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. Author manuscript; available in PMC: 2008 Jun 9.
Published in final edited form as: Mol Cell. 2008 Apr 11;30(1):98–107. doi: 10.1016/j.molcel.2008.02.016

Figure 2.

Figure 2

A 160 bp window was the only sequence of bub1-ade6 required for efficient integration. A. Deletion constructs that lacked portions of the bub1-ade6 sequence were tested with the plasmid assay for defects in integration frequency and targeting. The number of target plasmids screened for insertions is indicated by the number of AmpR colonies. The proportion of target plasmids that have an insertion is the number of KanR colonies divided by the AmpR colonies (integration frequency). The percent of the insertions that mapped to the intergenic sequence is in the column labeled targeting % in promoter. The strains used for this data were YHL1101 containing pHL414-2 (constr. A) or pHL2541, and the deleted versions of pHL2410 (Table S2). B. Blots of RNA from cells containing the deletion constructs were probed for mRNA produced by ade6 and actin. The ribosomal RNA stained with ethidium bromide is also shown. The RNA levels were quantified by phosphoimaging and the amount of ade6 mRNA is shown normalized to actin mRNA. C. RNA from the same strains was probed for bub1 mRNA and, as in B, the amounts were normalized to actin mRNA. D. Proteins bound to bub1-ade6 sequence were mapped by digesting chromatin fractions with increasing amounts of MNase. The positions of MNase cleavages were detected on DNA blots probed by indirect end label (black rectangle). The samples containing plasmids with the intact bub1-ade6 target (constr. A) are labeled WT (YHL8964) and the samples containing plamids with no target sequence are labeled empty vector (YHL9085). Naked DNA indicates the purified DNA that was treated with increasing amounts of MNase and pooled. M indicates the molecular weight markers and the triangles are the nucleosome specific cleavages. The supersensitive cleavages are marked with black circles. The distances between the MNase cleavages are shown in bp. The grey ovals on the diagram of the bub1-ade6 sequence represent nucleosomes. E. The insertions in the plasmid with bub1-ade6 (Fig. 1C) are shown in a histogram. The positions of the MNase sensitive cleavages are indicated in red with triangles.