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At least 397 records · Page 22Linked to original sources

PubMed1982

Replication of the 2-micrometer DNA plasmid of yeast.

The 2-micrometer DNA plasmid of yeast provides an useful probe for the analysis of the factors involved in the initiation of chromosomal DNA replication in the cell division cycle. Cell-free extracts prepared from growing yeast cells stimulate DNA replication directed by this plasmid. The plasmid replicating activity is subject to control in the yeast cell cycle. The 2-micrometer DNA plasmid can be isolated from logarithmically-growing cells in association with yeast folded chromosomes. The interaction of the plasmid with structures in the cell corresponding to the folded chromosome appears dynamic and cell cycle-dependent. Thus, not only the induction and activity of the proteins involved in replication, but also the intracellular locus of a replicon may be important factors in the control of initiation of DNA replication.

Cell Cycle↗

PubMed1987

Structural variability of 40-50 Mdal virulence plasmids from Yersinia enterocolitica. Geographical and ecological distribution of plasmid variants.

Restriction endonuclease analysis (BamHI and EcoRI) was used to investigate the structural variability of 40-50 Mdal plasmids from 129 Yersinia enterocolitica strains isolated from human patients, animals, and food in 12 countries in Europe, North America, and Asia. A total of 12 restriction patterns was detected among plasmids from the six serogroups examined. The DNA fragment profiles were found to vary, not only between serogroups, but also among plasmids isolated from strains with the same serogroup affiliation. Despite widespread geographical and ecological origin, the plasmids isolated from the 0:3 and 0:9 strains examined revealed a surprising stability, whereas plasmids from 0:8 and 0:5,27 showed substantial diversity. The genes associated with autoagglutination, calcium dependency, and mouse virulence were conserved in all 12 plasmid variants detected. Our finding that porcine and human isolates harboured plasmids with identical restriction patterns, provides additional support for the importance of pigs in the epidemiology of human Y. enterocolitica infection.

Animal Diseases↗

PubMedDate not supplied

Cyclodextrin-modified polyethylenimine polymers for gene delivery.

Linear and branched poly(ethylenimines), lPEI and bPEI, respectively, grafted with beta-cyclodextrin are prepared to give CD-lPEI and CD-bPEI, respectively, and are investigated as in vitro and in vivo nonviral gene delivery agents. The in vitro toxicity and transfection efficiency are sensitive to the level of cyclodextrin grafting. The cyclodextrin-containing polycations, when combined with adamantane-poly(ethylene glycol) (AD-PEG) conjugates, form particles that are stable at physiological salt concentrations. PEGylated CD-lPEI-based particles give in vitro gene expression equal to or greater than lPEI as measured by the percentage of EGFP expressing cells. Tail vein injections into mice of 120 microg of plasmid DNA formulated with CD-lPEI and AD-PEG do not reveal observable toxicities, and both nucleic acid accumulation and expression are observed in liver.

Animals↗

PubMed1984

Protoplast transformation of glutamate-producing bacteria with plasmid DNA.

A method for polyethylene glycol-induced protoplast transformation of glutamate-producing bacteria with plasmid DNA was established. Protoplasts were prepared from cells grown in the presence of penicillin by treatment with lysozyme in a hypertonic medium. The concentration of penicillin during growth affected the efficiency of formation, regeneration, and polyethylene glycol-induced DNA uptake of protoplasts. Regeneration of protoplasts was accomplished on a hypertonic agar medium containing sodium succinate and yeast extract. The spectinomycin and streptomycin resistance plasmid pCG4, originally from Corynebacterium glutamicum T250, could transform various glutamate-producing bacteria such as C. glutamicum, Corynebacterium herculis, Brevibacterium flavum, and Microbacterium ammoniaphilum. The plasmid was structurally unchanged and stably maintained in new hosts. The transformation frequency of most competent protoplasts with pCG4 DNA isolated from primary transformants was high (ca. 10(6) transformants per microgram of covalently closed circular DNA) but was still two orders of magnitude below the frequency of transfection with modified DNA of the bacteriophage phi CGI. The difference was ascribed to the involvement of regeneration in transformation.

