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Figure 1. Protein sequences deduced of both identified Amphioxus metallothionein genes, which are patently transcribed in adult organisms.(A) Aligned protein sequences of the two identified amphioxus MTs, BfMT1 and BfMT2, in silico translated from the rbfeg037o01 and bfne062f22 cDNA clones, respectively. Cysteine residues are highlighted in gray. Alignment was performed using the T-Coffee software. (B) RT-PCR amplification of BfMT1 and BfMT2 cDNA using adult B. lanceolatum total mRNA as a template. Both band sizes corresponded to that expected for the respective coding sequences (255 bp for BfMT1 and 150 bp for BfMT2). The amplified cDNAs were cloned, and their sequences were verified as exact matches with respect to the B. floridae library cDNA clones.
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Figure 2. The genomic localization, orientation and structure of the BfMT1 and BfMT2 genes coincide in a 100-Kb region of the B. floridae genome.Both are located in the negative strand of the B. floridae genome scaffold 398. Exact location in pair bases with respect to the scaffold sequence is represented below each gene. The distance between both genes, as well as their size, are also indicated. Exons are shown in gray, and numbered E1 to E4. Black arrows indicate the transcription direction and the metal response elements (MREs) identified in the promoter regions are represented as black boxes.
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Figure 3. The analysis of the BfMT1 and BfMT2 genomic sequences identify all the elements of these genes.Promoter regions (-1 kb) are included and the in silico identified regulatory sequences (MRE and ARE) are highlighted in bold. 5′and 3′UTR regions, corresponding to the cDNA sequences of the cDNA clones, are included and represented in italics and underlined. Coding sequences are boxed in their corresponding exons and translated protein sequences are included below each exon.
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Figure 4.
BfMT2 transcription proceeds through alternative splicing patterns.Schematic representation of the BfMT2 gene structure and the protein sequence corresponding to each exon (in the centre), and the protein sequences for each alternative cDNA. BfMT2 coding sequence used for translation was extracted from the NCBI Databank and corresponds to B. floridae strain S238N-H82. cDNA clone sequences were extracted and obtained from the B. floridae cDNA Database.
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Figure 5.
BlMT1 is essentially constitutive, while BlMT2 is an inducible gene, as revealed in metal and non-metal treated B. lanceolatum organisms.(A) Semi quantification by RT-PCR of transcription rates under Cu and Cd supplementation, and statistical comparison. Data for each gene and condition is normalized by the corresponding value for the constitutive gene β-actin, previously homogenized between organisms and groups. Data represent mean and standard deviation of six organisms. Significance was assessed using the Newman-Keuls statistical test. Stars denote statistical significance as follows: * as equivalent to a significance of P<0.05, ** equivalent to P<0.01, and *** to P<0.001. B) Best examples of RT-PCR bands representing the results observed in A). NT stands for non-treated organisms.
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Figure 6. ICP-measured Cd and Cu accumulation in treated lancelets follow different patterns.
B. lanceolatum organisms were treated with two different concentrations of Cd or Cu, or non-metal treated (NT). Data is represented as mean and standard deviation of six organisms per group. Statistical significance is represented as ** equivalent to P<0.01, and *** to P<0.001 according to the Newman-Keuls statistical test.
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Figure 7. The BfMT1 and BfMT2 peptides exhibit different metal binding abilities.Deconvoluted ESI-MS spectra, at neutral pH, corresponding to the BfMT1 and BfMT2 recombinant syntheses, obtained from bacterial cultures grown in Zn-, Cd- and Cu-supplemented. Insets correspond to the CD fingerprint of each preparation. The ESI-MS spectra at pH 2.4 is also included for the Cu-BfMT preparations obtained under normal aeration conditions of the cultures.
