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At least 721 records · Page 40Linked to original sources
PubMed1991

Characterization of pGA1, a new plasmid from Corynebacterium glutamicum LP-6.

A new plasmid, pGA1, has been isolated from Corynebacterium glutamicum LP-6, and its detailed restriction map has been prepared. The 4.9-kb plasmid has a G + C content of 57%. It replicates in C. glutamicum ATCC13032 and is compatible with the three other plasmids, pCC1, pBL1 and pHM1519, commonly used for vector construction for amino acid-producing corynebacteria. Fusions of pGA1 with different Escherichia coli replicons (transferred from E. coli to Corynebacterium via transformation of spheroplasts or by filter mating experiments with intact cells) are shown to be suitable as shuttle plasmids; some of them are highly stable in C. glutamicum, even when propagated without any selection pressure.

Base Composition↗
PubMed1995

Characterization of pPvu1, the autonomous plasmid from Proteus vulgaris that carries the genes of the PvuII restriction-modification system.

Plasmid pPvu1 from Proteus vulgaris carries the genes of the PvuII restriction-modification system [Blumenthal et al., J. Bacteriol. 164 (1985) 501-509]. This report focuses on physical and functional features of the 4.84-kb plasmid, which shows a composite genetic architecture. Plasmid pPvu1 has a replication origin and an incompatibility locus that each function in Escherichia coli, and an apparent cer recombination site. The replication origin includes a possible RNA I gene, and the incompatibility locus closely resembles a rom gene. These loci show substantial sequence similarity to corresponding loci from the E. coli plasmids P15A, ColEI and pSC101, and closely flank the PvuII genes. The close association between a recombinational locus and the PvuII genes has implications for their mobility.

Amino Acid Sequence↗
PubMed2006

The conjugates of uracil-cyclen Zn(II) complexes: synthesis, characterization, and their interaction with plasmid DNA.

As an important nucleobase in RNA, uracil was introduced into the side chain of cyclen (1,4,7,10-tetraazacyclododecane) by using phenylene dimethylene group as bridge. The target compounds 5 were obtained in high yields. Subsequent experiments demonstrated that the uracil-cyclen conjugates can bind Zn(2+) cation rapidly in water, and the catalytic activities of their Zn(II) complexes 6 in DNA cleavage were also studied. The results showed that Zn(II) complexes can catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA) (Form I) to produce nicked DNA (Form II and Form III) with high selectivity. In water solution, complex 6b may form a unique and stable supramolecular structure, which benefits the DNA cleavage process.

Base Sequence↗
PubMed2006

Chiral multinuclear macrocyclic polyamine complexes: synthesis, characterization and their interaction with plasmid DNA.

A series of multinuclear macrocyclic polyamine metal (Zn(2+), Cu(2+), Co(2+)) complexes containing chiral dipeptide linkage were synthesized and used as artificial nuclease enzyme model. The interaction between the complexes and plasmid DNA (pUC19) was studied, and the results revealed that these complexes could act as powerful catalysts for the cleavage of plasmid DNA under physiological conditions.

Catalysis↗
PubMed2005

Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida.

Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America. These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tc(r)) plasmids of approximately 58 kb. The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance (tet) gene carried [tet(A) or tet(B)], incompatibility of the plasmid [IncU or other], whether the plasmid carried the IS6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn1721, and their ability to transfer their Tc(r) plasmids to an Escherichia coli recipient at 15 degrees C. Five of the isolates, with genetically related Tc(r) plasmids, were able to transfer their plasmids to an E. coli recipient at frequencies ranging from 5.7x10(-4) to 2.8x10(-6) per recipient. The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate. The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E. coli recipient. Conjugal transfer at 15 degrees C is the lowest temperature that has been documented in bacteria.

Aeromonas salmonicida↗
PubMed2005

Characterization of a topologically aberrant plasmid population from pilot-scale production of clinical-grade DNA.

