Abstract:
:The ability to examine gene regulation in living cells has been greatly enabled by the development of chromatin immunoprecipitation (ChIP) methodology. ChIP captures a snapshot of protein-DNA interactions in vivo and has been used to study interactions in bacteria, yeast, and mammalian cell culture. ChIP conditions vary depending upon the organism and the nature of the DNA-binding proteins under study. Here, we describe a customized ChIP protocol to examine the genome-wide distribution of a mobile DNA-binding enzyme, Escherichia coli RNA Polymerase (RNAP) as well as the factors that dynamically associate with RNAP during different stages of transcription. We describe new data analysis methods for determining the association of a broadly distributed DNA-binding complex. Further, we describe our approach of combining small molecules and antibiotics that perturb specific cellular events with ChIP and genomic platforms to dissect mechanisms of gene regulation in vivo. The chemical genomic methods can be leveraged to map natural and cryptic promoters and transcription units, annotate genomes, and reveal coupling between different processes in regulation of genes. This approach provides the framework for engineering gene networks and controlling biological output in a desired manner.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Davis SE,Mooney RA,Kanin EI,Grass J,Landick R,Ansari AZdoi
10.1016/B978-0-12-385120-8.00020-6subject
Has Abstractpub_date
2011-01-01 00:00:00pages
449-71eissn
0076-6879issn
1557-7988pii
B978-0-12-385120-8.00020-6journal_volume
498pub_type
杂志文章abstract::Antibody unfolding and aggregation have been gaining increasing concern as the need for pharmaceutical antibodies with high quality has increased. Assays for continuous application of severe stress to antibodies, which are typically employed to evaluate tolerance to aggregation or to understand the mechanism of aggreg...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.02.021
更新日期:2019-01-01 00:00:00
abstract::Enzymatic processes have great potential for applications in industrial biocatalysis, biosensing, and biomedical engineering. However, poor stability and difficulty in the reuse of enzymes still create challenges for their use in catalysis and other applications. In recent years, enzyme-incorporated metal-organic fram...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.12.002
更新日期:2017-01-01 00:00:00
abstract::Covalent modification of proteins with ubiquitin plays an important role in a wide array of cellular processes (Hershko and Ciechanover, 1998; Pickart, 2004). For this reason an increasing number of investigators in diverse research fields are confronted with the question whether their favorite proteins are ubiquitina...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)99016-2
更新日期:2005-01-01 00:00:00
abstract::The identification and isolation of the soluble catalytic domains of adenylyl cyclase have provided investigators with useful reagents for the study of these enzymes. They have permitted detailed mechanistic investigation of the actions of forskolin, Gs alpha, and the inhibitory G protein, Gi alpha. They have served a...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)45012-4
更新日期:2002-01-01 00:00:00
abstract::Slp4-a/granuphilin-a is a member of the synaptotagmin-like protein (Slp) family and consists of an N-terminal Slp homology domain (SHD) and C-terminal tandem C2 domains. Slp4-a is specifically localized on secretory granules in some endocrine and exocrine cells through its SHD, and it attenuates Ca(2+)-dependent dense...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)03039-9
更新日期:2005-01-01 00:00:00
abstract::A neuron is able to seamlessly respond to a number of signals, in a timely and specific manner. This process, of integrating multiple inputs, relays on the orchestration of intracellular events by signaling networks. The inherent complexity of signaling networks has made computational modeling a useful approach to und...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-388448-0.00014-0
更新日期:2012-01-01 00:00:00
abstract::Here we describe an optimized recombineering protocol for the creation of conditional alleles. Recombineering is a DNA engineering methodology based on homologous recombination in Escherichia coli. It presents a number of advantages over conventional engineering methods, including the ability to place target sites for...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)77008-7
更新日期:2010-01-01 00:00:00
abstract::Nitric oxide formation in ischemia-reperfusion injury can be identified and measured directly using EPR of nitroso-heme complexes comprising NO bound to either Mb or Hb-Fe2+. This article described the successful use of this method to detect and quantify the generation of NO formed independently of nitric oxide syntha...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)59182-5
更新日期:2002-01-01 00:00:00
abstract::Concomitant with the rapid development in biomedical knowledge, including the methods of molecular biology and proteomics, and the manufacture of ever more precise optical instruments, powerful lasers, and sophisticated microcomputing hardware and software, laser microdissection systems have emerged which are now ente...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)56935-4
更新日期:2002-01-01 00:00:00
abstract::The RNA-guided, sequence-specific endonuclease Cas9 has been widely adopted as genome engineering tool due to its efficiency and ease of use. Derived from the microbial CRISPR (clustered regularly interspaced short palindromic repeat) type II adaptive immune system, Cas9 has now been successfully engineered for genome...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-801185-0.00008-8
更新日期:2014-01-01 00:00:00
abstract::Reliable detection of keratins in tissues is important for investigating their physiological role and for using keratin expression as a biomarker in medical diagnostics. A particular challenge for the detection of keratins by immunofluorescence microscopy or immunohistochemistry relates to the fact that keratin interm...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.08.003
更新日期:2016-01-01 00:00:00
abstract::Once the appropriate site has been selected for the attachment of GFP to the sarcomeric protein, it is quite remarkable that the large size of the GFP molecule does not appear to interfere with the localization of the fluorescent sarcomeric proteins into the sarcomeric regions of the myofibrils. A similar approach usi...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(99)02017-0
更新日期:1999-01-01 00:00:00
abstract::Aliphatic carbon-halogen (C-X) bonds are prevalent in modern pharmaceuticals and bioactive natural products. Three distinct chemical strategies are known in Nature to generate these structural motifs. The first is via the nucleophilic substitution at a prefunctionalized electrophilic carbon center with a halide anion ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.02.015
更新日期:2018-01-01 00:00:00
