Dual Plasmid Research Articles

Engineering of recombinant E. coli cells co‐expressing P450pyrTM monooxygenase and glucose dehydrogenase for highly regio‐ and stereoselective hydroxylation of alicycles with cofactor recycling

E. coli (P450pyrTM-GDH) with dual plasmids, pETDuet containing P450pyr triple mutant I83H/M305Q/A77S (P450pyrTM) and ferredoxin reductase (FdR) genes and pRSFDuet containing glucose dehydrogenase (GDH) and ferredoxin (Fdx) genes, was engineered to show a high activity (12.7 U g⁻¹ cdw) for the biohydroxylation of N-benzylpyrrolidine 1 and a GDH activity of 106 U g⁻¹ protein. The E. coli cells were used as efficient biocatalysts for highly regio- and stereoselective hydroxylation of alicyclic substrates at non-activated carbon atom with enhanced productivity via intracellular recycling of NAD(P)H. Hydroxylation of N-benzylpyrrolidine 1 with resting cells in the presence of glucose showed excellent regio- and stereoselectivity, giving (S)-N-benzyl-3-hydroxypyrrolidine 2 in 98% ee as the sole product in 9.8 mM. The productivity is much higher than that of the same biohydroxylation using E. coli (P450pyrTM)b without expressing GDH. E. coli (P450pyrTM-GDH) was found to be highly regio- and stereoselective for the hydroxylation of N-benzylpyrrolidin-2-one 3, improving the regioselectivity from 90% of the wild-type P450pyr to 100% and giving (S)-N-benzyl-4-hydroxylpyrrolidin-2-one 4 in 99% ee as the sole product. A high activity of 15.5 U g⁻¹ cdw was achieved and (S)-4 was obtained in 19.4 mM. E. coli (P450pyrTM-GDH) was also found to be highly regio- and stereoselective for the hydroxylation of N-benzylpiperidin-2-one 5, increasing the ee of the product (S)-N-benzyl-4-hydroxy-piperidin-2-one 6 to 94% from 33% of the wild-type P450pyr. A high activity of 15.8 U g⁻¹ cdw was obtained and (S)-6 was produced in 3.3 mM as the sole product. E. coli (P450pyrTM-GDH) represents the most productive system known thus far for P450-catalyzed hydroxylations with cofactor recycling, and the hydroxylations with E. coli (P450pyrTM-GDH) provide with simple and useful syntheses of (S)-2, (S)-4, and (S)-6 that are valuable pharmaceutical intermediates and difficult to prepare.

Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis

Background: Altering target cell apoptosis is one of the challenging ideas of biotechnological applications. There are several applications of over expressing Bcl-xL anti-apoptotic protein from recombinant protein production to DNA vaccination strategies. The aim of the present study is to evaluate the anti-apoptotic efficacy of Bcl-xL expressing dual promoter plasmid system as a candidate to be used for recombinant protein production and DNA vaccination approaches. For this purpose, Bcl-xL anti-apoptotic protein gene was inserted in a dual expressing vector system in frame with EGFP (enhanced green fluorescence protein) after IRES (internal ribosomal site). The plasmid has a multiple cloning site after CMV (cytomegalovirus promoter) left empty to be inserted a biopharmaceutical protein gene region or DNA vaccine antigens. Results: In order to determine the anti-apoptotic efficacy of Bcl-xL inserted dual expressing vector, BHK-21 cells were transfected both with this plasmid and empty vector as control. Apoptosis was stimulated by several apoptosis inducing agents and serum deprivation in the transfected cells for 48 hrs. Cells expressing Bcl-xL protein in frame with EGFP were determined by flow cytometry as an indicator of cell viability. Additionally, apoptosis were determined by intracellular cleaved Casp 3 staining in Bcl-xL expressing EGFP positive cells. The dual expression plasmid bearing Bcl-xL anti-apoptotic protein prolonged the cell survival rate and protected cells from apoptosis upon apoptosis induction by doxorubicin and camptothecin in which the anti-apoptotic efficacies are inhibited through over expressing of Bcl-xL. pIRES2EGFP/Bcl-xL transfected cell ratio was significantly higher compared to empty vector transfected cells (P Conclusion: In conclusion, it was shown that in vitro transient expression of Bcl-xL efficiently inhibited apoptosis induced by serum deprivation, doxorubicin and camptothecin. Thus, the dual expression plasmid bearing Bcl-xL anti-apoptotic protein could be a good candidate for recombinant protein production and DNA vaccination applications.

