Pharmaceutical Research

, 28:2863 | Cite as

Trial and Error: How the Unclonable Human Mitochondrial Genome was Cloned in Yeast

  • Brian W. Bigger
  • Ai-Yin Liao
  • Ana Sergijenko
  • Charles Coutelle
Research Paper

ABSTRACT

Purpose

Development of a human mitochondrial gene delivery vector is a critical step in the ability to treat diseases arising from mutations in mitochondrial DNA. Although we have previously cloned the mouse mitochondrial genome in its entirety and developed it as a mitochondrial gene therapy vector, the human mitochondrial genome has been dubbed unclonable in E. coli, due to regions of instability in the D-loop and tRNAThr gene.

Methods

We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination.

Results

Human mitochondrial DNA is unclonable in E. coli and cannot be retained in multi- or single-copy vectors under any conditions. It was, however, possible to clone and stably maintain the entire human mitochondrial genome in yeast as long as a single-copy centromeric plasmid was used. D-loop and tRNAThr were both stable and unmutated.

Conclusions

This is the first report of cloning the entire human mitochondrial genome and the first step in developing a gene delivery vehicle for human mitochondrial gene therapy.

KEY WORDS

gene therapy human mitochondrial DNA mitochondrial disease unclonable yeast 

ABBREVIATIONS

ARS

autonomous replicating sequence

BAC

bacterial artificial chromosome

D-loop

displacement loop

mtDNA

mitochondrial DNA

PAC

P1 phage artificial chromosome

TAR

transformation-associated recombination

TB

terrific broth

Supplementary material

11095_2011_527_MOESM1_ESM.docx (21 kb)
Supplementary Table 1PCR and sequencing primers (DOCX 21 kb)
11095_2011_527_MOESM2_ESM.docx (13 kb)
Supplementary Table 2PCR conditions (DOCX 12.8 kb)

