Separate Plasmids Research Articles

Specific recognition of supercoiled plasmid DNA in arginine affinity chromatography

Arginine chromatography was used to fully separate supercoiled and open circular plasmid DNA (pDNA) isoforms. The results show that the arginine matrix promotes multiple interactions with pDNA, including not only electrostatic and hydrophobic but also biorecognition of nucleotide bases by the arginine ligand. The strong interactions occurring with DNA backbone provide stability, conducting to high effectiveness of arginine support to bind pDNA at low ionic strength. The specific interaction of arginine with sc pDNA could be due to the ability of arginine matrix to be involved in complex interactions that are partly dependent on the conformation of the DNA molecule.

Coexpression of Flt3 ligand and GM-CSF genes modulates immune responses induced by HER2/neu DNA vaccine

DNA vaccine and dendritic cells (DCs)-based vaccine have emerged as promising strategies for cancer immunotherapy. Fms-like tyrosine kinase 3-ligand (Flt3L) and granulocyte-macrophage-colony-stimulating factor (GM-CSF) have been exploited for the expansion of DC. It was reported previously that combination of plasmid encoding GM-CSF with HER2/neu DNA vaccine induced predominantly CD4(+) T-cell-mediated antitumor immune response. In this study, we investigated the modulation of immune responses by murine Flt3L and GM-CSF, which acted as genetic adjuvants in the forms of bicistronic (pFLAG) and monocistronic (pFL and pGM) plasmids for HER2/neu DNA vaccine (pN-neu). Coexpression of Flt3L and GM-CSF significantly enhanced maturation and antigen-presentation abilities of splenic DC. Increased numbers of infiltrating DC at the immunization site, higher interferon-gamma production, and enhanced cytolytic activities by splenocytes were prominent in mice vaccinated with pN-neu in conjunction with pFLAG. Importantly, a potent CD8(+) T-cell-mediated antitumor immunity against bladder tumors naturally overexpressing HER2/neu was induced in the vaccinated mice. Collectively, our results indicate that murine Flt3L and GM-CSF genes coexpressed by a bicistronic plasmid modulate the class of immune responses and may be superior to those codelivered by two separate monocistronic plasmids as the genetic adjuvants for HER2/neu DNA vaccine.

A second large plasmid encodes conjugative transfer and antimicrobial resistance in O119:H2 and some typical O111 enteropathogenic Escherichia coli strains.

A novel and functional conjugative transfer system identified in O119:H2 enteropathogenic Escherichia coli (EPEC) strain MB80 by subtractive hybridization is encoded on a large multidrug resistance plasmid, distinct from the well-described EPEC adherence factor (EAF) plasmid. Variants of the MB80 conjugative resistance plasmid were identified in other EPEC strains, including the prototypical O111:NM strain B171, from which the EAF plasmid has been sequenced. This separate large plasmid and the selective advantage that it confers in the antibiotic era have been overlooked because it comigrates with the virulence plasmid on conventional gels.

Performance of a tetracycline-responsive transactivator system for regulating transgenes in the oomycete Phytophthora infestans

The oomycete genus Phytophthora includes many important plant pathogens for which extensive genome data exist, but lacking is an inducible expression system to study contributions of their genes to growth and pathogenicity. Here the adaptation of the reverse tetracycline transactivator (rtTA) system to P. infestans is described. Vectors were developed containing rtTA expressed from an oomycete promoter, and beta-glucuronidase (GUS) controlled by TetR binding sites fused to a minimal oomycete promoter. Transformants were obtained in which GUS was expressed in a dose-dependent manner by the rtTA inducer doxycycline, indicating that the gene switch functions in P. infestans. However, toxicity of rtTA hindered the isolation of transformants if expressed on the same plasmid as the nptII selection marker. Better results were obtained by cotransforming those genes on separate plasmids, with 92% of transformants acquiring both DNAs although only 4% expressed rtTA at detectable levels. Low levels of reporter activity were measured in such transformants, suggesting that rtTA activated transcription weakly. Also, significant variation in the sensitivity of isolates to doxycycline and tetracycline was observed. These results are useful both in terms of developing tools for functional genomics and understanding the fate of DNA during Phytophthora transformation.

