RNA Turnover in Bacteria, Archaea and Organelles

METHODS IN ENZYMOLOGY Editors-in-Chief JOHN N. ABELSON AND MELVIN I. SIMON Division of Biology California Institute of...

Author: Lynne E. Maquat | Cecilia Arraiano

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METHODS IN ENZYMOLOGY Editors-in-Chief

JOHN N. ABELSON AND MELVIN I. SIMON Division of Biology California Institute of Technology Pasadena, California, USA Founding Editors

SIDNEY P. COLOWICK AND NATHAN O. KAPLAN

Academic Press is an imprint of Elsevier 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK Copyright # 2008, Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (www.copyright.com), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2008 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0076-6879/2008 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, E-mail: permissions@elsevier. com. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Support & Contact’’ then ‘‘Copyright and Permission’’ and then “Obtaining Permissions.”

For information on all Elsevier Academic Press publications visit our Web site at elsevierdirect.com ISBN-13: 978-0-12-374377-0 PRINTED IN THE UNITED STATES OF AMERICA 08 09 10 11 9 8 7 6 5 4 3 2 1

CONTRIBUTORS

Hiroji Aiba Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya, Japan Soraya Aı¨t-Bara Laboratoire de Microbiologie et Geńe´tique Molećulaire, UMR 5100, Centre National de la Recherche Scientifique et Universite´ Paul Sabatier, Toulouse, France Mońica Amblar Unidad de Investigacioń Biome´dica, Instituto de Salud Carlos III (Campus de Majadahonda), Madrid, Spain Cecı´lia Maria Arraiano Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Oeiras, Portugal Ana Barbas Instituto de Tecnologia Quı´mica e Biolo´gica, Universidade Nova de Lisboa, Oeiras, Portugal David H. Bechhofer Mount Sinai School of Medicine of New York University, Department of Pharmacology and Systems Therapeutics, New York, USA Joel G. Belasco Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Microbiology, New York University School of Medicine, New York, USA Lionel Beńard CNRS UPR 9073 (affiliated with Universite´ de Paris 7; Denis Diderot), Institut de Biologie Physico-Chimique, Paris, France Jonathan A. Bernstein Department of Pediatrics, Stanford University, Stanford, CA 94305, USA Sandrine Boisset INSERM U851, Centre National de Re´fe´rence des Staphylocoques, Universite´ de Lyon I, Lyon, France

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Contributors

Douglas J. Briant Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada Agamemnon J. Carpousis Laboratoire de Microbiologie et Geńe´tique Molećulaire (LMGM), Centre National de la Recherche Scientifique (CNRS) and Universite´ Paul Sabatier, Toulouse, France Helena Celesnik Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Microbiology, New York University School of Medicine, New York, USA Cle´ment Chevalier Architecture et Reáctivite´ de l’ARN, CNRS, Universite´ de Strasbourg, Institut de Biologie Molećulaire et Cellulaire, Strasbourg, France Mikkel Christensen-Dalsgaard Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle, Newcastle, United Kingdom Ciarań Condon CNRS UPR 9073 (affiliated with Universite´ de Paris 7; Denis Diderot), Institut de Biologie Physico-Chimique, Paris, France Elena Conti Max-Planck Institute of Biochemistry, Martinsried, Germany Glen A. Coburn Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada Atilio Deana Kimmel Center for Biology and Medicine at the Skirball Institute and Department of Microbiology, New York University School of Medicine, New York, USA Hili Giladi Department of Genetics, University of Georgia, Athens, Georgia, USA Gianni Deho` Dipartimento di Scienze biomolecolari e Biotecnologie, Universita` degli Studi di Milano, Milan, Italy Gintaras Deikus Mount Sinai School of Medicine of New York University, Department of Pharmacology and Systems Therapeutics, New York, USA Murray P. Deutscher Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida, USA

