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1.
J Photochem Photobiol B ; 130: 241-53, 2014 Jan 05.
Article in English | MEDLINE | ID: mdl-24362320

ABSTRACT

By isolating putative binding partners through the two-hybrid system (THS) we further extended the characterization of the specific interstrand cross-link (ICL) repair gene PSO2 of Saccharomyces cerevisiae. Nine fusion protein products were isolated for Pso2p using THS, among them the Sak1 kinase, which interacted with the C-terminal ß-CASP domain of Pso2p. Comparison of mutagen-sensitivity phenotypes of pso2Δ, sak1Δ and pso2Δsak1Δ disruptants revealed that SAK1 is necessary for complete WT-like repair. The epistatic interaction of both mutant alleles suggests that Sak1p and Pso2p act in the same pathway of controlling sensitivity to DNA-damaging agents. We also observed that Pso2p is phosphorylated by Sak1 kinase in vitro and co-immunoprecipitates with Sak1p after 8-MOP+UVA treatment. Survival data after treatment of pso2Δ, yku70Δ and yku70Δpso2Δ with nitrogen mustard, PSO2 and SAK1 with YKU70 or DNL4 single-, double- and triple mutants with 8-MOP+UVA indicated that ICL repair is independent of YKu70p and DNL4p in S. cerevisiae. Furthermore, a non-epistatic interaction was observed between MRE11, PSO2 and SAK1 genes after ICL induction, indicating that their encoded proteins act on the same substrate, but in distinct repair pathways. In contrast, an epistatic interaction was observed for PSO2 and RAD52, PSO2 and RAD50, PSO2 and XRS2 genes in 8-MOP+UVA treated exponentially growing cells.


Subject(s)
DNA Damage , Endodeoxyribonucleases/genetics , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae Proteins/genetics , Cross-Linking Reagents/pharmacology , DNA-Binding Proteins/genetics , Methoxsalen/pharmacology , Rad52 DNA Repair and Recombination Protein/genetics , Saccharomyces cerevisiae , Two-Hybrid System Techniques , Ultraviolet Rays
2.
Protein Expr Purif ; 69(2): 132-6, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19747547

ABSTRACT

Mycoplasma hyopneumoniae, the etiological agent of swine enzootic pneumonia, is an important pathogen in the swine industry worldwide. Vaccination is the most cost-effective strategy for controlling and prevention of this disease. However, investigations on pathogenicity mechanisms as well as current serological detection methods and the development of new recombinant subunit vaccines are hampered by the lack of known and well characterized species-specific M. hyopneumoniae antigens. In this work, 54 predicted genes encoding proteins with potential to be used as subunit vaccine or antigens in diagnostic tests were selected, amplified by PCR and cloned into Escherichia coli expression vectors. Recombinant protein expression, solubility and yields were analyzed. The majority of the recombinant proteins were expressed in inclusion bodies. After solubilization with urea or N-lauroyl sarcosine, recombinant proteins were purified by Ni(2+) affinity chromatography. This approach allowed purification of thirty recombinant M. hyopneumoniae proteins which will be evaluated as vaccine candidates and/or as antigens to be used in diagnostic tests.


Subject(s)
Escherichia coli , Mycoplasma hyopneumoniae , Recombinant Proteins , Animals , Antigens, Bacterial/immunology , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Bacterial Vaccines/genetics , Bacterial Vaccines/immunology , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Mycoplasma hyopneumoniae/genetics , Mycoplasma hyopneumoniae/metabolism , Pneumonia of Swine, Mycoplasmal/microbiology , Pneumonia of Swine, Mycoplasmal/prevention & control , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Swine , Vaccines, Subunit/genetics , Vaccines, Subunit/immunology , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunology
3.
J Bacteriol ; 187(16): 5568-77, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16077101

ABSTRACT

This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.


Subject(s)
Genome, Bacterial , Mycoplasma Infections/microbiology , Mycoplasma hyopneumoniae/genetics , Mycoplasma synoviae/genetics , Pneumonia of Swine, Mycoplasmal/microbiology , Poultry Diseases/microbiology , Animals , Evolution, Molecular , Gene Rearrangement , Gene Transfer, Horizontal , Genomics , Molecular Sequence Data , Phylogeny , Poultry , Swine
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