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1 (Fig. 4), the culture was filtered, washed, and resuspended in isotopically normal minimal medium containing 1% sodium acetate, 0.5% ammonium sulfate, and α-factor, and split in half. Galactose (final concentration of 2%) was added to one portion; the other portion was the control, uninduced sample. After 5 hours, glucose was added to both portions (final concentration of 2%); 30 min thereafter, the cultures were shifted to 37°C and treated with Pronase, whereupon the cells progressed to the cdc7 block. The cells were released into S phase by shifting the culture to 23°C; samples were collected through S phase and processed for CsCl density gradient centrifugation (5). For excision at the nocodazole block, cells grown in isotopically dense medium were synchronized with α-factor as before, and nocadazole was added to the culture (final concentration of 10 μg/ ml) before release from the α-pheromone block. Once the culture had arrested as predominantly large-budded cells, it was split in two and galactose was added to one portion. After 5 hours, both portions were filtered, washed, and resuspended in isotopically normal minimal medium containing 2% glucose and incubated at 37°C until the cells reached the cdc7 block. The cells were then released into S phase and samples were collected as before. Small samples were also collected at various steps through the whole procedure and analyzed by flow cytometry to confirm the cell-cycle arrests (16).
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note
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We are grateful to M. Gartenberg and K. Kolor for gifts of plasmids and strains and to M. Hoang for technical assistance. We also thank A. Donaldson, K. Kolor, and A. van Brabant for their helpful comments on the manuscript. Supported by National Institutes of General Medical Sciences grant 18926 to B.J.B. and W.L.F.
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