Design of a trans protease lentiviral packaging system that produces high titer virus

BackgroundThe structural and enzymatic proteins of the human immunodeficiency virus (HIV) are initially generated as two long polyproteins encoded from overlapping reading frames, one producing the structural proteins (Gag) and the second producing both structural and enzymatic proteins (Gag-Pol). The Gag to Gag-Pol ratio is critical for the proper assembly and maturation of viral particles. To minimize the risk of producing a replication competent lentivirus (RCL), we developed a "super-split" lentiviral packaging system in which Gag was separated from Pol with minimal loss of transducibility by supplying protease (PR) in trans independently of both Gag and Pol.ResultsIn developing this "super-split" packaging system, we incorporated several new safety features that include removing the Gag/Gag-Pol frameshift, splitting the Gag, PR, and reverse transcriptase/integrase (RT/IN) functions onto separate plasmids, and greatly reducing the nucleotide sequence overlap between vector and Gag and between Gag and Pol. As part of the construction of this novel system, we used a truncated form of the accessory protein Vpr, which binds the P6 region of Gag, as a vehicle to deliver both PR and RT/IN as fusion proteins to the site of viral assembly and budding. We also replaced wt PR with a slightly less active T26S PR mutant in an effort to prevent premature processing and cytoxicity associated with wt PR. This novel "super-split" packaging system yielded lentiviral titers comparable to those generated by conventional lentiviral packaging where Gag-Pol is supplied intact (1.0 × 106 TU/ml, unconcentrated).ConclusionHere, we were able to create a true "split-function" lentiviral packaging system that has the potential to be used for gene therapy applications. This novel system incorporates many new safety features while maintaining high titers. In addition, because PR is supplied in trans, this unique system may also provide opportunities to examine viral protein processing and maturation.

Escherichia coli dinJ-yafQgenes act as a toxin-antitoxin module

Bacterial toxin-antitoxin (TA) systems are operons that code for a stable toxic protein and a labile antitoxin. TA modules are widespread on the chromosomes of free-living Bacteria and Archaea, where they presumably act as stress response elements. The chromosome of Escherichia coli K-12 encodes four known TA pairs, as well as the dinJ-yafQ operon, which is hypothesized to be a TA module based on operon organization similar to known TA genes. Induction of YafQ inhibited cell growth, but its toxicity was counteracted by coexpression of dinJ cloned on a separate plasmid. YafQ(His)(6) and DinJ proteins coeluted in Ni(2+)-affinity and gel filtration chromatography, implying the formation of a specific and stable YafQ-DinJ protein complex with an estimated molecular mass of c. 37.3 kDa. Induction of YafQ reduced protein synthesis up to 40% as judged by incorporation of [(35)S]-methionine, but did not influence the rates of DNA and RNA synthesis. Structure modelling of E. coli YafQ revealed its structural relationship with bacterial toxins of known structure suggesting that it might act as a sequence-specific mRNA endoribonuclease.

Fusion of antigen to Fas-ligand in a DNA vaccine enhances immunogenicity

DNA vaccines have considerable potential for the prophylaxis and therapy of a range of diseases, but their potential has not been realised largely due to poor immunogenicity. Fas ligand is a pro-apoptotic molecule, able to induce death of Fas expressing cells. We describe the construction of a DNA vaccine encoding a chimeric fusion between Fas ligand and a truncated version of HIV gp120 as a model antigen. The fusion DNA was used as a priming vaccine, along with boosting with recombinant gp120 protein. Priming with fusion protein DNA resulted in a powerful enhancement of immune responses to the protein boost, and, in the presence of aluminum phosphate, to a strong enhancement in T helper 2 type responses. Fas ligand delivered in a separate plasmid also had an adjuvant effect, although it was weaker than that delivered by the fusion protein.

Dispersion of the RmInt1 group II intron in the Sinorhizobium meliloti genome upon acquisition by conjugative transfer

RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase–maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (ΔORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic ΔORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature.

Molecular Evolution of Typical EnteropathogenicEscherichia coli: Clonal Analysis by Multilocus Sequence Typing and Virulence Gene Allelic Profiling

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Typical EPEC isolates are differentiated from other types of pathogenic E. coli by two distinctive phenotypes, attaching effacement and localized adherence. The genes specifying these phenotypes are found on the locus of enterocyte effacement (LEE) and the EPEC adherence factor (EAF) plasmid. To describe how typical EPEC has evolved, we characterized a diverse collection of strains by multilocus sequence typing (MLST) and performed restriction fragment length polymorphism (RFLP) analysis of three virulence genes (eae, bfpA, and perA) to assess allelic variation. Among 129 strains representing 20 O-serogroups, 21 clonal genotypes were identified using MLST. RFLP analysis resolved nine eae, nine bfpA, and four perA alleles. Each bfpA allele was associated with only one perA allele class, suggesting that recombination has not played a large role in shuffling the bfpA and perA loci between separate EAF plasmids. The distribution of eae alleles among typical EPEC strains is more concordant with the clonal relationships than the distribution of the EAF plasmid types. These results provide further support for the hypothesis that the EPEC pathotype has evolved multiple times within E. coli through separate acquisitions of the LEE island and EAF plasmid.