As part of a program to develop DNA vaccines for pharmaceutical applications, we recently established a manufacturing process for the production of clinical grade plasmid DNA. In an evaluation of two cell separation methods, the cell culture experienced a temperature spike in a new tangential flow filtration rig, resulting in an aberrant plasmid HPLC peak. Analysis by agarose gel electrophoresis and HPLC demonstrated that the aberrant plasmid material's overall primary structure, methylation pattern and topological integrity was indistinguishable from that of reference material. Transmission electron microscopy and high-resolution agarose gel electrophoresis revealed that the unknown plasmid form exhibited a very low level of supercoiling, whereas the normal supercoiled fraction contained highly twisted DNA. We hypothesized that an enzymatic process, induced by stress during the temperature spike, caused the distinct plasmid topology. This idea was supported by a lab-scale fermentation experiment, where plasmid topology was shown to be similarly altered by conditions designed to induce metabolic stress.

Chromatography, High Pressure Liquid↗
PubMed2006

Development of a rapid real-time PCR assay for detection and quantification of four familiar species of Chlamydiaceae.

Chlamydiae are one of the causative agents of various diseases in animals and human beings, which include abortion, pneumonia, gastroenteritis, encephalomyelitis, conjunctivitis, arthritis and sexually transmitted diseases. Much work has been carried out to attempt to develop an efficient pathogen detection strategy. Here, we presented a Chlamydiaceae-specific 23S rRNA-based real-time PCR assay for simultaneous detection and quantification of four members of Chlamydiaceae family, C. trachomatis, C. psittaci, C. pneumoniae and C. pecorum, using SYBR Green and Lightcycler. The assay was characterized using plasmid constructs of the bacteria and verified on standard strains of all four species of the Chlamydiaceae and a large cohort of clinical samples collected from human and animals by comparison with fluorescence immunohistochemistry method. The results showed that the present real-time PCR assay was of high specificity and sensitivity. It was capable of detecting as few as 250fg of chlamydial DNA (equivalent to 10(-1)IFU) and was applicable to both liquid cultures and clinical samples. This assay may therefore offer a rapid, economic and reliable means for screening of the chlamydiaceae pathogens.

Animals↗
PubMed2005

Dinuclear macrocyclic polyamine zinc(II) complexes: syntheses, characterization and their interaction with plasmid DNA.

The syntheses, characteristics of dinuclear macrocyclic polyamine zinc complexes and their interaction with plasmid DNA are reported. The two cyclen (1,4,7,10-tetraazacyclododecane) moieties are bridged by rigid and flexible linkages. The crystal structures of Zn2C27H43N8O15Cl4 [5c.(ClO4)3.2H2O] and Zn2C30H43N10O13Cl3 [5e.(ClO4)3.H2O] have been determined. The complexes crystallize in the monoclinic space group C2/c and P2(1)/c with the following unit cell parameters: 5c.(ClO4)3.2H2O: a=32.568(4)A, b=14.8593(17)A, c=19.443(2)A, alpha=90.00 degrees , beta=119.435(4) degrees , gamma=90.00 degrees , Dc=1.551 mg/m3, FW=956.71, F(000)=3932; 5e.(ClO4)3.H2O: a=15.807(2)A, b=16.756(2)A, c=16.161(2)A, alpha=90.00 degrees , beta=97.062(4) degrees , gamma=90.00 degrees , Dc=1.546 mg/m3, FW=988.83, F(000)=2032. The distance between the two Zn(II) ions is about 4.0 A. The structures show that two zinc ions can synergistically interact with the substrate DNA. With this novel structural characteristics, the dinuclear macrocyclic polyamine Zn(II) complexes via the synergetic effect between the two zinc ions can catalyze the cleavage of plasmid DNA (pUC18) with unprecedented speed at physiological conditions.

Crystallography, X-Ray↗
PubMed2004

The plasmids of Borrelia burgdorferi: essential genetic elements of a pathogen.

