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. 2020 Aug 8;38(1):215–228. doi: 10.1093/molbev/msaa203

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© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Fig. 5. — Functional validation and characterization of apple snail TR. (A) Direct telomerase activity assay of acorn worm telomerase reconstituted in vitro. (top) Template sequence of acorn worm TR (open box) with base-pairing of six permuted telomeric DNA primers a–f. Sequence and number of expected nucleotides added are depicted for each primer. (bottom) Direct primer-extension assay of apple snail telomerase. Apple snail telomerase was in vitro reconstituted from T7 transcribed PdiTR (408 nt) and PdiTERT synthesized in RRL. The reconstituted acorn worm telomerase was analyzed with six permuted telomeric DNA primers (lanes 1–6). A ³²P end-labeled 18-mer oligonucleotide was added to each reaction as recovery control (r.c.) prior to ethanol precipitation of DNA products. Numbers to the right of the gel indicate the number of repeats or nucleotides added to the primer. (B) Two essential fragments of PdiTR. The T-PK and CR4/5 fragments of PdiTR were synthesized separately and assembled with PdiTERT in RRL, followed by telomerase activity assay. The schematic secondary structures of the PdiTR T-PK (top) and eCR4/5 (bottom) fragments. The eCR4/5 domain consists of three stems, P4–P6. Nucleotide numbers denote the 5′- and 3′-ends of the T7 transcribed TR fragments, T-PK, P4/5/6, P5, and P6. (C) Minimal requirement of TR domains for telomerase activity. T7 transcribed PdiTR fragments, T-PK (nt 1–146), P4/5/6 (nt 162–335), P5 (nt 191–244), or P6 (nt 245–309), were assembled with in vitro synthesized PdiTERT and analyzed for activity using the primer (GGGTTA)₃. The PdiTR fragments included in each reaction are indicated above the gel. A ³²P end-labeled 18-mer oligonucleotide was added to each reaction prior to ethanol precipitation of DNA products as r.c. The number of nucleotides added to the primer is shown to the right of the gel. (D) The effect of P1.1 position on template boundary definition. Two PdiTR mutants, L1 and L2, with a single adenosine residue inserted immediately after positions 11 and 32, respectively, were assembled in vitro with PdiTERT and assayed for telomerase activity using primer (GGGTTA)₃.