Two new members of the Tetrahymena multi-stress-inducible metallothionein family: Characterization and expression analysis of T. rostrata Cd/Cu metallothionein genes

Amaro, Francisco; Lucas, María Pilar de; Martín‐González, Ana; Gutiérrez, Juan Carlos Restrepo

doi:10.1016/j.gene.2008.07.002

Cited by 17 publications

(50 citation statements)

References 35 publications

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“…Ciliate MTs have been included in the classification system of the MT superfamily, as family 7, and it has been divided in two main subfamilies: (28)(29)(30) subfamily 7a, at present composed of eight cadmium-binding metallothioneins (CdMTs) (mainly acting as cadmium/zinc-binding MTs); two T. pyriformis MTs (abbreviated TpyrMT-1 and TpyrMT-2), three T. thermophila MTs (TtheMTT1, TtheMTT3 and TtheMTT5), one T. tropicalis MT (abbreviated as TtroMTT1 in this article), one T. rostrata MT (TrosMTT1), and one T. pigmentosa MT (TpigMT-1); (28)(29)(30)(31)(32)(33)(34)(35) and subfamily 7b, constituted at present, by four CuMTs (principally acting as copper-binding MTs), two T. thermophila MTs (TtheMTT2 and TtheMTT4), one T. pigmentosa MT (TpigMT-2) and another from T. rostrata (TrosMTT2). (29,30,36,37) Both subfamilies differ in the induction patterns by heavy metals (Cd/Zn or Cu) and the pattern of Cys residue clustering.…”

Section: Tetrahymena Mts Classificationmentioning

confidence: 99%

“…(29,30,36,37) Both subfamilies differ in the induction patterns by heavy metals (Cd/Zn or Cu) and the pattern of Cys residue clustering. (29,30,31,34,37,38) The majority of Tetrahymena MT genes have been isolated as DNA complementary to RNA (cDNA) molecules, and from their alignment to the corresponding genomic DNA sequences, the absence of introns in all these MT genes has been revealed. On the other hand, introns have been described in several MTs from other organisms.…”

Section: Tetrahymena Mts Classificationmentioning

confidence: 99%

“…1). (29,30) All eight members of Tetrahymena MTsubfamily 7a are readily and unambiguously divided into segments initially defined by the criterion that every segment carries a CXCCK motif at its C terminus (with several exceptions) (Fig. 1); these have been named 'modules'.…”

Section: Tetrahymena Mts Classificationmentioning

confidence: 99%

“…organization based on CKCX 2-5 CXC repeats (where in a few cases K may be substituted by other amino acid) might be considered. (29) Another independent structural feature that also differentiates both Tetrahymena MT subfamilies is the physical relationship of Lys to Cys residues along the polypeptide backbone. The Lys residues of CuMTs have a strong tendency to be juxtaposed with Cys (CKC), as seen in mammalian MTs.…”

Section: Tetrahymena Mts Classificationmentioning

confidence: 99%

“…The Lys residues of CuMTs have a strong tendency to be juxtaposed with Cys (CKC), as seen in mammalian MTs. (29) In contrast, in subfamily 7a (CdMTs), Cys-juxtaposed Lys residues are rarer, and are concentrated in type 2 submodules. It has been reported that having an adjacent basic residue considerably decreases the pK value and, consequently, the reactivity of cysteinyl residues.…”

Section: Tetrahymena Mts Classificationmentioning

confidence: 99%

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From heavy metal‐binders to biosensors: Ciliate metallothioneins discussed

2009

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Metallothioneins (MTs) are ubiquitous proteins with the capacity to bind heavy metal ions (mainly Cd, Zn or Cu), and they have been found in animals, plants, eukaryotic and prokaryotic micro-organisms. We have carried out a comparative analysis of ciliate MTs (Tetrahymena species) to well-known MTs from other organisms, discussing their exclusive features, such as the presence of aromatic amino acid residues and almost exclusive cysteine clusters (CCC) present in cadmium-binding metallothioneins (CdMTs), higher heavy metal-MT stoichiometry values, and a strictly conserved modular-submodular structure. Based on this last feature and an extensive gene duplication, we propose a possible model for the evolutionary history of T. thermophila MTs. We also suggest possible functions for these MTs from consideration of their differential gene expressions and discuss the potential use of these proteins and/or their gene promoters for designing molecular or whole-cell biosensors for a fast detection of heavy metals in diverse polluted ecosystems.

