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Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing.
McCann TS, Guo Y, McDonald WH, Tansey WP
(2016) Proc Natl Acad Sci U S A 113: 1309-14
MeSH Terms: Chromatography, Affinity, Gene Silencing, Peptide Hydrolases, Saccharomyces cerevisiae Proteins, Telomere, Ubiquitin-Protein Ligases
Show Abstract · Added January 26, 2016
Ubiquitin, and components of the ubiquitin-proteasome system, feature extensively in the regulation of gene transcription. Although there are many examples of how ubiquitin controls the activity of transcriptional regulators and coregulators, there are few examples of core components of the transcriptional machinery that are directly controlled by ubiquitin-dependent processes. The budding yeast protein Asr1 is the prototypical member of the RPC (RING, PHD, CBD) family of ubiquitin-ligases, characterized by the presence of amino-terminal RING (really interesting new gene) and PHD (plant homeo domain) fingers and a carboxyl-terminal domain that directly binds the largest subunit of RNA polymerase II (pol II), Rpb1, in response to phosphorylation events tied to the initiation of transcription. Asr1-mediated oligo-ubiquitylation of pol II leads to ejection of two core subunits of the enzyme and is associated with inhibition of polymerase function. Here, we present evidence that Asr1-mediated ubiquitylation of pol II is required for silencing of subtelomeric gene transcription. We show that Asr1 associates with telomere-proximal chromatin and that disruption of the ubiquitin-ligase activity of Asr1--or mutation of ubiquitylation sites within Rpb1--induces transcription of silenced gene sequences. In addition, we report that Asr1 associates with the Ubp3 deubiquitylase and that Asr1 and Ubp3 play antagonistic roles in setting transcription levels from silenced genes. We suggest that control of pol II by nonproteolytic ubiquitylation provides a mechanism to enforce silencing by transient and reversible inhibition of pol II activity at subtelomeric chromatin.
0 Communities
2 Members
0 Resources
6 MeSH Terms
An efficient fluorescent protein-based multifunctional affinity purification approach in mammalian cells.
Ma H, McLean JR, Gould KL, McCollum D
(2014) Methods Mol Biol 1177: 175-91
MeSH Terms: Animals, Chromatography, Affinity, Cloning, Molecular, Humans, Protein Interaction Mapping, Proteomics, Recombinant Proteins, Tandem Mass Spectrometry
Show Abstract · Added January 20, 2015
Knowledge of an individual protein's modifications, binding partners, and localization is essential for understanding complex biological networks. We recently described a fluorescent protein-based (mVenus) multifunctional affinity purification (MAP) tag that can be used both to purify a given protein and determine its localization (Ma et al., Mol Cell Proteomics 11:501-511, 2012). MAP purified protein complexes can be further analyzed to identify binding partners and posttranslational modifications by LC-MS/MS. The MAP approach offers rapid FACS-selection of stable clonal cell lines based on the expression level/fluorescence of the MAP-protein fusion. The MAP tag is highly efficient and shows little variability between proteins. Here we describe the general MAP purification method in detail, and show how it can be applied to a specific protein using the human Cdc14B phosphatase as an example.
0 Communities
1 Members
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8 MeSH Terms
A structure-specific nucleic acid-binding domain conserved among DNA repair proteins.
Mason AC, Rambo RP, Greer B, Pritchett M, Tainer JA, Cortez D, Eichman BF
(2014) Proc Natl Acad Sci U S A 111: 7618-23
MeSH Terms: Adenosine Triphosphate, Animals, Chromatography, Affinity, Chromatography, Agarose, Chromatography, Gel, Chromatography, Ion Exchange, Cloning, Molecular, Crystallization, DNA Helicases, DNA Repair, Hydrolysis, Likelihood Functions, Mice, Models, Molecular, Nucleic Acids, Protein Structure, Tertiary, Scattering, Small Angle, X-Ray Diffraction
Show Abstract · Added May 19, 2014
SMARCAL1, a DNA remodeling protein fundamental to genome integrity during replication, is the only gene associated with the developmental disorder Schimke immuno-osseous dysplasia (SIOD). SMARCAL1-deficient cells show collapsed replication forks, S-phase cell cycle arrest, increased chromosomal breaks, hypersensitivity to genotoxic agents, and chromosomal instability. The SMARCAL1 catalytic domain (SMARCAL1(CD)) is composed of an SNF2-type double-stranded DNA motor ATPase fused to a HARP domain of unknown function. The mechanisms by which SMARCAL1 and other DNA translocases repair replication forks are poorly understood, in part because of a lack of structural information on the domains outside of the common ATPase motor. In the present work, we determined the crystal structure of the SMARCAL1 HARP domain and examined its conformation and assembly in solution by small angle X-ray scattering. We report that this domain is conserved with the DNA mismatch and damage recognition domains of MutS/MSH and NER helicase XPB, respectively, as well as with the putative DNA specificity motif of the T4 phage fork regression protein UvsW. Loss of UvsW fork regression activity by deletion of this domain was rescued by its replacement with HARP, establishing the importance of this domain in UvsW and demonstrating a functional complementarity between these structurally homologous domains. Mutation of predicted DNA-binding residues in HARP dramatically reduced fork binding and regression activities of SMARCAL1(CD). Thus, this work has uncovered a conserved substrate recognition domain in DNA repair enzymes that couples ATP-hydrolysis to remodeling of a variety of DNA structures, and provides insight into this domain's role in replication fork stability and genome integrity.