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Figure 8. The in vitro Cd(II) binding abilities of BfMT1 points to a partial preference for divalent metal ion coordination.(A) CD corresponding to the titration of a 10 µM solution of Zn-BfMT1 with Cd(II) at pH 7.0 until 8 Cd(II) eq added (B) Deconvoluted ESI-MS spectrum of an aliquot corresponding to the addition of 8 Cd(II) eq to Zn-BfMT1. (C) Comparison of the CD spectra of the recombinant Cd-BfMT1 preparation (solid line), the acidified and reneutralized sample (dashed line), and the latter after the addition of 4 S2− equivalents (dotted line). (D) Comparison of the CD spectra of the Cd-BfMT1 sample before (solid line) and after (dashed line) 28-days of evolution under inert atmosphere and ESI-MS spectrum of the final sample.
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Figure 9. The in vitro Cd(II) binding abilities of BfMT2 points to a partial preference for divalent metal ion coordination.(A) CD corresponding to the titration of a 10 µM solution of Zn-BfMT2 with Cd(II) at pH 7.0 from 0 to 4 and from 4 to 8 Cd(II) eq added. (B) Deconvoluted ESI-MS spectrum of an aliquot corresponding to the addition of 8 Cd(II) eq to Zn-BfMT2. (C) Comparison of the CD spectra of the recombinant Cd-BfMT2 preparation (solid line), the acidified and reneutralized sample (dashed line), and the latter after the addition of 4 sulfide equivalents (dotted line). (D) Comparison of the CD spectra of the Cd-BfMT2 sample before (solid line) and after (dashed line) 28-days of evolution under inert atmosphere and ESI-MS spectrum of the final sample.
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Figure 10. The in vitro Cu(I) binding abilities of BfMT peptides shows the preference of BfMT1 vs. BfMT2 to coordinate this metal ion.(A) CD corresponding to the titration of a 10 µM solution of Zn-BfMT1 with Cu(I) at pH 7.0 until 18 Cu(I) eq added. The arrows show the number of Cu(I) eq added at each stage of the titration. (B) Deconvoluted ESI-MS spectrum of several aliquots extracted from the solution at 6, 12, and 18 Cu(I) eq added to Zn-BfMT1. (C) Comparison of the CD spectra of the recombinant Cu-BfMT2 preparation (solid line), and that of the Zn-BfMT1 sample after the addition of 12 Cu(I) eq (dashed line).
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Figure 11. Protein distance analysis of MTs from organisms of different taxa positions Branchiostoma MTs at the base of vertebrates and close to Echinoderm MTs.The Maximum Likelihood tree was obtained by the Phylip method, and the corresponding bootstrap values (in percentages) obtained are included. Sequences and their Uniprot IDs are: Human MT-1A (P04731), Human MT-2A (P02795), Human MT-3 (P25713), Human MT-4 (P47944), Gallus gallus MT (P68497), Columba livia MT-2 (P15787), Podarcis sicula MT (Q708T3), Ictalurus punctatus MT (O93571), Scyliorhinus torazame MT (Q6J1T3), Ambystoma mexicanum MTA (O42152), Mytilus edulis 10 Ia (P80246), Crassostrea virginica MT (P23038), Mytilus edulis MT-20-II (P80252), Helix pomatia CdCuMT (D1LZJ8), Helix pomatia CdMT (P33187), Helix pomatia CuMT (P55947), Homarus americanus MT1 (P29499), Carcinus maenas MT (P55948), Potamon potamios MT (P55952), Drosophila melanogaster MtnB (P11956), Drosophila melanogaster MtnC (Q9VDN2), Drosophila melanogaster MtnD (Q8I9B4), Caenorhabditis elegans MT1 (P17511), Caenorhabditis elegans MT2 (P17512), Saccharomyces cerevisiae Cup1 (P0CX80), Saccharomyces cerevisiae CRS5 (P41902). The following sequences were obtained from GeneBank: Strongylocentrotus purpuratus MT1 (AAA30061.1), Ciona intestinalis MT1 (ACN32211.2), Herdmania curvata MT (AY314949.1). Drosophila melanogaster MtnE sequence was obtained from FlyBase (FlyBaseID: FBpp0293071).
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