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Comparative analysis of the ability of the polymerase complexes of influenza viruses type A, B and C to assemble into functional RNPs that allow expression and replication of heterotypic model RNA templates in vivo.

Influenza viruses type A, B, and C are human pathogens that share common structural and functional features, yet they do not form natural reassortants. To determine to what extent type-specific interactions of the polymerase complex with template RNA contribute to this lack of genotypic mixing, we investigated whether homotypic or heterotypic polymerase complexes support the expression and replication of model type A, B, or C RNA templates in vivo. A plasmid-based expression system, as initially described by Pleschka et al. [(1996) J. Virol. 70, 4188-4192] for influenza A virus, was developed for influenza viruses B/Harbin/7/94 and C/Johannesburg/1/66. The type A core proteins expressed heterotypic model RNAs with similar efficiencies as the homotypic RNA. The influenza B virus model RNA was efficiently expressed by all three types of polymerase complexes. Although no functional polymerase complex could be reconstituted with heterotypic P protein subunits, when the influenza A virus P proteins were expressed together with heterotypic nucleoproteins, significant, albeit limited, expression of RNA templates of all influenza virus types was detected. Taken together, our results suggest that less strict type-specific interactions are involved for the polymerase complex of influenza A compared with influenza B or C viruses.

Animals↗

Expression, antigenicity and studies on cell receptor binding of the hemagglutinin of infectious salmon anemia virus.

Infectious salmon anemia (ISA) virus belongs to the proposed genus Isavirus of Orthomyxoviridae and is an emerging pathogen in Atlantic salmon (Salmo salar) farming. The hemagglutinin-esterase (HE) of ISA virus share several characteristics with the influenza virus hemagglutinin. This study reports recombinant expression of different ISA virus HE mutants in fish cell lines. Some introduced mutations, representing minimal differences in single amino acid residues, resulted in remarkable effects on expression efficiency in general and on surface expression specifically. Receptor binding assays showed that amino acid residues in the N-terminal half part are important in receptor binding, either being directly involved in the binding, or by influencing the structure of the binding site. Deletion of the putative N-glycosylation sites of the ISA virus HE, located near the transmembrane region, did not influence expression, receptor binding properties or staining by either a neutralising MAb, or salmon convalescent sera. The humoral immune response of Atlantic salmon after ISA virus infection showed weak neutralising activity and the results indicated that it was directed against HE.

Amino Acid Substitution↗

Development of an antibody-based assay for determination of baculovirus titers in 10 hours.

The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein. For an efficient expression of the target protein, it is necessary to infect insect cells with an adequate amount of virus. However, current methods are time-consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene. A novel method is developed to yield virus titers in 10 h that is easy to perform using 96-well plates and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immunostaining. The titer is determined by counting foci produced as a result of infection of the virus under a fluorescent microscope. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post-infection time of 4 h. Therefore, 10 h was enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer. Titers determined using this immunological assay are comparable, both in value and validity, to those obtained using a traditional method, provided that the stocks have titers above 10(3) pfu/mL.

Animals↗

Translational control of retroviruses.

All replication-competent retroviruses contain three main reading frames, gag, pol and env, which are used for the synthesis of structural proteins, enzymes and envelope proteins respectively. Complex retroviruses, such as lentiviruses, also code for regulatory and accessory proteins that have essential roles in viral replication. The concerted expression of these genes ensures the efficient polypeptide production required for the assembly and release of new infectious progeny virions. Retroviral protein synthesis takes place in the cytoplasm and depends exclusively on the translational machinery of the host infected cell. Therefore, not surprisingly, retroviruses have developed RNA structures and strategies to promote robust and efficient expression of viral proteins in a competitive cellular environment.

Animals↗

Nucleotide sequence and expression of the cloned gene of bacteriophage SP6 RNA polymerase.

The coding region of the gene for bacteriophage SP6 RNA polymerase was cloned into pBR322, and its entire nucleotide sequence was deduced. The predicted amino acid sequence for the polymerase consists of 874 amino acid residues with a total molecular weight of 98,561 daltons. Comparison of the amino acid sequence with that of T7 RNA polymerase reveals that regions with partial homology are present along the sequence. The coding region of SP6 RNA polymerase was inserted into an E. coli expression vector. The polymerase gene was efficiently expressed in E. coli cells, and the enzymatic properties of the expressed polymerase were very similar to those of the enzyme synthesized in SP6 phage-infected Salmonella typhimurium cells.

Amino Acid Sequence↗

Microphthalmia-associated transcription factor as a regulator for melanocyte-specific transcription of the human tyrosinase gene.