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Section: Tetrahymena Mts Classificationmentioning

confidence: 99%

Section: Tetrahymena Mts Classificationmentioning

confidence: 99%

Section: Tetrahymena Mts Classificationmentioning

confidence: 99%

Section: Tetrahymena Mts Classificationmentioning

confidence: 99%

Section: Tetrahymena Mts Classificationmentioning

confidence: 99%

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From heavy metal‐binders to biosensors: Ciliate metallothioneins discussed

2009

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Isolation and characterization of a novel copper‐inducible metallothionein gene of a ciliate, Tetrahymena tropicalis lahorensis

Chaudhry

Shakoori

2010

J of Cellular Biochemistry

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The two isoforms of copper metallothionein (CuMT) gene of a copper resistant ciliate, Tetrahymena tropicalis lahorensis (Ttl), have been isolated and characterized. The molecular cloning and nucleotide sequencing of cDNAs coding for the two CuMT isoforms revealed that TtlCuMT1 gene has 300, while TtlCuMT2 has 327 nucleotides, both with ATG as the initiation codon and TGA as the translational termination codon. TAG codes for glutamine in TtlCuMT2 gene which is peculiar to Tetrahymena. The deduced or translated TtlCuMT1 and TtlCuMT2 peptide sequences contain 100 and 108 amino acid residues including 28 and 32 cysteine residues, respectively. The amino acid sequences of TtlCuMT1 and TtlCuMT2 have special features of two and three CXCXXCXCXXCXC intragenic tandem repeats with a conserved structural pattern of cysteine, respectively. The predicted tertiary structures of these two isoforms indicate two domains. Domain I and the initial part of domain II showed >98% homology with other Tetrahymena CuMT. On the basis of the differences in the domain II, the metallothionein subfamily 7b can be divided into two groups, one (TtlCuMT1) comprising >100 amino acids and the other (TtlCuMT2) comprising <100 amino acids. This is a novel finding of the present study as no such report on this type of classification exists at the moment. TtlCuMT1 has 95%, while TtlCuMT2 has 97% resemblance with the previously reported CuMT genes of Tetrahymena spp. SDS-PAGE analysis using fluorescent probe as well as coomassie brilliant blue staining also confirmed the presence of metallothionein.

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Molecular Characterization of a Copper Metallothionein Gene From a Ciliate Tetrahymena farahensis

Zahid

Shakoori

Zulifqar

et al. 2016

J of Cellular Biochemistry

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A new copper metallothionein (TfCuMT) gene has been identified from a locally isolated ciliate Tetrahymena farahensis. It contains 327 nucleotides encoding a peptide chain of 108 amino acids and belongs to class MTT2 and subfamily 7b. Amplification from both gDNA and mRNA confirmed the intronless nature of this gene. Like most of the metallohtioneins, cysteine residues contribute nearly 30% content with the specific CKC motifs. Structural repeats present in peptide sequence of TfCuMT indicate internal duplication of gene at some stage of gene evolution. The predicted model of copper metallothionein protein showed that copper ions are mainly chelated by thiol sulfur of cysteine residues and are embedded in the folds of polypeptide chain. For in vivo expression of TfCuMT in Escherichia coli host cells the classical stop codons, which coded for glutamine in the ciliate were mutated to CAA and CAG through site directed mutagenesis. The mutated gene showed higher expression in pET28a expression vector compared with pET21a. Optimum expression was obtained after 6-8 h of 0.1 mM IPTG induction. Stability of His tagged TfCuMT in 5% SDS was low, with half-life of about 104 min. Presence of 1.0 μM copper increased the expression level by 1.65-fold. Presence of 100 μM Cysteine in culture medium caused 2.4-fold increase in expression level. His tagged TfCuMT was purified through affinity chromatography using NTN-His binding resin in the presence of 0.1 M imidazole and NaCl. The modeled structure of the TfCuMT showed a cleft for Cu binding with correct orientation of Cys residues in the motif CKC. J. Cell. Biochem. 117: 1843-1854, 2016. © 2016 Wiley Periodicals, Inc.

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Two new members of the Tetrahymena multi-stress-inducible metallothionein family: Characterization and expression analysis of T. rostrata Cd/Cu metallothionein genes

Cited by 17 publications

References 35 publications

From heavy metal‐binders to biosensors: Ciliate metallothioneins discussed

From heavy metal‐binders to biosensors: Ciliate metallothioneins discussed

Isolation and characterization of a novel copper‐inducible metallothionein gene of a ciliate, Tetrahymena tropicalis lahorensis

Molecular Characterization of a Copper Metallothionein Gene From a Ciliate Tetrahymena farahensis

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