Enhancement of polylysine-mediated transferrinfection by nuclear localization sequences: polylysine does not function as a nuclear localization sequence.
Polylysine (pLy) has been used successfully as a DNA carrier in receptor-mediated gene transfer, enhancement of transfection having been proposed to be in part through efficient nuclear targeting stemming from the resemblance of pLy to the nuclear localization sequence (NLS) from simian virus SV40 large tumor antigen (T-ag). In this study we test whether pLy carrying covalently attached peptides comprising the T-ag NLS (the pLyP101 derivative) can enhance transferrin-pLy-mediated transfection ("transferrinfection"). Unlike pLy itself or a pLy derivative (pLyP101T) carrying cross-linked T-ag NLS mutant peptides, pLyP101 significantly enhanced transferrinfection of a beta-galactosidase-expressing reporter plasmid. The basis of this was shown to be the ability of the pLyP101-plasmid DNA complex to be recognized with high affinity by the NLS-binding importin subunits, in contrast to pLyP101T- and pLy-plasmid complexes. Confocal laser scanning microscopy was used to determine the nuclear import kinetics of fluorescently labeled pLyP101 and pLyP101T in the presence of complexed plasmid, indicating that pLyP101 and not pLyP101T complexes accumulated rapidly in the nucleus. We conclude that pLy itself does not function as an NLS and that the addition of exogenous NLSs conferring interaction with the cellular nuclear import machinery can increase transferrinfection by enhancing the nuclear targeting of pLy-DNA complexes.
Chan
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CK
Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra, ACT, Australia.
Jans
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DA
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001692:Biological Transport; D006528:Carcinoma, Hepatocellular; D002467:Cell Nucleus / Q000378:metabolism; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D028884:Karyopherins; D008113:Liver Neoplasms; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics*; D011107:Polylysine; D051381:Rats; D014162:Transfection; D014168:Transferrin; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D028884:Karyopherins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D014168:Transferrin; D011107:Polylysine; D001616:beta-Galactosidase