utilization phenotype in P. putida and P. aeruginosa involving at least six separate genes.

Alkanes↗

PubMed1989

Identification and characterization of a locus inhibiting extrachromosomal maintenance of the Streptomyces plasmid SLP1.

We report here the existence and initial characterization of a genetic locus (imp) that inhibits maintenance of SLP1-derived plasmids as extrachromosomal replicons in a manner distinct from normal incompatibility between autonomous SLP1 replicons. The trans-acting imp function has been localized to a 1.8 kb Eco47III restriction fragment present on integrated SLP1 elements. At least part of this DNA segment is absent from SLP1-derived plasmids. DNA sequence analysis of the imp region indicates that it contains three overlapping open reading frames (ORFs) that may constitute a polycistronic operon. The effects of insertions within the imp region indicate that uninterrupted transcription through all three ORFs is necessary for imp activity.

Amino Acid Sequence↗

PubMed2006

Characterization of the transcriptional activity of the cryptic plasmid pRN1 from Sulfolobus islandicus REN1H1 and regulation of its replication operon.

The plasmid pRN1 from Sulfolobus islandicus REN1H1 belongs to the crenarchaeal plasmid family pRN. The plasmids in this family encode three conserved proteins that participate in plasmid replication and copy number regulation, as suggested by biochemical characterization of the recombinant proteins. In order to deepen our understanding of the molecular biology of these plasmids, we investigated the transcriptional activity of the model plasmid pRN1. We detected five major transcripts present at about 2 to 15 copies per cell. One long transcriptional unit comprises the genes for the plasmid-copy-number control protein Orf56/CopG and the replication protein Orf904. A second transcript with a long 3'-untranslated region codes for the DNA binding protein Orf80. For both transcripts, we identified countertranscripts which could play a regulatory role. The function of the fifth transcript is unclear. For the five transcripts, we determined the start site, the transcript end, the stability, and the abundance in different growth phases. Reporter gene experiments demonstrated that the copy number control protein Orf56 represses transcription of the orf56-orf904 cotranscript in vivo.

Base Sequence↗

PubMed1984

Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR.

A 5.4-kilobase region containing the origin of replication and stability maintenance of the 44-kilobase Agrobacterium tumefaciens plasmid pTAR has been mapped and characterized. Within this region is a 1.3-kilobase segment that is capable of directing autonomous replication. The remaining segment contains the stability locus for maintenance of pTAR during nonselective growth. Approximately 35% of pTAR shares sequence homology with pAg119, a 44-kilobase cryptic plasmid in grapevine strain 1D1119. However, no homology was detected between pTAR DNA and several Ti plasmids or several other small cryptic plasmids in many A. tumefaciens strains. A recombinant plasmid containing the origin of replication and stability maintenance region of pTAR was compatible with pTiC58, pTi15955, and pTi119 and incompatible with pAg119. A new compatibility group, Inc Ag-1, is discussed.

Base Sequence↗

PubMed1991

Improved broad-host-range lac-based plasmid vectors for the isolation and characterization of protein fusions in Pseudomonas aeruginosa.

Several new broad-host-range vectors for the construction of protein fusions to the Escherichia coli lacZ gene have been developed. In all of the constructs, a multiple cloning site (MCS) containing unique restriction sites is located upstream of lac operon segments whose lacZ genes lack translational start signals. Some of the vectors (pPZ10, pPZ20 and pPZ30) also contain transcriptional terminators upstream of the MCS. The new vectors allow the fusion of genes to lacZ in all translational reading frames. Due to a higher copy number they allow direct screening in E. coli for weakly expressed foreign promoters. Their usefulness for gene analysis in Pseudomonas aeruginosa was demonstrated by construction and expression of a regA'::'lacZ-encoded protein fusion.

Amino Acid Sequence↗

PubMed1985

Characterization and sequence analysis of pilin from F-like plasmids.

