Showing papers in "Methods in Enzymology in 2002"

Getting started with yeast.

Abstract: Publisher Summary The yeast Saccharomyces cerevisiae is now recognized as a model system representing a simple eukaryote whose genome can be easily manipulated. Yeast has only a slightly greater genetic complexity than bacteria and shares many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal baker's yeast are commercially available and can provide a cheap source for biochemical studies. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Cloned yeast sequences, accompanied by foreign sequences on plasmids, can therefore be directed at will to specific locations in the genome.

Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method

Abstract: In this chapter we have provided instructions for transforming yeast by a number of variations of the LiAc/SS-DNA/PEG method for a number of different applications. The rapid transformation protocol is used when small numbers of transformants are required. The high efficiency transformation protocol is used to generate large numbers of transformants or to deliver DNA constructs or oligonucleotides into the yeast cell. The large-scale transformation protocol is primarily applicable to the analysis of complex plasmid DNA libraries, such as those required for the yeast two-hybrid system. The microtiter plate versions of the rapid and high efficiency transformation protocols can be applied to high-throughput screening technologies.

Saccharomyces Genome Database.

Abstract: Publisher Summary The goal of the Saccharomyces Genome Database (SGD) is to provide information about the genome of this yeast, the genes it encodes, and their biological functions. The genome sequence of S. cerevisiae provides the structure around which information in SGD is organized; value is added to the sequence by careful biological annotation drawn from a number of sources. SGD curates and stores information about budding yeast DNA and protein sequences, genetics, cell biology, and the associated community of researchers. SGD also provides search and analysis tools designed to help researchers mine the data for pieces or patterns of biological information relevant to their interests. A continuing challenge for the staff of SGD is to present up-to-date information about yeast genes in a format that is intuitive and useful to biomedical researchers, while responding to the needs of this community by providing resources and tools for exploring the data in new ways. This chapter describes the organization of SGD, the sources of the data stored in SGD, some methods for retrieving information from the database, connections SGD has with outside databases and non-yeast research communities, and SGD's repository of yeast community information.

Redox potential of GSH/GSSG couple: assay and biological significance.

Abstract: Publisher Summary Redox-signaling mechanisms function in cell regulation and growth control. Agents altering glutathione (GSH) concentration affect the transcription of detoxification enzymes, cell proliferation, and apoptosis. GSH, GSH S-conjugates, and its disulfide form (GSSG), or the redox state of the GSH/GSSG couple provide a mechanistic control or signal for functional changes. A loss or oxidation of GSH occurs in association with differentiation both in vitro and in vivo and during apoptosis. In contrast, increases in GSH and/or a reduction of the GSH/GSSG pool is associated with growth stimulation by nutrients and growth factors. Thus, the balance of GSH and GSSG may not only reflect oxidative stress but may reflect changes in redox signaling and control. The most widely used indicator of the redox state of the GSH pool is the ratio of GSH to GSSG. The cellular balance of GSH and GSSG provides a dynamic indicator of oxidative stress. Although GSH is synthesized continuously and has various fates in cells, these rates are generally slow relative to the turnover by the oxidation-reduction cycle. The activity of the GSSG reductase is dependent on GSSG concentration. During acute oxidative stress, GSH concentration decreases and the associated increase in GSSG concentration results in an increased turnover of the GSH/GSSG cycle.

RP4-based plasmids for conjugation between Escherichia coli and members of the vibrionaceae

Abstract: Publisher Summary The Vibrionaceae family includes both important pathogens and useful model organisms in the study of processes ranging from the regulation of light production by quorum sensing to the establishment of mutualistic animal-bacteria associations. The self-transmissible broad host-range IncPα plasmid RP4 provides the means for genetic manipulation of many diverse bacteria, including members of the Vibrionaceae. Derivatives of RP4 facilitate the conjugal transfer of vectors engineered using Escherichia coli ( E. coli ) as a host into other bacterial species of interest. The new RP4-based constructs described in the chapter are at least as efficient as existing tools for mediating conjugal transfer between E. coli and members of the Vibrionaceae. The tools and methods described in the chapter are also useful for mediating conjugation between E. coli and many other gram-negative bacteria. Helper plasmid pEVS 104 may be particularly advantageous for recipients, such as the Enterobacteriaceae, that replicate ColE1- based vectors or support transposition of IS903, Mu, Tn7, or E. coli IS elements.

Measurement of absolute oxygen levels in cells and tissues using oxygen sensors and 2-nitroimidazole EF5.