Tyrosinase is a rate-limiting enzyme in melanin biosynthesis and is specifically expressed in differentiated melanocytes. We have identified the enhancer element in the 5'-flanking region of the human tyrosinase gene that is responsible for its pigment cell-specific transcription and have termed it tyrosinase distal element (TDE) (positions -1861 to -1842). Transient expression assays showed that TDE confers efficient expression of a firefly luciferase reporter gene linked to the tyrosinase gene promoter in MeWo pigmented melanoma cells but not in HeLa cells, which do not express tyrosinase. TDE was specifically bound by nuclear proteins of MeWo and HeLa cells, the binding properties of which were indistinguishable in gel mobility shift assays. TDE contains the CATGTG motif in its center, and mutation analysis indicates that the CA dinucleotides of this motif are crucial for protein binding and pigment cell-specific enhancer function. The CATGTG motif is consistent with the consensus sequence recognized by a large family of transcription factors with a basic helix-loop-helix structure, which prompted us to examine the possible involvement of a ubiquitous transcription factor, USF, and a novel factor, microphthalmia-associated transcription factor (MITF), recently cloned as the human homolog of the mouse microphthalmia (mi) gene product. The mi phenotype is associated with a mutant mi locus and characterized by small eyes and loss of melanin pigments. Both USF and MITF are predicted to contain a basic helix-loop-helix structure and a leucine zipper structure. We provide evidence that USF binds to TDE, whereas we were unable to detect the DNA-binding activity of MITF. Transient coexpression assays showed that MITF specifically transactivates the promoter activity of the tyrosinase gene through the CATGTG motif of TDE but not the promoter of the ubiquitously expressed heme oxygenase gene, while USF is able to activate both promoters. These results indicate that MITF is a cell-type-specific factor that is capable of activating transcription of the tyrosinase gene.

Base Sequence↗

Comparison of the trans-activation capabilities of the human T-cell leukemia virus type I and II chi proteins.

The mechanism of cellular transformation by the human T-cell leukemia viruses (HTLVs) is thought to involve a novel retrovirus gene known as chi. The chi gene is essential for HTLV replication and acts by enhancing transcription from the viral long terminal repeat. By using the HTLV type I and II chi gene-coding regions inserted into a highly efficient expression vector, we directly compared the efficiencies of the two chi proteins to trans activate the HTLV type I and II long terminal repeats. We demonstrate that the two chi proteins have different patterns of trans activation. The patterns were highly reproducible in all mammalian cells tested. A different pattern of activation was observed in avian cells. These results suggest that the mechanism of trans activation involves specific cellular factors that are highly conserved throughout mammalian species but different in avian cells. Understanding the mechanism of trans activation by the chi gene product may provide insights into mechanisms of cellular transformation by HTLV.

Acetyltransferases↗

Effects of cell cycle status on the efficiency of liposome-mediated gene transfection in mouse fetal fibroblasts.

Methods for cell cycle synchronization of mouse fetal fibroblast cells (MFFCs) were first selected and optimized. When MFFCs were cooled at 5 C for different periods of time, the highest percentage of cells at the G0/G1 phase (75.4+/-2.9%), with 3.5+/-0.3% of apoptotic cells, was achieved after 5 h of treatment. Extended cooling increased the number of apoptotic cells significantly. When MFFCs were treated with different concentrations of roscovitine (ROS) for different periods of time, the highest percentage of G0/G1 cells (83.5+/-1.8%), with 9.2+/-0.6% apoptotic cells, was obtained after exposure to 10 microM ROS for 24 h. When the cells were cooled at 5 C for 5 h followed by incubation in 10 microM ROS for 12 h, 83.6+/-1.9% were synchronized at the G0/G1 stage, with 3.6% undergoing apoptosis. Cell cycle progression was then observed after release of the MFFCs from different synchronization blocks. The highest percentages of S and G2/M cells (81% and 75%) were achieved at 12 and 20 h, respectively, after release of the MFFCs from the cooling plus ROS treatment, and these percentages were significantly higher than those obtained after release from the cooling or ROS alone blocks. Finally, MFFCs were transfected with pEGFP-N1 plasmid at the peak of the G0/G1, S, and G2/M phases, respectively, after release from the different blocks and both the transient and stable transfection efficiencies were determined. The GFP gene expression was greatly enhanced when transfection was performed at the time when most cells were at the G2/M stage after release from cooling, ROS alone, and cooling plus ROS treatments. Statistical analysis revealed a close correlation between the rate of G2/M cells and the transient and stable GFP gene expression efficiencies. Together, the results indicated that (a) the best protocol for cell cycle synchronization of MFFCs was a 5-h cooling at 5 C followed by incubation in 10 microM ROS for 12 h which produced both a high rate of synchronization in the G0/G1 phase with acceptable apoptosis and a high rate of G2/M cells after release; and (b) that the cell cycle status had marked effects on the efficiency of liposome-mediated transfection in MFFCs, with the highest transfection efficiency obtained in cells at the G2/M stage.

Animals↗

Development of novel technology of DDS for gene therapy.

In the near future, not only "systemic pharmacokinetics" but also "intracellular pharmacokinetics" seems to be important in Drug Delivery System (DDS) research for gene therapy. Beyond the basic philosophy of DDS of "delivering the optimal amounts of drugs to a target site", it is now necessary to "express the gene (as a drug) efficiently in a target cell for a required period" in gene therapy. To achieve these objectives, vectors for introducing the gene into the target cell are being improved, and techniques to efficiently express the transgene and to regulate the transgene expression are being developed. DDS is expected to play a large part in achieving this goal. Here, we review a novel DDS technology to satisfy these criteria.

Journal Article↗

Establishment of an artificial beta-cell line expressing insulin under the control of doxycycline.

AIM: Artificial beta-cell lines may offer an abundant source of cells for the treatment of type I diabetes, but insulin secretion in beta-cells is tightly regulated in physiological conditions. The Tet-On system is a "gene switch" system, which can induce gene expression by administration of tetracycline (Tet) derivatives such as doxcycline (Dox). Using this system, we established 293 cells to an artificial cell line secreting insulin in response to stimulation by Dox. METHODS: The mutated proinsulin cDNA was obtained from plasmid pcDNA3.1/C-mINS by the polymerase chain reaction (PCR), and was inserted downstream from the promoter on the expression vector pTRE2, to construct a recombined expression vector pTRE2mINS. The promoter on pTRE2 consists of the tetracycline-response element and the CMV minimal promoter and is thus activated by the reverse tetracycline-controlled transactivator (rtTA) when Dox is administrated. pTRE2mINS and plasmid pTK-Hyg encoding hygromycin were co-transfected in the tet293 cells, which express rtTA stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Dox was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of Dox in culture medium on the expression of proinsulin gene was estimated respectively with Northern blot, RT-PCR, and radioimmunoassay. RESULTS: From the 28 hygromycin-resistant cell strains, we selected one cell strain (tet293/Ins6) secreting insulin not only automatically, but in response to stimulation by Dox. The amount on insulin secretion was dependent on the Dox dose (0,10,100,200,400,800 and 1000 microg.L(-1)), the level of insulin secreted by the cells treated with Dox (1000 microg.L(-1)) was 241.0pU.d(-1).cell(-1) , which was 25-fold that of 9.7pU.d(-1).cell(-1) without Dox treatment. Northern blot analyses and RT-PCR further confirmed that the transcription of insulin gene had already been up-regulated after exposing tet293/Ins6 cells to Dox for 15 minutes, and was also induced in a dose-dependent manner. However, the concentration of insulin in the media did not increase significantly until 5 hours following the addition of Dox. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability of gene therapy, suggesting that conditional establishment of artificial beta-cells may be a useful approach to develop cellular therapy for diabetes mellitus.

Cell Line↗

[Regulated expression of insulin gene in non-beta cell].

OBJECTIVE: To engineer 293 cells to an artificial beta cell line which secrets insulin in response to stimulation by doxycycline. METHODS: A recombined expression vector, pTRE2mINS, which contained both of the tetracycline response element and proinsulin gene, was constructed. This vector was co-transfected with plasmid pTK-Hyg encoding hygromycin in the tet293 cells, which express the reverse tetracycline-controlled transactivator stably. Following hygromycin screening, the survived cells expressing insulin were selected and enriched. Then, doxycycline one of tetracycline derivatives, was used to control the expression of insulin in these cells. At the levels of mRNA and protein, the regulating effect of doxycycline in culture medium on the expression of proinsulin gene was estimated respectively with Northern blotting, RT-PCR, and radioimmunoassay. RESULTS: Of the 28 hygromycin resistant cell strains, we selected one cell strain secreting insulin not only automatically, but in response to stimulation by doxycycline was selected. The cells secreted insulin at the rate of 9.7 U/24 h/1.0 x10(6) cells without doxycy cline treatment, and the amount of secreted insulin increased to 241.0 U/24 h/1.0 x 10(6) cells (25-fold) in the presence of doxycycline(1 000 ng/ml). I insulin secretion was induced by doxycycline in a dose dependent manner. CONCLUSION: Human proinsulin gene was transfected successfully and expressed efficiently in 293 cells, and the expression was modulated by tetracycline and its derivatives, improving the accuracy, safety, and reliability for the gene therapy of diabetes mellitus.

