scholarly journals Production of baculovirus defective interfering particles during serial passage is delayed by removing transposon target sites in fp25k

2012 ◽  
Vol 93 (2) ◽  
pp. 389-399 ◽  
Author(s):  
Lopamudra Giri ◽  
Michael G. Feiss ◽  
Bryony C. Bonning ◽  
David W. Murhammer
Keyword(s):  
Large Scale ◽  
Serial Passage ◽  
Autographa Californica ◽  
Wild Type ◽  
Systematic Method ◽  
Defective Interfering Particles ◽  
Extracellular Virus ◽  
Transposon Insertion ◽  
Target Sites ◽  
Insertion Sites

Accumulation of baculovirus defective interfering particle (DIP) and few polyhedra (FP) mutants is a major limitation to continuous large-scale baculovirus production in insect-cell culture. Although overcoming these mutations would result in a cheaper platform for producing baculovirus biopesticides, little is known regarding the mechanism of FP and DIP formation. This issue was addressed by comparing DIP production of wild-type (WT) Autographa californica multiple nucleopolyhedrovirus (AcMNPV) with that of a recombinant AcMNPV (denoted Ac-FPm) containing a modified fp25k gene with altered transposon insertion sites that prevented transposon-mediated production of the FP phenotype. In addition to a reduction in the incidence of the FP phenotype, DIP formation was delayed on passaging of Ac-FPm compared with WT AcMNPV. Specifically, the yield of DIP DNA in Ac-FPm was significantly lower than in WT AcMNPV up to passage 16, thereby demonstrating that modifying the transposon insertion sites increases the genomic stability of AcMNPV. A critical component of this investigation was the optimization of a systematic method based on the use of pulsed-field gel electrophoresis (PFGE) to characterize extracellular virus DNA. Specifically, PFGE was used to detect defective genomes, determine defective genome sizes and quantify the amount of defective genome within a heterogeneous genome population of passaged virus.

scholarly journals A Few-Polyhedra Mutant and Wild-Type Nucleopolyhedrovirus Remain as a Stable Polymorphism during Serial Coinfection in Trichoplusia ni

2003 ◽  
Vol 69 (4) ◽  
pp. 2052-2057 ◽  
Author(s):  
James C. Bull ◽  
H. C. J. Godfray ◽  
David R. O'Reilly
Keyword(s):  
Cell Culture ◽  
Population Genetics ◽  
Trichoplusia Ni ◽  
Serial Passage ◽  
Autographa Californica ◽  
Wild Type ◽  
Cell Infection ◽  
Occlusion Bodies ◽  
Budded Virus

ABSTRACT Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.

scholarly journals Genes Involved in Yellow Pigmentation of Cronobacter sakazakii ES5 and Influence of Pigmentation on Persistence and Growth under Environmental Stress

2009 ◽  
Vol 76 (4) ◽  
pp. 1053-1061 ◽  
Author(s):  
Sophia Johler ◽  
Roger Stephan ◽  
Isabel Hartmann ◽  
Kirsten A. Kuehner ◽  
Angelika Lehner
Keyword(s):  
Environmental Stress ◽  
Inorganic Ions ◽  
Cronobacter Sakazakii ◽  
Wild Type ◽  
Transposon Insertion ◽  
Food Borne ◽  
Increased Sensitivity ◽  
Yellow Pigmentation ◽  
Insertion Sites ◽  
Increased Susceptibility

ABSTRACT Cronobacter spp. are opportunistic food-borne pathogens that are responsible for rare but highly fatal cases of meningitis and necrotizing enterocolitis in neonates. While the operon responsible for yellow pigmentation in Cronobacter sakazakii strain ES5 was described recently, the involvement of additional genes in pigment expression and the influence of pigmentation on the fitness of Cronobacter spp. have not been investigated. Thus, the aim of this study was to identify further genes involved in pigment expression in Cronobacter sakazakii ES5 and to assess the influence of pigmentation on growth and persistence under conditions of environmental stress. A knockout library was created using random transposon mutagenesis. The screening of 9,500 mutants for decreased pigment production identified 30 colorless mutants. The mapping of transposon insertion sites revealed insertions in not only the carotenoid operon but also in various other genes involved in signal transduction, inorganic ions, and energy metabolism. To determine the effect of pigmentation on fitness, colorless mutants (ΔcrtE, ΔcrtX, and ΔcrtY) were compared to the yellow wild type using growth and inactivation experiments, a macrophage assay, and a phenotype array. Among other findings, the colorless mutants grew at significantly increased rates under osmotic stress compared to that of the yellow wild type while showing increased susceptibility to desiccation. Moreover, ΔcrtE and ΔcrtY exhibited increased sensitivity to UVB irradiation.