Abstract: We have established basic methods, using quantitative measures of EF5 binding, to estimate the actual pO2 of cells and tissues. In situations where the tissue can be dissociated into single cells, or for cell cultures, we can measure the distribution of cellular binding rates using flow cytometry and these can be compared with cells treated under pO2S controlled by the spinner vial or thin-film methods in vitro. The flow cytometer is calibrated by staining V79 cells treated with EF5 under "standard" conditions. For intact tissues treated with EF5 in vivo, we need to correct for possible variations in drug exposure (AUC). Frozen sections are stained for EF5 binding and are analyzed by a sensitive (cooled) CCD camera with linear output vs fluorescence [figure: see text] input. The camera has very consistent sensitivity, but the entire optical system, including the camera, can be calibrated by an absolute fluorescence standard (dye in hemocytometer). This system can also be used to measure the fluorescence of the flow cytometer standards, providing a direct link between the two assays. We can measure the maximum binding rate using the tissue cube method, but need to assume an "average" oxygen dependence of binding for intact tissues. The best-fit approximation for existing data is an inverse relationship between binding and pO2, with binding decreasing 50-fold between 0.1 and 10% oxygen. Using these methods, we routinely estimate the minimum pO2 (maximum binding) in experimental rodent and human tumors. In normal tissue models, an excellent correlation is found between near-maximal binding (severe hypoxia) and apoptosis (heart infarct and ductus arteriosus). Some normal tissues (e.g., skeletal muscle) are refractory to both cellular disaggregation and cube calibration methods. To extend the tissue imaging measurements to a complete two- or three-dimensional analysis of the distribution of tissue pO2s requires a substantial additional investment of imaging methods, which are currently being implemented.

ChIP-chip: a genomic approach for identifying transcription factor binding sites.

Abstract: Publisher Summary Transcription factors are key regulatory proteins that can influence the expression of hundreds of genes in response to a particular environmental condition or internal cue The collective set of genes regulated by a transcription factor therefore defines the state of a cell and can determine cell fate Among the 6200 predicted proteins in the yeast Saccharomyces cerevisiae, there are about 300 transcription factors Approximately 85% of the yeast transcription factors have been characterized to some extent and some of these are known to play a critical role in cell cycle initiation, pheromone response, mating type switching, pseudohyphal growth, and nutrient and stress response This chapter develops an approach in yeast that will comprehensively identify genomic sequences directly bound by transcription factors This approach has been used to successfully identify targets of the yeast transcription factors SBF and MBF Briefly, protein-DNA complexes are fixed in vivo with formaldehyde and lysed, and the lysate is sonicated to shear DNA The transcription factor of interest is purified by immunoprecipitation; the associated DNA is extracted and then amplified and labeled for hybridization to a yeast intergenic array Two important reagents for this method are specific immunoprecipitating antibodies and a yeast intergenic microarray

Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods.

Abstract: The Rho GTPases are members of the Ras superfamily and include several isoforms of Cdc42, Rac, and Rho. The Rho subfamily regulates a variety of signal transduction pathways in eukaryotic cells, including cell adhesion and migration, by modulating cytoskeletal dynamics. Cdc42 induces formation of filopodia, whereas Rac regulates actin polymerization at the plasma membrane where ruffles are formed. Both induce the formation of cytoskeletal/signaling aggregates known as focal complexes. Rho controls the assembly of focal adhesions and the reorganization of actin into stress fibers. More recently, it has been recognized that Rho GTPases also initiate signaling pathways that impact on gene expression and cell growth regulation, and that each of these GTPases is essential for Ras to induce malignant transformation. A specific role for Rac has been identified in phagocytic cells, such as neutrophils or monocytes, where it activates the NADPH Oxidase enzymatic complex. This chapter describes an assay developed for Rac and Cdc42 activation using the GTPase/p21-binding region of p21-activated kinase 1 (PAK1). Similar strategies developed as assays for Rho GTP are discussed.

Three-dimensional representations of G protein-coupled receptor structures and mechanisms.

Abstract: Publisher Summary G protein-coupled receptors (GPCRs) have been grouped into five somewhat distinct families: one resembling rhodopsin, another identified with the secretin receptor, a class related to the metabotropic glutamate receptor, another to the fungal pheromone receptor, and a class of CAMP receptors The recent breakthrough in determining the crystal structure of rhodopsin has confirmed the general expectation that GPCRs are composed of seven helical transmembrane segments connected by intracellular and extracellular loop segments, as well as the expected topology of an extracellular N terminus and an intracellular C terminus This chapter describes the construction, evaluation, and use of the three-dimensional (3D) molecular models of GPCRs from the various families reflects the general current understanding of their architecture This understanding is implemented in the 3D receptor models based on a variety of direct experimental data, as well as results from various approaches from computational genomics, biophysics, and bioinformatics

