Triple Gene Research Articles

Phylogenetic relationship of some "accessory" helicases of plant positive-stranded RNA viruses: toward understanding the evolution of triple gene block.

Recently, we hypothesized that silencing suppression activity gained by a viral replicative helicase led to the emergence of the second helicase possessing activity of the viral silencing suppressor and/or movement protein (MP). Our hypothesis accounted for the evolutionary origin of the specialized ‘triple gene block’ (TGB) in plant virus genomes encoding the MPs TGB1, TGB2, and TGB3 required for viral cell-to-cell transport through plasmodesmata. Here, we used public transcriptome databases to identify previously unrecognized viruses. The analysis of novel viral genomes further supported the previously proposed scenario of TGB origin and evolution, which included the following steps. First, the accessory helicase gene could have been acquired by horizontal gene transfer (HGT) presumably occured independently in different virus groups. Second, the TGB2 gene evolved by HGT or autonomization of the C-terminal transmembrane domain found in at least one TGB1 helicase. Third, the TGB3 gene has most likely emerged in the genomic block consisting of the TGB1 and TGB2 genes.

Assessment of the coordinated role of ST3GAL3, ST3GAL4 and ST3GAL6 on the α2,3 sialylation linkage of mammalian glycoproteins

In this research, we examined which genes are involved in N-linked sialylation in Chinese Hamster Ovary (CHO) cells using siRNA knockdown approaches. Three genes from the sialyltransferase family (ST3GAL3, ST3GAL4 and ST3GAL6) were chosen as knockdown targets with siRNA applied to reduce their expression. Single, double and triple gene knockdowns were investigated, and the reduction levels of sialylation on the total cell lysate were monitored by enzyme-linked lectin absorption assays (ELLA) and sialic acid quantification with high performance liquid chromatography (HPLC). All transfection groups showed effective reduction in 2,3-linked sialylation whereas the trend of reduction levels of triple siRNA transfection outweighed both the dual siRNA groups and single siRNA transfection groups. Next, this transfection approach was applied to CHO cells producing erythropoietin (EPO). Quantification of EPO sialylation showed similar result to total cell lysate except that the ST3GAL4 siRNA transfection exhibited the largest reduction according to the HPLC analysis as compared with other single siRNA transfections. Finally, the N-glycan released from the EPO transfected with ST3GAL4 siRNA showed a prominent reduction in sialyation level among the single siRNA transfections. From these experiments, we concluded that each of these three genes were involved in N-linked sialylation and ST3GAL4 may play the critical role in glycoprotein sialylation of recombinant proteins such as EPO.

SNPer: an R library for quantitative variant analysis on single nucleotide polymorphisms among influenza virus populations.

Influenza virus (IFV) can evolve rapidly leading to genetic drifts and shifts resulting in human and animal influenza epidemics and pandemics. The genetic shift that gave rise to the 2009 influenza A/H1N1 pandemic originated from a triple gene reassortment of avian, swine and human IFVs. More minor genetic alterations in genetic drift can lead to influenza drug resistance such as the H274Y mutation associated with oseltamivir resistance. Hence, a rapid tool to detect IFV mutations and the potential emergence of new virulent strains can better prepare us for seasonal influenza outbreaks as well as potential pandemics. Furthermore, identification of specific mutations by closely examining single nucleotide polymorphisms (SNPs) in IFV sequences is essential to classify potential genetic markers associated with potentially dangerous IFV phenotypes. In this study, we developed a novel R library called “SNPer” to analyze quantitative variants in SNPs among IFV subpopulations. The computational SNPer program was applied to three different subpopulations of published IFV genomic information. SNPer queried SNPs data and grouped the SNPs into (1) universal SNPs, (2) likely common SNPs, and (3) unique SNPs. SNPer outperformed manual visualization in terms of time and labor. SNPer took only three seconds with no errors in SNP comparison events compared with 40 hours with errors using manual visualization. The SNPer tool can accelerate the capacity to capture new and potentially dangerous IFV strains to mitigate future influenza outbreaks.

Genetic variability of blueberry scorch virus isolates from highbush blueberry in New York State.

The genetic variability of blueberry scorch virus (BlScV) isolates from New York was determined within a portion of the RNA-dependent RNA polymerase gene and the triple gene block and coat protein (CP) genes. Phylogenetic analysis of 19 New York isolates and other isolates for which sequence information is available in GenBank revealed two distinct clades, regardless of the coding region analyzed, and limited variability within (0.029 ± 0.007) and between (0.183 ± 0.032) phylogroups. Recombination events were identified in the CP gene of three New York isolates, and codons of the five BlScV genes characterized were found to be under neutral or negative selective pressure.