abstract::Neurons are probably the most highly polarized cell type and typically develop a single axon and several dendrites. The establishment of a polarized morphology and the functional specialization of axonal and dendritic compartments are essential steps in the differentiation of neurons. Primary cultures of dissociated h...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)06056-3
更新日期:2006-01-01 00:00:00
abstract::In eukaryotes, determination of the subcellular location of a novel protein encoded in genomic or transcriptomic data provides useful clues as to its possible function. However, experimental localization studies are expensive and time-consuming. As a result, accurate in silico prediction of subcellular localization fr...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(09)05002-2
更新日期:2009-01-01 00:00:00
abstract::Among posttranslational modifications of proteins entailed with signal transduction, the redox transition is today brought to the focus as a major biochemical event accounting for the signaling functions of reactive oxygen species. Thermodynamic and kinetic criteria highlight hydroperoxides and protein disulfides as s...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(10)73011-1
更新日期:2010-01-01 00:00:00
abstract::Technical innovations in structural probing have drastically advanced the field of RNA structure analysis. These advances have led to parallel approaches developed in separate labs for analyzing RNA structure and dynamics. With the wealth of methodologies available, it can be difficult to determine which is best suite...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.04.002
更新日期:2019-01-01 00:00:00
abstract::Recent advances have demonstrated the feasibility and robustness of chemical synthesis for the production of homogeneously glycosylated protein forms (glycoforms). By taking advantage of the unmatchable flexibility and precision provided by chemical synthesis, the quantitative effects of glycosylation were obtained us...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.02.030
更新日期:2019-01-01 00:00:00
abstract::In recent years the facile, yet powerful, genetics of the baker's yeast Saccharomyces cerevisiae has been appropriated for the study of amyloid toxicity. Several models of amyloid toxicity using this simple eukaryotic organism have been developed that faithfully recapitulate many disease-relevant phenotypes. Furthermo...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)12013-3
更新日期:2006-01-01 00:00:00
abstract::Experimental screening for protein-ligand interactions is a central task in drug discovery. Nuclear magnetic resonance (NMR) spectroscopy enables the determination of binding affinities, as well as the measurement of structural and dynamic parameters governing the interaction. With traditional liquid-state NMR relying...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.08.016
更新日期:2019-01-01 00:00:00
abstract::Hypoxia and inflammation are coincidental events in an array of diseased tissues, including chronically inflamed sites (e.g., inflammatory bowel disease, rheumatoid arthritis), growing tumors, myocardial infarcts, atherosclerotic plaques, healing wounds, and sites of bacterial infection (Murdoch et al., 2005). An unde...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(07)35025-8
更新日期:2007-01-01 00:00:00
abstract::Inducible expression systems represent the founding technology for the emergence of synthetic biology in mammalian cells. The core molecules in these systems are bacterial regulator proteins that bind to or dissociate from a cognate DNA operator sequence in response to an exogenous stimulus like a small-molecule induc...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-385075-1.00011-1
更新日期:2011-01-01 00:00:00
abstract::The specificity of CRISPR-Cas systems for nucleic acid targets is determined by a combination of binding and cleavage. Understanding the mechanisms by which Cas proteins specifically select their targets is critical for the development of CRISPR-Cas systems for biotechnology applications. Moreover, the specificity of ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.10.019
更新日期:2019-01-01 00:00:00
abstract::Protein (poly-)ubiquitination is a posttranslational modification that plays a key role in almost all cellular processes. It involves the installment of either single ubiquitin (Ub) moieties or one of eight different polyUb linkage types, each giving a distinct cellular outcome. Deubiquitinating enzymes (DUBs) reverse...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.12.037
更新日期:2019-01-01 00:00:00
abstract::The Gene Expression Omnibus (GEO) repository at the National Center for Biotechnology Information archives and freely distributes high-throughput molecular abundance data, predominantly gene expression data generated by DNA microarray technology. The database has a flexible design that can handle diverse styles of bot...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(06)11019-8
更新日期:2006-01-01 00:00:00
abstract::Increasing numbers of compounds, previously classified as antagonists, were shown to inhibit this spontaneous or constitutive receptor activity, instead of leave it unaffected as expected for a formal antagonist. In addition, some other antagonists did not have any effect by themselves, but prevented the inhibition of...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381296-4.00003-8
更新日期:2010-01-01 00:00:00
abstract::Measurement of catalysis by MnSOD using direct observation of the UV absorbance of superoxide allows determination of steady-state catalytic constants. Stabilizing superoxide in aprotic solvents such as dimethyl sulfoxide permits the use of stopped-flow spectrophotometry, although significant information is lost in th...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)49321-4
更新日期:2002-01-01 00:00:00
abstract::A- and B-type lamins support the nuclear envelope, contribute to heterochromatin organization, and regulate a myriad of nuclear processes. The mechanisms by which lamins function in different cell types and the mechanisms by which lamin mutations cause over a dozen human diseases (laminopathies) remain unclear. The id...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2015.08.008
更新日期:2016-01-01 00:00:00
abstract::Classifications and characterizations of specific proteins, such as enzymes, not only allow us to understand biosynthetic and metabolic pathways but they also help to drive our understanding of protein structure and function. How those characterizations are evaluated, however, may change our interpretations and lead u...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2017.06.024
更新日期:2017-01-01 00:00:00
abstract::Nucleic acid aptamers find widespread use as targeting and sensing agents in nature and biotechnology. Their ability to bind an extensive range of molecular targets, including small molecules, proteins, and ions, with high affinity and specificity enables their use in diverse diagnostic, therapeutic, imaging, and gene...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-801122-5.00019-2
更新日期:2014-01-01 00:00:00