bluuebunny/crossref_metadata_2025 huggingface dataset:
Probing RNA Structure and Metal‐Binding Sites Using Terbium(III) Footprinting
Authors: Harris Dinari A. , Walter Nils G.
Published: June 2004

The function of an RNA molecule is determined by its overall secondary and tertiary structure. The tertiary structure is facilitated and stabilized by the interaction with metal ions. The current chapter offers a detailed protocol on the use of the lanthanide metal ion terbium(III) as a powerful probe of RNA structure and metal‐binding properties. When incubating RNA with low (micromolar) concentrations of terbium(III), specific backbone scission by partially deprotonated aqueous terbium(III) complexes can be used to detect high‐affinity metal‐binding sites, while incubation with high (millimolar) terbium(III) concentrations cleaves the RNA backbone preferentially at structurally accessible regions, providing a footprint of the RNA secondary and tertiary structure.
Calorimetry of Nucleic Acids
Authors: Pilch Daniel S.
Published: June 2004

This unit describes the application of differential scanning and isothermal titration calorimetry (DSC and ITC) to the study of the thermodynamics of nucleic acid structure. DSC is used to study order‐disorder transitions. A single DSC profile provides a wealth of thermodynamic and extrathermodynamic information: transition enthalpy, entropy, free energy, heat capacity, the state of the transition (two‐state vs. multistate), and the size of the cooperative unit. ITC is used to study hybridization of nucleic acids at constant temperature. Results can be used to determine the stoichiometry of the association reaction, the enthalpy of association, the equilibrium association constant, and the free energy and entropy of association. A thorough discussion is presented of the details required to obtain meaningful results, as well as relevant methods for analyzing the data produced.
Characterization of DNA Structures by Circular Dichroism
Authors: Bishop G. Reid , Chaires Jonathan B.
Published: June 2004

Circular dichroism (CD) of nucleic acids is commonly used to provide a signature for a given secondary structure. CD is particularly powerful for monitoring structural changes resulting from changes in environmental conditions such as temperature, ionic strength, and pH. A number of commercially available instruments are available for the measurement of CD spectra. Since the details of instrumental operation differ for each machine, this unit provides a descriptive guide, rather than a strict protocol. It describes considerations that the experimentalist will face when attempting to examine nucleic acid structures by CD spectropolarimetry. Specific details are also given for cell filling and cleaning, and for instrumental calibration. Finally, several examples of CD spectra are given to provide useful reference points for a variety of nucleic acid structures, ranging from single‐stranded to triplex and quadruplex forms.
In Vitro Selection Using Modified or Unnatural Nucleotides
Authors: Knudsen Scott M. , Robertson Michael P. , Ellington Andrew D.
Published: June 2004

The use of modified nucleotides in an RNA or DNA pool to be used for in vitro selection offers many potential advantages, such as the increased stability of the selected nucleic acid against nuclease degradation. This unit provides useful information and protocols for in vitro selection using modified nucleotides. It includes a discussion of when to use modified nucleotides; protocols for preparing a modified RNA pool and verifying its suitability for in vitro selection; and protocols for selecting and amplifying a functionally enriched pool.
Polyacrylamide Gel Electrophoresis (PAGE) of Synthetic Nucleic Acids
Authors: Andrus Alex , Kuimelis Robert G.
Published: June 2004

Protocols are given for analysis of oligonucleotides by PAGE, using either methylene blue staining or radiolabeling to mark the oligonucleotide. In addition, a separate protocol is provided for purification by PAGE.
Analysis and Purification of Synthetic Nucleic Acids Using HPLC
Authors: Andrus Alex , Kuimelis Robert G.
Published: June 2004

HPLC is a powerful and popular method for analyzing and purifying biomolecules. Reversed‐phase HPLC allows a high‐capacity method for purification, and uses volatile buffer systems that simplify product recovery. Anion‐exchange HPLC provides better resolution and a more predictable elution pattern. This unit presents protocols that are optimized for HPLC of oligonucleotides. Because of the resolution limits of both reversed‐phase and anion‐exchange HPLC, it can be used for oligonucleotides of up to ˜50 nt in length.
Cartridge Methods for Oligonucleotide Purification
Authors: Andrus Alex , Kuimelis Robert G.
Published: June 2004

Protocols are given for purification of oligonucleotides by dimethoxytrityl‐sensitive and affinity desalting methods. The protocols are applicable for many of the convenient disposable products available for rapid oligonucleotide purification, clean‐up by selective adsorption, and elution on solid‐phase media. Many of these products are prepackaged, single‐use cartridges or columns filled with affinity or size‐exclusion media.
Denaturing Polyacrylamide Gel Electrophoresis
Authors: Albright Lisa M. , Slatko Barton E.
Published: June 2004

Thin polyacrylamide gels that contain a high concentration of urea as a denaturant are capable of resolving short (<500 nucleotides) single‐stranded fragments of DNA or RNA that differ in length by as little as one nucleotide. Such gels are uniquely suited for nucleic acid sequence analysis, which is required, for instance, for all footprinting protocols. Thicker gels are often used to purify oligonucleotides. This appendix describes the pouring, running, and processing of a typical sequencing gel, which is 40 cm long with a uniform thickness of 0.4 mm, containing 7 M urea and 4% to 8% acrylamide.
Column Chromatography
Authors: Meyers C.L.F.
Published: June 2004

