Screening of cobalamin metabolism in methotrexate-treated psoriatics.
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Adult↗
Biomedical subjects
C A Bunker
Publications and source records attributed to C A Bunker.
Activation domain-mediated enhancement of activator binding to chromatin in mammalian cells.
DNA binding by transcriptional activators is typically an obligatory step in the activation of gene expression. Activator binding and subsequent steps in transcription are repressed by genomic chromatin. Studies in vitro have suggested that overcoming this repression is an important function of some activation domains. Here we provide quantitative in vivo evidence that the activation domain of GAL4-VP16 can increase the affinity of GAL4 for its binding site on genomic DNA in mammalian cells. Moreover, the VP16 activation domain has a much greater stimulatory effect on expression from a genomic reporter gene than on a transiently transfected reporter gene, where factor binding is more permissive. We found that not all activation domains showed a greater activation potential in a genomic context, suggesting that only some activation domains can function in vivo to alleviate the repressive effects of chromatin. These data demonstrate the importance of activation domains in relieving chromatin-mediated repression in vivo and suggest that one way they function is to increase binding of the activator itself.
3T3 Cells↗
Repression and activation by multiprotein complexes that alter chromatin structure.
Recent studies have provided strong evidence that macromolecular complexes are used in the cell to remodel chromatin structure during activation and to create an inaccessible structure during repression, Although there is not yet any rigorous demonstration that modification of chromatin structure plays a direct, causal role in either activation or repression, there is sufficient smoke to indicate the presence of a blazing inferno nearby. It is clear that complexes that remodel chromatin are tractable in vitro; hopefully this will allow the establishment of systems that provide a direct analysis of the role that remodeling might play in activation. These studies indicate that establishment of functional systems to corroborate the elegant genetic studies on repression might also be tractable. As the mechanistic effects of these complexes are sorted out, it will become important to understand how the complexes are regulated. In many of the instances discussed above, the genes whose products make up these complexes were identified in genetic screens for effects on developmental processes. This implies a regulation of the activity of these complexes in response to developmental cues and further implies that the work to fully understand these complexes will occupy a generation of scientists.
Adenosine Triphosphate↗
Identification of a cDNA for SSRP1, an HMG-box protein, by interaction with the c-Myc oncoprotein in a novel bacterial expression screen.
We describe a system for screening cDNA expression libraries in Escherichia coli based on protein-protein interactions. The system utilizes fusion proteins containing the DNA binding domain of the lambda phage cl repressor and a heterologous dimerization domain, which is the target of the screen. Such chimeric proteins were functional as transcriptional repressors in E.coli; function was dependent on the presence of the heterologous dimerization domain, and function of the chimeras was disrupted by expression of excess dimerization domain. A screen was designed to identify factors that could interact with the heterologous dimerization domain and thereby inactivate the chimeric repressor. We used this screen to identify factors that could interact with the basic helix-loop-helix/leucine zipper domains of c-Myc, and isolated the cDNA for a previously characterized HMG domain protein that interacts specifically with c-Myc in this system. This screening method could be used with proteins that have the ability to homo- or heterodimerize.
3T3 Cells↗
Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells.
The Polycomb group (Pc-G) genes are essential for maintaining the proper spatially restricted expression pattern of the homeotic loci during Drosophila development. The Pc-G proteins appear to function at target loci to maintain a state of transcriptional repression. The murine oncogene bmi-1 has significant homology to the Pc-G gene Posterior sex combs (Psc) and a highly related gene, Suppressor two of zeste [Su(z)2]. We show here that the proteins encoded by bmi-1 and the Pc-G genes Polycomb (Pc) and Psc as well as Su(z)2 mediate repression in mammalian cells when targeted to a promoter by LexA in a cotransfection system. These fusion proteins repress activator function by as much as 30-fold, and the effect on different activation domains is distinct for each Pc-G protein. Repression is observed when the LexA fusion proteins are bound directly adjacent to activator binding sites and also when bound 1,700 bases from the promoter. These data demonstrate that the products of the Pc-G genes can significantly repress activator function on transiently introduced DNA. We suggest that this function contributes to the stable repression of targeted loci during development.
3T3 Cells↗
Secretion of Escherichia coli chloramphenicol acetyltransferase by mammalian cells.
We show here that expression of the Escherichia coli cat gene in mammalian cells results in accumulation of enzymatically active CAT in the culture media as well as in the cytoplasm. We call the extracellular product secreted CAT (sCAT). Three to four days after introduction of cat-expressing plasmids into mouse L cells by transient transfection, total extracellular sCAT activity exceeds total cytoplasmic CAT activity. As sCAT levels increase, substantially more CAT is found outside the cells than inside at later times. Comparison of different populations of cat-expressing cells shows that, at any given time, the level of sCAT is proportional to the level of intracellular CAT. Thus, assay of sCAT provides a convenient, non-invasive alternative to assay of intracellular CAT. The molecular sizes of sCAT and intracellular CAT are indistinguishable, suggesting that the protein is not cleaved or glycosylated during secretion. Several observations, including a lack of sensitivity to drugs which inhibit Golgi activity, suggest that CAT may be secreted via an unusual pathway.
Animals↗