abstract：:We have developed two screening systems for isolating inhibitors that target bacterial two-component signal transduction: (1) a differential growth assay using a temperature-sensitive yycF mutant (CNM2000) of Bacillus subtilis, which is supersensitive to histidine kinase inhibitors, and (2) a high-throughput genetic s...

journal_title：Methods in enzymology

authors： Okada A,Gotoh Y,Watanabe T,Furuta E,Yamamoto K,Utsumi R

更新日期：2007-01-01 00:00:00

abstract：:PYP-phytochrome (Ppr) is a unique photoreceptor that contains a blue light-absorbing photoactive yellow protein (PYP) domain, a red light-absorbing phytochrome domain, and a histidine kinase domain. This chapter describes overexpression of Ppr in a strain of Escherichia coli that allows covalent attachment of substoic...

journal_title：Methods in enzymology

authors： Chung YH,Masuda S,Bauer CE

更新日期：2007-01-01 00:00:00

abstract：:Advances in chemical synthesis and characterization of nucleic acids allows for atom-specific modification of complex RNAs, such as present in RNA editing substrates. By preparing substrates for ADARs by chemical synthesis, it is possible to subtly alter the structure of the edited nucleotide. Evaluating the effect th...

journal_title：Methods in enzymology

authors： Maydanovych O,Easterwood LM,Cui T,Véliz EA,Pokharel S,Beal PA

更新日期：2007-01-01 00:00:00

abstract：:Mitochondrial RNAs in the myxomycete Physarum polycephalum differ from the templates from which they are transcribed in defined ways. Most transcripts contain nucleotides that are not present in their respective genes. These "extra" nucleotides are added during RNA synthesis by an unknown mechanism. Other differences ...

journal_title：Methods in enzymology

authors： Byrne EM,Visomirski-Robic L,Cheng YW,Rhee AC,Gott JM

更新日期：2007-01-01 00:00:00

abstract：:Hematopoietic stem cells (HSCs) primarily reside in bone marrow, are defined by their ability to maintain blood homeostasis, and replenish themselves through self-renewal. Although HSC purification schemes vary from laboratory to laboratory, the resulting cell populations are similar, if not the same. This chapter wil...

journal_title：Methods in enzymology

authors： Lin KK,Goodell MA

更新日期：2006-01-01 00:00:00

abstract：:Human embryonic stem cells (hESCs) are derived from preimplantation embryos. Approximately 60% of human embryos are blocked during in vitro development. Although statistics are inconclusive, experience demonstrates that hESCs are more effectively derived from high-quality embryos. In this way, optimal human embryo cul...

journal_title：Methods in enzymology

authors： Mercader A,Valbuena D,Simón C

更新日期：2006-01-01 00:00:00

abstract：:Sulfatases hydrolyze sulfate esters on a variety of molecules including glycosaminoglycans, sulfoglycolipids, and cytosolic steroids. These enzymes are found in a wide range of organisms with their basic enzymatic mechanisms broadly conserved. In mammals, many of the sulfatases localize in the lysosome and exhibit enz...

journal_title：Methods in enzymology

authors： Uchimura K,Morimoto-Tomita M,Rosen SD

更新日期：2006-01-01 00:00:00

abstract：:Glycosaminoglycans (GAGs) are long unbranched polysaccharides, most of which are linked to a core protein to form proteoglycans. Depending on the nature of their backbone, one can discern galactosaminoglycans (chondroitin sulfate [CS] and dermatan sulfate [DS]) and glucosaminoglycans (heparan sulfate [HS], heparin, hy...

journal_title：Methods in enzymology

authors： Smits NC,Lensen JF,Wijnhoven TJ,Ten Dam GB,Jenniskens GJ,van Kuppevelt TH

更新日期：2006-01-01 00:00:00

abstract：:The lung is composed of two major anatomically distinct regions-the conducting airways and gas-exchanging airspaces. From a cell biology standpoint, the conducting airways can be further divided into two major compartments, the tracheobronchial and bronchiolar airways, while the alveolar regions of the lung make up th...

