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Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene.

Melo SC, Santos RX, Melgaço AC, Pereira AC, Pungartnik C, Brendel M - Int J Mol Sci (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Temperature and carbon source-dependent growth of S. cerevisiae transformants harboring either plasmid pRS313 or pLBF01. Lines (fiveserial 1:10 dilutions): (1) SM01; (2) SM02; (3) SM03; (4) SM04.
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ijms-16-12324-f005: Temperature and carbon source-dependent growth of S. cerevisiae transformants harboring either plasmid pRS313 or pLBF01. Lines (fiveserial 1:10 dilutions): (1) SM01; (2) SM02; (3) SM03; (4) SM04.

Mentions: Another typical phenotype of Scsod2Δ is its sensitivity to lactate at different temperatures. Medium with glycerol or lactate shows that respiration on non-fermentable carbon sources generates superoxide radicals that inhibit the growth of cells lacking mitochondrial dismutase [71]. When we exposed the four yeast transformants to lactate, glucose and glycerol containing media at different growth temperatures (Figure 5), survival of the transformed yeast strains is influenced by the carbon source in the growth medium and the temperature.


Altered Phenotypes in Saccharomyces cerevisiae by Heterologous Expression of Basidiomycete Moniliophthora perniciosa SOD2 Gene.

Melo SC, Santos RX, Melgaço AC, Pereira AC, Pungartnik C, Brendel M - Int J Mol Sci (2015)

Temperature and carbon source-dependent growth of S. cerevisiae transformants harboring either plasmid pRS313 or pLBF01. Lines (fiveserial 1:10 dilutions): (1) SM01; (2) SM02; (3) SM03; (4) SM04.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490446&req=5

ijms-16-12324-f005: Temperature and carbon source-dependent growth of S. cerevisiae transformants harboring either plasmid pRS313 or pLBF01. Lines (fiveserial 1:10 dilutions): (1) SM01; (2) SM02; (3) SM03; (4) SM04.
Mentions: Another typical phenotype of Scsod2Δ is its sensitivity to lactate at different temperatures. Medium with glycerol or lactate shows that respiration on non-fermentable carbon sources generates superoxide radicals that inhibit the growth of cells lacking mitochondrial dismutase [71]. When we exposed the four yeast transformants to lactate, glucose and glycerol containing media at different growth temperatures (Figure 5), survival of the transformed yeast strains is influenced by the carbon source in the growth medium and the temperature.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus