bluuebunny/crossref_metadata_2025_split huggingface dataset:
Yeast One‐Hybrid Screening for DNA‐Protein Interactions
Authors: Ouwerkerk Pieter B.F. , Meijer Annemarie H. 
Published: June 2004

AbstractOne‐hybrid screening in yeast is a powerful method to rapidly identify heterologous transcription factors that can interact with a specific regulatory DNA sequence of interest (the bait sequence). In this technique, the interaction between two proteins (bait and prey) is detected via in vivo reconstitution of a transcriptional activator that turns on expression of a reporter gene. Detection is based on the interaction of a transcription factor (prey) with a bait DNA sequence upstream of a reporter gene. To ensure that DNA binding results in reporter‐gene activation, cDNA expression libraries are used to produce hybrids between the prey and a strong trans‐activating domain. The advantage of cloning transcription factors or other DNA‐binding proteins via one‐hybrid screenings, compared to biochemical techniques, is that the procedure does not require specific optimization of in vitro conditions.
Yeast Vectors and Assays for Expression of Cloned Genes
Authors: Reynolds Ann , Lundblad Victoria , Dorris David , Keaveney Marie 
Published: June 2004

AbstractThis unit describes some of the most commonly used yeast vectors, as well as the cloned yeast genes that form the basis for these plasmids. Yeast vectors can be grouped into five general classes, based on their mode of replication in yeast: YIp, YRp, YCp, YEp, and YLp plasmids. With the exception of the YLp plasmids (yeast linear plasmids), all of these plasmids can be maintained in E. coli as well as in S. cerevisiae and thus are referred to as shuttle vectors. The nomenclature of different classes of yeast vectors, as well as details about their mode of replication in yeast are discussed.
Cloning Yeast Genes by Complementation
Authors: Lundblad Victoria 
Published: June 2004

AbstractThis unit presents a generalized protocol and describes the principles involved in cloning yeast genes by complementation in yeast. The protocol is presented using a hypothetical mutation of yeast, the cdc101‐1 mutation. This mutation was isolated as a cell cycle mutant and is both recessive and temperature‐sensitive for growth: it can grow relatively normally at 30oC but is unable to make a colony at 37oC. A genomic DNA clone that complements this mutation will be isolated by transforming the cdc101‐1 strain with a yeast genomic library and subsequently screening for temperature‐resistant colonies. Once isolated, two steps are necessary to prove that the insert present on the plasmid contains the wild‐type CDC101 gene. First, segregation of the complementing plasmid must result in co‐loss of both the plasmid‐borne selectable marker and the complementing phenotype, demonstrating that the observed complementation is plasmid‐specific and is not due to reversion of the
cdc101‐1 mutation. Second, it must be ruled out whether the cloned gene encodes a phenotypic suppressor of the mutation, rather than the wild‐type gene. This is done via a complementation test, which demonstrates whether or not a disruption of the cloned gene that is integrated into the genome can complement the original mutation.
Manipulation of Cloned Yeast DNA
Authors: Lundblad Victoria , Hartzog Grant , Moqtaderi Zarmik 
Published: June 2004

AbstractA major advantage of working with yeast is the ability to replace the wild‐type chromosomal copy of a gene with a mutant derivative that is constructed in vitro using a cloned copy of the gene. This techniqueunavailable in most other eukaryotesallows the phenotype of the mutation to be studied under accurate in vivo conditions, with the mutation present in single copy at its normal chromosomal location. In the first protocol, a plasmid harboring both a selectable marker and a cloned gene of interest is integrated at the chromosomal location of the cloned gene via homologous recombination (integrative trandformation). Four methods are described for constructing a mutation in vitro in a cloned gene and reintroducing this mutation at the correct chromosomal site. This allows assessment of the genetic consequences of a mutation, and is often used to determine whether or not a gene is essential (by determining if a complete gene deletion is viable). Two of these techniquesintegrative disruption and one‐step gene disruptiongenerate either insertion or deletion mutations. The third techniquetransplacementis more generally applicable: it can be used to introduce insertion or deletion mutations containing a selectable marker, but it can also be used to introduce nonselectable mutations, such as conditional lethal mutations in an essential gene. Protocols are also provided to allow creation of modified genes by one‐step integrative replacement, and also conditional alleles by a copper‐inducible double‐shutoff procedure.
Preparation of Protein Extracts from Yeast
Authors: Dunn Barbara , Wobbe C. Richard 
Published: June 2004

AbstractThree protocols are presented for preparing protein extracts; they differ primarily in the way the cells are broken. In the basic protocol, cells are enzymatically converted to spheroplasts, which are then lysed by a combination of osmotic shock and Dounce homogenization. A support protocol for isolating intact nuclei by differential centrifugation is also presented. An alternate protocol describes mechanical breakage of cells by vortexing in the presence of glass beads. In a second alternate protocol, growing cells are frozen immediately in liquid nitrogen and then lysed by grinding in an industrial‐strength blender in the presence of liquid nitrogen.
Overview of Schizosaccharomyces pombe
Authors: Forsburg Susan L. 
Published: June 2004

AbstractThe fission yeast S. pombe provides an attractive alternative system to budding yeast S. cerevisiae for studies of fundamental cell biology. Fission yeast is a particularly powerful model for studies of cell cycle, chromosome dynamics, and polarity. Other areas of study are also expanding. Conceptually similar genetic tools are available in both systems, and both organisms have completely sequenced genomes.
Growth and Manipulation of S. pombe
Authors: Forsburg Susan L. 
Published: June 2004

AbstractThis unit presents aspects specific to genetic and cytological manipulation of fission yeast, including mating type testing, crosses, preparingmaking diploids, and analysis of meiotic products, and basic methods of cell cycle synchronization and analysis. These methods are different from those used in budding yeast because they depend upon the distinct biology of S. pombe, particularly its unwillingness to be a diploid in normal growth conditions. Similarly, the different cell shape and growth behavior of S. pombe require different approaches to basic cell cycle analysis.
Introduction of DNA into S. pombe Cells
Authors: Forsburg Susan L. 
Published: June 2004

AbstractMethods of transformation rely upon conditioning cells to take up DNA, and growing them under selective conditions to establish and maintain the plasmid or integration. Different methods may be used, including electroporation, treatment with lithium cations, or protoplast treatment which removes the cell wall. Protoplasts prepared as for transformation can also be induced to fuse with each other and undergo karyogamy, which provides a means of mating sterile strains.
In Situ Hybridization to CellularRNA
Authors: Zeller Rolf , Rogers Melissa , Haramis Anna G. , Carrasceo André s E. 
Published: June 2004

AbstractIn situ hybridization to cellular RNA is used to determine the cellular localization of specific messages within complex cell populations and tissues. In this unit, protocols are described for hybridizing slide‐mounted paraffin sections or cryosections with labeled probes. Support protocols describe synthesis of 35S‐labeled riboprobes and dsDNA probes, which are then detected using film autoradiography or emulsion autoradiography. Another support protocol describes synthesis of digoxigenin‐labeled RNA probes, which are non‐radioactive and thus have several advantages. They are easily synthesized in large quantities, they are stable for several months, and they can be reused up to three times. An additional advantage of RNA versus DNA probes is that they result in cleaner signals because nonspecifically bound probe is removed during ribonuclease treatment.
Counterstaining and Mounting of Autoradiographed In Situ Hybridization Slides
Authors: Zeller Rolf , Rogers Melissa 
Published: June 2004

AbstractThe morphology of specimen sections and the identity of specific areas are defined by lightly counterstaining sections. After staining, slides are dehydrated, mounted with coverslips, hardened, and cleaned for examination under the microscope. In the protocols provided in this unit, Giemsa stains predominantly the nuclei, hematoxylin/eosin stain differentiates both the nuclei and cytoplasm, toluidine blue staining is a simpler procedure that lightly stains both nuclei and cytoplasm, and Hoechst staining of nuclei provides a fast, easy, and effective way to simultaneously view the entire tissue and the regions of hybridization.
Immunohistochemistry
Authors: Watkins Simon 
Published: June 2004

