TY - JOUR
T1 - Inducible expression of a dominant negative DNA polymerase-γ depletes mitochondrial DNA and produces a ρ0 phenotype
AU - Jazayeri, Mona
AU - Andreyev, Alexander
AU - Will, Yvonne
AU - Ward, Manus
AU - Anderson, Christen M.
AU - Clevenger, William
PY - 2003/3/14
Y1 - 2003/3/14
N2 - We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-γ to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-γ harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t1/2 = 2-3 days) when expression of the transgene was induced. Concurrent reduction of mitochondrial encoded mRNA and protein, decreased cellular growth rate, and compromised respiration and mitochondrial membrane potential were observed. mtDNA depletion was reversible, as demonstrated by restoration of mtDNA copy number to normal within 10 days when the expression of POLGdn was suppressed following a 3-day induction period. Long term (20 days) expression of POLGdn completely eliminated mtDNA from the cells, resulting in ρ0 cells that were respiration-deficient, lacked electron transport complex activities, and were auxotrophic for pyruvate and uridine. Fusion of the ρ0 cells with human platelets yielded clonal cybrid cell lines that were populated exclusively with donor-derived mtDNA. Respiratory function, mitochondrial membrane potential, and electron transport activities were restored to normal in the cybrid cells. Inducible expression of a dominant negative DNA polymerase-γ can yield mtDNA-deficient cell lines, which can be used to study the impact of specific mtDNA mutations on cellular physiology, and to investigate mitochondrial genome function and regulation.
AB - We report the inducible, stable expression of a dominant negative form of mitochondria-specific DNA polymerase-γ to eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA polymerase-γ harboring a D1135A substitution (POLGdn). The cells rapidly lost mtDNA (t1/2 = 2-3 days) when expression of the transgene was induced. Concurrent reduction of mitochondrial encoded mRNA and protein, decreased cellular growth rate, and compromised respiration and mitochondrial membrane potential were observed. mtDNA depletion was reversible, as demonstrated by restoration of mtDNA copy number to normal within 10 days when the expression of POLGdn was suppressed following a 3-day induction period. Long term (20 days) expression of POLGdn completely eliminated mtDNA from the cells, resulting in ρ0 cells that were respiration-deficient, lacked electron transport complex activities, and were auxotrophic for pyruvate and uridine. Fusion of the ρ0 cells with human platelets yielded clonal cybrid cell lines that were populated exclusively with donor-derived mtDNA. Respiratory function, mitochondrial membrane potential, and electron transport activities were restored to normal in the cybrid cells. Inducible expression of a dominant negative DNA polymerase-γ can yield mtDNA-deficient cell lines, which can be used to study the impact of specific mtDNA mutations on cellular physiology, and to investigate mitochondrial genome function and regulation.
UR - http://www.scopus.com/inward/record.url?scp=0037984268&partnerID=8YFLogxK
U2 - 10.1074/jbc.M211730200
DO - 10.1074/jbc.M211730200
M3 - Article
C2 - 12645575
AN - SCOPUS:0037984268
SN - 0021-9258
VL - 278
SP - 9823
EP - 9830
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 11
ER -