Skip to main content

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

Pharmaceutical Research volume 28, Article number: 2863 (2011) Cite this article

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.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3

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

REFERENCES

  1. Larsson NG, Luft R. Revolution in mitochondrial medicine. FEBS Lett. 1999;455(3):199–202.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  4. McFarland R, Taylor RW, Turnbull DM. A neurological perspective on mitochondrial disease. Lancet Neurol. 2010;9(8):829–40.

    PubMed  Article  CAS  Google Scholar 

  5. Bigger B, Collombet JM, Coutelle C. Tipping the scales in favour of mitochondrial gene therapy [comment]. Gene Ther. 1999;6(12):1909–10.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  15. Shuster RC, Rubenstein AJ, Wallace DC. Mitochondrial DNA in anucleate human red blood cells. Biochem Biophys Res Comm. 1988;155(3):1360–5.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  20. Newlon CS. Yeast chromosome replication and segregation. Microbiol Rev. 1988;52:568–601.

    PubMed  CAS  Google Scholar 

  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. 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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  24. Clarke L, Carbon J. Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature. 1980;287(5782):504–9.

    PubMed  Article  CAS  Google Scholar 

  25. Keith JM, Cochran DA, Lala GH, Adams P, Bryant D, Mitchelson KR. Unlocking hidden genomic sequence. Nucleic Acids Res. 2004;32(3):e35.

    PubMed  Article  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  27. Beggs JD. Transformation of yeast by a replicating hybrid plasmid. Nature. 1978;275(5676):104–9.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

  30. Palzkill TG, Newlon CS. A yeast replication origin consists of multiple copies of a small conserved sequence. Cell. 1988;53(3):441–50.

    PubMed  Article  CAS  Google Scholar 

  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.

    PubMed  Article  CAS  Google Scholar 

Download references

ACKNOWLEDGMENTS & DISCLOSURES

This work was supported by the Cystic Fibrosis Trust Muller Bequest to CC. BWB, AYL and AS are supported by the UK society for Mucopolysaccharide diseases and the Manchester Biomedical Research Centre.

Author information

Affiliations

  1. Stem Cell & Neurotherapies, Faculty of Medical and Human Sciences, University of Manchester, 3.721 Stopford Building, Manchester, M13 9PT, UK

    Brian W. Bigger, Ai-Yin Liao & Ana Sergijenko

  2. Gene Therapy Research Group, Sir Alexander Fleming Building, Imperial College London, London, SW7 5AZ, UK

    Charles Coutelle

Authors
  1. Brian W. Bigger
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ai-Yin Liao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ana Sergijenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Charles Coutelle
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Brian W. Bigger.

Electronic supplementary materials

Below is the link to the electronic supplementary material.

Supplementary Table 1

PCR and sequencing primers (DOCX 21 kb)

Supplementary Table 2

PCR conditions (DOCX 12.8 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bigger, B.W., Liao, AY., Sergijenko, A. et al. Trial and Error: How the Unclonable Human Mitochondrial Genome was Cloned in Yeast. Pharm Res 28, 2863 (2011). https://doi.org/10.1007/s11095-011-0527-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11095-011-0527-1

KEY WORDS

  • gene therapy
  • human mitochondrial DNA
  • mitochondrial disease
  • unclonable
  • yeast