Supplementary Material A New Noncanonical Nuclear Genetic Code: Translation of UAA into Glutamate

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1 Supplementary Material A New Noncanonical Nuclear Genetic Code: Translation of UAA into Glutamate S1 Rocío Sánchez-Silva, Eduardo Villalobo, Loïc Morin, and Antonio Torres Supplementary Experimental Procedures Culture Conditions and Nucleic Acid Preparations Vorticella microstoma, Opisthonecta henneguyi, and Opisthonecta matiensis, kindly provided by Dr. Almudena Guinea (Departamento de Microbiologia III, UCM, Spain), were grown at 25 C in cereal leaves medium (Sigma) inoculated with Acetobacter pasteurianum. Exponentially growing cells were sedimented and were subsequently used for nucleic acid manipulations. Total DNA was isolated by conventional methods, including lysis, phenol extraction, and ethanol precipitation. Total RNA was isolated by using TRIPURE (Roche) according to the supplier s instructions. cdna reaction was done in 20 l reactions by using the Enhanced Avian RT first strand synthesis kit (Sigma) according to the supplier s directions. which both bootstrap values were lower than 50% were eliminated, as they were nonsignificant, and the corresponding branches were joined to the main stem. Polymerase Chain Reaction Reactions were made by using ng template DNA in 50 l reactions, containing 10 mm Tris (ph 8.3), 50 mm KCl, 2 mm MgCl 2, 0.2 mm dntp, 25 pmol of each primer, and 2.5 U Taq polymerase (Bioline). Cycling conditions were: 94 C for 3 min, plus 35 cycles of denaturation (94 C for 30 s), annealing (40 C 60 C for 30 s, depending on Tm of the primers used), and polymerization (72 C for 1 3 min, depending on the size of the DNA fragment to be amplified). Total genomic DNA was used as template in PCR to amplify actins, tubulins, and trnas. Circularized DNA, previously digested with restriction enzymes, was used as template in inverse PCR to amplify 5 and 3 flanking actin regions. And finally, cdna was used as template in RT-PCR to demonstrate the presence of introns in actin genes. Reactions were run on agarose gels, and the desired amplicons were purified by using a QIAEX gel extraction kit (Qiagen) according to the supplier s instructions. Purified amplicons were subsequently used for cloning in pgem-t (Promega) and for sequencing by the dideoxi-chain termination method. Sequence Analyses The GCG software was used for the alignments shown in Figure 1. Extended alignments, used to calculate the percentage of conservation of amino acid positions of Table 1, were done with ClustalW software. Sets of actin and -tubulin amino acid sequences were retrieved under the accession numbers PS00406 and PS00227, respectively, from the EBI Database ( Only full-length sequences were used in alignments. The trnascan-se software ( was used to determine trna identity. For phylogenetic analysis, -tubulin amino acid sequences from diverse organisms (115 sequences) were retrieved from the GenBank database and were aligned with the ED program of the MUST package. Matrix homogeneity and outgroup composition were tested by using the RASA algorithm. Due to missing amino acids located in the carboxy- and aminoterminal parts of the sequences in several species, a stretch of 377 internal amino acids was used for the phylogenetic analysis. A distance matrix was calculated by considering all amino acid differences, without weighting the transition probabilities. This matrix was used to reconstruct a distance tree by using a neighborjoining method, and a 1000 bootstrap resampling analysis was carried out with the NJBOOT program from the MUST package. Parsimony analysis was performed by using the PROTPARS program from the PHYLIP package, version 3.6, 2 (see genetics.washington.edu/phylip/software.html) with 100 replicates, and maximum likelihood analysis by using the PROML program with the Jones-Taylor-Thornton model with a constant rate of change among sites and 50 replicates. A neighbor-joining tree was produced with the program TREEPLOT from the MUST package. Nodes for

