scholarly journals Generation of Synthetic Severe Acute Respiratory Syndrome Coronavirus Pseudoparticles: Implications for Assembly and Vaccine Production

2004 ◽  
Vol 78 (22) ◽  
pp. 12557-12565 ◽  
Author(s):  
Yue Huang ◽  
Zhi-yong Yang ◽  
Wing-pui Kong ◽  
Gary J. Nabel
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Buoyant Density ◽  
Open Reading Frames ◽  
Expression Vectors ◽  
Gene Encoding ◽  
Transmission Electron ◽  
Genes Encoding ◽  
Necessary And Sufficient ◽  
N Proteins

ABSTRACT The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) contains four structural genes, two replicase-transcriptase open reading frames, and more than five potential genes of unknown function. Despite this relative simplicity, the molecular regulation of SARS-CoV replication and assembly is not understood. Here, we report that two viral genes, encoding the SARS-CoV membrane (M) and nucleocapsid (N) proteins, are necessary and sufficient for formation of virus-like particles. Expression vectors encoding these two proteins were synthesized by using preferred human codons. When M and N expression plasmids were cotransfected into human 293 renal epithelial cells, pseudoparticles formed readily. The addition of a third gene, encoding the spike (S) glycoprotein, facilitated budding of particles that contained a corona-like halo resembling SARS-CoV when examined by transmission electron microscopy, with a buoyant density characteristic of coronaviruses. Specific biochemical interactions of these proteins were also shown in vitro. The S, M, and N proteins of the SARS-CoV are, therefore, necessary and sufficient for pseudovirus assembly. These findings advance the understanding of the morphogenesis of SARS-CoV and enable the generation of safe, conformational mimetics of the SARS virus that may facilitate the development of vaccines and antiviral drugs.

scholarly journals Ferrous Iron- and Sulfur-Induced Genes in Sulfolobus metallicus

2007 ◽  
Vol 73 (8) ◽  
pp. 2491-2497 ◽  
Author(s):  
Stephan Bathe ◽  
Paul R. Norris
Keyword(s):  
Reverse Transcription ◽  
Ferrous Iron ◽  
Subtractive Hybridization ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Induced Genes ◽  
Reading Frames ◽  
Sulfur Oxygenase Reductase

ABSTRACT Genes of Sulfolobus metallicus that appeared to be upregulated in relation to growth on either ferrous iron or sulfur were identified using subtractive hybridization of cDNAs. The genes upregulated during growth on ferrous iron were found in a cluster, and most were predicted to encode membrane proteins. Quantitative reverse transcription-PCR of cDNA showed upregulation of most of these genes during growth on ferrous iron and pyrite compared to results during growth on sulfur. The highest expression levels observed included those for genes encoding proteins with similarities to cytochrome c oxidase subunits and a CbsA-like cytochrome. The genes identified here that may be involved in oxidation of ferrous iron by S. metallicus are termed fox genes. Of three available genomes of Sulfolobus species (S. tokodaii, S. acidocaldarius, and S. solfataricus), only that of S. tokodaii has a cluster of highly similar open reading frames, and only S. tokodaii of these three species was also able to oxidize ferrous iron. A gene encoding sulfur oxygenase-reductase was identified as the source of the dominant transcript in sulfur-grown cells of S. metallicus, with the predicted protein showing high identities to the previously described examples from S. tokodaii and species of Acidianus.

scholarly journals Construction and Characterization of Shuttle Vectors for Succinic Acid-Producing Rumen Bacteria

2007 ◽  
Vol 73 (17) ◽  
pp. 5411-5420 ◽  
Author(s):  
Yu-Sin Jang ◽  
Young Ryul Jung ◽  
Sang Yup Lee ◽  
Ji Mahn Kim ◽  
Jeong Wook Lee ◽  
...  
Keyword(s):  
Succinic Acid ◽  
Shuttle Vector ◽  
Green Fluorescence Protein ◽  
Expression Vectors ◽  
Rumen Bacteria ◽  
Gene Encoding ◽  
Shuttle Vectors ◽  
Genes Encoding ◽  
Fluorescence Protein

