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The Tetrahymena metallothionein gene family: twenty-one new cDNAs, molecular characterization, phylogenetic study and comparative analysis of the gene expression under different abiotic stressors.

de Francisco P, Melgar LM, Díaz S, Martín-González A, Gutiérrez JC - BMC Genomics (2016)

Bottom Line: The Tetrahymena metallothionein family represents a quite conserved protein structure group with unique features with respect to standard MTs.Both Cd- and CuMT subfamilies present very defined and differentiated characteristics at several levels: cysteine patterns, modular structure, glutamine codon usage and gene expression under metal stress, among others.Gene duplication through evolution seems to be the major genetic mechanism for creating new MT gene isoforms and increasing their functional diversity.

View Article: PubMed Central - PubMed

Affiliation: Departamento Microbiología-III, Facultad de Biología. C/José Antonio Novais, 12, Universidad Complutense de Madrid (UCM), 28040, Madrid, Spain.

ABSTRACT

Background: Ciliate metallothioneins (MTs) are included in family 7 of the MT superfamily. This family has been divided into two main subfamilies: 7a or CdMTs and 7b or CuMTs. All ciliate MTs reported have been isolated from different Tetrahymena species and present unique features with regard to standard MTs. Likewise, an expression analysis has been carried out on some of MT genes under metal stress, corroborating their classification into two subfamilies.

Results: We isolated 21 new cDNAs from different Tetrahymena species to obtain a wider view of the biodiversity of these conserved genes. Structural analysis (cysteine patterns) and an updated phylogenetic study both corroborated the previous classification into two subfamilies. A new CuMT from a Tetrahymena-related species Ichthyophthirius multifiliis was also included in this general analysis. We detected a certain tendency towards the presentation of a CdMT tri-modular structure in Borealis group species with respect to Australis group. We report for the first time a semi-complete paralog duplication of a CdMT gene originating a new CdMT gene isoform in T. malaccensis. An asymmetry of the codon usage for glutamine residues was detected between Cd- and CuMTs, and the phylogenetic implications are discussed. A comparative gene expression analysis of several MT genes by qRT-PCR revealed differential behavior among them under different abiotic stressors in the same Tetrahymena species.

Conclusions: The Tetrahymena metallothionein family represents a quite conserved protein structure group with unique features with respect to standard MTs. Both Cd- and CuMT subfamilies present very defined and differentiated characteristics at several levels: cysteine patterns, modular structure, glutamine codon usage and gene expression under metal stress, among others. Gene duplication through evolution seems to be the major genetic mechanism for creating new MT gene isoforms and increasing their functional diversity.

No MeSH data available.


Related in: MedlinePlus

qRT-PCR analysis of six Tetrahymena MT genes after apoptotic or oxidative stress treatments. Relative expression levels for each MT gene are shown in the different histograms, after cellular treatment with different oxidative or apoptotic stressors, during exposures of 1 h (grey bars) or 24 h (black bars). Each bar of the histogram corresponds to an average value ± SD of two or three independent experiments. Asterisks indicate significant differences from control at p < 0.05. Numbers on each bar indicate the corresponding average fold induction value for each treatment. A gene expression induction is considered positive when the fold-induction value obtained is > 2 (indicated by the dashed line). See Table 1 for MT gene identification
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Fig7: qRT-PCR analysis of six Tetrahymena MT genes after apoptotic or oxidative stress treatments. Relative expression levels for each MT gene are shown in the different histograms, after cellular treatment with different oxidative or apoptotic stressors, during exposures of 1 h (grey bars) or 24 h (black bars). Each bar of the histogram corresponds to an average value ± SD of two or three independent experiments. Asterisks indicate significant differences from control at p < 0.05. Numbers on each bar indicate the corresponding average fold induction value for each treatment. A gene expression induction is considered positive when the fold-induction value obtained is > 2 (indicated by the dashed line). See Table 1 for MT gene identification

Mentions: MT gene expression, including both Tetrahymena Cd- and CuMT genes, is induced by oxidative stress originated by H2O2 or organic compounds, such as Paraquat (PQ) or Menadione (MD) [19–21, 23, 26]. However, the fold-induction values obtained after PQ treatment are generally very low or (Fig. 7). On the other hand, fold-induction values for MD (1 h treatment) are generally significantly higher than those obtained with PQ treatment (Fig. 7). Both compounds are pro-oxidants generating superoxide anions through redox cycling [62, 63], and they are known to potentially activate the transcription of some MTs [64], such as CUP1 from S. cerevisiae [65]. Certain antioxidant ability for both T. thermophila MTs (TtheMTT1 and TtheMTT2) has been suggested, because of the appearance of disulfide bonds in CdMT complexes after in vitro reaction with NO [40]. However, as shown by both previous results and our own (Fig. 7), other Tetrahymena CdMT genes (TpyrMT-2 and TrosMTT1) are not significantly induced by H2O2 or PQ [20, 23]. Likewise, the apoptosis inductor camptothecin (CAM) does not seem to be a good inducer of Tetrahymena MT gene expression, except in the case of the TborMTT7 gene, which is slightly induced (≈4.5- fold) after 24 h treatment (Fig. 7).Fig. 7


The Tetrahymena metallothionein gene family: twenty-one new cDNAs, molecular characterization, phylogenetic study and comparative analysis of the gene expression under different abiotic stressors.

de Francisco P, Melgar LM, Díaz S, Martín-González A, Gutiérrez JC - BMC Genomics (2016)

qRT-PCR analysis of six Tetrahymena MT genes after apoptotic or oxidative stress treatments. Relative expression levels for each MT gene are shown in the different histograms, after cellular treatment with different oxidative or apoptotic stressors, during exposures of 1 h (grey bars) or 24 h (black bars). Each bar of the histogram corresponds to an average value ± SD of two or three independent experiments. Asterisks indicate significant differences from control at p < 0.05. Numbers on each bar indicate the corresponding average fold induction value for each treatment. A gene expression induction is considered positive when the fold-induction value obtained is > 2 (indicated by the dashed line). See Table 1 for MT gene identification
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4862169&req=5

Fig7: qRT-PCR analysis of six Tetrahymena MT genes after apoptotic or oxidative stress treatments. Relative expression levels for each MT gene are shown in the different histograms, after cellular treatment with different oxidative or apoptotic stressors, during exposures of 1 h (grey bars) or 24 h (black bars). Each bar of the histogram corresponds to an average value ± SD of two or three independent experiments. Asterisks indicate significant differences from control at p < 0.05. Numbers on each bar indicate the corresponding average fold induction value for each treatment. A gene expression induction is considered positive when the fold-induction value obtained is > 2 (indicated by the dashed line). See Table 1 for MT gene identification
Mentions: MT gene expression, including both Tetrahymena Cd- and CuMT genes, is induced by oxidative stress originated by H2O2 or organic compounds, such as Paraquat (PQ) or Menadione (MD) [19–21, 23, 26]. However, the fold-induction values obtained after PQ treatment are generally very low or (Fig. 7). On the other hand, fold-induction values for MD (1 h treatment) are generally significantly higher than those obtained with PQ treatment (Fig. 7). Both compounds are pro-oxidants generating superoxide anions through redox cycling [62, 63], and they are known to potentially activate the transcription of some MTs [64], such as CUP1 from S. cerevisiae [65]. Certain antioxidant ability for both T. thermophila MTs (TtheMTT1 and TtheMTT2) has been suggested, because of the appearance of disulfide bonds in CdMT complexes after in vitro reaction with NO [40]. However, as shown by both previous results and our own (Fig. 7), other Tetrahymena CdMT genes (TpyrMT-2 and TrosMTT1) are not significantly induced by H2O2 or PQ [20, 23]. Likewise, the apoptosis inductor camptothecin (CAM) does not seem to be a good inducer of Tetrahymena MT gene expression, except in the case of the TborMTT7 gene, which is slightly induced (≈4.5- fold) after 24 h treatment (Fig. 7).Fig. 7

Bottom Line: The Tetrahymena metallothionein family represents a quite conserved protein structure group with unique features with respect to standard MTs.Both Cd- and CuMT subfamilies present very defined and differentiated characteristics at several levels: cysteine patterns, modular structure, glutamine codon usage and gene expression under metal stress, among others.Gene duplication through evolution seems to be the major genetic mechanism for creating new MT gene isoforms and increasing their functional diversity.

View Article: PubMed Central - PubMed

Affiliation: Departamento Microbiología-III, Facultad de Biología. C/José Antonio Novais, 12, Universidad Complutense de Madrid (UCM), 28040, Madrid, Spain.

ABSTRACT

Background: Ciliate metallothioneins (MTs) are included in family 7 of the MT superfamily. This family has been divided into two main subfamilies: 7a or CdMTs and 7b or CuMTs. All ciliate MTs reported have been isolated from different Tetrahymena species and present unique features with regard to standard MTs. Likewise, an expression analysis has been carried out on some of MT genes under metal stress, corroborating their classification into two subfamilies.

Results: We isolated 21 new cDNAs from different Tetrahymena species to obtain a wider view of the biodiversity of these conserved genes. Structural analysis (cysteine patterns) and an updated phylogenetic study both corroborated the previous classification into two subfamilies. A new CuMT from a Tetrahymena-related species Ichthyophthirius multifiliis was also included in this general analysis. We detected a certain tendency towards the presentation of a CdMT tri-modular structure in Borealis group species with respect to Australis group. We report for the first time a semi-complete paralog duplication of a CdMT gene originating a new CdMT gene isoform in T. malaccensis. An asymmetry of the codon usage for glutamine residues was detected between Cd- and CuMTs, and the phylogenetic implications are discussed. A comparative gene expression analysis of several MT genes by qRT-PCR revealed differential behavior among them under different abiotic stressors in the same Tetrahymena species.

Conclusions: The Tetrahymena metallothionein family represents a quite conserved protein structure group with unique features with respect to standard MTs. Both Cd- and CuMT subfamilies present very defined and differentiated characteristics at several levels: cysteine patterns, modular structure, glutamine codon usage and gene expression under metal stress, among others. Gene duplication through evolution seems to be the major genetic mechanism for creating new MT gene isoforms and increasing their functional diversity.

No MeSH data available.


Related in: MedlinePlus