The Asd + -DadB + Dual-Plasmid System Offers a Novel Means To Deliver Multiple Protective Antigens by a Recombinant Attenuated Salmonella Vaccine

We developed means to deliver multiple heterologous antigens on dual plasmids with non-antibiotic-resistance markers in a single recombinant attenuated vaccine strain of Salmonella enterica serotype Typhimurium. The first component of this delivery system is a strain of S. Typhimurium carrying genomic deletions in alr, dadB, and asd, resulting in obligate requirements for diaminopimelic acid (DAP) and d-alanine for growth. The second component is the Asd(+)-DadB(+) plasmid pair carrying wild-type copies of asdA and dadB, respectively, to complement the mutations. To evaluate the protection efficacy of the dual-plasmid vaccine, S. Typhimurium strain χ9760 (a strain with multiple attenuating mutations: Δasd Δalr ΔdadB ΔrecF) was transformed with Asd(+) and DadB(+) plasmids specifying pneumococcal antigens PspA and PspC, respectively. Both plasmids were stable in χ9760 for 50 generations when grown in nonselective medium. This was significantly (P < 0.05) greater than the stability seen in its recF(+) counterpart χ9590 and could be attributed to reduced interplasmid recombination in χ9760. Oral immunization of BALB/c mice with 1 × 10(9) CFU of χ9760 (carrying Asd(+)-PspA and DadB(+)-PspC plasmids) elicited a dominant Th1-type serum IgG response against both antigens and protected mice against intraperitoneal challenge with 200 50% lethal doses (LD(50)s) of virulent Streptococcus pneumoniae strain WU2 or intravenous challenge with 100 LD(50)s of virulent S. pneumoniae strain L81905 or intranasal challenge with a lethal dose of S. pneumoniae A66.1 in a pneumonia model. Protection offered by χ9760 was superior to that offered by the mixture of two strains, χ9828 (Asd(+)-PspA) and χ11026 (DadB(+)-PspC). This novel dual-plasmid system marks a remarkable improvement in the development of live bacterial vaccines.

Replacement of huntingtin exon 1 by trans-splicing

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). HD offers unique opportunities for promising RNA-based therapeutic approaches aimed at reducing mutant HTT expression, since the HD mutation is considered to be a "gain-of-function" mutation. Allele-specific strategies that preserve expression from the wild-type allele and reduce the levels of mutant protein would be of particular interest. Here, we have conducted proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically repair the HTT allele. We employed a dual plasmid transfection system consisting of a pre-mRNA trans-splicing module (PTM) containing HTT exon 1 and a HTT minigene to demonstrate that HTT exon 1 can be replaced in trans. We detected the presence of the trans-spliced RNA in which PTM exon 1 was correctly joined to minigene exons 2 and 3. Furthermore, exon 1 from the PTM was trans-spliced to the endogenous HTT pre-mRNA in cultured cells as well as disease-relevant models, including HD patient fibroblasts and primary neurons from a previously described HD mouse model. These results suggest that the repeat expansion of HTT can be repaired successfully not only in the context of synthetic minigenes but also within the context of HD neurons. Therefore, pre-mRNA trans-splicing may be a promising approach for the treatment of HD and other dominant genetic disorders.

Fluorescent protein engineering by in vivo site‐directed mutagenesis

AbstractIn vivo site‐directed mutagenesis by single‐stranded deoxyribonucleic acid recombineering is a facile method to change the color of fluorescent proteins (FPs) without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the Escherichia coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the FP. Eight different FPs (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza, and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo site‐directed mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive quantitative assay for recombination. A set of rules to simplify mutant isolation by recombineering is provided. © 2012 IUBMB IUBMB Life, 2012

Use of the Saccharomyces cerevisiae endopolygalacturonase promoter to direct expression in Escherichia coli