Low multiplicity of infection in vivo results in purifying selection against baculovirus deletion mutants

2008 ◽  
Vol 89 (5) ◽  
pp. 1220-1224 ◽  
Author(s):  
Mark P. Zwart ◽  
Eloy Erro ◽  
Monique M. van Oers ◽  
J. Arjan G. M. de Visser ◽  
Just M. Vlak
Keyword(s):  
Cell Culture ◽  
Purifying Selection ◽  
Serial Passage ◽  
Autographa Californica ◽  
Homologous Region ◽  
Deletion Mutants ◽  
Wild Type ◽  
Insect Larvae ◽  
Selection For

The in vivo fate of Autographa californica multiple nucleopolyhedrovirus deletion mutants originating from serial passage in cell culture was investigated by passaging a population enriched in these mutants in insect larvae. The infectivity of polyhedra and occlusion-derived virion content per polyhedron were restored within two passages in vivo. The frequency of occurrence of deletion mutants was determined by real-time PCR. The frequency of the non-homologous region origin (non-HR ori) of DNA replication was reduced to wild-type levels within two passages. The frequency of the polyhedrin gene did not increase and remained below wild-type levels. A low m.o.i. during the initial infection in insect larvae, causing strong purifying selection for autonomously replicating viruses, could explain these observations. The same virus population used in vivo was also passaged once at a different m.o.i. in cell culture. A similar effect (i.e. lower non-HR ori frequency) was observed at low m.o.i. only, indicating that m.o.i. was the key selective condition.

scholarly journals INTRACISTRONIC MAPPING OF ELECTROPHORETIC SITES IN DROSOPHILA MELANOGASTER: FIDELITY OF INFORMATION TRANSFER BY GENE CONVERSION

Genetics ◽  
1974 ◽  
Vol 76 (2) ◽  
pp. 289-299
Author(s):  
Margaret McCarron ◽  
William Gelbart ◽  
Arthur Chovnick
Keyword(s):  
Drosophila Melanogaster ◽  
Information Transfer ◽  
Large Scale ◽  
Genetic Basis ◽  
Xanthine Dehydrogenase ◽  
Specific Protein ◽  
Wild Type ◽  
Electrophoretic Variation ◽  
The Stability ◽  
Recombination Experiments

ABSTRACT A convenient method is described for the intracistronic mapping of genetic sites responsible for electrophoretic variation of a specific protein in Drosophila melanogaster. A number of wild-type isoalleles of the rosy locus have been isolated which are associated with the production of electrophoretically distinguishable xanthine dehydrogenases. Large-scale recombination experiments were carried out involving null enzyme mutants induced on electrophoretically distinct wild-type isoalleles, the genetic basis for which is followed as a nonselective marker in the cross. Additionally, a large-scale recombination experiment was carried out involving null enzyme rosy mutants induced on the same wild-type isoallele. Examination of the electrophoretic character of crossover and convertant products recovered from the latter experiment revealed that all exhibited the same parental electrophoretic character. In addition to documenting the stability of the xanthine dehydrogenase electrophoretic character, this observation argues against a special mutagenesis hypothesis to explain conversions resulting from allele recombination studies.

scholarly journals A Novel and Efficient High-Yield Method for Preparing Bacterial Ghosts

Toxins ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 420
Author(s):  
Yi Ma ◽  
Liu Cui ◽  
Meng Wang ◽  
Qiuli Sun ◽  
Kaisheng Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Expression System ◽  
High Yield ◽  
Wild Type ◽  
High Quality ◽  
Efficient Protection ◽  
Bacterial Ghosts ◽  
Cell Envelopes ◽  
Extracellular Structures ◽  
First Time

