Plasmid DNA purification via the use of a dual affinity protein

Methods are presented for the production, affinity purification and analysis of plasmid DNA (pDNA). Batch fermentation is used for the production of the pDNA, and expanded bed chromatography, via the use of a dual affinity glutathione S-transferase (GST) fusion protein, is used for the capture and purification of the pDNA. The protein is composed of GST, which displays affinity for glutathione immobilized to a solid-phase adsorbent, fused to a zinc finger transcription factor, which displays affinity for a target 9-base pair sequence contained within the target pDNA. A Picogreen™ fluorescence assay and/or anx ethidium bromide agarose gel electrophoresis assay can be used to analyze the eluted pDNA.

Process engineering approach to large-volume methacrylate monolith synthesis for plasmid purification

The construction of large?volume methacrylate monolithic columns for preparative-scale plasmid purification is obstructed by the enormous release of exotherms, thus introducing structural heterogeneity in the monolith pore system. A remarkable radial temperature gradient develops along the monolith thickness, reaching a terminal temperature that supersedes the maximum temperature required for the preparation of a structurally homogeneous monolith. A novel heat expulsion technique is employed to overcome the heat build-up during the synthesis process. The enormous heat build-up is perceived to encompass the heat associated with initiator decomposition and the heat released from free radical-monomer and monomer-monomer interactions. The heat resulting from the initiator decomposition was expelled along with some gaseous fumes before commencing polymerisation in a gradual addition fashion. Characteristics of a 50 mL monolith synthesized using this technique showed an improved uniformity in the pore structure radially along the length on the monolith. Chromatographic characterization of this adsorbent displayed a persistent binding capacity of 14.5 mg pDNA/mL of the adsorbent. The adsorbent was able to fractionate a clarified bacteria lysate in only 3 min (after loading) into RNA, protein and pDNA respectively. The pDNA fraction obtained was analyzed to be a homogeneous supercoiled pDNA.

A conjugative plasmid carrying the carbapenem resistance gene blaOXA-23 in AbaR4 in an extensively resistant GC1 Acinetobacter baumannii isolate

OBJECTIVES: To locate the acquired bla(OXA-23) carbapenem resistance gene in an Australian A. baumannii global clone 1 (GC1) isolate. METHODS: The genome of the extensively antibiotic-resistant GC1 isolate A85 harbouring bla(OXA-23) in Tn2006 was sequenced using Illumina HiSeq, and the reads were used to generate a de novo assembly. PCR was used to assemble relevant contigs. Sequences were compared with ones in GenBank. Conjugation experiments were conducted. RESULTS: The sporadic GC1 isolate A85, recovered in 2003, was extensively resistant, exhibiting resistance to imipenem, meropenem and ticarcillin/clavulanate, to cephalosporins and fluoroquinolones and to the older antibiotics gentamicin, kanamycin and neomycin, sulfamethoxazole, trimethoprim and tetracycline. Genes for resistance to older antibiotics are in the chromosome, in an AbaR3 resistance island. A second copy of the ampC gene in Tn6168 confers cephalosporin resistance and the gyrA and parC genes have mutations leading to fluoroquinolone resistance. An 86 335 bp repAci6 plasmid, pA85-3, carrying bla(OXA-23) in Tn2006 in AbaR4, was shown to transfer imipenem, meropenem and ticarcillin/clavulanate resistance into a susceptible recipient. A85 also contains two small cryptic plasmids of 2.7 and 8.7 kb. A85 is sequence type ST126 (Oxford scheme) and carries a novel KL15 capsule locus and the OCL3 outer core locus. CONCLUSIONS: A85 represents a new GC1 lineage identified by the novel capsule locus but retains AbaR3 carrying genes for resistance to older antibiotics. Resistance to imipenem, meropenem and ticarcillin/clavulanate has been introduced into A85 by pA85-3, a repAci6 conjugative plasmid carrying Tn2006 in AbaR4.

