scholarly journals Characterization of a Baculovirus Lacking the Alkaline Nuclease Gene

2004 ◽  
Vol 78 (19) ◽  
pp. 10650-10656 ◽  
Author(s):  
Kazuhiro Okano ◽  
Adam L. Vanarsdall ◽  
George F. Rohrmann
Keyword(s):  
Dna Recombination ◽  
Northern Analysis ◽  
Sf9 Cells ◽  
Wild Type ◽  
Reading Frame ◽  
Baculovirus Gene ◽  
Chloramphenicol Acetyltransferase Gene ◽  
Alkaline Nuclease

ABSTRACT The Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) alkaline nuclease (AN) associates with the baculovirus single-stranded DNA binding protein LEF-3 and possesses both a 5′→3′ exonuclease and an endonuclease activity. These activities are thought to be involved in DNA recombination and replication. To investigate the role of AN in AcMNPV replication, the λ Red system was used to replace the an open reading frame with a chloramphenicol acetyltransferase gene (cat) and a bacmid containing the AcMNPV genome in Escherichia coli. The AcMNPV an knockout bacmid (vAcAN-KO/GUS) was unable to propagate in Sf9 cells, although an an-rescued bacmid (vAcAN-KO/GUS-Res) propagated normally. In addition, the mutant did not appear to produce budded virions. These data indicated that an is an essential baculovirus gene. Slot blot and DpnI assays of DNA replication in Sf9 cells transfected with vAcAN-KO/GUS, vAcAN-KO/GUS-Res, and a wild-type bacmid showed that the vAcAN-KO/GUS bacmid was able to replicate to levels similar to those seen with the vAcAN-KO/GUS-Res and wild-type bacmids at early stages posttransfection. However, at later time points DNA did not accumulate to the levels seen with the repaired or wild-type bacmids. Northern analysis of Sf9 cells transfected with bacmid vAcAN-KO/GUS showed that transcription of late and very late genes was lower at later times posttransfection relative to the results seen with wild-type and vAcAN-KO/GUS-Res bacmids. These data suggest that the an gene might be involved in the maturation of viral DNA or packaging of the DNA into virions.

scholarly journals Characterization of a Baculovirus Alkaline Nuclease

2000 ◽  
Vol 74 (14) ◽  
pp. 6401-6407 ◽  
Author(s):  
Lulin Li ◽  
George F. Rohrmann
Keyword(s):  
Essential Gene ◽  
Point Mutations ◽  
Reading Frame ◽  
Baculovirus Gene ◽  
Salt Requirement ◽  
Low Levels ◽  
Alkaline Nuclease ◽  
Maximal Expression ◽  
Rule Out

ABSTRACT All baculovirus genomes sequenced to date encode a homolog of an alkaline nuclease that has been characterized in theHerpesviridae. In this report we describe the characterization of the alkaline nuclease (AN) homolog of theAutographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) (open reading frame 133). His-tagged AN constructs were expressed in recombinant baculoviruses and affinity purified, and then their enzymatic activity was characterized. AN was found to degrade linear DNA at alkaline pH, preferred Mg2+ over Mn2+, had optimal activity at 35°C, and did not appear to have a salt requirement. To rule out contamination by the endogenous baculovirus gene product or a cellular enzyme, point mutations were introduced into a highly conserved domain of the gene. These mutations were found to markedly reduce or eliminate most of the activity of the affinity-purified enzyme. An antibody generated against the protein was used to analyze its expression by Western blot analysis. AN was found to be expressed at low levels by 12 h postinfection, with maximal expression at 24 h postinfection. Attempts to generate a virus with this gene inactivated were unsuccessful, suggesting that AN may be encoded by an essential gene.

scholarly journals The Conserved Poxvirus L3 Virion Protein Is Required for Transcription of Vaccinia Virus Early Genes

2005 ◽  
Vol 79 (23) ◽  
pp. 14719-14729 ◽  
Author(s):  
Wolfgang Resch ◽  
Bernard Moss
Keyword(s):  
Vaccinia Virus ◽  
Promoter Sequence ◽  
Wild Type ◽  
Virion Protein ◽  
Reading Frame ◽  
Virus Particles ◽  
Mature Virus

ABSTRACT We provide the initial characterization of the product of the vaccinia virus L3L open reading frame (VACWR090), which is conserved in all sequenced members of the poxvirus family. The predicted polypeptide contains no motifs or other features that provided a clue to the role of the L3 protein, and no functional information was available regarding a homolog discovered in Plasmodium falciparum. The L3 protein was expressed following viral DNA replication, a finding consistent with a putative late promoter sequence, and was packaged as a non-membrane protein in mature virus particles. A recombinant virus, in which the L3L gene was regulated by the Escherichia coli lac operator/repressor system, had a conditional lethal phenotype. The virus replicated in the presence of inducer, but in its absence, the yields of infectious virus were reduced by 99%. When cells were infected without inducer, however, no defect in gene expression or morphogenesis was noted. Virus particles lacking L3, which assembled in the absence of inducer, were indistinguishable from wild-type virions with regard to morphology, major structural proteins, and DNA content but were noninfectious. L3-deficient virions were able to bind and penetrate cells but produced extremely small amounts of viral early mRNA. A defect in transcription was demonstrated by in vitro studies with permeabilized virions, but soluble extracts of L3-deficient virions showed normal levels of template-dependent transcriptional activity, indicating that only transcription of the packaged genome is impaired.

scholarly journals Isolation and characterization of SGE1: a yeast gene that partially suppresses the gal11 mutation in multiple copies.

