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Nucleic Acids Research, 1994, Vol. 22, No. 10 1861-1865
© 1994

METHODS

Highly efficient DNA synthesis in isolated mitochondria from rat liver

José A. Enríquez, Javier Ramos, Acisclo Pérez-Martos, Manuel J. López-Pérez and Julio Montoya*

Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Veterinaria, Universidad de Zaragoza Miguel Servet 177, 50013 Zaragoza, Spain

*To whom correspondence should be addressed

Received February 1, 1994. Revised April 12, 1994. Accepted April 12, 1994.

We have developed a highly efficient DNA-synthesizing system with isolated intact rat liver mitochondria. The ATP requirements for this in organello DNA synthesis are provided by endogenous synthesis in the presence of exogenous ADP and an oxidizable substrate. In this system, mitochondrial DNA synthesis strikingly proceeds at a constant rate for about 5 h at 37°C. Gel electrophoresis, hybridization and restriction enzyme analyses show that intact mitochondria synthesize nucleic acids with a size of 16.5 kb, that correspond to mitochondrial DNA, and that both DNA strands are replicated. This in organello DNA synthesis requires the supply of dNTPs and decreases at high ADP concentration in the incubation medium.


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