Conjugative pili are expressed by derepressed plasmids and initiate cell-to-cell contact during bacterial conjugation. They are also the site of attachment for pilus-specific phages (f1, f2, and QB). In this study, the number of pili per cell and their ability to retract in the presence of cyanide was estimated for 13 derepressed plasmids. Selected pilus types were further characterized for reactivity with anti-F and anti-ColB2 pilus antisera as well as two F pilus-specific monoclonal antibodies, one of which is specific for a sequence common to most F-like pilin types (JEL92) and one which is specific for the amino terminus of F pilin (JEL93). The pilin genes from eight of these plasmids were cloned and sequenced, and the results were compared with information on F, ColB2, and pED208 pilin. Six pilus groups were defined: I, was F-like [F, pED202(R386), ColV2-K94, and ColVBtrp]; IIA was ColB2-like in sequence but had a lowered sensitivity to f1 phage due to its decreased ability for pilus retraction [pED236(ColB2) and pED203(ColB4)]; IIB was ColB2-like but retained f1 sensitivity [pED200(R124) and pED207(R538-1)]; III contained R1-19, which had a ColB2-like amino terminus but had an additional lysine residue at its carboxy terminus which may affect its phage sensitivity pattern and its antigenicity; IV was R100-1-like [R100-1 and presumably pED241(R136) and pED204(R6)] which had a unique amino-terminal sequence combined with a carboxy terminus similar to that of F. pED208(Folac) formed group V, which was multipiliated and exhibited poor pilus retraction although it retained full sensitivity to f1 phage. The pED208 pilin gene could not be cloned at this time since it shared no homology with the pilin gene of the F plasmid.

Amino Acid Sequence↗

PubMed1997

Characterization of endemic strains of Shigella sonnei by use of plasmid DNA analysis and pulsed-field gel electrophoresis to detect patterns of transmission.

Shigellosis is hyperendemic in Utah. Most isolates are Shigella sonnei, making it difficult to identify epidemiologic clustering. To better define transmission, molecular markers and epidemiologic data were examined for 90 cases. Plasmid analysis and pulsed-field gel electrophoresis (PFGE) of the S. sonnei isolates identified 11 and 4 patterns, respectively. Plasmid pattern I infections occurred in 8 day care centers over a 6-month period, suggesting spread between centers. Plasmid pattern III was isolated from children at 3 additional centers and pattern IV was associated with another day care center, suggesting different outbreaks. By PFGE, plasmid groups I and XI appeared identical, as were plasmid groups II and V; plasmid group X had a unique pattern. Plasmid groups III, IV, and VII-IX were closely related PFGE subtypes. Both plasmid analysis and PFGE allow better characterization of S. sonnei transmission patterns of "endemic" strains and could lead to improved control measures.

DNA, Bacterial↗

PubMed1987

A variant of CRPV DNA preferentially maintained as a plasmid in NIH 3T3 cells and characterization of its transcripts in nude mouse tumors.

Rabbit and human papillomaviruses are strictly epitheliotropic and their DNA replicates extrachromosomally in benign lesions (warts) of their natural host. Their tissue and host specificity is thought to be genetically controlled and may account for the inefficiency in transforming heterologous cells. In our hands, CRPV DNA did not induce foci in NIH 3T3 cells and in cotransfections with a selectable marker only integrated DNA was found. The viral DNA appeared to be transcriptionally inactive since no transcripts could be detected and in cells were not tumorigenic for nude mice. In contrast to these results a spontaneously derived CRPV variant DNA was able to replicate extrachromosomally and a majority of focus-derived cell lines were tumorigenic for nude mice. In nude mouse tumors the variant DNA remained exclusively extrachromosomal and viral transcripts were detected. The sizes of the major transcripts were 2.0 and 1.3 kb and this suggested that E6 and E7 were expressed. The situation in rabbit tissue was different; both variant and wild-type DNA were maintained extrachromosomally. The extrachromosomal maintenance of the variant but not of wild-type DNA in mouse cells suggests that the variant lacks sequences which may play a role in the host and the tissue restriction of CRPV. The deletion in the variant DNA was located in the late region and included most or all of L1 and a carboxy terminal segment of L2. A second deletion eliminated some pBR322 sequences.