In Saccharomyces cerevisiae, an endopolygalacturonase encoded by the PGL1 gene catalyzes the random hydrolysis of the α-1,4 glycosidic linkages in polygalacturonic acid. To study the regulation of the PGL1 gene, we constructed a reporter vector containing the lacZ gene under the control of PGL1 promoter. Surprisingly, when Escherichia coli DH5α was transformed by this vector, cells harboring the constructed plasmid produced blue colonies. Sequence analysis of this promoter revealed that E. coli consensus sequences required to express an in-frame lacZ alpha product were present. We next decided to investigate how the PGL1 promoter is regulated in E. coli compared to yeast. In this study, we examined the modulation of the PGL1 promoter in E. coli, and the results indicated that its activity is greatly induced by saturated digalacturonic acid and is indirectly regulated by the transcriptional regulators the 2-keto-3-deoxygluconate repressor. Moreover, PGL1 expression is enhanced under aerobic conditions. We found that β-galactosidase activity in E. coli could reach 180 units, which is 40-fold greater than the activity produced in S. cerevisiae, and greater than recombinant protein expression previously reported by other researchers. We thus demonstrate that this vector can be considered as a dual expression plasmid for both E. coli and S. cerevisiae hosts. So far, no modulation of endoPG promoters expressed in E. coli has been reported.

Fluorescent protein engineering by in vivo site‐directed mutagenesis

In vivo site-directed mutagenesis by single-stranded deoxyribonucleic acid recombineering is a facile method to change the color of fluorescent proteins (FPs) without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the Escherichia coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the FP. Eight different FPs (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza, and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo site-directed mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive quantitative assay for recombination. A set of rules to simplify mutant isolation by recombineering is provided.

Plasmid-based Survivin shRNA and GRIM-19 carried by attenuated Salmonella suppresses tumor cell growth

Persistent activation of Survivin and its overexpression contribute to the formation, progression and metastasis of several different tumor types. Therefore, Survivin is an ideal target for RNA interference mediated-growth inhibition. Blockade of Survivin using specific short hairpin RNAs (shRNA) can significantly reduce prostate tumor growth. RNA interference does not fully ablate target gene expression, owing to the idiosyncrasies associated with shRNAs and their targets. To enhance the therapeutic efficacy of Survivin-specific shRNA, we employed a combinatorial expression of Survivin-specific shRNA and gene associated with retinoid-interferon-induced mortality-19 (GRIM-19). Then, the GRIM-19 coding sequences and Survivin-specific shRNAs were used to create a dual expression plasmid vector and were carried by an attenuated strain of Salmonella enteric serovar typhimurium (S. typhimurium) to treat prostate cancer in vitro and in vivo. We found that the co-expressed Survivin-specific shRNA and GRIM-19 synergistically and more effectively inhibited prostate tumor proliferation and survival, when compared with treatment with either single agent alone in vitro and in vivo. This study has provided a novel cancer gene therapeutic approach for prostate cancer.

Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase

BackgroundSolar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant Escherichia coli with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H2) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H2 production yield under light conditions.ResultsRecombinant E. coli BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from Hydrogenovibrio marinus produced ~1.3-fold more H2 in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m2·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H2 production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m2·s) led to an increase (~1.8-fold) in H2 production from 287.3 to 525.7 mL H2/L-culture in the culture of recombinant E. coli co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H2 achieved in this study was ~3.4%.ConclusionHere, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H2 production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant E. coli BL21(DE3) in a light intensity-dependent manner. These results demonstrate that E. coli can be applied as light-powered cell factories for biohydrogen production by introducing proteorhodopsin.