The spirochete Borrelia burgdorferi, the causative agent of Lyme disease, has an unusual genome comprised of a linear chromosome and the largest plasmid complement of any characterized bacterium. Certain plasmid-encoded elements are required for virulence and viability, both in vitro and in vivo. The genetic tools to manipulate B. burgdorferi are sufficiently developed for precise molecular genetic investigations. B. burgdorferi now represents a prime system with which to address basic questions of plasmid biology and plasmid contributions to bacterial virulence and disease pathogenesis.

Animals↗
PubMed2002

Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure.

The 10,877bp tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 was completely sequenced. The sequence revealed a composite structure containing DNA from up to four different sources. The replication region had homology to other plasmids of lactic acid bacteria while the tetracycline resistance region, containing a tet(M) gene, had high homology to sequences from Clostridium perfringens and Staphylococcus aureus. Within the tetracycline resistance region a Lactobacillus IS-element was found. The remaining part of the plasmid contained three open reading frames with unknown functions. The composite structure with several truncated genes suggests a recent assembly of the plasmid. This is the first sequence of an antibiotic resistance plasmid isolated from L. plantarum.

Base Sequence↗
PubMed2003

Characterization of a small cryptic plasmid, pHP51, from a Korean isolate of strain 51 of Helicobacter pylori.

The nucleotide sequence of a 3955-bp Helicobacter pylori plasmid, pHP51 was determined, and two open reading frames, ORF1 and ORF2, were identified. The deduced amino acid sequence of ORF1 was highly conserved (87-89%) among plasmid replication initiation proteins, RepBs. The function of ORF2 was not assigned because it lacked known functional domains or sequence similarity with other known proteins, although it had a HPFXXGNG motif that was also found in the cAMP-induced filamentation (fic) gene. Three kinds of repeats were present on the plasmid outside of the ORFs, including the R1 and R2 repeats that are common in H. pylori plasmids. One 100-bp sequence detected in the noncoding region of pHP51 was highly similar to the genomic sequence of H. pylori 26695.

Amino Acid Sequence↗
PubMed1997

Application of molecular methods to a nosocomial outbreak of Salmonella enteritidis phage type 4.

A nosocomial outbreak of Salmonella enteritidis phage type 4 occurred in July 1995. Seven definite cases were identified over 13 days affecting four wards in a London hospital. The outbreak strain was characterized by plasmid profile typing and pulsed-field gel electrophoresis (PFGE), and was unusual in that it did not possess a 38 MDa plasmid common to most isolates of S. enteritidis PT 4 made from humans and food animals in England and Wales. Seven asymptomatic excreters were identified on screening. No additional cases occurred on wards after standard isolation procedures were implemented. No common or continuing food or dietary source was identified. Results of epidemiological, microbiological and environmental investigations suggested that the outbreak was due to person-to-person transmission within the hospital. The source of the outbreak was not established but was probably due to admission of a patient with an unrecognized infection of S. enteritidis PT 4. The study highlights the importance of close collaboration between hospital staff, epidemiologists and microbiologists, and demonstrates the value of molecular techniques for strain subdivision in outbreak investigations.

Aged↗
PubMed1997

Construction and characterization of a one-plasmid system for the controlled expression of genes in mammalian cells by tetracycline.

The two-plasmid system of Gossen and Bujard [Gossen and Bujard (1992) Proc. Natl. Acad. Sci. USA 89, 5547-5551] to express mammalian genes in a tetracycline-repressed fashion was combined into a single-plasmid system. Two variants of this single-plasmid system that differ in the multiple cloning site (MCS) region are described. These vectors were used to stably transfect raf kinase domain into the normal rat kidney epithelial cell line (NRKE) to obtain a conditionally transformed cell line. These vectors were also used to stably transfect wild-type and mutant human p53 into the human osteosarcoma cell line, SAOS-2. Tetracycline repressed gene expression in both cell lines; about 12-fold in NRKE and about 80-fold in SAOS-2 cell line.