Logical transformation of genome-scale metabolic models for gene level applications and analysis.

In recent years, genome-scale metabolic models (GEMs) have played important roles in areas like systems biology and bioinformatics. However, because of the complexity of gene-reaction associations, GEMs often have limitations in gene level analysis and related applications. Hence, the existing methods were mainly focused on applications and analysis of reactions and metabolites. Here, we propose a framework named logic transformation of model (LTM) that is able to simplify the gene-reaction associations and enables integration with other developed methods for gene level applications. We show that the transformed GEMs have increased reaction and metabolite number as well as degree of freedom in flux balance analysis, but the gene-reaction associations and the main features of flux distributions remain constant. In addition, we develop two methods, OptGeneKnock and FastGeneSL by combining LTM with previously developed reaction-based methods. We show that the FastGeneSL outperforms exhaustive search. Finally, we demonstrate the use of the developed methods in two different case studies. We could design fast genetic intervention strategies for targeted overproduction of biochemicals and identify double and triple synthetic lethal gene sets for inhibition of hepatocellular carcinoma tumor growth through the use of OptGeneKnock and FastGeneSL, respectively. Source code implemented in MATLAB, RAVEN toolbox and COBRA toolbox, is public available at https://sourceforge.net/projects/logictransformationofmodel.

THE GENETIC DETERMINANTS OF SYMPTOMS INDUCTION BY PEPINO MOSAIC VIRUS IN TOMATO

The Pepino mosaic virus (PepMV) is one of the most important pathogens infecting tomato crops worldwide. Four major genotypes have been identified so far: Peruvian (LP), European (EU), Chilean 1 (CH1) and Chilean 2 (CH2). In Poland isolates representing the EU and CH2 genotypes have been found. The isolates belonging to the CH2 genotype induce a wide range of symptoms from very mild mosaic or even a lack of symptoms, through severe yellowing, up to necrosis of leaves. Therefore, the characterization of different genetic variants of PepMV is important to clarify processes of disease emergence caused by this virus. The necrotic and yellowing pathotypes representing the Chilean 2 genotype have been extensively studied. Development of infectious clones facilitates manipulation of the viral genome by site-directed mutagenesis. The studies performed made it possible to identify the viral genetic determinants responsible for the induction of different symptoms on infected plants. The introduction of single nucleotide substitutions in particular positions of triple gene block 3 (TGB3) resulted in changes of symptoms on tomato plants. Moreover, the mutations affect significantly viral accumulation, which has been measured by real-time RT-PCR. Single nucleotide substitutions introduced to the genome of genetically different European isolates also had an effect on biological properties and accumulation of the virus. Identified mutations in the viral genome are responsible for the development of symptoms and virus accumulation, and may play an important role during infection.

Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae

CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we report the development and successful application of a multiplex CRISPR/Cas9 system for genome engineering of up to 5 different genomic loci in one transformation step in baker's yeast Saccharomyces cerevisiae. To assess the specificity of the tool we employed genome re-sequencing to screen for off-target sites in all single knock-out strains targeted by different gRNAs. This extensive analysis identified no more genome variants in CRISPR/Cas9 engineered strains compared to wild-type reference strains. We applied our genome engineering tool for an exploratory analysis of all possible single, double, triple, quadruple and quintuple gene disruption combinations to search for strains with high mevalonate production, a key intermediate for the industrially important isoprenoid biosynthesis pathway. Even though we did not overexpress any genes in the mevalonate pathway, this analysis identified strains with mevalonate titers greater than 41-fold compared to the wild-type strain. Our findings illustrate the applicability of this highly specific and efficient multiplex genome engineering approach to accelerate functional genomics and metabolic engineering efforts.

