Plasmid Fragment Research Articles

Characteristics of IS401, a New Member of the IS3 Family Implicated in Plasmid Rearrangements in Pseudomonas cepacia

We have determined the nucleotide sequence of IS401, an insertion sequence implicated in rearrangements of a 170-kb cryptic plasmid from Pseudomonas cepacia . Our analysis focused on a 4066-bp plasmid fragment containing adjacent copies of IS401 and of IS408, an element reported previously to activate gene expression in P. cepacia. One objective was to determine if an apparent increase in the copy number of IS401 in strains carrying adjacent plasmid copies of these two elements might be due to readthrough transcription of an IS401 transposase gene from an outwardly directed promoter within IS408. This possibility was ruled out by nucleotide sequence analysis of the 4066-bp plasmid fragment, which indicated that the major open reading frames of IS401 were oriented in the direction of IS408. IS401 was 1316 bp in length and had 26-bp terminal inverted repeats flanked by 3-bp direct duplications of adjacent DNA. It was closely related to the IS3 family elements IS51 from P. savastanoi and IS3411 from Escherichia coli. Pertinent features of IS408 are also discussed.

Monofunctional Adducts of Platinum(II) Produce in DNA a Sequence-Dependent Local Denaturation

The effects on the conformation of DNA produced by the monofunctional adducts of chloro-(diethylenetriamine)platinum(II) chloride or cis-diamminemonoaquamonochloroplatinum(II) have been investigated by means of the single-strand-specific probe chloroacetaldehyde (CAA). The denatured sites to which CAA was bound and that were induced in DNA by the monofunctional adducts of the platinum complexes were characterized by means of three experimental approaches. These include measurement of the fluorescence of a plasmid fragment treated with CAA, analysis of oligonucleotides treated with CAA and cleaved by piperidine, and termination of duplex transcription on a fragment of plasmid DNA treated with CAA. The results indicate that the denaturational change preferentially occurs in the base pair containing the monoadducted deoxyriboguanosine in the trinucleotide sequence Py-deoxyriboguanosine-Py (Py is a pyrimidine deoxyribonucleoside). It was suggested that this conformational alteration facilitates in DNA the formation of minor bifunctional adducts of cis-diamminedichloroplatinum(II).

Amplification of plasmid DNA to detect plant pathogenic mycoplasmalike organisms

SummaryThe polymerase chain reaction (PCR) was employed to develop a specific assay for plant pathogenic mycoplasmalike organisms (MLOs). A cloned fragment of a plasmid from a severe strain of western aster yellows (AY)‐MLO was sequenced to identify oligonucleotide primers for PCR. Amplified DNA fragments of the predicted size were obtained from DNA extracted from plants and insects infected with pear decline MLO, beet leafhopper‐transmitted virescence agent, elm yellows MLO and several AY‐MLO strains. No amplification occurred from healthy leafhopper or plant DNA. The PCR‐based assay was over 500 times more sensitive than a _tilized_tion‐based assay which _tilized a cloned AY plasmid fragment as a probe. With the PCR‐based assay, MLOs could be detected using DNA samples of leafhoppers that were only crushed and boiled in buffer. Amplification of the target DNA was confirmed by digestion of the PCR product with Mbo I which yielded predicted sized fragments for all MLO strains except Bradford AY and eastern AY. Sequencing the PCR product from elm yellows and eastern AY‐MLOs revealed greater than 90% homology, and the failure to restrict the PCR product with Mbo I was due to a single base change in the restriction endonuclease site. The ability of the assay to detect a wide range of MLOs with minimal sample preparation and high sensitivity will be useful in epidemiological studies of MLO‐caused diseases.

AFM of phospholipid membranes: From ripples to domains to interdigitation

In the past few years, a great deal of effort was made in many laboratories in the development of suitable specimens and better instrumentation for biological applications of atomic force microscopy (AFM) for its promise of high spatial resolution imaging of biological samples under physiological conditions. This effort has resulted in the publication of a number of excellent and reproducible images of various bio-macromolecules. Most notables include plasmid DNA and DNA fragments at, gap junction plaques, and cholera toxin, among others. However, the majority of these work were aimed at the methodology and the characterization of the AFM. Very little information was actually revealed about the biological systems being studied. This has been one of the major criticisms from many biologists and electron microscopists, that nothing new was learned by biological AFM.This situation has started to change recently. Rees et al. showed by AFM in air that the DNA template at the site of DNA-RNA polymerase complex bent differently in the presence of open or elongation promoter.

Detection of the Bean Common Blight Bacteria,Xanthomonas campestrispv.phaseoliandX. c. phaseolivar.fuscans,Using the Polymerase Chain Reaction

A 3.4-kb plasmid DNA fragment (p7) of Xanthomonas campestris pv. phaseoli that hybridizes specifically to X. c. phaseoli was subcloned and partially sequenced to design primers for a polymerase chain reaction (PCR) assay for the detection of bacteria causing common blight in bean. In Southern hybridization experiments, p7X3 and p7X4 subfragments showed high specificity for total genomic DNA of pathogenic X. c. phaseoli, whereas the p7X2 segment also weakly hybridized to DNA extracted from some strains belonging to other X. campestris pathovars. For each of these p7 fragments, a set of G + C-rich oligonucleotide primers was devised and tested under high-stringency conditions in PCR assays

Probes for the study of mupirocin resistance in staphylococci.

Probes constructed from a 4.05-kb EcoRI digest fragment of a mupirocin resistance plasmid and a 751-bp internal part of this fragment hybridised with DNA from all of 36 independent high-level mupirocin-resistant staphylococci tested from seven centres; most were Staphylococcus aureus. In most instances the probes detected an EcoRI digest fragment of approximately 4 kb. Probes did not hybridise to DNA from low-level resistant strains, nor from strains sensitive to mupirocin.

Equilibrium association constants for oligonucleotide-directed triple helix formation at single DNA sites: linkage to cation valence and concentration.

The linkage between the energetics of oligonucleotide-directed triple helix formation and the cationic solution environment has been investigated in mixed-valence salt solutions. Equilibrium constants for formation of the local pyrimidine.purine.pyrimidine structure afforded by binding of the oligonucleotide 5'-d(T*TTTTCTCTCTCTCT)-3' to a single site within a 339-bp plasmid fragment were measured using quantitative affinity cleavage titrations at pH 7.0 and 22 degrees C in the presence of various concentrations of KCl, MgCl2, and spermine tetrahydrochloride (SpmCl4). In a solution containing 10 mM NaCl, 140 mM KCl, 1.0 mM MgCl2, and 1.0 mM SpmCl4, the measured binding constant was 3.3 (+/- 1.4) x 10(5) M-1. The equilibrium constant previously reported for the same association reaction in 100 mM NaCl and 1 mM SpmCl4 at the same temperature and pH was 10-fold higher [Singleton, S. F., & Dervan, P. B. (1992) J. Am. Chem. Soc. 114, 6957-6965]. Further study demonstrated that varying the potassium ion concentration between 5.0 and 140 mM (in the presence of 10 mM NaCl, 1.0 mM MgCl2, and 1.0 mM SpmCl4) resulted in an overall 100-fold decrease in the binding affinity from the lowest to the highest concentration. In contrast, measured binding constants increased 500-fold as the spermine concentration was increased from 0.40 to 4.0 mM (in the presence of 10 mM NaCl, 140 mM KCl, and 1.0 mM MgCl2). There was a modest effect on the binding constant (a 3-fold decrease) upon varying the magnesium ion concentration from 0.10 to 10 mM (in the presence of 10 mM NaCl, 140 mM KCl, and 1.0 mM SpmCl4).(ABSTRACT TRUNCATED AT 250 WORDS)

Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells.

A 31-kb fragment of the large virulence plasmid of Shigella flexneri is necessary for bacterial entry into epithelial cells in vitro. One locus of this fragment encodes the IpaA, -B, -C, and -D proteins, which are the dominant antigens of the humoral immune response during shigellosis. To address the role of the ipa genes, which are clustered in an operon, we constructed a selectable cassette that does not affect transcription of downstream genes and used this cassette to inactivate the ipaB, ipaC, and ipaD genes. Each of these nonpolar mutants was defective in entry and lysis of the phagocytic vacuole but was not impaired in adhesion to the cells. We showed that, like IpaB and IpaC, IpaD is secreted into the culture supernatant and that none of these proteins is necessary for secretion of the other two. This result differentiates the Ipa proteins, which direct the entry process, from the Mxi and Spa proteins, which direct secretion of the Ipa proteins. Moreover, lack of either IpaB or IpaD resulted in the release of larger amounts of the other Ipa polypeptides into the culture medium, which indicates that, in addition to their role in invasion, IpaB and IpaD are each involved in the maintenance of the association of the Ipa proteins with the bacterium.

