Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “plasmid characterization”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed1988

Characterization of plasmid pAZ1 and the type III dihydrofolate reductase gene.

The plasmid pAZ1, which determines trimethoprim and sulfonamide resistance, was characterized by restriction endonuclease mapping. The restriction map was identical to that of the incQ plasmid RSF1010 over a 5.1-kbp region. The type III dihydrofolate reductase gene was cloned, and the DNA sequence was determined. The predicted protein had 162 amino acid residues, and it was more closely related to the gram-negative bacterial chromosomal dihydrofolate reductases than to other plasmid or vertebrate dihydrofolate reductases. Sequence identity was 51% with the Escherichia coli enzyme and 44% with the Neisseria gonorrhoeae enzyme.

Amino Acid Sequence↗
PubMed1979

Characterization of plasmids from antibiotic-resistant Shigella isolates by agarose gell electrophoresis.

Gel electrophoresis of DNA from 95 clinical isolates of Shigella sonnei and Shigella flexneri resistant to antibiotics revealed a heterogeneous plasmid population. Most of the plasmids were smaller than 6 megadaltons (Mdal). Six S. sonnei isolates with the most common antibiotic resistance pattern were characterized. They had two plasmids in common: one was a self-transmissible Fi+ plasmid of 46 Mdal encoding resistance to streptomycin and sulphafurazole. In addition, several cryptic plasmids ranging in size from 1.0 to 24.5 Mdal were present. Mobilization of the 5.5 Mdal SuSm plasmid and a 1.0 Mdal cryptic plasmid was demonstrated with all six S. sonnei isolates during conjugation. This mobilization was mediated by the 46 Mdal self-transmissible Fi+ R plasmid and also by a 24.5 Mdal Fi- plasmid carrying no known drug resistance determinants.

Anti-Bacterial Agents↗
PubMed2006

Molecular characterization of plasmids encoding CTX-M-15 beta-lactamases from Escherichia coli strains in the United Kingdom.

OBJECTIVES: The UK, like other countries worldwide, has a growing problem with CTX-M beta-lactamase-producing Escherichia coli. Five major clonally related strains have been identified among CTX-M-15 producers. We characterize here the plasmids from clonal strains A and D. METHODS: Plasmids were extracted and transformed into E. coli DH5alpha; conjugative mating was attempted on agar. MICs were determined by agar dilution. beta-Lactamases were typed by isoelectric focusing; antibiotic resistance genes and integrons were identified by PCR and sequenced. Plasmid incompatibility groups were determined by replicon PCR. RESULTS: bla(CTX-M-15) was carried by a 150 kb plasmid in strain A and a 70 kb plasmid in strain D. Conjugative transfer of cefotaxime resistance was only achieved from strain D; plasmids from both strains were transferred by transformation. The plasmid from strain A additionally carried bla(TEM-1) (variably), bla(OXA-1), aac(6')-Ib-cr and tet(A), as well as a class 1 integron with the gene cassettes aadA5 and dfr(17); the plasmid from strain D carried bla(TEM-1) consistently, also bla(OXA-1), aac(6')-Ib-cr, aac3-IIa and tet(A). Both plasmids belonged to incompatibility group FII. CONCLUSIONS: bla(CTX-M-15) was plasmid-mediated in both strains A and D and was linked to other antibiotic resistance genes including aac(6')-Ib-cr, which encodes an acetyltransferase, not previously found in Europe, acting on both aminoglycosides and some fluoroquinolones. Although the plasmids from the two strains differed in size, both were related and conferred similar multi-drug resistance phenotypes, suggesting that they may share a similar genetic scaffold. Both shared features with plasmids encoding CTX-M-15 beta-lactamases in E. coli from Canada and India.

Anti-Bacterial Agents↗
PubMedDate not supplied

Characterization of the plasmid pMB1 from Bifidobacterium longum and its use for shuttle vector construction.

The nucleotide sequence of the 1847-bp Bifidobacterium longum B2577 cryptic plasmid pMB1 was determined. The plasmid had a G+C content of 62.0%, and contained two open reading frames, orf1 and orf2, likely arranged in an operon. The proteins encoded by orf1 and orf2 show the highest degree of similarity with similarly arranged peptide sequences translated from Corynebacterium glutamicum pXZ10142 and Mycobacterium fortuitum pAL5000 plasmids. Recombinant plasmids containing the pMB1 replicon were able to replicate in Bifidobacterium animalis MB209. The successful transformation of this strain with pMB1-based plasmids facilitated characterization of this replicon, results of which showed that both orf1 and orf2 are necessary for plasmid replication. A family of new Escherichia coli-B. animalis shuttle plasmids, based on the pMB1 replicon and expressing a cat and an ery gene, was constructed.

Amino Acid Sequence↗
PubMed2000

Characterization of plasmids and formulations for non-viral gene therapy.

Analytical methods are described that provide a platform for systematically evaluating the effect of formulation variables in the development of DNA-lipid non-viral gene therapy complexes. The methods encompass those used to characterize the components, i.e., plasmid DNA and liposomes, as well as those applied to the complexes themselves. The focus is on physicochemical methods so that their parameters can be assessed in relationship to the functionality of the DNA-lipid complexes in vivo.

Animals↗
PubMed2005

Molecular characterization of plasmid pBM300 from Bacillus megaterium QM B1551.

Strain QM B1551 of Bacillus megaterium contains seven compatible plasmids: two small rolling circle plasmids and five theta-replicating plasmids with cross-hybridizing replicons. To expand our understanding of these plasmids, the replicon region (6.7 kb) from pBM300 was cloned, sequenced, and functionally characterized. Sequence analysis showed that the replication protein (RepM300) was highly homologous to two other plasmid Rep proteins of the same strain but to no other known proteins. Furthermore, the location of the replication origin was within the RepM300 coding region, and the origin contained three 12-base direct repeats. Deletion analysis of the replicon confirmed the role of the Rep protein and showed that open reading frame 2 (ORF2) was required for stability. However, the protein encoded by ORF2 is entirely different from the replicon stability proteins encoded by the other two replicons. The entire plasmid was isolated from the plasmid array by integrating a spectinomycin resistance gene and transforming a plasmidless strain, PV361. Complete sequencing showed that pBM300 was 26,300 bp long, had a G+C content of 35.2%, and contained 20 ORFs, two of which encoded proteins that had no similarity to other proteins in the database. The proteins encoded by the plasmid ORFs had similarity to proteins for mobilization and transfer, an integrase, a rifampin resistance protein, a cell wall hydrolase, glutathione synthase, and a biotin carboxylase. The similarities were to several gram-positive genera and a few gram-negative genera and archaea. oriT and ssoT-like regions were detected near two mob genes. These results suggest that pBM300 is a mobilizable hybrid plasmid that confers increased metabolic and germination ability on its host. Its replicon also helps define a new plasmid family.

Bacillus megaterium↗
PubMed1989

Isolation and characterization of altered plasmids in mutant strains of Pseudomonas putida NCIB 9816.

The ability of P. putida NCIB 9816 to grow with naphthalene (Nah+) and salicylate (Sal+) is correlated with the presence of an 83 kilobase (kb) conjugative plasmid, pDTG1. Derivatives of pDTG1 were obtained from cells after exposure to halogenated analogs of naphthalene or salicylate. The selection of mutants having a Nah-Sal- or a Nah-Sal+ phenotype could be enhanced by the addition of triphenyltetrazolium chloride to the indicator medium. Structurally modified plasmids were characterized by restriction endonuclease digestion and Southern hybridization experiments. The region of pDTG1 DNA that encodes the enzymes responsible for the conversion of naphthalene to salicylate was identified. The structural changes in mutant plasmids were correlated with the absence of essential enzymatic activities.

