Limits...
Tetrahymena metallothioneins fall into two discrete subfamilies.

Díaz S, Amaro F, Rico D, Campos V, Benítez L, Martín-González A, Hamilton EP, Orias E, Gutiérrez JC - PLoS ONE (2007)

Bottom Line: We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5).Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs.EST evidence for alternative splicing in the 3' UTR of the MTT5 mRNA with potential regulatory activity is reported.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología-III, Facultad de Biología, Universidad Complutense (UCM), Spain.

ABSTRACT

Background: Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals.

Methodology/principal findings: We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3' UTR of the MTT5 mRNA with potential regulatory activity is reported.

Conclusion/significance: The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances.

Show MeSH

Related in: MedlinePlus

Relative expression levels of Tetrahymena thermophila MTT1, MTT3 and MTT5 genes obtained by quantitative RT-PCR.Panels A, B and C: fold-induction for each gene (MTT1, MTT3, and MTT5, respectively) after treatment by different heavy metals. Gene expression levels are shown relative to an untreated control (which is set at 1±0.0 for every gene). Normalization of expression was achieved against the amplification of an endogenous gene (α-tubulin). Each bar of the histogram corresponds to an average value ±SD of two or three independent experiments. Heavy metal concentrations are as reported in the Material & Methods; Cd+ refers to 27 µM Cd plus 80 µM Cu; treatments times were: 1 h (light bars) and 24 h (dark bars) for all heavy metals. See Table S2 for numerical values. Panel D. Relative expression levels for each gene, in the order MTT1 (blue bars), MTT3 (green bars) and MTT5 (red bars), after induction by stress treatments other than heavy metals. Temperature treatments were for 2h, pH and Paraquat treatments were for 24 h, while starvation treatments were for 24 hrs and 4 days.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1808422&req=5

pone-0000291-g006: Relative expression levels of Tetrahymena thermophila MTT1, MTT3 and MTT5 genes obtained by quantitative RT-PCR.Panels A, B and C: fold-induction for each gene (MTT1, MTT3, and MTT5, respectively) after treatment by different heavy metals. Gene expression levels are shown relative to an untreated control (which is set at 1±0.0 for every gene). Normalization of expression was achieved against the amplification of an endogenous gene (α-tubulin). Each bar of the histogram corresponds to an average value ±SD of two or three independent experiments. Heavy metal concentrations are as reported in the Material & Methods; Cd+ refers to 27 µM Cd plus 80 µM Cu; treatments times were: 1 h (light bars) and 24 h (dark bars) for all heavy metals. See Table S2 for numerical values. Panel D. Relative expression levels for each gene, in the order MTT1 (blue bars), MTT3 (green bars) and MTT5 (red bars), after induction by stress treatments other than heavy metals. Temperature treatments were for 2h, pH and Paraquat treatments were for 24 h, while starvation treatments were for 24 hrs and 4 days.

Mentions: Comparative gene expression analysis of the three T. thermophila subfamily 7a genes was carried out under various stress conditions using real-time quantitative RT-PCR. In Fig. 6 and Table S2 we show the relative mRNA expression levels of MTT1, MTT3 and MTT5 induced by various stressors, normalized against the level of α-tubulin (the reference gene). MTT5 consistently showed the highest induction levels, relative to α-tubulin, while MTT3 showed the weakest induction. Heavy metal treatments result in generally similar induction patterns for the three genes. Cd is a strong inducer of every gene, but Pb seems to preferentially induce MTT5. None of the MT genes showed significant induction by Ni. Regarding other types of stressors, MTT5 was strongly and preferentially induced by acid pH. It was also the only MT gene significantly induced by basic pH (pH 9) or oxidative stress (PQ) and was weakly induced by starvation (24 h treatment). Although there was some variability in our measurements, MTT5, like MTT1, clearly behaves as a multi-stress response protein, with MTT5 responding to the widest range of stressors.