Cell Line↗

Expression of human papillomavirus type 6 L1 and L2 isolated in China and self assembly of virus-like particles by the products.

To study variations of genome late region of human papillomavirus type 6 (HPV-6) isolated in China and assembling capabilities of the encoded capsid proteins, HPV-6 L1 and L2 sequences were cloned and used for expression in Bac-to-Bac baculovirus expression systems (Gibco BRL). Based upon L1 and L2 overlapping sequence two sequences (GenBank accession number AY015006, AY015008) of HPV-6 late region (2869 bp long) were assembled and classified into HPV-6b by phylogenetic analysis. Compared with prototype sequence, nine point mutations were found, including four missense mutations. L1, instead of L2, could self-assemble into virus-like particles (VLPs) in Sf9 nucleus. VLPs self-assembled by L1 alone (L1-VLPs) and by L1 plus L2 (L1+L2-VLPs) were purified and further characterized. Both types of VLPs were spherical particles with a diameter of approximately 50 nm. L1+L2 VLPs comprising L1 and L2 in the molar ratio of about 4:1 possessed the HPV-6 L1 VLP surface and conformational epitopes. In co-expression assay with a series of MOI combination of L1 and L2 recombinant baculoviruses (total MOI=10), existence of L2 of certain level enhanced L1 production by 0.8 fold and VLP production by 3 - 4 folds under experimental conditions. In conclusion, variation rate of HPV-6 genome late region is less than 0.28% and the substitutions A to G at position 7081 and G to A at 7099 may represent region characteristics. The cloned HPV-6 L1 and L2 sequences can be expressed efficiently in Sf9 cells, and the expressed products (L1 or L1+L2) can self-assemble into VLPs that resemble naturally occurring virions.

Animals↗

[In-vivo tracing of bone marrow stromal cell differentiating into chondrocytes by green fluorescent protein gene transfection].

AIM: To achieve highly efficient, stable and long-term expression of green fluorescent protein(GFP) reporter gene in bone marrow stromal cells(BMSCs) and to trace directly the distribution and differentiation of BMSCs in articular cartilage defects. METHODS: Recombinant RV-GFP expression vector was constructed and transfected into packaging cell PT67. After G418 screening and amplification, cell clones producing high level recombinant viruses were obtained and in-vitro expanded. The virus supernatant from infected PT67 cell culture was used to infect BMSCs directly. Autologous GFP-labeled BMSCs were seeded onto biodegradable polymer and implanted into porcine articular cartilage defects. 7 months later, the repaired tissue was evaluated by confocal microscope. RESULTS: The constructed recombinant expression vector RV-GFP was identified by restriction digestion. The transfection rates of PT67 cells reached 20%-50%, which reached 100% after G418 screening, bright green fluorescence could be seen under fluorescence microscope. BMSCs could be transfected successfully by the culture supernatant from infected PT67 cells and GFP was expressed efficiently in a long-term period. Confocal microscope revealed that GFP-labeled cells existed in many neocartilage lacunae after 7 months. CONCLUSIONS: The recombinant vector RV-GFP can provide a simple, sensitive and reliable tool to label BMSCs which can be used to study cytologic dynamic follow-up in-vivo. BMSCs can differentiate into chondrocytes and play an important role in repairing articular cartilage defects.

Animals↗

Codon optimization for high-level expression of human erythropoietin (EPO) in mammalian cells.

Codon bias has been observed in many species. The usage of selective codons in a given gene is positively correlated with its expression efficiency. As an experimental approach to study codon-usage effects on heterologous gene expression in mammalian cells, we designed two human erythropoietin (EPO) genes, one in which native codons were systematically substituted with codons frequently found in highly expressed human genes and the other with codons prevalent in yeast genes. Relative performances of the re-engineered EPO genes were evaluated with various combinations of promoters and signal leader sequences. Under the comparable set of combinations, mature EPO gene with human high-frequency codons gave a considerably higher level of expression than that with yeast high-frequency codons. However, the levels of EPO expression varied, depending on the alternate combinations. Since the promoters and the signal leader sequences that we used are known to be equally efficient in gene expression, we hypothesized that the varied expression levels were due to the linear sequence between the promoter and the coding gene sequence. To test this possibility, we designed the EPO gene with hybrid codon usage in which the 5'-proximal region of the EPO gene was synthesized with yeast-biased codons and the rest with human-biased codons. This codon-usage hybrid EPO gene substantially enhanced the level of EPO transcripts and proteins up to 2.9-fold and 13.8-fold, respectively, when compared to the level reached by the original counterpart. Our results suggest that the linear sequence between the promoter and the 5'-proximal region of a gene plays an important role in achieving high-level expression in mammalian cells.

