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Results for “Plasmid structure”

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At least 163 records · Page 9Linked to original sources

PubMed1993

A useful cloning vector for Bacillus subtilis.

We have constructed plasmid pDN1050, a new small cloning vector for Bacillus subtilis. pDN1050 harbors the origin of replication of Staphylococcus aureus plasmid pUB110 and the chloramphenicol resistance gene of S. aureus plasmid pC194. The plasmid is segregationally and structurally stable. Plasmid pDN1370, a low copy number mutant of pDN1050 was isolated and shown to harbor a mutation in the repA gene of the replication protein.

Bacillus subtilis↗

PubMed1990

Stimulation by gamma-carboline derivatives (simplified analogues of antitumor ellipticines) of site specific DNA cleavage by calf DNA topoisomerase II.

gamma-Carbolines are tricyclic aromatic compounds which intercalate into DNA base pairs and exhibit significant cytotoxic and antitumor activities. These compounds which are structurally related to ellipticine by deletion of an aromatic ring, induce DNA breaks in cultured L1210 cells. Since the mechanism of cytotoxic activity of ellipticines involves DNA topoisomerase II, this enzyme might also be a target for gamma-Carbolines. We have tested this hypothesis using an in vitro system containing purified enzyme and pBR322 DNA. The ability of nine derivatives to stabilize the DNA-enzyme covalent complex was studied and compared to their cytotoxicity. The four less cytotoxic compounds do not induce cleavable complex to a significant extent. In contrast, the two most cytotoxic gamma-Carbolines are the most efficient stabilizers of the cleavable complex. The last three compounds exhibit an intermediate cytotoxicity and cleavage activity. In the presence of gamma-Carbolines, cleavage occurs predominantly at a single site in pBR322 which is one of the cleavage sites observed with ellipticines. The cleavage position was determined at the nucleotide level. The increased DNA cleavage specificity observed with gamma-Carbolines suggests that a tricyclic system is as efficient as ellipticines for DNA topoisomerase II cleavage at DNA sequences involved specifically in cytotoxic response. The data presented support the hypothesis that DNA topoisomerase II is a target involved in the mechanisms of action of antitumor gamma-Carbolines.

Amsacrine↗

PubMed1991

Equilibrium binding of four anthracyclines to nucleic acids: thermodynamic properties and sequence selectivity.

The thermodynamic parameters of the interaction of the two anthracyclines 13-dihydrodaunomycin and marcellomycin with calf thymus DNA were examined by equilibrium binding studies. Enthalpy and entropy changes of the binding of both drugs show salt dependence profiles that cannot be rationalized by the polyelectrolyte theory. This feature is common to other anthracycline compounds. The nucleotide sequence binding preferences of daunomycin, adriamycin, 13-dihydrodaunomycin and marcellomycin have been studied by monitoring the degree of protection from cleavage by restriction endonucleases of linearized pBR322. Differential protection of pBR322 DNA against the cleavage of Bgl I and Ava II suggests that these drugs recognize changes in the sequences near the enzyme recognition site. Alterations of the electrophoretic restriction pattern of pBR322 in the presence of anthracyclines are dependent on time and on concentration. These results are discussed in relation to the existence of nucleotide sequences with different affinity for these drugs.

Antibiotics, Antineoplastic↗

PubMed2005

Generation of a single-chain Fv fragment for the monitoring of deoxycholic acid residues anchored on endogenous proteins.

A subset of lipophillic bile acids, including deoxycholic acid (DCA) and lithocholic acid (LCA), are thought to be biologically transformed into reactive intermediates forming covalently modified, "tissue-bound" bile acids that can exert several toxic effects. We have generated a single-chain Fv fragment (scFv) as a probe to monitor DCA residues anchored on proteins. DNA fragments encoding the variable heavy (V(H)) and light (V(L)) domains of a mouse antibody raised against a DCA hapten (Ab #88) were cloned by rapid amplification of cDNA 5'-ends. These sequences were combined via a common linker sequence coding (Gly(4)Ser)(3) to construct a single scFv gene with the gene segments in the following order: 5'-V(H)-linker-V(L)-3'. This construct was subcloned into an antibody-expression vector, pEXmide 5; soluble scFv protein was then expressed in the bacterial periplasm of the XLOLR Escherichia coli strain. In a competitive enzyme-linked immunosorbent assay using DCA-coated microtiter plates, the scFv provided a dose-response curve for free DCA ranging between 2 and 5000 pg/assay. The scFv reacts similarly with the l-lysine adduct of DCA (cross-reactivity, 72%), while bile acids having a modified DCA steroid skeleton were well-discriminated (cross-reactivity, <1%). This scFv could also monitor trace amounts of DCA residues anchored on a protein through DCA acyl adenylate reactions, the likely reactive intermediate. The present scFv may be a useful tool for trace characterization of tissue-bound bile acids; this usefulness may be significantly enhanced by fusion with signal-generating proteins, such as alkaline phosphatase or green fluorescent protein.

