Fluorescent in situ hybridization of mitochondrial DNA and RNA

• Authors: Lukáš Alán , Jaroslav Zelenka , Jan Ježek , Andrea Dlasková , Petr Ježek
• Languages of publication: EN

Abstracts

EN

To reveal nucleic acid localization in mitochondria, we designed molecular beacon fluorescent probes against: i) the light strand complementary to ND5 mitochondrial DNA (mtDNA) gene (annealing also to corresponding mRNA); ii) displacement (D) loop 7S DNA (annealing also to parallel heavy strand mtDNA and corresponding light strand transcript); iii) the proximal D-loop heavy strand displaced by the light strand promoter minor RNA. Confocal microscopy demonstrated ND5 probe spreading (less for other probes) in mitochondrial reticulum tubules but upon RNase A treatment all probes contoured mtDNA nucleoid localization. DNase I spread the signal over mitochondrial tubules. Future applications are discussed.

Keywords

EN

molecular beacon fluorescent hybridization probes; nucleoids of mitochondrial DNA; confocal microscopy; transcription and replication intermediates; mitochondrial DNA and RNA

• Publisher: Polish Biochemical Society
• Journal: Acta Biochimica Polonica
• Year: 2010
• Volume: 57
• Issue: 4
• Pages: 403-408

Dates

published

2010

received

2010-08-30

revised

2010-11-04

accepted

2010-11-25

(unknown)

