Cell-Free Transcription of SV40

The development of soluble cell-free systems that accurately transcribe genes has provided a means to investigate the mechanisms of eukaryotic transcription. The results of many studies have shown that specific DNA cis -acting elements, together with a complex array of protein factors, are required for transcription to occur. In particular, there has been an explosion of knowledge regarding the general transcription factors required by RNA polymerase II to function efficiently (1 -4 ). Our laboratory has been investigating the molecular mechanisms by which the genes of SV40 are transcriptionally regulated. To elucidate the biochemical factors involved in the transcriptional regulation of SV40, the development of a cell-free transcription system was required. In this chapter, we describe conditions for growing and harvesting cells that can serve as a source for the cell-free nuclear extract, a method for preparing the extract, conditions for transcribing the SV40 early and late promoters in this cell-free system, and assays for the analysis of the transcription products.