Identification of promoter elements required for TLC1 expression and function. (A) Schematic representation of the TLC1 chromosomal locus and the flanking upstream genes. The start codon of PDX3 as well as the 5′-ends of the mature snR161 and TLC1 RNAs are represented by arrows indicating the transcription direction. The identity of the conserved sequence motifs is indicated by letters. (B) Northern blot analysis of TLC1 RNA isolated from cells harboring different deletions in the TLC1 promoter. TLC1 RNA abundance was normalized to Act1 mRNA and is presented as an average of three experiments with a SD of ±20% or less. (C) Southern blot analysis of telomere length of the different strains examined in B. The DNA was extracted from individual yeast clones grown for 30 or 110 generations, digested with XhoI, and separated on an Agarose gel. The different fragments were visualized using a randomly labeled probe complementary to the telomeric sequence. Note that strains harboring vector only (Vector), a complete deletion of the intergenic sequences (ΔA-G), or a deletion of the E element (ΔE) could not be cultured beyond 40 generations, and thus, only DNA derived from cultures grown for 30 generations was analyzed. Changes in Y′ telomere length for cells grown for 110 generations are indicated at the bottom. S denotes senescing cells that have unstable telomere length.
