Rapid identification of microorganisms by circular-intensity differential scattering.

There is a pressing need in clinical medicine for rapid identification of microorganisms. We describe a method that has the potential for such rapid identification: circular-intensity differential scattering, which is based on the differential scattering of left and right circularly polarized light. The scanning time required to obtain the spectral signature of an organism is about 4 min. Using a commercial circular dichrograph modified to measure circular intensity differential scattering at 90 degrees, we obtained significantly different spectra for five different crude influenza viruses. Salmonella typhimurium TA98 and TA100, and Escherichia coli HB101, HB101(pBR322), and HB101(pMB9). Purified supercoiled plasmid pBR322 DNA was readily distinguishable from linear pBR322 DNA; such differences in nucleic acid packaging may be significant factors in the discriminatory power of this technique.     

Atomic force microscopic study on topological structures of pBR322 DNA

Plasmid pBR322 DNA (0.5mg/mL) isolated from Escherichia coli HB101 was suspended in Tris-HCl-EDTA (1 mol/L - 0.1 mol/L, pH8.5); then a drop of the above solution was deposited on freshly cleaved mica substrate. After adsorption for about 1 min, the sample was stained with phosphotungstic acid. The residua] solution was removed with a piece of filter paper. Afterwards the sample was imaged with a home-made atomic force microscope (AFM) in air. The AFM images of pBR322 DNA with a molecular resolution have been obtained. These images show that pBR322 DNA exists in several different topological structures: (i) relaxed circular DNA with a different diameter; (ii) supercondensed DNA with different particle sizes; (iii) dimeric catenane connected by one relaxed circular molecule and another dose-compacted molecule which might be either supercoiled or intramolecular knotted form; (iv) oligomeric catenane with multiple irregular molecules in which DNA is interlocked into a complex oligomer; (v) possibly-existing     

Fate in soil of a recombinant plasmid carrying aDrosophila gene

A recombinant plasmid (C357; 3.5 Mdal) containing heterologous DNA (pBR322 [2.6 Mdal] with cDNA for an egg yolk protein fromDrosophila grimshawi) inEscherichia coli strain HB101 survived in and was recovered on selective media from sterile and nonsterile soil during 27 days at frequencies similar to those of theE. coli(pBR322) system. In sterile saline, the numbers of all cells decreased during 34 days, but the numbers of the plasmidless host declined less. There was no selective loss of the heterologous DNA in either soil or saline, as determined by colony hybridization with a³²P-labeled DNA probe for the cDNA, but the HB101(C357) appeared to be less able than HB101(pBR322) to cope with conditions of starvation. These results suggested that nonessential eucaryotic DNA inserted into plasmid DNA has little effect on the survival in soil or saline of the bacterial host and the maintenance of the vector.     

Preservation of Xenopus laevis rDNA-containing plasmid, pXlr101A, injected into the fertilized egg of Xenopus laevis

A circular rDNA-containing recombinant plasmid, pXlr101A, and its vector pBR322 were microinjected into the cytoplasm of fertilized Xenopus laevis eggs. The DNAs extracted from injected embryos at various stages of development were analyzed by hybridization with 32P-labeled pBR322 as the probe. Neither pXlr101A nor pBR322 were replicated, but they were maintained until the tailbud stage, disappearing during the muscular response stage. When pXlr101A-injected embryos were raised until the 2-week-old tadpole stage, sequences homologous to pBR322 were detectable in two Eco RI fragments of the pXlr101A-injected tadpole DNA. The sizes of the Eco RI fragments suggested that the plasmid sequences were preserved most probably in the chromosome-integrated form.     

Enterotoxin-like activity produced by Campylobacter jejuni and Campylobacter coli isolated from patients and healthy controls in Algeria

Abstract In our earlier studies, hexammine ruthenium (III) chloride (HRC) was found to eliminate the pBR322 plasmid from E. coli HB101 with 100% frequency. However, this plasmid was not eliminated by other known curing agents, such as acridine orange, sodium dodecyl sulphate (SDS), rifampicin or temperature conditions. In our present communication, we report the sensitivity of a variant of plasmid pBR322 to curing agents which were not effective on the parent plasmid.     

A biochemical analysis of the interaction of DNA gyrase with the bacteriophage Mu, pSC101 and pBR322 strong gyrase sites: the role of DNA sequence in modulating gyrase supercoiling and biological activity

Replication of bacteriophage Mu DNA, a process requiring efficient synapsis of the prophage ends, takes place within the confines of the Escherichia coli nucleoid. Critical to ensuring rapid synapsis is the function of the SGS, a strong gyrase site, located at the centre of the Mu genome. Replacement of the SGS by the strong gyrase sites from pSC101 or pBR322 fails to support efficient prophage replication. To probe the unique SGS properties we undertook a biochemical analysis of the interaction of DNA gyrase with the Mu SGS, pSC101 and pBR322 sites. In binding and cleavage assays the order of efficacy was pSC101 > Mu SGS > pBR322. However, in supercoiling assays the Mu SGS (cloned into pUC19) exhibited a strong enhancement of gyrase-catalysed supercoiling over pUC19 alone; the pSC101 site showed none and the pBR322 site gave a moderate improvement. Most striking was the Mu SGS-dependent increase in processivity of the gyrase reaction. This highly processive supercoiling coupled with efficient binding may account for the unique biological properties of the SGS. The results emphasize the importance of the DNA substrate as an active component in modulating the gyrase supercoiling reaction, and in determining the biological roles of specialized gyrase sites.     

