Dunn J J, Krippl B, Bernstein K E, Westphal H, Studier F W
Biology Department, Brookhaven National Laboratory, Upton, NY 11973.
Gene. 1988 Sep 7;68(2):259-66. doi: 10.1016/0378-1119(88)90028-5.
Indirect immunofluorescence shows that purified T7 RNA polymerase, when microinjected into monkey kidney (Vero) cells, localizes predominantly in the cytoplasm. To direct active T7 RNA polymerase to the nucleus, we first created unique restriction sites at two locations within the cloned gene for T7 RNA polymerase, T7 gene 1 and then inserted into these sites a 36-bp synthetic nucleotide sequence encoding the SV40 T antigen nuclear location signal. Insertion of the nuclear location signal between codons 10 and 11 of T7 RNA polymerase has only minimal effect on transcription activity in Escherichia coli, but its insertion four codons from the C terminus abolishes activity. Fusion proteins having only foreign codons ahead of codon 11 also have transcription activity in E. coli. Such fusion proteins can be expressed transiently from plasmids microinjected into monkey cells, using SV40 expression signals, and detected by immunofluorescence. A fusion protein containing a nuclear location signal localized predominantly in the nucleus whereas those which lack the signal localize predominantly in the cytoplasm. Ability to direct T7 RNA polymerase to the nucleus may be an advantage in attempting to make this enzyme useful for selective transcription in eukaryotic cells.
间接免疫荧光显示，纯化的T7 RNA聚合酶显微注射到猴肾（Vero）细胞后，主要定位于细胞质中。为了将活性T7 RNA聚合酶导向细胞核，我们首先在克隆的T7 RNA聚合酶基因（T7基因1）的两个位置创建了独特的限制性酶切位点，然后在这些位点插入了一个36个碱基对的合成核苷酸序列，该序列编码SV40 T抗原核定位信号。在T7 RNA聚合酶的第10和11个密码子之间插入核定位信号对大肠杆菌中的转录活性影响极小，但在距C末端四个密码子处插入则会消除活性。仅在第11个密码子之前含有外源密码子的融合蛋白在大肠杆菌中也具有转录活性。利用SV40表达信号，此类融合蛋白可从显微注射到猴细胞中的质粒瞬时表达，并通过免疫荧光检测。含有核定位信号的融合蛋白主要定位于细胞核，而缺乏该信号的融合蛋白则主要定位于细胞质。将T7 RNA聚合酶导向细胞核的能力可能有助于使该酶用于真核细胞中的选择性转录。