Contributors

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Andre´ Dietrich Institut de Biologie Molećulaire des Plantes, Centre National de la Recherche Scientifique, Unite´ Propre de Recherche 2357, Universite´ de Strasbourg, France Marc Dreyfus Ećole Normale Supe´rieure, Laboratoire de Geńe´tique Molećulaire, Paris, France, and Centre National de la Recherche Scientifique (CNRS), Paris, France Daniel Karzai Dulebohn Department of Biochemistry and Cell Biology and Center for Infectious Diseases of Stony Brook University, Stony Brook, New York, USA Elena Evguenieva-Hackenberg Institut fu¨r Mikrobiologie und Molekularbiologie, University of Giessen, Giessen, Germany Pierre Fechter Architecture et Reáctivite´ de l’ARN, Universite´ de Strasbourg, CNRS, IBMC, Strasbourg, France Dominique Gagliardi Institut de Biologie Molećulaire des Plantes, Centre National de la Recherche Scientifique, Unite´ Propre de Recherche 2357, Universite´ de Strasbourg, France Thomas Geissmann Architecture et Reáctivite´ de l’ARN, Universite´ de Strasbourg, CNRS, IBMC, Strasbourg, France Zhiyun Ge Department of Biochemistry and Cell Biology and Center for Infectious Diseases of Stony Brook University, Stony Brook, New York, USA Kenn Gerdes Institute for Cell and Molecular Biosciences, Medical School, University of Newcastle, Newcastle, United Kingdom Pawel Golik Department of Genetics and Biotechnology, Warsaw University, Warsaw, Poland, and Institute of Biochemistry and Biophysics PAS, Warsaw, Poland Eliane Hajnsdorf CNRS UPR9073; Institut de Biologie Physico-Chimique; Universite´ Paris Diderot, Paris7, Paris, France Janet S. Hankins Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada

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Contributors

Narumi Hino-Shigi Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan Sarah Holec Institut de Biologie Molećulaire des Plantes, Centre National de la Recherche Scientifique, Unite´ Propre de Recherche 2357, Universite´ de Strasbourg, France Eric Huntzinger Max Planck Institute for Developmental Biology, Tu¨bingen, Germany Robert Jedrzejczak Synchrotron Radiation Research Section, MCL, National Cancer Institute, Argonne National Puchta Laboratory, Argonne, Illinois, USA Stefanie S. Jourdan Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom A. Wali Karzai Department of Biochemistry and Cell Biology and Center for Infectious Diseases of Stony Brook University, Stony Brook, New York, USA Vanessa Khemici Laboratoire de Microbiologie et Geńe´tique Molećulaire (LMGM), Centre National de la Recherche Scientifique (CNRS) and Universite´ Paul Sabatier, Toulouse, France Louise Kime Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom Gabriele Klug Institut fu¨r Mikrobiologie und Molekularbiologie, University of Giessen, Giessen, Germany Sidney R. Kushner Department of Genetics, University of Georgia, Athens, Georgia, USA Heike Lange Institut de Biologie Molećulaire des Plantes, Centre National de la Recherche Scientifique, Unite´ Propre de Recherche 2357, Universite´ de Strasbourg, France Pei-Hsun Lin Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan Sue Lin-Chao Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan

Contributors

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Zhongwei Li Department of Biomedical Science, Florida Atlantic University, Boca Raton, Florida, USA Esben Lorentzen Birkbeck College London, Institute of Structural Molecular Biology, London, United Kingdom George A. Mackie Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada Kimika Maki Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya, Japan Michal Malecki Department of Genetics and Biotechnology, Warsaw University, Warsaw, Poland Valerie F. Maples Department of Genetics, University of Georgia, Athens, Georgia , USA Nathalie Mathy CNRS, Institut de Biologie Physico-Chimique, Paris, France Pierluigi Mauri Istituto di Tecnologie Biomediche, Consiglio Nazionale delle Ricerche, Segrate (Milan), Italy Kenneth J. McDowall Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom Wenzhao Meng Departments of Chemistry, Temple University, Philadelphia, Pennsylvania, USA Xin Miao Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada Bijoy K. Mohanty Department of Genetics, University of Georgia, Athens, Georgia, USA Teppei Morita Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya, Japan Asuteka Nagao Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan

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Contributors

Lilian Nathania Departments of Chemistry, Temple University, Philadelphia, Pennsylvania, USA Rhonda H. Nicholson Departments of Biology, Temple University, Philadelphia, Pennsylvania, USA Allen W. Nicholson Departments of Biology and Chemistry, Temple University, Philadelphia, Pennsylvania, USA Irina A. Oussenko Mount Sinai School of Medicine of New York University, Department of Pharmacology and Systems Therapeutics, New York, USA Martin Overgaard Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark Olivier Pellegrini CNRS UPR 9073 (affiliated with Universite´ de Paris 7; Denis Diderot), Institut de Biologie Physico-Chimique, Paris, France Alexandre V. Pertzev Departments of Chemistry, Temple University, Philadelphia, Pennsylvania, USA Leonora Poljak Laboratoire de Microbiologie et Geńe´tique Molećulaire (LMGM), Centre National de la Recherche Scientifique (CNRS) and Universite´ Paul Sabatier, Toulouse, France Victoria Portnoy Department of Biology Technion, Israel Institute of Technology, Haifa, Israel Florence Proux Ećole Normale Supe´rieure, Laboratoire de Geńe´tique Molećulaire, Paris, France, and Centre National de la Recherche Scientifigure (CNRS), Paris, France Annie Prud’homme-Genereux Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada Olga Puchta Department of Genetics and Biotechnology, Warsaw University, Warsaw, Poland, and Institute of Biochemistry and Biophysics PAS, Warsaw, Poland Yulia Redko CNRS UPR 9073 (affiliated with Universite´ de Paris 7; Denis Diderot), Institut de Biologie Physico-Chimique, Paris, France

Contributors

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Philippe Re´gnier CNRS UPR9073; Institut de Biologie Physico-Chimique; Universite´ Paris Diderot, Paris7, Paris, France Jamie Richards Department of Biochemistry and Cell Biology and Center for Infectious Diseases of Stony Brook University, Stony Brook, New York, USA Pascale Romby Architecture et Reáctivite´ de l’ARN, Universite´ de Strasbourg, CNRS, IBMC, Strasbourg, France Gadi Schuster Department of Biology Technion, Israel Institute of Technology, Haifa, Israel Dharam Singh Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan Shimyn Slomovic Department of Biology Technion, Israel Institute of Technology, Haifa, Israel Piotr P. Stepien Department of Genetics and Biotechnology, Warsaw University, Warsaw, Poland, and Institute of Biochemistry and Biophysics PAS, Warsaw, Poland Leigh M. Stickney Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada Thomas Sundermeier Department of Biochemistry and Cell Biology and Center for Infectious Diseases of Stony Brook University, Stony Brook, New York, USA Tsutomu Suzuki Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan François Vandenesch INSERM U851, Centre National de Re´fe´rence des Staphylocoques, Universite´ de Lyon I, Lyon, France Steffen Wagner Institut fu¨r Mikrobiologie und Molekularbiologie, University of Giessen, Giessen, Germany Kristoffer Skovbo Winther Institute for Cell and Molecular Biosciences, The Medical School, University of Newcastle, Newcastle, United Kingdom

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Contributors

Mieko Yagi Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya, Japan Shiyi Yao Mount Sinai School of Medicine of New York University, Department of Pharmacology and Systems Therapeutics, New York, USA

