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內容簡介
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.
作者簡介
The editor of Nuclease Methods and Protocols, Catherine H. Schein, PhD, obtained degrees in chemistry (U. Pennsylvania), biochemical engineering (MIT) and technical microbiology (ETH Zurich). After pioneering work in producing recombinant proteins in bacteria, including the first large (100,000 l) scale production of human interferon for clinical studies, she started investigating the role of nucleases in cytokine mechanisms. Her work on protein solubility eventually lead her to computational and structural biology. She currently coordinates projects between medical research groups in the Sealy Center for Structural Biology at one of the largest medical research centers in the United States, the University of Texas Medical Branch at Galveston
The authors of the individual chapters are from academic and industrial research groups from four different continents.
目次
Part I. Characterizing Nuclease Activity Processivity of DNA Repair Enzymes R. Stephen Lloyd
Analysis by HPLC of Distributive Activities and the Synthetic (Back) Reaction of Pancreatic-Type Ribonucleases M. Victòria Nogués and Claudi M. Cuchillo.
lre1p: A Kinase and Site-Specific Endoribonuclease Tania N. Gonzalez and Peter Walter
Microtiter-Plate Assay and Related Assays for Nonspecific Endonucleases Gregor Meiss, Oleg Gimadutdinow, Peter Friedhoff, and Alfred M. Pingoud
Quantitating mRNAs with Relative and Competitive RT-PCR, Ellen A. Prediger
Expressing Self-Incompatibility RNases (S-RNases) in Transgenic Plants Brian Beecher and Bruce A. McClure
Molecular Cloning, Tissue Distribution, and Chromosomal Localization of the Human Homolog of the R2/Th/Stylar Ribonuclease Gene Family Francesco Acquati, Cinzia Nucci, Marco G. Bianchi, Tatiana Gorletta, and Roberto Taramelli
Part II. Inhibitors and Activators of Nucleases Ribonuclease Inhibitors Brittan L. Pasloske
Producing Soluble Recombinant RNases and Assays to Measure Their Interaction with Interferon-g In Vitro Catherine H. Schein
Assays for the Evaluation of HIV-1 Integrase Inhibitors Zeger Debyser, Peter Cherepanov, Wim Pluymers, and Erik De Clercq
The Direction of Ribonucleases H by Antisense Oligodeoxynucleotides Richard V. Giles and David M. Tidd
The 2-5A/RNase L Pathway and Inhibition by RNase L Inhibitor (RLI) Catherine Bisbal, Tamim Salehzada, Michelle Silhol, Camille Martinand, Florence Le Roy, and Bernard Lebleu Part III. Relating Nuclease Structure and Function
Crystallization and Crystal Structure Determination of Ribonuclease A-Ribonuclease Inhibitor Protein Complex Bostjan Kobe
Methods for Studying the Interaction of Barnase with Its Inhibitor Barstar Gideon Schreiber
Assaying In Vitro Refolding of RNases by Mass Spectrometry Gennaro Marino, Piero Pucci, and Margherita Ruoppolo
Dissecting Nucleases into Their Structural and Functional Domains: Mapping the RNA-Binding Surface of RNase III by NMR Andres Ramos and Annalisa Pastore
Using Electrostatics to Define the Active Site of Serratia Endonuclease Kurt L. Krause and Mitchell D. Miller
Homology Modeling and Simulations of Nuclease Structures Kizhake V. Soman, Catherine H. Schein, Hongyao Zhu, and Werner Braun
Methods for Determining Activity and Specificity of DNA Binding and DNA Cleavage by Class II Restriction Endonucleases Albert Jeltsch and Alfred M. Pingoud
Engineered Properties and Assays for Human DNase I Mutants Clark Q. Pan, Dominick V. Sinicropi, and Robert A. Lazarus Part IV. Nucleases in the Clinic
Assays for Human DNase I Activity in Biological Matrices Dominick V. Sinicropi and Robert A. Lazarus
Evaluation of Retroviral Ribonuclease H Activity Jennifer T. Miller, Jason W. Rausch, and Stuart F. J. Le Grice
Assays for Detection of RNase A Superfamily Ribonucleases Helene F. Rosenberg and Joseph B. Domachowske
Assay for Antitumor and Lectin Activity in RNase Homologs Kazuo Nitta
Isolation and Enzymatic Activity of Angiogenin James F. Riordan and Robert Shapiro Preparation and Preclinical Characterization of RNase-Based Immunofusion Proteins Dianne L. Newton and Susanna M. Rybak Part V. Assays Using Nucleases Restriction Endonucleases and Their Uses Raymond J. Williams Isolation and Characterization of an Unknown Restriction Endonuclease Raymond J. Williams
Gene Modification with Hapaxoterministic Restriction Enzymes: Easing the Way, Shelby L. Berger
Techniques to Measure Nucleic Acid-Protein Binding and Specificity: Nuclear Extract Preparations, DNase I
Footprinting, and Mobility Shift Assays Richard A. Rippe, David A. Brenner, and Antonio Tugores
Analyzing the Developmental Expression of Sigma Factors with S1-Nuclease Mapping Jan Kormanec
Detection and Quantitation of mRNAs Using Ribonuclease Protection Assays Ellen A. Prediger
Index.
