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. 2013 Apr 17;41(11):5799–5816. doi: 10.1093/nar/gkt273

Figure 3.

Figure 3.

Effects of overproducing DIN7 on DSBs at ori5 and mtDNA copy number. (A) Example of a gel analysis of DSBs at ori5 and mtDNA copy number in the indicated cells, with or without Din7 overproduction. DIN7 expression under control of the GAL1 promoter was induced in wild-type and Δmhr1 HS ρ− cells, both of which harbored the DIN7 expression vector, pYES2/CT-DIN7, by growing the cells for 12 h at 30°C in galactose-containing RGal medium. Control cells contain the empty pYES2/CT vector. Cellular DNA (∼10 µg) was isolated from cells, digested with BglII and analyzed by 2% agarose gel electrophoresis and Southern hybridization, using 32P-labeled HS ρ− mtDNA and the NUC1 gene as probes. (B) Changes in DSBs at ori5 and mtDNA copy number in wild-type and Δmhr1 HS ρ− cells during growth under Din7 overproduction. The HS ρ− wild-type and Δmhr1 cells harboring either the DIN7 expression vector or the empty vector were grown for the indicated times after the induction of DIN7 expression. Whole cellular DNA was isolated and subjected to Southern hybridization analysis as in (A). The amount of DSBs at ori5 in HS ρ− mtDNA and the mtDNA copy number were measured and calculated as in Figure 2B. Top, DSBs at ori5; bottom, copy number of HS ρ− mtDNA. ○, HS ρ− wild-type cells harboring pYES2/CT; •, HS ρ− wild-type cells harboring pYES2/CT-DIN7; ▵, HS ρ− Δmhr1 cells harboring pYES2/CT; ▴, HS ρ− Δmhr1 cells harboring pYES2/CT-DIN7. Each symbol represents the results of at least two independent experiments. (C) Immunoblot analysis of Din7 and Mhr1 expression in DIN7-induced cells. Cell-free extracts were prepared from HS ρ− wild-type and Δmhr1 cells both of which harbor either the empty pYES2/CT vector or the DIN7 expression vector pYES2/CT-DIN7 and grown for the indicated times under the conditions that induced DIN7 expression. Din7 with a C-terminal 6× His tag, Mhr1 and porin (a constitutively expressed control protein) was detected using an anti-His-tag antibody, an anti-Mhr1 serum (12) and a monoclonal anti-porin antibody, respectively.