Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a method for the genetic analysis of the DNA-binding properties of Xenopus transcription factor IIIA (TFIIIA). In this approach, a transcriptional activator with the DNA-binding specificity of Xenopus TFIIIA is expressed in yeast cells, where it specifically activates expression of a beta-galactosidase reporter gene containing one or more Xenopus 5 S rRNA genes that function as upstream activator sequences. This transcription-promoting activity was used as the basis for a genetic assay of Xenopus TFIIIA's DNA-binding function in yeast, an assay that we show can be calibrated quantitatively to allow the affinity of the Xenopus TFIIIA-5 S rRNA gene interaction to be deduced from measurements of beta-galactosidase activity. We have combined this genetic assay with a simple and efficient method of mutagenesis that makes use of error-prone PCR and homologous recombination to generate and screen large numbers of TFIIIA mutants for those with altered 5 S rRNA gene-binding affinity. Over 30 such mutants have been identified and partially characterized. The mutants we have obtained provide strong support for the application to intact TFIIIA of recent structural models of the N-terminal zinc fingers of the protein bound to fragments of the 5 S rRNA gene. Other mutants permit identification of important residues in more C-terminal zinc fingers of TFIIIA for which high-resolution structural information is not currently available. Finally, our results have interesting implications with respect to the mechanism of activation of transcription by RNA polymerase II in yeast.
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PMID:Genetic analysis of Xenopus transcription factor IIIA. 987 52

Prothymosin alpha is a small, unfolded, negatively charged, poorly antigenic mammalian protein with a potent nuclear localization signal. Although it is apparently essential for growth, its precise function is unknown. We examined the location and behavior of the protein bearing different epitope tags using in situ immunolocalization in COS-1 and NIH3T3 cells. Tagged prothymosin alpha appeared to be punctate and widely dispersed throughout the nucleus, with the exception of the nucleolus. A tiny cytoplasmic component, which persisted in the presence of cycloheximide and actinomycin D during interphase, became pronounced immediately before, during, and after mitosis. When nuclear uptake was abrogated, small tagged prothymosin alpha molecules, but not prothymosin alpha fused to beta-galactosidase, accumulated significantly in the cytoplasm. Tagged prothymosin alpha shared domains with mobile proteins such as Ran, transportin, and karyopherin beta, which also traverse the nuclear membrane, and co-localized with active RNA polymerase II. Mild digitonin treatment resulted in nuclei devoid of prothymosin alpha. The data do not support tight binding to any nuclear component. Therefore, we propose that prothymosin alpha is a highly diffusible bolus of salt and infer that it facilitates movement of charged molecules in highly charged environments within and near the nucleus.
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PMID:Mobility within the nucleus and neighboring cytosol is a key feature of prothymosin-alpha. 1099 Apr 88

Set2 methylates Lys36 of histone H3. We show here that yeast Set2 copurifies with RNA polymerase II (RNAPII). Chromatin immunoprecipitation analyses demonstrated that Set2 and histone H3 Lys36 methylation are associated with the coding regions of several genes that were tested and correlate with active transcription. Both depend, as well, on the Paf1 elongation factor complex. The C terminus of Set2, which contains a WW domain, is also required for effective Lys36 methylation. Deletion of CTK1, encoding an RNAPII CTD kinase, prevents Lys36 methylation and Set2 recruitment, suggesting that methylation may be triggered by contact of the WW domain or C terminus of Set2 with Ser2-phosphorylated CTD. A set2 deletion results in slight sensitivity to 6-azauracil and much less beta-galactosidase produced by a reporter plasmid, resulting from a defect in transcription. In synthetic genetic array (SGA) analysis, synthetic growth defects were obtained when a set2 deletion was combined with deletions of all five components of the Paf1 complex, the chromodomain elongation factor Chd1, the putative elongation factor Soh1, the Bre1 or Lge1 components of the histone H2B ubiquitination complex, or the histone H2A variant Htz1. SET2 also interacts genetically with components of the Set1 and Set3 complexes, suggesting that Set1, Set2, and Set3 similarly affect transcription by RNAPII.
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PMID:Methylation of histone H3 by Set2 in Saccharomyces cerevisiae is linked to transcriptional elongation by RNA polymerase II. 1277 64

Atherosclerosis develops in distinct regions of the arterial tree. Defining patterns and mechanisms of endothelial cell gene expression in different regions of normal arteries is key to understanding the initial molecular events in atherogenesis. In this study, we demonstrated that the expression of endothelial nitric-oxide synthase (eNOS), an atheroprotective gene, and its phosphorylation on Ser(1177), a marker of activity, were lower in regions of the normal mouse aorta that are predisposed to atherosclerosis. The same expression pattern was observed in mouse strains that are both susceptible and resistant to atherosclerosis, and the topography of eNOS expression was inverse to p65, the main nuclear factor-kappaB subunit. Modeling of disturbed and uniform laminar flow in vitro reproduced the expression patterns of eNOS and p65 that were found in vivo. Heterogeneous nuclear RNA expression and RNA polymerase II chromosome immunoprecipitation studies demonstrated that regulation of transcription contributed to increased eNOS expression in response to shear stress. In vivo, the transcription of eNOS was reduced in regions of the mouse aorta predisposed to atherosclerosis, as defined by reporter gene expression in eNOS promoter-beta-galactosidase reporter transgenic mice. These data suggest that disturbed hemodynamic patterns found at arterial branches and curvatures uniquely modulate endothelial cell gene expression by regulating transcription, potentially explaining why these regions preferentially develop atherosclerosis when risk factors such as hypercholesterolemia are introduced.
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PMID:Relative reduction of endothelial nitric-oxide synthase expression and transcription in atherosclerosis-prone regions of the mouse aorta and in an in vitro model of disturbed flow. 1798 33

The positive transcription elongation factor b (P-TEFb) is essential for the elongation of transcription and cotranscriptional processing by RNA polymerase II. In mammals, it contains predominantly the C-type cyclin cyclin T1 (CycT1) or CycT2 and cyclin-dependent kinase 9 (Cdk9). To determine if these cyclins have redundant functions or affect distinct sets of genes, we genetically inactivated the CycT2 gene (Ccnt2) using the beta-galactosidase-neomycin gene (beta-geo) gene trap technology in the mouse. Visualizing beta-galactosidase during mouse embryogenesis revealed that CycT2 is expressed abundantly during embryogenesis and throughout the organism in the adult. This finding was reflected in the expression of CycT2 in all adult tissues and organs. However, despite numerous matings of heterozygous mice, we observed no CycT2(-/-) embryos, pups, or adult mice. This early lethality could have resulted from decreased expression of critical genes, which were revealed by short interfering RNAs against CycT2 in embryonic stem cells. Thus, CycT1 and CycT2 are not redundant, and these different P-TEFb complexes regulate subsets of distinct genes that are important for embryonic development.
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PMID:Cyclin T2 is essential for mouse embryogenesis. 1936 21


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