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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

Sensitivity of stationary phase (STAT) cultures of yeast transformants after exposure to (A) H2O2; (B) Paraquat (PAQ); (C) DEN; and (D) UVC. Symbol legend: □ = WT(pRS313); ■ = WT(pLBF01); ○ = Scsod2Δ(pRS313); ● = Scsod2Δ(pLBF01). Statistical analyses of the influence of MpSOD2 on the sensitivity phenotype of (E) WT and Scsod2Δ mutant compared amongst themselves in each treatment; (F) WT compared to Scsod2Δ mutant in each treatment. ns = not significant; * p < 0.04; *** p < 0.001.
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ijms-16-12324-f004: Sensitivity of stationary phase (STAT) cultures of yeast transformants after exposure to (A) H2O2; (B) Paraquat (PAQ); (C) DEN; and (D) UVC. Symbol legend: □ = WT(pRS313); ■ = WT(pLBF01); ○ = Scsod2Δ(pRS313); ● = Scsod2Δ(pLBF01). Statistical analyses of the influence of MpSOD2 on the sensitivity phenotype of (E) WT and Scsod2Δ mutant compared amongst themselves in each treatment; (F) WT compared to Scsod2Δ mutant in each treatment. ns = not significant; * p < 0.04; *** p < 0.001.

Mentions: Exposure of the four transformants to different mutagens allowed checking for phenotype complementation (Figure 4). The presence of MpSOD2 had no significant influence on the WT sensitivity phenotype (SM02) except when exposing the transformant to Paraquat (PAQ). Although the Scsod2Δ mutant is only slightly sensitive to H2O2 when compared to the WT (SM03 vs. SM01) the change of mutagen-sensitivity phenotype of pLBF01-transformed Scsod2Δ mutant (SM04) was always highly significant, except for UVC (Figure 4D, two-way analyses of variance—ANOVA). The MpSOD2-containing WT transformant had a significantly altered phenotype after exposure to PAQ and DEN (Figure 4B). WT and sod2Δ mutant transformed with empty pRS313 (SM01 and SM03) had the same phenotypes as non-transformed cells to all tested mutagens.


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)

Sensitivity of stationary phase (STAT) cultures of yeast transformants after exposure to (A) H2O2; (B) Paraquat (PAQ); (C) DEN; and (D) UVC. Symbol legend: □ = WT(pRS313); ■ = WT(pLBF01); ○ = Scsod2Δ(pRS313); ● = Scsod2Δ(pLBF01). Statistical analyses of the influence of MpSOD2 on the sensitivity phenotype of (E) WT and Scsod2Δ mutant compared amongst themselves in each treatment; (F) WT compared to Scsod2Δ mutant in each treatment. ns = not significant; * p < 0.04; *** p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-12324-f004: Sensitivity of stationary phase (STAT) cultures of yeast transformants after exposure to (A) H2O2; (B) Paraquat (PAQ); (C) DEN; and (D) UVC. Symbol legend: □ = WT(pRS313); ■ = WT(pLBF01); ○ = Scsod2Δ(pRS313); ● = Scsod2Δ(pLBF01). Statistical analyses of the influence of MpSOD2 on the sensitivity phenotype of (E) WT and Scsod2Δ mutant compared amongst themselves in each treatment; (F) WT compared to Scsod2Δ mutant in each treatment. ns = not significant; * p < 0.04; *** p < 0.001.
Mentions: Exposure of the four transformants to different mutagens allowed checking for phenotype complementation (Figure 4). The presence of MpSOD2 had no significant influence on the WT sensitivity phenotype (SM02) except when exposing the transformant to Paraquat (PAQ). Although the Scsod2Δ mutant is only slightly sensitive to H2O2 when compared to the WT (SM03 vs. SM01) the change of mutagen-sensitivity phenotype of pLBF01-transformed Scsod2Δ mutant (SM04) was always highly significant, except for UVC (Figure 4D, two-way analyses of variance—ANOVA). The MpSOD2-containing WT transformant had a significantly altered phenotype after exposure to PAQ and DEN (Figure 4B). WT and sod2Δ mutant transformed with empty pRS313 (SM01 and SM03) had the same phenotypes as non-transformed cells to all tested mutagens.

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