Enhancing functional production of a chaperone-dependent lipase in Escherichia coli using the dual expression cassette plasmid

sBackgroundThe lipase subfamilies I.1 and I.2 show more than 33% homology in the amino acid sequences and most members share another common property that their genes are clustered with the secondary genes whose protein products are required for folding the lipase into an active conformation and secretion into the culture medium. In previous studies, the lipase (LipA) and its chaperone (LipB) from Ralstonia sp. M1 were overexpressed in E. coli and the lipase was successfully refolded in vitro. The purpose of this study was to enhance the production of the active lipase LipA from Ralstonia sp. M1 in the heterologous host E. coli without in vitro refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems.ResultsTo produce more active lipase from Ralstonia sp. M1 in E. coli without in vitro refolding process but with the help of overexpression of the chaperone (LipB1 and LipB3 corresponding to 56-aa truncated and 26-aa truncated chaperone LipB), six different expression systems including 2 two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1a, BL21/pELipAB-LipB3a, BL21/pELipA-LipB1a, and BL21/pELipA-LipB3a) were constructed. The two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system E. coli BL21/pELipABa did. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1a and BL21/pELipAB-LipB3a yielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system E. coli BL21/pELipABa, respectively. Although the lipase amount was equally expressed in all these expression systems (40% of total cellular protein) and only a small fraction of the overexpressed lipase was folded in vivo into the functional lipase in soluble form whereas the main fraction was still inactive in the form of inclusion bodies. Another controversial finding was that the dual expression cassette plasmid systems E. coli BL21/pELipAB-LipB1a and E. coli/pELipAB-LipB3a secreted the active lipase into the culture medium of 51 and 29 times as high as the single expression cassette plasmid system E. coli pELipABa did, respectively, which has never been reported before. Another interesting finding was that the lipase form LipA6xHis (mature lipase fused with 6× histidine tag) expressed in the dual expression cassette plasmid systems (BL21/pELipA-LipB1a and BL21/pELipA-LipB3a) showed no lipase activity although the expression level of the lipase and two chaperone forms LipB1 and LipB3 in these systems remained as high as that in E. coli BL21/pELipABa + pELipB1k, BL21/pELipABa + pELipB3k, BL21/pELipAB-LipB1a, and BL21/pELipAB-LipB3a. The addition of Neptune oil or detergents into the LB medium increased the lipase production and secretion by up to 94%.ConclusionsOur findings demonstrated that a dual expression cassette plasmid system E. coli could overproduce and secrete the active chaperone-dependent lipase (subfamilies I.1 and I.2) in vivo and an improved dual expression cassette plasmid system E. coli could be potentially applied for industrial-scale production of subfamily I.1 and I.2 lipases.

Quantitative Co-Expression of Proteins at the Single Cell Level – Application to a Multimeric FRET Sensor

BackgroundCo-expression of proteins is generally achieved by introducing two (or more) independent plasmids into cells, each driving the expression of a different protein of interest. However, the relative expression levels may vary strongly between individual cells and cannot be controlled. Ideally, co-expression occurs at a defined ratio, which is constant among cells. This feature is of particular importance for quantitative single cell studies, especially those employing bimolecular Förster Resonance Energy Transfer (FRET) sensors.Methodology/Principal FindingsFour co-expression strategies based on co-transfection, a dual promotor plasmid, an internal ribosome entry site (IRES) and a viral 2A peptide were selected. Co-expression of two spectrally separable fluorescent proteins in single living cells was quantified. It is demonstrated that the 2A peptide strategy can be used for robust equimolar co-expression, while the IRES sequence allows expression of two proteins at a ratio of approximately 3:1. Combined 2A and IRES elements were used for the construction of a single plasmid that drives expression of three individual proteins, which generates a FRET sensor for measuring heterotrimeric G-protein activation. The plasmid drives co-expression of donor and acceptor tagged subunits, with reduced heterogeneity, and can be used to measure G-protein activation in single living cells.Conclusions/SignificanceQuantitative co-expression of two or more proteins can be achieved with little cell-to-cell variability. This finding enables reliable co-expression of donor and acceptor tagged proteins for FRET studies, which is of particular importance for the development of novel bimolecular sensors that can be expressed from single plasmid.