The v-MFG test: Investigating maternal, offspring and maternal-fetal genetic incompatibility effects on disease and viability

The MFG test is a family-based association test that detects genetic effects contributing to disease in offspring, including offspring allelic effects, maternal allelic effects and MFG incompatibility effects. Like many other family-based association tests, it assumes that the offspring survival and the offspring-parent genotypes are conditionally independent provided the offspring is affected. However, when the putative disease-increasing locus can affect another competing phenotype, for example, offspring viability, the conditional independence assumption fails and these tests could lead to incorrect conclusions regarding the role of the gene in disease. We propose the v-MFG test to adjust for the genetic effects on one phenotype, e.g., viability, when testing the effects of that locus on another phenotype, e.g., disease. Using genotype data from nuclear families containing parents and at least one affected offspring, the v-MFG test models the distribution of family genotypes conditional on offspring phenotypes. It simultaneously estimates genetic effects on two phenotypes, viability and disease. Simulations show that the v-MFG test produces accurate genetic effect estimates on disease as well as on viability under several different scenarios. It generates accurate type-I error rates and provides adequate power with moderate sample sizes to detect genetic effects on disease risk when viability is reduced. We demonstrate the v-MFG test with HLA-DRB1 data from study participants with rheumatoid arthritis (RA) and their parents, we show that the v-MFG test successfully detects an MFG incompatibility effect on RA while simultaneously adjusting for a possible viability loss.

Draft genome and plasmid sequences of Chlamydia pneumoniae strain B21 from an Australian endangered marsupial, the western barred bandicoot

Chlamydia pneumoniae is a ubiquitous intracellular pathogen, first associated with human respiratory disease and subsequently detected in a range of mammals, amphibians, and reptiles. Here we report the draft genome sequence for strain B21 of C. pneumoniae, isolated from the endangered Australian marsupial the western barred bandicoot.

The effect of different levels of inbreeding on self-incompatibility and inbreeding depression in pineapple

A high level of heterozygosity in domesticated pineapple is one of the main obstacles hindering the efficient development of new varieties. Selfing has been proposed as a strategy to minimise this heterozygosity through the development of parentals with a greater level of homozygosity. Selfing and a range of lesser levels of inbreeding were evaluated for their effects on seed production and inbreeding depression in the early growth of seedlings. Selfing produced few seeds, and very few viable seedlings. The paternal backcross and several half-sib combinations exhibited minimal effects on seed development or early growth and in some cases were similar to the outcross. Sibcrosses were generally unsuccessful. The highest inbreeding coefficient that was not associated with severe inbreeding depression was approximately 0.25. The effect of inbreeding depression and the level of homozygosity on several quantitative traits including those related to fruit quality within the inbred populations, is now being assessed.

Formation of linear plasmid multimers promoted by the phage lambda Red-system in lon mutants of Escherichia coli

We report here the formation of plasmid linear multimers promoted by the Red-system of phage lambda using a multicopy plasmid comprised of lambda red alpha and red beta genes, under the control of the lambda cI857 repressor. Our observations have revealed that the multimerization of plasmid DNA is dependent on the red beta and recA genes, suggesting a concerted role for these functions in the formation of plasmid multimers. The formation of multimers occurred in a recBCD+ sbcB+ xthA+ lon genetic background at a higher frequency than in the isogenic lon+ host cells. The multimers comprised tandem repeats of monomer plasmid DNA. Treatment of purified plasmid DNA with exonuclease III revealed the presence of free double-chain ends in the molecules. Determination of the size of multimeric DNA, by pulse field gel electrophoresis, revealed that the bulk of the DNA was in the range 50-240 kb, representing approximately 5-24 unit lengths of monomeric plasmid DNA. We provide a conceptual framework for Red-system-promoted formation and enhanced accumulation of plasmid linear multimers in lon mutants of E. coli.

An improved representation of vehicle incompatibility in frontal NCAP tests using a modified rigid barrier

