Figure 5.

SIR3 and HDA1 mRNA levels in age-synchronized cells. (A) Northern blot. RNA was prepared from 2, 5, 8, 11, 14, and 17 generation-old cells. Total RNA (10 μg) from each RNA sample was loaded in each lane of a gel. The same blot was used repeatedly by hybridizing with one DNA probe at a time and stripping it. The probe DNA to detect 5.8S rRNA (158 bp) was prepared by labeling the 5′ end of an oligonucleotide (5′-CATTTCGCTGCGTTCTTCATC-3′) with ³²P. The DNA probe to detect TLC1 RNA (1.3 kb) was the 1.28-kb XhoI fragment isolated from pBlue61 (Singer and Gottschling, 1994). The DNA probe for HDA1 mRNA (2.5 kb) was the 1.8-kb XhoI– XbaI fragment isolated from pskB93 (Rundlett et al., 1996). The DNA probe specific to SIR3 mRNA (3 kb) was the 2.3-kb ClaI–XhoI fragment isolated from pJR517 (provided by J. Rine, University of California, Berkeley, CA). (B) Quantitation of mRNA. The amount of mRNA present in each lane was quantitated by phosphorimaging and normalized to the amount of TLC1 RNA present in the same lane to obtain age-specific relative amounts of individual mRNA species. The amount of TLC1 RNA, which is a component of the yeast telomerase, remains relatively constant compared with 5.8S rRNA.