scholarly journals Expression of Foreign Proteins by Poliovirus Polyprotein Fusion: Analysis of Genetic Stability Reveals Rapid Deletions and Formation of Cardioviruslike Open Reading Frames

1998 ◽  
Vol 72 (1) ◽  
pp. 20-31 ◽  
Author(s):  
Steffen Mueller ◽  
Eckard Wimmer
Keyword(s):  
Fluorescent Protein ◽  
Growth Cycle ◽  
Open Reading Frames ◽  
Expression Vectors ◽  
Green Fluorescent Protein Gene ◽  
Published Data ◽  
Gene Encoding ◽  
Genomic Rnas ◽  
Reading Frame ◽  
Green Fluorescent

ABSTRACT Using a strategy developed by R. Andino, D. Silvera, S. D. Suggett, P. I. Achacoso, C. J. Miller, D. Baltimore, and M. B. Feinberg (Science 265:1448–1451, 1994), we constructed recombinant polioviruses by fusing the open reading frame (ORF) of the green fluorescent protein gene (gfp) of Aequorea victoria or the gag gene (encoding p17-p24) of human immunodeficiency virus type 1 (HIV-1) to the N terminus of the poliovirus polyprotein. All poliovirus expression vectors constructed by us and those obtained from Andino et al. were found to be severely impaired in viral replication and genetically unstable. Upon replication, inserted sequences were rapidly deleted as early as the first growth cycle in HeLa cells. However, the vector viruses did not readily revert to the wild-type sequence but rather retained some of the insert plus the artificial 3Cpro/3CDprocleavage site, engineered between the heterologous sequence and the poliovirus polyprotein, to give rise to genotypes reminiscent of cardioviruses. These virus variants that carry a small leader polypeptide were now relatively stable, and they grew better than their progenitor strains. Reverse transcription followed by PCR and sequence analysis of the genomic RNAs reproducibly revealed a few preferred genotypes among the isolated deletion variants. The remaining truncated inserts were retained through subsequent passages. In the immediate vicinity of the deletion borders, we observed short direct sequence repeats that we propose are involved in aligning RNA strands for illegitimate (nonhomologous) RNA recombination during minus-strand synthesis. On the basis of our results, which are at variance with published data, the utility of poliovirus vectors to express proteins >10 kDa in size through fusion with the polyprotein needs to be reevaluated.

scholarly journals Essential and Dispensable Virus-Encoded Replication Elements Revealed by Efforts To Develop Hypoviruses as Gene Expression Vectors

2000 ◽  
Vol 74 (16) ◽  
pp. 7568-7577 ◽  
Author(s):  
Nobuhiro Suzuki ◽  
Lynn M. Geletka ◽  
Donald L. Nuss
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Cryphonectria Parasitica ◽  
Expression Vectors ◽  
Green Fluorescent Protein Gene ◽  
Reading Frame ◽  
Chestnut Blight Fungus ◽  
Virulence Attenuation ◽  
Foreign Genes ◽  
Green Fluorescent

ABSTRACT We have investigated whether hypoviruses, viral agents responsible for virulence attenuation (hypovirulence) of the chestnut blight fungusCryphonectria parasitica, could serve as gene expression vectors. The infectious cDNA clone of the prototypic hypovirus CHV1-EP713 was modified to generate 20 different vector candidates. Although transient expression was achieved for a subset of vectors that contained the green fluorescent protein gene from Aequorea victoria, long-term expression (past day 8) was not observed for any vector construct. Analysis of viral RNAs recovered from transfected fungal colonies revealed that the foreign genes were readily deleted from the replicating virus, although small portions of foreign sequences were retained by some vectors after months of replication. However, the results of vector viability and progeny characterization provided unexpected new insights into essential and dispensable elements of hypovirus replication. The N-terminal portion (codons 1 to 24) of the 5′-proximal open reading frame (ORF), ORF A, was found to be required for virus replication, while the remaining 598 codons of this ORF were completely dispensable. Substantial alterations were tolerated in the pentanucleotide UAAUG that contains the ORF A termination codon and the overlapping putative initiation codon of the second of the two hypovirus ORFs, ORF B. Replication competence was maintained following either a frameshift mutation that caused a two-codon extension of ORF A or a modification that produced a single-ORF genomic organization. These results are discussed in terms of determinants of hypovirus replication, the potential utility of hypoviruses as gene expression vectors, and possible mechanisms by which hypoviruses recognize and delete foreign sequences.

