Nuclear extract transcription

Figure 5.5 Detection of PNA binding-mediated GFP transcription in HeLa nuclear extract. Plasmid DNA encoding green fluorescent protein (pUSAG3) was linearized and used as a template for in vitro transcription in a HeLa nuclear extract transcription system. Lane 1, CMV promoter-driven 372 nt RNA transcript as a positive control lane 2, pUSAG3 plasmid DNA alone lane 3, pUSAG3 plasmid DNA in the presence of PNA-1 lane 4, pUSAG3 plasmid DNA in the presence of PNA-2 lane 5, pUSAG3 plasmid DNA in the presence of both PNA-1 and PNA-2.

Fig. 12.3 The +294T/C polymorphic site in PPARD influences the binding of transcription factor Spl (24). Electromobihty shift assays (EMSAs) of nnclear extract derived from hnman monocytic U937 cells using double-stranded 25-mer oligonucleotides corresponding to the sequence from position -1-281 to -1-305 of the common T allele and the rare C allele. Arrow refers to the -I-294C allele-predominant factor. F denotes free DNA. Lane 1, without extract lanes 2 to 4,12, with 10 ag nuclear extract in the absence of competitor lanes 5 to 7,13 to 15, with 10 ag nuclear extract in the presence of 150-fold excess of unlabeled DNA as competitor. Competitors used were -I-294C probe (lanes 5, 13 indicated with C) -I-294T probe (lanes 6, 14 indicated with T) and a nonspecific probe (lanes 7, 15 indicated with X). Spl complex. Lanes 8 to 11, with lOng nuclear extract in the presence of antibodies directed against Spl, p50, p65, and c-Rel, respectively...

Detection of PNA binding-mediated GFP transcription in HeLa nuclear extract 71... 

Figure 2.10. Electorphoretic mobility shift assay (EMSA). C/EBPP is a basic leucine transcription factor. An DNA oligomer consisting of the C/EBP binding site was labeled with 32P and incubated with a nuclear extract (lanes 1 and 2). The same oligomer and nuclear extract were incubated in the presence of a C/EBPP antibody (lanes 4 and 5). In lanes 1 and 2, the unbound free probe can be seen at the bottom of the autoradiogram, while probe protein complexes can be visualized in the upper section. In lanes 4 and 5 the presence of the antibody resulted in the supershift of the protein-probe complex the ternary complex consisting of the probe, C/EBPP protein, and C/EBPP antibody are now visualized at the top of the autoradiogram. The bands that were supershifted with the antibody represent C/EBPp.

Both methods involve swelling of the cells in hypotonic solution, disruption of the plasma membrane, and extraction of the nuclei in a high salt buffer. The extracts support a number of nuclear functions and have been extremely useful in studying transcription and splicing (see Section 5.1). Nuclear extracts are also a useful starting point for purification of snRNAs, snRNPs, hnRNP proteins and SR-proteins (Section 3.1.2). 

Lee, K.A., Bindereif, A. and Green, M.R. (1988). A small-scale procedure for preparation of nuclear extracts that support efficient transcription and pre-mRNA splicing. Gene. Anal. Tech. 5, 22-31. 

Figure 17.5 Transcription past DB[o,/] PDE-dC adducts. Template preparation and reaction conditions have been published [136]. For RNA Pol II, these were performed as follows. Briefly, HeLa nuclear extracts, DNA template, and lOmM each of ATP,...

Higher eukaryotes also contain multiprotein complexes with homology to the yeast SWI/SNF complex. These complexes isolated from nuclear extracts of mammalian and Drosophila cells have been found to assist binding of transcription factors to their cognate sites in nucleosomal DNA in an ATP-requiring process. The experiment shown in Figure... 

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