journal_title：Methods in enzymology

authors： Liu X,Driskell RR,Engelhardt JF

更新日期：2006-01-01 00:00:00

abstract：:Extracellular signals dictate the biological processes of neural stem cells (NSCs) both in vivo and in vitro. The intracellular response elicited by these signals is dependent on the context in which the signal is received, which in turn is decided by previous and concurrent signals impinging on the cell. A synthesis ...

journal_title：Methods in enzymology

authors： Rajan P,Snyder E

更新日期：2006-01-01 00:00:00

abstract：:Embryonic stem (ES) cells are cells derived from the inner cell mass of a blastocyst stage embryo. These self-renewing multipotent cells are able to differentiate to the three embryonic germ layers, the endoderm, ectoderm, and mesoderm, and are thus able to produce virtually all cell types. The ES cell capacity to gen...

journal_title：Methods in enzymology

authors： Goldberg-Cohen I,Beck G,Ziskind A,Itskovitz-Eldor J

更新日期：2006-01-01 00:00:00

abstract：:Human embryonic stem cells hold great promise in furthering our treatment of disease and increasing our understanding of early development. This chapter describes protocols for the derivation and maintenance of human embryonic stem cells. In addition, it summarizes briefly several alternative methods for the culture o...

journal_title：Methods in enzymology

authors： Akutsu H,Cowan CA,Melton D

更新日期：2006-01-01 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

authors： Giorgini F,Muchowski PJ

更新日期：2006-01-01 00:00:00

abstract：:Amyloid aggregates are associated with a number of mammalian neurodegenerative diseases. Infectious aggregates of the mammalian prion protein PrP(sc) are hallmarks of transmissible spongiform encephalopathies in humans and cattle (Griffith, 1967; Legname et al., 2004; Prusiner, 1982; Silveira et al., 2004). Likewise, ...

journal_title：Methods in enzymology

authors： Bagriantsev SN,Kushnirov VV,Liebman SW

更新日期：2006-01-01 00:00:00

abstract：:High hydrostatic pressure (HHP) modulates protein-protein and protein-solvent interactions through volume changes and thereby affects the equilibrium of protein conformational species between native and denatured forms as well as monomeric, oligomeric, and aggregated forms without the addition of chemicals or use of h...

journal_title：Methods in enzymology

authors： Kim YS,Randolph TW,Seefeldt MB,Carpenter JF

更新日期：2006-01-01 00:00:00

abstract：:Nonnative protein aggregation has been classically treated as an amorphous process occurring by colloidal coagulation kinetics and proceeding to an essentially irreversible endpoint often ascribed to a chaotic tangle of unfolded chains. However, some nonnative aggregates, particularly amyloid fibrils, exhibit ordered ...

journal_title：Methods in enzymology

authors： O'Nuallain B,Thakur AK,Williams AD,Bhattacharyya AM,Chen S,Thiagarajan G,Wetzel R

更新日期：2006-01-01 00:00:00

abstract：:Changes in chromatin structure emanating from DNA breaks are among the most initiating events in the damage response of the cell. In higher eukaryotes, poly(ADP-ribose) polymerase-1 (PARP-1) translates the occurrence of DNA breaks detected by its zinc-finger domain into a signal, poly ADP-ribose, synthesized and ampli...

journal_title：Methods in enzymology

authors： Dantzer F,Amé JC,Schreiber V,Nakamura J,Ménissier-de Murcia J,de Murcia G

更新日期：2006-01-01 00:00:00

abstract：:In vivo genome manipulation through site-directed mutagenesis and chromosome rearrangements has been hindered by the difficulty in achieving high frequencies of targeting and the intensive labor required to create altered genomes that do not contain any heterologous sequence. Here we describe our approach, referred to...

journal_title：Methods in enzymology

authors： Storici F,Resnick MA

更新日期：2006-01-01 00:00:00

abstract：:When yeast are faced with irreparable DNA damage, they will first arrest in G2/M, via the DNA damage checkpoint pathway, but will subsequently adapt to that arrest and resume division. Here, we summarize assays that we have used to examine checkpoint adaptation. Specifically, we discuss the merits of inducing DNA dama...