Column chromatography is used extensively in nucleic acid chemistry to purify or characterize products from chemical reactions. This appendix focuses on separations using silica gel or alumina. The procedures described include loading the column, assembling the apparatus, separating compounds and collecting fractions, and optimizing chromatography conditions.
Imaging Dural Sinus Thrombosis
Authors: Midyett F. Allan , Mukherji Suresh , Fisher Laurie
Published: June 2004

The acute onset of irreversible neurological deficit is referred to as a “stroke” and is the third leading cause of death and the major cause of adult long‐term disability in the United States. Obstruction of the venous sinuses by thrombosis accounts for ˜1% of strokes. Thrombosis of the superior saggital sinus is most frequent, with involvement of transverse, sigmoid, and cavernous sinuses occurring less often. Venous thrombosis may involve the dural sinuses, the deep venous system, and the superficial cortical veins separately or in any combination. This unit describes a for imaging dural sinuses by time‐of‐flight magnetic resonance venography (MRV).
Pituitary
Authors: Hallam Danial K.
Published: June 2004

Benefiting from superior tissue contrast, multi‐planar capability and lack of bone artifact, MRI readily depicts complex anatomy in and about the pituitary gland. This unit presents three basic protocols for common indications relating to pathology of the sella and parasellar region. The protocols differ in emphasis more than in concept, and share a basic theme of thin slice high‐resolution imaging including the use of gadolinium. With the possible exception of dynamic imaging, all protocols may be readily performed on any MR scanner.
Paradigms for Clinical fMRI
Authors: Thulborn Keith R. , Davis Denise
Published: June 2004

This unit presents description of four basic clinical paradigms for clinical fMRI of language cmprehension, eye movement (visually guidede saccades), motor cortex (finger‐thumb apposition), and visual cortex. Given the neurosurgical concern with preservation of eloquent cortex, brain functions of particular interest are primary sensory (e.g., visual, auditory) and motor functions, and high level processing of language comprehension and expression. Compromise of these functions is usually readily apparent clinically and the quality of life of the patient is severely diminished. Functional MRI offers a means to locate these functions, thereby allowing the surgeon to plan on preserving these functions, or prepare the patient appropriately.
Cranial Nerve I
Authors: Evers Robert W. , Yousem David M.
Published: June 2004

This unit presents the basic protocol for imaging cranial nerve I. The olfactory bulbs and tracts mediate the sense of smell from the nasal cavity to the brain. Unfortunately they are located in a precarious position for MR imaging, above the air‐filled nasal cavity and ethmoid sinuses at a bone‐air‐soft tissue interface. This creates problems with susceptibility artifact. This issue, plus the very small size of the structures to be studied and the superimposed eye motion artifact makes imaging of the olfactory system a technical challenge.
Cranial Nerves III to VI
Authors: Evers Robert W. , Yousem David M.
Published: June 2004

Cranial nerves III, IV, V, and VI are small structures that travel in a reproducible manner from the midbrain and pons to the cavernous sinus and then to the orbit. While there are branches that course through other foramina of the skull, the emphasis in MRI is to evaluate the brainstem, the cavernous sinus, and the pericavernous regions for pathology. This unit present a basic protocol for imaging cranial nerves III to VI. Because the nerves run from a posterior to an anterior position, coronal scanning is ideal for visualizing the nerves in cross‐section. Thin sections and contrast enhancement are required to best visualize the diseases that affect these nerves. An alternate protocol is also discussed for the case when demyelinating etiologies for the cranial nerve deficits are considered.
Spinal Primary Neoplasia/Metastasis
Authors: Jinkins J. Randy , Stark David D.
Published: June 2004

This unit presents a basic protocol for conventional and fast spin echo imaging of spine for visualization of the spinal compartment to assess the extent and degree of spinal cord or cauda equina compression frequently associated with primary and metastatic neoplasia. An alternate protocol is presented for the cases e.g., neurofibromatosis with multiple bilateral neoplasms extending through the neural foramina, where a coronal acquisition may be helpful to analyze the perispinal tissues for tumor extension either inward to, or outward from, the central spinal canal.
Spinal Trauma
Authors: Jinkins J. Randy , Stark David D.
Published: June 2004

This unit presents a basic protocol for conventional and fast spin echo imaging of spine for detecting spinal trauma. MR demonstrates traumatic change quite well within the spinal cord and epidural tissues. An alternate protocol is presented based on contrast enhanced acquisitions where MRI scan that has findings that do not match the clinical findings.
Myocardial Perfusion and Viability
Authors: Jerosch‐Herold Michael , Stillman Arthur E.
Published: June 2004