Classical mutagenesis techniques

Abstract: Publisher Summary Generating mutants, to identify new genes and to study their properties, is the starting point for much of molecular biology. Forward mutations and metabolic suppressors obtained by reversion can provide powerful insights into the functions and relationships of normal gene products. Similarly, mutations and intragenic revertants provide the raw material for the analysis of gene product structure-function relationships. Site-specific mutagenesis and other methods based on recombinant DNA techniques are increasingly used for these purposes, and they are clearly the methods of choice where particular changes in specified genes or genetic sites are needed. Nevertheless, classical methods, in which cells are treated with mutagens, are likely to remain the chief means for inducing mutations in many circumstances because they require no prior knowledge of gene or product and are generally applicable: the user need to only specify an appropriate alteration in phenotype. However, unless selection for the desired strain is possible, hunting for mutants can be extremely laborious and analyzing the material obtained even more so.

Roles of Nrf2 in activation of antioxidant enzyme genes via antioxidant responsive elements.

Abstract: Publisher Summary This chapter outlines the role of Nrf2 in the activation of antioxidation enzyme via antioxidant responsive element/electrophile responsive element (ARE/EpRE). The role is studied using experiments on mice and those involving the cell culture system. The experiments suggest that kelch-like ECH-associated protein 1 (Keap 1) and Nrf2 comprise the primary sensor in the cellular response to oxidative stress. The cytoplasmic protein Keap 1 interacts with Nrf2 and inhibits its translocation to nucleus. Electrophiles and ROS disrupt the binding between Nrf2 and Keap 1, such that activated Nrf2 can translocate to the nucleus, Nrf2 forms a heterodimer with a small Maf protein and activates gene transcription via ARE/EpRE. In macrophages exposed to oxidative stress agents, Nrf2 also plays a major role in the transcriptional activation of genes encoding A170, HO-1, and xCT.

Gene expression and thiol redox state.

Abstract: Publisher Summary This chapter reviews the different practical approaches that are commonly used to analyze eukaryotic transcription factors whose activation and DNA binding are controlled by the thiol redox status of the cell. It focuses on two well-defined transcription factors: NF−κB (nuclear factor κB) and HSF1, which confer redox state-dependent inducible gene expression. The more frequent form of NF−κB is a heterodimeric complex that contains p50 and p65 polypeptides. Except for B lymphocytes, where it is constitutively active, NF−κB is cytoplasmic in most cells and needs extracellular signals to be activated and to migrate in the nucleus where it binds KB sites (5'-GGGAN/NYYCC-3') on the DNA. Heat shock factor 1 (HSF1) is a transcription factor that regulates the expression of stress (heat shock) genes. HSF1 cytoplasmic activation is mediated by stresses, such as heat shock, or numerous different agents or conditions that generate abnormally folded proteins. Transcription of heat shock (or stress) genes requires the formation of a homotrimeric HSF1 complex that binds to the heat shock promoter element (HSE). HSE is characterized as multiple adjacent and inverse iterations of the 5'-nGAAn-3' motif.

Alterations in membrane cholesterol that affect structure and function of caveolae.

Abstract: Most of the available methods for modifying caveolae structure and function depend on altering the cholesterol content of caveolae. The most important aspect of each method is to ensure the reagents are working in the cells that are being studied. The idiosyncrasies of each method are such that they cannot be universally applied without carefully optimizing the conditions. When used correctly, these methods are accepted as a specific way to perturb the structure and function of caveolae.

Nucleic-acid programmable protein arrays

Abstract: Arrays of polypeptides are generated by translation of nucleic acid sequences encoding the polypeptides at a plurality of addresses on the array.

Analysis of prion factors in yeast.

Abstract: Publisher Summary This chapter presents an analysis of prion factors in yeast. Prions are unique proteins that can adopt two or more distinctly different conformational states in vivo . The chapter reviews the principal techniques used for genetic, cell biological, and biochemical characterization of yeast prions. It focuses on [ PSI +]; however, [ URE3 ] and composite prions are also discussed for comparison. Many approaches are available to identify and study prion proteins in yeast. These include (1) approaches based on phenotypic changes, such as nonsense suppression or growth, (2) approaches based on genetic criteria, such as non-Mendelian (cytoplasmic) inheritance, curing by certain stress-inducing agents and by chaperone alterations, and induction de novo by the overproduction of the protein determinant, and (3) approaches based on biochemical criteria, such as aggregation and proteinase resistance in vivo and in vitro . Because no approach is sufficient to establish prion-like behavior, a collection of various approaches should be used.