1 Communities
2 Members
0 Resources
18 MeSH Terms
Simple sample processing enhances malaria rapid diagnostic test performance.
Davis KM, Gibson LE, Haselton FR, Wright DW
(2014) Analyst 139: 3026-31
MeSH Terms: Chromatography, Affinity, Humans, Malaria, Sensitivity and Specificity
Show Abstract · Added May 27, 2014
Lateral flow immunochromatographic rapid diagnostic tests (RDTs) are the primary form of medical diagnostic used for malaria in underdeveloped nations. Unfortunately, many of these tests do not detect asymptomatic malaria carriers. In order for eradication of the disease to be achieved, this problem must be solved. In this study, we demonstrate enhancement in the performance of six RDT brands when a simple sample-processing step is added to the front of the diagnostic process. Greater than a 4-fold RDT signal enhancement was observed as a result of the sample processing step. This lowered the limit of detection for RDT brands to submicroscopic parasitemias. For the best performing RDTs the limits of detection were found to be as low as 3 parasites per μL. Finally, through individual donor samples, the correlations between donor source, WHO panel detection scores and RDT signal intensities were explored.
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4 MeSH Terms
Polycyclic aromatic hydrocarbons: determinants of urinary 1-hydroxypyrene glucuronide concentration and risk of colorectal cancer in the Shanghai Women's Health Study.
Hofmann JN, Liao LM, Strickland PT, Shu XO, Yang G, Ji BT, Li HL, Rothman N, Kamangar F, Gao YT, Zheng W, Chow WH
(2013) BMC Cancer 13: 282
MeSH Terms: Case-Control Studies, Chromatography, Affinity, Colorectal Neoplasms, Female, Glucuronates, Humans, Middle Aged, Polycyclic Aromatic Hydrocarbons, Pyrenes, Risk Factors
Show Abstract · Added May 4, 2017
BACKGROUND - Associations between polycyclic aromatic hydrocarbons (PAHs) and colorectal cancer have been reported previously but few studies have characterized PAH exposure using biological measurements. We evaluated colorectal cancer risk in relation to urinary concentration of 1-hydroxypyrene glucuronide (1-OHPG), a polycyclic aromatic hydrocarbon (PAH) metabolite, and assessed determinants of PAH exposure among controls in the Shanghai Women's Health Study (SWHS).
METHODS - Concentrations of 1-OHPG were measured in spot urine samples collected from 343 colorectal cancer cases and 343 individually matched controls. Questionnaires were administered to collect information on demographic characteristics and reported exposures. Odds ratios were calculated for risk of colorectal cancer in relation to quartiles of urinary 1-OHPG concentration. Potential determinants of natural log-transformed urinary 1-OHPG concentration were evaluated among a combined sample of controls from this study and another nested case-control study in the SWHS (N(total)=652).
RESULTS - No statistically significant differences in risk of colorectal cancer by urinary 1-OHPG levels were observed. Among controls, the median (interquartile range) urinary 1-OHPG concentration was 2.01 pmol/mL (0.95-4.09). Active and passive smoking, using coal as a cooking fuel, eating foods that were cooked well done, and recent consumption of fried dough (e.g., yóutiáo) were associated with elevated levels of 1-OHPG, though only active smoking and fried dough consumption achieved statistical significance in multivariate analyses.
CONCLUSIONS - This study does not provide evidence of an association between urinary levels of 1-OHPG and risk of colorectal cancer among women. Several environmental and dietary sources of PAH exposure were identified. Overall, the levels of 1-OHPG in this population of predominantly non-smoking women were considerably higher than levels typically observed among non-smokers in Europe, North America, and other developed regions.
