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Ribonucleases, Part A: Functional Roles and Mechanisms of Action
1st Edition, Volume 341 - September 18, 2001
Editor: Allen W. Nicholson
Language: English
Hardback ISBN:9780121822422
9 7 8 - 0 - 1 2 - 1 8 2 2 4 2 - 2
eBook ISBN:9780080496917
9 7 8 - 0 - 0 8 - 0 4 9 6 9 1 - 7
This first of two volumes provides up-to-date, methods-related information on ribonuclease functions, assays, and applications. Chapter topics include the identification of,…Read more
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This first of two volumes provides up-to-date, methods-related information on ribonuclease functions, assays, and applications. Chapter topics include the identification of, characterization of, and assays for secreted ribonucleases; viral ribonucleases, artificial and engineered ribonucleases, and ribozymes.The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today--truly an essential publication for researchers in all fields of life sciences.
Biochemists, Molecular Biologists, Microbiologists, Cell Biologists, and Biomedical Researchers.
Contributors to volume 341
Preface
Volume in series
Section I: Ribonuclease Classification and Review
[1]: A Natural Classification of Ribonucleases
Introduction and Classification Approach
Evolutionary Classification of Ribonucleases
Functional Systems and Evolutionary History of Ribonucleases
[2]: The Ribonuclease T1 Family
Introduction
Similarities and Differences among RNase T1 Family Members
RNase Assay Method
Purification
[3]: Ribonuclease T2
Classification
Protein Structure
Mechanism of Action
Base Specificity
Physiological Roles
Enzyme Assay
Enzyme Units
Preparation of Fungal T2–like RNase
Preparation of RNase Trv from Commercial Enzyme Preparation “Cellulase T-AP” Produced from Trichoderma viride
Preparation of Oyster (Crassostrea gigas) RNase
Inhibitors
[4]: The Ribonuclease P Family
Introduction
RNase P Subunit Composition
RNase P Structure and Function
Universal Features of RNase P
Acknowledgments
Section II: Ribunucleases Assays
[5]: Fast, Facile, Hypersensitive Assays for Ribonucleolytic Activity
Assays for Ribonucleolytic Activity
Design and Synthesis of Fluorogenic Substrates
Preparation of Reagents
Assay Procedures and Data Analysis
Applications
[6]: Activity Staining for Detection of Ribonucleases Using Dried Agarose Film Overlay Method After Isoelectric Focusing
Introduction
Zymogram Method for Detection of RNase Activities after Isoelectric Focusing by Means of Dried Agarose Film Overlay Method
Materials and Analytical Methods
Specific Detection of Pancreatic-Type Ribonucleases Based on Polycytidylic Acid/Ethidium Bromide Fluorescence Following IEF
pH Gradient Electrophoresis of Basic Ribonucleases in Sealed Slab Polyacrylamide Gels Followed by DAFO Zymogram Detection
Acknowledgments
[7]: Gel Renaturation Assay for Ribonucleases
Introduction
Advantages and Limitations of Activity Gels Applied to Ribonucleases
General Guidelines for in Situ Recovery of Enzymatic Activity
Preparing, Casting (Pouring), and Running an Activity Gel
Processing of Gel for Renaturation, and in Situ Detection of Ribonuclease Activity
Interpretation of Results
[8]: Analysis of Ribonucleases following Gel Electrophoresis
Zymograms
One-Dimensional Zymograms
Notes
One-Dimensional Zymograms using p29 and RNase A
Two-Dimensional Zymograms
2D Zymogram using p29
Elution of Proteins from SDS–Polyacrylamide Gels
Recovery of p29 following SDS–PAGE
Acknowledgments
[9]: Ribonuclease Assays Utilizing Toluidine Blue Indicator Plates, Methylene Blue, or Fluorescence Correlation Spectroscopy
Introduction
Toluidine Blue O Indicator Plates
Methylene Blue Assay
FCS Assay
Acknowledgments
[10]: Ribonuclease Activities of Trypanosome RNA Editing Complex Directed to Cleave Specifically at a Chosen Site
Background
Trypanosoma brucei RNA Editing Complex
gRNA-Directed Endonuclease
gRNAs for Cleavage at Natural A6 Editing Sites
Design of Minimal Anchor “gRNA” to Cleave Heterologous RNAs
U-Specific Exonuclease
Additional Relevant Assays
Conclusion
[11]: Ribonuclease YI*, RNA Structure Studies, and Variable Single-Strand Specificities of RNases
Introduction
Materials and Methods
Results
Discussion
Section III: Secreted Ribonucleases
[12]: Bovine Pancreatic Ribonuclease A: Oxidative and Conformational Folding Studies
Introduction
Preparative Methods
Native Structure of RNase A
Equilibrium Unfolding Transitions of RNase A
Reductive Unfolding and Oxidative Folding of RNase A
Effect of Proline cis/trans-Isomers in Disulfide-Intact Folding
Conformational Folding of RNase A
Methodology of Reductive Unfolding and Oxidative Folding Studies
[13]: Purification of Engineered Human Pancreatic Ribonuclease
Introduction
Heterologous Production of HP-RNase
Obtaining High Levels of HP-RNase
Native Conformation: From Solubilization to Purification
Characterization: Analysis of Protein Homogeneity and Structural Integrity
Future Prospects and Applications of HP-RNase Engineering
Acknowledgments
[14]: Degradation of Double-Stranded RNA by Mammalian Pancreatic-Type Ribonucleases
Introduction
Properties of Double-Stranded RNA
Mechanism of Double-Stranded RNA Degradation
Influence of Ionic Strength on dsRNA Degradation by Mammalian ptRNases
Experimental Procedure
Acknowledgment
[15]: Seminal Ribonuclease: Preparation of Natural and Recombinant Enzyme, Quaternary Isoforms, Isoenzymes, Monomeric Forms; Assay for Selective Cytotoxicity of the Enzyme
Introduction
Experimental Procedures
[16]: Angiogenin
Introduction
Methods of Assay
Isolation of Angiogenin
Biological Function
[17]: Eosinophil-Derived Neurotoxin
1 Background
2 GenBank Accession Numbers for EDN
3 Recombinant EDN from Bacterial Expression Systems
4 Recombinant EDN from Baculovirus
5 Recombinant EDN from Eukaryotic Cell Culture
6 Natural Forms of EDN
7 Ribonuclease Assay
8 Biological Assays: Antiviral Activity
[18]: Eosinophil Cationic Protein
Purification of ECP
RNase Activity
ECP Biological Properties
Quantification of ECP Levels in Biological Fluids
Acknowledgments
[19]: Deciphering the Mechanism of RNase T1
Introduction
Reactivity of Scissile Bond
Chemical Nature of Transition State
Experimental Basis for In-Line Acid–Base Catalysis
Thio Substitutions to Map Catalytic Interactions with Nonbridging Oxygens
Triester-Like Mechanism Involving Internal Proton Transfer
A Three-Centered Hydrogen Bond for Concerted Phosphoryl Transfer
Nucleophile Activation by Cooperative Hydrogen Bonding
Positioning of Proper Imidazole Tautomer Contributes to Leaving Group Activation
Transition State Stabilization by Specific Solvation/Desolvation
Subsite-Binding Effects on Turnover
Perfect Match between RNase T1 and Transition State
[20]: Mitogillin and Related Fungal Ribotoxins
Introduction
Assay Methods
Overexpression and Purification of Mitogillin
Conformational Analysis of Mitogillin
Immunological Properties
Procedures for Detection of IgE Antibodies against Asp f 1
Concluding Remarks and Future Directions
Acknowledgments
[21]: RNase U2 and α-Sarcin: A Study of Relationships
Introduction
Acknowledgments
[22]: Secretory Acid Ribonucleases from Tomato, Lycopersicon esculentum Mill.
Introduction
Source of Enzymes
Assay Methods
Purification Procedures
Subcellular Localization of Intracellular Ribonucleases
Properties of Tomato Ribonucleases LE, LX, LV-1, LV-2, and LV-3
Acknowledgment
[23]: Leczyme
Introduction
Materials and Cells
Tumor Cell Agglutination and Growth Inhibition
Leczyme-Induced Apoptosis and Expression of Apoptosis-Related Antigens
Conclusion
Acknowledgments
Section IV: Ribonucleases H
[24]: Prokaryotic Type 2 RNases H
Introduction
Acknowledgment
[25]: RNase H1 of Saccharomyces cerevisiae: Methods and Nomenclature
Roles for RNases H in Cells
Solution-Based Assay
Gel-Renaturation Assay
Complementation of Temperature-Sensitive Growth Defect of rnhA Mutants of Escherichia coli
Northwestern Assay
Substrate Preparation
Northwestern Procedure
[26]: Ribonucleases H of the Budding Yeast, Saccharomyces cerevisiae
Introduction
Assays for Determining RNase H Activity
Enzyme Purification Procedures
Evolutionarily Related Proteins from Organisms Other than S. cerevisiae by Alignments of Predicted Amino Acid Sequences
Acknowledgments
[27]: Human RNases H
Introduction
Molecular Cloning and Expression of Human RNases H
Purification of Human RNase H Proteins
Preparation of Heteroduplex Substrate
RNase H1 Cleavage Assay
Determination of Binding Affinity
Conclusions
[28]: Assays for Retroviral RNase H
Assays for Retroviral RNase H