Bacterial ghosts (BGs) are empty cell envelopes possessing native extracellular structures without a cytoplasm and genetic materials. BGs are proposed to have significant prospects in biomedical research as vaccines or delivery carriers. The applications of BGs are often limited by inefficient bacterial lysis and a low yield. To solve these problems, we compared the lysis efficiency of the wild-type protein E (EW) from phage ΦX174 and the screened mutant protein E (EM) in the Escherichia coli BL21(DE3) strain. The results show that the lysis efficiency mediated by protein EM was improved. The implementation of the pLysS plasmid allowed nearly 100% lysis efficiency, with a high initial cell density as high as OD600 = 2.0, which was higher compared to the commonly used BG preparation method. The results of Western blot analysis and immunofluorescence indicate that the expression level of protein EM was significantly higher than that of the non-pLysS plasmid. High-quality BGs were observed by SEM and TEM. To verify the applicability of this method in other bacteria, the T7 RNA polymerase expression system was successfully constructed in Salmonella enterica (S. Enterica, SE). A pET vector containing EM and pLysS were introduced to obtain high-quality SE ghosts which could provide efficient protection for humans and animals. This paper describes a novel and commonly used method to produce high-quality BGs on a large scale for the first time.

A Systematic Method for the Selection of Independent Variables in the Investigation of Complex Systems

1988 ◽  
Vol 32 (17) ◽  
pp. 1179-1182 ◽  
Author(s):  
P. Jay Merkle ◽  
Douglas B. Beaudet ◽  
Robert C. Williges ◽  
David W. Herlong ◽  
Beverly H. Williges
Keyword(s):  
Large Scale ◽  
Subjective Ratings ◽  
Systematic Method ◽  
Independent Variables ◽  
Integrated Strategy ◽  
Literature Reviews ◽  
Systematic Methodology ◽  
Research Problems ◽  
Inquiry System ◽  
Selection Of

This paper describes a systematic methodology for selecting independent variables to be considered in large-scale research problems. Five specific procedures including brainstorming, prototype interface representation, feasibility/relevance analyses, structured literature reviews, and user subjective ratings are evaluated and incorporated into an integrated strategy. This methodology is demonstrated in the context of designing the user interface for a telephone-based information inquiry system. The procedure was successful in reducing an initial set of 95 independent variables to a subset of 19 factors that warrant subsequent detailed analysis. These results are discussed in terms of a comprehensive sequential research methodology useful for investigating human factors problems.

scholarly journals Large-Scale Synonymous Substitutions in Cucumber Mosaic Virus RNA 3 Facilitate Amino Acid Mutations in the Coat Protein

2018 ◽  
Vol 92 (22) ◽  
Author(s):  
Tomofumi Mochizuki ◽  
Rie Ohara ◽  
Marilyn J. Roossinck
Keyword(s):  
Amino Acid ◽  
Secondary Structure ◽  
Viral Genome ◽  
Large Scale ◽  
Viral Rna ◽  
Wild Type ◽  
Competitive Fitness ◽  
Content Type ◽  
Synonymous Substitutions ◽  
Cp Gene

ABSTRACTThe effect of large-scale synonymous substitutions in a small icosahedral, single-stranded RNA viral genome on virulence, viral titer, and protein evolution were analyzed. The coat protein (CP) gene of the Fny stain of cucumber mosaic virus (CMV) was modified. We created four CP mutants in which all the codons of nine amino acids in the 5′ or 3′ half of the CP gene were replaced by either the most frequently or the least frequently used synonymous codons in monocot plants. When the dicot host (Nicotiana benthamiana) was inoculated with these four CP mutants, viral RNA titers in uninoculated symptomatic leaves decreased, while all mutants eventually showed mosaic symptoms similar to those for the wild type. The codon adaptation index of these four CP mutants against dicot genes was similar to those of the wild-type CP gene, indicating that the reduction of viral RNA titer was due to deleterious changes of the secondary structure of RNAs 3 and 4. When two 5′ mutants were serially passaged inN. benthamiana, viral RNA titers were rapidly restored but competitive fitness remained decreased. Although no nucleic acid changes were observed in the passaged wild-type CMV, one to three amino acid changes were observed in the synonymously mutated CP of each passaged virus, which were involved in recovery of viral RNA titer of 5′ mutants. Thus, we demonstrated that deleterious effects of the large-scale synonymous substitutions in the RNA viral genome facilitated the rapid amino acid mutation(s) in the CP to restore the viral RNA titer.IMPORTANCERecently, it has been known that synonymous substitutions in RNA virus genes affect viral pathogenicity and competitive fitness by alteration of global or local RNA secondary structure of the viral genome. We confirmed that large-scale synonymous substitutions in the CP gene of CMV resulted in decreased viral RNA titer. Importantly, when viral evolution was stimulated by serial-passage inoculation, viral RNA titer was rapidly restored, concurrent with a few amino acid changes in the CP. This novel finding indicates that the deleterious effects of large-scale nucleic acid mutations on viral RNA secondary structure are readily tolerated by structural changes in the CP, demonstrating a novel part of the adaptive evolution of an RNA viral genome. In addition, our experimental system for serial inoculation of large-scale synonymous mutants could uncover a role for new amino acid residues in the viral protein that have not been observed in the wild-type virus strains.