Transfection of Human Mesothelial Cells Mediated by Different Asbestos Fiber Types

Several different asbestos fiber types mediate transfection of human mesothelial cells by exogenous DNA. We have employed the human MeT-5A mesothelial cell line, which allows the use of DNA replication as an assay for entry of DNA when plasmids bearing the SV4O origin of replication are used for transfection. We find that Canadian chrysotile, Calidria chrysotile, amosite, and crocidolite are each capable of introducing plasmid pSVod DNA into MeT-5A cells followed by subsequent replication of a fraction of the plasmid DNA. A significant fraction of the input plasmid DNA associated with the cells in the presence of asbestos is fragmented, and this fragmentation is particularly evident with crocidolite. Each of the fiber types is highly cytotoxic for the MeT-5A cells, and these cells actively accumulate the added fibers from the surrounding environment as visualized by phase-contrast microscopy. MeT-5A cells were transfected at higher efficiency with calcium phosphate than were several other primate cell lines. Calcium phosphate, however, did not induce fragmentation of the input plasmid DNA. Compared with several different mineral agents, including glass fibers, kaolin, and talc, Calidria chrysotile fibers were most effective at mediating transfection of the MeT-5A cells. Results provide a mechanism by which transfection can contribute to mutagenicity of asbestos fibers and indicate that this mechanism can operate in human mesothelial cells.

DNA interstrand cross-links of trans-diamminedichloroplatinum(II) are preferentially formed between guanine and complementary cytosine residues.

Bases in the opposite strands of DNA cross-linked by clinically ineffective trans-diamminedichloroplatinum(II) (trans-[Pt(NH3)2Cl2]) have been identified by means of three experimental approaches. These include HPLC analysis of enzymatic digests of synthetic oligonucleotide duplexes containing the interstrand cross-link, footprinting experiments on the interstrand cross-linked oligonucleotide duplexes, and termination of the duplex transcription on trans-[Pt(NH3)2Cl2]-treated fragments of plasmid DNA. The results reveal that deoxyguanine and complementary deoxycytosine residues are preferential binding sites of trans-[Pt(NH3)2Cl2] in the interstrand adducts. The interstrand cross-linking reaction was studied by means of gel electrophoresis for the cis and trans isomers. The rate of formation of interstrand cross-links was lower for the trans isomer; however, trans-[Pt(NH3)2Cl2] formed about twice the amount of interstrand cross-links as compared with the cis isomer after 48 hr. The present results are suggested to be relevant to differences in clinical activity of the two platinum(II) isomers.

Natural Transformation of Acinetobacter calcoaceticus by Plasmid DNA Adsorbed on Sand and Groundwater Aquifer Material

It is known that plasmid DNA and linear duplex DNA molecules adsorb to chemically purified mineral grains of sand and to particles of several clay fractions. It seemed desirable to examine whether plasmid DNA would also adsorb to nonpurified mineral materials taken from the environment and, particularly, whether adsorbed plasmid DNA would be available for natural transformation of bacteria. Therefore, microcosms consisting of chemically pure sea sand plus buffered CaCl(2) solution were compared with microcosms consisting of material sampled directly from a groundwater aquifer (GWA) plus groundwater (GW) with respect to the natural transformation of Acinetobacter calcoaceticus by mineral-associated DNA. The GWA minerals were mostly sand with inorganic precipitates and organic material plus minor quantities of silt and clay (illite and kaolinite). The amount of plasmid DNA which adsorbed to GWA (in GW) was about 80% of the amount which adsorbed to purified sand (in buffered CaCl(2) solution). Plasmid DNA adsorbed on sand transformed A. calcoaceticus significantly less efficiently than did plasmid DNA in solution. In contrast, the transformation by sand-adsorbed chromosomal DNA was as high as that by DNA in solution. In GWA/GW microcosms, the efficiency of transformation by chromosomal DNA was similar to that in sand microcosms, whereas plasmid transformation was not detectable. However, plasmid transformants were found at a low frequency when GWA was loaded with both chromosomal and plasmid DNA. Reasons for the low transformation efficiency of plasmid DNA adsorbed to mineral surfaces are discussed. Control experiments showed that the amounts of plasmid and chromosomal DNA desorbing from sand during incubation with a cell-free filtrate of a competent cell suspension did not greatly contribute to transformation in sand microcosms, suggesting that transformation occurred by direct uptake of DNA from the mineral surfaces. Taken together, the observations suggest that plasmid DNA and chromosomal DNA fragments which are adsorbed on mineral surfaces in a sedimentary or soil habitat may be available (although with different efficiencies for the two DNA species) for transformation of a naturally competent gram-negative soil bacterium.

Positive and negative modulation of Jun action by thyroid hormone receptor at a unique AP1 site.

We have characterized the putative AP1 site in the backbone of pUC plasmids and found unique regulatory effects. The site, which mapped to a 19-bp region around nucleotide 37, conferred transcriptional activation by Jun or Jun/Fos that was boosted up to fivefold by unliganded thyroid hormone receptor (TR). Thyroid hormone changed potentiation of the Jun response by TR into repression. Although the plasmid sequence is a near-perfect consensus AP1 site, the perfect consensus AP1 site from the human collagenase promoter did not show the same effects. Deletion of the ligand binding domain of the TR eliminated the ability of the receptor to boost Jun activity, and deletion, mutation, or changes in specificity of the DNA binding domain eliminated both its ability to potentiate Jun activity and repress with hormone. In vitro Jun/Fos complexes bound the operative plasmid fragment, and the presence of TR interfered very little with Jun/Fos binding activity. Protein interaction studies in the absence of DNA showed that TR bound Jun protein in solution either in the presence or in the absence of hormone. These observations suggest a mechanism for synergy and repression by TR through modulation of Jun activity: positive when TR is unliganded, and negative when hormone is bound. They also suggest that the presence of the plasmid element can confound studies of the regulation of linked promoters.

Positive and Negative Modulation of Jun Action by Thyroid Hormone Receptor at a Unique AP1 Site

We have characterized the putative AP1 site in the backbone of pUC plasmids and found unique regulatory effects. The site, which mapped to a 19-bp region around nucleotide 37, conferred transcriptional activation by Jun or Jun/Fos that was boosted up to fivefold by unliganded thyroid hormone receptor (TR). Thyroid hormone changed potentiation of the Jun response by TR into repression. Although the plasmid sequence is a near-perfect consensus AP1 site, the perfect consensus AP1 site from the human collagenase promoter did not show the same effects. Deletion of the ligand binding domain of the TR eliminated the ability of the receptor to boost Jun activity, and deletion, mutation, or changes in specificity of the DNA binding domain eliminated both its ability to potentiate Jun activity and repress with hormone. In vitro Jun/Fos complexes bound the operative plasmid fragment, and the presence of TR interfered very little with Jun/Fos binding activity. Protein interaction studies in the absence of DNA showed that TR bound Jun protein in solution either in the presence or in the absence of hormone. These observations suggest a mechanism for synergy and repression by TR through modulation of Jun activity: positive when TR is unliganded, and negative when hormone is bound. They also suggest that the presence of the plasmid element can confound studies of the regulation of linked promoters.

Evidence for plasmid-encoded virulence factors in the phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382.

The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt, harbors two plasmids pCM1 (27.5 kb) and pCM2 (72 kb). After curing of the plasmids, bacterial derivatives were still proficient in the ability to colonize the host plant and in the production of exopolysaccharides but exhibited a reduced virulence. When one of the two plasmids is lost, there is a significant delay in the development of wilting symptoms after infection and a plasmid-free derivative is not able to induce disease symptoms. By cloning of restriction fragments of both plasmids in the plasmid-free strain CMM100, two DNA fragments which restored the virulent phenotype were identified. Further analysis suggested that a fragment of plasmid pCM1 encodes an endocellulase which is involved in the expression of the pathogenic phenotype.