2010-11-29

Contributors

author

Lukáš Alán

• Department No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

author

Jaroslav Zelenka

• Department No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

author

Jan Ježek

• Department No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

author

Andrea Dlasková

• Department No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

author

Petr Ježek

• Department No. 75, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic

References

• Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N (1999) Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 23: 147.
• Bogenhagen DF, Rousseau D, Burke S (2008) The layered structure of human mitochondrial DNA nucleoids. J Biol Chem 283: 3665-3675.
• Bowmaker M, Yang MY, Yasukawa T, Reyes A, Jacobs HT, Huberman JA, Holt IJ (2003) Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone. J Biol Chem 278: 50961-50969.
• Brown TA, Clayton DA (2006) Genesis and wanderings: origins and migrations in asymmetrically replicating mitochondrial DNA. Cell Cycle 5: 917-921.
• Brown TA, Cecconi C, Tkachuk AN, Bustamante C, Clayton DA (2005) Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism. Genes Dev 19: 2466-2476.
• Brown TA, Tkachuk AN, Clayton DA (2008) Native R-loops persist throughout the mouse mitochondrial DNA genome. J Biol Chem 283: 36743-36751.
• Chang DD, Clayton DA (1985) Priming of human mitochondrial DNA replication occurs at the light-strand promoter. Proc Natl Acad Sci USA 82: 351-355.
• Chang DD, Hixson JE, Clayton DA (1986) Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally. Mol Cell Biol 6: 294-301.
• Clayton DA (2003) Mitochondrial DNA replication: what we know. IUBMB Life 55: 213-217.
• Cree LM, Samuels DC, de Sousa Lopes SC, Rajasimha HK, Wonnapinij P, Mann JR, Dahl HH, Chinnery PF (2008) A reduction of mitochondrial DNA molecules during embryogenesis explains the rapid segregation of genotypes. Nat Genet 40: 249-254.
• Falkenberg M, Larsson NG, Gustafsson CM (2007) DNA replication and transcription in mammalian mitochondria. Annu Rev Biochem 76: 679-699.
• Fusté JM, Wanrooij S, Jemt E, Granycome CE, Cluett TJ, Shi Y, Atanassova N, Holt IJ, Gustafsson CM, Falkenberg M (2010) Mitochondrial RNA polymerase is needed for activation of the origin of light-strand DNA replication. Mol Cell 37: 67-78.
• Hell SW (2007) Far-field optical nanoscopy. Science 316: 1153-1158.
• Holt IJ (2009) Mitochondrial DNA replication and repair: all a flap. Trends Biochem Sci 34: 358-365.
• Holt IJ (2010) Zen and the art of mitochondrial DNA maintenance. Trends Genet 26: 103-109.
• Holt IJ, Lorimer HE, Jacobs HT (2000) Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100: 515-524.
• Holt IJ, He J, Mao C-C, Boyd-Kirkup JD, Martinsson P, Sembongi H, Reyes A, Spelbrink JN (2007) Mammalian mitochondrial nucleoids: organizing an independently minded genome. Mitochondrion 7: 311-321.
• Hyvärinen AK, Pohjoismäki JL, Reyes A, Wanrooij S, Yasukawa T, Karhunen PJ, Spelbrink JN, Holt IJ, Jacobs HT (2007) The mitochondrial transcription termination factor mTERF modulates replication pausing in human mitochondrial DNA. Nucleic Acids Res 35: 6458-6474.
• Kajander OA, Karhunen PJ, Holt IJ, Jacobs HT (2001) Prominent mitochondrial DNA recombination intermediates in human heart muscle. EMBO Rep 2: 1007-1012.
• Koopman WJH, Hink MA, Verkaart S, Visch HJ, Smeitink JAM, Willems PHGM (2007) Partial complex I inhibition decreases mitochondrial motility and increases matrix protein diffusion as revealed by fluorescence correlation spectroscopy. Biochim Biophys Acta 1767: 940-947.
• Lu F, Selak M, O'Connor J, Croul S, Lorenzana C, Butunoi C, Kalman B. J. (2000) Oxidative damage to mitochondrial DNA and activity of mitochondrial enzymes in chronic active lesions of multiple sclerosis. J Neurol Sci 177: 95-103.
• Pohjoismäki JL, Holmes JB, Wood SR, Yang MY, Yasukawa T, Reyes A, Bailey LJ, Cluett TJ, Goffart S, Willcox S, Rigby RE, Jackson AP, Spelbrink JN, Griffith JD, Crouch RJ, Jacobs HT, Holt IJ (2010) Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid. J Mol Biol 397: 1144-1155.
• Reyes A, Yang MY, Bowmaker M, Holt IJ (2005) Bidirectional replication initiates at sites throughout the mitochondrial genome of birds. J Biol Chem 280: 3242-3250.
• Rossignol R, Gilkerson R, Aggeler R, Yamagata K, Remington SJ, Capaldi RA (2004) Energy substrate modulates mitochondrial structure and oxidative capacity in cancer cells. Cancer Res 64: 985-993.
• Santangelo PJ, Nitin N, Bao G (2005) Direct visualization of mRNA colocalization with mitochondria in living cells using molecular beacons. J Biomed Opt 10: 044025-1-16.
• Scaglia F, Wong L-JC (2008) Human mitochondrial transfer RNAs: Role of pathogenic mutation in disease. Muscle Nerve 37: 150-171.
• Shadel GS, Clayton DA (1997) Mitochondrial DNA maintenance in vertebrates. Ann Rev Biochem 66: 409-435.
• Stewart JB, Treter C, Elson JL, Wredenberg A, Cansu Z, Trifunovic A, Larsson N-G (2008) Strong purifying selection in transmission of mammalian mitochondrial DNA. PLoS Biology 6: e10.
• Tuppen HA, Blakely EL, Turnbull DM, Taylor RW (2010) Mitochondrial DNA mutations and human disease. Biochim Biophys Acta 1797: 113-128.
• Xu B, Clayton DA (1996) RNA-DNA hybrid formation at the human mitochondrial heavy-strand origin ceases at replication start sites: an implication for RNA-DNA hybrids serving as primers. EMBO J 15: 3135-3143.
• Yang MY, Bowmaker M, Reyes A, Vergani L, Angeli P, Gringeri E, Jacobs HT, Holt IJ (2002) Biased incorporation of ribonucleotides on the mitochondrial L-strand accounts for apparent strand-asymmetric DNA replication. Cell 111: 495-505.
• Yasukawa T, Reyes A, Cluett TJ, Yang MY, Bowmaker M, Jacobs HT, Holt IJ (2006) Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand. EMBO J 25: 5358-5371.
• Zhadanov SI, Grechanina EY, Grechanina YB, Gusar VA, Fedoseeva NP, Lebon S, Munnich A, Schurr TG (2007) Fatal manifestation of a de novo ND5 mutation: Insights into the pathogenetic mechanisms of mtDNA ND5 gene defects. Mitochondrion 7: 260-266.