TARGETING BACTERIOPHAGE-T7 RNA-POLYMERASE TO THE MAMMALIAN-CELL NUCLEUS
TARGETING BACTERIOPHAGE-T7 RNA-POLYMERASE TO THE MAMMALIAN-CELL NUCLEUS
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DOI：
10.1016/0378-1119(88)90028-5
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发表时间：
1988-09-07
期刊：
影响因子：
3.5
通讯作者：
STUDIER, FW
中科院分区：
文献类型：
--
作者：
DUNN, JJ;KRIPPL, B;STUDIER, FW
间接免疫荧光显示，纯化的T7 RNA聚合酶，当显微注射到猴肾（Vero）细胞，主要定位在细胞质中。为了将活性T7 RNA聚合酶引导至细胞核，我们首先在T7 RNA聚合酶的克隆基因T7基因1内的两个位置处创建独特的限制性位点，然后将编码SV 40 T抗原核定位信号的36-bp合成核苷酸序列插入这些位点。在T7 RNA聚合酶的密码子10和11之间插入核定位信号对大肠杆菌中的转录活性仅具有最小的影响，但其从C末端插入四个密码子则废除活性。在密码子11之前仅具有外源密码子的融合蛋白在大肠杆菌中也具有转录活性。杆菌这样的融合蛋白可以使用SV 40表达信号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在核中，而缺乏信号的融合蛋白主要定位在细胞质中。将T7 RNA聚合酶导向细胞核的能力可能是试图使这种酶用于真核细胞中的选择性转录的优势。
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 localizes 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.