Plasmid distribution in .GAMMA.-polyglutamate-producing Bacillus strains isolated from "dan-douchi," a "natto"-like non-salty fermented soybean food in China.

Four γ-polyglutamate (γ-PGA)-producing Bacillus strains were isolated from a dan-douchi in China, all of which indicated biotin requirement for growth and a considerably high γ-glutamyltranspeptidase (γ-GTP) productivity. These strains carried a single plasmid species, and their molecular sizes and restriction patterns differed completely. Some of HindIII DNA fragments of dan-douchi plasmids strongly hybridized with a 1.7-kb TaqI DNA fragment of natto plasmid, pUH1, which encodes γ-GTP gene responsible for γ-PGA production, and their molecular sizes of homologous fragments were found to be similar. The hybridization analysis revealed that the structure gene of γ-GTP encoded on dan-douchi plasmid was found to greatly resemble that on natto plasmid. Furthermore, dan-douchi plasmid had a high degree of homology with DNA segment encoding the replication protein of pUH1. These results, therefore, strongly suggest that dan-douchi plasmid might be a functional plasmid as well as natto plasmid, and it should be considered that both natto and dan-douchi plasmids might develop from a common ancestral molecule.

A new trinuclear complex of platinum and iron efficiently promotes cleavage of plasmid DNA.

The compound [[Pt(trpy)]2Arg-EDTA]+ is synthesized in five steps, purified, and characterized by 1H, 13C, and 195Pt NMR spectroscopy, mass spectrometry, UV-vis spectrophotometry, and elemental analysis. The binuclear [[(Pt(trpy)]2Arg]3+ moiety binds to double-stranded DNA, and the chelating EDTA moiety holds metal cations. In the presence of ferrous ions and the reductant dithiothreitol, the new compound cleaves DNA. It cleaves a single strand in the pBR322 plasmid nearly as efficiently as methidiumrpropyl-EDTA (MPE), and it cleaves a restriction fragment of the XP10 plasmid nonselectively and more efficiently than [Fe(EDTA)]2-. The mechanism of cleavage was studied in control experiments involving different transition-metal ions, superoxide dismutase, catalase, glucose oxidase with glucose, metal-sequestering agents, and deaeration. These experiments indicate that adventitious iron and copper ions, superoxide anion, and hydrogen peroxide are not involved and that dioxygen is required. The cleavage apparently is done by hydroxyl radicals generated in the vicinity of the DNA molecule. The reagent [[Pt(trypy)]2Arg-EDTA]+ differs from methidiumpropyl-EDTA in not containing an intercalator. This difference in binding modes between the binuclear platinum(II) complex and the planar heterocycle may cause useful differences between the two reagents in cleavage of nucleic acids.

Polymerase chain reaction with the 30-kb circular plasmid of Borrelia burgdorferi B31 as a target for detection of the Lyme borreliosis agents in cerebrospinal fluid

The polymerase chain reaction (PCR) was developed for use in the detection of Borrelia burgdorferi sensu lato, the Lyme disease agent. A 333-bp fragment of the 30-kbp circular plasmid from Borrelia burgdorferi B31 was amplified and PCR products were analysed by DNA-DNA hybridization. Sensitivity was enhanced by addition of a carrier to the samples before treatment and enabled detection of as few as 1 to 10 bacteria. Specific products were obtained only with the lyme disease agents, but not with other spirochetes or unrelated bacteria. B. burgdorferi sensu lato was detected in cerebrospinal fluid (CSF) from 11 out of 45 patients with confirmed Lyme neuroborreliosis. In a prospective study, 20 out of 315 CSF samples from potential patients were PCR-positive. Forty uninfected patients were PCR-negative.