Both first‐pass perfusion studies and imaging of delayed hyperenhancement can be combined in one patient exam. The MRI protocols for myocardial perfusion and viability assessment are presented together in this unit due to the complimentary information that is obtained with both protocols. Also, the imaging techniques used for both protocols are closely related. The first protocol assesses the functional severity of coronary artery lesions, while the second, used in addition to first basic protocol, is used in patient presents with symptoms of myocardial infarction or post‐coronary revascularization. The procedures are useful in determining the presence and extent of nonviable myocardium.
Nulling Signal of a Slice, Slice Select Profile, and Radiofrequency Power
Authors: Cheng Yu‐Chung Norman , Haacke E. Mark
Published: June 2004

This unit discusses nulling of remaining signal from a RF pulse prior to the next RF pulse. This is to avoid mixing leftover signal with the signal generated from the next RF pulse. The slice select profile is also discussed in detail. The power of the RF pulse and its limit imposed by the Food and Drug Administration (FDA) is also discussed.Normal.
Short‐TR, Spoiled, Gradient Echo Imaging
Authors: Norman Cheng Yu‐Chung , Haacke E. Mark
Published: June 2004

When a spin system is repeatedly disturbed by a fast repetition of RF pulses, the transverse magnetization after each new RF pulse approaches a steady‐state value which is smaller than the thermal equilibrium value. The spin system takes a finite number of pulses before this steady‐state is reached in a time that depends on both the T1 of the tissue and the flip angle of the RF pulse. Sequences utilizing a steady‐state approach can be broadly classified as steady‐state incoherent (SSI) and steady‐state coherent (SSC) sequences. The main difference between the two lies in whether the transverse magnetization is forced to zero or allowed to go naturally to steady‐state between successive RF pulses. As the nomenclature suggests, the SSI sequences are based on the elimination, or the “spoiling,” of any remnant transverse magnetization prior to the occurrence of each new RF pulse. This unit discusses the SSI sequences in detail.
Signal and Noise
Authors: Celik Azim , Lin Weili
Published: June 2004

Signal and Noise (Azim Celik and Weili Lin, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina). This unit discusses the basic signal detection technique in magnetic resonance imaging based on quadrature (i.e., measured in two orthogonal channels) technique. The detected signal is demodulated with respect to the Larmor frequency and digitized. The measured MR signal also includes noise. The noise in MR generally derives from the random fluctuations in the receive coil electronics and from the sample. Even though there are other sources of signal fluctuations such as digitization noise and pseudo‐random ghosting due to moving spins, these sources are minimized in an ideal experiment. The effect of these noises in MRI image reconstruction is explained in this unit.
Signal‐to‐Noise Ratio as a Function of Imaging Parameters
Authors: Celik Azim , Lin Weili
Published: June 2004

Signal‐to‐Noise Ratio as a Function of Imaging Parameters (Azim Celik, General Electric Company, Milwaukee, Wisconsin and Weili Lin, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina). The degree to which noise affects a measurement is generally characterized by the signal‐to‐noise ratio (SNR, as measured by the ratio of the voxel signal to the noise standard deviation). This unit describes the importance of SNR in describing image quality. SNR is the key parameter for determining the quality of any given imaging experiment. If the SNR is not high enough, it becomes impossible to differentiate tissues from one another or the background. The dependence of SNR on imaging parameters such as the number of repetitions, the number of k‐space samples (Nx, Ny, and Nz), the readout bandwidth, and voxel dimensions (Dx, Dy, and Dz) is explained in detail.
Fixation, Embedding, and Sectioning of Tissues, Embryos, and Single Cells
Authors: Zeller Rolf
Published: June 2004

This unit describes selected methods for fixing and sectioning various forms of biological materials ranging from tissues to single cells. Sections prepared according to these protocols can then be used to examine cell and tissue morphology and in studies involving in situ hybridization, immunohistochemistry, and enzyme histochemistry. The basic protocol describes how tissues and embryos can be prepared for sectioning by fixing in paraformaldehyde followed by embedding in wax, while the alternate protocol describes fixation of suspended or cultured cells. Two support protocols cover preserving and fixing organs by perfusion of whole animals with paraformaldehyde and a procedure for sectioning wax blocks of fixed tissue, plus the subsequent mounting of sections onto prepared or “subbed” glass slides.
Minipreps of Plasmid DNA
Authors: Engebrecht JoAnne , Brent Roger , Kaderbhai Mustak A.
Published: June 2004

Although there are a large number of protocols for the isolation of small quantities of plasmid DNA from bacterial cells (minipreps), this unit presents four procedures based on their speed and success: the alkaline lysis prep, a modification of the alkaline lysis prep that is performed in 1.5‐ml tubes or 96‐well microtiter dishes, the boiling method, and a lithium‐based procedure. A support protocol provides information on storing plasmid DNA.
Plating Lambda Phage to Generate Plaques
Authors: Lech Karen , Brent Roger
Published: June 2004

This unit provides detailed protocols for isolating a single plaque by titering serial dilutions or streaking on a lawn of cells. A discussion of phage transfection and in vitro packaging is also included.
Preparing and Using M13‐Derived Vectors
Authors: Greenstein David , Besmond Claude
Published: June 2004