Export of Erwinia chrysanthemi (EC16) protease by Escherichia coli

Abstract During exponential growth, Erwinia chrysanthemi (EC16) exports 99% of the protease (PRT) into the growth medium. By screening an EC16 genomic library in Escherichia coli HB101, several Prt⁺ clones were identified. A 16-kb Eco RI fragment, carrying the prt gene, was subcloned into pBR322 (pAKC326). E. coli HB101[pAKC326] cells exported PRT into the growth medium during exponential growth. PRT export was not accompanied by periplasmic leakage. E. coli HB101 carrying EC16 prt and pel genes (encoding pectate lyase) exported PRT but retained PEL in the periplasm. These findings indicate the occurrence of a PRT-specific export system in EC16, which is also functional in an E. coli strain carrying the prt ⁺ DNA segment.     

The effect of polymyxin B nonapeptide (PMBN) on transformation

The effect of the outer membrane permeabilizing polycation, polymyxin B nonapeptide (PMBN) on the transformation of E. coli HB101 with pBR322 plasmid DNA was investigated. Pretreatment of cells with PMBN (followed by suspending the cells in PMBN-free medium) did not stimulate the development of competence induced by the calcium heat pulse. In the absence of calcium-ions, a high PMBN concentration (1 mM) was able to induce a low transformation frequency provided that PMBN was not removed before the addition of DNA.     

竹红菌甲素敏化质粒pBR 322 DNA光氧化——使封闭环DNA转变为开环DNA

甲素可敏化质粒pBR 322 DNA光氧化断链的使其封闭环DNA转变为开环DNA。甲素敏化pBR 322 DNA光氧化反应可被单线态氧淬灭剂-NaN_3抑制,证明此光敏氧化机制属Ⅱ型过程。     

Electro-stimulated transformation of E. coli cells pretreated by EDTA solution

The phenomenon of transformation of E. coli cells under electric treatment has been studied. The cells of strains MH 1, HB 101 and DH 1 after EDTA treatment in an isotonic medium were transformed with DNA pBR322 by applying a single exponential pulse (E = 10 kV/cm, T = 1.5 ms) to the suspension. The maximum transformation efficiency obtained was 4 X 10(6) colonies/micrograms DNA. The maximum transformation frequency was 0.4% at a DNA concentration of 15 micrograms/ml.     

Photosensitized inactivation of plasmid and bacteriophage lambda DNA: relative contribution to the lethal effect of monoadducts and diadducts of 8-methoxypsoralen and their repair in SOS-induced Escherichia coli cells

The curves of UV (254 nm)-inactivation and inactivation by furocoumarin derivatives + UVA radiation (PUVA) of bacteriophage lambda and biologically active plasmid pBR322 were measured using Escherichia coli K12 bacteria with different defects of DNA repair system as a ghost. The ratio of mono- and diadducts (interstrand cross-links) of 8-methoxypsoralen was determined that are formed after treating the DNA of pBR322 and bacteriophage lambda with PUVA. It is shown that, on the average, about five monoadducts per one diadduct are formed in DNA of pBR322, and about 0.9 monoadducts per one diadduct are formed in lambda phage DNA. An increased (up to 50%) efficiency of SOS-repair of monoadducts of 8-methoxypsoralen in DNA of pBR322 and lambda in the presence of plasmid pKM101 muc+ (incN) was found.     

Nicking activity on pBR322 DNA of ribosome inactivating proteins from Phytolacca dioica L. leaves

Ribosome-inactivating proteins isolated from Phytolacca dioica L. leaves are rRNA-N-glycosidases, as well as adenine polynucleotide glycosylases. Here we report that some of them cleave supercoiled pBR322 dsDNA, generating relaxed and linear molecules. PD-L1, the glycosylated major form isolated from the winter leaves of adult P . dioica plants, produces both free 3'-OH and 5'-P termini randomly distributed along the DNA molecule, as suggested by labelling experiments with [alpha- 32P]dCTP and [gamma- 32 P]dATP. Moreover, when the reaction is carried out under low-salt conditions, cleavage is observed mainly at a specific site, located downstream of the ampicillin resistance gene (close to position 3200), ending with the deletion of a fragment of approximately 70 nucleotides. This cleavage pattern is similar to that obtained under the same conditions with mung bean nuclease, a single-strand endonuclease. Furthermore, pBR322 DNA treated with PD-L1 shows reduced transforming activity with E . coli HB101 competent cells in comparison to untreated control plasmid DNA.     