REFERENCES

  1. 1.
    Larsson NG, Luft R. Revolution in mitochondrial medicine. FEBS Lett. 1999;455(3):199–202.PubMedCrossRefGoogle Scholar
  2. 2.
    Chinnery PF, Johnson MA, Wardell TM, Singh-Kler R, Hayes C, Brown DT, et al. The epidemiology of pathogenic mitochondrial DNA mutations. Ann Neurol. 2000;48(2):188–93.PubMedCrossRefGoogle Scholar
  3. 3.
    Elliott HR, Samuels DC, Eden JA, Relton CL, Chinnery PF. Pathogenic mitochondrial DNA mutations are common in the general population. Am J Hum Genet. 2008;83(2):254–60.PubMedCrossRefGoogle Scholar
  4. 4.
    McFarland R, Taylor RW, Turnbull DM. A neurological perspective on mitochondrial disease. Lancet Neurol. 2010;9(8):829–40.PubMedCrossRefGoogle Scholar
  5. 5.
    Bigger B, Collombet JM, Coutelle C. Tipping the scales in favour of mitochondrial gene therapy [comment]. Gene Ther. 1999;6(12):1909–10.PubMedCrossRefGoogle Scholar
  6. 6.
    Doyle SR, Chan CK. Mitochondrial gene therapy: an evaluation of strategies for the treatment of mitochondrial DNA disorders. Hum Gene Ther. 2008;19(12):1335–48.PubMedCrossRefGoogle Scholar
  7. 7.
    Kyriakouli DS, Boesch P, Taylor RW, Lightowlers RN. Progress and prospects: gene therapy for mitochondrial DNA disease. Gene Ther. 2008;15(14):1017–23.PubMedCrossRefGoogle Scholar
  8. 8.
    Tapper DP, Van Etten RA, Clayton DA. Isolation of mammalian mitochondrial DNA and RNA and cloning of the mitochondrial genome. Methods Enzymol. 1983;97:426–34.PubMedCrossRefGoogle Scholar
  9. 9.
    Bigger B, Tolmachov O, Collombet JM, Coutelle C. Introduction of chloramphenicol resistance into the modified mouse mitochondrial genome: cloning of unstable sequences by passage through yeast. Anal Biochem. 2000;277(2):236–42.PubMedCrossRefGoogle Scholar
  10. 10.
    Bigger BW, Tolmachov O, Collombet JM, Fragkos M, Palaszewski I, Coutelle C. An araC-controlled bacterial cre expression system to produce DNA minicircle vectors for nuclear and mitochondrial gene therapy. J Biol Chem. 2001;276(25):23018–27.PubMedCrossRefGoogle Scholar
  11. 11.
    Wheeler VC, Aitken M, Coutelle C. Modification of the mouse mitochondrial genome by insertion of an exogenous gene. Gene. 1997;198(1–2):203–9.PubMedCrossRefGoogle Scholar
  12. 12.
    Katrangi E, D’Souza G, Boddapati SV, Kulawiec M, Singh KK, Bigger B, et al. Xenogenic transfer of isolated murine mitochondria into human rho0 cells can improve respiratory function. Rejuvenation Res. 2007;10(4):561–70.PubMedCrossRefGoogle Scholar
  13. 13.
    Mita S, Monnat Jr RJ, Loeb LA. Resistance of HeLa cell mitochondrial DNA to mutagenesis by chemical carcinogens. Cancer Res. 1988;48(16):4578–83.PubMedGoogle Scholar
  14. 14.
    Mita S, Monnat Jr RJ, Loeb LA. Direct selection of mutations in the human mitochondrial tRNAThr gene: reversion of an ‘uncloneable’ phenotype. Mutat Res. 1988;199(1):183–90.PubMedCrossRefGoogle Scholar
  15. 15.
    Shuster RC, Rubenstein AJ, Wallace DC. Mitochondrial DNA in anucleate human red blood cells. Biochem Biophys Res Comm. 1988;155(3):1360–5.PubMedCrossRefGoogle Scholar
  16. 16.
    Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM, Howell N. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA [letter]. Nat Genet. 1999;23(2):147.PubMedCrossRefGoogle Scholar
  17. 17.
    Ioannou PA, Amemiya CT, Garnes J, Kroisel PM, Shizuya H, Chen C, et al. A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nat Genet. 1994;6(1):84–9.PubMedCrossRefGoogle Scholar
  18. 18.
    Larionov V, Kouprina N, Eldarov M, Perkins E, Porter G, Resnick MA. Transformation-associated recombination between diverged and homologous DNA repeats is induced by strand breaks. Yeast. 1994;10(1):93–104.PubMedCrossRefGoogle Scholar
  19. 19.
    Larionov V, Kouprina N, Graves J, Chen X-N, Korenberg JR, Resnick MA. Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination. Proc Natl Acad Sci USA. 1996;93(1):491–6.PubMedCrossRefGoogle Scholar
  20. 20.
    Newlon CS. Yeast chromosome replication and segregation. Microbiol Rev. 1988;52:568–601.PubMedGoogle Scholar
  21. 21.
    Stinchcomb DT, Mann C, Selker E, Davis RW. DNA sequences that allow the replication and segregation of yeast chromosomes ICN-UCLA Symp. Mol Cell Biol. 1981;22:473.Google Scholar
  22. 22.
    Huang RY, Kowalski D. A DNA unwinding element and an ARS consensus comprise a replication origin within a yeast chromosome. EMBO J. 1993;12(12):4521–31.PubMedGoogle Scholar
  23. 23.
    Kouprina N, Annab L, Graves J, Afshari C, Barrett JC, Resnick MA, et al. Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence. Proc Natl Acad Sci USA. 1998;95(8):4469–74.PubMedCrossRefGoogle Scholar
  24. 24.
    Clarke L, Carbon J. Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature. 1980;287(5782):504–9.PubMedCrossRefGoogle Scholar
  25. 25.
    Keith JM, Cochran DA, Lala GH, Adams P, Bryant D, Mitchelson KR. Unlocking hidden genomic sequence. Nucleic Acids Res. 2004;32(3):e35.PubMedCrossRefGoogle Scholar
  26. 26.
    Botstein D, Falco SC, Stewart SE, Brennan M, Scherer S, Stinchcomb DT, et al. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979;8(1):17–24.PubMedCrossRefGoogle Scholar
  27. 27.
    Beggs JD. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978;275(5676):104–9.PubMedCrossRefGoogle Scholar
  28. 28.
    Kazakova TBM, Babich SG, Golovina GI, Mel’nikova MP, Tsymbalenko NV. [Autonomous replication of plasmid pBR322 containing a mitochondrial DNA fragment of animal origin in the cells of bacteria mutant for DNA- polymerase I]. Genetika. 1983;19(3):381–7.PubMedGoogle Scholar
  29. 29.
    Zakian VA. Origin of replication from Xenopus laevis mitochondrial DNA promotes high-frequency transformation of yeast. Proc Natl Acad Sci USA. 1981;78(5):3128–32.PubMedCrossRefGoogle Scholar
  30. 30.
    Palzkill TG, Newlon CS. A yeast replication origin consists of multiple copies of a small conserved sequence. Cell. 1988;53(3):441–50.PubMedCrossRefGoogle Scholar
  31. 31.
    Rashid MB, Shirahige K, Ogasawara N, Yoshikawa H. Anatomy of the stimulative sequences flanking the ARS consensus sequence of chromosome VI in Saccharomyces cerevisiae. Gene. 1994;150(2):213–20.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Brian W. Bigger
    • 1
  • Ai-Yin Liao
    • 1
  • Ana Sergijenko
    • 1
  • Charles Coutelle
    • 2
  1. 1.Stem Cell & Neurotherapies, Faculty of Medical and Human SciencesUniversity of ManchesterManchesterUK
  2. 2.Gene Therapy Research Group, Sir Alexander Fleming BuildingImperial College LondonLondonUK

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