Many vectors in current use are derived from filamentous phages. These vectors are used because DNA inserted into them can be recovered in two forms: double‐stranded circles and single‐stranded circles. This overview unit describes the lifecycle of filamentous phages along with the development and use of filamentous phage vectors.
Preparation of Genomic DNA from Plant Tissue
Authors: Richards Eric , Reichardt Mark , Rogers Sharon
Published: June 2004

This unit describes two methods for preparing genomic DNA from plant tissue. In the first method, plant cells are lysed with ionic detergent, treated with protease, and subsequently purified by cesium chloride (CsCl) density gradient centrifugation. The second method is based upon a series of treatments with the nonionic detergent cetyltrimethylammonium bromide (CTAB) to lyse cells and purify nucleic acid. Nucleic acid is recovered from the final CTAB solution by isopropanol or ethanol precipitation. The first method, although somewhat more lengthy, results in highly purified nucleic acid. The second method requires fewer manipulations, results in very high yields (˜10‐fold higher per gram fresh tissue depending on species and condition of starting material), and produces DNA that is less pure but nonetheless suitable in quality for use in many molecular biology manipulations.
DNA‐Dependent RNA Polymerases
Authors: Tabor Stanley
Published: June 2004

There are two types of DNA‐dependent RNA polymerases commonly used for nucleic acid research: E. coli RNA polymerase and the bacteriophage T7, T3, and SP6 RNA polymerases. This unit describes the uses and limitations of each class of RNA polymerase along with specific reaction conditions and applications.
Constructing Recombinant DNA Molecules by the Polymerase Chain Reaction
Authors: Elion Elaine A.
Published: June 2004

This unit describes the use of PCR to construct hybrid DNA molecules. The main objective is to give an overview of how PCR can be exploited to accomplish numerous cloning strategies; it is assumed that the reader is already familiar with basic molecular biology techniques including PCR amplification and subcloning. The outlines the PCR amplification and cloning strategies. A troubleshooting guide for problems most frequently encountered in PCR cloning, and three specific examples of this technique‐‐for creating (1) in‐frame fusion proteins, (2) recombinant DNA products, and (3) deletions and inversions by inverse PCR‐‐are also presented.
Amplification of Cosmid and Plasmid Libraries
Authors: Weis John H.
Published: June 2004

The purpose of amplification is to provide a reagent library that can be used many times. In this protocol, cacteria containing the recombinant clones are grown on agar plates, washed off the plates, and stored in glycerol. An amplified cosmid or plasmid library will contain one library's equivalence in 10 to 100 µl. By producing 100 µl of such a library frozen in 100 1‐ml tubes, over 1000 platings can be expected.
Recombination‐Based Assay (RBA) for Screening Bacteriophage Lambda Libraries
Authors: Kurnit David M.
Published: June 2004

The recombination‐based assay represents a convenient way to screen a complex library constructed in bacteriophage l for homology to a given sequence cloned into a specially designed plasmid. The technique serves to screen a bacteriophage library rapidly and efficiently with a sequence cloned into a plasmid; counterselection then yields the gene product of interest with its plasmid carrier deleted. Because 106 to 107 plaque‐forming units (pfu) may be screened using several petri dishes, and the homology for crossing‐over need only be greater than 25 bp, the RBA represents an efficient way to screen complex l libraries rapidly for homology to a given sequence. In this procedure, a l library is screened using a specially designed plasmid carrying the desired target sequence. Recombinants arising from cross‐over events between the plasmid and a bacteriophage carrying a corresponding region of homology are selected by their ability to grow on strain DM21. Growth of l on DM21 requires the presence of an allele encoded on the plasmid to suppress an amber mutation in the host strain that prevents l propagation. Recovery of the original phage carrying the target sequence requires a reversal of the homologous recombination event. This reversal occurs spontaneously, and is detected by PCR amplification using primers that flank the cloning site in the l vector.
Transfection Using DEAE‐Dextran
Authors: Gulick Tod
Published: June 2004

Transfection of cultured mammalian cells using diethylaminoethyl (DEAE)‐dextran/DNA can be an attractive alternative to other transfection methods in many circumstances. The major advantages of the technique are its relative simplicity and speed, limited expense, and remarkably reproducible interexperimental and intraexperimental transfection efficiency. Disadvantages include inhibition of cell growth and induction of heterogeneous morphological changes in cells. Furthermore, the concentration of serum in the culture medium must be transiently reduced during the transfection. In general, DEAE‐dextran DNA transfection is ideal for transient transfections with promoter/reporter plasmids in analyses of promoter and enhancer functions, and is suitable for overexpression of recombinant protein in transient transfections or for generation of stable cell lines using vectors designed to exist in the cell as episomes. This unit presents a general description of DEAE‐dextran transfection, as well as two more specific protocols for typical experimental applications. The is suitable for transfection of anchorage‐dependent (attached) cells. For cells that grow in suspension, electroporation or lipofection is usually preferred, although DEAE‐dextran‐mediated transfection can be used.
Selection of Transfected Mammalian Cells
Authors: Mortensen Richard , Chesnut Jonathan D. , Hoeffler James P. , Kingston Robert E.
Published: June 2004

