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基本説明
Developed to introduce the scientists to cell-characterized protein nucleases, and the methods used to determine their activity, structure, interaction with other molecules and their physiological role.
Full Description
Nucleases, enzymes that restructure or degrade nucleic acid polymers, are vital to the control of every area of metabolism. They range from "housekeeping" enzymes with broad substrate ranges to extremely specific tools (1). Many types of nucleases are used in lab protocols, and their commercial and clinical uses are expanding. The purpose of Nuclease Methods and Protocols is to introduce the reader to some we- characterized protein nucleases, and the methods used to determine their activity, structure, interaction with other molecules, and physiological role. Each chapter begins with a mini-review on a specific nuclease or a nuclease-related theme. Although many chapters cover several topics, they were arbitrarily divided into five parts: Part I, "Characterizing Nuclease Activity," includes protocols and assays to determine general (processive, distributive) or specific mechanisms. Methods to assay nuclease products, identify cloned nucleases, and determine their physiological role are also included here. Part II, "Inhibitors and Activators of Nucleases," summarizes assays for measuring the effects of other proteins and small molecules. Many of these inhibitors have clinical relevance. Part III, "Relating Nuclease Structure and Function," provides an overview of methods to determine or model the 3-D structure of nucleases and their complexes with substrates and inhibitors. A 3-D structure can greatly aid the rational design of nucleases and inhibitors for specific purposes. Part IV, "Nucleases in the Clinic," summarizes assays and protocols suitable for use with t- sues and for nuclease based therapeutics.
Contents
Characterizing Nuclease Activity.- Processivity of DNA Repair Enzymes.- Analysis by HPLC of Distributive Activities and the Synthetic (Back) Reaction of Pancreatic-Type Ribonucleases.- Ier1 p: A Kinase and Site-Specific Endoribonuclease.- Microtiter-Plate Assay and Related Assays for Nonspecific Endonucleases.- Quantitating mRNAs with Relative and Competitive RT-PCR.- Expressing Self-Incompatibility RNases (S-RNases) in Transgenic Plants.- Molecular Cloning, Tissue Distribution, and Chromosomal Localization of the Human Homolog of the R2/Th/Stylar Ribonuclease Gene Family.- Inhibitors and Activators of Nucleases.- Ribonuclease Inhibitors.- Producing Soluble Recombinant RNases and Assays to Measure Their Interaction with Interferon-? In Vitro?.- Assays for the Evaluation of HIV-1 Integrase Inhibitors.- The Direction of Ribonucleases H by Antisense Oligodeoxynucleotides.- The 2-5A/RNase L Pathway and Inhibition by RNase L Inhibitor (RLI).- Relating Nucleas Structure and Function.- Crystallization and Crystal Structure Determination of Ribonuclease A-Ribonuclease Inhibitor Protein Complex.- Methods for Studying the Interaction of Barnase with Its Inhibitor Barstar.- Assaying In Vitro Refolding of RNases by Mass Spectrometry.- Dissecting Nucleases into Their Structural and Functional Domains.- Using Electrostatics to Define the Active Site of Serratia Endonuclease.- Homology Modeling and Simulations of Nuclease Structures.- Methods for Determining Activity and Specificity of DNA Binding and DNA Cleavage by Class II Restriction Endonucleases.- Engineered Properties and Assays for Human DNase I Mutants.- Nucleases in the Clinic.- Assays for Human DNase I Activity in Biological Matrices.- Evaluation of Retroviral Ribonuclease H Activity.- Assays for Detection of RNase ASuperfamily Ribonucleases.- Assay for Antitumor and Lectin Activity in RNase Homologs.- Isolation and Enzymatic Activity of Angiogenin.- Preparation and Preclinical Characterization of RNase-Based Immunofusion Proteins.- Assays Using Nucleases.- Restriction Endonucleases and Their Uses.- Isolation and Characterization of an Unknown Restriction Endonuclease.- Gene Modification with Hapaxoterministic Restriction Enzymes.- Techniques to Measure Nucleic Acid-Protein Binding and Specificity.- Analyzing the Developmental Expression of Sigma Factors with S1-Nuclease Mapping.- Detection and Quantitation of mRNAs Using Ribonuclease Protection Assays.
