FIGURE 7.

Telomerase assay to determine the essential SpuTR fragments necessary for activity. (A) Outline of two putative helices forming the SpuTR central domain. Nucleotide numbers denote the 5′- and 3′-ends of the T7 transcribed RNA fragments, P4.1 (191–291 nt), P4.2 (312–420 nt), and five P4.2 truncated fragment (A–E), of the central domain (186–456 nt). (B) Direct primer-extension assay of telomerase reconstituted from various T7 transcribed SpuTR fragments and in vitro-synthesized SpuTERT. The SpuTR fragments included in each reaction are indicated above the gel. The primer-extension reactions were performed in the presence of radioactive [α-³²P]dGTP and 1 µM telomeric primer (TTAGGG)₃. A ³²P-end-labeled 18-mer oligonucleotide was included as the loading control (LC). Numbers to the left indicate the number of nucleotides added to the primer. Relative activity (%) of each reaction was normalized to the reaction with the entire central domain (186–456 nt) and is indicated below the gel.