Nonspecific RNase H Assays
Specific RNase H Cleavages
Section V: Synthetic Ribonucleases
[29]: Sequence-Selective Artificial Ribonucleases
Significance of Artificial Ribonucleases
Molecular Design
Methods
Prospect
Acknowledgments
[30]: RNA Cleavage by 1,4-Diazabicyclo[2.2.2]octane–Imidazole Conjugates
Introduction
Design of RNA Cleaving Conjugates
Comparison of Ribonuclease Activities of the Designed Conjugates
Mechanism of RNA Cleavage by Imidazole Conjugates
RNA Cleavage by Conjugates ABL3Cm
Specificity of RNA Cleavage with Conjugates ABL3Cm; Comparison with RNase A
Specific Features of RNA Cleavage by Conjugates of 1,4-Diazabicyclo[2.2.2]octane and Imidazole
Materials and Methods
Structural Probing of RNA with Conjugates ABL3Cm
Analysis of Cleaved RNA and Quantitation of Data
Acknowledgment
[31]: Preparation and Use of ZFY-6 Zinc Finger Ribonuclease
Introduction
Synthesis and Purification of ZFY-6 Zinc Finger Peptide
Preparation of ZFY-6 Zinc Finger Homodimer
Formation of Homodimer
Preparation and Purification of Oligoribonucleotide Substrate
32P Labeling of Oligoribonucleotide Substrate
Cleavage Assay
pH Dependence
Conclusions
Section VI: Ribonucleolytic Nucleic Acids
[32]: RNA Cleavage by the 10-23 DNA Enzyme
Introduction
10-23 Motif
Kinetic Properties
Substrate Specificity
Target Site Selection
Delivery to Cells
Cellular Uptake
Conclusions
Acknowledgments
[33]: Leadzyme
Introduction
Active Structure Determination
Catalysis and Ions
Computational Studies
Conclusion
Acknowledgment
[34]: Hammerhead Ribozyme Structure and Function in Plant RNA Replication
Introduction
Structure of Hammerhead Ribozymes
Function of Hammerhead Ribozymes in Their Natural Habitat
In Vitro Assays to Test Hammerhead-Mediated RNA Cleavage
Conclusions and Perspectives
Acknowledgments
[35]: Kinetic Analysis of Bimolecular Hepatitis delta Ribozyme
Design of Bimolecular delta Ribozyme System
Materials and Methods
Results and Discussion
Perspectives
Conclusion
Acknowledgment
[36]: Catalytic and Structural Assays for the Hairpin Ribozyme
Introduction
Ribozyme Reaction Chemistry
Structure
Catalytic Assays
Structural Analysis
[37]: Intracellular Applications of Ribozymes
Catalytic RNAs
Target Accessibility
Intracellular Ribozyme Expression
Colocalization of Ribozyme and RNA Target
Ribozyme Delivery
Intracellular in Vivo Ribozyme Activity
Conclusions
Acknowledgments
Section VII: Ribonuclease Inhibitor
[38]: Barnase–Barstar Interaction
Introduction
Barnase–Inhibitor Equilibria
Simultaneous Randomization of Groups of Structurally Related Residues of Barstar and Selection for Activity
[39]: Cytoplasmic Ribonuclease Inhibitor
Introduction
Physicochemical Properties and Primary Structure
Specificity
Structural Basis for Tight Binding
Biological Role of RI
Isolation of RI
Stability of RI
Quantification of RI
Kinetic Characterization of RI Complexes
Acknowledgments
[40]: Small Molecule Inhibitors of RNase A and Related Enzymes
Introduction
Binding Subsites on RNase A
Pancreatic RNase Superfamily
Inhibitors of RNase A
Inhibitors of Human RNase-2, RNase-4, and Angiogenin
Practical Considerations for Use or Assay of RNase Inhibitors
Preparation and Characterization of ppA-3′-p, ppA-2′-p, pdUppA-3′-p, and pTppA-3′-p
Structures of Complexes of RNase A with Adenosine 5′-Pyrophosphate Derivatives
Acknowledgments
[41]: Ribonuclease-Resistant RNA Controls and Standards
Introduction
Ribonuclease-Resistant RNA Controls and Standards
In Vitro Transcription using Modified NTPs
Section VIII: Nonenzymatic Cleavage of RNA
[42]: Nonenzymatic Cleavage of Oligoribonucleotides
Introduction
Materials and Methods
Features of Oligoribonucleotide Cleavage
Effects of Polyamines on Cleavage of Oligoribonucleotides
Effect of Oligoribonucleotide Structure on Cleavage
Mechanism of Cleavage, and External Factors Affecting Cleavage
Stability of Biologically Active RNA Molecules
Biological Implication of Nonenzymatic RNA Cleavage
Conclusions
Author Index
Subject Index
No. of pages: 734
Language: English
Edition: 1
Volume: 341
Published: September 18, 2001
Imprint: Academic Press
Hardback ISBN: 9780121822422
eBook ISBN: 9780080496917
AN
Allen W. Nicholson
Affiliations and expertise
Wayne State University, Detroit, U.S.A.
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