Granulocyte-macrophage colony-stimulating factor DNA prime-protein boost strategy to enhance efficacy of a recombinant pertussis DNA vaccine

To investigate a new strategy to enhance the efficacy of a recombinant pertussis DNA vaccine. The strategy is co-injection with cytokine plasmids as prime, and boosted with purified homologous proteins. A recombinant pertussis DNA vaccine containing the pertussis toxin subunit 1 (PTS1), fragments of the filamentous hemagglutinin (FHA) gene and pertactin (PRN) gene encoding filamentous hemagglutinin and pertactin were constructed. Balb/c mice were immunized with several DNA vaccines and antigen-specific antibodies anti-PTS1,anti-PRN, anti-FHA,cytokines interleukin (IL)-10, IL-4, IFN-gamma,TNF-alpha,and splenocyte-proliferation assay were used to describe immune responses. The recombinant DNA vaccine could elicit similar immune responses in mice as that of separate plasmids encoding the 3 fragments, respectively. Mice immunized with DNA and boosted with the corresponding protein elicited more antibodies than those that received DNA as boost. In particular, when the mice were co-immunized with murine granulocyte-macrophage colony-stimulating factor plasmids and boosted with proteins, all 4 cytokines and the 3 antigen-specific antibodies were significantly increased compared to the pVAX1 group. Anti-PTS1, anti- FHA, IL-4 and TNF-alpha elicited in the colony stimulating factor (CSF) prime-protein boost group showed significant increase compared to all the other groups. This prime and boost strategy has proven to be very useful in improving the immunogenicity of DNA vaccines against pertussis.

Co-expression of antibody fab heavy and light chain genes from separate evolved compatible replicons in E. coli

Antibody molecules bind to antigen with six complementary determining region (CDR) loops, three of which are located on each variable heavy (V H) and light (V L) chains. Discovery and optimization of antibodies that bind antigen using in vitro techniques require diversification of one or more of these CDRs. Since antibodies are dimeric, simultaneous diversification of heavy and light chains on separate genetic elements would allow “chain shuffling” to occur simply and efficiently. Efficient expression of antibody V H and V L requires that the two separate replicons be compatible with one another, but also have similar properties, such as copy number in E. coli. Standard plasmids that are compatible with one another in E. coli exist at widely variable copy numbers. Recently we described the isolation of ColE1 mutants that have similar copy numbers but different incompatibility characteristics. Thus, new compatibility groups in the ColE1 family were established. Herein we describe the E. coli expression of V H and V L genes to form a functional Fab. The ability to express antibody heavy and light chains from separate but compatible high copy plasmids should allow new opportunities in antibody engineering, such as rapid chain shuffling and generation of more complex antibody libraries.

Efficient expression of recombinant human monoclonal antibodies in Drosophila S2 cells

We have explored the Drosophila S2 cell line for expression of Ig molecules isolated as Fab or scFv cDNA from phage-displayed libraries. We present a series of vectors for inducible expression and secretion of human Ig heavy (HC) and light chains (LC), both on separate plasmids and in combination constructs. Both HC (tested as human γ 1) and LC (human κ) could be expressed separately and were secreted into the medium, confirming previous reports. When the combination vector carrying both the HC and LC cDNA, as well as when the HC and LC vectors were co-transfected, complete IgG1 was found in the medium. Transient transfection resulted in production levels of 0.5–1 mg/l. Stable cell lines could be established within 2–3 weeks. After 10–12 days of expression from such cell lines, Ig molecules accumulated and the medium contained typically 5–35 mg/l of IgG1. The IgG in these preparations was purified to more than 90% purity on protein G columns. Binding characteristics for IgG of the same clone expressed in S2 cells or mammalian cells were indistinguishable. The main advantages with this system compared to mammalian expression were its robustness and the much faster establishment of stable, high level producing cell lines.

Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct

Small fish are a popular laboratory model for studying gene expression and function by transgenesis. If, however, the transgenes are not readily detectable by visual inspection, a large number of embryos must be injected, raised and screened to identify positive founder fish. Here, we describe a strategy to efficiently generate and preselect transgenic lines harbouring any transgene of interest. Co-injection of a selectable reporter construct (e.g., GFP), together with the transgene of interest on a separate plasmid using the I-SceI meganuclease approach, results in co-distribution of the two plasmids. The quality of GFP expression within the F0 generation therefore reflects the quality of injection and allows efficient and reliable selection of founder fish that are also positive for the second transgene of interest. In our experience, a large fraction (up to 50%) of GFP-positive fish will also be transgenic for the second transgene, thus providing a rapid (within 3-4 months) and efficient way to establish transgenic lines for any gene of interest in medaka and zebrafish.

Efficient site-specific integration in Plasmodium falciparum chromosomes mediated by mycobacteriophage Bxb1 integrase

Here we report an efficient, site-specific system of genetic integration into Plasmodium falciparum malaria parasite chromosomes. This is mediated by mycobacteriophage Bxb1 integrase, which catalyzes recombination between an incoming attP and a chromosomal attB site. We developed P. falciparum lines with the attB site integrated into the glutaredoxin-like cg6 gene. Transfection of these attB(+) lines with a dual-plasmid system, expressing a transgene on an attP-containing plasmid together with a drug resistance gene and the integrase on a separate plasmid, produced recombinant parasites within 2 to 4 weeks that were genetically uniform for single-copy plasmid integration. Integrase-mediated recombination resulted in proper targeting of parasite proteins to intra-erythrocytic compartments, including the apicoplast, a plastid-like organelle. Recombinant attB x attP parasites were genetically stable in the absence of drug and were phenotypically homogeneous. This system can be exploited for rapid genetic integration and complementation analyses at any stage of the P. falciparum life cycle, and it illustrates the utility of Bxb1-based integrative recombination for genetic studies of intracellular eukaryotic organisms.

Identification of Mn2+-binding Aspartates from α, β, and γ Subunits of Human NAD-dependent Isocitrate Dehydrogenase

The human NAD-dependent isocitrate dehydrogenase (IDH), with three types of subunits present in the ratio of 2alpha:1beta:1gamma, requires a divalent metal ion to catalyze the oxidative decarboxylation of isocitrate. With the aim of identifying ligands of the enzyme-bound Mn(2+), we mutated aspartates on the alpha, beta, or gamma subunits. Mutagenesis target sites were based on crystal structures of metal-isocitrate complexes of Escherichia coli and pig mitochondrial NADP-IDH and sequence alignments. Aspartates replaced by asparagine or cysteine were 206, 230, and 234 of the alpha subunit and those corresponding to alpha-Asp-206: 217 of the beta subunit and 215 of the gamma subunit. Each expressed, purified mutant enzyme has two wild-type subunits and one subunit with a single mutation. Specific activities of WT, alpha-D206N, alpha-D230C, alpha-D234C, beta-D217N, and gamma-D215N enzymes are 22, 29, 1.4, 0.2, 7.3 and 3.7 micromol of NADH/min/mg, respectively, whereas alpha-D230N and alpha-D234N enzymes showed no activity. The K(m,Mn(2+)) for alpha-D230C and gamma-D215N are increased 32- and 100-fold, respectively, along with elevations in K(m,isocitrate). The K(m,NAD) of alpha-D230C is increased 16-fold, whereas that of beta-D217N is elevated 10-fold. For all the mutants K(m,isocitrate) is decreased by ADP, indicating that these aspartates are not needed for normal ADP activation. This study demonstrates that alpha-Asp-230 and alpha-Asp-234 are critical for catalytic activity, but alpha-Asp-206 is not needed; alpha-Asp-230 and gamma-Asp-215 may interact directly with the Mn(2+); and alpha-Asp-230 and beta-Asp-217 contribute to the affinity of the enzyme for NAD. These results suggest that the active sites of the human NAD-IDH are shared between alpha and gamma subunits and between alpha and beta subunits.

