F-cores were incubated with the microtubule depolymerizing agent nocodazole. In nocodazole treated extracts, chromosome condensation began on schedule and discrete chromosome threads formed 65 min, as in Factor Xa cancer extracts of contr If you do not have the ZM. Although chromosomes in nocodazole treated extracts to a certain degree w Grouped during mitosis, they remained were condensed at the same time in extracts of contr down on, and some chromosomal son recognizable. This contrasts with the appearance of the chromosomes in the ZM treated extracts, not to discrete chromosomal F To, instead, underwent premature chromosome decondensation. These results suggest that defects in chromosome morphology and premature chromosome decondensation in ZM treated extracts was observed not only a consequence of the absence of mitotic spindles.
ZM Inhibits the formation of the integrity of its spindle checkpoint, but not its maintenance, if the concentration exceeds the nuclei in extracts 9,000 / l, extracting sensitive spindle checkpoint: in the presence of nocodazole or other agents to prevent the polymerization Fingolimod 162359-56-0 of microtubules, cyclin activity remains B/Cdc2 t remain highly condensed and chromosomes. To ask whether ZM with the control st Rt Integrity T of the spindle, 10,000 cells / l and rhodamine tubulin to cycling extracts at time 30 min on ice. Parts of the extract were either complements with DMSO, nocodazole, or ZM erg, And after 10 minutes, 21 exp Rmt to the resumption of cell cycle adjusted to erm. Controlled in the extract On the histone H1 kinase activation was seen by 90 min and decreased after the 110th As Figure 5 should.
ZM reduces spindle assembly in extracts in M phase by CSF extract or nondegradable cyclin B protein, the extracts were driven arrested metaphase II eggs and cooked erg complements With rhodamine-labeled tubulin and sperm nuclei Of. The extracts were entered Born in interphase by the addition of calcium chloride. Sixty minutes after the addition of calcium, or DMSO ZM added. Twenty minutes later Ter, the controller ZM and the extracts were treated entered into M-phase Born by the addition of CSF extract containing either DMSO or ZM, respectively. The samples were incubated at 80 after the start of M phase and adjusted by means of fluorescence microscopy. The number of bipolar spindles, spindles, which were not obviously bipolar, or cores, the microtubule-F Lacked detectable staining determined.
The images in panel I shows the different categories that were quantified in Panel II. The inhibitory effect of ZM on spindle assembly varies between experiments. Quantification of spindle structures of two independent Ngigen experiments is shown in Table II. More than 100 structures for each experiment were gez Hlt. Bar, 15 m. CSF egg extract was treated as in A, au He added that the non-degradable cyclin B protein to drive the extract into mitosis, instead of CSF extract. Spindle structures were as in A. gez Hlt bar, 15 m. More than 100 structures were gez in this experiment Hlt. The graph shows the percentage of spindle structures observed in extracts from DMSO treated and ZM a repr Sentative for three independent Independent experiments. BB Gadea and JV Ruderman 1312 Molecular Biology of the Cell for nocodazole treated extracts, histone H1 kinase