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. 2007 Jan 30;35(4):1119–1133. doi: 10.1093/nar/gkl1162

Figure 1.

Figure 1.

Physical interactions between Spo11 proteins. (A) Immunoblotting analysis for Gal4BD-fused Spo11 and Spo11-3FLAG expressed in meiotic cells. Whole-cell extracts (WCEs) of meiotic cells were prepared from cells cultured for 3.5 h. Upper panel: anti-Gal4BD; lower panel: anti-FLAG immunoblotting. Arrows indicate Gal4BD–Spo11 (upper panel) or Spo11-3FLAG (lower panel) proteins. ‘WB’ means Western blotting. (B) Immunoprecipitation (IP) of Spo11-3FLAG with Gal4BD–Spo11 (or Gal4BD–spo11Y135F). YHS113 (Spo11-3FLAG), ORD5806 (Gal4BD–Spo11), YHS173 (Gal4BD–Spo11/Spo11-3FLAG) and YHS550 (Gal4BD–spo11Y135F/Spo11-3FLAG) cells were transferred to a sporulation medium (SPM). Meiotic cells were disrupted under non-denaturing conditions. Then, WCEs were immunoprecipitated with anti-FLAG antibody as described in the Materials and methods section. Gal4BD-fused proteins were detected by immunoblotting with anti-Gal4BD antibody. Strains expressing Spo11-3FLAG alone or Gal4BD–Spo11 alone were used as negative controls. In these experiments, all SPO11-derived genes were expressed under the control of the native SPO11 promoter. Lanes marked WCE and IP represent WCEs and immunoprecipitates, respectively. Western blotting of IP fraction with anti-FLAG is shown in lanes 9–12 as a control (lane 9: Spo11-3FLAG, lane 10: Gal4BD–Spo11, lane 11: Gal4BD–Spo11/Spo11-3FLAG, lane 12: Gal4BD–spo11Y135F/Spo11-3FLAG) (C) Comparison of the interactions between Spo11 subunits during premeiosis and meiosis. Two aliquots of the culture were taken after incubation of the YHS425 strain in premeiotic SPS medium. One aliquot was processed to prepare premeiotic WCEs and the rest was further incubated in SPM for 3.5 h to prepare meiotic WCEs. IP experiments were conducted as described in (B). The asterisk indicates a band attributable to a non-specific reaction. The lower panel shows immunoprecipitated Spo11-3FLAG detected with anti-FLAG by immunoblotting analysis. (D) Treatment of the Spo11 complex with DNase I. Immunoprecipitates were treated with DNase I as described in the Materials and methods section. The double asterisk represents the mouse IgG heavy chain and the arrow indicates immunoprecipitated Gal4BD–Spo11 protein. The lower graph shows quantification of the band intensity for Gal4BD–Spo11 protein in upper panel. The data are averages of three independent experiments. YHS425 strain was used in this experiment. (E) Interactions between Spo11 in DSB-defective mutants. WCEs were prepared from wild-type (YHS425), rec102Δ (YHS620), rec103Δ (YHS639), rec104Δ (YHS612), rec107Δ (YHS615), rec114Δ (YHS616) and mei4Δ (YHS618) strains. The upper panel shows the polyacrylamide gel stained with Coomassie brilliant blue as a loading control for each mutant and wild-type WCE. The middle panel shows the results of immunoblotting of each WCE with anti-Gal4BD and anti-FLAG antibodies. The arrow in the lower panel indicates Gal4BD–Spo11 immunoprecipitated by anti-FLAG. This experiment was repeated three times using independent cultures. The bottom graph indicates quantification of the intensity of each band normalized with reference to those of Gal4BD–Spo11 in WCE.

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