DNA-dependent transcription of adenovirus genes in a soluble whole-cell extract.

Abstract

We have developed a cell-free system for studying the synthesis of mRNA in mammalian cells. The system consists of a dialyzed and concentrated whole-cell extract derived from HeLa cells, small molecules and cofactors needed for transcription, and exogenously added DNA. Accurate transcription by RNA polymerase II is entirely dependent upon addition of promoter-containing eukaryotic DNA. At optimal DNA and extract concentrations, transcription initiation from the adenovirus serotype 2 late promoter is readily detectable, and specific transcripts over 4000 nucleotides in length are observed. The (4,5). Recently, Weil et al. (6) demonstrated that accurate initiation of transcription occurs on exogenously added DNA at a known viral transcription start site when purified RNA polymerase II is mixed with a cytoplasmic extract. Here we describe a simple system consisting primarily of a soluble whole-cell extract that accurately initiates transcription on exogenously added DNA templates. We feel that this system will prove useful for studying the biochemical mechanisms involved in the control of transcription and gene expression in mammalian cells.MATERIALS AND METHODS Preparation of DNA. Adenovirus serotype 2 (Ad2) virus and DNA were prepared as described (7). Restriction endonuclease DNA fragments Bal I-D [21.5-28.3 map units (m.u.)] and Bal I-E (14.7-21.5 m.u.) were inserted into plasmid pBR322 with BamHI linkers and cloned (8). Sma I-F fragment (2.9-11.3 m.u.) was similarly inserted into pBR322 but with EcoRI linkers (gift of Kathleen Berkner and Frank Laski). Plasmid DNA was prepared by standard protocols (9).Preparation of HeLa Cell Extracts. Cell lysates were prepared according to the method of Sugden and Keller (10 decanted so as not to disturb the pellet and precipitated by addition of solid (NH4)2SO4 (0.33 g/ml of suspension). After the (NH4)2SO4 was dissolved, 0.01 ml of 1 M NaOH per 10 g of (NH4)2SO4 was added, and the suspension was stirred for an additional 30 min. The precipitate was collected by centrifugation at 15,000 X g for 20 min and resuspended with 'Ao the volume of the high-speed supernatant into a buffer containing 50mM Tris-HCI, pH 7.9,6mM MgCl2, 40 mM (NH4)2SO4, 0.2 mM EDTA, 1 mM dithiothreitol, and 15% glycerol. This suspension was dialyzed for 4-8 hrs against each of two changes of 100 vol of the same buffer. The dialysate was centrifuged at 10,000 X g for 10 min and the supernatant was quick frozen in small samples in liquid N2. Samples stored at -80°C retained full activity for at least 2 months. In some later experiments (e.g., the one shown in Fig. 5) the dialysis buffer was 20 mM Hepes, pH 7.9/100 mM KC1/12.5 mM MgCl2/0.1 mM EDTA/2 mM dithiothreitol/17% glycerol.In Vitro Incubations 3855

The Prp19/Pso4 core complex undergoes ubiquitylation and structural alterations in response to DNA damage

“…HeLa whole cell extracts were prepared and fractionated as described [21,22]. For responses to DNA damage, HeLa cells were transfected with the indicated plasmid, and MMS was added to the medium 24 hours later.…”

Macrophage Effector Functions Controlled by Bruton’s Tyrosine Kinase Are More Crucial Than the Cytokine Balance of T Cell Responses for Microfilarial Clearance

“…Western blot analysis was used to compare the induction of both cellular iNOS protein levels and nuclear levels of proteins of the NF-B family in macrophages stimulated with LPS (10 g/ml) for 36 -48 h. Cell lysates or nuclear extracts were prepared as described (34,35), centrifuged at 20,000 rpm (Beckman Coulter, Fullerton, CA), dialyzed, and stored at Ϫ70°C. Cell lysates or nuclear extracts (20 g protein/lane) were separated in a 10% SDS-PAGE under reducing conditions.…”

Transcription of two classes of rat growth hormone gene-associated repetitive DNA: differences in activity and effects of tandem repeat structure

“…Whole cell lysates were prepared from cultured HeLa cells as described by Manley et al (19). The standard assay was performed as previously described (19) Figure 3B and Table I FR (lane 1, Fig.…”

“…In the current study we used the cell-free transcription system of Manley et al (19) and the unique organization of the Alu-like sequences associated with the rGH gene to: 1) determine the Pol III activity of the two major rat repetitive DNA families; 2) map the rGH gene Pol III transcription units; 3) determine if the Pol III promoters tandemly arranged within IB behave as a single transcription unit; and, 4) determine, by competition studies, if the rat Alu-like repeats and tRNA genes share Pol III transcription factors.…”