scholarly journals Affinity chromatography of thiol-containing purines and ribonucleic acid

1977 ◽  
Vol 168 (3) ◽  
pp. 595-597 ◽  
Author(s):  
W T Melvin ◽  
H M Keir
Keyword(s):  
Gene Expression ◽  
Affinity Chromatography ◽  
Ribonucleic Acid ◽  
Specific Interaction ◽  
Rna Metabolism ◽  
New Method

Evidence is presented for the specific interaction of 6-mercaptopurine with mercurated cellulose. Following from this, a new method is described for the affinity chromatography of thiol-containing molecules and of RNA containing incorporated 6-thioguanosine on columns of mercurated cellulose. This technique may find application in the study of RNA metabolism and gene expression.

A new method for the analysis of plasmin and plasminogen. Application of high-performance affinity chromatography.

1985 ◽  
Vol 16 (1) ◽  
pp. 23-25
Author(s):  
Kenichi KASAI ◽  
Kiyohito SHIMURA ◽  
Naofumi ITO ◽  
Kohji NOGUCHI ◽  
Mutsuyoshi KAZAMA
Keyword(s):  
Affinity Chromatography ◽  
High Performance ◽  
New Method ◽  
High Performance Affinity Chromatography

Identification of a C/EBP-Rel complex in avian lymphoid cells

1994 ◽  
Vol 14 (10) ◽  
pp. 6635-6646
Author(s):  
J A Diehl ◽  
M Hannink
Keyword(s):  
Gene Expression ◽  
Affinity Chromatography ◽  
Protein Interactions ◽  
Binding Proteins ◽  
Regulation Of Gene Expression ◽  
Lymphoid Cells ◽  
Cytokine Gene Expression ◽  
Protein Protein Interactions ◽  
Dna Complex ◽  
Dna Affinity Chromatography

Protein-protein interactions between the CCAAT box enhancer-binding proteins (C/EBP) and the Rel family of transcription factors have been implicated in the regulation of cytokine gene expression. We have used sequence-specific DNA affinity chromatography to purify a complex from avian T cells that binds to a consensus C/EBP motif. Our results provide evidence that Rel-related proteins are components of the C/EBP-DNA complex as a result of protein-protein interactions with the C/EBP proteins. A polyclonal antiserum raised against the Rel homology domain of v-Rel and antisera raised against two human RelA-derived peptides specifically induced a supershift of the C/EBP-DNA complex in mobility shift assays using the affinity-purified C/EBP. In addition, several kappa B-binding proteins copurified with the avian C/EBP complex through two rounds of sequence-specific DNA affinity chromatography. The kappa B-binding proteins are distinct from the C/EBP proteins that directly contact DNA containing the C/EBP binding site. The identification of a protein complex that binds specifically to a consensus C/EBP site and contains both C/EBP and Rel family members suggests a novel mechanism for regulation of gene expression by Rel family proteins.

scholarly journals Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

2020 ◽  
Author(s):  
Daniel M. Sapozhnikov ◽  
Moshe Szyf
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Promoter Region ◽  
Dna Methyltransferase ◽  
Dna Demethylation ◽  
New Method ◽  
Inducible Promoters ◽  
Specific Targeting ◽  
Main Effect

AbstractAlthough associations between DNA methylation and gene expression were established four decades ago, the causal role of DNA methylation in gene expression remains unresolved. Different strategies to address this question were developed; however, all are confounded and fail to disentangle cause and effect. We developed here a highly effective new method using only deltaCas9(dCas9):gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, enabling examination of the role of DNA methylation per se in living cells. We show that the extensive induction of gene expression achieved by TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using our new method, we show that in several inducible promoters, the main effect of DNA methylation is silencing basal promoter activity. Thus, the effect of demethylation of the promoter region in these genes is small, while induction of gene expression by different inducers is large and DNA methylation independent. In contrast, targeting demethylation to the pathologically silenced FMR1 gene targets robust induction of gene expression. We also found that standard CRISPR/Cas9 knockout generates a broad unmethylated region around the deletion, which might confound interpretation of CRISPR/Cas9 gene depletion studies. In summary, this new method could be used to reveal the true extent, nature, and diverse contribution to gene regulation of DNA methylation at different regions.