ABSTRACT Shuttle vectors carrying the origins of replication that function in Escherichia coli and two capnophilic rumen bacteria, Mannheimia succiniciproducens and Actinobacillus succinogenes, were constructed. These vectors were found to be present at ca. 10 copies per cell. They were found to be stably maintained in rumen bacteria during the serial subcultures in the absence of antibiotic pressure for 216 generations. By optimizing the electroporation condition, the transformation efficiencies of 3.0 × 106 and 7.1 × 106 transformants/μg DNA were obtained with M. succiniciproducens and A. succinogenes, respectively. A 1.7-kb minimal replicon was identified that consists of the rep gene, four iterons, A+T-rich regions, and a dnaA box. It was found that the shuttle vector replicates via the theta mode, which was confirmed by sequence analysis and Southern hybridization. These shuttle vectors were found to be suitable as expression vectors as the homologous fumC gene encoding fumarase and the heterologous genes encoding green fluorescence protein and red fluorescence protein could be expressed successfully. Thus, the shuttle vectors developed in this study should be useful for genetic and metabolic engineering of succinic acid-producing rumen bacteria.

scholarly journals Identification of Genes Encoding Exported Mycobacterium tuberculosis Proteins Using a Tn552′phoA In Vitro Transposition System

2000 ◽  
Vol 182 (10) ◽  
pp. 2732-2740 ◽  
Author(s):  
Miriam Braunstein ◽  
Thomas J. Griffin ◽  
Jordan I. Kriakov ◽  
Sarah T. Friedman ◽  
Nigel D. F. Grindley ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protective Immunity ◽  
Cell Envelope ◽  
Open Reading Frames ◽  
Signal Peptides ◽  
Genomic Libraries ◽  
Target Dna ◽  
Genes Encoding ◽  
Associated Proteins

ABSTRACT Secreted and cell envelope-associated proteins are important to both Mycobacterium tuberculosis pathogenesis and the generation of protective immunity to M. tuberculosis. We used an in vitro Tn552′phoA transposition system to identify exported proteins of M. tuberculosis. The system is simple and efficient, and the transposon inserts randomly into target DNA. M. tuberculosis genomic libraries were targeted with Tn552′phoA transposons, and these libraries were screened in M. smegmatis for active PhoA translational fusions. Thirty-two different M. tuberculosis open reading frames were identified; eight contain standard signal peptides, six contain lipoprotein signal peptides, and seventeen contain one or more transmembrane domains. Four of these proteins had not yet been assigned as exported proteins in the M. tuberculosisdatabases. This collection of exported proteins includes factors that are known to participate in the immune response of M. tuberculosis and proteins with homologies, suggesting a role in pathogenesis. Nine of the proteins appear to be unique to mycobacteria and represent promising candidates for factors that participate in protective immunity and virulence. This technology of creating comprehensive fusion libraries should be applicable to other organisms.

scholarly journals The Open Reading Frame 3a Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Promotes Membrane Rearrangement and Cell Death

2009 ◽  
Vol 84 (2) ◽  
pp. 1097-1109 ◽  
Author(s):  
Eric C. Freundt ◽  
Li Yu ◽  
Cynthia S. Goldsmith ◽  
Sarah Welsh ◽  
Aaron Cheng ◽  
...  
Keyword(s):  
Cell Death ◽  
Severe Acute Respiratory Syndrome ◽  
Vero Cells ◽  
Small Gtpase ◽  
Open Reading Frames ◽  
Accessory Proteins ◽  
Reading Frame ◽  
Golgi Fragmentation

ABSTRACT The genome of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains eight open reading frames (ORFs) that encode novel proteins. These accessory proteins are dispensable for in vitro and in vivo replication and thus may be important for other aspects of virus-host interactions. We investigated the functions of the largest of the accessory proteins, the ORF 3a protein, using a 3a-deficient strain of SARS-CoV. Cell death of Vero cells after infection with SARS-CoV was reduced upon deletion of ORF 3a. Electron microscopy of infected cells revealed a role for ORF 3a in SARS-CoV induced vesicle formation, a prominent feature of cells from SARS patients. In addition, we report that ORF 3a is both necessary and sufficient for SARS-CoV-induced Golgi fragmentation and that the 3a protein accumulates and localizes to vesicles containing markers for late endosomes. Finally, overexpression of ADP-ribosylation factor 1 (Arf1), a small GTPase essential for the maintenance of the Golgi apparatus, restored Golgi morphology during infection. These results establish an important role for ORF 3a in SARS-CoV-induced cell death, Golgi fragmentation, and the accumulation of intracellular vesicles.

