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Volumn 288, Issue 5467, 2000, Pages 863-867

Template boundary in a yeast telomerase specified by RNA structure

Author keywords

[No Author keywords available]

Indexed keywords

RNA; TELOMERASE;

EID: 0034608073     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5467.863     Document Type: Article
Times cited : (117)

References (34)
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    • E. H. Blackburn and C. W. Greider, Eds. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
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    • (1995) Telomeres , pp. 11-34
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    • Tesmer, V.M.1
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    • note
    • Kluyveromyces lactis TER was cloned previously (16). Cloning of the other TER genes will be described elsewhere.
  • 15
    • 0342649724 scopus 로고    scopus 로고
    • note
    • 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.
  • 16
    • 0343520004 scopus 로고    scopus 로고
    • note
    • 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′.
  • 22
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    • note
    • 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.
  • 24
    • 0343083861 scopus 로고    scopus 로고
    • note
    • 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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    • R. F. Gesteland, T. R. Cech, J. F. Atkins, Eds. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
    • E. H. Blackburn, in The RNA World, R. F. Gesteland, T. R. Cech, J. F. Atkins, Eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999), pp. 609-636.
    • (1999) The RNA World , pp. 609-636
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    • M. Zuker, Science 244, 48 (1989).
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    • note
    • 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).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.