Maroteaux L, Kahana C, Mory Y, Groner Y, Revel M
Department of Virology, The Weizmann Institute of Science, Rehovot, Israel.
EMBO J. 1983;2(3):325-32. doi: 10.1002/j.1460-2075.1983.tb01426.x.
The human genomic EcoRI fragment of 1.83 kb containing the interferon (IFN) gene IFN-beta1 with 285 nucleotides of 5'-flanking sequences was transfected into monkey kidney CV-1 cells as part of an SV40-pML2 vector. Induction of the monkey cells to produce IFN led to a rapid accumulation of IFN-beta1 RNA whose 5' ends were identical to the IFN-beta1 mRNA of human fibroblasts. This induction occurred with all recombinants tested. Expression from the SV40 late promoter was also seen in non-induced cells. We conclude that the regulation of the IFN-beta1 gene is retained in the replicating episomal SV40 vectors with high copy number, even when the gene is being transcribed from an external promoter. When the 5'-flanking sequences were deleted to leave only 40 bp before the presumed cap site of the IFN-beta1 gene, inducible formation of IFN-RNA with authentic 5' ends could still be demonstrated. However, inducibility and expression depended on the position of the deleted IFN-beta1 gene in the vector. We conclude that the sequences around the TATAA box and cap site on the IFN gene are involved in the regulation of its expression. Regulated short-term expression of the human IFN-beta1 gene in SV40 vectors provides a defined system in which the structures required to maintain the regulation and the influence of known external transcription signals can be examined.
将含有干扰素（IFN）基因IFN-β1及285个核苷酸5'侧翼序列的1.83 kb人类基因组EcoRI片段，作为SV40-pML2载体的一部分转染至猴肾CV-1细胞。诱导猴细胞产生IFN导致IFN-β1 RNA迅速积累，其5'末端与人成纤维细胞的IFN-β1 mRNA相同。所有测试的重组体均出现这种诱导现象。在未诱导的细胞中也可见到来自SV40晚期启动子的表达。我们得出结论，即使该基因由外部启动子转录，IFN-β1基因的调控在具有高拷贝数的复制型附加体SV40载体中仍得以保留。当5'侧翼序列被删除，仅在IFN-β1基因假定的帽位点之前留下40 bp时，仍可证明能诱导形成具有真实5'末端的IFN-RNA。然而，诱导性和表达取决于载体中删除的IFN-β1基因的位置。我们得出结论，IFN基因上TATAA框和帽位点周围的序列参与其表达的调控。在SV40载体中对人类IFN-β1基因进行受调控的短期表达，提供了一个明确的系统，在其中可以研究维持调控所需的结构以及已知外部转录信号的影响。