Amino Acid Sequence↗

PubMed2002

Path of DNA within the Mu transpososome. Transposase interactions bridging two Mu ends and the enhancer trap five DNA supercoils.

The phage Mu transpososome is assembled by interactions of transposase subunits with the left (L) and right (R) ends of Mu and an enhancer (E) located in between. A metastable three-site complex LER progresses into a more stable type 0 complex in which a tetrameric transposase is poised for DNA cleavage. "Difference topology" has revealed five trapped negative supercoils within type 0, three contributed by crossings of E with L and R, and two by crossings of L with R. This is the most complex DNA arrangement seen to date within a recombination synapse. Contrary to the prevailing notion, the enhancer appears not to be released immediately following type 0 assembly. Difference topology provides a simple method for determining the ordered sequestration of DNA segments within nucleoprotein assemblies.

Bacteriophage mu↗

PubMed1991

Synthesis of water-soluble (aminoalkyl)camptothecin analogues: inhibition of topoisomerase I and antitumor activity.

Water-soluble analogues of the antitumor alkaloid camptothecin (1) were prepared in which aminoalkyl groups were introduced into ring A or B. Most of the analogues were prepared by oxidation of camptothecin to 10-hydroxycamptothecin (2) followed by a Mannich reaction to give N-substituted 9-(aminomethyl)-10-hydroxycamptothecins (4-12) or by subsequent modification of Mannich product 4 (13, 15, 17, 19, 21). Others were obtained by modification of the hydroxyl group of 2 (25,26) or by total synthesis (35,42,43). These analogues, as well as some of their synthetic precursors, were evaluated for inhibition of topoisomerase I, cytotoxicity, and antitumor activity. Although there was not a quantitative correlation between these assays, compounds that inhibited topoisomerase I were also cytotoxic and demonstrated antitumor activity in vivo. Further evaluation of the most active water-soluble analogue led to the selection of 9-[(dimethylamino)methyl]-10-hydroxycamptothecin (4, SK&F 104864) for development as an antitumor agent. In addition to its water solubility, ease of synthesis from natural camptothecin, and high potency, 4 demonstrated broad-spectrum activity in preclinical tumor models and is currently undergoing Phase I clinical trials in cancer patients.

Animals↗

PubMed1991

Synthesis and evaluation of DNA-targeted spatially separated bis(aniline mustards) as potential alkylating agents with enhanced DNA cross-linking capability.

DNA-targeted separated bis-mustards were synthesized by attaching aniline mono-mustards at the 4- and 9-positions of the DNA-intercalating ligand 9-aminoacridine-4-carboxamide, with the intention of improving the low cross-link to monoadduct ratio found with most alkylating agents. The geometry of these compounds requires that, when the acridine binds to DNA by intercalation, one alkylating moiety is delivered to each DNA groove. Gel electrophoretic studies show that only one arm of these compounds (probably that attached to the 9-position) alkylates DNA, such alkylation occurring specifically in the major groove at the N7 of guanines. Cell-line studies confirm that the mode of cytotoxicity of these compounds (unlike that of untargeted aniline bis-mustards of comparable reactivity) is due to bulky DNA monoadduct formation. It is concluded that more information is required about the exact orientation of the initial monoadducts before ligands with specific DNA cross-linking ability can be designed.

Alkylating Agents↗

PubMed2001

Synthesis and DNA cleavage activity of a novel bleomycin A(5) glycoconjugate.