Molecular evolution of Pepino mosaic virus during long‐term passaging in different hosts and its impact on virus virulence

AbstractIn this study the effect of host changes and multiple passages on Pepino mosaic virus (PepMV) evolution was analysed. A population of a mild isolate of PepMV was used to generate five independent evolution lineages on three tomato cultivars, which differ in rate of appearance of symptoms and their severity during viral infection (Beta Lux, Moneymaker and Malinowy Ożarowski) and on Datura inoxia. Twenty serial passages were performed over a period of 217–220 days. Symptom severity was monitored along the entire experiment. After the last series of passages total RNAs from each lineage and host were isolated and the triple gene block 3 (TGB3) and coat protein (CP) were amplified, cloned and 10 clones for each gene sequenced. Among the 400 clones for both genes, 143 individual mutations (61 synonymous and 82 nonsynonymous) were identified, with the largest number of nonsynonymous mutations being observed for the tomato cultivars Malinowy Ożarowski and Beta Lux. In two of the lineages evolving in the most susceptible variety of tomato (Beta Lux) necrotic changes in leaf blades appeared after 17 passages, leading to death of the plants. In these two lineages the mutation responsible for necrotic symptoms was K67E in TGB3. The appearance of this convergent mutation in independently evolving lineages may suggest that selection in this experimental set up favours more aggressive PepMV variants. We found a positive association between the severity of symptoms and the amount of genetic variability contained on viral populations. Indeed, the severity of symptoms turned out to be a good predictor for several indices of molecular variability. In addition, mapping all observed mutations in CP and TGB3 protein structures revealed that most were located on the surface, indicating a possible implication in viral–viral or viral–host interactions.

Importin-α-mediated nucleolar localization of potato mop-top virus TRIPLE GENE BLOCK1 (TGB1) protein facilitates virus systemic movement, whereas TGB1 self-interaction is required for cell-to-cell movement in Nicotiana benthamiana.

Recently, it has become evident that nucleolar passage of movement proteins occurs commonly in a number of plant RNA viruses that replicate in the cytoplasm. Systemic movement of Potato mop-top virus (PMTV) involves two viral transport forms represented by a complex of viral RNA and TRIPLE GENE BLOCK1 (TGB1) movement protein and by polar virions that contain the minor coat protein and TGB1 attached to one extremity. The integrity of polar virions ensures the efficient movement of RNA-CP, which encodes the virus coat protein. Here, we report the involvement of nuclear transport receptors belonging to the importin-α family in nucleolar accumulation of the PMTV TGB1 protein and, subsequently, in the systemic movement of the virus. Virus-induced gene silencing of two importin-α paralogs in Nicotiana benthamiana resulted in significant reduction of TGB1 accumulation in the nucleus, decreasing the accumulation of the virus progeny in upper leaves and the loss of systemic movement of RNA-CP. PMTV TGB1 interacted with importin-α in N. benthamiana, which was detected by bimolecular fluorescence complementation in the nucleoplasm and nucleolus. The interaction was mediated by two nucleolar localization signals identified by bioinformatics and mutagenesis in the TGB1 amino-terminal domain. Our results showed that while TGB1 self-interaction is needed for cell-to-cell movement, importin-α-mediated nucleolar targeting of TGB1 is an essential step in establishing the efficient systemic infection of the entire plant. These results enabled the identification of two separate domains in TGB1: an internal domain required for TGB1 self-interaction and cell-to-cell movement and the amino-terminal domain required for importin-α interaction in plants, nucleolar targeting, and long-distance movement.

A Triple Gene Mutant of BoHV-1 Administered Intranasally in Lambs Replicates Efficiently in the Nasal Epithelium and Induces Neutralizing Antibody

Bovine herpes virus 1 (BoHV-1) causes respiratory infections and abortions in cattle, and is an important component of Bovine respiratory disease complex (BRDC). BoHV-1 has also been isolated from sheep with respiratory disorder. Experimentally, sheep and goats are infected productively with BoHV-1 after intranasal inoculation and they generate immune response against BoHV-1. We have engineered a triple gene- mutated bovine herpes virus type 1 (BoHV-1 tmv) vaccine virus which is protective against BoHV-1 in calves. In the present study, we have tested BoHV-1 tmv infectivity and serum neutralizing antibody titers in lambs after intranasal inoculation. The results showed that BoHV-1 tmv replicated well in the nasal mucosa with moderate titers for 7 days and the infected lambs generated serum neutralizing titers.

A novel virus transmitted through pollination causes ring-spot disease on gentian (Gentiana triflora) ovaries.