With the objective of better understanding the significance of New Car Assessment Program (NCAP) tests conducted by the National Highway Traffic Safety Administration (NHTSA), head-on collisions between two identical cars of different sizes and between cars and a pickup truck are studied in the present paper using LS-DYNA models. Available finite element models of a compact car (Dodge Neon), midsize car (Dodge Intrepid), and pickup truck (Chevrolet C1500) are first improved and validated by comparing theanalysis-based vehicle deceleration pulses against corresponding NCAP crash test histories reported by NHTSA. In confirmation of prevalent perception, simulation-bascd results indicate that an NCAP test against a rigid barrier is a good representation of a collision between two similar cars approaching each other at a speed of 56.3 kmph (35 mph) both in terms of peak deceleration and intrusions. However, analyses carried out for collisions between two incompatible vehicles, such as an Intrepid or Neon against a C1500, point to the inability of the NCAP tests in representing the substantially higher intrusions in the front upper regions experienced by the cars, although peak decelerations in cars arc comparable to those observed in NCAP tests. In an attempt to improve the capability of a front NCAP test to better represent real-world crashes between incompatible vehicles, i.e., ones with contrasting ride height and lower body stiffness, two modified rigid barriers are studied. One of these barriers, which is of stepped geometry with a curved front face, leads to significantly improved correlation of intrusions in the upper regions of cars with respect to those yielded in the simulation of collisions between incompatible vehicles, together with the yielding of similar vehicle peak decelerations obtained in NCAP tests.

Plasmid DNA mediated transfer of the herpes simplex virus thymidine kinase gene to a new bromodeoxyuridine resistant variant of human primary lung carcinoma cells

A human primary lung carcinoma cell line (HPL-R1) established from the tumor biopsy of a lung cancer patient, lacking in cytochrome P1-450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)], was cloned and used to obtain variants deficient in the expression of thymidine-kinase via treatment with 5-bromo-2'-deoxyuridine, and selection for drug resistance phenotype. The variant cell line, precharacterized for thymidine kinase negative phenotype, was transfected with the thymidine kinase gene bearing p R-tk and px1-tk plasmids. Transfections from both the plasmids, demonstrated a frequency of 5.5 X 10(-5). The transfectants showed a 76-100% retention of the transferred phenotype. These data suggest that transfection in variant human cells can approach significant levels of stability observed with rodent cell recipients.

Genetic transformation of Brassica nigra by agrobacterium based vector and direct plasmid uptake

Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection medium. All of these expressed B-glucuronidase gene on histochemical staining. Protoplasts isolated from hypocotyl tissues of seedlings could be transformed with a plasmid vector by FEG mediated uptake of vector DNA. A number of fertile kanamycin resistant plants were obtained using both the methods, and their transformed nature was confirmed by Southern blot analysis and histochemical staining for GUS. Backcrossed and selfed progenies of these transformed plants showed the presence of npt and gus genes.

Translational Up-Regulation and High-Level Protein Expression from Plasmid Vectors by mTOR Activation via Different Pathways in PC3 and 293T Cells

Background: Though 293T cells are widely used for expression of proteins from transfected plasmid vectors, the molecular basis for the high-level expression is yet to be understood. We recently identified the prostate carcinoma cell line PC3 to be as efficient as 293T in protein expression. This study was undertaken to decipher the molecular basis of high-level expression in these two cell lines. Methodology/Principal Findings: In a survey of different cell lines for efficient expression of platelet-derived growth factor-B (PDGF-B), beta-galactosidase (beta-gal) and green fluorescent protein (GFP) from plasmid vectors, PC3 was found to express at 5-50-fold higher levels compared to the bone metastatic prostate carcinoma cell line PC3BM and many other cell lines. Further, the efficiency of transfection and level of expression of the reporters in PC3 were comparable to that in 293T. Comparative analyses revealed that the high level expression of the reporters in the two cell lines was due to increased translational efficiency. While phosphatidic acid (PA)-mediated activation of mTOR, as revealed by drastic reduction in reporter expression by n-butanol, primarily contributed to the high level expression in PC3, multiple pathways involving PA, PI3K/Akt and ERK1/2 appear to contribute to the abundant reporter expression in 293T. Thus the extent of translational upregulation attained through the concerted activation of mTOR by multiple pathways in 293T could be achieved through its activation primarily by the PA pathway in PC3. Conclusions/Significance: Our studies reveal that the high-level expression of proteins from plasmid vectors is effected by translational up-regulation through mTOR activation via different signaling pathways in the two cell lines and that PC3 is as efficient as 293T for recombinant protein expression. Further, PC3 offers an advantage in that the level of expression of the protein can be regulated by simple addition of n-butanol to the culture medium.

Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90

Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy. Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium. The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2. The plasmid pMAB2 was found to have undergone a deletion Of a 20-kb fragment of pMAB1. The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation. Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase. After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment. The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different. The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine.