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16
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3543095296
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note
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s] was by Y. Kassir, and derivatives of SK1 [ura3, leu2, hisG trp1-hisG, lys2, ho-LYS2, his4] by A. Mitchell and N. Kleckner. L numbers refer to strains in our own collection.
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17
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0028879611
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L. Dirick, unpublished data
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The SBF and MBF deletion mutants were created by integration of a pGAL1-10::RME1 construct in K1107 (L43) and multiple crosses to swi4Δ, swi6Δ and mbp1Δ strains. swi4Δ/Δ swi6Δ/Δ (L44) and swi4Δ/Δ mbp1Δ/Δ (L453) cultures were enabled to grow mitotically by overexpression of the RME1 gene [W. M. Toone et al., EMBO J. 14, 5824 (1995); L. Dirick, unpublished data]. The triple CLN-deleted strain (L49), kept alive by CLN3 under pGAL1-10 promoter, comes from a cross between K2759 and K3130.
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EMBO J.
, vol.14
, pp. 5824
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Toone, W.M.1
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19
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3543098836
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note
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clb5Δ and clb6Δ deletion constructs were as described in (1). Transformation of all three clb5Δ strains with the WT CLB5 gene on a CEN plasmid [Ycplac111::CLB5 (LEU2); D74] restored sporulation.
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20
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3543096492
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note
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For fluorescence-activated cell sorter (FACS) analysis, 1-ml aliquots of cultures were fixed in 70% ethanol, treated overnight with ribonuclease (RNase) A (300 μg/ml) at 37°C, rinsed in phosphate-buffered saline (PBS), and stained with propidium iodide (40 μg/ml in PBS); 10,000 cells per sample were analyzed on a Becton-Dickinson FACSscan. The same propidium iodide-stained cells were used to record chromosome segregation by fluorescence microscopy. RNA extraction and RNA blotting conditions were as described in (2).
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23
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0027158083
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Protein immunoblot analysis of Sic1 was done as described [U. Surana et al., EMBO J. 12, 1969 (1993); A. Amon et al., Cell 77, 1037 (1994)] with a 1:2000 dilution of polyclonal antibody to Sic1 (M. Tyers). Proteins were detected with the enhanced chemiluminescence detection system (ECL; Amersham). Kinase assays were done as described in [U. Surana et al., EMBO J. 12, 1969 (1993)] starting from 140 μg of protein, except for ime2Δ in YPD where 70 μg was used. The monoclonal antibody to hemagglutinin (HA), 12CA5, was used at a 1:100 dilution to immunoprecipitate the HA3-tagged Clb5 proteins (8). Specificity was confirmed on an SK1 WT strain carrying untagged CLB5 (L111).
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(1993)
EMBO J.
, vol.12
, pp. 1969
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Surana, U.1
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24
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0028240049
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Protein immunoblot analysis of Sic1 was done as described [U. Surana et al., EMBO J. 12, 1969 (1993); A. Amon et al., Cell 77, 1037 (1994)] with a 1:2000 dilution of polyclonal antibody to Sic1 (M. Tyers). Proteins were detected with the enhanced chemiluminescence detection system (ECL; Amersham). Kinase assays were done as described in [U. Surana et al., EMBO J. 12, 1969 (1993)] starting from 140 μg of protein, except for ime2Δ in YPD where 70 μg was used. The monoclonal antibody to hemagglutinin (HA), 12CA5, was used at a 1:100 dilution to immunoprecipitate the HA3-tagged Clb5 proteins (8). Specificity was confirmed on an SK1 WT strain carrying untagged CLB5 (L111).
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(1994)
Cell
, vol.77
, pp. 1037
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Amon, A.1
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25
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0027158083
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Protein immunoblot analysis of Sic1 was done as described [U. Surana et al., EMBO J. 12, 1969 (1993); A. Amon et al., Cell 77, 1037 (1994)] with a 1:2000 dilution of polyclonal antibody to Sic1 (M. Tyers). Proteins were detected with the enhanced chemiluminescence detection system (ECL; Amersham). Kinase assays were done as described in [U. Surana et al., EMBO J. 12, 1969 (1993)] starting from 140 μg of protein, except for ime2Δ in YPD where 70 μg was used. The monoclonal antibody to hemagglutinin (HA), 12CA5, was used at a 1:100 dilution to immunoprecipitate the HA3-tagged Clb5 proteins (8). Specificity was confirmed on an SK1 WT strain carrying untagged CLB5 (L111).
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(1993)
EMBO J.
, vol.12
, pp. 1969
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Surana, U.1
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C. J. Sherr, Cell 79, 551 (1994).
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(1994)
Cell
, vol.79
, pp. 551
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Sherr, C.J.1
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note
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We thank Y. Kassir, N. Kleckner, A. Mitchell, and K. Nasmyth for strains and plasmids; M. Tyers for the Sic1p antibody; and D. Lydall, S. Piatti, D. Toczysk, and W. Fangman for useful comments on the manuscript. Supported by NIH grant GM18541 (B.B.) and the Austrian Fonds zur Forderung der Forschung (G. A.). L.D. is with the FNRS, Belgium.
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