FIGURE 3:

Profiles of tRNA fragments in Rnt2 KO strains. (A) Southern blot analysis confirming complete replacement of macronuclear RNT2 genes with the resistance cassette neo2 or bsr2. Genomic DNA was digested with the indicated restriction enzymes to resolve wild-type (wt) from KO chromosomes as schematized above each blot. Two or three independent clonal cell lines are compared with wt for each RNT2 locus targeting. (B) Total RNA from control and KO strains grown in defined complete synthetic medium without or with transfer to starvation medium for a subsequent 3 h. RNA was analyzed by SYBR Gold stain (top) and by Northern blot hybridization with a probe against the 3′ end of tRNA Gly (bottom; only tRNA-relevant blot sections are shown). The positions of mature tRNA and tRNA halves are indicated. An arrow highlights the Rnt2 C KO lane with an altered size of tRNA halves. (C) RNA from cells grown in complex, rich medium NEFF with or without subsequent transfer to starvation medium. RNA was analyzed by Northern blot hybridization with probes against tRNA Gly 5′ and 3′ ends.