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14
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0342649723
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note
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Kluyveromyces lactis TER was cloned previously (16). Cloning of the other TER genes will be described elsewhere.
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15
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0342649724
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note
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Secondary structures were predicted by the computer program mfold version 2.3 (28). The analyses were performed on the full-length wild-type and mutant TER sequences using the default parameters of the program except for the folding temperature, which was defined as 30°C.
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16
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0343520004
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note
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Cells were grown in minimal medium to an optical density (OD) of 1 (plasmid-encoded TER alleles), then diluted 1:4 in yeast extract, peptone, and dextrose (YPD) medium, and grown to an OD of 2; or they were grown directly in YPD medium to an OD of 2 (integrated TER alleles). Telomerase activity in extracts from the homogenized cells (9) was partially purified and assayed as described (74). The primer used for all telomerase assays was 5′-GTGGTGTACGGA-3′.
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22
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0343520002
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note
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Cloning of telomere fragments was modified from the ligation-anchored PCR strategy to clone RNA 5′ ends (29) as follows. Genomic DNA (0.5 μg) was ligated to a 5′-phosphorylated, 3′-NH2-modified anchor oligonucleotide (Operon, Alameda, CA) at 37°C for 2 hours, followed by heat inactivation at 70°C for 15 min, then digestion with Eco RI restriction endonuclease, and purification with QIAquick PCR kit (Qiagen, Valencia, CA). The ligated DNA (150 ng) was amplified by PCR with an upper primer composed of a subtelomeric sequence present internally to 11 out of the 12 K. lactis telomeres (75) and an Apa I restriction site (5′-GACCGGGCCCAGCAGGACCAAG-3′), and a lower primer complementary to the anchor primer and containing an Eag I restriction site (5′-CGACGCGGCCGCTTATTAACCCT-3′). PCR products were extracted from an agarose gel, cloned into a Bluescript vector, and sequenced.
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24
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0343083861
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note
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In the D1′ mutant, read-through of up to 3 nt would copy the D1′ mutation, resulting in the incorporation of 5′-TCA-3′. However, the same sequence would be incorporated at the beginning of another round of synthesis by the Bd I-marked telomerase (see Fig. 3C). Therefore, limited read-through by the D1′ mutant enzyme may also have occurred in vivo but could not be distinguished from normal Bcl I telomeric repeats.
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28
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0032528327
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G. P. Harrison, M. S. Mayo, E. Hunter, A. M. Lever, Nucleic Acids Res. 26, 3433 (1998); B. I. Klasens, H. T. Huthoff, A. T. Das, R. E. Jeeninga, B. Berkhout, Biochim. Biophys. Acta 1444, 355 (1999).
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34
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0343955796
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note
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We thank R. Andino, C. Autexier, T. Cech, S. Chan, C. Gross, A. Krauskopf, J. Lin, Y. Mandel-Gutfreund, M. McEachern, S. Nautiyal, J. Shlomai, H. Wang, and H. Zehavi for critical reading of the manuscript and useful advice; the members of the Blackburn lab for stimulating discussions; and M. Lachance for kindly providing Kluyveromyces species. Supported by an NIH grant to E.H.B. (GM26259), a Human Frontier Science Program fellowship to Y.T. (LT-415/96), and an NIH training grant to T.B.F. (T32CA09270).
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