Amino Acid Sequence↗

ATM protein purified from vaccinia virus expression system: DNA binding requirements for kinase activation.

The ataxia-telangiectasia mutated (ATM) gene product plays a role in responding to double stand DNA breaks. Some biochemical studies of ATM function have been hampered by lack of an efficient expression system and abundant purified ATM protein. We report the construction of a vaccinia virus expressing ATM, vWR-ATM, which was used to produce large amounts of functional FLAG-tagged ATM protein (FLAG-ATM) in HeLa cells. Kinase activity of the purified FLAG-ATM was dependent on manganese and inhibited with wortmannin. Using the FLAG-ATM recombinant protein, GST-p53 serine 15 phosphorylation increased in the presence of damaged DNA. PHAS-1 phosphorylation was found to be DNA independent. Purified FLAG-ATM was recovered in the autophosphorylated form, as demonstrated by phosphorylation of ATM serine 1981. As shown by atomic force microscopy, FLAG-ATM bound to linear DNA both at broken ends and in mid-strands. Vaccinia virus is the most efficient ATM expression system described to date.

Ataxia Telangiectasia Mutated Proteins↗

Restriction fragment length polymorphism-coupled domain-directed differential display: a highly efficient technique for expression analysis of multigene families.

In this paper, a reverse-transcriptase PCR-based protocol suitable for efficient expression analysis of multigene families is presented. The method combines restriction fragment length polymorphism (RFLP) technology with a gene family-specific version of mRNA differential display and hence is called "RFLP-coupled domain-directed differential display. "With this method, expression of all members of a multigene family at many different developmental stages, in diverse tissues and even in different organisms, can be displayed on one gel. Moreover, bands of interest, representing gene family members, are directly accessible to sequence analysis, without the need for subcloning. The method thus enables a detailed, high-resolution expression analysis of known gene family members as well as the identification and characterization of new ones. Here the technique was used to analyze differential expression of MADS-box genes in male and female inflorescences of maize (Zea mays ssp. mays). Six different MADS-box genes could be identified, being either specifically expressed in the female sex or preferentially expressed in male or female inflorescences, respectively. Other possible applications of the method are discussed.

Amino Acid Sequence↗

RNA primary sequence or secondary structure in the translational initiation region controls expression of two variant interferon-beta genes in Escherichia coli.

Efficient expression in Escherichia coli (E. coli) of the human interferon-beta gene (IFN-beta) gene and of a chemically synthesized IFN-beta gene variant (506 base pairs; synIFN-beta) adapted to the E. coli codon usage, both fused to the E. coli atpE ribosome-binding site, is controlled either by primary sequence or by mRNA secondary-structure in the translational initiation region. High level expression of the natural human atpE/IFN-beta gene fusion is governed by the nucleotide composition preceding the initiator codon AUG. A single U----C exchange in the -2 or -1 position preceding the initiator codon AUG reduces the translational efficiency from 18% of total cellular protein to only 8% or 4%, respectively, while both U----C substitutions reduce IFN-beta expression below 1%. These sequence alterations interfere with efficient ribosome binding as revealed by toeprinting. They provide further evidence for the influence of the anticodon-flanking regions of tRNA(fMet) upon the initiation rate of translation. In contrast, translation of the synthetic variant atpE/synIFN-beta gene fusion is controlled by a moderately stable stem-loop structure (delta G = -4 kcal/mol; 37 degrees C) located within the coding region and overlapping the 30 S ribosomal subunit attachment site. That the stability of the hairpin interferes with the initiation of translation is inferred from site-directed mutagenesis and toeprint analyses. mRNA half-life in these variants is positively correlated with the rate of translation and involves two major endonucleolytic cleavage site 5'-upstream of the Shine-Dalgarno region.

Base Sequence↗

Structural features of the lac promoter affecting gusA expression in Lactobacillus casei.

With the aim of designing efficient expression systems for Lactobacillus casei, different factors affecting gene expression from the strong and highly modulated lac promoter have been systematically analyzed, by using the Escherichia coli beta-glucuronidase gene (gusA) as reporter. The activity of this enzyme (GUS) was quantified when plac:: gusA fusions were cloned in plasmids with different copy number or when gusA was inserted in the chromosomal lactose operon (single copy). Results showed a clear gene dosage effect and a positive influence of the native lac operon transcription and translation signals on GUS expression.

Amino Acid Sequence↗