The two-nucleotide junction J4 is required for telomerase function in vivo. (A) Deletion of the two nucleotides in J4 causes senescence in vivo. Indicated junction mutations in Mini-T(460), expressed from 2μ plasmids, were serially passaged for 275 generations. (B) All Mini-T(460) junction mutants, when expressed from 2μ plasmids, are detectable by northern blotting. The abundance represents the average amount of mature RNA from two biological duplicates, with the error below indicating the range of expression from those duplicates. Sizes of full-length Mini-T RNAs (filled triangle) vary consistently with the predicted length of the RNA. (C) Telomere length of each junction mutant correlates with the in vitro activity (Figure 6C). A Southern blot probed for telomeric DNA and the chromosome IV loading control (LC) is shown. Note that J4Δ mutation causes senescence and so genomic DNA for that sample is from 50 generations, whereas others are from 300 generations, as listed above each lane (with senescence indicated by underlining).