Rapid targeting of plasmid DNA to zebrafish embryo nuclei by the nuclear localization signal of SV40 T antigen.
Binding SV40 T antigen nuclear localization signals (NLSs) to plasmid DNA promotes transgene expression following injection of DNA-NLS complexes into the cytoplasm of zebrafish eggs. We now demonstrate that NLS peptides mediate import of DNA from the cytoplasm into embryo nuclei, under conditions in which naked DNA is not imported. Plasmid DNA was localized by polymerase chain reaction (PCR) in isolated nuclei, and relative amounts were quantified by densitometry. Binding DNA to NLSs, but not to nuclear-import-deficient peptides, promoted rapid targeting of DNA-NLS complexes to nuclei, and transport across the nuclear envelope. Import of DNA-NLS complexes was competed by co-injected albumin-NLS conjugates. NLS, but not reverse NLS, was detected on blots of nuclei probed with 32P-labeled DNA. The results suggest that NLS-mediated DNA transfer into nuclei may constitute a valuable tool for several gene transfer applications.
Collas
P
P
Department of Biochemistry, Norwegian College of Veterinary Medicine, Oslo, Norway.
Aleström
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D000957:Antigens, Viral, Tumor / Q000096:biosynthesis*; D001483:Base Sequence; D002467:Cell Nucleus / Q000502:physiology*; D017931:DNA Primers; D004279:DNA, Viral / Q000096:biosynthesis; D004625:Embryo, Nonmammalian / Q000502:physiology*; D005260:Female; D017930:Genes, Reporter; D008156:Luciferases / Q000096:biosynthesis; D008297:Male; D009865:Oocytes / Q000502:physiology*; D010446:Peptide Fragments / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011865:Radioisotope Dilution Technique; D011994:Recombinant Proteins / Q000096:biosynthesis; D013539:Simian virus 40 / Q000235:genetics; D015027:Zebrafish / Q000196:embryology
D000957:Antigens, Viral, Tumor; D017931:DNA Primers; D004279:DNA, Viral; D010446:Peptide Fragments; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D008156:Luciferases