Figure 5.

Generation of a T.thermophilus mutant lacking TrmI activity, by homologous recombination. (A) Plasmid construction in E.coli for T.thermophilus trmI gene inactivation (see Materials and Methods for details). The trmI gene is represented by a solid bar, the thermostable knt gene (Km^r) by an open bar and the 16S promoter by a horizontal arrow. Restriction sites are indicated as follows: B, Bsu36I; E, EcoRI; M, MluI. (B) Autoradiography of 2-dimensional chromatograms of P1 hydrolysates of [α-³²P]ATP-labeled T.thermophilus tRNA^Asp transcripts incubated for 1 h at 60°C in the presence of a crude extract of the T.thermophilus wild-type strain (HB27) or trmI mutant (RD1). Circles of dotted lines show the migration of the pG, pC and pU nucleotides used as UV markers.