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At least 451 records · Page 25Linked to original sources
PubMed1998

Characterization of a theta plasmid replicon with homology to all four large plasmids of Bacillus megaterium QM B1551.

A replicon from one of an array of seven indigenous compatible plasmids of Bacillus megaterium QM B1551 has been cloned and sequenced. The replicon hybridized with all four of the large plasmids (165, 108, 71, and 47 kb) of strain QM B1551. The cloned 2374-bp HindIII fragment was sequenced and contained two upstream palindromes and a large (>419-amino-acid) open reading frame (ORF) truncated at the 3' end. Unlike most plasmid origins, a region of four tandem 12-bp direct repeats was located within the ORF. The direct repeats alone were incompatible with the replicon, suggesting that they are iterons and that the plasmid probably replicates by theta replication. The ORF product was shown to act in trans. A small region with similarity to the B. subtilis chromosomal origin membrane binding region was detected as were possible binding sites for DnaA and IHF proteins. Deletion analysis showed the minimal replicon to be a 1675-bp fragment containing the incomplete ORF plus 536 bp upstream. The predicted ORF protein of >48 kDa was basic and rich in glutamate + glutamine (16%). There was no significant amino acid similarity to any gene, nor were there any obvious motifs present in the ORF. The data suggest that this is a theta replicon with an expressed rep gene required for replication. The replicon contains its iterons within the gene and has no homology to reported replicons. It is the first characterization of a B. megaterium replicon.

Amino Acid Sequence↗
PubMed1997

Characterization of pBP614, a putative rolling-circle plasmid from Bacillus popilliae.

A plasmid, pBP614 (5.647 kb), has been isolated from the New Zealand Bacillus popilliae strain 17 and characterized by physical mapping, cloning, and sequencing. An open reading frame was found which could encode a protein with homology to the replication (Rep) proteins of rolling-circle plasmids. The predicted B. popilliae Rep protein shows closest homology to Rep proteins from Bacillus plasmids of the pC194 family. Sequences homologous to plus- and minus-strand replication origins were found. The minus-strand origin shows similarities to the pal-T type family. Taken together, these observations suggest that pBP614 is a rolling-circle plasmid, the first such plasmid characterized from B. popilliae.

Amino Acid Sequence↗
PubMed1999

Stabilized plasmid-lipid particles: construction and characterization.

A detergent dialysis procedure is described which allows encapsulation of plasmid DNA within a lipid envelope, where the resulting particle is stabilized in aqueous media by the presence of a poly(ethyleneglycol) (PEG) coating. These 'stabilized plasmid-lipid particles' (SPLP) exhibit an average size of 70 nm in diameter, contain one plasmid per particle and fully protect the encapsulated plasmid from digestion by serum nucleases and E. coli DNase I. Encapsulation is a sensitive function of cationic lipid content, with maximum entrapment observed at dioleoyldimethylammonium chloride (DODAC) contents of 5 to 10 mol%. The formulation process results in plasmid-trapping efficiencies of up to 70% and permits inclusion of 'fusigenic' lipids such as dioleoylphosphatidylethanolamine (DOPE). The in vitro transfection capabilities of SPLP are demonstrated to be strongly dependent on the length of the acyl chain contained in the ceramide group used to anchor the PEG polymer to the surface of the SPLP. Shorter acyl chain lengths result in a PEG coating which can dissociate from the SPLP surface, transforming the SPLP from a stable particle to a transfection-competent entity. It is suggested that SPLP may have utility as systemic gene delivery systems for gene therapy protocols.

Animals↗
PubMed1996

Cloning, characterization and properties of plasmids containing CGG triplet repeats from the FMR-1 gene.