Animals↗
PubMed2001

Cationized human serum albumin as a non-viral vector system for gene delivery? Characterization of complex formation with plasmid DNA and transfection efficiency.

Cationized human serum albumin (cHSA) could serve as a potential non-viral vector system for gene delivery. Native human serum albumin was cationized by covalent coupling of hexamethylenediamine to the carboxyl groups resulting in a shift of the isoelectric point from pH 4-5 to 7-9. The cationized albumin underwent spontaneous self-assembly with DNA as demonstrated by retardation of CMV-nlacZ plasmid in agarose gel electrophoresis. Photon correlation spectroscopy showed a decrease of complex size with increasing cHSA/plasmid ratios. Under optimized conditions complexes were formed with 230-260 nm mean diameter and a homogenous, narrow size distribution. At room temperature complexes were stable in 0.9% sodium chloride solution pH 7.4 for 1 h without aggregation. Process parameters such as albumin concentration, incubation time, temperature, pH, order of reagent addition, the presence of bivalent ions and the ionic strength of the complexation medium all influenced the complex size. Confocal laser scanning microscopy showed interactions of a Texas Red labeled cationized albumin with cell membranes of ECV 304 cells and an enhanced endocytic uptake compared to native albumin. The potential for introducing exogeneous DNA into cells was shown using NIH 3T3 fibroblasts. Successful, albeit low reporter gene expression could be achieved in the presence of chloroquine. Under in vitro conditions no toxic effect could be observed. In conclusion, cationized albumin may have promise as a non-toxic vector for gene delivery, especially for DNA vaccination.

DNA↗
PubMed1998

Purification and in vitro reconstitution of the essential protein components of an aromatic polyketide synthase.

A minimal set of proteins which catalyze the synthesis of aromatic polketides from malonyl CoA has been purified and partially characterized. Plasmid-encoded actinorhodin (act) ketosynthase/chain-length factor (KS/CLF) complex was purified from Streptomyces coelicolor CH999/pSEK38, and assayed with purified aromatic PKS holo-ACPs which were overproduced and purified from Escherichia coli and phosphopantetheinylated in vitro using purified E. coli holo-ACP synthase. When highly purified preparations of KS/CLF, and holo-ACP failed to catalyze polyketide biosynthesis, a fourth protein was sought and purified from the S. coelicolor CH999 host on the basis of its ability to complement KS, CLF, and holo-ACP in polyketide synthesis. N-terminal sequencing identified this protein as the fatty acid synthase (fabD) malonyl CoA:ACP transacylase (MAT), recruited from primary metabolism. A alpha2/beta2 structure was shown for the act KS/CLF complex, and three malonyl-enzyme biosynthetic intermediates were identified, defining an escorted path followed by malonyl groups en route from CoA to polyketide.

Acyltransferases↗
PubMed1989

Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification.

The molecular analysis of many genetic diseases requires the isolation of probes for defined human chromosome regions. Existing techniques such as the screening of chromosome-specific libraries, subtractive DNA cloning and chromosome jumping are either tedious or not generally applicable. Microdissection and microcloning has successfully been applied to various chromosome regions in Drosophila and mouse, but conventional microtechniques are too coarse and inefficient for analysis of the human genome. Because microdissection has previously been used on unbanded chromosomes only, cell lines in which the chromosome of interest could be identified without banding had to be used. At least one hundred chromosomes were needed for dissection and lambda vectors used to achieve maximum cloning efficiency. Recombinant phage clones are, however, more difficult to characterize than plasmid clones. Here we describe the dissection of the Langer-Giedion syndrome region on chromosome 8 from GTG-banded metaphase chromosomes (G-banding with trypsin-Giemsa) and the universal enzymatic amplification of the dissected DNA. Eighty per cent of clones from this library (total yield 20,000) identify single-copy DNA sequences. Fifty per cent of clones detect deletions in two patients with Langer-Giedion syndrome. Although the other clones have not yet been mapped, this result demonstrates that thousands of region-specific probes can be isolated within ten days.

Base Sequence↗