Tetrahymena metallothioneins fall into two discrete subfamilies.

Díaz S, Amaro F, Rico D, Campos V, Benítez L, Martín-González A, Hamilton EP, Orias E, Gutiérrez JC - PLoS ONE (2007)

Relative expression levels of Tetrahymena thermophila MTT1, MTT3 and MTT5 genes obtained by quantitative RT-PCR.Panels A, B and C: fold-induction for each gene (MTT1, MTT3, and MTT5, respectively) after treatment by different heavy metals. Gene expression levels are shown relative to an untreated control (which is set at 1±0.0 for every gene). Normalization of expression was achieved against the amplification of an endogenous gene (α-tubulin). Each bar of the histogram corresponds to an average value ±SD of two or three independent experiments. Heavy metal concentrations are as reported in the Material & Methods; Cd+ refers to 27 µM Cd plus 80 µM Cu; treatments times were: 1 h (light bars) and 24 h (dark bars) for all heavy metals. See Table S2 for numerical values. Panel D. Relative expression levels for each gene, in the order MTT1 (blue bars), MTT3 (green bars) and MTT5 (red bars), after induction by stress treatments other than heavy metals. Temperature treatments were for 2h, pH and Paraquat treatments were for 24 h, while starvation treatments were for 24 hrs and 4 days.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1808422&req=5

pone-0000291-g006: Relative expression levels of Tetrahymena thermophila MTT1, MTT3 and MTT5 genes obtained by quantitative RT-PCR.Panels A, B and C: fold-induction for each gene (MTT1, MTT3, and MTT5, respectively) after treatment by different heavy metals. Gene expression levels are shown relative to an untreated control (which is set at 1±0.0 for every gene). Normalization of expression was achieved against the amplification of an endogenous gene (α-tubulin). Each bar of the histogram corresponds to an average value ±SD of two or three independent experiments. Heavy metal concentrations are as reported in the Material & Methods; Cd+ refers to 27 µM Cd plus 80 µM Cu; treatments times were: 1 h (light bars) and 24 h (dark bars) for all heavy metals. See Table S2 for numerical values. Panel D. Relative expression levels for each gene, in the order MTT1 (blue bars), MTT3 (green bars) and MTT5 (red bars), after induction by stress treatments other than heavy metals. Temperature treatments were for 2h, pH and Paraquat treatments were for 24 h, while starvation treatments were for 24 hrs and 4 days.
Mentions: Comparative gene expression analysis of the three T. thermophila subfamily 7a genes was carried out under various stress conditions using real-time quantitative RT-PCR. In Fig. 6 and Table S2 we show the relative mRNA expression levels of MTT1, MTT3 and MTT5 induced by various stressors, normalized against the level of α-tubulin (the reference gene). MTT5 consistently showed the highest induction levels, relative to α-tubulin, while MTT3 showed the weakest induction. Heavy metal treatments result in generally similar induction patterns for the three genes. Cd is a strong inducer of every gene, but Pb seems to preferentially induce MTT5. None of the MT genes showed significant induction by Ni. Regarding other types of stressors, MTT5 was strongly and preferentially induced by acid pH. It was also the only MT gene significantly induced by basic pH (pH 9) or oxidative stress (PQ) and was weakly induced by starvation (24 h treatment). Although there was some variability in our measurements, MTT5, like MTT1, clearly behaves as a multi-stress response protein, with MTT5 responding to the widest range of stressors.

Bottom Line: We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5).Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs.EST evidence for alternative splicing in the 3' UTR of the MTT5 mRNA with potential regulatory activity is reported.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Microbiología-III, Facultad de Biología, Universidad Complutense (UCM), Spain.

ABSTRACT

Background: Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals.

Methodology/principal findings: We report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3' UTR of the MTT5 mRNA with potential regulatory activity is reported.

Conclusion/significance: The small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances.

Show MeSH
Related in: MedlinePlus