DNA protection with the DNA methylase M . BbvI from Bacillus brevis var. GB against cleavage by the restriction endonucleases PstI and PvuII

BamHI fragments of the Bacillus brevis var. GB plasmid pAD1 have been cloned in Escherichia coli HB101 using pBR322 plasmid as a vector. The analysis of the recombinant plasmids showed that additional PstI sites had appeared in cloned fragments of pAD1. Methylation of the recombinant plasmids in vitro by enzymes from B. brevis GB cells blocks cleavage at these additional PstI sites of cloned pAD1 fragments and at the PstI site of pBR322. Among DNA methylases of B. brevis GB, the cytosine DNA methylase M . BbvI is the most likely agent modifying the recognition sequences of PstI. The methylase can modify cytosine residues in PstI or PvuII sites if these recognition sequences are linked to G at 5'- or to C at 3'-termini. In particular, in vitro methylation of the SV40 DNA by B. brevis GB methylases protects one of the two PstI sites and two of the three PvuII sites. The described effect of the protection of the specific PstI and PvuII sites may be used for physical mapping of genomes and DNA cloning.     

Cloning and characterization of a plasmid DNA from anacystis nidulans 6301

A plasmid DNA of Anacystis nidulans 6301 was isolated by CsCl-EtBr centrifugation. The Mr of the plasmid, named pBA1, was estimated to be 5.04 +/- 0.26 X 10(6) by electron microscopic analysis and 5.2 X 10(6) by agarose gel electrophoresis. The pBA1 DNA was opened at a unique site with BamHI and cloned in pBR322 vector propagated in Escherichia coli HB101 cells. The recombinant plasmid, named pBAS18, was digested with various restriction endonucleases and its cleavage map was constructed. Based on this result, the cleavage map of the pBA1 plasmid is presented.     

Electron microscopic mapping of Thermus thermophilus RNA polymerase binding sites on plasmid pBR322

The binding of RNA polymerase from the extreme thermophile T. thermophilus HB8 to plasmid pBR322 was measured by electron microscopy. DNA-protein complexes were prepared at 35 and 60 degrees C. At both temperatures the enzyme binds strongly to sites which coincide with promoters P1, P2, P3 and P4 present in pBR322. At 60 degrees C, an additional binding site appears, which is located between P3 and P4. There is a high degree of correlation between RNA polymerase binding sites and the location of A-T rich regions on pBR322 DNA.     

Human placental endonuclease cleaves Holliday junctions

A partially purified endonuclease from human placenta cleaves cruciform structures. The placental enzyme is active both on extruded cruciform structures from negatively supercoiled covalently closed circular plasmid DNA and on synthetic X-junctions formed by reannealing short oligonucleotides. Plasmids containing natural or cloned palindromes such as pBR322 and pHD101-3 were used as substrates. The synthetic X-junction tetramer DNA formed by reannealing short oligonucleotides, was converted into dimer form by the enzyme. This is the first report of an enzyme activity involved in resolution of recombination intermediates in higher eukaryotes and second report of a cellular enzyme.     

Cloning and characterization of the yjeA gene, encoding a novel deoxyribonuclease, from Bacillus subtilis

The yjeA gene, encoding a secreted protein, YjeA, of Bacillus subtilis, was cloned and characterized. A derivative of YjeA, the recombinant YjeA-H, which contained a C-terminal His(6)-tag, was purified from Escherichia coli for functional studies. YjeA-H was shown to be an endonuclease, which hydrolyses both single-stranded and double-stranded DNA, but not RNA. Covalently closed circular pBR322 DNA incubated with YjeA-H was shown by gel electrophoresis to be first nicked to an open circular form, and then to a linearized structure on a background of DNA smear, and finally to small species of linear molecules that accumulated gradually. When (32)P-labelled pBR322 DNA was used as substrate, YjeA-H was shown to progressively nick both DNA strands in a random fashion, creating intermediates of various structures, as well as DNA smears comprising linear molecules of different sizes. The final products were found to consist essentially of degraded species of DNA. The detection of a putative signal peptide at the N-terminus of YjeA, together with the purification of YjeA-H from the culture supernatants of E. coli yjeA-H clones, and the identification of YjeA in the culture medium of Bacillus subtilis, supports the conclusion that YjeA is a secretory protein of Bacillus subtilis.     

Cloning of a restriction-modification system from Proteus vulgaris and its use in analyzing a methylase-sensitive phenotype in Escherichia coli.

A 4.84-kilobase-pair plasmid was isolated from Proteus vulgaris (ATCC 13315) and cloned into the plasmid vector pBR322. Plasmid pBR322 contains substrate sites for the restriction endonucleases PvuI and PvuII. The recombinant plasmids were resistant to in vitro cleavage by PvuII but not PvuI endonuclease and were found to cause production of PvuII endonuclease or methylase activity or both in Escherichia coli HB101. The approximate endonuclease and methylase gene boundaries were determined through subcloning, Bal 31 resection, insertional inactivation, DNA-dependent translation, and partial DNA sequencing. The two genes are adjacent and appear to be divergently transcribed. Most E. coli strains tested were poorly transformed by the recombinant plasmids, and this was shown by subcloning and insertional inactivation to be due to the PvuII methylase gene. At a low frequency, stable methylase-producing transformants of a methylase-sensitive strain were obtained, and efficiently transformed cell mutants were isolated from them.