Collas P, Aleström P
Department of Biochemistry, Norwegian College of Veterinary Medicine, Oslo, Norway.
Mol Mar Biol Biotechnol. 1997 Mar;6(1):48-58.
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.
将SV40 T抗原核定位信号（NLSs）与质粒DNA结合，在将DNA-NLS复合物注入斑马鱼卵细胞质后可促进转基因表达。我们现在证明，在未导入裸DNA的条件下，NLS肽介导DNA从细胞质导入胚胎细胞核。通过聚合酶链反应（PCR）在分离的细胞核中定位质粒DNA，并通过光密度法对相对量进行定量。将DNA与NLSs而非核输入缺陷肽结合，可促进DNA-NLS复合物快速靶向细胞核并穿过核膜。共注射白蛋白-NLS偶联物可竞争DNA-NLS复合物的导入。在用32P标记的DNA探测的细胞核印迹上检测到了NLS，而非反向NLS。结果表明，NLS介导的DNA转移到细胞核中可能成为几种基因转移应用的有价值工具。
