|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 11, 9823-9830, March 14, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From MitoKor, Inc., San Diego, California 92121
We report the inducible, stable expression of a
dominant negative form of mitochondria-specific DNA polymerase-
Inducible Expression of a Dominant Negative DNA Polymerase-
Depletes Mitochondrial DNA and Produces a 
0
Phenotype*,
, and
to
eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA
polymerase- 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 
0 cells that were
respiration-deficient, lacked electron transport complex activities,
and were auxotrophic for pyruvate and uridine. Fusion of the
0 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- 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.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains videos that accompany Fig. 10 of
the main text.
To whom correspondence should be addressed: MitoKor, 11494 Sorrento Valley Rd., San Diego, CA 92121. Tel.: 858-509-5613;
Fax: 858-793-7805; E-mail: andersonc@mitokor.com.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Amaral, J. Ramalho-Santos, and J. C. St John The expression of polymerase gamma and mitochondrial transcription factor A and the regulation of mitochondrial DNA content in mature human sperm Hum. Reprod., June 1, 2007; 22(6): 1585 - 1596. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Wanrooij, S. Goffart, J. L.O. Pohjoismaki, T. Yasukawa, and J. N. Spelbrink Expression of catalytic mutants of the mtDNA helicase Twinkle and polymerase POLG causes distinct replication stalling phenotypes Nucleic Acids Res., May 11, 2007; 35(10): 3238 - 3251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Khan, R. M. Smigrodzki, and R. H. Swerdlow Cell and animal models of mtDNA biology: progress and prospects Am J Physiol Cell Physiol, February 1, 2007; 292(2): C658 - C669. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Vidal, J. C. Domingo, J. Guallar, M. Saumoy, B. Cordobilla, R. Sanchez de la Rosa, M. Giralt, M. L. Alvarez, M. Lopez-Dupla, F. Torres, et al. In Vitro Cytotoxicity and Mitochondrial Toxicity of Tenofovir Alone and in Combination with Other Antiretrovirals in Human Renal Proximal Tubule Cells Antimicrob. Agents Chemother., November 1, 2006; 50(11): 3824 - 3832. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. R. Stuart, J. H. Santos, M. K. Strand, B. Van Houten, and W. C. Copeland Mitochondrial and nuclear DNA defects in Saccharomyces cerevisiae with mutations in DNA polymerase {gamma} associated with progressive external ophthalmoplegia Hum. Mol. Genet., January 15, 2006; 15(2): 363 - 374. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Lee, C.-H. Kim, D. K. Simon, L. R. Aminova, A. Y. Andreyev, Y. E. Kushnareva, A. N. Murphy, B. E. Lonze, K.-S. Kim, D. D. Ginty, et al. Mitochondrial Cyclic AMP Response Element-binding Protein (CREB) Mediates Mitochondrial Gene Expression and Neuronal Survival J. Biol. Chem., December 9, 2005; 280(49): 40398 - 40401. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. Bayona-Bafaluy, B. Blits, B. J. Battersby, E. A. Shoubridge, and C. T. Moraes Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease PNAS, October 4, 2005; 102(40): 14392 - 14397. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. E. Nagiec, L. Wu, S. M. Swaney, J. G. Chosay, D. E. Ross, J. K. Brieland, and K. L. Leach Oxazolidinones Inhibit Cellular Proliferation via Inhibition of Mitochondrial Protein Synthesis Antimicrob. Agents Chemother., September 1, 2005; 49(9): 3896 - 3902. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Ferrari, E. Lamantea, A. Donati, M. Filosto, E. Briem, F. Carrara, R. Parini, A. Simonati, R. Santer, and M. Zeviani Infantile hepatocerebral syndromes associated with mutations in the mitochondrial DNA polymerase-{gamma}A Brain, April 1, 2005; 128(4): 723 - 731. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Del Bo, A. Bordoni, M. Sciacco, A. Di Fonzo, S. Galbiati, M. Crimi, N. Bresolin, and G. P. Comi Remarkable infidelity of polymerase {gamma}A associated with mutations in POLG1 exonuclease domain Neurology, October 14, 2003; 61(7): 903 - 908. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |