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. 2006 Jan 30;172(3):347–362. doi: 10.1083/jcb.200507003

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Copyright © 2006, The Rockefeller University Press

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Figure 7. — AgClb1/2p levels do not diminish at mitotic exit, and cyclin mutants lacking D-box sequences have normal nuclear division. (A) Mitotic AgClb1/2-13mycp was visualized in young germling cells (ASG43) that were arrested and released from nocodazole. Clb1/2-13myc protein was evaluated by immunofluorescence and (B) on a Western blot, where tubulin was used as a loading control. Arrows highlight anaphase/telophase nuclei with mitotic cyclin. In overlays, DNA is blue, tubulin is green, and cyclins are red, leading to purple nuclei containing cyclins. (C) Wild-type strains were transformed with plasmids containing either AgCLB1/2, Agclb1/2-db1 (pAKH47), Agclb1/2-db2 (pAKH45), or Agclb1/2-dbd (pAKH46) and grown in liquid media containing G418 to maintain the plasmid in most nuclei. A subset of asynchronous cultures grown for 16 h were fixed and processed for antitubulin immunofluorescence, and nuclei were evaluated according to spindle stage (t = 0). The remaining cultures were then arrested and released from nocodazole, and percent nuclei in mitosis were evaluated at 120 min, the point at which wild-type cultures have just returned to normal asynchronous levels of mitosis. (D) Spores from cells containing AgCLB1/2, Agclb1/2-db1, Agclb1/2-db2, or Agclb1/2-dbd were inoculated on plates, and radial growth was evaluated after 5 d at 30°C. Similar growth rates were also observed when alleles are integrated in the genome at the ADE2 locus (not depicted). (E) Spores from cells containing AgCLB1/2-13myc, Agclb1/2-db1-13myc, Agclb1/2-db2-13myc, or Agclb1/2-dbd-13myc were inoculated into liquid media containing 150 μg/ml G418 for selection, grown for 16 h at 30°C, and lysed and processed for an anti-myc Western blot. The asterisk is a nonspecific cross-reacting band for a loading control. Molecular mass is shown in E in kilodaltons.