Characterization of the licensing inhibitor in metaphase extracts
a, Metaphase-arrested extract was mixed with either geminin bead-purified RLF-B or interphase extract. The mixture was incubated for 20 min with “6-DMAP chromatin”, and licensing subsequently assessed. b, Metaphase extract was immunodepleted with anti-geminin antibodies (filled triangles) or non-immune antibodies (open triangles). Serial dilutions in LFB2/50 buffer of depleted extract or untreated metaphase extract (open circles) were then assayed for their ability to inhibit licensing of “6-DMAP chromatin” by an equal volume of interphase extract. Inset: extract depleted with control antibodies (lane 1) or anti-geminin antibodies (lane 2) immunoblotted for geminin. c, Histone H1 kinase activity of interphase extract (1), metaphase extract (2), geminin-depleted metaphase extract (3) or control-depleted metaphase extract (4). d, Morphology of sperm nuclei incubated in metaphase extract (i, ii), geminin-depleted metaphase extract (iii, iv), or interphase extract (v, vi), stained with Hoechst 33258 and viewed under UV fluorescence (i, iii, v) or phase contrast optics (ii, iv, vi). Scale bar 10 μm. e, The indicated dilution of depleted metaphase extract, or untreated metaphase or interphase extract was incubated for 20 min with “6-DMAP chromatin” and licensing subsequently assessed.