Quorum-sensing system of Agrobacterium plasmids: Analysis and utility

Abstract: Publisher Summary This chapter presents strategies and protocols for studying the quorum-sensing system used by Agrobacterium. tumefaciens to regulate conjugal transfer of its Ti plasmids from one bacterium to another as well as the applications of these protocols to quorum-sensing systems of other bacteria. The Ti plasmids code for the uptake and catabolism of the opines produced by the crown gall tumors. These plasmids code for two independent transfer systems; one, called vir, is responsible for processing and transfer of the T-region from the bacterium to host plant cells. The quorum-sensing system of the Ti plasmid conjugal transfer apparatus, as well as the components of this system, has provided broadly applicable models and tools for the study of this gene regulatory paradigm in other bacteria. The chapter describes the physiological, genetic, biochemical, and structural studies of TraR and the target genes it regulates by quorum sensing have contributed to our understanding of the nature of these systems as well as the molecular mechanisms by which acylhomoserine lactone signals are translated to gene regulation through LuxR-like transcription factors. The chapter describes a number of these tools and assays. In each case the chapter presents a brief rationale for the assay or technique, and then outlines in general form the method. It indicates the general applicability of, and, where appropriate, the strengths, limitations, and pitfalls of each test.

Cloning-free genome alterations in Saccharomyces cerevisiae using adaptamer-mediated PCR.

Abstract: Summary Each of the adaptamer-directed genome manipulation methods is predicated on the fact that recombination between two DNAs is enhanced by increasing the length of homology. Many of the current PCR-based genome manipulation techniques rely on very short homologies to promote recombination. In these cases homology length is dictated by the technical limits of oligonucleotide synthesis. Adaptamers circumvent this problem since long homology regions are produced in a first round of PCR, and then fused to the selectable marker in a second round of PCR via complementary sequence tags on the adaptamers. Furthermore, many of the techniques described here rely on preexisting and commercially available adaptamer sets that can be obtained inexpensively rather than designing new primers for every experiment. Although a cost is incurred when performing multiple PCR amplifications, the increase in recombination efficiency is dramatic. Finally, the adaptamer-mediated PCR fusion methodology is versatile and can be applied to varied genome manipulations.

Reporter transgenes for study of oxidant stress in Caenorhabditis elegans.

Abstract: Publisher Summary For many studies of the effects of oxidant stress on cells, it can be advantageous to visualize the transcriptional response of the cell in vivo in real time. In optically transparent model systems, gene expression can be directly visualized by the construction of reporter transgenes expressing green fluorescent protein (GFP), as originally demonstrated by Chalfie and colleagues. This chapter describes both the general considerations involved in the construction of GFP reporter transgenes responsive to oxidative stress and the specific details of constructing a representative transgenic reporter in the model nematode worm Caenorhabditis elegans. Although the details of the representative reporter transgene apply specifically to C. elegans , the general approach should be applicable to many model systems. For genetic model systems such as C. elegans , oxidative stress-dependent induction of GFP expression represents a phenotype that can be the basis of forward genetic screens. These screens can potentially identify genes involved in the induction of oxidative stress response genes or genes that can be mutated to preemptively block oxidative stress in the first place.

Identification of cysteine sulfenic acid in AhpC of alkyl hydroperoxide reductase.

Abstract: C165S AhpC in its sulfenate (Cys-SO-) and presumed thiolate (Cys-S-) forms at pH 7 (pKa for sulfenic acid about pH 6.1) exhibit low extinction absorbance bands around 367 and 324 nm, respectively. Sulfenic acid content of the protein can be assessed by its reactivity with the chromophoric TNB anion. Using this technique, H2O2 titrations of C165S AhpC give a maximum of about 1 SOH per subunit on addition of 1.0 to 1.2 equivalents of H2O2. Cys46-SO- is moderately air stable at neutral pH and room temperature and is oxidized at a steady rate of about 10% per half hour. Cys46-SO- of C165S AhpC is reduced in the presence of catalytic amounts of AhpF by approximately 1 equivalent of NADH to regenerate the Cys46-S- species. NBD chloride is extremely useful as a trapping agent for cysteine sulfenic acid. The Cys46-S(O)-NBD adduct absorbs maximally at 347 nm and is 16 amu larger than the Cys46-S-NBD adduct (lambda max = 420 nm) as shown by ESI-MS. Other electrophilic thiol reagents also react with Cys46-SO-; however, iodoacetamide and N-ethylmaleimide reactivities are much lower with Cys46-SO- than with Cys46-S-. These methods are applicable to other sulfenic acid-containing proteins, although in some cases the proteins must be denatured in order to provide accessibility of this species toward labeling agents.