Characterization of DNA polymerase β from Danio rerio by overexpression in E. coli using the in vivo/in vitro compatible pIVEX plasmid

BackgroundEukaryotic DNA polymerase β (pol β), the polymerase thought to be responsible for DNA repair synthesis, has been extensively characterized in rats and humans. However, pol β has not been purified or enzymatically characterized from the model fish species Danio rerio (zebrafish). We used the in vitro/in vivo dual expression system plasmid, pIVEX, to express Danio rerio pol β (Danio pol β) for biochemical characterization.ResultsDanio pol β encoded by the in vitro/in vivo-compatible pIVEX plasmid was expressed in E. coli BL21(DE3), BL21(DE3)pLysS, and KRX, and in vitro as a C-terminal His-tagged protein. Danio pol β expressed in vitro was subject to proteolysis; therefore, bacterial overexpression was used to produce the protein for kinetic analyses. KRX cells were preferred because of their reduced propensity for leaky expression of pol β. The cDNA of Danio rerio pol β encodes a protein of 337 amino acids, which is 2-3 amino acids longer than other pol β proteins, and contains a P63D amino acid substitution, unlike mammalian pol βs. This substitution lies in a hairpin sequence within an 8-kDa domain, likely to be important in DNA binding. We performed extensive biochemical characterization of Danio pol β in comparison with rat pol β, which revealed its sensitivity to metal ion activators (Mn2+ and Mg2+), its optimum salt concentration (10 mM KCl and 50 mM NaCl), alkaline pH optimum (pH 9.0), and low temperature optimum (30°C). Substituting Mn2+ for Mg2+ resulted in 8.6-fold higher catalytic efficiency (kcat/Km).ConclusionsOur characterization of pol β from a model fish organism contributes to the study of the function and evolution of DNA polymerases, which are emerging as important cellular targets for chemical intervention in the development of anticancer agents.

Efficient GLP-1 gene delivery using two-step transcription amplification plasmid system with a secretion signal peptide and arginine-grafted bioreducible polymer

Glucagon-like peptide (GLP-1) encoding dual plasmid (pDNA) system (TSTA (SP-GLP-1)) which is composed of pβ-Gal4-p65 and pUAS-SP-GLP-1 was constructed to improve the production and secretion of expressed GLP-1 by combining the advantages of signal peptide (SP) and two-step transcription amplification (TSTA) system. Its potential for GLP-1 gene delivery system was investigated with employment of arginine-grafted bioreducible polymer (ABP) as a gene carrier. Their polyplexes have about 140nm-sizes and 20mV Zeta-potential values. ABP showed no cytotoxicity contrary to PEI25k. It was found in RT-PCR experiments that TSTA-SP pDNA systems showed increased GLP-1 gene transcription level in comparison with mono pDNA system (pβ-GLP-1). It was also observed in GLP-1 ELISA that GLP-1 secretion level of TSTA (SP-GLP-1) pDNA system was 2.7–3.4 times higher than those of pβ-GLP-1 and 1.5–1.7 times than TSTA (GLP-1). Additionally, 2.5–3.5 folds increased level of GLP-1 secretion was found in ABP gene carrier system in comparison with PEI25k. When transfection medium containing secreted GLP-1 was transferred to NIT-1 insulinoma cells, the highest secretion level of insulin was induced in ABP/TSTA (SP-GLP-1) polyplex medium-treated cells. Therefore, this novel system could be utilized as a safe and efficient GLP-1 gene delivery system for type 2 diabetes therapy.

Suppression of bovine viral diarrhea virus replication by single and dual short hairpin RNA-mediated RNA interference

Bovine viral diarrhea virus (BVDV) is one of the most important pathogens to the cattle industry, causing a significant economic loss throughout the world. Despite the wide use of various control measures for BVDV, the disease remains prevalent. In this study, we achieved an efficient inhibition of NADL strain replication by plasmid-mediated shRNA targeting conserved regions of the viral genome. To further enhance the inhibiting efficiency, a dual shRNA expression plasmid, which could simultaneously express two different shRNA, was established and showed stronger inhibitory effects on virus replication. Moreover, the antiviral activity induced by the dual shRNA expression system was also evident on other BVDV-1 subgenotypes (BVDV-1a, BVDV-1b and BVDV-1c). Therefore, the dual shRNA system provides a more powerful strategy for inhibiting BVDV replication in a cross-resistance manner.

