A plasmid based assay for integration targets. A. Transposition was induced by activating transcription of Tf1 from an expression plasmid. The Tf1-neo mRNA was packaged into virus like particles, reverse transcription occurred, and IN inserted the Tf1-neo cDNA. A target plasmid present in the same cells had the potential to be the target of Tf1 integration. The target plasmid shown contained bub1-ade6 sequence as a potential region for integration. LEU2d was used to select for cells with high copy numbers of the target plasmid. B. Cells with inserts of Tf1-neo were identified on medium containing G418. Frameshift mutations that block expression of PR and RT (PRfs) or IN (INfs) inhibit transposition. The position of insertions in target plasmids are shown with the plasmid coordinates. The plasmids contained bub1-ade6 (C), fbp1 (D), nup124 (E), SPCC4F11.03c (F), SPBC365.14c (G), and the 173 bp insertion window of bub1-ade6 (H). Restriction sites for Xma I (Xm), Sbf I (Sb), Bmg BI (Bm), Spe I (Sp), Hind III (H), and Bam HI (B) are shown.

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 Amp^R colonies. The proportion of target plasmids that have an insertion is the number of Kan^R colonies divided by the Amp^R 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.

Targeted integration in the promoter of fbp1 requires UAS1 and Atf1p. A. The positions of Tf1 integration in the plasmid that contained fbp1 (Fig. 1D) were represented on a histogram of the promoter. The height of each line is the percent of all the insertions in the plasmid that occurred at the specified position. B. The target plasmid assay was used to map Tf1 integration in fbp1 that contained a six bp mutation in UAS1. C. The target plasmid assay was used to map integration in fbp1 in a strain that lacked atf1 (YHL9526/pHL2541+pHL2543). The positions of insertion were represented on a histogram of the promoter. D. Insertion sites were mapped in bub1-ade6 in the strain that lacked atf1 (YHL9526/pHL2541+pHL2410). E. The insertions in fbp1 in the strain lacking atf1 are shown on a map of the plasmid. F. The insertions in bub1-ade6 in the strain lacking atf1 are shown on a map of the plasmid. G. Immunoprecipitation was performed on extracts from cells expressing Atf1p-FLAG. The whole cell extract (WCE) and the protein precipitated with anti-FLAG antibody were immunoblotted and probed with an anti-IN antibody. H. The binding of Atf1p to the UAS1 of fbp1 and to the target window of ade6 was measured by ChIP. The amount of fbp1 and ade6 precipitated was measured relative to the amount of the actin gene and was normalized using the ratio of fbp1 or ade6 from the whole cell extract (WCE) divided by actin. The quantified results from three separate experiments were averaged and are shown with error bars indicating one standard deviation.

Integration of Tf1-neo activates the transcription of ade6 and bub1. A. Plasmids derived from construct A (A) include: construct A with an insertion of Tf1-neo in the left orientation at position 4573 (A-L), construct A with an insertion of Tf1-neo in the right orientation at position 4573 (A-R), construct A with a deletion from position 4573 up to the ATG of bub1 (M), construct M with an insertion of Tf1-neo in the left orientation at position 4573 (M-L), and construct M with an insertion of Tf1-neo in the right orientation at position 4573 (M-R). Neo is indicated by a grey rectangle and the direction of its transcription is indicated by an arrow. B. The levels of ade6, actin, and ribosomal RNA produced by strains (YHL9435-YHL9441) with these construct plasmids was measured on an RNA blot. The RNA levels from four independent yeast transformants were quantified by phosphoimaging and averaged. The amount of ade6 mRNA was normalized to actin mRNA and adjusted for differences in plasmid copy number (Fig. S2 and S3). The variation in mRNA levels between transformants is depicted with error bars indicating one standard deviation. C. The levels of bub1 and actin mRNA were measured and normalized as described for ade6.