SEARCH · PubMed Health

Results for “Plasmid structure”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 361 records · Page 20Linked to original sources

PubMed1989

Deletion hot spots in chimeric Escherichia coli plasmids.

Deletions form frequently in chimeric plasmids composed of M13mp2, pBR322, and pC194 (B. Michel and S. D. Ehrlich, Proc. Natl. Acad. Sci. USA 83:3386-3390, 1986). They are generated by joining of the nucleotide neighboring the nick site in the M13 replication origin to a nonadjacent nucleotide. This nucleotide is most often located within particular short plasmid regions, named deletion hot spots. Three natural hot spots were present in the chimeric plasmids. Two were active only when the DNA replication initiated at the M13 origin was allowed to progress; the third was active only in the presence of wild-type amounts of DNA ligase. Three artificial hot spots were generated by creating palindromic sequences in the plasmids.

Base Sequence↗

PubMed1991

Structure of the gas vesicle plasmid in Halobacterium halobium: inversion isomers, inverted repeats, and insertion sequences.

Halobacterium-halobium NRC-1 harbors a 200-kb plasmid, pNRC100, which contains a cluster of genes for synthesis of buoyant gas-filled vesicles. Physical mapping of pNRC100 by using pulsed-field gel electrophoresis showed the presence of a large (35 to 38-kb) inverted repeat (IR) sequence. Inversion isomers of pNRC100 were demonstrated by Southern hybridization analysis using two restriction enzymes, AflII and SfiI, that cut asymmetrically within the intervening small single-copy region and the large single-copy region, respectively, but not within the large IRs. No inversion isomers were observed for a deletion derivative of pNRC100 lacking one IR, which suggests that both copies are required for inversion to occur. Additionally, the identities and approximate positions of 17 insertion sequences (IS) in pNRC100 were determined by Southern hybridization and limited nucleotide sequence analysis across the IS element-target site junctions: ISH2, a 0.5-kb element, was found in four copies; ISH3, a 1.4-kb heterogeneous family of elements, was present in seven copies; ISH8, a 1.4-kb element, was found in five copies; and ISH50, a 1.0-kb element, was present in a single copy. The large IRs terminated at an ISH2 element at one end and an ISH3 element at the other end. pNRC100 is similar in structure to chloroplast and mitochondrial genomes, which contain large IRs and other large halobacterial and prokaryotic plasmids that are reservoirs of IS elements but lack the large IRs.

Base Sequence↗

PubMed1986

Cloning and sequencing of the gene encoding cytochrome c3 from Desulfovibrio vulgaris (Hildenborough).

The gene encoding the redox protein cytochrome c3 from Desulfovibrio vulgaris (Hildenborough) has been cloned using two synthetic oligonucleotides (one 17-mer and one 18-mer), designed to recognize the structural gene. Plasmid pCYC3 was derived from the clone and contains a 7.5 X 10(3)-base EcoRI-HindIII insert of D. vulgaris DNA in pUC9. A 674-base-pair fragment of this insert was sequenced with the dideoxy-chain-termination procedure and found to contain the entire structural gene encoding cytochrome c3 bracketed by apparent Escherichia coli consensus sequences for initiation and termination of transcription. The amino acid sequence of 107 residues, derived from protein sequencing [Trousil, E. B. and Campbell, L. L. (1974) J. Biol. Chem. 249, 386-393], is confirmed by the nucleic acid sequence, which shows in addition that it is preceded by a hydrophobic, positively charged signal sequence of 21 residues. This amino-terminal extension functions in the export of cytochrome c3, which is thought to reside in the periplasm of D. vulgaris.

Amino Acid Sequence↗

PubMed1988

High-voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA.

Electroporation permits the uptake of DNA by mammalian cells and plant protoplasts because it induces transient permeability of the cell membrane. We investigated the utility of high-voltage electroporation as a method for genetic transformation of intact bacterial cells by using the enteric pathogen Campylobacter jejuni as a model system. This report demonstrates that the application of high-voltage discharges to bacterial cells permits genetic transformation. Our method involves exposure of a Campylobacter cell suspension to a high-voltage exponential decay discharge (5-13 kV/cm) for a brief period of time (resistance-capacitance time constant = 2.4-26 msec) in the presence of plasmid DNA. Electrical transformation of C. jejuni results in frequencies as high as 1.2 x 10(6) transformants per microgram of DNA. We have investigated the effects of pulse amplitude and duration, cell growth conditions, divalent cations, and DNA concentration on the efficiency of transformation. Transformants of C. jejuni obtained by electroporation contained structurally intact plasmid molecules. In addition, evidence is presented that indicates that C. jejuni possesses DNA restriction and modification systems. The use of electroporation as a method for transforming other bacterial species and guidelines for its implementation are also discussed.

