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0342479264
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note
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GFP-Lacl expression driven by CVC1 promoter, pAFS 152 (38).
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11
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0343348780
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note
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If only one homolog is marked with GFP, normal segregation of the two sister chromatids in meiosis II will produce two spores that have a single copy of the GFP-marked chromosomes. Nondisjunction in meiosis II will lead to a single spore that contains two copies of the GFP marked chromosome. The measured frequencies of nondisjunction in wild-type strains are higher than those reported from genetic tests (39). We believe that this discrepancy is due to a low frequency of recombination events that reduce the number of Lac operator repeats below the threshold required for detection.
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12
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0342913560
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note
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All strains are in the W303 background, unless otherwise noted: MAT a/MATα, ura3-1 leu2, 3-172, his3-11, trp1-1, ade2-1, can1-100. All mutations are homozygous unless otherwise noted. GFP tagging of chromosome IV (MAS 386, 327; wild-type and mad7Δ, respectively) and III (MAS 401, 424) is described in (9). Strains MAS 559, 561 were constructed by integrating pMAS72 near the centromere of chromosome VII. Plasmid pMAS72 contains a Bam Hl-Sac I fragment spanning nucleotides 478360 through 479162 of chromosome VII in plasmid pAFS149 (38) which contains 128 repeats of LacO. Strains MAS 656, 658 were constructed by integrating pMAS79 near the centromere of chromosome VIII. Plasmid pMAS79 contains a Bam Hl-Sac I fragment spanning nucleotides 123761 through 124110 on chromosome VIII in pAFS149. All experiments were repeated at least twice with similar results. In all experiments, at least 200 cells were counted.
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13
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0343784503
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note
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We used the SK1 strain background for all experiments requiring a rapid, synchronous meiosis. Synchronous sporulation in 2% potassium acetate as in (74). All SK1 strains derived from NKY 611:leu2::hisG ura3, ho::LYS2. lys2. SK1 wild-type: MAS 120, SK1 mad2A: MAS 403. SK1 mad2Δ mutants also undergo meiosis I nondisjunction, but at higher levels than W303.
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17
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0030448251
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O. Cohen-Fix, J. M. Peters, M. W. Kirschner, D. Koshland, Genes Dev. 10, 3081 (1996).
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Koshland, D.4
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18
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0343784478
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note
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4 formed dyads at reduced efficiency compared to those sporulated in the absence of copper. Sister chromatid separation occurs in only 4% of divisions for both wild type and mad2Δ.
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19
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0342913518
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note
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SK1 cells were sporulated for 4 hours at room temperature in 2% potassium acetate as in (13), then resuspended in 2% potassium acetate plus 60 μg/ml benomyl, prepared by adding a 30 mg/ml stock of benomyl in dimethyl sulfoxide to boiling media and slowly cooling to room temperature. Cells were fixed in 40% ethanol and 0.1 M sorbitol.
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21
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F. Klein et al., Cell 98, 91 (1999).
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Klein, F.1
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24
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0343784421
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note
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We determined the position of chromosome III homologs on the long meiosis I spindle in spo11Δ (SK1 strain MAS 645: LacO:LEU2 GFP-Lacl spo11Δ) by indirect immunofluorescence against Tub1 and GFP-Lacl. We find that the marked centromere is always pulled to a spindle pole, suggesting that homologs are attached to the spindle.
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29
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0030968972
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A. M. Galbraith, S. A. Bullard, K. Jiao, J. J. Nau, R. E. Malone, Genetics 146, 481 (1997).
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Nau, J.J.4
Malone, R.E.5
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0034007469
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R. Cha, B. Weiner, S. Keeney, J. Dekker, N. Kleckner, Genes Dev. 14, 493 (2000).
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Cha, R.1
Weiner, B.2
Keeney, S.3
Dekker, J.4
Kleckner, N.5
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38
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0342913511
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unpublished data
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A. Straight, unpublished data.
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Straight, A.1
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41
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0032473568
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M. Shirayama, W. Zachariae, R. Ciosk, K. Nasmyth, EMBO J. 17, 1336 (1998).
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Shirayama, M.1
Zachariae, W.2
Ciosk, R.3
Nasmyth, K.4
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42
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0342479163
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note
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Plasmid pMAS68 was constructed by inserting a fragment of the TUB1 promoter into pAFS160 (38) which contains a fusion between TUB4 and GFP. Plasmid was integrated at URA3 to create SK1 strain MAS 601.
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43
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0343348684
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note
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SKI wild-type (MAS608), spo11Δ (MAS613), spo11Δ mad2Δ (MAS613), and spo11Δ spo13Δ (MAS 659) homozygous diploids all express the Pds1 protein tagged with 18 copies of the Myc epitope (47).
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44
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0003529272
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Cold Spring Harbor Press, Cold Spring Harbor, NY
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M. Rose, F. Winston, P. Heiter, Methods in Yeast Genetics (Cold Spring Harbor Press, Cold Spring Harbor, NY, 1990).
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Rose, M.1
Winston, F.2
Heiter, P.3
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45
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0342913504
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note
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We thank M. Winey for sharing unpublished information and S. Roeder, S. Hawley, B. Nicklas, and members of the Murray lab for critical reading of the manuscript. We are grateful to the Kleckner lab for SK1 strains. Supported by grants from NIH and the Human Frontiers In Science Program.
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