Abstract
The metallothionein (MT) gene superfamily consists of metal-binding proteins involved in various metal detoxification and storage mechanisms. The evolution of this gene family in vertebrates has mostly been studied in mammals using sparse taxon or gene sampling. Genomic databases and available data on MT protein function and expression allow a better understanding of the evolution and functional divergence of the different MT types. We recovered 77 MT coding sequences from 20 representative vertebrates with annotated complete genomes. We found multiple MT genes, also in reptiles, which were thought to have only one MT type. Phylogenetic and synteny analyses indicate the existence of a eutherian MT1 and MT2, a tetrapod MT3, an amniote MT4, and fish MT. The optimal gene-tree/species-tree reconciliation analyses identified the best root in the fish clade. Functional analyses reveal variation in hydropathic index among protein domains, likely correlated with their distinct flexibility and metal affinity. Analyses of functional divergence identified amino acid sites correlated with functional divergence among MT types. Uncovering the number of genes and sites possibly correlated with functional divergence will help to design cost-effective MT functional and gene expression studies. This will permit further understanding of the distinct roles and specificity of these proteins and to properly target specific MT for different types of functional studies. Therefore, this work presents a critical background on the molecular evolution and functional divergence of vertebrate MTs to carry out further detailed studies on the relationship between heavy metal metabolism and tolerances among vertebrates.
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Acknowledgments
We are thankful to M Fonseca, N. Galtier, JM Lourenço, B Nabholz, S Rocha, D Salvi, and Z Yang for feedback on this work. We are grateful to J-P Doyon for his help with the reconciliation analysis and his comments on this part. We are thankful to an anonymous reviewer for comments on an early version of this manuscript. YC was partially financially supported by a FCT (Fundação para Ciência e Tecnologia, Portugal) postdoctoral fellowship SFRH/BDP/73515/2010.
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239_2014_9612_MOESM1_ESM.xls
Online Resource 1 Vertebrate metallothionein CDS dataset used in this work (see Materials and Methods for further details). “Annotation” reflects names according to the database used to retrieve this sequence. “Sequence Code” refers to names used for this work. “Length (bp-aa)” refers to length of the full exon when known (CDS+UTR or CDS) in number of base pairs (bp) and the number of amino acids; “Gene Type – Ensembl” with the classification “known” if there is a sequence match to the CDS or protein for the same species, “novel” if there is no match and “known by projection” classification if the transcript is a match for another species. “RefSeq Status – NCBI” as defined according to www.ncbi.nlm.nih.gov/RefSeq/key.html#status; “Location” indicates the interval of basepairs where the CDS is located in the chromosome; “Direction” means the direction of expression of the CDS in the double stranded DNA chromosome. Acession numbers of the sequences are indicated as full gene in Ensembl and NCBI (Gene Acess No and NCBI annotation) and as CDS in Ensembl (Transcript Acess No.). (XLS 73 kb)
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Online Resource 2 List of sequences removed, edited or replaced after checking them on the Ensembl and NCBI genomic databases. (PDF 148 kb)
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Online Resource 3 Bootstrap and posterior probability support values for all phylogenetic analyses represented on the unrooted Bayesian consensus tree (50% majority-rule, one model of evolution) topology obtained with nucleotide data. Numbers on the tree refer to nodes. Bootstrap for ML (in % and only when above 60%) and Posterior Probability (pp only for values above 0.70) values are given for each node (indicated here by the numbers in bold) in the following order: ML nucleotide/ML amino acid/Bayesian nucleotide no partition/Bayesian nucleotide partition/Bayesian amino acid. Values replaced by “-” when below 60% bootstrap or 0.70 pp. Values not shown when clade is not recovered by a specific analysis are indicated with “#”. 1. 96/81/1/1/0.97; 2. 100/65/1/1/1; 3. -/#/-/0.75/#; 4. 75/-/0.88/-/#; 5. -/-/-/#/0.71; 6. 89/-/0.99/1/-; 7. -/-/0.97/0.97/1; 8. 68/74/0.98/0.96/1; 9. 71/-/0.88/0.99/#; 10. -/#/0.73/0.86/#; 11. #/76/-/0.71/#; 12. -/-/0.91/0.99/#; 13. 100/75/0.98/1/0.95; 14. #/#/1/1/#; 15. #/#/0.74/-/#; 16. 87/89/1/1/0.97; 17. -/-/0.97/0.99/0.75; 18. -/-/0.74/0.82/1; 19. 100/98/1/1/0.97; 20. 100/98/1/1/1; 21. 100/98/1/1/1; 22. #/#/-/0.91/#; 23. 100/97/1/1/1; 24. 95/-/1/1/99; 25. #/#/0.75/0.72/#; 26. 95/99/1/1/1; 27. 99/-/0.99/0.96/#; 28. 78/#/0.90/0.85/#; 29. 100/94/1/1/1; 30. -/-/-/0.95/0.96; 31. 78/-/0.79/1/-; 32. -/-/-/-/#; 33. #/#/0.85/#/#; 34. 83/79/0.94/1/1; 35. #/#/0.85/0.96/#; 36. 74/#/0.94/-/#; 37. 76/#/0.94/0.79/#; 38. #/#/-/#/#; 39. 98/96/1/1/1; 40. 99/94/1/1/1; 41. 98/92/0.89/0.99/0.99; 42. 79/-/1/1/0.96; 43. 91/#/1/#/#; 44. -/#/0.66/#/#; 45. 98/92/1/1/0.85; 46. 99/100/1/1/1; 47. 99/79/1/1/0.92; 48. 39/#/-/-/#; 49. 62/#/0.85/0.71/0.99; 50. -/-/-/-/#; 51. 81/84/0.98/0.96/1; 52. 87/84/0.98/1/1; 53. 93/99/1/1/1; 54. 65/-/1/#/#; 55. -/-/0.99/0.88/#; 56. -/#/-/#/#. (PDF 146 kb)
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Online Resource 4 Rooted gene trees resulting from the optimal reconciliation. Gene duplication is indicated with (D), while gene loss is indicated by a dashed line. a) and b) indicate the reconciliation results obtained using the Baysian amino acid and nucletotide gene trees, respectively, as input. (PDF 1895 kb)
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Online Resource 5 Functional divergence alignments (Type I divergence). Vertical bar represents amino acid sites indicated as functionally divergent among the indicated clades for a cut-off value = 0.9. (PDF 375 kb)
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Serén, N., Glaberman, S., Carretero, M.A. et al. Molecular Evolution and Functional Divergence of the Metallothionein Gene Family in Vertebrates. J Mol Evol 78, 217–233 (2014). https://doi.org/10.1007/s00239-014-9612-5
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DOI: https://doi.org/10.1007/s00239-014-9612-5