Bacteriological Techniques↗

PubMed1998

Extrachromosomal unequal homologous recombination and gene conversion in simian kidney cells: effects of UV damage.

Shuttle plasmid vectors containing the SV40 origin of replication and tandem neo genes with distally placed non-overlapping deletions were used to study the effects of DNA damage on extrachromosomal homologous recombination in simian kidney cells. DNA was introduced into COS7 cells by a lipofectin-mediated transfection procedure and recombination was assessed by analyzing the structure of plasmids. Recombinational events observed included unequal homologous recombination (triplication), gene conversion, double reciprocal recombination, deletion (pop-outs), gene amplification (4-6 copies), and multimerization. Triplication, an event that previously had not been reported in association with extrachromosomal recombination, predominated in experiments with undamaged vectors. The recombination frequency (NeoR/AmpR) of vectors randomly damaged by UV irradiation was essentially unchanged; however, the relative number of triplication events decreased significantly. Selective damage in one of the two neo genes increased the relative frequency of gene conversion. The experimental system developed for use in this study detects all major homologous recombination events observed in chromosomal direct repeat sequences in mammalian cells and yeast and should prove valuable for future studies of homologous recombination in mammalian cells.

Animals↗

PubMed1998

Monte Carlo simulation of DNA strand-break induction in supercoiled plasmid pBR322 DNA from indirect effects.

We present a new Monte Carlo simulation code system (DBREAK) of the detailed events that occur when ionizing radiation interacts with water and DNA molecules. The model treats the initial energy deposition by radiation, the formation of chemically active species, subsequent diffusion-controlled chemical reactions, and induction of DNA strand breaks. DBREAK assumes one-hit single-strand break (SSB) and two-hit double-strand break (DSB) mechanisms. A high-resolution model of plasmid DNA structure has been introduced. The calculated results are compared with the results of previously performed experiments of the same type. Under aerobic conditions, 89.4% of the DNA damage was attributed to OH-radical and 10.5% and 0.1% to eaq- and H, respectively. We also compared the differences between liquid-water track structure and gas-phase-water track structure. The calculated yield of SSBs by liquid-water track structure exceeded that of gas-phase-water track structure by a factor of 1.2.

Aerobiosis↗

PubMed1981

Structure of a naturally occurring plasmid with genes for enterotoxin production and drug resistance.

A physical map of the 117-kilobase conjugative plasmid pCG86 was constructed using electron microscope heteroduplex analysis. This plasmid carries the genes elt, for heat-labile enterotoxin, and estA, for heat-stable enterotoxin, as well as the genes for resistance to tetracycline, streptomycin, sulfonamides, and mercury. These genes were mapped using deletions and Tn5 insertions as physical markers. Analysis of a heteroduplex between pCG86 and a previously described enterotoxin plasmid (EntP307) showed a 48-kilobase region of complete homology which included the genes elt and estA. An 8.8-kilobase BamHI fragment of EntP307 carrying elt, cloned by others, was also shown to be completely homologous with pCG86. The position of elt on the fragment was verified, and it was shown to carry estA as well. A 44-kilobase region of pCG86 showed partial homology with the region of EntP307 previously shown to contain conjugal transfer genes. The gene for tetracycline resistance is carried on a stem-loop structure with the dimensions of Tn10, and the genes for the other drug resistance markers are carried on a 14.6-kilobase segment that forms an insertion loop in heteroduplexes with EntP307. These studies suggest that pCG86 arose either by recombination between an enterotoxin plasmid of incompatibility group FI, like EntP307, and a multiple resistance factor of incompatibility group FII, or by transposition into EntP307 of two transposons.

Cloning, Molecular↗

PubMed2005

Optimizing plasmid-based gene transfer for investigating skeletal muscle structure and function.

Intramuscular injection of naked plasmid DNA is a less cytotoxic alternative to viral vectors for delivering genetic material to skeletal muscle in vivo. However, the low efficiency of plasmid-based gene transfer limits its potential therapeutic efficacy and/or its use for many experimental applications. Current strategies to enhance transfection efficiency (i.e., electroporation) can cause significant muscle damage, confounding physiological assessments such as muscle contractility. Optimizing protocols to limit damage is critical for accurate physiological, biochemical, and molecular measurements. Following extensive testing, we developed an electroporation protocol that enhances transfection efficiency in skeletal muscles without causing muscle damage. Pretreating mouse tibialis anterior muscles with hyaluronidase and electroporation at 75 V/cm (using 50% vol/vol saline as a vehicle for plasmid DNA) resulted in 22 +/- 5% of the muscle fibers expressing a reporter gene. This protocol did not compromise contractile function of skeletal muscles assessed at both the intact (whole) muscle and the cellular (single fiber) level. Furthermore, ectopic expression of insulin-like growth factor I to levels that induced muscle fiber hypertrophy without causing tissue damage or compromising muscle function highlights the therapeutic potential of these methods for myopathies, muscle wasting disorders, and other pathophysiologic conditions.