[structure in text] To explore the possibility of modifying bleomycin in a fashion that could alter its physiological distribution in a therapeutic setting, a new analogue of bleomycin has been prepared. This analogue is intended to target the asialoglycoprotein receptor on liver cells. Critically, despite the large C-substituent, the bleomycin conjugate was found to degrade DNA in the same fashion as bleomycin A(5) itself, and with only modestly decreased efficiency.

Bleomycin↗

PubMed1998

Identification of two linear plasmids in the actinomycete Planobispora rosea.

Two linear plasmids (pPR1, 27.5 kb, and pPR2, 16 kb) were identified in Planobispora rosea, an actinomycete that produces the antibiotic GE2270, an inhibitor of the elongation factor Tu. Strains lacking both plasmids still produce and are resistant to GE2270. The two plasmids share an internal region of high similarity, but no cross-hybridization was detected between their telomeric regions or between plasmid and chromosomal DNA. The 5' ends of the plasmids appear to be linked to terminal proteins. The telomeric regions of pPR2 were cloned after 3'-end homopolymer tailing and PCR amplification. The approximately 650 nt telomeric DNA sequences of pPR2 are repeated in inverted orientation and are rich in direct and inverted repeats; the 350 bp terminal region is less G + C-rich than the rest of the plasmid. The structural organization of these plasmids appears to be similar to Streptomyces linear replicons.

Actinomycetales↗

PubMed1995

Membrane topology analysis of the Escherichia coli cytosine permease.

The Escherichia coli codBA operon encodes cytosine permease (CodB) and cytosine deaminase (CodA). CodB mediates uptake of exogenously supplied cytosine, and CodA catalyses the hydrolytic deamination of cytosine to uracil and ammonia. The hydropathic profile of CodB indicates that it is an integral cytoplasmic membrane protein possessing several transmembrane-spanning domains. The membrane topology of CodB was investigated by using gene fusions containing varying lengths of the amino-terminus of CodB fused to either alkaline phosphatase (AP) or beta-galactosidase (BG). The AP activities expressed by the CodB-AP fusions are consistent with a topological model in which the amino- and the carboxy-termini of CodB are located in the cytoplasm, and in which CodB possesses 12 membrane-spanning segments. The enzyme activities of most of the CodB-BG fusions support the model. However, the results obtained with some of the CodB-BG fusions illustrate the limitations of using BG as a reporter protein in studies of membrane protein topology.

Alkaline Phosphatase↗

PubMed2005

Novel and specific inhibitors of a poxvirus type I topoisomerase.

Vaccinia DNA topoisomerase (vTopo) is a prototypic pox virus family topoisomerase that shares extensive structural and mechanistic properties with the human type IB enzyme (hTopo) and is important for viral replication. Despite their far-reaching similarities, vTopo and hTopo have surprisingly distinct pharmacological properties. To further exploit these differences, we have developed recently the first high-throughput screen for vTopo, which has allowed rapid screening of a 1990-member small-molecule library for inhibitors. Using this approach, 21 compounds were identified with IC(90) values less than 10 muM, and 19 of these were also found to inhibit DNA supercoil relaxation by vTopo. Four of the most potent compounds were completely characterized and are structurally novel topo I inhibitors with efficacies at nanomolar concentrations. These inhibitors were highly specific for vTopo, showing no inhibition of the human enzyme even at 500- to 2000-fold greater concentrations. We describe a battery of efficient experiments to characterize the unique mechanisms of these vTopo inhibitors and discuss the surprising promiscuity of this enzyme to inhibition by structurally diverse small molecules.

Antiviral Agents↗

PubMed1994

The interferon-gamma receptor extracellular domain. Non-identical requirements for ligand binding and signaling.

The human interferon-gamma receptor (hIFN-gamma R) extracellular domain interacts in a species-specific manner with both its ligand and an accessory factor encoded on human chromosome 21. Mutant interferon-gamma receptors were constructed by homolog-scanning mutagenesis, replacing segments of the human extracellular domain with the corresponding murine sequence. Replacement of hIFN-gamma R amino acids 1-100, 100-132, 134-183, or 183-245 abolished binding to human interferon-gamma (hIFN-gamma). However, replacement of hIFN-gamma R amino acids 134-209, 183-209, 134-153, 153-167, or 167-183 or deletion of residues 156-165 affected hIFN-gamma binding only partially or not at all. Receptors that bound hIFN-gamma were tested for their ability to signal a functional response, induction of major histocompatibility complex class I antigen expression. Replacement of residues 134-209 greatly reduced the ability of the receptor to signal. This signaling defect could not be attributed solely to a reduction in affinity for ligand and could not be localized to any subregion.