journal_title：Methods in enzymology

authors： Toczyski DP

更新日期：2006-01-01 00:00:00

abstract：:Two lymphoid cell-specific proteins, called RAG-1 and RAG-2, initiate the process of antigen receptor gene rearrangement, termed V(D)J recombination, by assembling a protein-DNA complex with two recombination signal sequences (RSSs), each of which adjoins a different receptor gene segment, and then introducing a DNA d...

journal_title：Methods in enzymology

authors： Bergeron S,Anderson DK,Swanson PC

更新日期：2006-01-01 00:00:00

abstract：:This chapter discusses experimental methods and protocols for the purification and preliminary characterization of DNA polymerases that are specialized for the replicative bypass (translesion DNA synthesis) of base or other types of DNA damage that typically arrest high-fidelity DNA synthesis, with particular emphasis...

journal_title：Methods in enzymology

authors： Fischhaber PL,McDaniel LD,Friedberg EC

更新日期：2006-01-01 00:00:00

abstract：:Recent advances in functional genomics have enabled genome-wide genetic studies in mammalian cells. These include the establishment of high-throughput transfection and viral propagation methodologies, the production of large-scale cDNA and siRNA libraries, and the development of sensitive assay detection processes and...

journal_title：Methods in enzymology

authors： Rines DR,Tu B,Miraglia L,Welch GL,Zhang J,Hull MV,Orth AP,Chanda SK

更新日期：2006-01-01 00:00:00

abstract：:Nucleotide excision repair is a multicomponent, multistep enzymatic system that removes a wide spectrum of DNA damage by dual incisions in the damaged strand on both sides of the lesion. The basic steps are damage recognition, dual incisions, resynthesis to replace the excised DNA, and ligation. Each step has been stu...

journal_title：Methods in enzymology

authors： Reardon JT,Sancar A

更新日期：2006-01-01 00:00:00

abstract：:Intracellular calcium (Ca2+) is involved in the regulation of a variety of biological functions in cancer cells, including growth inhibition, tumor invasiveness, and drug resistance. To gain insight into the possible role played by Ca2+ in the development of drug resistance in breast cancer, we performed a comparative...

journal_title：Methods in enzymology

authors： DeBernardi MA,Brooker G

更新日期：2006-01-01 00:00:00

abstract：:As new organisms are being sequenced on a daily basis, new DNA glycosylases that recognize DNA damage can be easily identified in an effort to understand both their phylogenetics and substrate specificities. As a practical matter, existing bacterial and human homologs need to be readily available as laboratory reagent...

journal_title：Methods in enzymology

authors： Bandaru V,Blaisdell JO,Wallace SS

更新日期：2006-01-01 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

authors： Haasen D,Schnapp A,Valler MJ,Heilker R

更新日期：2006-01-01 00:00:00

abstract：:The Ras gene family has been implicated in the development of many human epithelial cancers. Mutations in K-ras or its downstream mediator BRAF have been detected in about two thirds of low-grade serous carcinomas and borderline serous tumors; mutations in K-ras are also often present in benign and invasive mucinous o...

journal_title：Methods in enzymology

authors： Rosen DG,Yang G,Bast RC Jr,Liu J

更新日期：2006-01-01 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

authors： Huxford T,Ghosh G

更新日期：2006-01-01 00:00:00

abstract：:Small GTPases of the Ras and Rho families are among the most studied signaling proteins and represent promising therapeutic targets for human neoplastic disease. Despite the high level of interest in these proteins, direct analysis of most aspects of Ras protein biology in living cells has not been possible, because m...

journal_title：Methods in enzymology

authors： Westwick JK,Michnick SW

更新日期：2006-01-01 00:00:00

abstract：:The Ras-regulatory network is a loosely defined composition of numerous Ras family members and effector pathways that couple to critical cell-regulatory processes. Investigators are increasingly turning to RNAi-mediated inhibition of gene expression as an effective tool to help generate authentic portraits of Ras prot...