Animals↗

PubMed1989

Bovine papillomavirus type 1 encodes two forms of a transcriptional repressor: structural and functional analysis of new viral cDNAs.

Genetic and biochemical evidence has established that the E2 open reading frame (ORF) of bovine papillomavirus type 1 encodes at least two different site-specific DNA-binding proteins, one which activates and the other which represses expression from a viral promoter (P. F. Lambert, B. A. Spalholz, and P. M. Howley, Cell 50:69-78, 1987). We have obtained data which show that a second form of the repressor gene is expressed in transformed cells harboring stable viral plasmids. The structural details of this gene have been discerned by cDNA cloning, by RNase protection, and by primer extension analysis of in vivo RNA. Moreover, data from in vitro transcription experiments support the notion that this form of the E2 repressor is expressed from a novel viral promoter and that a small exon from another ORF is linked to an active repressor domain in E2. Thus, two different forms of the repressor are expressed from different promoters and might be independently regulated either in the cell cycle or in different tissue types. We show by functional in vivo assays utilizing a cDNA vector encoding this gene that the trans-acting factor has in vivo activities similar to those of the known repressor. Our screen of a cDNA library for cDNA clones representing bovine papillomavirus transcripts has also revealed a number of other novel structures defining new donor and acceptor RNA-processing sites. Notably, clones which conceptually can be translated to yield an E7 protein, the viral M gene, and the entire E2 ORF have been characterized. Finally, truncated versions of putative E8 cDNAs were also obtained.

Bovine papillomavirus 1↗

PubMed2001

Conditional-replication, integration, excision, and retrieval plasmid-host systems for gene structure-function studies of bacteria.

We have developed a series of powerful and versatile conditional-replication, integration, and modular (CRIM) plasmids. CRIM plasmids can be replicated at medium or high copy numbers in different hosts for making gene (or mutant) libraries. They can be integrated in single copies into the chromosomes of Escherichia coli and related bacteria to study gene function under normal physiological conditions. They can be excised from the chromosome, e.g., to verify that phenotypes are caused by their presence. Furthermore, they can be retrieved singly or en masse for subsequent molecular analyses. CRIM plasmids are integrated into the chromosome by site-specific recombination at one of five different phage attachment sites. Integrants are selected as antibiotic-resistant transformations. Since CRIM plasmids encode different forms of resistance, several can be used together in the same cell for stable expression of complex metabolic or regulatory pathways from diverse sources. Following integration, integrants are stably maintained in the absence of antibiotic selection. Each CRIM plasmid has a polylinker or one of several promoters for ectopic expression of the inserted DNA. Their modular design allows easy construction of new variants with different combinations of features. We also report a series of easily curable, low-copy-number helper plasmids encoding all the requisite Int proteins alone or with the respective Xis protein. These helper plasmids facilitate integration, excision ("curing"), or retrieval of the CRIM plasmids.

Attachment Sites, Microbiological↗

PubMed2002

Entrapment and condensation of DNA in neutral reverse micelles.

DNA condensation and compaction is induced by a variety of condensing agents such as polycations. The present study analyzed the structure of plasmid DNA (DNA) in the small inner space of reverse micelles formed from nonionic surfactants (isotropic phase). Spectroscopic studies indicated that DNA was dissolved in an organic solvent in the presence of a neutral detergent. Fluorescent quenching of ethidium bromide and of rhodamine covalently attached to DNA suggested that the DNA within neutral, reverse micelles was condensed. Circular dichroism indicated that the DNA structure was C form (member of B family) and not the dehydrated A form. Concordantly, NMR experiments indicated that the reverse micelles contained a pool of free water, even at a ratio of water to surfactant (Wo) of 3.75. Electron microscopic analysis also indicated that the DNA was in a ring-like structure, probably toroids. Atomic force microscopic images also revealed small, compact particles after the condensed DNA structures were preserved using an innovative cross-linking strategy. In the lamellar phase, the DNA was configured in long strands that were 20 nm in diameter. Interestingly, such DNA structures, reminiscent of "nanowires," have apparently not been previously observed.

Circular Dichroism↗

PubMed1994

Isolation and characterization of novel plasmid-encoded umuC mutants.