Improvement of TA Cloning Method to Facilitate Direct Directional Cloning of PCR Products

2014 ◽  
Vol 565 ◽  
pp. 3-8 ◽  
Author(s):  
Ji Gang Li ◽  
Guo Ying Han ◽  
Xiu Min Li ◽  
Jiao Jiao Sun ◽  
Ke Jing Song ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Pcr Primers ◽  
Expression Vectors ◽  
Green Fluorescent Protein Gene ◽  
Reaction Products ◽  
Major Disadvantage ◽  
Cloning Method ◽  
Directional Cloning ◽  
Pcr Products ◽  
Green Fluorescent

Directional cloning is a prerequisite for the construction of expression vectors in molecular biology laboratories. Although TA cloning is widely used to clone unmodified PCR (polymerase chain reaction) products, a major disadvantage of this technique is that cloning is not directional. Here we reported a novel PCR products cloning vector with one deoxythymidine overhang and one deoxycytidine overhang at two 3'-ends respectively. With the choice of nucleotides of 5'-ends of PCR primers, PCR products can be cloned to this vector both directly and directionally. The feasibility and efficacy of this cloning method were confirmed by using a pET-17b derivative vector and a green fluorescent protein gene (EGFP) and a red fluorescent protein reporter (Ds-Red) gene. This cloning strategy may be useful in the high-throughput construction of expression vectors and could be viewed as an interesting improvement of existing TA cloning method.

scholarly journals Expression of the Totivirus Helminthosporium victoriae 190S Virus RNA-Dependent RNA Polymerase from Its Downstream Open Reading Frame in Dicistronic Constructs

2000 ◽  
Vol 74 (2) ◽  
pp. 997-1003 ◽  
Author(s):  
Ana I. Soldevila ◽  
Said A. Ghabrial
Keyword(s):  
Rna Polymerase ◽  
Fluorescent Protein ◽  
Eukaryotic Cell ◽  
Open Reading Frames ◽  
Rna Dependent Rna Polymerase ◽  
Reading Frame ◽  
Virus Rna ◽  
Helminthosporium Victoriae ◽  
Green Fluorescent ◽  
Overlapping Open Reading Frames

ABSTRACT The undivided double-stranded RNA (dsRNA) genome ofHelminthosporium victoriae 190S virus (Hv190SV) (genusTotivirus) consists of two large overlapping open reading frames (ORFs). The 5′-proximal ORF encodes a capsid protein (CP), and the downstream, 3′-proximal ORF encodes an RNA-dependent RNA polymerase (RDRP). Unlike the RDRPs of some other totiviruses, which are expressed as a CP-RDRP (Gag-Pol-like) fusion protein, the Hv190SV RDRP is detected only as a separate, nonfused polypeptide. In this study, we examined the expression of the RDRP ORF fused in frame to the coding sequence of the green fluorescent protein (GFP) in bacteria andSchizosaccharomyces pombe cells. The GFP fusions were readily detected in bacteria transformed with the monocistronic construct RDRP:GFP; expression of the downstream RDRP:GFP from the dicistronic construct CP-RDRP:GFP could not be detected. However, fluorescence microscopy and Western blot analysis indicated that RDRP:GFP was expressed at low levels from its downstream ORF in the dicistronic construct in S. pombe cells. No evidence that the RDRP ORF was expressed from a transcript shorter than the full-length dicistronic mRNA was found. A coupled termination-reinitiation mechanism that requires host or eukaryotic cell factors is proposed for the expression of Hv190SV RDRP.

scholarly journals Purification, Characterization, and Gene Analysis of a Chitosanase (ChoA) from Matsuebacter chitosanotabidus3001

1999 ◽  
Vol 181 (21) ◽  
pp. 6642-6649 ◽  
Author(s):  
Jae Kweon Park ◽  
Kumiko Shimono ◽  
Nobuhisa Ochiai ◽  
Kazutaka Shigeru ◽  
Masako Kurita ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Glycol Chitosan ◽  
Gene Encoding ◽  
Reading Frame ◽  
Acid Protein ◽  
Green Fluorescent ◽  
Potential Inhibitors

ABSTRACT The extracellular chitosanase (34,000 M r) produced by a novel gram-negative bacterium Matsuebacter chitosanotabidus 3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40°C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag+. Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire choA gene encoding chitosanase. Sequencing of the choA gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in Escherichia coli. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.

scholarly journals Identification of 5′ and 3′ cis-Acting Elements of the Porcine Reproductive and Respiratory Syndrome Virus: Acquisition of Novel 5′ AU-Rich Sequences Restored Replication of a 5′-Proximal 7-Nucleotide Deletion Mutant

2006 ◽  
Vol 80 (2) ◽  
pp. 723-736 ◽  
Author(s):  
Yu-Jeong Choi ◽  
Sang-Im Yun ◽  
Shien-Young Kang ◽  
Young-Min Lee
Keyword(s):  
Reverse Genetics ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Heterologous Gene Expression ◽  
Artificial Chromosome ◽  
Expression Vectors ◽  
Respiratory Syndrome Virus ◽  
Genomic Rnas ◽  
Cis Acting ◽  
Green Fluorescent

ABSTRACT We here demonstrate the successful engineering of the RNA genome of porcine reproductive and respiratory syndrome virus (PRRSV) by using an infectious cDNA as a bacterial artificial chromosome. Runoff transcription from this cDNA by SP6 polymerase resulted in capped synthetic RNAs bearing authentic 5′ and 3′ ends of the viral genome that had specific infectivities of >5 × 105 PFU/μg of RNA. The synthetic viruses recovered from the transfected cells were genotypically and phenotypically indistinguishable from the parental virus. Using our system, a series of genomic RNAs with nucleotide deletions in their 5′ ends produced viruses with decreased or no infectivity. Various pseudorevertants were isolated, and acquisition of novel 5′ sequences of various sizes, composed predominantly of A and U bases, restored their infectivities, providing a novel insight into functional elements of the 5′ end of the PRRSV genome. In addition, our system was further engineered to generate a panel of self-replicating, self-limiting, luciferase-expressing PRRSV viral replicons bearing various deletions. Analysis of these replicons revealed the presence and location of a 3′ cis-acting element in the genome that was required for replication. Moreover, we produced enhanced green fluorescent protein-expressing infectious viruses, which indicates that the PRRSV cDNA/viral replicon/recombinant virus can be developed as a vector for the expression of a variety of heterologous genes. Thus, our PRRSV reverse genetics system not only offers a means of directly investigating the molecular mechanisms of PRRSV replication and pathogenesis but also can be used to generate new heterologous gene expression vectors and genetically defined antiviral vaccines.

scholarly journals A Propionate-Inducible Expression System for Enteric Bacteria

2005 ◽  
Vol 71 (11) ◽  
pp. 6856-6862 ◽  
Author(s):  
Sung Kuk Lee ◽  
Jay D. Keasling
Keyword(s):  
Fluorescent Protein ◽  
Expression System ◽  
Enteric Bacteria ◽  
Expression Vectors ◽  
Gene Encoding ◽  
Basal Expression ◽  
Expression Of Genes ◽  
Catabolic Genes ◽  
Wide Range ◽  
Green Fluorescent

ABSTRACT A series of new expression vectors (pPro) have been constructed for the regulated expression of genes in Escherichia coli. The pPro vectors contain the prpBCDE promoter (P prpB ) responsible for expression of the propionate catabolic genes (prpBCDE) and prpR encoding the positive regulator of this promoter. The efficiency and regulatory properties of the prpR-P prpB system were measured by placing the gene encoding the green fluorescent protein (gfp) under the control of the inducible P prpB of E. coli. This system provides homogenous expression in individual cells, highly regulatable expression over a wide range of propionate concentrations, and strong expression (maximal 1,500-fold induction) at high propionate concentrations. Since the prpBCDE promoter has CAP-dependent activation, the prpR-P prpB system exhibited negligible basal expression by addition of glucose to the medium.

scholarly journals Molecular Analysis of Sucrose Metabolism ofErwinia amylovora and Influence on Bacterial Virulence

2000 ◽  
Vol 182 (19) ◽  
pp. 5351-5358 ◽  
Author(s):  
Jochen Bogs ◽  
Klaus Geider
Keyword(s):  
Flow Cytometry ◽  
Fire Blight ◽  
Fluorescent Protein ◽  
Sucrose Concentration ◽  
Sucrose Metabolism ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Green Fluorescent Protein Gene ◽  
E Coli ◽  
Green Fluorescent

ABSTRACT Sucrose is an important storage and transport sugar of plants and an energy source for many phytopathogenic bacteria. To analyze regulation and biochemistry of sucrose metabolism of the fire blight pathogen Erwinia amylovora, a chromosomal fragment which enabled Escherichia coli to utilize sucrose as sole carbon source was cloned. By transposon mutagenesis, the scrregulon of E. amylovora was tagged, and its nucleotide sequence was determined. Five open reading frames, with the genesscrK, scrY, scrA, scrB, and scrR, had high homology to genes of the scrregulons from Klebsiella pneumoniae and plasmid pUR400.scrB and scrR of E. amylovora were fused to a histidine tag and to the maltose-binding protein (MalE) ofE. coli, respectively. ScrB (53 kDa) catalyzed the hydrolysis of sucrose with a Km of 125 mM. Binding of a MalE-ScrR fusion protein to an scrYAB promoter fragment was shown by gel mobility shifts. This complex dissociated in the presence of fructose but not after addition of sucrose. Expression of the scr regulon was studied with an scrYABpromoter-green fluorescent protein gene fusion and measured by flow cytometry and spectrofluorometry. The operon was affected by catabolite repression and induced by sucrose or fructose. The level of gene induction correlated to the sucrose concentration in plant tissue, as shown by flow cytometry. Sucrose mutants created by site-directed mutagenesis did not produce significant fire blight symptoms on apple seedlings, indicating the importance of sucrose metabolism for colonization of host plants by E. amylovora.

scholarly journals P34.8 (GP37) is not essential for baculovirus replication

2001 ◽  
Vol 82 (2) ◽  
pp. 299-305 ◽  
Author(s):  
Xiao-Wen Cheng ◽  
Peter J. Krell ◽  
Basil M. Arif
Keyword(s):  
Northern Blot Analysis ◽  
Fluorescent Protein ◽  
Pcr Amplification ◽  
Restriction Endonuclease Analysis ◽  
Gene Encoding ◽  
Conserved Domains ◽  
Reading Frame ◽  
Viral Plaque ◽  
Green Fluorescent ◽  
Endonuclease Analysis

Previous reports have indicated that p34.8 (gp37) may be essential for the replication of Autographa californica nucleopolyhedrovirus (AcMNPV) because no virus with inactivated p34.8 was isolated. We have ascertained the requirement for this gene by attempting to inactivate it with a large insertion [the gene encoding GFP (green fluorescent protein)] or by deleting all the conserved domains from the open reading frame (ORF). The gene encoding GFP was inserted into the NotI site of the p34.8 ORF and a viral plaque containing the insertion was propagated in SF-21 cells. Similarly, 531 bp (NotI–XbaI) containing all conserved domains were deleted from the ORF. All mutants were authenticated by PCR amplification, restriction endonuclease analysis, DNA sequencing, and Southern and Northern blot analysis. It was found that inactivation of p34.8 of AcUW1-LacZ (AcMNPV containing a lacZ gene in the p10 locus) had no effect on the biological property of virus, such as virulence and kinetics. These two independent methods showed that p34.8 is not essential for replication and that this locus could provide another site for the engineering of baculoviruses.

scholarly journals DNA Microarray-Based Identification of Genes Associated with Glycopeptide Resistance in Staphylococcus aureus

2005 ◽  
Vol 49 (8) ◽  
pp. 3404-3413 ◽  
Author(s):  
Longzhu Cui ◽  
Jian-Qi Lian ◽  
Hui-min Neoh ◽  
Ethel Reyes ◽  
Keiichi Hiramatsu
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Gene Knockout ◽  
Open Reading Frames ◽  
Transport Systems ◽  
Green Fluorescent Protein Gene ◽  
Beta Lactam ◽  
Glycopeptide Resistance ◽  
Transcription Profiles ◽  
Green Fluorescent

ABSTRACT Six pairs of transcription profiles between glycopeptide-intermediate S. aureus (GISA [or vancomycin-intermediate S. aureus; VISA]) and glycopeptide-susceptible S. aureus (vancomycin-susceptible S. aureus [VSSA], including glycopeptide-susceptible isogenic mutants from VISA) strains were compared using a microarray. Ninety-two open reading frames which were or tended to be increased in transcription in VISA in at least five out of six array combination pairs were evaluated for their effects on glycopeptide susceptibility by introducing these genes one by one into VSSA strain N315 to construct an overexpression library. By screening the library, 17 genes including 8 novel genes were identified as associated with glycopeptide resistance since their experimental overexpression reduced vancomycin and/or teicoplanin susceptibility of N315. The raised MICs of vancomycin and teicoplanin were 1.25 to 3.0 and 1.5 to 6.0 mg/liter, respectively, as compared to 1.0 mg/liter of N315. Three of these genes, namely graF, msrA2, and mgrA, also raised the oxacillin MIC from 8.0 mg/liter for N315 to 64 to ∼128 mg/liter when they were overexpressed in N315. Their contribution to vancomycin and beta-lactam resistance was further supported by gene knockout and trans-complementation assay. By using a plasmid-based promoter-green fluorescent protein gene (gfp) transcriptional fusion system, graF promoter-activated cells were purified, and subsequent susceptibility tests and Northern blot analysis demonstrated that the cells with up-regulated activity of graF promoter showed reduced susceptibility to vancomycin, teicoplanin, and oxacillin. In addition, cell morphology studies showed that graF and msrA2 overexpression increased cell wall thickness of N315 by factors of 23.91 and 22.27%, respectively, accompanied by glycopeptide MIC increments of 3- to 6-fold, when they were overexpressed in N315. Moreover, extended experiments and analyses indicate that many of the genes identified above are related to the cell wall biosynthetic pathway, including active nutrient transport systems. We propose that the genes which raise glycopeptide resistance in S. aureus function toward altering the cell wall metabolic pathway.

Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

2008 ◽  
Vol 410 (2) ◽  
pp. 291-299 ◽  
Author(s):  
Fui-Ching Tan ◽  
Qi Cheng ◽  
Kaushik Saha ◽  
Ilka U. Heinemann ◽  
Martina Jahn ◽  
...  
Keyword(s):  
Expression Vector ◽  
Fluorescent Protein ◽  
Sequence Similarity ◽  
Homology Search ◽  
Protein Database ◽  
Gene Encoding ◽  
Reading Frame ◽  
Green Fluorescent

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.

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