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 site-specific recombinases at exactly chosen positions. Consequently, complicated designs, such as conditional alleles, can be rapidly achieved. We have applied these advantages to establish a pipeline method for the fluent generation of conditional alleles which employs Cre, Flp, and Dre recombinase target sites.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Fu J,Teucher M,Anastassiadis K,Skarnes W,Stewart AFdoi
10.1016/S0076-6879(10)77008-7subject
Has Abstractpub_date
2010-01-01 00:00:00pages
125-44eissn
0076-6879issn
1557-7988pii
S0076-6879(10)77008-7journal_volume
477pub_type
杂志文章abstract：:Multiprotein machines drive virtually all primary cellular processes. Modular multidomain proteins are widely distributed within these dynamic complexes because they provide the flexibility needed to remodel structure as well as rapidly assemble and disassemble components of the machinery. Understanding the functional...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2017.03.017
更新日期：2017-01-01 00:00:00
abstract：:RNA modifications, with over one hundred known so far, are commonly proposed to fine-tune the structure and function of RNA. While modifications in rRNA and tRNA are used to modulate RNA folding and decoding properties, little is known about the function of internal modifications in mRNA/lncRNA, which includes N(6)-me...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2015.03.005
更新日期：2015-01-01 00:00:00
abstract：:Although the function of histone deacetylases (HDACs) have primarily been associated with influencing transcription through chromatin remodeling, the capacity of these enzymes to interface with a diverse array of biologic processes by modulating a growing list of nonhistone substrates has gained recent attention. Rece...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-385114-7.00010-6
更新日期：2011-01-01 00:00:00
abstract：:Differential scanning calorimetry (DSC) detects protein thermal unfolding by directly measuring the heat absorbed. Simple DSC experiments that require relatively small amounts of pure material can provide a wealth of information related to structure, especially with respect to domain architecture, without the need for...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2015.08.014
更新日期：2016-01-01 00:00:00
abstract：:High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(06)14008-2
更新日期：2006-01-01 00:00:00
abstract：:Peroxiredoxins (Prdxs) are a family of proteins which catalyze the reduction of H2O2 through the interaction of active site cysteine residues. Conserved within all plant and animal kingdoms, the function of these proteins is related to protection from oxidation or participation of signaling through degradation of H2O2...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-405882-8.00008-8
更新日期：2013-01-01 00:00:00
abstract：:Molecular mechanisms of dark-to-light state transitions in flavoprotein photoreceptors have been the subject of intense investigation. Blue-light sensing flavoproteins fall into three general classes that share aspects of their activation processes: LOV domains, BLUF proteins, and cryptochromes. In all cases, light-in...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2019.03.023
更新日期：2019-01-01 00:00:00
abstract：:Ubiquitin-like protein (Ubl) ligation is common to diverse archaea and targets many cellular pathways, including those associated with sulfur mobilization, and also tags proteins as substrates for degradation by the proteasome. Here we highlight protocols to assay proteasome function and Ubl ligation in archaea. A cha...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2018.12.036
更新日期：2019-01-01 00:00:00
abstract：:Histone variants and posttranslational modifications (PTMs) are essential for epigenetic regulation of transcriptional expression. Single and/or combinatorial PTMs of histones play important roles in development and disease formation. Mass spectrometry (MS) has been a powerful tool to study histone variants and PTMs, ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-391940-3.00001-9
更新日期：2012-01-01 00:00:00
abstract：:Currently, over a hundred high-resolution structures of serpins are available, exhibiting a wide range of conformations. However, our understanding of serpin dynamics and conformational change is still limited, mainly due to challenges of monitoring structural changes and characterizing transient conformations using e...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-385950-1.00014-6
更新日期：2011-01-01 00:00:00
abstract：:Optical imaging of reporter molecules such as firefly luciferase has become a popular method of tracking and visualizing cells in living animals. Many biological processes involve ubiquitin ligases, which target specific proteins for destruction under specific sets of conditions. Importantly, the motifs recognized by ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(05)99036-8
更新日期：2005-01-01 00:00:00
abstract：:The breaking of RNA strands by 2'-O-transphosphorylation is a ubiquitous reaction in biology, and enzymes that catalyze this reaction play key roles in RNA metabolism. The mechanisms of 2'-O-transphosphorylation in solution are relatively well studied, but complex and can involve different transition states depending ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2017.07.017
更新日期：2017-01-01 00:00:00
abstract：:Posttranslational modifications (PTMs) of proteins perform crucial roles in regulating the biology of the cell. PTMs are enzymatic, covalent chemical modifications of proteins that typically occur after the translation of mRNAs. These modifications are relevant because they can potentially change a protein's physical ...
journal_title：Methods in enzymology
pub_type： 杂志文章,评审
doi：10.1016/S0076-6879(09)63040-8
更新日期：2009-01-01 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：:Cholestatic syndromes are inborn or acquired disorders of bile formation. In recent years, several inherited cholestatic syndromes were characterized at the molecular level: progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC). Both PFIC and BRIC were divided phenot...