Brevibacterium↗

PubMed1992

Nucleotide sequence of the R721 shufflon.

The shufflon is a DNA region that undergoes complex rearrangement mediated by the product of a putative site-specific recombinase gene, rci. The DNA sequences of the shufflon region and the rci gene of IncI2 plasmid R721 were determined. The R721 shufflon consists of three invertible DNA segments that are homologous to the shufflon segments found in IncI1 plasmid R64. Structural analysis of open reading frames indicated that the R721 shufflon possibly functions as a biological switch for selecting one of the six pilV genes in which the N-terminal region is constant and the C-terminal region is variable. The R721 rci gene was shown to encode a basic protein of 374 amino acid residues.

Amino Acid Sequence↗

PubMed1997

A broad-host-range mobilizable shuttle vector for the construction of transcriptional fusions to beta-galactosidase in gram-positive bacteria.

A low-copy-number vector designated pTCV-lac has been constructed to provide a convenient system to analyze regulatory elements in Gram-positive bacteria. The main components of this vector are: (i) the origins of replication of pACYC184 and of the broad-host-range enterococcal plasmid pAM beta 1, (ii) erythromycin- and kanamycin-resistance-encoding genes for selection in Gram-negative and Gram-positive bacteria, (iii) the transfer origin of the IncP plasmid RK2, and (iv) a promoterless beta-galactosidase-encoding lacZ gene with a Gram-positive ribosome binding site. This 12 kb plasmid is present in Gram-positive hosts in three to five copies per chromosome equivalent and contains three unique cloning sites (EcoRI, SmaI, BamHI) for cloning of DNA inserts upstream of the lacZ gene. Plasmid pTCV-lac and derivatives carrying different promoter fragments have been transferred by conjugation from an Escherichia coli IncP mobilizing donor strain to Bacillus subtilis, Listeria monocytogenes, Enterococcus faecalis, and Streptococcus agalactiae. These plasmids were structurally stable in these hosts and the corresponding promoter activities, quantitated by the determination of the beta-galactosidase specific activities, were found to cover at least a 100-fold range in beta-galactosidase values. These results indicate that pTCV-lac should be useful for analysis of gene regulation in a wide range of Gram-positive bacteria.

Bacillus subtilis↗

PubMed1998

Terplex DNA delivery system as a gene carrier.

PURPOSE: To characterize the physical and biochemical properties of the DNA terplex delivery system, which has previously been shown to deliver and express pSV-beta-gal plasmid efficiently in cultured smooth muscle cells (SMC) (1). METHODS: Atomic force microscopy (AFM), zeta-potential measurement (ZP), gel electrophoresis (GE), circular dichroism (CD), fluorescence quenching and 1H-NMR spectrometry were used. RESULTS: AFM showed that the plasmid DNA of about 600 nm long in its extended state was condensed to the size of about 100 nm by terplex formation. The DNA condensing effect of the terplex system was as good as unmodified PLL, as shown by an ethidium bromide displacement assay. Zeta-potential measurement showed that the terplex system exerts a slightly positive surface charge (+2 mV) at a 1:1:1 weight ratio of DNA:LDL:stearyl-PLL, which showed the best transfection efficiency on SMC. GE indicated that electrophoretic mobility of the terplex system decreased with increasing amounts of stearyl-PLL, indicating that the surface charge of the terplex system became more positive as more stearyl-PLL was added. Results from CD showed that there was no significant changes in tertiary structure of plasmid DNA from the terplex formation. Presence of strong hydrophobic interaction between stearyl-PLL and LDL was confirmed by 1H-NMR, where about a 30% decrease in epsilon-methylene peak of PLL backbone was observed when stearyl-PLL was mixed with LDL, but this phenomenon was not observed when unmodified PLL was used. CONCLUSIONS: Our results indicate that the plasmid DNA, when formulated with the stearyl-PLL and LDL, forms a stable and hydrophobicity/charge balanced terplex system of optimal size for efficient cellular uptake and the DNA is still intact after the terplex formation. This information is expected to be utilized for the development of much improved transfection vectors for in vivo gene therapy.

Animals↗

PubMed1994

Construction of plasmids, estimation of plasmid stability, and use of stable plasmids for the production of poly(3-hydroxybutyric acid) by recombinant Escherichia coli.

Plasmids containing the Alcaligenes eutrophus poly(3-hydroxybutyric acid) (PHB) biosynthetic genes were constructed for the production of PHB in Escherichia coli and plasmid stability was investigated by repeated subculturing without antibiotic pressure. Both pSYL101 (high copy) and pSYL102 (medium copy) were unstable during the subcultures. Higher instability was observed when cells were accumulating PHB. Segregational instability was aggravated by the faster growth of plasmid-free cells and by appearance of non-dividing cells harboring large amount of PHB during the fed-batch culture. Two derivatives, pSYL103 and pSYL104, were then developed by cloning the parB locus of plasmid R1 into pSYL102 and pSYL101, respectively. They showed 100% stability even during PHB synthesis and accumulation over 110 generations. All four plasmids were structurally stable. The final cell mass, PHB concentration, and PHB per dry cell weight (P/X, w/w, %) of 101.4 g l-1, 81.2 g l-1, and 80.1%, respectively, were obtained in 39 h by high cell density culture of XL1-Blue (pSYL104). The final PHB concentration was lower using XL1-Blue (pSYL103), which suggested that high gene dosage was required for the synthesis and accumulation of PHB to a high concentration in E. coli.

Escherichia coli↗

PubMed1992

Supercoiled DNA wraps around the bacteriophage phi 29 head-tail connector.

Supercoiled pBR322 DNA wraps around the outside of the isolated Bacillus subtilis bacteriophage phi 29 head-tail connector, the crux of the DNA packaging machine of the viral precursor capsid or prohead. The contour length of the supercoiled DNA, determined by EM, decreased by approximately 180 base pairs for each connector bound. Mass and radial density determinations by scanning transmission EM showed that the increased mass of the connector-DNA complex relative to the connector alone was equivalent to approximately 170 base pairs of DNA and was located around the outside of the connector. Topoisomerase I treatment of the complexes followed by deproteinization suggested that supercoils were restrained by the connectors. Connectors bound linear and open-circular plasmid DNAs inefficiently but were not wrapped by these DNAs. The wrapping of supercoiled DNA around the isolated phi 29 connector is hypothesized to reflect the initiation phase of the normal process of DNA packaging. Packaging substrates would be supercoiled, wrapped by the connector, linearized, and translocated by rotation of the connector relative to the viral capsid with the aid of ATP hydrolysis.

Bacillus Phages↗

PubMed1989

Accumulation of the insecticidal crystal protein of Bacillus thuringiensis subsp. kurstaki in post-exponential-phase Bacillus subtilis.

A gene from Bacillus thuringiensis subsp. kurstaki that codes for a Lepidoptera-specific insecticidal toxin (delta-endotoxin) was engineered for expression in Bacillus subtilis. A low-copy-number plasmid vector that replicates in Escherichia coli and B. subtilis was constructed to transform B. subtilis with gene fusions first isolated and characterized in E. coli. Naturally occurring promoter sequences from B. subtilis (43, veg, ctc, and spoVG) were inserted upstream from the plasmid-borne structural gene. In the most prolific case, when the sporulation-specific spoVG promoter was fused to the heterologous toxin gene, the toxin product accumulated during postexponential growth to greater than 25% of the total cell protein. However, the resulting specific activity of the insecticidal toxin product was not commensurate with the abundance of the protein.

Animals↗

PubMedDate not supplied

Effects of acridines on bacterial plasmid replication and endotoxin.

Different amino- and imino-acridines were systematically synthesized. The antibacterial, antiplasmid, antimotility and endotoxin complexing effect of acridines were studied, when antibacterial effect of the compounds was compared on E. coli. Aminoderivatives were more active than imino-acridines. The N-heptyl-9-imino-acridine was able to select lon minus mutants in the E. coli culture, however, the other acridines tested were ineffective in this respect. The iminoacridines inhibited the motility of Proteus vulgaris more effectively than aminoderivatives. The antimotility action of the acridines was also dependent on the ionic content of the media. The antiplasmid effect was measured on an F-prime plasmid of E. coli LE140 strain. Iminoacridines had a more powerful antiplasmid effect than the amino-substituted derivatives. The majority of the compounds inhibited the intercellular plasmid transfer from E. coli. Kmr donor to a Na-azide resistant recipient. In this test the aminosubstituted derivatives were shown to be more effective inhibitors of conjugation than the imino-substituted compounds. Endotoxin formed complexes with N-butylamino. N-propy-lamino and imidoderivatives. However, complex formation of N-ethyl-, N-heptyl-, N-diethylaminoethyl- and N-diethylamino-propyl-acridines were different. Correlations between molecular orbitals and the antibacterial effects are also discussed.

Acridines↗

PubMed1988

Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene.

The gene encoding lipoamide dehydrogenase from Azotobacter vinelandii has been cloned in Escherichia coli. Fragments of 9-23 kb from Azotobacter vinelandii chromosomal DNA obtained by partial digestion with Sau3A were ligated into the BamHI site of plasmid pUC9. E. coli TG2 cells were transformed with the resulting recombinant plasmids. Screening for clones which produced A. vinelandii lipoamide dehydrogenase was performed with antibodies raised against the purified enzyme. A positive colony was found which produced complete chains of lipoamide dehydrogenase as concluded form SDS gel electrophoresis of the cell-free extract, stained for protein or used for Western blotting. After subcloning of the 14.7-kb insert of this plasmid the structural gene could be located on a 3.2-kb DNA fragment. The nucleotide sequence of this subcloned fragment (3134 bp) has been determined. The protein-coding sequence of the gene consists of 1434 bp (478 codons, including the AUG start codon and the UAA stop codon). It is preceded by an intracistronic region of 85 bp and the structural gene for succinyltransferase. A putative ribosome-binding site and promoter sequence are given. The derived amino acid composition is in excellent agreement with that previously published for the isolated enzyme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three-dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483-496], it appears that essential residues in all four domains have been conserved. The enzyme is strongly expressed, although expression does not depend on the vector-encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.

Amino Acid Sequence↗

PubMed1997

In the presence of subunit A inhibitors DNA gyrase cleaves DNA fragments as short as 20 bp at specific sites.

A key step in the supercoiling reaction is the DNA gyrase-mediated cleavage and religation step of double-stranded DNA. Footprinting studies suggest that the DNA gyrase binding site is 100-150 bp long and that the DNA is wrapped around the enzyme with the cleavage site located near the center of the fragment. Subunit A inhibitors interrupt this cleavage and resealing cycle and result in cleavage occurring at preferred sites. We have been able to show that even a 30 bp DNA fragment containing a 20 bp preferred cleavage sequence from the pBR322 plasmid was a substrate for the DNA gyrase-mediated cleavage reaction in the presence of inhibitors. This DNA fragment was cleaved, although with reduced efficiency, at the same sites as a 122 bp DNA fragment. A 20 bp DNA fragment was cleaved with low efficiency at one of these sites and a 10 bp DNA fragment was no longer a substrate. We therefore propose that subunit A inhibitors interact with DNA at inhibitor-specific positions, thus determining cleavage sites by forming ternary complexes between DNA, inhibitors and DNA gyrase.

Benzimidazoles↗

PubMed1994

Plasmid integration, amplification and cytogenetics in CHO cells: questions and comments.

This paper addresses seven questions with respect to the transfer, integration, amplification and genetic stability of recombinant DNA in mammalian genomes. Most of these questions were raised with those issues in mind which are frequently discussed in the context of the manufacture of biologicals of therapeutic value on the basis of recombinant cell lines. For the most part, a high degree of ignorance has to be acknowledged and only very limited fragments of information are available. The reason for this ignorance is, as will become clear from this article, the sheer size and complexity of the mammalian genome and the inadequacy of presently available tools to unravel this complexity.

Animals↗

PubMed1992

Microbial oxidation of adamantanone by Pseudomonas putida carrying the camphor catabolic plasmid.

