Figure 5.

Validation of the in vitro telomerase assay. Cell extracts prepared from wild-type cells with untagged Trt1p (CF199) and cells expressing myc₉Trt1p fusion protein (CF830) were subject to immunopurification on monoclonal anti-c-myc antibodies conjugated to agarose beads. After incubation with (+) and without (−) RNase A, telomerase activity was assayed. A ³²P-5′-end labeled 100-mer oligonucleotide (Upper) was added to test that no reaction products were lost during phenol-chloroform extraction or ethanol precipitation. Products were separated by sequencing gel electrophoresis. As length markers, PBoli 14 oligonucleotide was elongated by using [α-³²P]dGTP (M ladder) or [α-³²P]ddGTP (M+1), respectively, by terminal deoxynucleotidyltransferase. Because electrophoretic mobility depends on nucleotide composition, the M ladder does not align with the telomerase extension products.