scholarly journals Use of a Beet necrotic yellow vein virus RNA-5-derived replicon as a new tool for gene expression

2005 ◽  
Vol 86 (2) ◽  
pp. 463-467 ◽  
Author(s):  
Laure Schmidlin ◽  
Didier Link ◽  
Jérôme Mutterer ◽  
Hubert Guilley ◽  
David Gilmer
Keyword(s):  
Gene Expression ◽  
Recombinant Proteins ◽  
Expression System ◽  
Green Fluorescence Protein ◽  
Yellow Vein ◽  
Expression Levels ◽  
Virus Rna ◽  
New Gene ◽  
Infected Cells ◽  
Fluorescence Protein

A new gene-expression system based on RNA-5 of Beet necrotic yellow vein virus (BNYVV) was constructed to allow the expression of recombinant proteins in virally infected cells. Replication and expression levels of the RNA-5-based replicon containing the green fluorescence protein (GFP) gene were compared with those obtained with the well-characterized RNA-3-derived replicon (Rep-3). When RNA-3 and/or RNA-4 BNYVV RNAs were added to the inoculum, the expression levels of RNA-5-encoded GFP were considerably reduced. To a lesser extent, RNA-3-derived GFP expression was also affected by the presence of RNA-4 and -5. Both RNA-3- and RNA-5-derived molecules were able to express proteins within the same infected cells. Together with Rep-3, the RNA-5-derived replicon thus provides a new tool for the co-expression of different recombinant proteins. In Beta macrocarpa, Rep-5-GFP was able to move in systemic tissues in the presence of RNA-3 and thus provides a new expression system that is not restricted to the inoculated leaves.

scholarly journals A tunable anthranilate-inducible gene expression system for Pseudomonas species

2020 ◽  
Vol 105 (1) ◽  
pp. 247-258
Author(s):  
Lena Hoffmann ◽  
Michael-Frederick Sugue ◽  
Thomas Brüser
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Ant System ◽  
Plant Host ◽  
Inducible Gene Expression ◽  
Gene Expression System ◽  
Expression Levels ◽  
Key Points ◽  
Wide Range ◽  
Inducible Gene

Abstract Pseudomonads are among the most common bacteria in soils, limnic ecosystems, and human, animal, or plant host environments, including intensively studied species such as Pseudomonas aeruginosa, P. putida, or P. fluorescens. Various gene expression systems are established for some species, but there is still a need for a simple system that is suitable for a wide range of pseudomonads and that can be used for physiological applications, i.e., with a tuning capacity at lower expression levels. Here, we report the establishment of the anthranilate-dependent PantA promoter for tunable gene expression in pseudomonads. During studies on P. fluorescens, we constructed an anthranilate-inducible AntR/PantA-based expression system, named pUCP20-ANT, and used GFP as reporter to analyze gene expression. This system was compared with the rhamnose-inducible RhaSR/PrhaB-based expression system in an otherwise identical vector background. While the rhamnose-inducible system did not respond to lower inducer concentrations and always reached high levels over time when induced, expression levels of the pUCP20-ANT system could be adjusted to a range of distinct lower or higher levels by variation of anthranilate concentrations in the medium. Importantly, the anthranilate-inducible expression system worked also in strains of P. aeruginosa and P. putida and therefore will be most likely useful for physiological and biotechnological purposes in a wide range of pseudomonads. Key points • We established an anthranilate-inducible gene expression system for pseudomonads. • This system permits tuning of gene expression in a wide range of pseudomonads. • It will be very useful for physiological and biotechnological applications.

A new gene expression system based on a fructose-dependent promoter from Rhodobacter capsulatus

Gene ◽  
1994 ◽  
Vol 145 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Catherine Duport ◽  
Christine Meyer ◽  
Isabelle Naud ◽  
Yves Jouanneau
Keyword(s):  
Gene Expression ◽  
Rhodobacter Capsulatus ◽  
Expression System ◽  
Gene Expression System ◽  
New Gene

scholarly journals Generation of a GFP Reporter Akabane Virus with Enhanced Fluorescence Intensity by Modification of Artificial Ambisense S Genome

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 634
Author(s):  
Akiko Takenaka-Uema ◽  
Shin Murakami ◽  
Nanako Ushio ◽  
Tomoya Kobayashi-Kitamura ◽  
Masashi Uema ◽  
...  
Keyword(s):  
Imaging Study ◽  
Green Fluorescence Protein ◽  
Histopathological Study ◽  
Akabane Virus ◽  
Gfp Reporter ◽  
The Central Nervous System ◽  
Infected Cells ◽  
Fluorescence Protein

We previously generated a recombinant reporter Akabane virus expressing enhanced green fluorescence protein (eGFP-AKAV), with an artificial S genome encoding eGFP in the ambisense RNA. Although the eGFP-AKAV was able to detect infected cells in in vivo histopathological study, its fluorescent signal was too weak to apply to in vivo imaging study. Here, we successfully generated a modified reporter, eGFP/38-AKAV, with 38-nucleotide deletion of the internal region of the 5′ untranslated region of S RNA. The eGFP/38-AKAV expressed higher intensity of eGFP fluorescence both in vitro and in vivo than the original eGFP-AKAV did. In addition, eGFP/38-AKAV was pathogenic in mice at a comparable level to that in wild-type AKAV. In the mice infected with eGFP/38-AKAV, the fluorescent signals, i.e., the virus-infected cells, were detected in the central nervous system using the whole-organ imaging. Our findings indicate that eGFP/38-AKAV could be used as a powerful tool to help elucidate the dynamics of AKAV in vivo.

scholarly journals Predicting susceptibility to tuberculosis based on gene expression profiling in dendritic cells

2017 ◽  
Author(s):  
John D. Blischak ◽  
Ludovic Tailleux ◽  
Marsha Myrthil ◽  
Cécile Charlois ◽  
Emmanuel Bergot ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dendritic Cells ◽  
Differentially Expressed Genes ◽  
Differentially Expressed ◽  
Healthy Individuals ◽  
Data Set ◽  
Expression Levels ◽  
Infected Cells ◽  
Latent Tb ◽  
Gene Expression Levels

ABSTRACTTuberculosis (TB) is a deadly infectious disease, which kills millions of people every year. The causative pathogen, Mycobac-terium tuberculosis (MTB), is estimated to have infected up to a third of the world’s population; however, only approximately 10% of infected healthy individuals progress to active TB. Despite evidence for heritability, it is not currently possible to predict who may develop TB. To explore approaches to classify susceptibility to TB, we infected with MTB dendritic cells (DCs) from putatively resistant individuals diagnosed with latent TB, and from susceptible individuals that had recovered from active TB. We measured gene expression levels in infected and non-infected cells and found hundreds of differentially expressed genes between susceptible and resistant individuals in the non-infected cells. We further found that genetic polymorphisms nearby the differentially expressed genes between susceptible and resistant individuals are more likely to be associated with TB susceptibility in published GWAS data. Lastly, we trained a classifier based on the gene expression levels in the non-infected cells, and demonstrated decent performance on our data and an independent data set. Overall, our promising results from this small study suggest that training a classifier on a larger cohort may enable us to accurately predict TB susceptibility.

scholarly journals Functional characterization of the Beet necrotic yellow vein virus RNA-5-encoded p26 protein: evidence for structural pathogenicity determinants

2005 ◽  
Vol 86 (7) ◽  
pp. 2115-2125 ◽  
Author(s):  
Didier Link ◽  
Laure Schmidlin ◽  
Audrey Schirmer ◽  
Elodie Klein ◽  
Mathieu Erhardt ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Chenopodium Quinoa ◽  
Yellow Vein ◽  
Virus Rna ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Necrotic Symptoms

A Beet necrotic yellow vein virus isolate containing a fifth RNA is present in the Pithiviers area of France. A full-length cDNA clone of RNA-5 was obtained and placed under the control of a T7-RNA-pol promoter that allowed the production of infectious transcripts. ‘Pithiviers' isolate-specific necrotic symptoms were obtained on Chenopodium quinoa when RNA-5-encoded p26 was expressed either from RNA-5 or from an RNA-3-derived replicon. By using haemagglutinin- and green fluorescent protein-tagged constructs, virally expressed p26-fusion proteins induced the same necrotic local lesions on host plants and were localized mainly in the nucleus of infected cells. Deletion mutagenesis permitted identification of two domains, responsible respectively for nuclear export and cytoplasmic retention of the p26 mutated proteins. By using a yeast two-hybrid system, Gal4DB–p26 protein self-activated transcription of the His3 reporter gene. The p26 transcription-activation domain was located within its first 55 aa and has been studied by alanine scanning. Resulting p26 mutants were tested for their capability to induce necrotic symptoms and to localize in the nuclear compartment.

Development of clover yellow vein virus as an efficient, stable gene-expression system for legume species

2000 ◽  
Vol 23 (4) ◽  
pp. 539-546 ◽  
Author(s):  
Chikara Masuta ◽  
Toshikazu Yamana ◽  
Yoko Tacahashi ◽  
Ichiro Uyeda ◽  
Masanao Sato ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression System ◽  
Yellow Vein ◽  
Stable Gene ◽  
Legume Species ◽  
Gene Expression System ◽  
Clover Yellow Vein Virus ◽  
Stable Gene Expression

scholarly journals Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins

2021 ◽  
Vol 22 (16) ◽  
pp. 8583
Author(s):  
Hao-Wen Cheng ◽  
Wei-Ting Tsai ◽  
Yi-Ying Hsieh ◽  
Kuan-Chun Chen ◽  
Shyi-Dong Yeh
Keyword(s):  
Recombinant Proteins ◽  
Protein Interactions ◽  
Expression System ◽  
Zucchini Yellow Mosaic Virus ◽  
Green Fluorescence Protein ◽  
High Sensitivity ◽  
Yellow Mosaic Virus ◽  
Epitope Tag ◽  
Protein Protein Interaction ◽  
Detection And Diagnosis

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein–protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein–protein interactions.

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