Blotting, Southern↗
PubMed1984

Isolation of uvrA mutation on a multicopy plasmid: preliminary characterization of the mutant protein.

A new uvrA mutation (uvrA276) has been isolated on a multicopy plasmid and shown to reside within the region of the uvrA gene defined by the KpnI to SalI endonuclease sites. The protein produced by the uvrA276 mutant gene is identical in size to the wild-type protein and binds to single-stranded DNA under the same conditions as the wild-type protein. However, extracts prepared from strains containing this mutant are deficient at incision of DNA that has been irradiated with UV light.

Bacterial Proteins↗
PubMed1986

Mapping and cloning of Corynebacterium diphtheriae plasmid pNG2 and characterization of its relatedness to plasmids from skin coryneforms.

The relationship of plasmid pNG2, isolated from an erythromycin-resistant strain of Corynebacterium diphtheriae, to plasmids isolated from skin coryneforms was examined. The extent of homology between plasmids from erythromycin-resistant and -susceptible skin coryneforms and pNG2 varied, but in aggregate homology was observed with all six BstEII fragments of pNG2. The data support the hypothesis that pNG2 originated in skin coryneforms. Intact plasmid pNG2 and some of its restriction fragments were cloned into Escherichia coli JM109. The erythromycin resistance phenotype was expressed in clones carrying intact pNG2 as well as in some of its fragments and appeared to depend on a C. diphtheriae promoter for expression. A 2.5-megadalton EcoRI fragment, the smallest expressing resistance, contained the 1.2-megadalton region of pNG2 which is deleted when the erythromycin-resistant strain of C. diphtheriae reverts spontaneously to the susceptible state.

Chromosome Mapping↗
PubMed1978

Characterization of plasmids that encode for the K88 colonization antigen.

K88 antigen, and important virulence factor in porcine enteropathogenic Escherichia coli (EEC), can be transferred along with the ability to ferment the trisaccharide raffinose (Raf). The plasmids from a number of EEC strains that encode these two properties were isolated and characterized. In most strains the K88 and Raf genes were found on a single nonconjugative plasmid approximately 50 x 10(6) daltons in size. This plasmid core was conserved with only slight variation among the strains tested. In some transconjugants, larger conjugative plasmids were observed that were apparently recombinants between the Raf/K88 plasmid and a transfer fa(tor. Occasionally plasmids carrying only the raffinose fermentation genes arose by deletion of a deoxyribonucleic acid segment of about 20 x 10(6) daltons that included the K88 antigen gene(s).

Animals↗
PubMed1993

Molecular characterization of plasmids in Salmonella enteritidis phage types.

Plasmids in selected type strains of 26 of the Salmonella enteritidis phage types have been characterized by restriction enzyme fingerprinting and by DNA-DNA hybridization with oligonucleotide probes for Salmonella plasmid virulence (Spv) genes. With one exception, the fingerprints of the 38 MDa plasmids studied were homogeneous but there was heterogeneity in the fingerprints of 59 MDa plasmids found in 4 of the type strains. However all 38 MDa and 59 MDa plasmids were related as was a 45 MDa plasmid identified in the type strain of phage type 19. A 3.5 kb fragment homologous to SpvC was conserved in Hind III digests of all 38 MDa and 59 MDa plasmids, and in the related 45 MDa plasmid. In contrast a 65 MDa plasmid found in the type strain of phage type 10 was not related to these three plasmid molecular weight groups and did not carry the SpvC gene.

Base Sequence↗
PubMed1987

The origin of transfer (oriT) of the conjugative plasmid R46: characterization by deletion analysis and DNA sequencing.

