Elimination of D. discoideum mtDNA by introduction of mitochondrion-targeted Eco RI. (a) Diagram of Wt-mtDNA indicating the positions of five Eco RI sites. The yellow region represents the dia3 gene cluster (about 6.5 kb) including the rps4 gene (red region). A gray arrow "1" corresponds to the rps4 gene-specific oligonucleotide probe used for Southern blot analysis. (b) Dose-response analysis of Tet-regulated Eco RI mRNA expression. LpCEco and parental MB35 cells were separately grown by shake culture for 24 hours in PS medium containing the designated concentrations of tetracycline (Tet; μg/ml), and the total RNA was extracted for Northern blot analysis using the pCoxIV-Eco RI-specific probe. It is evident that the Eco RI mRNA (about 1 kb) is expressed in LpCEco cells only under the Tet-minus condition. In the bottom of each lane, the amounts of ribosomal 17S and 26S RNA stained with EtBr are shown as loading control. (c) Kinetics of Eco RI mRNA induction during 24 hours of incubation without Tet. MB35 and LpCEco cells were grown in Tet-minus PS medium for the indicated times (hr), followed by Northern blotting. The Eco RI mRNA begins to be expressed in LpCEco cells 12 hours after removal of Tet and increase to the peak level after 24 hours of incubation. (d) Mitochondrially tagged Eco RI leads to almost complete degradation of mtDNA. Total DNAs were extracted from MB35 and LpCEco cells 48 hours after removal of Tet and digested with the indicated restriction enzymes (H, Hin dIII; N, Nde I), followed by electrophoretic size-fractionation and Southern blot analysis using either the 32P-labeled nuclear DNA-specific probe (dd-trap1) or the mtDNA-specific probe (rps4). It is clear that LpCEco cells grown without Tet are almost completely devoid of mtDNA. (e) Staining patterns of MB35, LpCEco, LpCEG, and LpCGE cells with DAPI. Cells were cultured in PS medium in the presence (+Tet) or absence (-Tet) of Tet for 48 hours. Under the Tet-plus conditions, the nuclei and mitochondria are stained well with DAPI as large dots and surrounding small granules respectively, in all of the cells examined. Under the Tet-minus conditions, however, the DAPI-staining of mitochondria disappears almost completely from LpCEco, LpCEG and LpCGE cells but not from parental MB35 cells, though the staining of the nucleus is retained well after removal of Tet. Bars, 10 μm.