Analysis of gene function frequently requires the formation of mammalian cell lines that contain the studied gene in a stably integrated form. Approximately one in 104 cells in a transfection will stably integrate DNA (the efficiency can vary depending on the cell type). Therefore, a dominant, selectable marker is used to permit isolation of stable transfectants. In the first part of this unit, the procedure for determining selection conditions and the resulting stable transfection is presented and the most commonly used selectable markers are discussed. The second protocol includes conditions for thirteen markers commonly used for selection of mammalian cells. A third protocols describes selection of transfected cells from the total population soon after transfection with plasmids that express both the gene of interest and a selection tag. Optimization of transfection conditions can be facilitated by a simple staining assay detailed in a .
Use of the A. Victoria Green Fluorescent Protein to Study Protein Dynamics in Vivo
Authors: Kahana Jason A. , Silver Pam A.
Published: June 2004

Fluorescent molecules serve as valuable tools for the detection of a variety of biochemical phenomena. Such reagents have been employed for protein localization, quantitation of gene expression, detection of nucleic acids, cell sorting, and determination of chemical concentrations. Although fluorescence is a useful tool for detecting molecules within cells, its application in vivo has heretofore been limited. The ideal vital fluorescent tag should (1) be detectable without causing cytological damage, (2) be able to label a wide variety of cell types readily, and (3) be able to be targeted to virtually any subcellular region. The recently cloned green fluorescent protein (GFP) from the jellyfish Aequorea victoria is such a molecule. This overview describes the use of this proteinaceous fluorophore for in vivo observation of cellular phenomena, including applications and problems with the use of GFP, a discussion of mutant GFPs with altered fluorescence characteristics, and also some details on microscopy requirements.
Spectrophotometric and Colorimetric Determination of Protein Concentration
Authors: Simonian Michael H. , Smith John A.
Published: June 2004

This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measurement at 280 nm is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein. An alternate protocol uses absorbance at 205 nm to calculate the protein concentration. Both methods can be used to quantitate total protein in crude lysates and purified or partially purified protein. Use of a spectrofluorometer or a filter fluorometer to measure the intrinsic fluorescence emission of a sample solution is also described. The measurement is compared with the emissions from standard solutions to determine the concentration of purified protein. The Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to an unknown protein, is also presented, as is the Lowry method, which measures colorimetric reaction of tyrosyl residues in an unknown.
Quantitative Amino Acid Analysis
Authors: Dunn Ben
Published: June 2004

This overview unit describes the information that can be derived from quantitative amino acid analysis, presents some details on sample preparation, and gives examples of the calculations that are needed.
Freezing and Recovery of Hybridoma Cell Lines
Authors: Fuller Steven A. , Takahashi Miyoko , Hurrell John G.R.
Published: June 2004

Liquid nitrogen storage is the method of choice for long‐term safekeeping of hybridoma cell lines. Frozen aliquots of originally isolated hybridomas provide insurance against loss of antibody production and vigor during culture. There are many variations of cell freezing methods in use. However, for freezing and recovering hybridomas and lymphoid cells in general, the protocol described in this unit is simple and has been successful.
Preparation of Nuclear and Cytoplasmic Extracts from Mammalian Cells
Authors: Abmayr Susan M. , Workman Jerry L.
Published: June 2004

Extracts prepared from the isolated nuclei of cultured cells are functional in accurate in vitro transcription and mRNA processing. Thus, such extracts can be used directly for functional studies and as the starting material for purification of the proteins involved in these processes. In this unit, reproducible extracts from different preparations of nuclei are generated. The conditions described are optimized to produce transcriptionally active extracts from HeLa cells. A support protocol describes how to optimize the basic procedure to increase the yield of specific proteins or activities from different cell types, and a second support protocol describes the preparation of the cytoplasmic (S‐100) fraction.
Analysis of DNA‐Protein Interactions Using Proteins Synthesized In Vitro from Cloned Genes
Authors: Struhl Kevin
Published: June 2004

To detect DNA binding activity, radiolabeled protein is incubated with specific DNA fragments, and protein‐DNA complexes are separated from free protein by electrophoresis in native acrylamide gels. Unlike the more conventional mobility shift assay which utilizes
32P‐labeled DNA and unlabeled protein, the assay described here generally utilizes 35S‐labeled protein and unlabeled DNA. Major advantages of this method are that any desired mutant protein can be tested for its DNA‐binding properties simply by altering the DNA template, and the subunit structure (e.g., dimer, tetramer) can be determined.
Preparation of Yeast Media
Authors: Treco Douglas A. , Lundblad Victoria
Published: June 2004

Preparation of sterile media of consistently high quality is essential for the genetic manipulation of yeast. Recipes for media needed in the protocols in this chapter are provided in this unit. Specific suppliers are recommended for specific ingredients.
Yeast Cloning Vectors and Genes
Authors: Lundblad Victoria
Published: June 2004

This 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.
In situ Polymerase Chain Reaction and Hybridization to Detect Low‐Abundance Nucleic Acid Targets
Authors: Bagasra Omar , Seshamma Thikkavarapu , Pomerantz Roger , Hanson John
Published: June 2004