The origin of transfer (oriT) is the sequence within which conjugal transfer of plasmid DNA is initiated, and is absolutely required in cis for plasmid mobilization. We have cloned oriT from the 52 kb IncN plasmid R46 on a 600 bp fragment, and mapped the limits of the relevant sequence by deletion analysis and transposon mutagenesis. The nucleotide sequence of the oriT region contains 13 direct repeats of an 11 bp consensus sequence, 3 different pairs of 10 bp inverted repeats, and a segment that is extremely A-T rich. The direct repeats are within a region required for high frequency transfer and their sequence is such that their periodic alignment along the helix may induce curvature of the DNA. Analysis of Tn1725 insertions within the sequenced fragment of R46 revealed that, unlike most other transposons, transposition of Tn1725 can cause target sequence duplications of three different sizes.

Base Sequence↗
PubMedDate not supplied

Isolation, characterization, and plasmid pUPI126-mediated indole-3-acetic acid production in acinetobacter strains from rhizosphere of wheat.

Thirty-seven strains of Acinetobacter isolated and characterized from rhizosphere of wheat were screened for indole-3-acetic acid (IAA) production. Only eight Acinetobacter strains showed IAA production. The genus Acinetobacter was confirmed by chromosomal DNA transformation assay. Biotyping of eight strains was carried out and they were found to be genospecies of A. junii, A. baumannii, A. genospecies 3, and A. haemolyticus. Five of eight strains produced IAA at the early stationary phase: A. haemolyticus (A19), A. baumannii (A18, A16, A13), and Acinetobactergenospecies 3 (A15). A. junii A6 showed maximum IAA production at log phase and A. genospecies 3 and A. baumannii (A28, A30) at late stationary phase. IAA was extracted by ethyl acetate and purified by preparative thin-layer chromatography. Purified IAA was confirmed by 1H-nuclear magnetic resonance and infrared spectrum analysis. Pot experiments showed a significant increase in plant growth inoculated with eight Acinetobacter genospecies as compared to control plants. IAA production was found to be encoded by plasmid pUPI126. All eight strains of Acinetobacter contain a plasmid pUPI126 with a molecular weight of 40 kb. Plasmid pUPI126 was transformed into Escherichia coli HB101 at a frequency of 5 x 10(-5), and E. coli HB101 (pUPI126) transformants also showed IAA activity. PUPI126 also encoded resistance to selenium, tellurium, and lead. This is the first report of plasmid-encoded IAA production in the genus Acinetobacter.

Acinetobacter↗
PubMed1999

Characterization and plasmid profile of an inhibitory strain of Erwinia herbicola isolated from Phaseolous vulgaris in Egypt.

Erwinia herbicola strain 48 was isolated from diseased phaseolous seedlings and characterized by biochemical properties, cellular fatty acid analysis and SDS-PAGE of the soluble cell protein. Although cellular fatty acid profile and the soluble cellular protein pattern showed high degree of similarity in comparison to those from E. herbicola strain 347417, obtained from the International Mycological Institute U.K., plasmid profiles were different. Both strains harbor a 23.1 kb plasmid, in addition, E. herbicola 48 contains 2 more plasmids (26.8 and 32.5 kb). The antagonism of E. herbicola 48 against a number of Gram-negative and Gram-positive bacteria was tested in vitro. Only Gram-negative bacteria were inhibited, suggesting that the inhibitory factor is likely to be bacteriocin.

Antibiosis↗
PubMed1992

Characterization of plasmids with antimicrobial resistant genes in Pasteurella haemolytica A1.

Two R plasmids, pYFC1 and pYFC2, from Pasteurella haemolytica A1 encoding sulfonamide, streptomycin (pYFC1), and ampicillin (pYFC2) resistances have been characterized by restriction endonuclease digestions, subcloning or DNA sequencing. pYFC1 consists of 4225 bp and is 51.9% in AT content. Physical mapping indicated a highly conserved region of restriction sites among pYFC1, RSF1010, pGS05, pFM739, pHD148 and pGS03B. pYFC1 encoded a dihydropteroate synthase (29.8 kDa), and streptomycin kinase (29.6 kDa) which is homologous in nucleotide sequences or deduced amino acid sequence to that encoded by a broad-host range IncQ plasmid RSF1010. Based on the primary structure of pYFC1, the sulfonamide and streptomycin genes are derived from the same ancestor of RSF1010. pYFC2 is similar to the plasmid from P. haemolytica LNPB51 isolated in France by partial restriction enzyme mapping. pYFC1 and pYFC2 have the same size of 4.2 kbp.