journal_title：Methods in enzymology

authors： Whitehurst AW,White MA

更新日期：2006-01-01 00:00:00

abstract：:Several genetically encoded fluorescent biosensors for Ras family GTPases have been developed that permit spatiotemporal analysis of the activation of these signaling molecules in living cells. We describe here the use of the simplest of these probes, the Ras binding domain (RBD) of selected effectors fused with green...

journal_title：Methods in enzymology

authors： Bivona TG,Quatela S,Philips MR

更新日期：2006-01-01 00:00:00

abstract：:Ras proteins are small GTPases that exhibit high-affinity binding to GDP and GTP and hydrolyze bound GTP to GDP. The intrinsic GTPase activity of Ras proteins is accelerated by GTPase activating proteins (GAPs), which act to attenuate GTPase signaling by accelerating the conversion of bound GTP to bound GDP. Tumor-ass...

journal_title：Methods in enzymology

authors： Shutes A,Der CJ

更新日期：2006-01-01 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

authors： Barrett T,Edgar R

更新日期：2006-01-01 00:00:00

abstract：:Powerful specialized software is essential for managing, quantifying, and ultimately deriving scientific insight from results of a microarray experiment. We have developed a suite of software applications, known as TM4, to support such gene expression studies. The suite consists of open-source tools for data managemen...

journal_title：Methods in enzymology

authors： Saeed AI,Bhagabati NK,Braisted JC,Liang W,Sharov V,Howe EA,Li J,Thiagarajan M,White JA,Quackenbush J

更新日期：2006-01-01 00:00:00

abstract：:Galectins are a family of animal lectins with affinity for beta-galactosides. By using recombinant proteins, a number of galectins have been shown to interact with cell-surface and extracellular matrix glycoconjugates through lectin-carbohydrate interactions. Through this action, they can affect a variety of cellular ...

journal_title：Methods in enzymology

authors： Hsu DK,Yang RY,Liu FT

更新日期：2006-01-01 00:00:00

abstract：:The identification of novel forms of O-linked glycosylation on epidermal growth factor and thrombospondin type 1 repeats, and their emerging functional significance, require the development of new methods for their analysis. This chapter describes detailed methods to analyze both the structure and the site of modifica...

journal_title：Methods in enzymology

authors： Nita-Lazar A,Haltiwanger RS

更新日期：2006-01-01 00:00:00

abstract：:We have developed an array-based whole-genome genotyping (WGG) assay (Infinium) using our BeadChip platform that effectively enables unlimited multiplexing and unconstrained single nucleotide polymorphism (SNP) selection. A single tube whole-genome amplification reaction is used to amplify the genome, and loci of inte...

journal_title：Methods in enzymology

authors： Gunderson KL,Steemers FJ,Ren H,Ng P,Zhou L,Tsan C,Chang W,Bullis D,Musmacker J,King C,Lebruska LL,Barker D,Oliphant A,Kuhn KM,Shen R

更新日期：2006-01-01 00:00:00

abstract：:Since its major launch into academia in the mid-1990s, spotted DNA microarray technology has expanded and matured into an important mainstream tool for genomic-scale gene expression studies across many species with many applications. Based on the principles of enzymatic nucleic acid labeling and DNA hybridization, the...

journal_title：Methods in enzymology

authors： Hager J

更新日期：2006-01-01 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

authors： Schwamborn JC,Li Y,Püschel AW

更新日期：2006-01-01 00:00:00

abstract：:The breakthrough discovery that double-stranded RNA of 21 nucleotides in length (referred to as short or small interfering RNA; siRNA) can trigger sequence-specific gene silencing in mammalian cells has led to the development of a powerful new approach to study gene function (Dillon et al., 2005; Dykxhoorn et al., 200...

journal_title：Methods in enzymology

authors： Janas J,Skowronski J,Van Aelst L

更新日期：2006-01-01 00:00:00