Sequence-specific protein interaction with a transcriptional enhancer involved in the autoregulated expression of cAMP receptor 1 in Dictyostelium

Development ◽  
1998 ◽  
Vol 125 (18) ◽  
pp. 3689-3698
Author(s):  
X. Mu ◽  
B. Lee ◽  
J.M. Louis ◽  
A.R. Kimmel
Keyword(s):  
Gene Expression ◽  
Early Development ◽  
Specific Interaction ◽  
Early Gene ◽  
Specific Gene ◽  
Critical Element ◽  
Molecular Response ◽  
Continuous Exposure ◽  
Camp Receptor ◽  
Extracellular Camp

Major stages of Dictyostelium development are regulated by secreted, extracellular cAMP through activation of a serpentine receptor family. During early development, oscillations of extracellular cAMP mobilize cells for aggregation; later, continuous exposure to higher extracellular cAMP concentrations downregulates early gene expression and promotes cytodifferentiation and cell-specific gene expression. The cAMP receptor 1 gene CAR1 has two promoters that are differentially responsive to these extracellular cAMP stimuli. The early CAR1 promoter is induced by nM pulses of cAMP, which in turn are generated by CAR1-dependent activation of adenylyl cyclase (AC). Higher, non-fluctuating concentrations of cAMP will adapt this AC stimulus-response, repress the activated early promoter and induce the dormant late promoter. We now identify a critical element of the pulse-induced CAR1 promoter and a nuclear factor with sequence-specific interaction. Mutation of four nucleotides within the element prevents both in vitro protein binding and in vivo expression of an otherwise fully active early CAR1 promoter and multimerization of the wild-type, but not mutant, sequence will confer cAMP regulation to a quiescent heterologous promoter. These cis and trans elements, thus, constitute a part of the molecular response to the cAMP transmembrane signal cascade that regulates early development of Dictyostelium.

Assessment of Alterations in Gene Expression in Recurrent Malignant Glioma after Radiotherapy Using Complementary Deoxyribonucleic Acid Microarrays

Neurosurgery ◽  
2001 ◽  
Vol 48 (1) ◽  
pp. 195-202 ◽  
Author(s):  
Tatsuhiro Joki ◽  
Rona S. Carroll ◽  
Ian F. Dunn ◽  
Jianping Zhang ◽  
Toshiaki Abe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factors ◽  
Growth Factor ◽  
Glioblastoma Multiforme ◽  
Ribonucleic Acid ◽  
Deoxyribonucleic Acid ◽  
Platelet Derived Growth Factor ◽  
Recurrent Tumors ◽  
Complementary Deoxyribonucleic Acid ◽  
Autocrine Loops

Abstract OBJECTIVE We used complementary deoxyribonucleic acid expression microarrays to assess the effects of radiotherapy on gene expression in glioblastoma multiforme. We hypothesized that postradiation recurrent tumors may demonstrate alterations in gene expression from the primary tumor specimen. METHODS Patients were diagnosed with glioblastoma multiforme at resection of the initial tumor, and they received 60 Gy of fractionated radiotherapy before recurrence. Ribonucleic acid samples from both the primary and the postradiation recurrent tumor in each patient were screened and compared using complementary deoxyribonucleic acid expression arrays and Northern blot analysis. RESULTS Messenger ribonucleic acid levels of growth factors participating in paracrine loops, such as vascular endothelial growth factor and platelet-derived growth factor receptor β, were decreased in postradiation recurrent tumors as compared with primary tumors in three of four patients. However, messenger ribonucleic acid levels of growth factors involved in autocrine loops, such as epidermal growth factor receptor, platelet-derived growth factor α, platelet-derived growth factor A, and basic fibroblast growth factor, were decreased in two of four, two of four, three of four, and three of four patients' recurrent tumors, respectively. Microvessel counts demonstrated that blood vessel growth was decreased significantly in postradiation recurrent tumor specimens. CONCLUSION After radiotherapy of glioblastoma multiforme, levels of paracrine-acting growth factors are diminished in correspondence with the reduction in vascular density. In contrast, growth factors that participate in autocrine loops demonstrate elevated levels of gene expression. These results suggest that maintenance of autocrine loops may be important in tumor regrowth after radiotherapy.

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