Amino Acid Sequence↗
PubMed1996

Characterization of plasmid DNA transfer into mouse skeletal muscle: evaluation of uptake mechanism, expression and secretion of gene products into blood.

The expression of naked plasmid DNA coding for firefly luciferase (pRSVluc) or a secreted protein, human-alpha-1-antitrypsin (pRcCMVhAAT) in mouse skeletal muscle was characterized following administration by an improved intramuscular injection technique. Injection guided by intense illumination along the longitudinal axis of the mouse quadriceps muscle and parallel to the myofibers yielded 200-fold higher levels of luciferase expression than perpendicular injection. Luciferase expression was inhibited by an excess of non-coding DNA or dextran sulfate suggesting that muscle DNA uptake mechanism(s) can be saturated. Injected plasmid DNA was rapidly eliminated from the muscle as evidenced by tissue distribution studies of radiolabeled hAAT plasmid and Southern analysis. However, PCR analysis demonstrated that hAAT cDNA persisted in the muscle for at least 1 month after injection. Immunohistochemistry techniques indicated that the hAAT gene was expressed by the muscle fibers. ELISA analysis of serum samples collected from intramuscularly injected mice demonstrated that secreted hAAT protein concentration peaked in serum by day 7, started to decline by day 14 and was barely detectable 21 days post-injection. RT-PCR analysis demonstrated that hAAT transcript persisted at the site of injection for at least 1 month indicating that the decline of serum hAAT concentration 21 days post-injection was not due to the absence of hAAT transcript. However, the decline of hAAT protein concentration in the serum was inversely correlated with accumulation of murine anti-hAAT antibodies in circulation.

Adenosine Triphosphate↗
PubMed1978

[Characterization of plasmid DNA in a lecithinase-positive and in a lecithinase-negative strain of Clostridium perfringens (author's transl)].

A non pathogenic variant of Clostridium perfringens and the wild type strain were characterized. The strains agreed in most of the biochemical properties, in susceptibility against antibiotics and in bacteriocin production. Contrary to the wild type the variant did not produce lecithinase and gelatinase. In deoxyribonucleic acid (DNA) of both strains centrifuged in cesiumchlorid-ethidiumbromide equilibrium there was found a satellite peak containing three distinct, covalently closed circular (CCC) DNA elements. The sum of the average molecular weight or contour length of the two small circular molecules was equal to the average molecular weight or contour length of the third. The presence of the plasmids in the variant indicated that the synthesis of lecithinase might not be coded by a plasmid.

Bacteriocins↗
PubMed1981

The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein.

(1) Total poly(A)-containing RNA isolated from human thyroid medullary carcinoma tissue was shown to direct the synthesis in the wheat germ cell-free system of a major (Mr 21000) and several minor forms of human calcitonin precursor polyproteins. Evidence for processing of these precursor(s) by the wheat germ cell-free system is also presented. (2) A small complementary DNA (cDNA) plasmid library has been constructed in the PstI site of the plasmid pAT153, using total human thyroid medullary carcinoma poly(A)-containing RNA as the starting material. (3) Plasmids containing abundant cDNA sequences were selected by hybridization in situ, and two of these (ph T-B3 and phT-B6) were characterized by hybridization--translation and restriction analysis. Each was shown to contain human calcitonin precursor polyprotein cDNA sequences. (4) RNA blotting techniques demonstrate that the human calcitonin precursor polyprotein is encoded within a mRNA containing 1000 bases. (5) The results demonstrate that human calcitonin is synthesized as a precursor polyprotein.

Calcitonin↗