scholarly journals Construction of artificial microRNA expression vectors for inhibition of Minc16281 gene in root-knot nematode Meloidogyne incognita

2019 ◽  
Vol 18 (4) ◽  
pp. 62-69
Author(s):  
Phong V. Nguyen
Keyword(s):  
Meloidogyne Incognita ◽  
Parasitic Nematode ◽  
Expression Vectors ◽  
Parasitic Nematodes ◽  
Plant Parasitic Nematodes ◽  
Root Knot Nematode ◽  
Gene Encoding ◽  
Soybean Field ◽  
Genes Encoding ◽  
Plant Parasitic

Effectors have been identified to play a very important role in the parasitism of plant-parasitic nematode. To cope with this type of pathogen, many approaches of silencing genes encoding for effectors have been studied and promise to be an effective tool to create plant varieties resistant to plant-parasitic nematodes. In this study, the Minc16281 gene encoding a pioneer effector with unknown function was determined and cloned from a Meloidogyne incognita population isolated from soybean field (ID: MH315945.1). The nucleotide sequence of this gene showed 97% identity to its homolog in GenBank (ID: JK287445.1) used as the control strain in our research. To generate host-induced gene silencing constructs which can potentially silence the expression of Minc16281 gene, two artificial microRNAs were synthesized based on the miR319a structure of Arabidopsis thaliana and inserted into an expression vector in soybean. These microRNAs can be introduced into soybean to investigate the function of Minc16281 on parasitism of root-knot nematode.

scholarly journals Physiological Control and Regulation of the Rhodobacter capsulatus cbb Operons

1998 ◽  
Vol 180 (16) ◽  
pp. 4258-4269 ◽  
Author(s):  
George C. Paoli ◽  
Padungsri Vichivanives ◽  
F. Robert Tabita
Keyword(s):  
Rhodobacter Capsulatus ◽  
Pentose Phosphate ◽  
Open Reading Frames ◽  
Physiological Control ◽  
Gene Encoding ◽  
Bisphosphate Carboxylase ◽  
Promoter Fusion ◽  
Fructose 1,6 Bisphosphatase ◽  
Mutant Strains ◽  
Genes Encoding

ABSTRACT The genes encoding enzymes of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway in Rhodobacter capsulatus are organized in at least two operons, each preceded by a separate cbbR gene, encoding potential LysR-type transcriptional activators. As a prelude to studies ofcbb gene regulation in R. capsulatus, the nucleotide sequence of a 4,537-bp region, which includedcbbR II, was determined. This region contained the following open reading frames: a partial pgmgene (encoding phosphoglucomutase) and a complete qorgene (encoding NADPH:quinone oxidoreductase), followed by cbbR II, cbbF (encoding fructose 1,6-bisphosphatase), cbbP (encoding phosphoribulokinase), and part of cbbT (encoding transketolase). Physiological control of the CBB pathway and regulation of the R. capsulatus cbb genes were studied by using a combination of mutant strains and promoter fusion constructs. Characterization of mutant strains revealed that either form I or form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), encoded by the cbbLS andcbbM genes, respectively, could support photoheterotrophic and autotrophic growth. A strain with disruptions in both cbbL and cbbM could not grow autotrophically and grew photoheterotrophically only when dimethyl sulfoxide was added to the culture medium. Disruption ofcbbP resulted in a strain that did not synthesize form II RubisCO and had a phenotype similar to that observed in the RubisCO-minus strain, suggesting that there is only onecbbP gene in R. capsulatus and that this gene is cotranscribed with cbbM. Analysis of RubisCO activity and synthesis in strains with disruptions in eithercbbR I orcbbR II, and β-galactosidase determinations from wild-type and mutant strains containing cbb Ip- andcbb IIp-lacZ fusion constructs, indicated that the cbb I andcbb II operons of R. capsulatus are within separate CbbR regulons.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Protein 6 Accelerates Murine Coronavirus Infections