AbstractThis unit surveys immunohistochemical techniques for protein localization. The first four protocols focus on indirect immunofluorescence which is the easiest and most widely used method of optical detection. Additional protocols present methods suitable for bright‐field microscopy, such as immunoperoxidase and immunogold labeling. The commentary contains an extensive troubleshooting guide with a list of suitable routine controls. The chosen method of tissue or cellular preparation must be suitable to answer the fundamental objective of the protocol (i.e., where the protein is localized within the system). Furthermore, the choice of fluorochrome and the method of labeling are also important to optimize the sensitivity of antigen localization. The basic protocol is therefore divided into two sections: (1) cellular preparations (monolayers of adherent cells and cells grown in suspension) and (2) tissue section labeling.
Whole‐Mount In Situ Hybridization and Detection ofRNAs in Vertebrate Embryos and Isolated Organs
Authors: Pizard Anne , Haramis Anna , Carrasco Andrés E. , Franco Paula , López Silvia , Paganelli Alejandra 
Published: June 2004

AbstractNonisotopic in situ hybridization using intact embryos or organs is an important method for determining the spatial distribution of RNAs. Because it allows the analysis of large numbers of samples, it is amenable to temporal expression studies and comparison between different genotypes. It offers sensitivity and reproducibility. In addition, histological details are not lost during the staining process. The protocols in this unit can be used for whole‐mount in situ hybridization inXenopus, mouse, and chicken embryos, as well as dissected organs from mouse and chicken. Preparation of digoxigenin‐labeled riboprobes is also described.
Basic Confocal Microscopy
Authors: Smith Carolyn L. 
Published: June 2004

AbstractConfocal microscopy produces sharp images of structures within relatively thick specimens (up to several hundred microns). It is particularly useful for examining fluorescent specimens, such as fluorescent‐stained cells or intact organisms (e.g., Drosophila or zebrafish embryos), and can localize intracellular antigens in dissociated cells. Its sensitivity even allows the monitoring of fluorescence in living organisms, making it possible to follow the movements of fluorescent probes such as GFP or dyes for specific organelles. This unit presents the theory of optical sectioning, describes the instrumentation of the technique, and offers practical guidelines for sample preparation and analysis.
Direct DNA Sequencing of PCR Products
Authors: Dorit Robert L. , Ohara Osamu , Hwang Charles B‐C. , Kim Jae Bum , Blackshaw Seth 
Published: June 2004

AbstractPCR products can be sequenced using either the dideoxy (Sanger) or chemical (Maxam‐Gilbert) approaches. In the dideoxy methods presented here, the target sequence is amplified and an excess of one strand of the target sequence (relative to its complement) is then generated by “asymmetric PCR,” where one primer is present in vast excess over the other. This single‐stranded product serves as the template for conventional dideoxy sequencing methods. Another procedure prepares PCR products for use as templates fes for characterizing unlabeled product by genomic sequencing and chemical sequencing of end‐labeled products are also presented.
Enzymatic Amplification of RNA by PCR (RT‐PCR)
Authors: Beverly Stephen M. 
Published: June 2004

AbstractMethods for enzymatic amplification of RNA by the polymerase chain reaction (RT‐PCR) are highlighted in this unit. The is especially useful for rare RNAs because all steps (annealing, reverse transcription, and amplification) are performed under optimal conditions, thereby maximizing efficiency and recovery. These are also important considerations when amplifying heterogeneous RNA populations or large RNAs. Although there are many commercially available kits for this procedure, the protocols listed here are good solid protocols that will work and that have withstood the test of time.
cDNA Amplification Using One‐Sided (Anchored) PCR
Authors: Dorit Robert L. , Ohara Osamu 
Published: June 2004

AbstractThe polymerase chain reaction (PCR) offers an opportunity to directly select, amplify, and isolate a message of interest. This unit describes anchored PCR, a modification of the standard reaction that allows amplification of full‐length mRNA when only a small amount of sequence information is available. Anchored PCR can be employed when only a small region of sequence lying within the mRNA is known in advance, unlike the amplification of RNA or cDNA by conventional PCR which requires prior knowledge of the sequences flanking the region of interest to design the PCR primers.
Overview of Protein Expression in E. coli
Authors: Schendel Paul F. 
Published: June 2004

