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Volumn 286, Issue 5442, 1999, Pages 1146-1149

An adenosine deaminase that generates inosine at the wobble position of tRNAs

Author keywords

[No Author keywords available]

Indexed keywords

ADENOSINE DEAMINASE; CYTIDINE DEAMINASE; INOSINE; TRANSFER RNA;

EID: 0033527628     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5442.1146     Document Type: Article
Times cited : (270)

References (35)
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    • Cells were transformed with PCR-amplified fragments bearing the TRP1 marker gene flanked by TAD2 or TAD3 sequences, respectively. Tryptophan prototrophs were selected and correct genomic integration of the TRP1 gene was verified by PCR. In tad2Δ cells the coding sequence from amino acids 74 to 210 of TAD2 was replaced by the TRP1 marker, and in tad3Δ cells, amino acids 152 to 233 were replaced.
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    • 6 were passed twice over 400 μl of an anti-M2-FLAG affinity matrix (Kodak) and eluted with buffer B [buffer A containing 25 mM KCl, 0.1 mM EDTA, and 0.1 mM dithiothreitol (DTT) instead of β-mercaptoethanol] supplemented with FLAG-peptide (50 μg/ ml) (Kodak). Fractions containing tagged Tad2p were loaded on a MonoQ column (PC 1.6/5; Smart System, Pharmacia) that had been equilibrated in buffer C (buffer B containing 0.2 mM EDTA and 1 mM DTT) and developed with a 2-ml gradient from 25 to 300 mM KCl. Fifty-microliter fractions were collected. Fractions with tRNA editing activity eluted at 145 mM KCl (∼4 μg).
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    • 2) is not the correct start codon but part of a second intron. To confirm this, we amplified the TAD3 ORF with primers YL8 (5′-GGAC-TAGTTAAGAAAGTTAATAATCCGC-3′) and YL2 (5′-CGACTAGTCCGCAGCAGACATCCCGGTCAAC-3′) on S. cerevisiae cDNA. Spe I sites for subcloning are underlined. TAD3 with introns was obtained by PCR on genomic DNA with primers YLreg1 (5′-GGTCTG-TAGATCAATGTCAAGC-3′) annealing 237 bp upstream and YLreg2 (5′-GTTCAAGCAGCAACTA-CAGTCG-3′) hybridizing 386 bp downstream of the TAD3 ORF. The fragment was further cloned into pFL38 and pFL36, respectively (27).
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    • We thank H. Langen (Hoffmann La-Roche) for the mass spectrometry analysis; H. Grosjean, J. Atkins, and H. Himeno for the tRNA templates; and members of our laboratory and M. A. O'Connell for critical reading of the manuscript Supported by Boehringer Ingelheim Fonds (A.P.G.), the University of Basel, the Swiss National Science Foundation, the Human Frontiers Science Program, and the Louis-Jeantet Foundation for Medicine.


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