Animals↗

PubMed1999

Preparation and characterization of poly (D,L-lactide-co-glycolide) microspheres for controlled release of poly(L-lysine) complexed plasmid DNA.

PURPOSE: To produce and characterize controlled release formulations of plasmid DNA (pDNA) loaded in poly (D,L-lactide-co-glycolide) (PLGA) microspheres both in free form and as a complex with poly (L-lysine). METHODS: Poly (L-lysine) (PLL) was used to form pDNA/PLL complexes with complexation ratio of 1:0.125 and 1:0.333 w/w to enhance the stability of pDNA during microsphere preparation and protect pDNA from nuclease attack. pDNA structure, particle size, zeta potential, drug loading, in vitro release properties, and protection from DNase I were studied. RESULTS: The microspheres were found to be spherical with average particle size of 3.1-3.5 microm. Drug loading of 0.6% was targeted. Incorporation efficiencies of 35.1% and 29.4-30.6% were obtained for pDNA and pDNA/PLL loaded microspheres respectively. Overall, pDNA release kinetics following the initial burst did not correlate with blank microsphere polymer degradation profile suggesting that pDNA release is convective diffusion controlled. The percentage of supercoiled pDNA in the pDNA and pDNA/PLL loaded microspheres was 16.6 % and 76.7-85.6% respectively. Unencapsulated pDNA and pDNA/PLL degraded completely within 30 minutes upon the addition of DNase I. Encapsulation of DNA/PLL in PLGA microspheres protected pDNA from enzymatic degradation. CONCLUSIONS: The results show that using a novel process, pDNA can be stabilized and encapsulated in PLGA microspheres to protect pDNA from enzymatic degradation.

Biocompatible Materials↗

PubMed1996

Plasmid-mediated formaldehyde resistance in Escherichia coli: characterization of resistance gene.

The formaldehyde resistance mechanisms in the formaldehyde-resistant strain Escherichia coli VU3695 were investigated. A large (4.6-kb) plasmid DNA fragment encompassing the formaldehyde resistance gene was sequenced. A single 1,107-bp open reading frame encoding a glutathione- and NAD-dependent formaldehyde dehydrogenase was identified and sequenced, and the enzyme was expressed in an in vitro assay and purified. Amino acid sequence homology studies showed 62.4 to 63.2% identity with class III alcohol dehydrogenases isolated from horse, human, and rat livers. We demonstrated that the resistance mechanism in the formaldehyde-resistant strain E. coli VU3695 and in other formaldehyde-resistant members of the family Enterobacteriaceae is based on the enzymatic degradation of formaldehyde by a formaldehyde dehydrogenase.

Aldehyde Oxidoreductases↗

PubMed1997

Isolation and characterization of a ColE1-like plasmid from Enterobacter agglomerans with a novel variant of rom gene.

Complete nucleotide sequence of a plasmid isolated from Enterobacter agglomerans has been determined. The plasmid, called pPIGDM1, consists of 2495 base pairs. The analysis of its nucleotide sequence suggested that pPIGDM1 may be a ColE1-like replicon. We confirmed this hypothesis by constructing a pPIGDM1-derived plasmid harboring the cat gene (pBW4), which could be introduced into Escherichia coli cells, and demonstrating that pBW4 cannot replicate in the absence of the polA function and that its copy number is significantly decreased in the pcnB mutant. Like some other ColE1-type replicons (e.g., pBR322), pPIGDM1-derived plasmids can be amplified both by chloramphenicol method and in isoleucine-starved relA mutants but not in relA+ bacteria. Inactivation of the putative rom gene by insertion of an amplicillin-resistance gene resulted in significant increase in pPIGDM1-derived plasmid copy number in E. coli-despite the fact that amino acid sequence of the putative RNA 1 modulator (Rom) protein is only 55.7% identical to the ColE1 analog. The pPIGDM1-derived rom-like coding sequence is also homologous to the rom-like gene present in the Proteus vulgaris plasmid pPvul. We suggest to group all these gene products into a new family called ROMS (RNA one modulators). Since a pPIGDM1-derived plasmid is compatible with other ColE1-like replicons (pMB1-, p15A, RSF1030-, and CloDF13-derived) in E. coli, one may consider pPIGDM1 as a progenitor of new cloning vehicles compatible with most (if not all) of currently used plasmid vectors. Moreover, this plasmid may serve as a source of the new rom-like gene coding for a protein useful in investigation of RNA-protein interactions. A role for the pPIGDM1 plasmid in the host strain is not known.