Most inducible mutagenesis in Escherichia coli is dependent upon the activity of the UmuDC proteins. The role of UmuC in this process is poorly understood, possibly because of the limited number of genetically characterized umuC mutants. To better understand the function of the UmuC protein in mutagenic DNA repair, we have isolated several novel plasmid-encoded umuC mutants. A multicopy plasmid that expressed UmuC at physiological levels was constructed and randomly mutagenized in vitro by exposure to hydroxylamine. Mutated plasmids were introduced into the umu tester strain RW126, and 16 plasmids that were unable to promote umuC-dependent spontaneous mutator activity were identified by a colorimetric papillation assay. Interestingly, these plasmid mutants fell into two classes: (i) 5 were expression mutants that produced either too little or too much wild-type UmuC protein, and (ii) 11 were plasmids with structural changes in the UmuC protein. Although hydroxylamine mutagenesis was random, most of the structural mutants identified in the screen were localized to two regions of the UmuC protein; four mutations were found in a stretch of 30 amino acids (residues 133 to 162) in the middle of the protein, while four other mutations (three of which resulted in a truncated UmuC protein) were localized in the last 50 carboxyl-terminal amino acid residues. These new plasmid umuC mutants, together with the previously identified chromosomal umuC25, umuC36, and umuC104 mutations that we have also cloned, should prove extremely useful in dissecting the genetic and biochemical activities of UmuC in mutagenic DNA repair.

Bacterial Proteins↗

PubMed2001

Purification of plasmids for gene therapy and DNA vaccination.

This chapter covers the different aspects of the production and purification of plasmids for gene therapy and DNA vaccination. Process issues are extensively covered and complemented with information related to plasmid DNA structure, vector construction, product specifications and quality assurance and control.

Animals↗

PubMed2000

Substrate-binding clusters of the K+-transporting Kdp ATPase of Escherichia coli investigated by amber suppression scanning mutagenesis.

The Kdp-ATPase of Escherichia coli is a four-subunit P-type ATPase that accumulates K(+) with high affinity and specificity. Residues clustered in four regions of the KdpA subunit of Kdp were implicated as critical for K(+) binding from the analysis of mutants with reduced affinity for K(+) (Buurman, E., Kim, K.-T., and Epstein, W. (1995) J. Biol. Chem. 270, 6678-6685). K(+) binding by this pump has been analyzed in detail by site-directed mutagenesis. We have examined 83 of the 557 residues in KdpA, from 11 to 34 residues in each of four binding clusters known to affect K(+) binding. Amber mutations were constructed in a plasmid carrying the kdpFABC structural genes. Transferring these plasmids to 12 suppressor strains, each inserting a different amino acid at amber codons, created 12 different substitutions at the mutated sites. This study delineates the four clusters and confirms that they are important for K(+) affinity but have little effect on the rate of transport. At only 21 of the residues studied did at least three substitutions alter affinity for K(+), an indication that a residue is in or very near a K(+) binding site. At many residues lysine was the only substitution that altered its affinity. The effect of lysine is most likely a repulsive effect of this cationic residue on K(+) and thus reflects the effective distance between a residue and the site of binding or passage of K(+) in KdpA. Once a crystallographic structure of Kdp is available, this measure of effective distance will help identify the path of K(+) as it moves through the KdpA subunit to cross the membrane.

Adenosine Triphosphatases↗

PubMed1989

Cationic metal-specific structures adopted by the poly(dG) region and the direct repeats in the chicken adult beta A globin gene promoter.

Naturally occurring contiguous deoxyguanine residues and their surrounding sequences in the chicken adult beta A globin gene promoter were analyzed for their inherent potential to adopt non-B DNA structures in supercoiled plasmid DNA. In particular, cationic effects on structure were studied by treating the supercoiled plasmid DNA harboring the chicken adult beta A globin 5' flanking sequence with an unpaired DNA base-specific probe, chloroacetaldehyde in the presence of either Mg++, Cu++, Zn++, Ca++ or Co++ ions. The chloroacetaldehyde-reactive bases were mapped at a single base resolution by a chemical cleavage method that specifically cleaves DNA at the chloroacetaldehyde modified sites. These experiments revealed that while Mg++ and Ca++ ions induce a dG.dG.dC triple helix structure at the contiguous dG residues, Zn++, Cu++ and Co++ ions induce yet another structure at the direct repeats immediately 5' of the dG residues. When Mg++ and Zn++ ions are both present, Zn++ inhibits the dG.dG.dC triplex at the contiguous dG residues and induces a particular non-B DNA structure at the adjacent direct repeats. The specific induction of non-B DNA structures by metal ions at the two adjacent sequences within the promoter region may be of biological significance.

