title: Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene.
abstract: Heterologous expression of a putative manganese superoxide dismutase gene (SOD2) of the basidiomycete Moniliophthora perniciosa complemented the phenotypes of a Saccharomyces cerevisiae sod2Δ mutant. Sequence analysis of the cloned M. perniciosa cDNA revealed an open reading frame (ORF) coding for a 176 amino acid polypeptide with the typical metal-binding motifs of a SOD2 gene, named MpSOD2. Phylogenetic comparison with known manganese superoxide dismutases (MnSODs) located the protein of M. perniciosa (MpSod2p) in a clade with the basidiomycete fungi Coprinopsis cinerea and Laccaria bicolor. Haploid wild-type yeast transformants containing a single copy of MpSOD2 showed increased resistance phenotypes against oxidative stress-inducing hydrogen peroxide and paraquat, but had unaltered phenotype against ultraviolet-C (UVC) radiation. The same transformants exhibited high sensitivity against treatment with the pro-mutagen diethylnitrosamine (DEN) that requires oxidation to become an active mutagen/carcinogen. Absence of MpSOD2 in the yeast sod2Δ mutant led to DEN hyper-resistance while introduction of a single copy of this gene restored the yeast wild-type phenotype. The haploid yeast wild-type transformant containing two SOD2 gene copies, one from M. perniciosa and one from its own, exhibited DEN super-sensitivity. This transformant also showed enhanced growth at 37 °C on the non-fermentable carbon source lactate, indicating functional expression of MpSod2p. The pro-mutagen dihydroethidium (DHE)-based fluorescence assay monitored basal level of yeast cell oxidative stress. Compared to the wild type, the yeast sod2Δ mutant had a much higher level of intrinsic oxidative stress, which was reduced to wild type (WT) level by introduction of one copy of the MpSOD2 gene. Taken together our data indicates functional expression of MpSod2 protein in the yeast S. cerevisiae.
authors.affiliation: Departamento de Ciências Biológicas, Laboratório de Biologia de Fungos, Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz (UESC), Rodovia Jorge Amado, km 16, Ilhéus, Bahia CEP 45662-900, Brazil. scmelo@uesc.br.
authors.forename: Sônia C
authors.initials: SC
authors.lastname: Melo
authors.affiliation: Departamento de Ciências Naturais, Universidade Estadual do Sudoeste da Bahia (UESB), Estrada do Bem Querer, km 4, Vitória da Conquista, Bahia CEP 45083-900, Brazil. sxneide@gmail.com.
authors.forename: Regineide X
authors.initials: RX
authors.lastname: Santos
authors.affiliation: Departamento de Ciências Biológicas, Laboratório de Biologia de Fungos, Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz (UESC), Rodovia Jorge Amado, km 16, Ilhéus, Bahia CEP 45662-900, Brazil. anaclarame@hotmail.com.
authors.forename: Ana C
authors.initials: AC
authors.lastname: Melgaço
authors.affiliation: Departamento de Ciências Biológicas, Laboratório de Biologia de Fungos, Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz (UESC), Rodovia Jorge Amado, km 16, Ilhéus, Bahia CEP 45662-900, Brazil. bellefp@gmail.com.
authors.forename: Alanna C F
authors.initials: AC
authors.lastname: Pereira
authors.affiliation: Departamento de Ciências Biológicas, Laboratório de Biologia de Fungos, Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz (UESC), Rodovia Jorge Amado, km 16, Ilhéus, Bahia CEP 45662-900, Brazil. cpungartnik@yahoo.com.br.
authors.forename: Cristina
authors.initials: C
authors.lastname: Pungartnik
authors.affiliation: Departamento de Ciências Biológicas, Laboratório de Biologia de Fungos, Centro de Biotecnologia e Genética, Universidade Estadual de Santa Cruz (UESC), Rodovia Jorge Amado, km 16, Ilhéus, Bahia CEP 45662-900, Brazil. martinbrendel@yahoo.com.br.
authors.forename: Martin
authors.initials: M
authors.lastname: Brendel
mesh_terms: D001487:Basidiomycota / Q000145:classification / Q000235:genetics*; D004052:Diethylnitrosamine / Q000494:pharmacology; D024881:Drug Resistance, Bacterial; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009154:Mutation; D018384:Oxidative Stress / Q000187:drug effects; D010269:Paraquat / Q000494:pharmacology; D010802:Phylogeny; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development*; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D004052:Diethylnitrosamine; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D010269:Paraquat
keywords: diethylnitrosamine super-sensitivity; functional heterologous expression; manganese superoxide dismutase; reactive oxygen species