AbstractThis overview unit introduces general considerations and strategies for expressing proteinsin E. coli. E. coli has two characteristics that make it ideally suited as an expression system for many kinds of proteins: it is easy to manipulate and it grows quickly in inexpensive media. These characteristics, coupled with more than 10 years' experience with expression of foreign genes, have established E. coli as the leading host organism for most scientific applications of protein expression. Despite a growing literature describing successful protein expression from cloned genes, each new gene still presents its own unique expression problems. There is no single set of methods that can guarantee successful production of every protein in a useful form. Nevertheless, the vast body of accumulated knowledge has led to a general approach that often helps to solve specific expression problems.
Expression Using the T7 RNA Polymerase/Promoter System
Authors: Tabor Stanley 
Published: June 2004

AbstractThis unit describes the expression of genes by placing them under the control of the bacteriophage T7 RNA polymerase. T7 RNA polymerase is a very active enzyme: it synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently; in fact, its transcription can circumnavigate a plasmid, resulting in RNA several times the plasmid length in size. T7 RNA polymerase is also highly selective for initiation at its own promoter sequences and is resistant to antibiotics such as rifampicin that inhibit E. coli RNA polymerase. Consequently, the addition of rifampicin to cells that are producing T7 RNA polymerase results in the exclusive expression of genes under the control of a T7 RNA polymerase promoter (pT7). In the , two plasmids are maintained within the same E. coli cell. One (the expression vector) contains pT7 upstream of the gene to be expressed. The second contains the T7 RNA polymerase gene under the control of a heat‐inducible E. coli promoter. Upon heat induction, the T7 RNA polymerase is produced and initiates transcription on the expression vector, resulting in turn in the expression of the gene(s) under the control of pT7. If desired, the gene products can be uniquely labeled by carrying out the procedure in minimal medium, adding rifampicin to inhibit the E. coli RNA polymerase, and then labeling the proteins with [35S]methionine.
Expression Using Vectors with Phage λ Regulatory Sequences
Authors: Shatzman Allan R. , Gross Mitchell S. , Rosenberg Martin 
Published: June 2004

AbstractIn the expression system described here, plasmids (pSKF) utilize regulatory signals‐such as the powerful promoter pL ‐from the bacteriophage λ. Transcription from pL can be fully repressed and plasmids containing it are thus stabilized by the λ repressor, cI. The repressor is supplied by an E. coli host which contains a integrated copy of a portion of the λ genome. This so‐called defective lysogen supplies the λ regulatory proteins cI and N but does not provide the lytic components that would normally lead to cell lysis. Thus, cells carrying these plasmids can be grown initially to high density without expression of the cloned gene and subsequently induced to synthesize the product upon inactivation of the repressor. This system also ensures that pL‐directed transcription efficiently traverses any gene insert, which is accomplished by providing the phage λ antitermination function, N, to the cell and by including on the pL transcription unit a site necessary for N utilization (Nut site). The N protein interacts with and modifies the RNA polymerase at the Nut site so as to block transcription termination at distal sites in the transcription unit. In order to express the coding sequence, efficient ribosome‐recognition and translation‐initiation sites have been engineered into the pL transcription unit. Expression occurs after temperature or chemical induction inactivates the repressor (see first and second basic protocols). Restriction endonuclease sites for insertion of the desired gene have been introduced both upstream and downstream from an ATG initiation codon. Thus, the system allows either direct expression or indirect expression (via protein fusion) of any coding sequence, thereby potentially allowing expression of any gene insert. Protocols describe direct expression of “authentic“ gene products, as well as heterologous genes fused to highly expressed gene partners generates chimeric proteins that differ from the native form. In the latter case, the fusion partner can be removed to obtain an unfused version of the gene product.
Expression and Purification of Glutathione‐S‐Transferase Fusion Proteins
Authors: Smith Donald B. , Corcoran Lynn M. 
Published: June 2004