Development of Dual Control Allotopic Expression System for Subunit 8 of Yeast Saccharomyces cerevisiae Mitochondrial ATP Synthase

The yeast mitochondrial F1F0-ATP synthase is a multisubunit complex that contains at least 17 different subunits. Subunit 8 of yeast mitochondrial ATP synthase is a hydrophobic protein of 48 amino acids encoded by the mitochondrial ATP8 gene. A dual control allotopic expression system for subunit 8 has been developed. The strategy involves maintenance of two different compatible yeast expression vectors each utilizing a different inducible promoter in the same host cells. The system thus enables cloning and allotopic expression of two different forms of subunit 8 gene. The goal of the developed strategy is to permit allotopic expression of functional wildtype subunit 8 gene under a conditional promoter system and subunit 8 variant gene under the control of a different promoter system. The system is potentially useful for accurate assessment of assembly behavior of functionally defective subunit 8 variants in vivo. The strategy relies on the ability to conditionally regulate the expression of the two genes. A set of functionally defective subunit 8 variants has been cloned under an inducible yeast promoter system and dual plasmid harboring strains for dual control allotopic expression of each variant have been constructed.

Poly(C)-Binding Protein 2 Interacts with Sequences Required for Viral Replication in the Hepatitis C Virus (HCV) 5′ Untranslated Region and Directs HCV RNA Replication through Circularizing the Viral Genome

Sequences in the 5' untranslated region (5'UTR) of hepatitis C virus (HCV) RNA is important for modulating both translation and RNA replication. The translation of the HCV genome depends on an internal ribosome entry site (IRES) located within the 341-nucleotide 5'UTR, while RNA replication requires a smaller region. A question arises whether the replication and translation functions require different regions of the 5'UTR and different sets of RNA-binding proteins. Here, we showed that the 5'-most 157 nucleotides of HCV RNA is the minimum 5'UTR for RNA replication, and it partially overlaps with the IRES. Stem-loops 1 and 2 of the 5'UTR are essential for RNA replication, whereas stem-loop 1 is not required for translation. We also found that poly(C)-binding protein 2 (PCBP2) bound to the replication region of the 5'UTR and associated with detergent-resistant membrane fractions, which are the sites of the HCV replication complex. The knockdown of PCBP2 by short hairpin RNA decreased the amounts of HCV RNA and nonstructural proteins. Antibody-mediated blocking of PCBP2 reduced HCV RNA replication in vitro, indicating that PCBP2 is directly involved in HCV RNA replication. Furthermore, PCBP2 knockdown reduced IRES-dependent translation preferentially from a dual reporter plasmid, suggesting that PCBP2 also regulated IRES activity. These findings indicate that PCBP2 participates in both HCV RNA replication and translation. Moreover, PCBP2 interacts with HCV 5'- and 3'UTR RNA fragments to form an RNA-protein complex and induces the circularization of HCV RNA, as revealed by electron microscopy. This study thus demonstrates the mechanism of the participation of PCBP2 in HCV translation and replication and provides physical evidence for HCV RNA circularization through 5'- and 3'UTR interaction.

Novel, fluorescent, SSB protein chimeras with broad utility

The Escherichia coli single-stranded DNA binding protein (SSB) is a central player in DNA metabolism where it organizes genome maintenance complexes and stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing. Due to the importance of SSB and to facilitate real-time studies, we developed a dual plasmid expression system to produce novel, chimeric SSB proteins. These chimeras, which contain mixtures of histidine-tagged and fluorescent protein(FP)-fusion subunits, are easily purified in milligram quantities and used without further modification, a significant enhancement over previous methods to produce fluorescent SSB. Chimeras retain the functionality of wild type in all assays, demonstrating that SSB function is unaffected by the FPs. We demonstrate the power and utility of these chimeras in single molecule studies providing a great level of insight into the biochemical mechanism of RecBCD. We also utilized the chimeras to show for the first time that RecG and SSB interact in vivo. Consequently, we anticipate that the chimeras described herein will facilitate in vivo, in vitro and single DNA molecule studies using proteins that do not require further modification prior to use.

Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication

The programmed −1 ribosomal frameshifting (−1 PRF) utilized by eukaryotic RNA viruses plays a crucial role for the controlled, limited synthesis of viral RNA replicase polyproteins required for genome replication. The viral RNA replicase polyproteins of severe acute respiratory syndrome coronavirus (SARS-CoV) are encoded by the two overlapping open reading frames 1a and 1b, which are connected by a −1 PRF signal. We evaluated the antiviral effects of antisense peptide nucleic acids (PNAs) targeting a highly conserved RNA sequence on the – PRF signal. The ribosomal frameshifting was inhibited by the PNA, which bound sequence-specifically a pseudoknot structure in the −1 PRF signal, in cell lines as assessed using a dual luciferase-based reporter plasmid containing the −1 PRF signal. Treatment of cells, which were transfected with a SARS-CoV-replicon expressing firefly luciferase, with the PNA fused to a cell-penetrating peptide (CPP) resulted in suppression of the replication of the SARS-CoV replicon, with a 50% inhibitory concentration of 4.4 μM. There was no induction of type I interferon responses by PNA treatment, suggesting that the effect of PNA is not due to innate immune responses. Our results demonstrate that −1 PRF, critical for SARS-CoV viral replication, can be inhibited by CPP-PNA, providing an effective antisense strategy for blocking −1 PRF signals.

Construction and application of a built-in dual luciferase reporter for microRNA functional analysis

Background: As key gene regulators, microRNAs post-transcriptionally modulate gene expression via binding to partially complementary sequence in the 3' UTR of target mRNA. An accurate, rapid and quantitative tool for sensing and validation of miRNA targets is of crucial significance to decipher the functional implications of miRNAs in cellular pathways. Results: Taking advantage of an improved restriction-free cloning method, we engineered a novel built-in dual luciferase reporter plasmid where Firefly and Renilla luciferase genes were assembled in a single plasmid named pFila. This design eliminates the transfection of a separate control plasmid and thus minimizes the time and labor required for miRNA-target sensing assays. pFila consistently produces Firefly and Renilla luciferase activities when transfected into human-, monkey- and mouse-derived mammalian cell systems. Moreover, pFila is capable of recapitulating the interaction of miR-16 and its known target CCNE1 in Hela cells. Additionally, pFila is shown to be a sensitive miR-biosensor by evaluating the inhibition efficiency of endogenous miRNA. Conclusions: pFila would facilitate miRNA target identification and verification in a rapid and simplified manner. Also, pFila is a sensitive biosensor for active miRNA profiling in vivo .

Genetically modified human umbilical cord blood cells expressing vascular endothelial growth factor and fibroblast growth factor 2 differentiate into glial cells after transplantation into amyotrophic lateral sclerosis transgenic mice

Current therapy of a number of neuropsychiatric maladies has only symptomatic modality. Effective treatment of these neuro-degenerative diseases, including amyotrophic lateral sclerosis (ALS), may benefit from combined gene/stem-cell approaches. In this report, mononuclear fraction of human umbilical cord blood cells (hUCBCs) were transfected by electroporation with dual plasmid constructs, simultaneously expressing vascular endothelial growth factor 165 (VEGF(165)) and human fibroblast growth factor 2 (FGF(2)) (pBud-VEGF-FGF(2)). These genetically modified hUCBCs were injected retro-orbitally into presymptomatic ALS transgenic animal models ((G)93(A) mice). Lumbar spinal cords of rodents were processed for immunofluoresent staining with antibodies against human nuclear antigen (HNA), oligodendrocyte-specific protein, S100, iba1, neuronal β(3)-tubulin and CD34. Co-localization of HNA and S100 was found in the spinal cord of mice after transplantation of genetically modified hUCBCs over-expressing VEGF-FGF(2). Double staining in control animals treated with unmodified hUCBCs, however, revealed HNA+ cells expressing iba1 and CD34. Neuron-specific β(3)-tubulin or oligodendrocyte-specific protein were not expressed in hUCBCs in either control or experimental mice. These results demonstrate that genetically naïve hUCBCs may differentiate into endothelial (CD34+) and microglial (iba1+) cells; however when over-expressing VEGF-FGF(2), hUCBCs transform into astrocytes (S100+). Autocrine regulation of VEGF and FGF(2) on hUCBCs, signal molecules from dying motor neurons in spinal cord, as well as self-differentiating potential may provide a unique microenvironment for the transformation of hUCBCs into astrocytes that eventually serve as a source of growth factors to enhance the survive potential of surrounding cells in the diseased regions.