Animals↗

PubMed2003

The mosaic structure of the symbiotic plasmid of Rhizobium etli CFN42 and its relation to other symbiotic genome compartments.

BACKGROUND: Symbiotic bacteria known as rhizobia interact with the roots of legumes and induce the formation of nitrogen-fixing nodules. In rhizobia, essential genes for symbiosis are compartmentalized either in symbiotic plasmids or in chromosomal symbiotic islands. To understand the structure and evolution of the symbiotic genome compartments (SGCs), it is necessary to analyze their common genetic content and organization as well as to study their differences. To date, five SGCs belonging to distinct species of rhizobia have been entirely sequenced. We report the complete sequence of the symbiotic plasmid of Rhizobium etli CFN42, a microsymbiont of beans, and a comparison with other SGC sequences available. RESULTS: The symbiotic plasmid is a circular molecule of 371,255 base-pairs containing 359 coding sequences. Nodulation and nitrogen-fixation genes common to other rhizobia are clustered in a region of 125 kilobases. Numerous sequences related to mobile elements are scattered throughout. In some cases the mobile elements flank blocks of functionally related sequences, thereby suggesting a role in transposition. The plasmid contains 12 reiterated DNA families that are likely to participate in genomic rearrangements. Comparisons between this plasmid and complete rhizobial genomes and symbiotic compartments already sequenced show a general lack of synteny and colinearity, with the exception of some transcriptional units. There are only 20 symbiotic genes that are shared by all SGCs. CONCLUSIONS: Our data support the notion that the symbiotic compartments of rhizobia genomes are mosaic structures that have been frequently tailored by recombination, horizontal transfer and transposition.

Anaerobiosis↗

PubMed2003

Eclipse period during replication of plasmid R1: contributions from structural events and from the copy-number control system.

The eclipse period (the time period during which a newly replicated plasmid copy is not available for a new replication) of plasmid R1 in Escherichia coli was determined with the classic Meselson-Stahl density-shift experiment. A mini-plasmid with the wild-type R1 replicon and a mutant with a thermo-inducible runaway-replication phenotype were used in this work. The eclipses of the chromosome and of the wild-type plasmid were 0.6 and 0.2 generation times, respectively, at temperatures ranging from 30 degrees C to 42 degrees C. The mutant plasmid had a similar eclipse at temperatures up to 38 degrees C. At 42 degrees C, the plasmid copy number increased rapidly because of the absence of replication control and replication reached a rate of 350-400 plasmid replications per cell and cell generation. During uncontrolled replication, the eclipse was about 3 min compared with 10 min at controlled replication (the wild-type plasmid at 42 degrees C). Hence, the copy-number control system contributed significantly to the eclipse. The eclipse in the absence of copy-number control (3 min) presumably is caused by structural requirements: the covalently closed circular plasmid DNA has to regain the right degree of superhelicity needed for initiation of replication and it takes time to assemble the initiation factors.

Centrifugation, Density Gradient↗

PubMed1976

Structural evolution of bacterial plasmids: role of translocating genetic elements and DNA sequence insertions.

Recent evidence suggests that plasmids have evolved by site-specific recombinational events involving translocation and insertion of discretely defined DNA segments. The role of translocating genetic elements and repeated DNA sequences in the formation and structural evolution of bacterial plasmids, and in the control of plasmid gene expression, is the subject of this brief review. Insertion sequence (IS) regions are discrete segments of DNA that are known to cause strongly polar mutations in the genes of Escherichia coli and several bacteriophages as a consequence of their insertion into bacterial or phage genomes. Recent investigations have identified three separate kinds of IS segments on plasmids, and have indicated that such regions may have a role in 1) site-specific reversible dissociation of antibiotic resistance plasmids into their component segments, 2) recombination of certain plasmids with the bacterial chromosome, and 3) translocation of segments of plasmid DNA onto other replicons, or onto different sites of the same replicon. In addition, such DNA sequences, which may be repeated on plasmid genomes in either direct or reverse orientation, are involved in the control of plasmid gene expression. Inverted repeats other than the genetically characterized IS segments also appear to be involved in recA-independent, recombination and translocation of plasmid DNA segments. These inverted repeats contain palindromic nucleotide sequences on each strand of DNA and are detectable as hairpin-loop structures by electron microscope heteroduplex analysis. Such palindromes resemble the recognition sites for restriction endonucleases, some of which are encoded by plasmids, suggesting that similar endonucleolytic enzymes may be involved in the translocation of plasmid DNA segments.