AbstractThis unit describes how pGEX vectors can be used in bacterial systems to express foreign polypeptides as fusions with glutathione‐S‐transferase (GST). In general, such fusion proteins are soluble and are easily purified from lysed cells under nondenaturing conditions by absorption with glutathione‐agarose beads, followed by elution in the presence of free glutathione. Potential applications of the pGEX vectors include the expression and purification of individual polypeptides (including short peptides) for use as immunogens and as biochemical and biological reagents, and in the construction of cDNA expression libraries. This protocol describes production and screening of pGEX transformants and purification of milligram quantities of fusion proteins from 1‐liter cultures. The commentary describes several modifications to the expression and purification protocol that may be useful in cases where fusion proteins are insoluble or unstable.
Expression of Proteins Using Semliki Forest Virus Vectors
Authors: Liljeström Peter , Garoff Henrik 
Published: June 2004

AbstractSemliki Forest virus (SFV) vectors have been developed to provide a convenient system to express protein‐encoding sequences in virtually any animal cell. This unit presents two strategies for protein expression using SFV vectors. In both cases the protein‐coding sequence of interest is cloned into a plasmid vector, which is subsequently used to produce recombinant RNA in vitro. This RNA, which is of positive polarity, is transfected into cells and there is amplified by virtue of its self‐encoded RNA replicase. The same replicase also produces a shorter RNA species that encodes the protein of interest. In the first protocol, cells are transfected (either by electroporation or liposome‐mediated transfection) and directly analyzed for expression of the heterologous protein. Accompanying support protocols provide methods for checking expression and transfection through galactosidase assays of transfected cells and cell lysates. The other strategy employs in vivo packaging of the RNA into SFV particles; recombinant RNA is cotransfected with a special helper RNA that codes for the structural proteins needed for virus assembly. SFV particles carrying only recombinant RNA are formed and are used to infect cells for analysis of protein expression. Accompanying support protocols describe methods for titrating and purifying recombinant virus stocks. Although the protocols presented here are designed for use with BHK (baby hamster kidney) cells, the virus has a very broad host range and can be used with many different cell types.
Special Considerations for Glycoproteins and Their Purification
Authors: Freeze Hudson H. 
Published: June 2004

AbstractThis unit begins by describing some properties of glycoproteins (e.g., subcellular location and solubility) that may be useful in determining which purification techniques to try. This discussion is followed by two protocols describing preparative glycoprotein purification using lectin‐affinity chromatography, as well as an outline for a small‐scale pilot procedure designed to check lectin binding and elution conditions. Lectins are often used for purifying glycoproteins because, in contrast to conventional purification procedures (e.g., gel filtration and ion‐exchange chromatography) that exploit general physical properties of glycoproteins, lectins recognize specific three‐dimensional structures created by a cluster of sugar residues. Conventional purification procedures are generally tried before applying lectin‐affinity chromatography.
Special Considerations for Glycolipids and Their Purification
Authors: Smith David F. , Prieto Pedro A. 
Published: June 2004

AbstractThis unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. A generalized system for inducing in vitro antibody production is presented along with a procedure for quantifying the number of antibody‐producing cells by plaque‐forming cell (PFC) assays: the Cunningham‐Szenberg technique and the Jerne‐Nordin technique. The assay can be modified as described to measure all classes of antibodies or to enumerate total immunoglobulin‐secreting B cells. A protocol for preparing the resting B cells by Percoll gradient centrifugation is also described.
Detection and Analysis of Proteins Modified by O‐Linked N‐Acetylglucosamine
Authors: Zachara Natasha E. , Hart Gerald W. , Cole Robert N. , Gao Yuan 
Published: June 2004

AbstractThe modification of Ser and Thr residues with O‐linked b‐N‐acetyl glucosamine (O‐GlcNAc) is a common and essential modification of nuclear and cytoplasmic proteins, and it is thought that O‐GlcNAc performs a regulatory role in the cell. This unit concentrates on the techniques for the detection and analysis of proteins modified by O‐GlcNAc as well as methods for the analysis of enzymes responsible for the addition and removal of this group.