scholarly journals OmpR and LeuO Positively Regulate the Salmonella enterica Serovar Typhi ompS2 Porin Gene

2004 ◽  
Vol 186 (10) ◽  
pp. 2909-2920 ◽  
Author(s):  
Marcos Fernández-Mora ◽  
José Luis Puente ◽  
Edmundo Calva
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Phosphorylation Site ◽  
Regulatory Region ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Upstream Regulatory Region ◽  
Wild Type ◽  
Transposon Insertion

ABSTRACT The Salmonella enterica serovar Typhi ompS2 gene codes for a 362-amino-acid outer membrane protein that contains motifs common to the porin superfamily. It is expressed at very low levels compared to the major OmpC and OmpF porins, as observed for S. enterica serovar Typhi OmpS1, Escherichia coli OmpN, and Klebsiella pneumoniae OmpK37 quiescent porins. A region of 316 bp, between nucleotides −413 and −97 upstream of the transcriptional start point, is involved in negative regulation, as its removal resulted in a 10-fold increase in ompS2 expression in an S. enterica serovar Typhi wild-type strain. This enhancement in expression was not observed in isogenic mutant strains, which had specific deletions of the regulatory ompB (ompR envZ) operon. Furthermore, ompS2 expression was substantially reduced in the presence of the OmpR D55A mutant, altered in the major phosphorylation site. Upon random mutagenesis, a mutant where the transposon had inserted into the upstream regulatory region of the gene coding for the LeuO regulator, showed an increased level of ompS2 expression. Augmented expression of ompS2 was also obtained upon addition of cloned leuO to the wild-type strain, but not in an ompR isogenic derivative, consistent with the notion that the transposon insertion had increased the cellular levels of LeuO and with the observed dependence on OmpR. Moreover, LeuO and OmpR bound in close proximity, but independently, to the 5′ upstream regulatory region. Thus, the OmpR and LeuO regulators positively regulate ompS2.

scholarly journals AcrAB Multidrug Efflux Pump Is Associated with Reduced Levels of Susceptibility to Tigecycline (GAR-936) in Proteus mirabilis

2003 ◽  
Vol 47 (2) ◽  
pp. 665-669 ◽  
Author(s):  
Melissa A. Visalli ◽  
Ellen Murphy ◽  
Steven J. Projan ◽  
Patricia A. Bradford
Keyword(s):  
Proteus Mirabilis ◽  
Efflux Pump ◽  
Wild Type ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Transposon Insertion ◽  
Spectrum Activity ◽  
Insertion Mutants ◽  
Broad Spectrum Activity ◽  
Increased Susceptibility

ABSTRACT Tigecycline has good broad-spectrum activity against many gram-positive and gram-negative pathogens with the notable exception of the Proteeae. A study was performed to identify the mechanism responsible for the reduced susceptibility to tigecycline in Proteus mirabilis. Two independent transposon insertion mutants of P. mirabilis that had 16-fold-increased susceptibility to tigecycline were mapped to the acrB gene homolog of the Escherichia coli AcrRAB efflux system. Wild-type levels of decreased susceptibility to tigecycline were restored to the insertion mutants by complementation with a clone containing a PCR-derived fragment from the parental wild-type acrRAB efflux gene cluster. The AcrAB transport system appears to be associated with the intrinsic reduced susceptibility to tigecycline in P. mirabilis.

scholarly journals Single-Primer PCR Procedure for Rapid Identification of Transposon Insertion Sites

BioTechniques ◽  
2000 ◽  
Vol 28 (6) ◽  
pp. 1078-1082 ◽  
Author(s):  
Andrey V. Karlyshev ◽  
Mark J. Pallen ◽  
Brendan W. Wren
Keyword(s):  
Rapid Identification ◽  
Transposon Insertion ◽  
Insertion Sites ◽  
Single Primer
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