Plasmid content and localisation of the STal (STaP) gene in enterotoxigenic Escherichia coli with a non-radioactive polynucleotide gene probe

Enterotoxigenic Escherichia coli (ETEC) strain P2200 of porcine origin possessed eight possibly plasmid-determined characters (K88+ Raf+ Hly+ Col+ Smr Tcr Su(r) STa+) and six plasmid DNA bands of 4.2-93 kb. Analysis of the spontaneous loss of characters and the results of matings with other E. coli strains revealed that the K88, Raf, Hly, Smr, Tcr and Su(r) characters could be transferred, and that the presence of the K88 and Raf characters was associated with an 83-kb plasmid. The presence and location of the STaI gene was investigated in several ETEC strains of bovine or porcine origin. Hybridisation with a non-radioactive polynucleotide probe associated the STaI gene with a plasmid in each strain; these plasmids were of 32-142 kb. In contrast, plasmids from a P2200 STa- variant and plasmids from two STa- variants of the bovine ETEC strain B41* (strain B41 obtained from a different source) did not hybridise with the probe. One of the B41*STa- variants had lost the STa plasmid, whereas the second variant retained a plasmid of the same size which did not hybridise. In contrast, a third B41*STa- variant retained a plasmid of the same size that still hybridised with the STaI probe. Plasmid DNA restriction fragment analysis, followed by hybridisation with the STaI probe, showed that the STaI gene was associated with 8.3-, 6.8- and 3.5-kb plasmid fragments in strain B41, and with 4.9-, 6.8- and 3.5-kb plasmid fragments in strain B41*, following digestion with EcoRI, BamHI, or EcoRI + BamHI, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamics of oligodeoxyribonucleotide-directed triple helix formation: an analysis using quantitative affinity cleavage titration

The free energy for oligodeoxyribonucleotide-directed triple helix formation at a single site on a DNA plasmid fragment has been analyzed using quantitative affinity cleavage titration. Measurement of the amount of site-specific cleavage of a 339-bp radiolabeled DNA duplex produced at 24-degrees-C (100 mM Na+, 1 mM spermine-4HCl, 50 mM Tris-acetate, pH 7.0) over a concentration change of four orders of magnitude for oligodeoxyribonucleotide-EDTA.Fe 1.Fe, (5'-T*TTTTCTCTCTCTCT-3') yields an equilibrium binding constant, K(T) = 3.7 +/- 1.1 x 10(6) M-1 (DELTA-G(T) = -9.0 +/- 0.2 kcal.mol-1). Quantitative affinity cleavage titration affords association constants that are identical within experimental uncertainty with those obtained from quantitative DNase I footprint titration of the same oligonucleotide with and without EDTA.In (1.In and 6, respectively). Removal of one thymidine and one cytidine residue from the 3' end of 1.Fe reduces the free energy of binding by 0.5 kcal.mol-1, and removal of two thymidine and two cytidine residues from its 3' terminus decreases the binding free energy by 1.1 kcal.mol-1 at pH 7.0. Single internal base triplet mismatches result in a destabilization of the local triple-helical structure by 2.5-3.0 kcal.mol-1. Quantitative affinity cleavage titration is a general method which should allow for the measurement of equilibrium constants for the association of many DNA-binding molecules to single sites on relatively large DNA under a broad range of solution conditions.

Characterization of the Virulence Regions in the Plasmids of Three Live Salmonella Vaccines

Three live Salmonella vaccines, Zoosaloral "Dessau", Bovisaloral "Dessau", and Suisaloral "Dessau" successfully used in veterinary medicine in eastern Germany were analysed for their plasmid DNA content. Plasmids of 60 MDa (S. typhimurium), 55 MDa and 26 MDa (S. dublin) and 34 MDa (S. choleraesuis) were found. The three large plasmids contained a virulence region as shown by restriction enzyme analysis and hybridization studies with virulence-region-specific DNA probes. Restriction enzyme pattern analysis of the whole plasmids revealed differences between vaccine strains and parental strains. We wanted to know if there were alterations within the virulence region of the vaccine strain plasmids compared with parental strains caused by the attenuation procedure. Therefore, the 6 kb-Cla I fragments of several plasmids were cloned and characterized by restriction enzyme fingerprinting. The expression of the Cla I fragment-encoded proteins was analysed in the minicell-producing strain DS 410. The experiments revealed that the restriction pattern of the 6 kb-Cla I fragments of Zoosaloral and Bovisaloral as well as the PAGE pattern of virulence-region-encoded proteins were in complete accordance with one another, between vaccine strains and the parental strain of Zoosaloral, and with a wild-type strain of S. typhimurium.