Fig. 2. Assay for yeast mtDNA maintenance activity of the mouse Tfam protein in yeast mitochondria.

(A) Construction of a yeast cell containing the mouse Tfam gene and full yeast mtDNA. The karyogamy-defective mutation (kar1- 1) and selection markers (URA3 and TRP1) were used to screen yeast cells (Tfam, ρ⁺) that maintained active mitochondrial respiratory function after a genetic cross between the wild-type mtDNA donor (ABF2, ρ⁺) and Tfam ρ⁰ recipient cells. (B) Yeast cell growth on the two different growth media. The ABF2 wild-type, abf2 deletion mutant (abf2Δ) and Tfam strains were cultured in glucose medium (YPD) and ethanol medium (SEG/Ura-), respectively. The abf2Δ strain could not grow on ethanol medium because the strain lacked active mitochondrial function to use ethanol as the carbon source for growth due to the loss of mtDNA. In contrast, the Tfam strain was able to grow on ethanol medium because the strain recovered the active mitochondrial function due to the mtDNA maintenance activity of the Tfam protein. (C) PCR analysis. Insertion of the Tfam gene and deletion of the ABF2 gene were verified by PCR. As a control, a PCR assay for the yeast MGM1 gene was performed.