title: Tetrahymena metallothioneins fall into two discrete subfamilies.
abstract: BACKGROUND
  Metallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Departamento de Microbiología-III, Facultad de Biología, Universidad Complutense (UCM), Spain.
authors.forename: Silvia
authors.initials: S
authors.lastname: Díaz
authors.forename: Francisco
authors.initials: F
authors.lastname: Amaro
authors.forename: Daniel
authors.initials: D
authors.lastname: Rico
authors.forename: Virginia
authors.initials: V
authors.lastname: Campos
authors.forename: Laura
authors.initials: L
authors.lastname: Benítez
authors.forename: Ana
authors.initials: A
authors.lastname: Martín-González
authors.forename: Eileen P
authors.initials: EP
authors.lastname: Hamilton
authors.forename: Eduardo
authors.initials: E
authors.lastname: Orias
authors.forename: Juan C
authors.initials: JC
authors.lastname: Gutiérrez
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D008668:Metallothionein / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013768:Tetrahymena / Q000187:drug effects / Q000235:genetics*; D016808:Tetrahymena thermophila / Q000737:chemistry / Q000235:genetics
chemical_list: D020413:3' Untranslated Regions; D015800:Protozoan Proteins; D012333:RNA, Messenger; D002104:Cadmium; D003300:Copper; D008668:Metallothionein