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note
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Micronuclear phenotypic quantitation was performed in over 700 cells from 14 independent transformant cell lines in two separate experiments from 5 to 7 days after transformation. On average, 55 random micronuclei per cell line per time point were assessed for anaphase by 4′,6′-diamidino-2-phenylindole (DAPI) stain. Cells with no visible micronuclei were excluded from the determination. Length was judged by eye to be roughly equivalent to that observed in WT (elongated) or at least 50% longer than that found in WT, with no apparent separation at the midzone (hyperelongated). The proportion of micronuclei in anaphase varied with respect to cell line and time point (standard deviations for elongated and hyperelongated micronuclei at 7 days were ±7% and ±16%, respectively).
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In the ter1-43AA mutant, the macronucleus was never observed to divide while the micronucleus was arrested in anaphase. Hence, the onset of macronuclear division may require completion of micronuclear division. Therefore, although mutant telomeres also impair macronuclear division (6), possibly independently of the micronuclear telomere phenotype, such impairment of macronuclear division may not become evident while the micronucleus is mitotically blocked.
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Cells were also imaged under wet buffer conditions without drying and were found to be quantitatively and qualitatively identical to dried cells: Anaphase micronuclei lengths were on average 10.46 μm by 2.06 μm by 2.49 μm for nondried (n = 7) and 10.95 μm by 1.98 μm by 2.23 μm (n = 3) for dried; non-dried chromosomes were 0.41 ± 0.09 μm wide (n = 69) versus 0.40 ±0.07 μm wide for dried chromosomes (n = 35).
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1842402126
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We thank J. C. Prescott, A. Bhattacharyya, and H. W. Bass for manuscript review. Supported by NIH grant GM26259 (to E.H.B.), an American Cancer Society postdoctoral fellowship (to K.E.K.), and by an NSF predoctoral fellowship (to B.P.H.).
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