Fig. 1.
Centromere tethering and NEC segregation are perturbed in zip1, zip3, and mad3 mutants. (A) Nondisjunction (NDJ) of chromosome V at meiosis I was analyzed using the LacO/LacI-GFP system in tetrads containing four GFP foci. (B) The frequencies of NEC nondisjunction are shown. In both wild type and zip1, <5% of tetrads display precocious separation of sister chromatids or meiosis II nondisjunction. (C–H) Centromere tethering was assessed for different stages of meiosis I by staining surface-spread meiotic nuclei with antibodies against tubulin, GFP (neCEN5), and Zip1. (C) Midprophase spreads from wild type and mutants were identified by screening DAPI-stained chromatin for condensed, worm-like chromosomes. [In wild type, 96% of such nuclei exhibit linear Zip1 staining, indicative of the pachytene stage (n = 101).] (E) Diplotene spreads have a single nucleus with two separate tubulin foci. (G) Metaphase I spreads have short spindles (∼2 μm). Arrows indicate the positions of the neCEN5 centromeres. (Scale bars, 2 μm.) (D, F, and H) Shown are the frequencies of tethered neCEN5s for the different stages of meiosis. The asterisks denote P-values < 0.017 compared to wild type. Strains: Y712, Y787, Y790, Y1010, Y784, Y1155 (see Table S1).