Amino Acid Sequence↗

PubMed1987

Molecular cloning and physical characterization of a Brassica linear mitochondrial plasmid.

A linear mitochondrial plasmid reported to be associated with cytoplasmic male sterility in the genus Brassica was analyzed. A protein was found to be associated with the 5' ends of the plasmid. The entire plasmid was cloned by the homopolymer tailing technique via free hydroxyl groups present at its 3' ends. DNA sequence analysis of the cloned plasmid revealed a perfect terminal inverted repeat of 325 base pairs. Southern hybridization and restriction enzyme mapping analysis confirmed colinearity of the native plasmid and the clone, which showed significant homology with organelle DNA but not with nuclear DNA. Under high-stringency hybridization conditions, an internal 4.6 kb fragment of the 11.5 kb plasmid hybridized to the main mitochondrial genome in several species. Although the hybridization signal was weaker, the chloroplast genome also showed homology to the mitochondrial plasmid. The plasmid was undetectable at a molar ratio of less than 1/10 000 of the main mitochondrial genome in some lines of Brassica and Raphanus that contain the Ogura male sterile cytoplasm (cms). The absence of the plasmid in these sterile lines demonstrates that the plasmid is not required for the expression and maternal inheritance of male sterility.

Base Sequence↗

PubMed1981

Isolation and characterization of new copy mutants of plasmid R1, and identification of a polypeptide involved in copy number control.

Site-specific deletions and insertions in the replication region of plasmid R1 have generated a new class of copy mutants that are present in the cell with 10-15-fold increased copy number. All mutations described inactivate a copy number control gene which is distinct from another cop inc gene that was identified previously (Molin and Nordström 1980). Insertion of the lac operon lacking the normal lac promoter has been used to determine the direction of transcription of this cop gene. The mutants may all be complemented by wild-type plasmid derivatives and are thus recessive. In incompatibility tests with wild-type R1 plasmids, these mutants are indistinguishable from the wild-type plasmid. It therefore seems that this cop function does not play an important role for the incompatibility function. A polypeptide, molecular weight 11,000, has been identified as being the product of this cop gene.

Bacterial Proteins↗

PubMed1987

Nucleotide sequence and molecular characterization of a maize mitochondrial plasmid-like DNA.

The mitochondrial genome of Black Mexican Sweet maize consists of the principal genome, a 2.3 kb minilinear DNA, a 1,913 bp (1.9 kb) and a 1,445 bp (1.4 kb) minicircular DNA. The three extrachromosomal DNAs exhibit characteristics of autonomous replication in cell suspension culture. The complete sequence of the 1.4 kb minicircle was determined. It has 61 bp of near perfect sequence homology to the 1.9 kb minicircle. Both minicircular DNAs are transcriptionally active; the longest open reading frame of the 1.4 kb minicircle was 231 bp. A putative origin of replication was identified as a high A + T sequence. These minicircles were present in some but not all of 20 maize lines surveyed. None of the lines examined carried the 1.4 kb minicircle without the 1.9 kb minicircle. Nuclear DNA of one line of the seven examined carried homology to both DNAs.

Cells, Cultured↗