We describe a method by which complex protein mixtures are fractionated by standard one-dimensional NaDodSO4polyacrylamide gel electrophoresis or O'Farrell two-dimensional gel electrophoresis and then are efficiently and rapidly transferred electrophoretically to diazobenzyloxymethyl-or diazophenylthioether-paper and analyzed by immunoautoradiography. The method is illustrated with protein extracts of human KB cells infected with adenovirus type 2. Proteins were transferred from gels without decrease in resolution and with an increase in the sensitivity of detection by autoradiography when [35S]-methionine-labeled proteins were used. When unlabeled proteins were transferred, low levels of virus encoded proteins could be detected by sequential treatment of diazobenzloxymethyl-paper with anti-adenovirus type 2 virion or anti-73,000 DNA binding protein and 'MI-labeled Staphyloccus aurets protein A. Covalently bound viral proteins retained immunologic reactivity after dissociation of the protein A and antibody. By one-dimensional gel transfer/immunoautoradiography, seven virion proteins were detected as prominent bands and several others as weaker bands. By two-dimensional gel transfer/immunoautoradiography, several additional viral proteins were detected. By use of anti-DNA binding protein serum, the Mr 73,000 protein and Mr 41,000-48,000 subspecies were detected. A protein present at a concentration of approximately 1 part in 100,000 of the total protein can be identified in cell extracts. This method may be applicable to various biological problems requiring resolution and detection of small amounts of specific proteins that can be recognized immunologically or that can be detected by binding to specific radiolabeled DNA or RNA sequences or hormones.The detection and analysis of small amounts of specific proteins are important in elucidating the mechanisms ofvirus replication and cell transformation as well as of many other biological processes. Two-dimensional (2D) gel electrophoresis ofcell extracts (1) followed by protein staining or autoradiography makes it possible to detect more than a thousand proteins as well-resolved spots in a single slab gel. However, these procedures do not identify the proteins.Immunoprecipitation of radiolabeled cell extracts permits the sensitive detection ofspecific proteins in complex mixtures. However, unrelated proteins may coprecipitate, leading to erroneous conclusions. To avoid this, unlabeled protein mixtures have been separated on a slab gel and then specific proteins were identified by incubation with antiserum followed by a radiolabeled second antibody (2). The high background, extensive washing required, and the fact that only protein present near the surface will react with antibody are drawbacks. Renart et al. (3) found that transferring proteins from gels to diazobenzyloxymethyl (DBM)-paper by the Southern blotting procedure greatly improved the potential of this approach. The covalently bound proteins are still capable ofreacting with antibodies, and the ...
The mechanism by which human immunodeficiency virus type 1 Tat transactivates the long terminal repeat promoter is not understood. It is generally believed that Tat has one or more transcription factors as its cellular target. One might expect a cellular target for Tat to possess several properties, including (i) the ability to bind to the Tat activation region, (ii) the possession of a transcriptional activation domain, and (iii) the ability to contact the cellular transcription machinery. Here we describe the cloning, expression, and characterization of a human protein, termed TAP (Tat-associated protein), which possesses some of these properties. TAP is highly conserved in eukaryotes and is expressed in a variety of human tissues. The major intracellular species of TAP is a highly acidic 209-amino-acid protein that likely is formed by removal of a highly basic 70-amino-acid N-terminal segment from a primary translation product. By deletion analysis, we have identified a TAP C-terminal region rich in acidic amino acids and leucine residues which acts as a strong transcriptional activator when bound through GAL4 sites upstream of the core long terminal repeat promoter, as well as flanking sequences that mask the activation function. Amino acid substitution of two leucine residues within the core activation region results in loss of the TAP activation function. Two lines of evidence suggest that Tat interacts with TAP in vivo. First, promoter-bound Tat can recruit a TAP/VP16 fusion protein to the promoter. Second, transiently expressed Tat is found associated with endogenous TAP, as demonstrated by coimmunoprecipitation analysis. As shown in an accompanying report, the TAP activation region binds the Tat core activation region and general transcription factor TFIIB (L. Yu, P. M. Loewenstein, Z. Zhang, and M. Green, J. Virol. 69:3017-3023, 1995). These combined results suggest the hypothesis that TAP may function as a coactivator that bridges Tat to the general transcription machinery of the cell via TFIIB.
The human adenovirus E1A 243R protein (243 residues) transcriptionally represses a set of cellular genes that regulate cellular growth and differentiation. We describe two lines of evidence that E1A repression does not require cellular protein synthesis but instead involves direct interaction with a cellular protein(s). First, E1A 243R protein represses an E1A-repressible promoter in the presence of inhibitors of protein synthesis, as shown by cell microinjection-in situ hybridization. Second, E1A 243R protein strongly represses transcription in vitro from promoters of the E1A-repressible genes, human collagenase, and rat insulin type II. Repression in vitro is promoter-specific, and an E1A polypeptide containing only the N-terminal 80 residues is sufficient for strong repression both in vivo and in vitro. By use of a series of E1A 1-80 deletion proteins, the E1A repression function was found to require two E1A sequence elements, one within the nonconserved E1A N terminus, and the second within a portion of conserved region 1 (40 -80). These domains have been reported to possess binding sites for several cellular transcription regulators, including p300, Dr1, YY1, and the TBP subunit of TFIID. The in vitro transcription-repression system described here provides a powerful tool for the further analysis of molecular mechanism and the possible role of these cellular factors.Group C adenovirus (Ad) 1 E1A encodes two multifunctional regulatory proteins of 243 and 289 amino acid residues (243R and 289R). The E1A proteins are involved in diverse cellular functions, including transcriptional activation, transcriptional repression, induction of cellular DNA synthesis, cell immortalization, cell transformation, as well as inhibition of metastasis and of cell differentiation (for reviews, see Refs. 1-5). E1A 243R differs from E1A 289R only by conserved region 3 (CR3), a 46-amino acid domain unique to 289R. E1A is the first viral gene expressed during productive infection of permissive human cells and is required to activate transcriptionally early viral genes. CR3 is essential (6 -10) and sufficient (11-12) for transactivation of early viral genes.The 243R protein encodes domains required for the growth regulatory functions of E1A. An intriguing function of 243R is its ability to repress transcriptionally a set of cellular genes involved in growth regulation and differentiation (13-17), as well as several viral promoters including those of SV40, polyoma virus, and human immunodeficiency virus type 1 (18 -20). How the E1A repression function interfaces with its growth regulatory properties is not known. Furthermore, the molecular mechanism of transcriptional repression is not understood. Elucidation of mechanism would inform our understanding of the biological roles of E1A transcriptional repression.One can imagine several mechanisms by which E1A might repress the activity of a target gene (for review, see Ref. 21). For example, E1A could (i) bind to promoter DNA and block access by a transcriptional activator; (ii) sequest...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.