Base Sequence↗

PubMed1984

An immune strain of Halobacterium halobium carries the invertible L segment of phage PhiH as a plasmid.

The structure of the circular prophage genome of PhiH varies with high frequency in single colony progeny of the defective lysogen Halobacterium halobium R(1)-3. As in linear PhiH DNA, a segment flanked by two copies of the insertion element ISH1.8 is inverted frequently. This L segment can also circularize to a plasmid of 12 kilobase pairs with simultaneous loss of the remaining phage DNA. Strain R(1)-L, which contains this plasmid, is immune to phage infection. A phage variant, PhiHL1, is able to grow on R(1)-L and carries an insertion of 1 kilobase pair in its L segment. PhiHL1 does not grow on normal lysogens. This shows that the plasmid confers to R(1)-L only part of the immunity of normal lysogens.

Journal Article↗

PubMed1982

Recombinant plasmids carrying the glutamate dehydrogenase structural gene from Escherichia coli K-12.

Plasmid pRSP1, isolated from the Escherichia coli gene bank of Clarke and Carbon (1975), has been shown to complement the gdh-1 mutation that affects the synthesis of glutamate dehydrogenase (GDH) in the E. coli strain PA340. The GDH structural gene of E. coli is present on a 2.3-Md DNA fragment from the hybrid plasmid pRSP1 and certain of its derivatives. Polypeptides synthesized by minicells carrying some of these plasmids, enabled us to make a preliminary determination of the direction of transcription of the gdh gene on the cloned fragments.

Chromosome Mapping↗

PubMed1997

Structural and fusogenic properties of cationic liposomes in the presence of plasmid DNA.

The structural and fusogenic properties of large unilamellar vesicles (LUVs) composed of the cationic lipid N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dioleoyl-3-phosphatidylethanotamine (DOPE) have been examined in the presence of pCMV5 plasmid and correlated with transfection potency. It is shown, employing lipid mixing fusion assays, that pCMV5 plasmid strongly promotes fusion between DOTMA/DOPE (1:1) LUVs and DOTMA/1,2-dioleoyl-3-phosphatidylcholine (DOTMA/DOPC) (1:1) LUVs such that at a cationic lipid-to-DNA charge ratio of 3.0, approximately 80% fusion is observed. The anions citrate and chloride can also trigger fusion, but at much higher concentrations. Freeze-fracture electron microscopy studies demonstrate the tendency of cationic vesicles to form clusters at low pCMV5 content, whereas macroscopic fused aggregates can be observed at higher plasmid levels. 31P NMR studies of the fused DNA-DOTMA/DOPE (1:1) complexes obtained at high plasmid levels (charge ratio 1.0) reveal narrow "isotropic" 31P NMR resonances, whereas the corresponding DOPC containing systems exhibit much broader "bilayer" 31P NMR spectra. In agreement with previous studies, the transfection potency of the DOPE-containing systems is dramatically higher than for the DOPC-containing complexes, indicating a correlation between transfection potential and the motional properties of endogenous lipids. Interestingly, it was found that the complexes could be separated by centrifugation into a pellet fraction, which exhibits superior transfection potencies, and a supernatant fraction. Again, the pellet fraction in the DOPE-containing system exhibits a significantly narrower 31P NMR resonance than the corresponding DOPC-containing system. It is suggested that the 31P NMR characteristics of complexes exhibiting higher transfection potencies are consistent with the presence of nonbilayer lipid structures, which may play a direct role in the fusion or membrane destabilization events vital to transfection.

Animals↗