2006 ◽  
Vol 81 (3) ◽  
pp. 1220-1229 ◽  
Author(s):  
Chandra Tangudu ◽  
Heidi Olivares ◽  
Jason Netland ◽  
Stanley Perlman ◽  
Thomas Gallagher
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Open Reading Frames ◽  
Viral Rna ◽  
Protein Accumulation ◽  
Sars Coronavirus ◽  
Viral Rnas ◽  
Growth Properties ◽  
Murine Coronavirus

ABSTRACT One or more of the unique 3′-proximal open reading frames (ORFs) of the severe acute respiratory syndrome (SARS) coronavirus may encode determinants of virus virulence. A prime candidate is ORF6, which encodes a 63-amino-acid membrane-associated peptide that can dramatically increase the lethality of an otherwise attenuated JHM strain of murine coronavirus (L. Pewe, H. Zhou, J. Netland, C. Tangudu, H. Olivares, L. Shi, D. Look, T. Gallagher, and S. Perlman, J. Virol. 79:11335-11342, 2005). To discern virulence mechanisms, we compared the in vitro growth properties of rJ.6, a recombinant JHM expressing the SARS peptide, with isogenic rJ.6-KO, which has an inactive ORF containing a mutated initiation codon and a termination codon at internal position 27. The rJ.6 infections proceeded rapidly, secreting progeny about 1.5 h earlier than rJ.6-KO infections did. The rJ.6 infections were also set apart by early viral protein accumulation and by robust expansion via syncytia, a characteristic feature of JHM virus dissemination. We found no evidence for protein 6 operating at the virus entry or assembly stage, as virions from either infection were indistinguishable. Rather, protein 6 appeared to operate by fostering viral RNA and protein synthesis, as RNA quantifications by reverse transcription-quantitative PCR revealed viral RNA levels in the rJ.6 cultures that were five to eight times higher than those lacking protein 6. Furthermore, protein 6 coimmunoprecipitated with viral RNAs and colocalized on cytoplasmic vesicles with replicating viral RNAs. The SARS coronavirus encodes a novel membrane protein 6 that can accelerate replication of a related mouse virus, a property that may explain its ability to increase in vivo virus virulence.

scholarly journals Identification of Substrates and Chaperone from the Yersinia enterocolitica 1B Ysa Type III Secretion System

2003 ◽  
Vol 71 (1) ◽  
pp. 242-253 ◽  
Author(s):  
Boris Foultier ◽  
Paul Troisfontaines ◽  
Didier Vertommen ◽  
Marie-Noëlle Marenne ◽  
Mark Rider ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Shigella Flexneri ◽  
Secretion System ◽  
Open Reading Frames ◽  
Target Cells ◽  
Type Iii ◽  
Genes Encoding ◽  
Reading Frames

ABSTRACT All pathogenic Yersinia enterocolitica strains carry the pYV plasmid encoding the Ysc-Yop type III secretion (TTS) system, which operates at 37°C. In addition, biovar 1B Y. enterocolitica strains possess a second, chromosomally encoded, TTS system called Ysa, which operates, at least in vitro, under low-temperature and high-salt (LTHS) conditions. Six open reading frames, sycB, yspB, yspC, yspD, yspA, and acpY, neighbor the ysa genes encoding the Ysa TTS apparatus. Here we show that YspA, YspB, YspC, and YspD are secreted by the Ysa TTS system under LTHS conditions. SycB is a chaperone for YspB and YspC and stabilizes YspB. YspB, YspC, and SycB share some similarity with TTS substrates and the chaperone encoded by the Mxi-Spa locus of Shigella flexneri and SPI-1 of Salmonella enterica. In addition, Ysa also secretes the pYV-encoded YopE under LTHS conditions, indicating that YopE is a potential effector of both Y. enterocolitica TTS systems. YspC could also be secreted by S. flexneri, but no functional complementation of ipaC was observed, which indicates that despite their similarity the Ysa and the Mxi-Spa systems are not interchangeable. When expressed from the yopE promoter, YspB and YspC could also be secreted via the Ysc injectisome. However, they could not form detectable pores in eukaryotic target cells and could not substitute for YopB and YopD for translocation of Yop effectors.

scholarly journals Colocation of Genes Encoding a tRNA-mRNA Hybrid and a Putative Signaling Peptide on Complementary Strands in the Genome of the Hyperthermophilic Bacterium Thermotoga maritima