This unit presents a novel approach for detecting low‐abundance nucleic acid targets in nuclear and cytoplasmic regions by amplification of specific target sequences using an in situ polymerase chain reaction (ISPCR). If the target sequence is RNA, ISPCR is preceded by in situ reverse transcription. Following ISPCR, in situ hybridization is performed. An describes a variant in situ method for simultaneously reverse transcribing and amplifying RNA transcripts using recombinant Thermos thermophilos (rTth) polymerase. In situ hybridization and detection of amplified targets is also described.
Overview of Image Analysis, Image Importing, and Image Processing using Freeware
Authors: Bearer E.L.
Published: June 2004

Quantitative image analysis turns microscopic data that might otherwise be merely descriptive into reliable mechanistic information. This unit provides an overview of the types of analysis that have been useful in the past, as well as descriptions of emerging applications. In addition, two protocols are included for importing data and for the first steps of data manipulation in the more common analytical applications of the freeware, NIH Image and ImageJ, as well as commercially available imaging software, Adobe Photoshop.
Ligation‐Mediated PCR for Genomic Sequencing and Footprinting
Authors: Mueller Paul R. , Wold Barbara , Garrity Paul A.
Published: June 2004

This unit describes how PCR can be used to exponentially amplify segments of DNA located between two specified primer hybridization sites. A single‐sided PCR method is used that initially requires specification of only one primer hybridization site; the second is defined by the ligation‐based addition of a unique DNA linker. This linker, together with the flanking gene‐specific primer, allows exponential amplification of any fragment of DNA. Because a defined, discrete‐length sequence is added to every fragment, complex populations of DNA such as sequence ladders can be amplified intact with retention of single‐base resolution. The ligation‐based protocol was specifically designed for genomic footprinting and direct sequencing reactions, and is described in this context; it can, however, be used for other applications.
Molecular Cloning of PCR Products
Authors: Finney Michael , Nisson Paul E. , Rashtchian Ayoub
Published: June 2004

It is often desirable to clone PCR products to establish a permanent source of cloned DNA for hybridization studies, to obtain high‐quality DNA sequencing results, or to separate products when PCR amplification yields a complex mixture. The efficiency of direct cloning of PCR products can be improved by generating suitable ends on the amplified fragments. This unit describes the strategies for generating and manipulating suitable ends on the PCR fragments.
Quantitation of Rare DNAs by PCR
Authors: Coen Donald M.
Published: June 2004

This unit presents a protocol that uses the polymerase chain reaction (PCR) to quantitate the numbers of a particular DNA sequence, from 1 to 20,000 molecules per sample. In addition, it helps assess the presence of contaminating sequences, which can seriously affect the outcome of the procedure.
Expression and Purification of Thioredoxin Fusion Proteins
Authors: McCoy John , LaVallie Edward
Published: June 2004

This unit describes a gene fusion expression system that uses thioredoxin, the product of the Escherichia coli trxA gene, as the fusion partner. The system is particularly useful for high‐level production of soluble fusion proteins in the E. coli cytoplasm; in many cases heterologous proteins produced as thioredoxin fusion proteins are correctly folded and display full biological activity. Protein fusions to His‐patch Trx can usually be purified in a single step from cell lysates. Additional protocols describe E. coli cell lysis using a French pressure cell and fractionation, osmotic release of thioredoxin fusion proteins from the E. coli cytoplasm, and heat treatment to purify some thioredoxin fusion proteins.
Expression and Purification of Epitope‐Tagged Multisubunit Protein Complexes from Mammalian Cells
Authors: Wu Shwu‐Yuan , Chiang Cheng‐Ming
Published: June 2004

Biochemical characterization and functional studies of mammalian proteins are often hampered by the availability of the purified protein, in particular, when the functional entity is present as a multisubunit protein complex in the cell. To overcome the difficulties in the purification of multisubunit protein complexes from mammalian cells, one may create stable cell lines containing epitope‐tagged protein. The first protocol in this unit describes the procedures involved in the establishment of a stable cell line constitutively expressing the FLAG‐tagged protein by retrovirus‐mediated gene transfer and immunoaffinity purification of the epitope‐tagged multisubunit protein complex. The next protocol outlines the steps involved in the establishment of an inducible cell line conditionally expressing the FLAG‐tagged protein by a tetracycline‐regulated system, and the one‐step immunoaffinity purification of the multisubunit protein complex. An alternate protocol provides an excellent example for the purification of different forms of human RNA polymerase II complexes, achieved simply by choosing the appropriate starting material and by varying wash conditions. The isolation of various human TATA‐binding protein (TBP)‐containing complexes is described in a support protocol, and is a good example of combining the P11 column and immunoaffinity purification. These protocols, collectively, illustrate a powerful methodology in applying epitope tagging and stable cell line approaches for the purification of multisubunit protein complexes from mammalian cells.
Metabolic Radiolabeling of Animal Cell Glycoconjugates
Authors: Diaz Sandra , Varki Ajit
Published: June 2004