journal_title：Methods in enzymology
pub_type： 杂志文章,评审
doi：10.1016/S0076-6879(05)00031-5
更新日期：2005-01-01 00:00:00
abstract：:Expressed protein ligation (EPL) combines two methods to ligate a synthetic peptide to a recombinant protein. Native chemical ligation (NCL) is a process in which two synthesized peptides are ligated by reaction of a C-terminal thioester on one peptide with an N-terminal cysteine residue of another protein. The chemis...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-420070-8.00009-X
更新日期：2014-01-01 00:00:00
abstract：:Neutron reflectometry (NR) is uniquely positioned in structural biology, because of its ability to characterize biomolecular interfacial architectures like lipid membranes and membrane-associated proteins nondestructively and in their native environment. Mimicking biological processes, samples can be manipulated and t...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2015.05.019
更新日期：2016-01-01 00:00:00
abstract：:The microbial production of commodity, fine, and specialty chemicals is a driving force in biotechnology. An essential requirement is to introduce biosynthetic pathways to the target compound(s) into chassis organisms. First suitable enzymes must be selected and characterized, and then genetic pathways must be designe...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2018.04.011
更新日期：2018-01-01 00:00:00
abstract：:We have developed a genetic system, termed IVET (in vivo expression technology), designed to identify bacterial genes that are induced when a pathogen infects its host. A subset of these induced genes should include those that encode virulence factors, products specifically required for the infection process. The syst...
journal_title：Methods in enzymology
pub_type： 杂志文章,评审
doi：10.1016/0076-6879(94)35164-3
更新日期：1994-01-01 00:00:00
abstract：:In order to produce a full quantitative description of yeast metabolism, a number of kinetic parameters of enzymes that are important for energy metabolism must be determined experimentally. We aim to determine the prospective in vivo kinetic properties of a range of yeast-purified isoenzymes that are important in ene...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-385118-5.00012-8
更新日期：2011-01-01 00:00:00
abstract：:In the presence of specialized proteins or peptides, a biological membrane can spontaneously restructure itself to allow communication between the intracellular and the extracellular sides. Examples of these proteins include cell-penetrating peptides and antimicrobial peptides (AMPs), which interact with cell membrane...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-381268-1.00016-1
更新日期：2011-01-01 00:00:00
abstract：:The functional properties of proteins are decided not only by their relatively rigid overall structures, but even more importantly, by their dynamic properties. In a protein, some regions of structure exhibit highly correlated or anti-correlated motions with others, some are highly dynamic but uncorrelated, while othe...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2020.04.020
更新日期：2020-01-01 00:00:00
abstract：:Endocytosis and autophagy are key vesicular pathways involved in degradation and recycling of cellular material. Both degradative pathways finally fuse with lysosome but are indeed interconnected at several levels. In particular, the fusion between endosomes and autophagosomes can generate intermediate vesicles named ...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-397926-1.00006-8
更新日期：2014-01-01 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：:Current methodologies available to quantify changes in mitochondrial turnover are limited to pulse-chase assays or specific assays that quantify mitophagy. Accordingly, new tools that can assess mitochondrial turnover are needed for the study of cellular, subcellular, and spatial parameters of mitochondrial turnover a...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/B978-0-12-801415-8.00002-3
更新日期：2014-01-01 00:00:00
abstract：:kappaB-Ras1 and kappaB-Ras2 are two small proteins that display similarity at the amino acid level to Ras-like small GTPases. Although little is known about the function of the kappaB-Ras proteins, they have been shown to interfere with activation of transcription factor NF-kappaB. They accomplish this by binding to I...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/S0076-6879(05)07042-4
更新日期：2006-01-01 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 long-pulse spallation source of the European Spallation Source-a facility under construction in Lund, Sweden-is well suited for macromolecular crystallography experiments. We review briefly the particular properties of the long-pulse source and the associated high-brilliance moderators from the point of view of in...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2020.01.005
更新日期：2020-01-01 00:00:00
abstract：:Portable, nanoparticle (NP)-enhanced enzyme sensors have emerged as powerful devices for qualitative and quantitative analysis of a variety of analytes for biomedicine, environmental applications, and pharmaceutical fields. This chapter describes a method for the fabrication of a portable, paper-based, inexpensive, ro...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2016.03.006
更新日期：2016-01-01 00:00:00
abstract：:Lymphocytes proliferate in response to several stimuli. In many situations, a rapid lymphocyte expansion, or the identification of a slow dividing cell subpopulation may be of great interest. Thus, it is necessary to perform reliable assays to study and compare lymphocyte subsets proliferation. For this purpose, carbo...
journal_title：Methods in enzymology
pub_type： 杂志文章
doi：10.1016/bs.mie.2019.05.020
更新日期：2020-01-01 00:00:00
