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. 2005 Jan 15;19(2):255–269. doi: 10.1101/gad.321105

Figure 2.

Figure 2.

Spo11-Myc foci form independently of DSBs and colocalize with Rad50 and Mre11 in rad50S cells. (A) A spread nucleus from early zygotene (z) and one from pachytene (p) are displayed side by side. Black and white panels show Spo11, Zip1, and DAPI separately, while the color panel presents Spo11 (red) and Zip1 (green) superimposed. The abundance of foci in both stages fluctuates considerably, suggesting dynamics in which foci are formed during zygotene and removed during pachytene. (B) The graphs show the appearance of Spo11-Myc foci during meiotic time courses in wild type, rad50Δ, rad50S, rec102Δ, rec104Δ, and rec114Δ cells. One-hundred cells were scored at each time point. The red symbols represent the fraction of nuclei with >20 foci, empty symbols the fraction containing less than five foci, and the blue symbols the fraction of cells past MI (determined by DAPI staining). (C) Typical spread meiotic nuclei from spo11-Y135F, rec102Δ, and rec114Δ mutant strains, stained with antibodies to Spo11-Myc (red) and to Zip1 (green). (D) Nuclear localization of Spo11 is relaxed in rec102Δ and rec104Δ mutants. Quantification of in situ staining of whole cells of rec102Δ and rec104Δ mutants at 6 h in meiosis stained with antibodies to Spo11-Myc (red) and to Rec8-Ha (green). One-hundred cells with nuclear Rec8 were counted in each experiment. Rec8 is an early meiosis-specific protein localizing to the nucleus, which serves to exclude errors from faulty staining or from nonmeiotic cells. Staining was classified as being “exclusively”, “predominantly”, or “equally” nuclear compared with the cytoplasm. These three classes are illustrated in the right panel, with the red and the green channel slightly horizontally dislocated. Under “absent” we counted cells with no detectable Spo11, but normal Rec8 staining. “AnaI” and “AnaII” denote meiotic anaphase stages, in which Spo11 is nuclear, even in the two mutants. (E) Spo11 and Rad50, and Spo11 and Mre11 foci colocalize in rad50S meiotic prophase nuclei. Cells from a 6-h time point were spread and stained with antibodies against Spo11-Myc (red), Rad50 (green), and Mre11 (green) and with DAPI. Regions of overlap between Spo11 and Rad50, or between Spo11 and Mre11, appear yellow in the merged images. A significant fraction but never all of the foci colocalize.