Fig. 4

Identification of genes on ChrXII suppressing ulp2Δ aneuploidy. a Strategy used to identify specific regions of ChrXII that suppress ulp2Δ aneuploidy. A ulp2Δ/YCplac33-ULP2 strain (MHY1379) was transformed with the indicated pGP564 (LEU2)-based ChrXII library plasmids. Cells were struck on SD–Leu + FOA twice in succession to evict YCplac33-ULP2. After the putative candidate plasmids were selected by qPCR-based aneuploidy assays of ChrI, ChrIII, and ChrXII, the copy number of ChrI, ChrV, ChrVIII, ChrX, and ChrXII was determined in ulp2Δ cells bearing the selected plasmids (b) or plasmids including specific ChrXII fragments (d). b qPCR ploidy assays of ulp2Δ cells with the high-copy plasmids from the first round of selection. Copy number of the five tested chromosomes was normalized to the euploid control strain (MHY1379 + pGP564) and compared with ulp2Δ cells expressing empty pGP564 vector. Error bars indicate the SD calculated from two independent genomic DNA preparations. Asterisks indicate plasmids showing a reduction of ChrXII disomy. c Schematic diagrams of plasmids pGP564-11-b5, pGP564-11-c5, pGP564-11-e11 and pGP564-11-f11. Locations of ORFs (arrows or boxes) and gene names are indicated. Subclones used in d are shown as bars below the ChrXII regions. Full-length genes contained within subclones are listed below the bars. d qPCR ploidy assays of ulp2Δ cells with the high-copy plasmids shown in c. Chromosome copy number was determined as in b. Error bars indicate the SD calculated from two independent experiments. Asterisks denote plasmids showing a loss of ChrXII disomy. e qRT-PCR analysis of RPL12B and RPS18B in ulp2Δ cells containing a plasmid with snoRNAs (11-e11) as in Fig. 2g. The data of (–) is identical with the (–) signal shown in Fig. 2g. The error bars represent the SD from three experiments. f ChIP analysis of Rap1-TAP and Ulp2-Flag strains as in Fig. 2a. The error bars indicate the SD from analyses of two independent chromatin preparations. g SUMO levels measured by ChIP assay in the WT and ulp2Δ strains expressing His-tagged Smt3 (SUMO) as in Fig. 2b. The error bars indicate the SD from two independent samples