FIGURE 3:

MAD2/mad2Δ cells do not arrest in response to short linear chromosomes. (A) A diagram of the effects of short linear chromosomes on the spindle checkpoint. CDC20-127 is a dominant allele of CDC20 that the spindle checkpoint cannot inhibit; in strains in which this allele is expressed from a tetracycline-regulated promoter (P_tet), the response to the spindle checkpoint can be manipulated. In the absence of doxycycline (left) the checkpoint is turned off: CDC20-127 is expressed, activation of the APC is resistant to signals from the checkpoint, and cells execute anaphase and proliferate normally. In contrast, in the presence of doxycycline (right) the checkpoint is turned on: expression of CDC20-127 is repressed, allowing the short linear chromosomes to activate the checkpoint and cause an arrest in mitosis that prevents cell proliferation. (B) Short linear chromosome–dependent activation of the spindle checkpoint. The indicated strains were tested for their ability to proliferate in the absence (CDC20-127 expressed; left) or presence (CDC20-127 repressed; right) of the spindle checkpoint. Serial fourfold dilutions of all strains were spotted onto plates containing 0 μg/ml doxycycline (left) or 10 μg/ml doxycycline (right) and grown for 2 d. (C) MAD2/mad2Δ heterozygotes do not have a mitotic delay in response to loss of tension. Exponentially dividing cultures of MAD2/MAD2 and MAD2/mad2Δ cells containing P_tet-CDC20-127, CDC28-VF, and short linear chromosomes were arrested in S phase with hydroxyurea. Cells were released into fresh media and treated with 10 μg/ml doxycycline to repress expression of P_tet-CDC20-127, allowing short linear chromosomes to activate the spindle checkpoint. The percentage of small, budded cells after release was determined by light microscopy at the indicated time points. Error bars represent the SD of three separate trials.