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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.

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Nucleotide sequence of the MTT5 gene and flanking regions.Motifs shared with MTT1 and MTT3 are indicated as in Figure 1. Additional features: a segment of partial red and blue overlap between two inverted copies of MTCM1 is shaded lavender; a 476-bp duplicated region (one duplicate is underlined, the other italicized); a 63 bp intron in the 3′UTR is highlighted yellow, with blue underscoring to indicate a 32-nucleotide potential stem-and-loop structure; Numbered arrows: PolyA addition sites inferred, from GenBank cDNA and EST submissions, as the junction between genomic sequence and a run of at least 5 As absent from it. The physical addition site is ambiguous when the genomic sequence contains one or more adenines at the right of the junction, as defined. (Accession numbers corresponding to each arrow: 1: CX576461, CX576877 and DQ517936; 2: CX577509; 3: CX576383; 4: CX572435; 5: CX573352 and CX578296; 6: CX581879, CX590118, and our unpublished observations; 7: CX574235.
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pone-0000291-g002: Nucleotide sequence of the MTT5 gene and flanking regions.Motifs shared with MTT1 and MTT3 are indicated as in Figure 1. Additional features: a segment of partial red and blue overlap between two inverted copies of MTCM1 is shaded lavender; a 476-bp duplicated region (one duplicate is underlined, the other italicized); a 63 bp intron in the 3′UTR is highlighted yellow, with blue underscoring to indicate a 32-nucleotide potential stem-and-loop structure; Numbered arrows: PolyA addition sites inferred, from GenBank cDNA and EST submissions, as the junction between genomic sequence and a run of at least 5 As absent from it. The physical addition site is ambiguous when the genomic sequence contains one or more adenines at the right of the junction, as defined. (Accession numbers corresponding to each arrow: 1: CX576461, CX576877 and DQ517936; 2: CX577509; 3: CX576383; 4: CX572435; 5: CX573352 and CX578296; 6: CX581879, CX590118, and our unpublished observations; 7: CX574235.

Mentions: The 297 bp coding region of MTT5 contains an open reading frame that encodes a predicted protein of 99 amino acids (Fig. 2). Alignment of our cDNA sequence to the genomic sequence revealed the existence of a 63 bp intron, beginning at position +30 downstream of the TGA stop codon, thus well outside the protein-coding sequence (Fig. 2). The intron has canonical donor and acceptor splice sites and represents the first intron to be described in a Tetrahymena MT transcript. Inspection of the cDNA submitted to GenBank by Santovito et al. and 17 ESTs downloaded by us from NCBI also confirm this intron. Surprisingly, four independent ESTs in the NCBI EST database (CX573730, CX576461, DY683553, and CX579679) retain the intron; they align perfectly with the MTT5 genomic DNA. The presence of both the spliced and unspliced versions of the MTT5 RNA was further validated by RT-PCR (Figure S1). We consider this strong evidence for alternative splicing of the MTT5 transcript – to our knowledge the first case of alternative splicing reported in T. thermophila.


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)

Nucleotide sequence of the MTT5 gene and flanking regions.Motifs shared with MTT1 and MTT3 are indicated as in Figure 1. Additional features: a segment of partial red and blue overlap between two inverted copies of MTCM1 is shaded lavender; a 476-bp duplicated region (one duplicate is underlined, the other italicized); a 63 bp intron in the 3′UTR is highlighted yellow, with blue underscoring to indicate a 32-nucleotide potential stem-and-loop structure; Numbered arrows: PolyA addition sites inferred, from GenBank cDNA and EST submissions, as the junction between genomic sequence and a run of at least 5 As absent from it. The physical addition site is ambiguous when the genomic sequence contains one or more adenines at the right of the junction, as defined. (Accession numbers corresponding to each arrow: 1: CX576461, CX576877 and DQ517936; 2: CX577509; 3: CX576383; 4: CX572435; 5: CX573352 and CX578296; 6: CX581879, CX590118, and our unpublished observations; 7: CX574235.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0000291-g002: Nucleotide sequence of the MTT5 gene and flanking regions.Motifs shared with MTT1 and MTT3 are indicated as in Figure 1. Additional features: a segment of partial red and blue overlap between two inverted copies of MTCM1 is shaded lavender; a 476-bp duplicated region (one duplicate is underlined, the other italicized); a 63 bp intron in the 3′UTR is highlighted yellow, with blue underscoring to indicate a 32-nucleotide potential stem-and-loop structure; Numbered arrows: PolyA addition sites inferred, from GenBank cDNA and EST submissions, as the junction between genomic sequence and a run of at least 5 As absent from it. The physical addition site is ambiguous when the genomic sequence contains one or more adenines at the right of the junction, as defined. (Accession numbers corresponding to each arrow: 1: CX576461, CX576877 and DQ517936; 2: CX577509; 3: CX576383; 4: CX572435; 5: CX573352 and CX578296; 6: CX581879, CX590118, and our unpublished observations; 7: CX574235.
Mentions: The 297 bp coding region of MTT5 contains an open reading frame that encodes a predicted protein of 99 amino acids (Fig. 2). Alignment of our cDNA sequence to the genomic sequence revealed the existence of a 63 bp intron, beginning at position +30 downstream of the TGA stop codon, thus well outside the protein-coding sequence (Fig. 2). The intron has canonical donor and acceptor splice sites and represents the first intron to be described in a Tetrahymena MT transcript. Inspection of the cDNA submitted to GenBank by Santovito et al. and 17 ESTs downloaded by us from NCBI also confirm this intron. Surprisingly, four independent ESTs in the NCBI EST database (CX573730, CX576461, DY683553, and CX579679) retain the intron; they align perfectly with the MTT5 genomic DNA. The presence of both the spliced and unspliced versions of the MTT5 RNA was further validated by RT-PCR (Figure S1). We consider this strong evidence for alternative splicing of the MTT5 transcript – to our knowledge the first case of alternative splicing reported in T. thermophila.

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