2006 ◽  
Vol 188 (19) ◽  
pp. 6802-6807 ◽  
Author(s):  
Clemente I. Montero ◽  
Derrick L. Lewis ◽  
Matthew R. Johnson ◽  
Shannon B. Conners ◽  
Elizabeth A. Nance ◽  
...  
Keyword(s):  
Thermotoga Maritima ◽  
Normal Growth ◽  
Growth Conditions ◽  
Open Reading Frames ◽  
Transcriptional Responses ◽  
Gene Encoding ◽  
Hyperthermophilic Bacterium ◽  
Genes Encoding ◽  
Original Genome ◽  
Opposite Strand

ABSTRACT In the genome of the hyperthermophilic bacterium Thermotoga maritima, TM0504 encodes a putative signaling peptide implicated in population density-dependent exopolysaccharide formation. Although not noted in the original genome annotation, TM0504 was found to colocate, on the opposite strand, with the gene encoding ssrA, a hybrid of tRNA and mRNA (tmRNA), which is involved in a trans-translation process related to ribosome rescue and is ubiquitous in bacteria. Specific DNA probes were designed and used in real-time PCR assays to follow the separate transcriptional responses of the colocated open reading frames (ORFs) during transition from exponential to stationary phase, chloramphenicol challenge, and syntrophic coculture with Methanococcus jannaschii. TM0504 transcription did not vary under normal growth conditions. Transcription of the tmRNA gene, however, was significantly up-regulated during chloramphenicol challenge and in T. maritima bound in exopolysaccharide aggregates during methanogenic coculture. The significance of the colocation of ORFs encoding a putative signaling peptide and tmRNA in T. maritima is intriguing, since this overlapping arrangement (tmRNA associated with putative small ORFs) was found to be conserved in at least 181 bacterial genomes sequenced to date. Whether peptides related to TM0504 in other bacteria play a role in quorum sensing is not yet known, but their ubiquitous colocalization with respect to tmRNA merits further examination.

scholarly journals Identification of a Novel Dioxygenase Involved in Metabolism of o-Xylene, Toluene, and Ethylbenzene by Rhodococcus sp. Strain DK17

2004 ◽  
Vol 70 (12) ◽  
pp. 7086-7092 ◽  
Author(s):  
Dockyu Kim ◽  
Jong-Chan Chae ◽  
Gerben J. Zylstra ◽  
Young-Soo Kim ◽  
Seong-Ki Kim ◽  
...  
Keyword(s):  
Aromatic Ring ◽  
Single Point ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Sulfur Protein ◽  
Benzene Toluene ◽  
Iron Sulfur Protein ◽  
Dioxygenase Genes

ABSTRACT Rhodococcus sp. strain DK17 is able to grow on o-xylene, benzene, toluene, and ethylbenzene. DK17 harbors at least two megaplasmids, and the genes encoding the initial steps in alkylbenzene metabolism are present on the 330-kb pDK2. The genes encoding alkylbenzene degradation were cloned in a cosmid clone and sequenced completely to reveal 35 open reading frames (ORFs). Among the ORFs, we identified two nearly exact copies (one base difference) of genes encoding large and small subunits of an iron sulfur protein terminal oxygenase that are 6 kb apart from each other. Immediately downstream of one copy of the dioxygenase genes (akbA1a and akbA2a ) is a gene encoding a dioxygenase ferredoxin component (akbA3), and downstream of the other copy (akbA1b and akbA2b ) are genes putatively encoding a meta-cleavage pathway. RT-PCR experiments show that the two copies of the dioxygenase genes are operonic with the downstream putative catabolic genes and that both operons are induced by o-xylene. When expressed in Escherichia coli, AkbA1a-AkbA2a-AkbA3 transformed o-xylene into 2,3- and 3,4-dimethylphenol. These were apparently derived from an unstable o-xylene cis-3,4-dihydrodiol, which readily dehydrates. This indicates a single point of attack of the dioxygenase on the aromatic ring. In contrast, attack of AkbA1a-AkbA2a-AkbA3 on ethylbenzene resulted in the formation of two different cis-dihydrodiols resulting from an oxidation at the 2,3 and the 3,4 positions on the aromatic ring, respectively.

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