Animals↗

PubMed2024

Putative evolution of Myxococcus fulvus 124B02 plasmid pMF1 from a chromosomal segment in another Myxococcus species.

Myxobacteria or order Myxococcales (old nomenclature) or phylum Myxococcota (new terminology) are fascinating organisms well known for their diverse peculiar physiological, taxonomic, and genomic properties. Researchers have long sought to identify plasmids within these organisms, yet thus far, only two organisms from different families have been found to harbor a plasmid. This study delves into the putative evolution of one of these plasmids, i.e., pMF1 present in Myxococcus fulvus 124B02 in the suborder Cystobacterineae and family Myxococcaceae. Here, we first reannotated the pMF1 plasmid genome sequence and identified two additional open reading frames or putative genes which were not annotated until now. We further reported that all pMF1 plasmid genes depict homology with Myxococcus stipitatus CYD1 draft genome (contig 28) and a chromosomal segment of M. stipitatus DSM14675 in a syntenic manner, implying the presence of plasmid-like structure in M. stipitatus CYD1, integrated into its chromosome. To comprehend the relationship among these three species, we conducted phylogenetic analyses using 16S and concatenated housekeeping genes and genome-to-genome distance calculator (GGDC) analysis, which confirmed that M. stipitatus CYD1 is a distinct and novel species within the genus Myxococcus. Overall, this comparative genomic study sheds light on the putative emergence of the pMF1 plasmid from a common ancestor of closely related yet distinct species, M. stipitatus CYD1, possibly through the partition from its chromosome as a segment.IMPORTANCEMyxobacteria are not well known to have plasmids. Until now, only two organisms have been shown to have plasmids, raising a pertinent question about how these plasmids evolved randomly within the phylum Myxococcota. The study presented in this manuscript delves into the emergence of the pMF1 plasmid found in Myxococcus fulvus 124B02, a member of the suborder Cystobacterineae and family Myxococcaceae. Our research addresses this intriguing topic of plasmid identification and evolution within myxobacteria, which are a group of fascinating organisms that have garnered significant interest due to their diverse physiological, taxonomic, and genomic properties.

Plasmids↗

PubMed1987

Virulence-plasmid is associated with the inhibition of opsonization in Yersinia enterocolitica and Yersinia pseudotuberculosis.

Plasmid-cured variants of virulent strains of Yersinia enterocolitica and Y. pseudotuberculosis were obtained by selection after growth in calcium-deficient medium. To obtain antigen preparations consisting of whole bacteria the original plasmid-containing strains and the plasmid-cured variants were grown in conditions favouring expression of the temperature-inducible outer membrane proteins of Yersinia (YOP) (37 degrees C, calcium-deficient culture medium). The presence or absence of the YOP on the bacteria was verified by immunoblotting. Opsonophagocytosis of YOP-negative Yersinia preparations (YOP-) was compared to that of YOP-containing ones (YOP+) in human polymorphonuclear leukocyte (PMN) chemiluminescence (CL) assay. The attachment of complement C3b on the surface of the bacteria after opsonization with normal human serum was determined by using a fluorescent anti-C3c-antibody and flow cytometry. YOP+ bacteria resisted opsonization in the absence of specific antibodies, as indicated by diminished C3b-fixation on bacteria and weaker CL response. This implies that virulence-plasmid-coded structures provide Y. enterocolitica and Y. pseudotuberculosis with an ability to avoid complement-mediated opsonization and phagocytosis.

Bacterial Outer Membrane Proteins↗

PubMed1980

Association of the 2-micron DNA plasmid with yeast folded chromosomes.

The 2-micron DNA plasmid cosedimented in sucrose gradients with the folded chromosome on centrifugation of lysates from logarithmically growing yeast cells. It banded with both the slow-sedimenting g1 form derived from prereplicative cells and the fast-sedimenting g2 form from postreplicative cells. When cells were induced to withdraw from the cell cycle by nitrogen starvation, the folded chromosome was converted to the g0 form, which sedimented more slowly than the g1 form, and only 1/10th the amount of 2-micron DNA cosedimented with this g0 form as compared to the logarithmic-phase forms, g1 and g2. Expression of the temperature-sensitive cell division cycle (cdc) 28 mutation, which defines the "start" of the cell cycle, resulted in a rapid alteration in the folded chromosome sedimentation pattern, and less than 20% of the 2-micron DNA cosedimented with the folded chromosome. These results raise the possibility that association of the 2-micron DNA plasmid with structures in the cell corresponding to the folded chromosome is subject to cell division cycle control.

Cell Cycle↗