Genetics ◽  
1993 ◽  
Vol 134 (3) ◽  
pp. 675-683
Author(s):  
H Amakasu ◽  
Y Suzuki ◽  
M Nishizawa ◽  
T Fukasawa
Keyword(s):  
Carbon Sources ◽  
Northern Analysis ◽  
Wild Type ◽  
Reading Frame ◽  
Recessive Mutations ◽  
Isolation And Characterization ◽  
Multiple Copies ◽  
The Right ◽  
Inducible Genes

Abstract Recessive mutations of GAL11 in Saccharomyces cerevisiae cause pleiotropic defects that include weak fermentation of galactose, alpha-specific sterility and slow growth on nonfermentable carbon sources. Recent experiments suggest that Gal11P functions as a "co-activator" that links transcriptional activators, such as Gal4p and Grf1p/Rap1p/Tuf1p, with the basic transcription machinery. In the present experiments we isolated a gene, SGE1, that suppresses gal11 for growth on ethidium bromide/galactose agar when the gene was present in two or more copies. The other gal11 phenotypes were not suppressed by SGE1 in the multiple-copy state. Multiple copies of SGE1 increased expression of galactose-inducible genes in gal11 yeast, presumably at the level of transcription. When SGE1 was disrupted in wild-type yeast, the expression of galactose-inducible genes decreased to 50-60% of the wild-type level, presumably due to effect on transcription. Complete DNA sequence analysis revealed that SGE1 encodes a predicted protein of 543 amino acids. SGE1-specific mRNA of 1.8 kilonucleotides was detected by Northern analysis along the direction of the open reading frame. The gene mapped near RAD56, at the right end of chromosome 16.

Characterization of the Role of the FluG Protein in Asexual Development of Aspergillus nidulans

Genetics ◽  
2001 ◽  
Vol 158 (3) ◽  
pp. 1027-1036 ◽  
Author(s):  
Cletus A D'Souza ◽  
Bee Na Lee ◽  
Thomas H Adams
Keyword(s):  
Aspergillus Nidulans ◽  
Negative Influence ◽  
Asexual Development ◽  
Wild Type ◽  
Significant Similarity ◽  
Developmental Defects ◽  
Asexual Sporulation ◽  
Type Strains

Abstract We showed previously that a ΔfluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG (∼400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of ΔfluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from ΔflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

scholarly journals Genetic analysis of the Arabidopsis TIR1/AFB auxin receptors reveals both overlapping and specialized functions

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Michael J Prigge ◽  
Matthieu Platre ◽  
Nikita Kadakia ◽  
Yi Zhang ◽  
Kathleen Greenham ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Early Embryo ◽  
Wild Type ◽  
The Family ◽  
Dependent Inhibition ◽  
Root Gravitropism ◽  
Specialized Function ◽  
Mutant Lines

The TIR1/AFB auxin co-receptors mediate diverse responses to the plant hormone auxin. The Arabidopsis genome encodes six TIR1/AFB proteins representing three of the four clades that were established prior to angiosperm radiation. To determine the role of these proteins in plant development we performed an extensive genetic analysis involving the generation and characterization of all possible multiply-mutant lines. We find that loss of all six TIR1/AFB proteins results in early embryo defects and eventually seed abortion, and yet a single wild-type allele of TIR1 or AFB2 is sufficient to support growth throughout development. Our analysis reveals extensive functional overlap between even the most distantly related TIR1/AFB genes except for AFB1. Surprisingly, AFB1 has a specialized function in rapid auxin-dependent inhibition of root growth and early phase of root gravitropism. This activity may be related to a difference in subcellular localization compared to the other members of the family.

scholarly journals Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

2013 ◽  
Vol 57 (11) ◽  
pp. 5658-5664 ◽  
Author(s):  
Soo-Jin Yang ◽  
Nagendra N. Mishra ◽  
Aileen Rubio ◽  
Arnold S. Bayer
Keyword(s):  
Staphylococcus Aureus ◽  
Single Nucleotide Polymorphisms ◽  
Point Mutations ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Single Nucleotide ◽  
Content Type ◽  
Reading Frame ◽  
A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

Effect of temperature on transition and transversion mutagenesis: characterization of wild type and a mutator T4 DNA polymerase mutant

1984 ◽  
Vol 26 (3) ◽  
pp. 386-389 ◽  
Author(s):  
Linda J. Reha-Krantz ◽  
Sükran Parmaksizoglu
Keyword(s):  
Dna Polymerase ◽  
Low Temperatures ◽  
Bacteriophage T4 ◽  
Effect Of Temperature ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Mutational Site