Intact cells of (+/-)camphor-grown Pseudomonas putida, ATCC17453(CAM), have been shown to oxidize readily the monoketone derivative of cage hydrocarbon adamantane, forming oxygenated products indicative of both biological Baeyer-Villiger and hydroxylation reactions. Formed products were identified as 4-oxahomoadamantan-5-one, 5-hydroxyadamantan-2-one and 1-hydroxy-4-oxahomoadamantan-5-one. Minor products formed as a result of secondary reactions were tentatively identified as syn- and anti-1,4-dihydroxyadamantanes and bicyclo[3.3.1]nonan-3-ol. Adamantanone initial concentrations determined whether 1-hydroxy-4-oxahomoadamantan-5-one was the sole product (below 120 mg/l) or 4-oxahomoadamantan-5-one was the principle (up to 92%) product (240-600 mg/l). Formation of 1-hydroxy-4-oxahomoadamantan-5-one appears to occur by two routes determined by the sequence of lactonization and hydroxylation.

Adamantane↗

PubMed1990

Subcloning of the enterobactin biosynthetic gene entB: expression, purification, characterization, and substrate specificity of isochorismatase.

The Escherichia coli entB gene, coding for the enterobactin biosynthetic enzyme isochorismatase, has been subcloned into the multicopy plasmid pKK223-3 under the control of the tac promoter. The resulting recombinant plasmid pFR1 expresses isochorismatase amounting to over 50% of the total cellular protein. The enzyme has been purified to homogeneity and a convenient assay developed. The enzyme has a Km for isochorismate of 14.7 microM and a turnover number of 600 min-1. By use of 1H NMR spectroscopy, the progress of the reaction was followed with the expected formation of 2,3-dihydro-2,3-dihydroxybenzoate product. Several substrate analogues were also utilized by the enzyme including chorismic acid, the immediate precursor to isochorismic acid in the enterobactin biosynthetic pathway.

Base Sequence↗

PubMed1996

Direct expression of Der f2, a major house dust mite allergen, in Escherichia coli.

Der f2 protein in a highly antigenic form was directly expressed in bacteria. Plasmid pFLU11 derived from pKK233-2 was designed to express methionyl-Der f2 under the control of the trc promoter and it has the replication origin of pUC118 instead of its original to increase the copy number. This expression plasmid directed the synthesis of recombinant Der f2 (rDer f2) protein in an insoluble form of inclusion bodies in Escherichia coli cells. The high copy number plasmid pFLU11 conferred the efficient production of the Der f2 protein in E. coli, when compared to a nonchanged origin material. rDer f2 inclusion bodies were easily solubilized in urea and renatured by dialysis to assume the active form. The rDer f2 protein was purified by means of anion exchange and gel filtration chromatography. This expression system yielded about 10 mg of purified rDer f2 protein from the 1L culture. Purified rDer f2 protein reacted with IgE from patient sera almost identically to the native Der f2 in the RAST enzyme immunoassay and skin prick test.

Allergens↗

PubMed1997

Dynamics in the isomerization of intramolecular DNA triplexes in supercoiled plasmids.

We report here kinetic and thermodynamic studies on differential isomerization of intramolecular Pyr*Pur.Pyr triplexes in supercoiled plasmids. Two structural isomers of the triplex exist: one with the 3'-half of the Pyr strand as the third strand (H-y3 form) and the other with the 5'-half as the third strand (H-y5 form). The relative populations of the two triplex isomers was determined using the chemical probe with diethyl pryrocarbonate as a function of incubation time. The results demonstrated that triplexes were formed rapidly after a pH change from pH 8.0 to 5.0 and that the initial population of the two isomers exponentially changed with incubation time to reach true thermodynamic equilibrium with a time constant of 0.6-10 h, depending on temperature and the presence of Mg2+. The results clearly demonstrated that interconversion occurs between the two isomers and that the presence of Mg2+ generally retarded the interconversion rates. Kinetic and thermodynamic analyses of the relative populations of the two isomers revealed that the apparent energy barrier for transition from duplex to the H-y3 form is higher than that to the H-y5 form, but H-y3 is more stable in enthalpy terms than H-y5. Therefore, H-y3 is kinetically inferior but thermodynamically favored at higher supercoil levels in plasmids. The presence of Mg2+ resulted in both a kinetic and a thermodynamic preference for H-y5 formation, relative to the H-y3 form.

DNA↗