Figure 5.

The pattern of mtDNA labeling in cells arrested at G1 and the fate of the labeled mtDNA in dividing (budding) cells after the release of the G1-arrest. (A) Outline of the experiment. Nascent (newly synthesized) mt-pMK2 DNA, in AFS98-pMK2 cells arrested at G1 by α-factor, was specifically labeled by [¹⁴C]thymidine for 8 h at 30°C. Then, the radioactive thymidine was removed, excess unlabeled thymidine (0.5 mg/ml) was added, and the incubation was continued for 1 h. After the α-factor was removed to release the G1 arrest, the cells were allowed to grow synchronously at 30°C for 1.5 h in YPD medium. The cells were collected, and mother cells and buds were separated. After the separation of mother cells and buds in each preparation, we confirmed by microscopic observation that the buds were enriched to >85% of the total cells. (B) Gel profile by staining with ethidium bromide. DNA was prepared from whole cells, mother cells, and buds, separately (lanes 4–9) and was analyzed for circular monomeric mt-pMK2 DNA. For the analyses for total mt-pMK2 DNA as linear monomers, to avoid possible overlap of the ¹⁴C incorporated in nuclear DNA after the release of the G1-arrest (see lanes 8 and 14 in C: dividing ρ⁰ cells), an aliquot of each DNA sample was treated with the restriction endonuclease SalI, which cleaves mt-pMK2 DNA at a single site (lanes 10–15; Whole DNA/SalI). The DNA samples were electrophoresed through a 0.5% agarose gel, which was stained with ethidium bromide. Lane 1, 5-kb ladder as a size marker. Lane 2, ¹⁴C-labeled pMK2 DNA, from E. coli, digested with SalI. Lane 3, ¹⁴C-labeled pMK2 DNA, from E. coli. (C) Profile of ¹⁴C-labeled DNA. After electrophoresis, the gel was dried and the ¹⁴C from only the area of the band of pMK2-DNA monomers in the gel was detected and quantified. (D) Profile of mtDNA detected by a [³²P] pMK2 DNA-probe. To estimate the amount of monomers of mt-pMK2 DNA, the gel was subjected to Southern hybridization by using the [³²P] pMK2 DNA-probe, and the amount of ³²P within the same area used in the measurements for ¹⁴C was quantified. (E) Relative ¹⁴C-densities in mtDNA. The net ¹⁴C signal and the ³²P signal of circular monomeric mt-pMK2 DNA in cells (or buds), in lanes 4 through 7 of the gels shown in C and D, respectively, were calculated with the correction described in Figure 2E. The net ¹⁴C signals and ³²P signals of the total mt-pMK2 DNA (i.e., circular monomers plus concatemers in cells, measured as SalI-produced linear monomers) are the gross ¹⁴C signals and ³²P signals, respectively, from linear monomers of endonuclease SalI-treated mt-pMK2 DNA in lanes 10 through 15 (in C for ¹⁴C and D for ³²P) minus the gross ¹⁴C signals and ³²P signals, respectively, from the corresponding gel areas for the DNA of p⁰ cells (lanes 14 and 15) as the background. The ¹⁴C density in each DNA species was calculated by dividing the net ¹⁴C signals by the net ³²P signals. Note that the linear monomers are the major species of endonuclease SalI-treated mt-pMK2 DNA, and the other minor products (see lane 4 of Figure 2B) were ignored to avoid the overlap of the ¹⁴C signals from nuclear DNA (see lane 14 in Figure 5B), assuming the ¹⁴C density is homogeneous throughout the concatemers. As for raw data and calculations, see the Supplementary Table 1. Note that in these experimental conditions, circular monomers and linear monomers were not well separated, and were not distinguished when counting the signals. The relative values of the ¹⁴C density of each DNA species to the total mtDNA in whole growing cells (grown for 1.5 h; black bar) were plotted. Gray bars, total mt-pMK2 DNA; open bars, circular monomeric mt-pMK2 DNA. Each bar indicates the average of two independent experiments.