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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

(A) Alignment of the MpSOD2 deduced amino acid sequence with MnSOD2 polypeptides from other organisms; Conserved residues are boxed in black frame. Fe/MnSod2p (a) N-terminal α hairpin domain; (b) C-terminal domain. Protein domains are in a black frame, full alignment not shown; (B) Nucleotide sequence of gene MpSOD2 and its protein sequence. (a) Highlighted are upstream a region defined as sequence of thymine-adenine-thymine-adenine TATA box (marked in bold letters), Kozak motif with start and stop codon (marked in bold letters). The highlighted gray sequence is the CDS (codon DNA sequence); (b) Highlighted are upstream region with the following sequence of aminoacids MANTLP (methionine, adenine, asparagine, threonine, leucine, proline) in basidiomycete fungi marked in bold letters from M. perniciosa and in a box frame from other organism.
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ijms-16-12324-f001: (A) Alignment of the MpSOD2 deduced amino acid sequence with MnSOD2 polypeptides from other organisms; Conserved residues are boxed in black frame. Fe/MnSod2p (a) N-terminal α hairpin domain; (b) C-terminal domain. Protein domains are in a black frame, full alignment not shown; (B) Nucleotide sequence of gene MpSOD2 and its protein sequence. (a) Highlighted are upstream a region defined as sequence of thymine-adenine-thymine-adenine TATA box (marked in bold letters), Kozak motif with start and stop codon (marked in bold letters). The highlighted gray sequence is the CDS (codon DNA sequence); (b) Highlighted are upstream region with the following sequence of aminoacids MANTLP (methionine, adenine, asparagine, threonine, leucine, proline) in basidiomycete fungi marked in bold letters from M. perniciosa and in a box frame from other organism.

Mentions: Sequence analysis of a putative MpSOD2 clone revealed that it contained a 748 bp insert with an ORF encoding a predicted polypeptide of 176 amino acids (aa) (Genbank accession No. XM2395448). Searches of GenBank using the BLASTP algorithm indicated that the predicted protein was similar to several proteins within the family of manganese-type superoxide dismutases (MnSODs, Figure 1A). Accordingly, the M. perniciosa gene was named MpSOD2. A Kozak consensus sequence [49] surrounds the putative SOD2 start codon (Figure 1B). There is an upstream consensus TATA box motif, but a 3ʹ AT-rich region (a region rich in residues of adenine and timine) is missing. The MpSOD2 cDNA contains a 132 bp 3ʹ-untranslated region. A consensus polyadenylation signal (AATAAA; [15]) is absent.


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)

(A) Alignment of the MpSOD2 deduced amino acid sequence with MnSOD2 polypeptides from other organisms; Conserved residues are boxed in black frame. Fe/MnSod2p (a) N-terminal α hairpin domain; (b) C-terminal domain. Protein domains are in a black frame, full alignment not shown; (B) Nucleotide sequence of gene MpSOD2 and its protein sequence. (a) Highlighted are upstream a region defined as sequence of thymine-adenine-thymine-adenine TATA box (marked in bold letters), Kozak motif with start and stop codon (marked in bold letters). The highlighted gray sequence is the CDS (codon DNA sequence); (b) Highlighted are upstream region with the following sequence of aminoacids MANTLP (methionine, adenine, asparagine, threonine, leucine, proline) in basidiomycete fungi marked in bold letters from M. perniciosa and in a box frame from other organism.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-12324-f001: (A) Alignment of the MpSOD2 deduced amino acid sequence with MnSOD2 polypeptides from other organisms; Conserved residues are boxed in black frame. Fe/MnSod2p (a) N-terminal α hairpin domain; (b) C-terminal domain. Protein domains are in a black frame, full alignment not shown; (B) Nucleotide sequence of gene MpSOD2 and its protein sequence. (a) Highlighted are upstream a region defined as sequence of thymine-adenine-thymine-adenine TATA box (marked in bold letters), Kozak motif with start and stop codon (marked in bold letters). The highlighted gray sequence is the CDS (codon DNA sequence); (b) Highlighted are upstream region with the following sequence of aminoacids MANTLP (methionine, adenine, asparagine, threonine, leucine, proline) in basidiomycete fungi marked in bold letters from M. perniciosa and in a box frame from other organism.
Mentions: Sequence analysis of a putative MpSOD2 clone revealed that it contained a 748 bp insert with an ORF encoding a predicted polypeptide of 176 amino acids (aa) (Genbank accession No. XM2395448). Searches of GenBank using the BLASTP algorithm indicated that the predicted protein was similar to several proteins within the family of manganese-type superoxide dismutases (MnSODs, Figure 1A). Accordingly, the M. perniciosa gene was named MpSOD2. A Kozak consensus sequence [49] surrounds the putative SOD2 start codon (Figure 1B). There is an upstream consensus TATA box motif, but a 3ʹ AT-rich region (a region rich in residues of adenine and timine) is missing. The MpSOD2 cDNA contains a 132 bp 3ʹ-untranslated region. A consensus polyadenylation signal (AATAAA; [15]) is absent.

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