Expression of recombinant enhanced green fluorescent protein provides insight into foreign gene‐expression differences betweenMut+andMutSstrains ofPichia pastoris

Yeast ◽  
2019 ◽  
Vol 36 (5) ◽  
pp. 285-296 ◽  
Author(s):  
Chrispian W. Theron ◽  
Julio Berrios ◽  
Sébastien Steels ◽  
Samuel Telek ◽  
Renaud Lecler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Foreign Gene ◽  
Foreign Gene Expression ◽  
Green Fluorescent ◽  
Insight Into

scholarly journals Generation of a Recombinant Akabane Virus Expressing Enhanced Green Fluorescent Protein

2015 ◽  
Vol 89 (18) ◽  
pp. 9477-9484 ◽  
Author(s):  
Akiko Takenaka-Uema ◽  
Yousuke Murata ◽  
Fumihiro Gen ◽  
Yukari Ishihara-Saeki ◽  
Ken-ichi Watanabe ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Rift Valley Fever Virus ◽  
Brain Slices ◽  
Foreign Gene ◽  
Akabane Virus ◽  
Sense Strand ◽  
Green Fluorescent

ABSTRACTWe generated a recombinant Akabane virus (AKAV) expressing enhanced green fluorescence protein (eGFP-AKAV) by using reverse genetics. We artificially constructed an ambisense AKAV S genome encoding N/NSs on the negative-sense strand, and eGFP on the positive-sense strand with an intergenic region (IGR) derived from the Rift Valley fever virus (RVFV) S genome. The recombinant virus exhibited eGFP fluorescence and had a cytopathic effect in cell cultures, even after several passages. These results indicate that the gene encoding eGFP in the ambisense RNA could be stably maintained. Transcription of N/NSs and eGFP mRNAs of eGFP-AKAV was terminated within the IGR. The mechanism responsible for this appears to be different from that in RVFV, where the termination sites for N and NSs are determined by a defined signal sequence. We inoculated suckling mice intraperitoneally with eGFP-AKAV, which resulted in neurological signs and lethality equivalent to those seen for the parent AKAV. Fluorescence from eGFP in frozen brain slices from the eGFP-AKAV-infected mice was localized to the cerebellum, pons, and medulla oblongata. Our approach to producing a fluorescent virus, using an ambisense genome, helped obtain eGFP-AKAV, a fluorescent bunyavirus whose viral genes are intact and which can be easily visualized.IMPORTANCEAKAV is the etiological agent of arthrogryposis-hydranencephaly syndrome in ruminants, which causes considerable economic loss to the livestock industry. We successfully generated a recombinant enhanced green fluorescent protein-tagged AKAV containing an artificial ambisense S genome. This virus could become a useful tool for analyzing AKAV pathogenesis in host animals. In addition, our approach of using an ambisense genome to generate an orthobunyavirus stably expressing a foreign gene could contribute to establishing alternative vaccine strategies, such as bivalent vaccine virus constructs, for veterinary use against infectious diseases.

scholarly journals Construction and Evaluation of Plasmid Vectors Optimized for Constitutive and Regulated Gene Expression in Burkholderia cepacia Complex Isolates

2002 ◽  
Vol 68 (12) ◽  
pp. 5956-5964 ◽  
Author(s):  
Matthew D. Lefebre ◽  
Miguel A. Valvano
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Burkholderia Cepacia ◽  
Enhanced Green Fluorescent Protein ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Burkholderia Cepacia Complex ◽  
Green Fluorescent ◽  
Regulated Gene Expression

ABSTRACT Genetic studies with Burkholderia cepacia complex isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. Also, some of the promoters widely employed for gene expression in Escherichia coli are inefficient in B. cepacia. In this study, we have utilized the backbone of the vector pME6000, a derivative of the pBBR1 plasmid that was originally isolated from Bordetella bronchiseptica, to construct a set of vectors useful for gene expression in B. cepacia. These vectors contain either the constitutive promoter of the S7 ribosomal protein gene from Burkholderia sp. strain LB400 or the arabinose-inducible P BAD promoter from E. coli. Promoter sequences were placed immediately upstream of multiple cloning sites in combination with the minimal sequence of pME6000 required for plasmid maintenance and mobilization. The functionality of both vectors was assessed by cloning the enhanced green fluorescent protein gene (e-gfp) and determining the levels of enhanced green fluorescent protein expression and fluorescence emission for a variety of clinical and environmental isolates of the B. cepacia complex. We also demonstrate that B. cepacia carrying these constructs can readily be detected intracellularly by fluorescence microscopy following the infection of Acanthamoeba polyphaga.

scholarly journals Role of Untranslated Regions in Regulation of Gene Expression, Replication, and Pathogenicity of Newcastle Disease Virus Expressing Green Fluorescent Protein

2009 ◽  
Vol 84 (5) ◽  
pp. 2629-2634 ◽  
Author(s):  
Shin-Hee Kim ◽  
Siba K. Samal
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Newcastle Disease Virus ◽  
Newcastle Disease ◽  
Fluorescent Protein ◽  
Untranslated Regions ◽  
Foreign Gene ◽  
Gfp Expression ◽  
Green Fluorescent

ABSTRACT To gain insight into the role of untranslated regions (UTRs) in regulation of foreign gene expression, replication, and pathogenicity of Newcastle disease virus (NDV), a green fluorescent protein (GFP) gene flanked by 5′ and 3′ UTRs of each NDV gene was individually expressed by recombinant NDVs. UTRs of each gene modulated GFP expression positively or negatively. In particular, UTRs of the M and F genes enhanced levels of GFP expression at the junction of the P and M genes without altering replication of NDV, suggesting that UTRs could be used for enhanced expression of a foreign gene by NDV.

scholarly journals Yeast-enhanced green fluorescent protein (yEGFP): a reporter of gene expression in Candida albicans

Microbiology ◽  
1997 ◽  
Vol 143 (2) ◽  
pp. 303-311 ◽  
Author(s):  
B. P. Cormack ◽  
G. Bertram ◽  
M. Egerton ◽  
N. A. R. Gow ◽  
S. Falkow ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent

scholarly journals Tracking Fluorescence-Labeled Rabies Virus: Enhanced Green Fluorescent Protein-Tagged Phosphoprotein P Supports Virus Gene Expression and Formation of Infectious Particles

2004 ◽  
Vol 78 (22) ◽  
pp. 12333-12343 ◽  
Author(s):  
Stefan Finke ◽  
Krzysztof Brzózka ◽  
Karl-Klaus Conzelmann
Keyword(s):  
Gene Expression ◽  
Green Fluorescent Protein ◽  
Rabies Virus ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Live Cell ◽  
Fluorescence Labeling ◽  
Extracellular Virus ◽  
P Gene ◽  
Green Fluorescent

ABSTRACT Rhabdoviruses such as rabies virus (RV) encode only five multifunctional proteins accomplishing viral gene expression and virus formation. The viral phosphoprotein, P, is a structural component of the viral ribonucleoprotein (RNP) complex and an essential cofactor for the viral RNA-dependent RNA polymerase. We show here that RV P fused to enhanced green fluorescent protein (eGFP) can substitute for P throughout the viral life cycle, allowing fluorescence labeling and tracking of RV RNPs under live cell conditions. To first assess the functions of P fusion constructs, a recombinant RV lacking the P gene, SAD ΔP, was complemented in cell lines constitutively expressing eGFP-P or P-eGFP fusion proteins. P-eGFP supported the rapid accumulation of viral mRNAs but led to low infectious-virus titers, suggesting impairment of virus formation. In contrast, complementation with eGFP-P resulted in slower accumulation of mRNAs but similar infectious titers, suggesting interference with polymerase activity rather than with virus formation. Fluorescence microscopy allowed the detection of eGFP-P-labeled extracellular virus particles and tracking of cell binding and temperature-dependent internalization into intracellular vesicles. Recombinant RVs expressing eGFP-P or an eGFP-P mutant lacking the binding site for dynein light chain 1 (DLC1) instead of P were used to track interaction with cellular proteins. In cells expressing a DsRed-labeled DLC1, colocalization of DLC1 with eGFP-P but not with the mutant P was observed. Fluorescent labeling of RV RNPs will allow further dissection of virus entry, replication, and egress under live-cell conditions as well as cell interactions.

scholarly journals Enhanced Green Fluorescent Protein (GFP) fluorescence after polyelectrolyte caging

2006 ◽  
Vol 14 (21) ◽  
pp. 9815 ◽  
Author(s):  
Alberto Diaspro ◽  
Silke Krol ◽  
Barbara Campanini ◽  
Fabio Cannone ◽  
Giuseppe Chirico
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Gfp Fluorescence ◽  
Green Fluorescent

scholarly journals Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 632
Author(s):  
Yingyun Cai ◽  
Shuiqing Yu ◽  
Ying Fang ◽  
Laura Bollinger ◽  
Yanhua Li ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Lines ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Infectious Clone ◽  
Hemorrhagic Fever ◽  
Simian Hemorrhagic Fever Virus ◽  
Hemorrhagic Fever Virus ◽  
Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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