In this study, we identified a novel virus from gentian (Gentiana triflora) that causes ring-spots on ovaries. Furthermore, the virus causes unusual symptoms, ring-spots that appear specifically on the outer surface of the ovarian wall after pollination. Pollen grains carrying the virus were used to infect host plants by hand-pollination. RNA extracted from purified virions indicated that the virus had two segments, RNA1 and RNA2. The full-length cDNA sequence indicated that RNA1 had two ORFs: ORF1 had methyltransferase and helicase motifs, and ORF2 had an RNA-dependent RNA polymerase motif. RNA2 had five ORFs encoding a coat protein, triple gene block proteins 1-3 and a cysteine-rich protein. The length of RNA1 was 5519 bases and that of RNA2 was 3810 bases not including a polyU/polyA region between the first and second ORFs. Viral RNA does not have a polyA tail at the 3' end. Sequence similarity and phylogenetic analysis suggested that the virus is closely related to members of the genera Pecluvirus and Hordeivirus but distinct from them. These combined results suggest that the causal agent inducing ring-spot symptoms on gentian ovaries is a new virus belonging to the family Virgaviridae but not to any presently known genus. We tentatively name the virus gentian ovary ring-spot virus.

Novel triple fusion reporter gene for labeling and tracking human mesenchymal stem cells in vitro

Objective To evaluate the feasibility of labeling and tracing human bone marrow mesenchymal stem cells (hMSC) by a triple fusion reporter gene imaging.Methods A lentivirus vector,the pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP,which contained optical reporter gene-enhanced green fluorescent protein (EGFP),bioluminescent reporter gene-luciferase2 and MR reporter gene-ferritin,was constructed by gateway technology.After the accuracy of gene sequence was validated,the lentivirus was packaged and infected hMSC,and a stable transformed hMSC cell line was established by fluorescence-activated cell sorter.The expression of EGFP was inspected with fluoresence microscope; the bioluminescence photon number was counted by optical imaging system in 1 s after D-luciferin was given; the iron content of the cells was detected by prussian blue iron stain; T2 values of the T2-mapping of MRI were measured.After an extended series of 3 to 5 times repeated experiment,results were compared between transfected hMSC and un-transfected hMSC to explore the efficiency of the labeling and tracking hMSC of the reporter genes.The positive rate of expression of the EGFP,the difference in iron stain and MR signal (T2) between the two groups was measured by t test.The bioluminescence photon detected in two gradient groups was count to set a Pearson correlation analysis.Results The gene sequence of pLenti7.3-ferritin-IRES-luciferase2-SV40-EGFP was confirmed by DNA sequencing analysis.The lentivirus vector was successfully constructed and infected hMSC.A stable transformed hMSC cell line,with triple fusion reporter genes,was successfully established for the follow-up test.Single hMSC expressed EGFP could be detected by fluorescence microscope.The optical signal of 2.00× 106,1.00× 106,5.00× 105,2.50× 105,1.25 × 102,6.25 × 104/ml cells could be detected as 7.972× 107,3.061 × 107,1.547 × 107,7.805 × 106,4.256 × 106,1.411 × 106 bioluminescence photon after D-luciferin was given,and there was linear positive correlation between the intensity of optical signal and the number of cells (r=0.993,P＜0.01),while no optical signal was captured under the same experimental condition.The iron stain rate of transfection cells was (81.6± 5.1)％,which was significantly higher than that of un-transfection cells (21.6±3.6)％ (t=20.97,P＜0.01).MR showed the difference of T2 value of 2.5× 106 cells per milliliter between the transfection (628.0±23.1)ms andun-transfection cells (646.5±17.2)ms,but the difference was not significant (t=1.286,P=0.246).However,the difference was obvious when ammonium ferric citrate was given,(488.3± 10.7)ms in the transfeetion cells and (520.8 ± 21.0)ms in the un-transfected cells (t=2.76,P=0.033).Conclusion A triple fusion reporter gene imaging,including EGFP,luciferase2 and ferritin,is feasible,which can successfully be transfected into hMSC and effectively expressed in the daughter cells. Key words: Human bone marrow mesenchymal stem cells ; Gene imaging; Comparative study

A Next-generation Genetically Attenuated Plasmodium falciparum Parasite Created by Triple Gene Deletion