The FMR-1 gene for the human fragile-chi syndrome, a mental retardation disease inherited by non-Mendelian transmission, contains a genetically unstable CGG region in the 5' non-translated region. The severity of the disease is correlated with the length of the CGG tract. The cloning of 28 stable plasmids containing (CGG)n inserts (where n = 6 to 240) with different extents and types of sequence interruptions (polymorphisms), and in different orientations was accomplished by three strategies in Escherichia coli. Some shorter tracts were prepared by the direct cloning of synthetic oligonucleotides, and longer runs were clones of multimers of (CGG)61, (CGG)11AGG(CGG)60CAG(CGG)8, from a cDNA from a fragile-chi patient or from expansions or deletions of these sequences in E. coli. The genetic stability of the inserts, especially for the longer tracts, was dependent on the sequence length, the presence of polymorphisms, the host cell genotypes, the orientation of the inserts in the vector and the position of cloning in a vector. Two-dimensional agarose gel electrophoresis studies on fully methylated and on non-methylated plasmids as well as chemical probe studies revealed the absence of underwound structures or accessible base-pairs. These DNAs enable a range of genetic and biochemical investigations into the molecular basis of the fragile-chi syndrome.

Base Sequence↗
PubMed1993

Isolation and characterization of a linear plasmid from the entomopathogenic fungus Ascosphaera apis.

An extrachromosomal DNA plasmid was isolated from both mating types of the entomopathogenic fungus Ascosphaera apis and named pAaL. The subculture in which pAaL was first identified originated from mummified honey bee larvae from an apiary in Wyoming. Very similar, homologous plasmids were found in 9 out of 10 of isolates collected from diverse geographic locations. The plasmid is found inside the mitochondria, has the same buoyant density as mitochondrial DNA in bisbenzamide--CsCl gradients, and does not contain sequences homologous to either mitochondrial DNA or genomic DNA. The plasmid is linear, double-stranded, of 12 kilobase pair, and has a higher copy number than the mitochondrial DNA. Endonuclease and exonuclease digestions suggest that an inverted repeat is probably present at each terminus and that pAaL has two blocked 5' ends, probably due to the presence of terminal binding proteins. Restriction site data showed pAaL to be AT-rich. There were no apparent differences in the growth rate, culture appearance, and reproductive cycle of plasmid-bearing or plasmid-free A. apis isolates. pAaL was stably inherited in the plasmid-free strains, but it was lost in the progeny of crosses and reciprocal crosses between the plasmid-free strain and plasmid-bearing strains. The biological function of this plasmid has not yet been determined.

Ascomycota↗
PubMed1994

Molecular characterization of a plasmid-borne (pGT633) erythromycin resistance determinant (ermGT) from Lactobacillus reuteri 100-63.

Lactobacillus reuteri 100-63 contains a 9.8 kb plasmid (pGT633) encoding resistance to the macrolide-lincosamide-streptogramin (MLS) type B antibiotics. The restriction endonucleases AccI, BamHI, BclI, EcoRI, EcoRV, HhaI, HindII, HpaI, KpnI, SalI, and SspI were employed to establish a physical map of pGT633. Next, genetic transfer experiments were conducted to establish the host range of pGT633. The results revealed that pGT633 replicated autonomously and encoded constitutive resistance to erythromycin in a variety of bacterial hosts, including Bacillus subtilis BD170, Streptococcus sanguis DL1, Staphylococcus aureus RN4220, and Enterococcus faecalis 19433, as well as several Lactobacillus spp. A 2.2 kb BamHI fragment of pGT633 containing the genetic determinant (ermGT) encoding resistance to erythromycin was cloned into Escherichia coli. Determination of the nucleotide sequence of ermGT revealed a methylase gene 731 bp in length that was highly related (ca. 81% nucleotide and ca. 78% amino acid identity) to the ermC gene from S. aureus plasmid pE194. The leader sequences of ermGT and ermC were identical except for a single base change at nt 51.

Amino Acid Sequence↗
PubMed1996

Molecular characterization of a plasmid-borne (pTC82) chloramphenicol resistance determinant (cat-TC) from Lactobacillus reuteri G4.