This unit describes metabolic labeling techniques that provide specific information about the structure, sequence, and distribution of the sugar chains of glycoconjugates. Although these techniques provide less information than complete sequencing of the sugar chains, the partial structural information derived is sufficient for many purposes. In the basic procedure presented in this unit, actively growing cell cultures are grown through several population doublings in complete medium supplemented with radiolabeled glycoconjugate precursors to reach a steady‐state level of incorporation. In alternate protocols, cells are cultured for a short period of time in a deficient medium that contains a high concentration of radiolabeled precursor. A pulse or pulse‐chase labeling procedure can be used to analyze precursor‐product relationships. With sequential pulse‐labeling, it is possible to obtain quantities of labeled glycoconjugates with the use of a minimal amount of labeled precursor by using the same medium to pulse‐label a series of cultures. A support protocol describes the preparation of multiply deficient medium (MDM) for use in making appropriate deficient media.
Inhibition of N‐Linked Glycosylation
Authors: Powell Leland D.
Published: June 2004

Treatment of cells with inhibitors of the enzymes that synthesize N‐linked oligosaccharide chains results in production of glycoproteins containing missing or altered chains. This approach is useful for examining potential functional role(s) of this class of oligosaccharides on specific proteins or intact cells. This unit describes the use of inhibitors to prevent N‐linked glycosylation of proteins in cultured cells. First, the optimal concentration of inhibitor for the experiment (i.e., highest nontoxic concentration) is determined by monitoring [35S]methionine incorporation as a measure of protein biosynthesis. The inhibitor's ability to inhibit oligosaccharide processing is then determined by analyzing cells labeled with [HH]mannose using TCA precipitation or endo H digestion (UNIT 13). Further suggestions are given on how to use methods for identifying a specific glycoprotein (if available) to measure the effect of the inhibitor on its N‐linked oligosaccharide chains. A support protocol details a method for concentrating proteins by acetone precipitation.
Synthetic Glycosides as Primers of Oligosaccharide Biosynthesis and Inhibitors of Glycoprotein and Proteoglycan Assembly
Authors: Esko Jeffrey D. , Montgomery Rebecca I.
Published: June 2004

With the exception of hyaluronic acid, all mammalian saccharides assemble while attached to a lipid or protein primer. Several cases are now known in which oligosaccharide synthesis will occur on synthetic glycoside primers added to cells. A protocol is described in this unit in which b‐D‐xylosides initiate glycosaminoglycan (GAG) synthesis by substituting for endogenous xylosylated core proteins. At high concentration xylosides will also prime oligosaccharides that resemble glycolipids. N‐acetyl‐a‐D‐galactosaminides initiate the synthesis of O‐linked oligosaccharides found on mucins and other glycoproteins in an analogous manner. Even disaccharides, such as peracetylated N‐acetyllactosaminide, can act as primers. Because these primers compete with endogenous substrates, they also act as inhibitors of proteoglycan (PG) and glycoprotein synthesis. Thus, primers have utility for studying the biological activity of glycoconjugates in cells, tissues, and animals. This unit describes procedures for using glycoside primers in cell culture.
Total Compositional Analysis by High‐Performance Liquid Chromatography or Gas‐Liquid Chromatography
Authors: Manzi Adriana E.
Published: June 2004

Once the presence of carbohydrate in a glycoprotein has been confirmed, the next step is to determine the precise molar ratio of its monosaccharide constituents. The analysis involves two major phases. The release of the individual monosaccharides is achieved by methanolysis (described here), total acid hydrolysis, or enzymatic release of sialic acids. The resulting mixtures of monosaccharides are then analyzed by methods described in this unit: fractionation, characterization, and quantitation by high‐performance liquid chromatography using anion‐exchange chromatography with pulsed amperometric detection (HPAEC‐PAD) and other HPLC systems or by gas‐liquid chromatography with flame ionization detection. The identity of the individual monosaccharides is determined by comparison with known standards processed and analyzed in the same way.
Internet Basics for Biologists
Authors: Baxevanis Andreas D. , Ouellette B.F. Francis
Published: June 2004

Before embarking on any practical discussion of computational methods in solving biological problems, it is necessary to lay the common groundwork that will enable users to both access and implement the algorithms and tools discussed in this book. This unit begins with a review of the Internet and its terminology, and also discusses major classes of Internet protocols, without becoming overly engaged in the engineering minutiae underlying these protocols. This unit also discusses matters of connectivity, ranging from simple modem connections to digital subscriber lines (DSL). Finally, one of the most common problems that has arisen with the proliferation of Web pages throughout the world is addressed, i.e., finding useful information on the World Wide Web.
Affinity Purification of Proteins Binding to GST Fusion Proteins
Authors: Swaffield Jonathan C. , Johnston Stephen Albert
Published: June 2004

This unit describes the use of proteins fused to glutathione‐S‐transferase (GST fusion proteins) to affinity purify other proteins, a technique also known as GST pulldown purification. The describes a strategy in which a GST fusion protein is bound to agarose affinity beads and the complex is then used to assay the binding of a specific test protein that has been labeled with [35S]methionine by in vitro translation. However, this method can be adapted for use with other types of fusion proteins; for example, His6, biotin tags, or maltose‐binding protein fusions (MBP), and these may offer particular advantages. A describes preparation of an E. coli extract that is added to the reaction mixture with purified test protein to reduce nonspecific binding.
Two‐Hybrid Dual Bait System
Authors: Kotova Elena , Serebriiskii Ilya , Coleman Thomas
Published: June 2004