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7 S7 Figure S1. Alignment of Nucleotide Sequences of Actin and -Tubulin Accession numbers (in parentheses) and abbreviations are: for actin: Rabact, rabbit (NM_007392); Athaact, Arabidopsis thaliana (M20016); Creact, Chlamydomonas reinhardtii (D ); Tthact, Tetrahymena thermophila (M13939); Pteact, Paramecium tetraurelia (our unpublished data); VmiactI, Vorticella microstoma (AY64417); VmiactII, V. microstoma (AY101353); Oheact, Opisthonecta henneguyi (AY101354); OmaactI, Opisthonecta matiensis (AY101356); OmaactII, Opisthonecta matiensis (AY101355); Ofaact, Oxytricha fallax (J01163); Otriact, Oxytricha trifallax (U18940); Hipoact, Hypotrichida sp. (AF508055); Hcavact, Histriculus cavicola (Y12047); Gsteact, Gastrostyla steinii (AF508047); EvaactI, Euplotes vannus (AF273753); SmyactI, Stylonychia mytilus (AF508051); OnovactI, Oxytricha nova (M22480); Upisact, Uroleptus pisces (AF508053); Hgarnact, Halteria grandinella (AF508048); Ugranact, Urostyla grandis (AF508054), Diophrys sp (AY004214); Tpyact, T. pyriformis (X05195); for -tubulin, Mmustub, Mus musculus (BC ); Athatub, A. thaliana (AY ); Cretub, C. reinhardtii (M ); Tthtub, T. thermophila (M86723); Ptetub, P. tetraurelia (X99489); Vmtub, V. microstoma (AY101350); Ohtub, Opisthonecta henneguyi (AY101352); Omatub, Opisthonecta matiensis (AY101351); Eaetub, E. aediculatus (Z49851); Eoctotub, E. octocarinatus (X69466); Evantub, E. vannus (Z11769); Scoertub, Stentor coeruleus (Z49849); Slemtub, S. lemnae (X01746); Entotub, Entodinium sp. (Z49853); Spatub, Spathidium sp. (Z49848); Zosterotub, Zosterograptus sp. (Z49855); Lstritub, Loxodes striatus (Z49852); Cmagtub, Condylostoma magnum (Z49850). The black and gray shadowed triplets represent UAR codons in ciliates encoding glutamate and glutamine residues, respectively. Figure S2. Alignment of trna Nucleotide Sequences The small bold letters indicate the three nucleotides of the anticodon. Abbreviations for trnas: TtUAA, Tetrahymena thermophila supressorlike trna UUA (accession number M35402); OmUAA, Opisthonecta matiensis supressor-like trna UUA ; TtGAA, mitochondrial Tetrahymena thermophila glu-trna UUC (accession number X13117); OmGAA, Opisthonecta matiensis glu-trna UUC.

8 S8 Table S1. Characteristics of Actin and -Tubulin Genes Sequenced in V. microstoma, O. matiensis, and O. henneguyi and Putative trnas Sequenced in O. matiensis Gene Given Accession ORF A T 110 (-1)-Intron (I) 231 (-1)-Intron (II) Position and Nature of UAR Species Name Number Size (nt/aa) Content Size/A T Content Size/A T Content In-Frame Codons Vorticella VmactI AY /363 60% 5 -GTACCAATATCTTATTTTAG-3 5 -GTAAAAATAATTTAATTTAAG-3 59(UAA); 216(UAA); 228(UAA); 272(UAA) microstoma 20 nt/77.2% 21 nt/91% VmactII AY / % 5 -GTACCAATATCTTATTTTAG-3 5 -GTAAAAATAATTTAATTTAAG-3 59(UAA); 216(UAA); 228(UAA); 272(UAA) 20 nt/77.2% 21 nt/91% Vmtub AY / % No intron No intron 411(UAA) Opisthonecta Ohact AY / % 5 -GTAATCTAAGTATTTTAATTTAG-3 5 -GTAAAAATTGTTTTAATTTCAAG-3 5(UAA); 216(UAA); 272(UAA) henneguyi 23 nt/82.6% 23 nt/82.6% Ohetub AY /394 58% No intron No intron 77 (UAA); 297 (UAA); 411 (UAA) Opisthonecta OmactI AY / % 5 -GTATTAGTGTTTGGATTCAG-3 No intron 127(UAG); 197(UAA); 313(UAG) matiensis 20 nt/65% OmactII AY / % No intron 5 -GTAATTTATTTTTTTATCATAG-3 272(UAA) 22 nt/86.4% Omatub AY / % No intron No intron No UAR OmtRNA UUC AF % No intron No intron GAA* OmtRNA UUA AF % No intron No intron UAA* Amino acid positions are referred to as rabbit actin (GenBank accession number NM_007392) and Mus musculus -tubulin (GenBank accession number BC ), respectively, for actin (denoted as act) and -tubulin (denoted as tub) sequences. The asterisks denote that the codon recognized by the trna is shown in the cell.

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