Lactobacillus reuteri G4 contains a 7.0-kb plasmid (pTC82) encoding resistance to chloramphenicol (Cm). Determination of the nucleotide sequence of the genetic determinant (cat-TC) encoding resistance to Cm on pTC82 revealed an open reading frame for a 238-amino-acid Cm acetyltransferase (CAT) monomer. This structural cat gene, 714 bp in length, was highly related (ca. 95% nucleotide and ca. 81% amino acid identity) to the 648-bp cat gene from Staphylococcus aureus plasmid pC194. A total of 6 bp transversions and 4 bp deletions was observed along the whole DNA sequence of cat-TC compared to that of cat-pC194. To determine the activity of the putative cat-TC gene, recombinant plasmid pUC8217 containing the cat determinant from pTC82 was subjected to a maxicell analysis. The observed molecular mass of the synthesized protein, based on electrophoretic mobility, was in reasonable agreement with the 27.3 kDa predicted from the DNA sequence. This is the first reported nucleotide sequence of a Cm-resistance determinant from L. reuteri and also the first evidence of adding Lactobacillus to the list of versatile bacterial genera which naturally acquire the cat-pC194 gene in the microbial ecological system.

Amino Acid Sequence↗
PubMed1997

Characterization of the cryptic plasmids of the Pseudomonas alcaligenes type strain.

The species type strain of Pseudomonas alcaligenes contains three small cryptic plasmids (designated pECB1, 2, and 3) of 7740, 4480, and 2700 bp, respectively. Partial restriction enzyme maps have been constructed for pECB1 and 2 which on this basis do not appear to be related. pECB3 proved refractile to cutting with commonly used restriction enzymes, though it was completely rendered by those enzymes which recognize 4-bp sequences containing only G + C. This suggested that pECB3 is especially rich in these bases. Hybridization studies using labeled pECB2 as probe revealed homology with pECB3 and with regions of the bacterial chromosome, but not with pECB1. A 1214-bp region of pECB2 showed great sequence similarity to the basic replicon of pPS10, a 10-kb Pseudomonas-specific plasmid isolated from Pseudomonas syringae pv. savastonoi. The putative replicon (including the gene for a replication protein) was subcloned and both DNA strands were sequenced. Introduction of the putative replicon into the Escherichia coli plasmid pUC19 created a recombinant vector able to replicate in both E. coli and Pseudomonas spp. Minicell analyses did not reveal any peptides which could be attributed to the remaining region of pECB2 or to pECB1--a finding supported by sequencing studies. Attempts at plasmid curing were unsuccessful. A phenotypic comparison with a non-plasmid-harboring strain of P. alcaligenes, based on nutritional versatility and antibiotic susceptibility, revealed a single difference of note: the type strain alone was able to utilize benzoate for growth. Transformation of the non-harboring strain with pECB1-3, followed by selection on a minimal medium containing benzoate, gave no colonies. The advantages gained by possession of pECB1-3, if any, are at present unknown.

Amino Acid Sequence↗
PubMed1999

Sequencing and characterization of Salmonella typhi plasmid R27 (incompatibility group HI1) trhC, a transfer gene encoding a potential nucleoside triphosphate-binding domain.

IncHI1 plasmids are one of the few plasmids known to mediate multiple antibiotic resistance in Salmonella typhi. These plasmids are temperature-sensitive for transfer and R27 is the prototype plasmid. DNA sequencing within the Tra2 region of R27, encoding genes involved in mating pair formation, identified trhC encoding the TrhC protein of 90,000 Da, which was visualized using an in vitro transcription/translation system. Expression of the TrhC protein also identified two smaller protein products of approximately 23 and 25 kDa which are predicted to be protease digestion products. The migration of these smaller products changed when the reactions were run at 28 vs 37 degrees C. The TrhC protein contained a putative nucleotide triphosphate-binding protein and exhibited sequence similarities with several other proteins implicated in bacterial conjugation, including TraC (F), TraB (pKM101), VirB4 (Ti), TrbE (RP4). Phylogenetic analysis showed TrhC was most closely related to TraC. Mini-Tn10 insertions in trhC generated transfer defective mutants, and no pili were specified by the trhC mutants. The trhC gene appeared to be a hot spot for transposon insertion as 37.5% mapped into this open reading frame. One trhC mutant, pDT2990, was able to be complemented by a cloned trhC gene giving a transfer frequency of 1 x 10(-3) transconjugants per recipient in an 18-h mating, whereas the wild-type transfer frequency of R27 was 1 x 10(-2) transconjugants per recipient.

Amino Acid Sequence↗
PubMed1999

Characterization of the Acinetobacter plasmid, pRAY, and the identification of regulatory sequences upstream of an aadB gene cassette on this plasmid.

Primer extension analyses carried out to identify the transcription start site of an aadB gene, which is part of a gene cassette recombined at a secondary site on an Acinetobacter plasmid, pRAY, suggest that transcription control signals in Acinetobacter are similar but not identical to their counterparts in Escherichia coli. pRAY was sequenced. An AT-rich region, containing eight copies of the consensus sequence, AAAAAATAT, previously shown to be present in the origins of replication of other Acinetobacter plasmids, was predicted to be the origin of pRAY. The translation product of one of the 10 open reading frames identified on pRAY shows homology to the mobilization protein, MbeA.

Acinetobacter↗
PubMed2000

Sequencing and characterization of the cryptic plasmid QpRS from Coxiella burnetii.

Plasmid QpRS from Coxiella burnetii, isolate Priscilla Q177, phase I, has been completely sequenced. DNA for sequencing was amplified with "extra-long" (XL) PCR. The size of the QpRS plasmid sequence was determined to be 39,280 bp, with a G + C content of 39.3%. Putative proteins associated with replication and recombination were identified. The sequence of QpRS plasmid was analyzed for shared and unique regions among C. burnetii plasmids.

Base Sequence↗
PubMed1978

Isolation and characterization of a ColE1 plasmid containing the entire bio gene cluster of Escherichia coli K12.

ColE1amp plasmids carrying the entire bio gene cluster were constructed in vitro using ColE1amp as the cloning vehicle and a lambda transducing phage, lambdaatt2, as the source of bio DNA. Restriction endonuclease EcoRI digests of ColE1amp and lambdaatt2 DNA were joined by polynucleotide ligase and plasmids bearing the entire bio gene cluster were selected, after transformation, in bio deletion strains of E. coli. Recombinant DNA molecules contained one ColE1amp fragment (7.4 X 10(6) daltons) and one lambdaatt2 DNA fragment (5.4 X 10(6) daltons). Clones carrying ColE1 amp-bio plasmids produce elevated levels of biotin and biotin synthetase activity.

Biotin↗
PubMed1981

Vectors for cloning in cyanobacteria: construction and characterization of two recombinant plasmids capable of transformation of Escherichia coli K12 and Anacystis nidulans R2.

Two plasmids were constructed consisting of the E. coli vector pACYC184 and the cyanobacterial plasmid pUC1. These recombinants, designated pUC104 and pUC105, can be transformed to E. coli K12 as well as to the cyanobacterium Anacystis nidulans R2 and in both hosts they express their antibiotic markers. pUC104 and pUC105 differ with respect to the location and the orientation of the pACYC184 segment in pUC1. pUC104 was found to be stable under all circumstances. Transformation of pUC105 to A. nidulans R2 gave intact plasmids when chloramphenicol was the selective agent, but upon ampicillin selection a deletion derivative was produced identical to pUC1. Further characteristics of pUC104 and pUC105 are described and their usefulness as cloning vectors is discussed.

Chloramphenicol↗
PubMed1992

Characterization of the F plasmid bifunctional conjugation gene, traG.

The Escherichia coli F plasmid gene, traG, is required for two stages of the conjugation process: pilus biosynthesis and mating aggregate stabilization. The nucleotide sequence of traG has been determined and the topology of its product in the cytoplasmic membrane analysed using protease accessibility experiments. Complementation analysis employing plasmid deletions revealed a correlation between an N-terminal periplasmic segment of the protein product (TraGp) and its pilus assembly activity. Production of an anti-TraGp antiserum has facilitated the detection of TraGp*, a possible internal cleavage product of TraGp. Although its function is unknown. TraGp* is located in the periplasm and has been shown to possess sequences required for aggregate stabilization. The detection of TraGp* raises the possibility that the two functions of traG are carried out by separate products.

Amino Acid Sequence↗
PubMed1991

Characterization of circular mitochondrial plasmids in three Pythium species.

Four circular plasmids, with a monomer size ranging from 3.2 to 4.94 kb, have been identified in isolates of P. aphanidermatum (two different plasmids), P. torulosum, and an unidentified echinulate isolate. The mitochondrial location has been confirmed for three of the plasmids. Each fungal isolate contained a single plasmid, present in both monomeric and oligomeric forms; plasmid monomers were present as open circles and as supercoiled forms. Restriction maps of the plasmids were dissimilar. Hybridization studies using cloned plasmids revealed no DNA sequence similarity among the different plasmids or between the plasmids and the nuclear or mitochondrial genome of the isolates from which they were recovered. Hybridization of labeled plasmid DNA to Northern transfers of mitochondrial RNA for two isolates indicate that what appears to be the predominant RNA transcript is unit length in size. For three isolates, the plasmid was retained following subculturing and was present in all asexual and sexual single-spore progeny evaluated. For one isolate of P. aphanidermatum the plasmid was unstable and was lost during subculturing.

Blotting, Northern↗
PubMed1980

Characterization and cloning of plasmid like DNA of the ascomycete Podospora anserina.

The previously reported existence of plasmid-like (pl) DNA in senescent mycelia of Podospora anserina was confirmed using new methodology. Detailed anaysis of bulk DNA has further shown a possible relationship between pl DNA and mt DNA. According to biophysical and electron microscopic experiments the pl DNA was found to consist of oligomeres having a basic unit with a contour length of 0.75 micrometer corresponding to 2.4 kb. To overcome the handicap that pl DNA is only produced in rather small amounts in the aging mycelia, this DNA was cloned in E. coli after insertion into a bacterial plasmid vector, pBR 322. It was possible to isolate a stable hybrid plasmid consisting of the vector and only one integrated monomere of pl DNA. The composition of this hybrid plasmid was confirmed by restriction endonuclease analysis and heteroduplex formation. A restriction map of the pl DNA is presented and its insertion site onto pBR 322 indicated.

Ascomycota↗
PubMed1987

Characterization of chromosome and plasmid transformation in Bacillus subtilis using gently lysed protoplasts.

Competent cells of Bacillus subtilis were transformed with DNA from gently lysed protoplasts. Significant linkages among markers separated by distances of approximately 2.3% of the total chromosome were found, which have not been detected for conventional transformation. In comparison to previous reports, enhanced plasmid transformation was observed [4.0 X 10(7) transformants per microgram DNA (one transformant per 5 X 10(4) molecules added)], when competent cells were transformed with DNA from lysed protoplasts harboring pUB110.

Bacillus subtilis↗
PubMed1992

Physical and genetic characterization of linear DNA plasmids from the heterothallic yeast Saccharomycopsis crataegensis.

Five strains of the heterothallic yeast Saccharomycopsis crataegensis have been previously shown to contain DNA and/or RNA plasmidlike molecules (Shepherd et al. 1987). Three DNA plasmids, designated pScrl-1, -2 and -3, were found in strain NRRL Y-5902, while two were identified in each of NRRL strains Y-5903 and Y-5904. DNA plasmids were not identified in S. crataegensis strains Y-5910 or YB-192. Four S. crataegensis strains (Y-5903, Y-5904, Y-5910 and YB-192) were also shown to possess double-stranded RNA (dsRNA) molecules not found in strain Y-5902 (Shepherd et al. 1987). Hybridization studies now demonstrate the DNA plasmids in Y-5903 and Y-5904 to be highly homologous to their respective size counterparts (pScrl-1 and pScrl-2) in Y-5902 and to show some homology to pScrl-3. Restriction endonuclease mapping studies confirm the linear nature of each plasmid and establish identical restriction maps for a 1.4 kilobase (kb) region in pScrl-2 and -3. This 1.4 kb region accounts for the hybridization homology of pScrl-2 and pScrl-3 noted by Shepherd et al. (1987) and for homology of the plasmids of Y-5903 and Y-5904 to pScrl-3 of Y-5902. The pScrl plasmids show no homology to the dsRNA molecules of S. crataegensis, the 2 microM circular DNA of Staccharomyces cerevisiae, the 'killer' plasmids of Kluyveromyces lactis, or the linear DNA plasmids of Pichia inositovora. In crosses between linear DNA plasmid-containing and dsRNA-containing strains, only progeny containing the pScrl plasmids were recovered. Poor spore viability and a lack of complete tetrad recovery limited the extent of the analysis, but the findings suggest a cytoplasmic mode of inheritance for these linear DNAs.

Blotting, Northern↗