Asymmetric reduction of ketones using recombinant E. coli cells that produce a versatile carbonyl reductase with high enantioselectivity and broad substrate specificity

The gene encoding a versatile biocatalyst that shows high enantioselectivity for a variety of ketones, SCR ( Saccharomyces cerevisiae carbonyl reductase), has been identified, cloned, and expressed in Escherichia coli . Two types of expression systems with high NADPH-regenerating capacities have been constructed. One is the tandem system, where the genes encoding SCR and GDH (glucose dehydrogenase) are located in the same plasmid, and the other is the two-plasmid system, where each of the SCR and GDH genes is located in separate plasmids that can coexist in one E. coli cell. Asymmetric reduction of ketones with the recombinant E. coli cells gave synthetically useful 20 alcohols, 11 of which were enantiomerically pure. The productivity of one of these products was as high as 41 g/L.

Novel Role of Cold/Menthol-sensitive Transient Receptor Potential Melastatine Family Member 8 (TRPM8) in the Activation of Store-operated Channels in LNCaP Human Prostate Cancer Epithelial Cells

Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the prostate and in the prostate cancer-derived epithelial cell line, LNCaP. In this study, we show that despite such expression, LNCaP cells respond to cold/menthol stimulus by membrane current (I(cold/menthol)) that shows inward rectification and high Ca(2+) selectivity, which are dramatically different properties from "classical" TRPM8-mediated I(cold/menthol). Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both I(cold/menthol) and TRPM8 protein in LNCaP cells. We demonstrate that these puzzling results arise from TRPM8 localization not in the plasma, but in the endoplasmic reticulum (ER) membrane of LNCaP cells, where it supports cold/menthol/icilin-induced Ca(2+) release from the ER with concomitant activation of plasma membrane (PM) store-operated channels (SOC). In contrast, GFP-tagged TRPM8 heterologously expressed in HEK-293 cells target the PM. We also demonstrate that TRPM8 expression and the magnitude of SOC current associated with it are androgen-dependent. Our results suggest that the TRPM8 may be an important new ER Ca(2+) release channel, potentially involved in a number of Ca(2+)- and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis.

A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors

Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination.

Neuroblastoma Cells Transiently Transfected to Simultaneously Express the Co-Stimulatory Molecules CD54, CD80, CD86, and CD137L Generate Antitumor Immunity in Mice

The goal of this study was to show that nonviral gene transfection technology can be used to genetically modify neuroblastoma cells with immune stimulatory molecules, and that the modified cells can generate an antitumor immune response. The authors found that an electroporation-based gene transfection method, nucleofection, could be used to modify mouse AGN2a (an aggressive variant of Neuro-2a) neuroblastoma cells to simultaneously express as many as four different immune stimulatory molecules encoded by separate plasmid vectors. Within 18 hours after nucleofection, greater than 60% of the cells typically expressed the transfected gene products, and the percentages of cells expressing the products often exceeded 96%. High levels of plasmid in cell nuclei immediately after nucleofection documented instantaneous availability of gene vectors to the transcriptional machinery. AGN2a cells nucleofected to express the co-stimulatory molecules CD80 and CD86 expressed higher levels of these molecules than cells that had been permanently transfected with these same plasmid vectors, and the nucleofected cells were as effective as the permanently transfected cells at inducing an antitumor response in vivo in a tumor prevention model. AGN2a cells nucleofected with four separate plasmid vectors encoding CD54, CD80, CD86, and CD137L induced a T-cell immune response in vitro and served as a potent tumor vaccine in the tumor prevention model. These data show that transient transfection using a nonviral based method, nucleofection, can be used to rapidly generate novel cell-based tumor vaccines.

Reduced cellular immune responses following immunization with a multi-gene HIV-1 vaccine

We investigated the effects of immunizing with several genes and subtypes of HIV-1. The genes used as immunogens were: gp160 envelope (env subtypes A, B and C), p37gag (gag subtypes A and B), rev (subtype B) and reverse transcriptase (RT subtype B). The different genes are all carried by separate plasmids. C57BL/6 and BALB/c mice were immunized with different combinations of the genes together with recombinant cytokine granulocyte macrophage-colony stimulating factor. The env genes injected alone induced significantly stronger cellular responses to envelope in both strains of mice than when env genes were injected together with gag and RT genes. In the C57BL/6 mice, the envelope specific responses were significantly increased after spatial separation of env genes from gag and RT genes as compared to when all vaccine genes were injected as a mixture. The gag responses were strong in gag-immunized animals and were not significantly affected by the spatial separation of gag and RT genes from the env genes. Our results illustrate the importance of being cautious when formulating multivalent genetic vaccines and that it might be possible to overcome lost immune responses through spatial separation of vaccine antigens.