The effect of temperature on genetically well-defined mutational pathways was examined in the bacteriophage T4. The mutational site was a T4 rII ochre mutant which could revert to rII+ via a transversion or to the amber convertant via a transition. Temperature did not strongly affect any of the pathways examined in a wild-type background; however, increased temperature reduced the mutational activity of a mutator DNA polymerase mutant. Possible models to explain the role of temperature in mutagenesis are discussed as well as the significance of low temperatures for in vitro mutagenesis reactions.Key words: bacteriophage T4, mutator, transition, transversion, temperature effects.

scholarly journals The AC4 Protein of a Cassava Geminivirus Is Required for Virus Infection

2019 ◽  
Vol 32 (7) ◽  
pp. 865-875 ◽  
Author(s):  
Kegui Chen ◽  
Behnam Khatabi ◽  
Vincent N. Fondong
Keyword(s):  
Plant Viruses ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Type Virus ◽  
Wild Type ◽  
Knockout Mutant ◽  
East African ◽  
Reading Frame ◽  
Cell Membrane Binding

Geminiviruses (family Geminiviridae) are among the most devastating plant viruses worldwide, causing severe damage in crops of economic and subsistence importance. These viruses have very compact genomes and many of the encoded proteins are multifunctional. Here, we investigated the role of the East African cassava mosaic Cameroon virus (EACMCV) AC4 on virus infectivity in Nicotiana benthamiana. Results showed that plants inoculated with EACMCV containing a knockout mutation in an AC4 open reading frame displayed symptoms 2 to 3 days later than plants inoculated with wild-type virus, and these plants recovered from infection, whereas plants inoculated with the wild-type virus did not. Curiously, when an additional mutation was made in the knockout mutant, the resulting double mutant virus completely failed to cause any apparent symptoms. Interestingly, the role of AC4 on virus infectivity appeared to be dependent on an encoded N-myristoylation motif that mediates cell membrane binding. We previously showed that EACMCV containing the AC4T38I mutant produced virus progeny characterized by second-site mutations and reversion to wild-type virus. These results were confirmed in this study using additional mutations. Together, these results show involvement of EACMCV AC4 in virus infectivity; they also suggest a role for the combined action of mutation and selection, under prevailing environmental conditions, on begomovirus genetic variation and diversity.

scholarly journals Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

2015 ◽  
Vol 59 (8) ◽  
pp. 4669-4679 ◽  
Author(s):  
Nilmar Silvio Moretti ◽  
Leonardo da Silva Augusto ◽  
Tatiana Mordente Clemente ◽  
Raysa Paes Pinto Antunes ◽  
Nobuko Yoshida ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Drug Targets ◽  
Wild Type ◽  
Content Type ◽  
Damage Repair ◽  
Lysine Deacetylases

ABSTRACTAcetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD+-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation ofTrypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion byT. cruziat concentrations that did not affect host cell viability. In addition,in vivoinfection was partially controlled upon administration of salermide. There are two sirtuins inT. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed inEscherichia coliwith a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

scholarly journals Characterization of Biofilm Formation and the Role of BCR1 in Clinical Isolates of Candida parapsilosis

2013 ◽  
Vol 13 (4) ◽  
pp. 438-451 ◽  
Author(s):  
Srisuda Pannanusorn ◽  
Bernardo Ramírez-Zavala ◽  
Heinrich Lünsdorf ◽  
Birgitta Agerberth ◽  
Joachim Morschhäuser ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Candida Parapsilosis ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type ◽  
Initial Adhesion ◽  
High Level ◽  
Increased Susceptibility

ABSTRACT In Candida parapsilosis , biofilm formation is considered to be a major virulence factor. Previously, we determined the ability of 33 clinical isolates causing bloodstream infection to form biofilms and identified three distinct groups of biofilm-forming strains (negative, low, and high). Here, we establish two different biofilm structures among strains forming large amounts of biofilm in which strains with complex spider-like structures formed robust biofilms on different surface materials with increased resistance to fluconazole. Surprisingly, the transcription factor Bcr1, required for biofilm formation in Candida albicans and C. parapsilosis , has an essential role only in strains with low capacity for biofilm formation. Although BCR1 leads to the formation of more and longer pseudohyphae, it was not required for initial adhesion and formation of mature biofilms in strains with a high level of biofilm formation. Furthermore, an additional phenotype affected by BCR1 was the switch in colony morphology from rough to crepe, but only in strains forming high levels of biofilm. All bcr1 Δ/Δ mutants showed increased proteolytic activity and increased susceptibility to the antimicrobial peptides protamine and RP-1 compared to corresponding wild-type and complemented strains. Taken together, our results demonstrate that biofilm formation in clinical isolates of C. parapsilosis is both dependent and independent of BCR1 , but even in strains which showed a BCR1 -independent biofilm phenotype, BCR1 has alternative physiological functions.

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