0 Communities
1 Members
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10 MeSH Terms
Diagnostic performance of the BinaxNow Influenza A&B rapid antigen test in ED patients.
Self WH, McNaughton CD, Grijalva CG, Zhu Y, Chappell JD, Williams JV, Talbot HK, Shay DK, Griffin MR
(2012) Am J Emerg Med 30: 1955-61
MeSH Terms: Adolescent, Adult, Antigens, Viral, Child, Child, Preschool, Chromatography, Affinity, Cross-Sectional Studies, Emergency Service, Hospital, Female, Humans, Influenza A virus, Influenza B virus, Influenza, Human, Male, Middle Aged, Prospective Studies, Real-Time Polymerase Chain Reaction, Sensitivity and Specificity, Young Adult
Show Abstract · Added December 10, 2013
OBJECTIVE - The objective of this study is to evaluate the diagnostic performance of the BinaxNow Influenza A&B rapid antigen test (RAT) in emergency department (ED) patients.
METHODS - We prospectively enrolled a systematic sample of ED patients older than 6 months with acute respiratory symptoms or nonlocalizing fever during 3 consecutive influenza seasons (2008-2011). Nasal and throat swabs collected by research personnel were tested for influenza by real-time reverse transcription-polymerase chain reaction (RT-PCR). Clinicians independently ordered RATs during clinical care; these specimens were collected by clinical staff and tested for influenza using the BinaxNow RAT. Patients with both a research RT-PCR and clinical RAT were included in the study. Rapid antigen test diagnostic performance was evaluated using RT-PCR as a criterion standard, with preplanned, stratified analysis for subject age, duration of symptoms, influenza subtype, and polymerase chain reaction cycle threshold, which provides a semiquantitative estimate of viral load.
RESULTS - Of 561 subjects enrolled, 131 (23.4%) had a positive RT-PCR, and 37 (6.6%) had a positive RAT. Overall, RAT performance included sensitivity of 24.4% (95% confidence interval [CI], 17.5%-32.9%), specificity of 98.8% (95% CI, 97.1%-99.6%), positive predictive value of 86.5% (95% CI, 70.4%-94.9%), negative predictive value of 81.1% (95% CI, 77.4%-84.3%). Rapid antigen test sensitivities were low for all categories of subject age, symptom duration, influenza subtype, and cycle threshold.
CONCLUSION - The BinaxNow RAT demonstrated high specificity and poor sensitivity in ED patients selected by treating clinicians for influenza testing. A negative RAT is a poor predictor for the absence of influenza in the ED and should not be used as a criterion to withhold antiviral medications.
Copyright © 2012 Elsevier Inc. All rights reserved.
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2 Members
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19 MeSH Terms
Affinity capture of TAP-tagged protein complexes: affinity purification step 2.
Link AJ, Weaver C, Farley A
(2011) Cold Spring Harb Protoc 2011: pdb.prot5607
MeSH Terms: Antibodies, Fungal, Chromatography, Affinity, Complex Mixtures, Fungal Proteins, Immunoglobulin G, Protein Interaction Mapping, Recombinant Fusion Proteins, Yeasts
Added February 20, 2015
0 Communities
1 Members
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8 MeSH Terms
IgG affinity capture of TAP-tagged protein complexes from cell extracts: affinity purification step 1.
Link AJ, Weaver C, Farley A
(2011) Cold Spring Harb Protoc 2011: pdb.prot5606
MeSH Terms: Antibodies, Fungal, Chromatography, Affinity, Complex Mixtures, Fungal Proteins, Immunoglobulin G, Protein Interaction Mapping, Recombinant Fusion Proteins, Yeasts
Added February 20, 2015
0 Communities
1 Members
0 Resources
8 MeSH Terms
Making yeast cell extracts for purifying TAP-tagged protein complexes.
Link AJ, Weaver C, Farley A
(2011) Cold Spring Harb Protoc 2011: pdb.prot5605
MeSH Terms: Chromatography, Affinity, Complex Mixtures, Fungal Proteins, Protein Interaction Mapping, Recombinant Fusion Proteins, Yeasts
Added February 20, 2015
0 Communities
1 Members
0 Resources
6 MeSH Terms
Growing and harvesting TAP-tagged yeast cells.
Link AJ, Weaver C, Farley A
(2011) Cold Spring Harb Protoc 2011: pdb.prot5604
MeSH Terms: Chromatography, Affinity, Fungal Proteins, Protein Interaction Mapping, Recombinant Fusion Proteins, Yeasts
Added February 20, 2015
0 Communities
1 Members
0 Resources
5 MeSH Terms