Immunization with live-attenuated Plasmodium sporozoites completely protects against malaria infection. Genetic engineering offers a versatile platform to create live-attenuated sporozoite vaccine candidates. We previously generated a genetically attenuated parasite (GAP) by deleting the P52 and P36 genes in the NF54 wild-type (WT) strain of Plasmodium falciparum (Pf p52(-)/p36(-) GAP). Preclinical assessment of p52(-)/p36(-) GAP in a humanized mouse model indicated an early and severe liver stage growth defect. However, human exposure to >200 Pf p52(-)/p36(-) GAP-infected mosquito bites in a safety trial resulted in peripheral parasitemia in one of six volunteers, revealing that this GAP was incompletely attenuated. We have now created a triple gene deleted GAP by additionally removing the SAP1 gene (Pf p52(-)/p36(-)/sap1(-) GAP) and employed flippase (FLP)/flippase recognition target (FRT) recombination for drug selectable marker cassette removal. This next-generation GAP was indistinguishable from WT parasites in blood stage and mosquito stage development. Using an improved humanized mouse model transplanted with human hepatocytes and human red blood cells, we show that despite a high-dose sporozoite challenge, Pf p52(-)/p36(-)/sap1(-) GAP did not transition to blood stage infection and appeared to be completely attenuated. Thus, clinical testing of Pf p52(-)/p36(-)/sap1(-) GAP assessing safety, immunogenicity, and efficacy against sporozoite challenge is warranted.

Expressional Comparison Between Episomal and Stable Transfection of a Selected tri-fusion Protein in Leishmania tarentolae

Introduction: Leishmania tarentolae (L. tarentolae ) is a nonpathogenic species of Leishmania genus that can be used as an expression system to produce immunogenic proteins or epitopes in vivo acting as an efficient and safe recombinant live vector in vaccinology. Cysteine proteases (CPs) are one group of Family C1 peptidases in Leishmania that are important for survival of the parasite within the host and are introduced as potential vaccine candidates. Two types of cysteine proteases, CPA and CPB, play a critical role in Leishmania . Previously, it has indicated that immunization with two genes or recombinant proteins of CPA and CPB individually or fused together with various adjuvant or combined with liposome are able to elicit a protective immune response against L. major in BALB/c mice. Methods: In this study, for the first time, two lines of recombinant nonpathogenic Leishmania were generated by transfection of a heterologous triple fusion gene encoding cpa/cpb/egfp . For this purpose, two different expressions approaches were taken; episomal expression using rDNA promoter, and integrative expression from rRNA locus of genome. Results: After genotype confirmation, the fluorescence intensity was monitored in different phases of parasite growth by both fluorescence microscopy and FACS analysis.Western blot and RT-PCR analysis showed that cpa/cpb/egfp tri-fused genes were specifically expressed in both lines of transgenic parasites. Discussion: In this study a single fused gene was expressed in both systems and the results showed that the expression of integrated gene was higher than episomal. In addition, the present data suggest that L. tarentolae expressing cpa/cpb/egfp is a promising carrier as vectored vaccine in future research. Vac Res , 2014, 1 (1): 9 Pages

A triple gene mutant of BoHV-1 administered intranasally is significantly more efficacious than a BoHV-1 glycoprotein E-deleted virus against a virulent BoHV-1 challenge

Bovine herpesvirus 1 (BoHV-1) causes respiratory infections and abortions in cattle, and is an important component of bovine respiratory disease complex, which causes a considerable economic loss worldwide. Several efforts have been made to produce safer and more effective vaccines. One of these vaccines is a glycoprotein E (gE)-deleted marker vaccine which is currently mandated for use in EU countries. In the present study, we have constructed a three-gene-mutated BoHV-1 vaccine virus (UL49.5 luminal domain residues 30–32 and cytoplasmic tail residues 80–96 deleted, gE cytoplasmic tail- and entire Us9-deleted) and compared its protective vaccine efficacy in calves after intranasal vaccination with that of a gE-deleted virus. Following vaccination, both the triple mutant and gE-deleted vaccine virus replicated well in the nasal epithelium of the calves. The vaccinated calves did not show any clinical signs. Four weeks post-vaccination, the animals were challenged intranasally with a virulent BoHV-1 wild-type virus. Based on clinical signs, both the gE-deleted and triple mutant group were protected equally against the virulent BoHV-1 challenge. However, based on the quantity and duration of nasal viral shedding, virus neutralizing antibody and cellular immune responses, the triple mutant virus vaccine induced a significantly better protective immune response than the gE-deleted virus vaccine. Notably, after the virulent BoHV-1 challenge, the triple mutant virus vaccinated group cleared the challenge virus three days earlier than the BoHV-1 gE-deleted virus vaccinated group.

Intraperitoneal administration of a tumor-associated antigen SART3, CD40L, and GM-CSF gene-loaded polyplex micelle elicits a vaccine effect in mouse tumor models.

Polyplex micelles have demonstrated biocompatibility and achieve efficient gene transfection in vivo. Here, we investigated a polyplex micelle encapsulating genes encoding the tumor-associated antigen squamous cell carcinoma antigen recognized by T cells-3 (SART3), adjuvant CD40L, and granulocyte macrophage colony-stimulating factor (GM-CSF) as a DNA vaccine platform in mouse tumor models with different types of major histocompatibility antigen complex (MHC). Intraperitoneally administrated polyplex micelles were predominantly found in the lymph nodes, spleen, and liver. Compared with mock controls, the triple gene vaccine significantly prolonged the survival of mice harboring peritoneal dissemination of CT26 colorectal cancer cells, of which long-term surviving mice showed complete rejection when re-challenged with CT26 tumors. Moreover, the DNA vaccine inhibited the growth and metastasis of subcutaneous CT26 and Lewis lung tumors in BALB/c and C57BL/6 mice, respectively, which represent different MHC haplotypes. The DNA vaccine highly stimulated both cytotoxic T lymphocyte and natural killer cell activities, and increased the infiltration of CD11c+ DCs and CD4+/CD8a+ T cells into tumors. Depletion of CD4+ or CD8a+ T cells by neutralizing antibodies deteriorated the anti-tumor efficacy of the DNA vaccine. In conclusion, a SART3/CD40L+GM-CSF gene-loaded polyplex micelle can be applied as a novel vaccine platform to elicit tumor rejection immunity regardless of the recipient MHC haplotype.

Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.

Hypoxia-targeted triple suicide gene therapy radiosensitizes human colorectal cancer cells.

The hypoxic microenvironment, an important feature of human solid tumors but absent in normal tissue, may provide an opportunity for cancer-specific gene therapy. The purpose of the present study was to investigate whether hypoxia-driven triple suicide gene TK/CD/UPRT expression enhances cytotoxicity to ganciclovir (GCV) and 5-fluorocytosine (5-FC), and sensitizes human colorectal cancer to radiation in vitro and in vivo. Stable transfectant of human colorectal HCT8 cells was established which expressed hypoxia-inducible vectors (HRE-TK/eGFP and HRE-CD/UPRT/mDsRed). Hypoxia-induced expression/function of TK, CD and UPRT was verified by western blot analysis, flow cytometry, fluorescent microscopy and cytotoxicity assay of GCV and 5-FC. Significant radiosensitization effects were detected after 5-FC and GCV treatments under hypoxic conditions. In the tumor xenografts, the distribution of TK/eGFP and CD/UPRT/mDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC and GCV in mice in combination with local irradiation resulted in tumor regression, as compared with prodrug or radiation treatments alone. Our data suggest that the hypoxia-inducible TK/GCV+CDUPRT/5-FC triple suicide gene therapy may have the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor.

RNA silencing plays an important antiviral role in plants and invertebrates. To counteract antiviral RNA silencing, most plant viruses have evolved viral suppressors of RNA silencing (VSRs). TRIPLE GENE BLOCK PROTEIN1 (TGBp1) of potexviruses is a well-characterized VSR, but the detailed mechanism by which it suppresses RNA silencing remains unclear. We demonstrate that transgenic expression of TGBp1 of plantago asiatica mosaic virus (PlAMV) induced developmental abnormalities in Arabidopsis thaliana similar to those observed in mutants of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) required for the trans-acting small interfering RNA synthesis pathway. PlAMV-TGBp1 inhibits SGS3/RDR6-dependent double-stranded RNA synthesis in the trans-acting small interfering RNA pathway. TGBp1 interacts with SGS3 and RDR6 and coaggregates with SGS3/RDR6 bodies, which are normally dispersed in the cytoplasm. In addition, TGBp1 forms homooligomers, whose formation coincides with TGBp1 aggregation with SGS3/RDR6 bodies. These results reveal the detailed molecular function of TGBp1 as a VSR and shed new light on the SGS3/RDR6-dependent double-stranded RNA synthesis pathway as another general target of VSRs.

Correction: Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production

Correction: Triple Immunoglobulin Gene Knockout Transchromosomic Cattle: Bovine Lambda Cluster Deletion and Its Effect on Fully Human Polyclonal Antibody Production