Random insertion of injected DNA molecules into the macronuclear chromosome of <i>Paramecium caudatum</i>

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  • Takenaka Yasuhiro
    Department of Biochemistry, National Institute of Agrobiological Sciences Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST)
  • Yanagi Akira
    Department of Biotechnology, Ishinomaki Senshu University
  • Masuda Hiromi
    VALWAY Technology Center, NEC Soft, Ltd.
  • Mitsui Youji
    Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University
  • Mizuno Hiroshi
    Department of Biochemistry, National Institute of Agrobiological Sciences
  • Haga Nobuyuki
    Department of Biotechnology, Ishinomaki Senshu University

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  • Random insertion of injected DNA molecules into the macronuclear chromosome of Paramecium caudatum

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Abstract

<p>Linear DNA molecules injected into the macronucleus of Paramecium caudatum became integrated into genomic DNA. The plasmid harboring fusion gene of P. caudatum histone H2B and a yellow fluorescent protein (YFP) named PcVenus was linearized and microinjected into the macronucleus of P. caudatum. Southern blots of total cellular DNA from three fluorescence-positive transformant clones probed with the PcVenus sequence revealed that the injected DNA had become randomly inserted into the chromosome. Free linear monomers or multimers of injected DNA molecules as seen in P. primaurelia and P. tetraurelia were not evident even though the plasmid possessed extant telomeric repeats at its both extremities. We also cloned fragments containing the integration site of genomic DNA from transformant cells by plasmid rescue. Sequence analysis of the flanking DNA confirmed random insertion of the linear plasmid into the chromosomal DNA with extant telomere repeats at the fusion junction. Therefore, P. caudatum maintains introduced DNA in a unique manner by non-homologous or illegitimate, rather than homologous recombination.</p>

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