Cell-free transcription in Xenopus egg extract

Abstract

Soluble extracts prepared from Xenopus eggs have been used extensively to study various aspects of cellular and developmental biology. During early egg development, transcription of the zygotic genome is suppressed. As a result, traditional extracts derived from unfertilized and early stage eggs possess little or no intrinsic transcriptional activity. In this study, we show that Xenopus nucleoplasmic extract (NPE) supports robust transcription of a chromatinized plasmid substrate. Although prepared from eggs in a transcriptionally inactive state, the process of making NPE resembles some aspects of egg fertilization and early embryo development that lead to transcriptional activation. With this system, we observed that promoter-dependent recruitment of transcription factors and RNA polymerase II leads to conventional patterns of divergent transcription and pre-mRNA processing, including intron splicing and 3' cleavage and polyadenylation. We also show that histone density controls transcription factor binding and RNA polymerase II activity, validating a mechanism proposed to regulate genome activation during development. Together, these results establish a new cell-free system to study the regulation, initiation, and processing of mRNA transcripts.

Keywords: RNA processing; Xenopus; cell-free system; gene regulation; genome activation; histone; in vitro assay; nucleoplasmic extract; plasmid; transcription.