Price D H, Sluder A E, Greenleaf A L
J Biol Chem. 1987 Mar 5;262(7):3244-55.
Drosophila Kc cells were utilized to prepare nuclear extracts in which promoter-containing DNA templates were efficiently transcribed by RNA polymerase II. A combination of fractionation schemes was used to identify and partially purify seven activities (factors) which affected the transcription of four different genes in vitro. Reconstructing specific transcription required exogenous RNA polymerase II in addition to these factors. Moreover, the high efficiency of transcription characteristic of the crude extract was preserved in reconstruction reactions. The methods used are presented in detail. Functions were assigned to several of the factors. One essential factor appeared to affect initiation and displayed chromatographic properties unlike any other Drosophila transcription factor previously described. Two factors specifically affected RNA chain elongation. Another activity was a DNase inhibitor required to preserve template integrity in the fractionated system. The remaining three factors were not absolutely essential but affected the specific in vitro transcription either qualitatively or quantitatively. A comparison of these transcription factors with other Drosophila and mammalian transcription factors is made.
利用果蝇Kc细胞制备核提取物，其中含启动子的DNA模板可被RNA聚合酶II有效转录。采用多种分级分离方案来鉴定并部分纯化了七种活性物质（因子），这些因子在体外影响四个不同基因的转录。除了这些因子外，重建特异性转录还需要外源RNA聚合酶II。此外，粗提取物高效转录的特性在重建反应中得以保留。详细介绍了所使用的方法。为其中几个因子赋予了功能。一种必需因子似乎影响起始过程，其色谱特性与之前描述的任何果蝇转录因子都不同。两种因子特异性影响RNA链的延伸。另一种活性物质是一种DNase抑制剂，在分级分离系统中用于保持模板完整性。其余三种因子并非绝对必需，但在质量或数量上影响特异性体外转录。对这些转录因子与其他果蝇和哺乳动物转录因子进行了比较。