The yeast two‐hybrid system, or interaction trap, is one of the most versatile methods available with which to identify and establish protein‐protein interactions. The dual bait system is one adaptation of the classic approach. This system facilitates the simultaneous comparison of two distinct baits with one prey. One protein of interest is expressed as a fusion to the DNA binding protein LexA (bait 1), while a second protein of interest is expressed as a fusion to the DNA binding protein cI (bait 2). Strains of yeast engineered for screening of these dual baits possess four separate reporter genes. A plasmid expressing an activation domain‐fused protein (prey), which can be either a defined protein interactor or a cDNA library, is also expressed to allow dual hybrid‐mediated transcriptional activation.
Gene Silencing by RNAi in Mammalian Cells
Authors: John Matthias , Geick Anke , Hadwiger Philipp , Vornlocher Hans‐Peter , Heidenreich Olaf
Published: June 2004

This unit provides information how to use short interfering RNA (siRNA) for sequence specific gene silencing in mammalian cells. Several ways for siRNA generation and optimisation, as well as recommendations for cell transfection are presented.
Safe Use of Radioisotopes
Authors: Meisenhelder Jill , Semba Kentaro
Published: June 2004

This appendix presents useful basic information, including common abbreviations, useful measurements and data, characteristics of amino acids and nucleic acids, information on radioactivity and the safe use of radioisotopes and other hazardous chemicals, conversions for centrifuges and rotors, characteristics of common detergents, and common conversion factors.
Parenteral Injections
Authors: Donovan John , Brown Patricia
Published: June 2004

This unit describes the techniques for the following routes of injection for mice, rats, hamsters and rabbits: intramuscular, intradermal, subcutaneous, intravenous, intraperitoneal, footpad, and intrathymic. Guidelines are also given regarding injection volumes and temperatures, and the use of proper restraints.
Removal of Lymphoid Organs
Authors: Reeves J.P. , Reeves P.A.
Published: June 2004

Although for most experiments lymphoid tissue is removed from the freshly sacrificed animal, there are several circumstances which require the surgical removal, under anesthesia, of either the thymus or spleen, and the recovery of the animal for further investigation. This unit describes survival surgery for removal of the adult spleen as well as the adult and neonatal thymus of the mouse.
Assessment of Lymphocyte Development in Radiation Bone Marrow Chimeras
Authors: Spangrude Gerald J.
Published: June 2004

The utilizes congenic strains of mice that differ at the CD45 (common leukocyte antigen) locus. Bone marrow‐derived cells from these strains of mice can be identified by monoclonal antibodies specific for the two allelic variants of the CD45 molecule. Because this antigen is expressed on all nucleated blood cells, it provides a convenient genetic marker for following the progeny of transplanted bone marrow cells. Transplantation of marrow between mouse strains congenic for CD45 after lethal irradiation establishes hematopoiesis driven by genetically marked cells in recipient animals. The first support protocol can be used to establish appropriate radiation doses for use in the . After several weeks, peripheral blood or primary and secondary lymphoid organs of transplant recipients can be evaluated for the presence of donor‐derived cells using the second support protocol. Two‐ or three‐color flow cytometry can be used to identify the progeny of transplanted cells, to document their cell‐surface phenotypes, and to follow development of T, B, and myeloid lineages in vivo.
Measurement of Intracellular Ions by Flow Cytometry
Authors: June Carl H. , Abe Ryo , Rabinovitch Peter S.
Published: June 2004

The recent development of a number of new fluorescent probes makes it possible to measure the concentrations of various intracellular free ions in single living cells. Among these ions are calcium, magnesium, sodium, potassium, and hydrogen (pH). This unit describes flow cytometric protocols using the dyes Indo‐1 AM, Fluo‐3, and Fura Red AM to measure intracellular calcium concentration. Support protocols detail the use of calcium buffers to calibrate a flow cytometric calcium assay, and methods to facilitate dye loading; an alternate protocol describes the use of a spectrofluorimeter to measure intracellular calcium for those investigators without access to a flow cytometer.
Measurement of Intercellular Conjugates by Flow Cytometry
Authors: Segal David M.
Published: June 2004

Analysis of fluorescently labeled cellular aggregates can be accomplished quickly and accurately with the technique of flow cytometry. With a dual‐laser flow cytometer, the excitation and emission spectra of fluorophores used to label cells are widely separated, and there is no spillover of emission from one fluorophore into the detector measuring emission from the other. Consequently, conjugates can be measured by a dual‐laser instrument when the extent of labeling two cell types with fluorophores are very different. The more commonly available single‐laser cytometers can also be used to measure multicellular conjugates, but due to overlaps in emission spectra, the extent of labeling cells with fluorophores must be controlled much more carefully when the single‐laser machines are used. This unit describes the labeling of cells and analysis of conjugates with either dual‐laser or single laser flow cytometers.