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18
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10244276519
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est mutant
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est mutant.
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19
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10244275194
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note
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371 → Ser change (10).
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20
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10244276830
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note
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ts strain at 23°C, as previously observed (9). This implies that some aspect of negative regulation of telomere length control has been affected; we do not yet know the mechanism.
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23
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10244281008
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-
note
-
For preparation of extracts, 2 liters of late log-phase culture were harvested and resuspended in an equal volume of lysis buffer (26); 10 ml of cell suspension was then mixed with an equal volume of glass beads in a 50-ml screw-cap centrifuge lube. The mixtures were vortexed repeatedly for 20 s at maximum speed followed by 20 s of cooling in an ice-water bath. The total vortexing time was ∼10 min. Extracts were clarified by centrifugation at 10,000g for 10 min and then at 200,000g for 1 hour. Telomerase-containing fractions were derived from a POROS 50 HS column (PerSeptive Biosystems).
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24
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10244273895
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note
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6 epitope (27) confirmed that the purified protein was Cdc13p (20).
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-
-
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26
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10244269147
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-
note
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3 oligomer in the presence of 1:1 and 10:1 molar excess of yeast, human, and Oxytricha cold competitor DNAs; data were quantitated by Phosphorlmager analysis.
-
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27
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10244281167
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-
note
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ts mutation, and suppression is specific for the ESTI gene, in that overexpression of TLC1 or EST2 has no effect (12).
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0023037564
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10244281166
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note
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ts/CDC13, tlc1-Δ::LEU2/TLC1 dipbids constructed by gene replacement of a single wild-type diploid parental strain; all haploid strains used in Fig. 1 were isogenic. For Southern blot analysis, strains were grown from freshly germinated spores to saturation, followed by two successive subculturings, representing a total of ∼50 generations of growth, before preparation of DNA.
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0025233744
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10244273090
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note
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The length of three individual telomeres (a 2.8-kb fragment corresponding to the left telomere of chromosome V-L and the 2.3- and 2.0-kb telomeres indicated by the lower two arrows in Rg. 1A) was measured by simultaneously probing with a telomere-specific probe and aλ-specific probe to detect diluted λHind III + λSty I markers present in each lane. The data are presented as the change between the "1x" and "3x" culture, rather than the change per cell generation, because of the rapid increase in dying cells in later cultures (which can still contribute DNA to the Southern profile). To ensure that similar numbers of cells were assayed over this time period, we assayed total cell counts for each successive culture; the variation between the three strains at each time point was no more than 20%.
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42
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10244272426
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note
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A assays, reactions were incubated on ice for 30 to 60 min and gels were run at 4°C; reactions for substrates 1 to 9 did not contain poly(dl-dC), Protein concentration was determined by amino acid analysis and by quantitative comparison to bovine serum albumin standards using SDS-polyacrylamide gel electrophoresis (PAGE) followed by Coomassie staining, which gave comparable results (within 15%). Coomassie gels and gel shifts were quantitated with a scanning densitometer or Phosphorlmager, respectively, and the Imagequant software package (Molecular Dynamics).
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43
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45
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10244275193
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note
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We thank H. Zentgraf for monoclonal antibody 13/ 45/31, A. Salinger and K. Shah for technical assistance, and S. Elledge and S. Plon for critical review of the manuscript. Supported by grants from NIH and Geron Corporation and by an American Cancer Society Junior Faculty Research Award to V.L. T.R.H. is supported by an NIH predoctoral fellowship.
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