PREFACE

The cellular cleavage of RNA has remarkably diverse biologic consequences. With growing recognition that RNA turnover has a profound impact on gene expression and that rates of RNA decay can be modulated in response to environmental and developmental signals, significant progress has been made toward explaining cleavage mechanisms and how such mechanisms are regulated. In view of research developments over the past 20 years, it is surprising that a Methods in Enzymology on RNA turnover is not a well-worn tome on our bookshelves. To fill the void, this volume, which addresses RNA turnover in bacteria, archaea, and organelles, and two companion volumes (Volumes 448 and 449), which focus on RNA turnover in eukaryotes, are hoped to serve both as useful references for specialists in the field and as a helpful guides for the broader community of research scientists wishing to initiate studies of RNA decay. Fundamental principles govern RNA decay and how to study it. By incorporating this information for a diverse array of organisms, we hope to stimulate the cross-fertilization of concepts and techniques. A wide range of methods and reagents are presented, often in the context of answering important biologic questions. The authors have offered detailed rationalizations for and descriptions of their work, endeavoring to ensure that important technical points are made clearly. We wish to express our appreciation to the authors for their thoughtful contributions and willingness to share expertise. Most chapters provide not only methodologic recipes but also short reviews that place the methods in a proper biologic framework, taking into account the interplay between RNA turnover and other cellular processes. Some degree of overlap between chapters on related topics is unavoidable. This allows each chapter to be read and understood as an independent unit while permitting the expression of diverse viewpoints on alternate methods. Chapters are written to emphasize the characterization of RNA elements, protein factors, and cellular processes that mediate and/or regulate RNA turnover. RNA elements, including primary sequences and, frequently, higher-order structures, can influence the decay functions of a variety of protein factors such as exoribonucleases, endoribonucleases, helicases, and other types of RNA-binding proteins, all of which often exist in multiprotein complexes. Cellular processes having the potential to affect RNA turnover xxiii

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include transcription, translation, and polyadenylation. Many experimental approaches for analyzing RNA decay are presented, each generally focusing on a specific organism and a particular class of RNA such as mRNA, a stable RNA, or a type of small noncoding RNA. Techniques presented include isolating total-cell or organelle RNA by different methods, determining RNA half-lives, establishing in vitro and in vivo RNA degradation assays, mapping full-length RNAs and decay intermediates, and purifying and characterizing ribonucleases and RNA binding proteins. Global approaches are also discussed, such as genomic analyses of RNA decay with DNA microarrays and proteomic analyses of the RNA degradosome with two-dimensional chromatography coupled to tandem mass spectrometry. The knowledge collected makes clear how far our understanding of RNA degradation has come in the past few years and how much about this important regulatory process remains to be discovered. This volume should be of lasting value in providing techniques and tools for studying RNA turnover mechanisms. Applications of this knowledge to medicine and biotechnology are underway. It is our hope that this volume will reflect the excitement currently held by RNA turnover specialists and will serve as a source of inspiration for scientists entering this rapidly moving and important field. Lynne E. Maquat and Cecilia M. Arraiano

METHODS IN ENZYMOLOGY

VOLUME I. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME II. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME III. Preparation and Assay of Substrates Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME IV. Special Techniques for the Enzymologist Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME V. Preparation and Assay of Enzymes Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VI. Preparation and Assay of Enzymes (Continued) Preparation and Assay of Substrates Special Techniques Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VII. Cumulative Subject Index Edited by SIDNEY P. COLOWICK AND NATHAN O. KAPLAN VOLUME VIII. Complex Carbohydrates Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG VOLUME IX. Carbohydrate Metabolism Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation Edited by RONALD W. ESTABROOK AND MAYNARD E. PULLMAN VOLUME XI. Enzyme Structure Edited by C. H. W. HIRS VOLUME XII. Nucleic Acids (Parts A and B) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XIII. Citric Acid Cycle Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids Edited by J. M. LOWENSTEIN VOLUME XV. Steroids and Terpenoids Edited by RAYMOND B. CLAYTON xxv

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VOLUME XVI. Fast Reactions Edited by KENNETH KUSTIN VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B) Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME XIX. Proteolytic Enzymes Edited by GERTRUDE E. PERLMANN AND LASZLO LORAND VOLUME XX. Nucleic Acids and Protein Synthesis (Part C) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXI. Nucleic Acids (Part D) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques Edited by WILLIAM B. JAKOBY VOLUME XXIII. Photosynthesis (Part A) Edited by ANTHONY SAN PIETRO VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B) Edited by ANTHONY SAN PIETRO VOLUME XXV. Enzyme Structure (Part B) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVI. Enzyme Structure (Part C) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVII. Enzyme Structure (Part D) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVIII. Complex Carbohydrates (Part B) Edited by VICTOR GINSBURG VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXXI. Biomembranes (Part A) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXII. Biomembranes (Part B) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B) Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK


VOLUME XXXV. Lipids (Part B) Edited by JOHN M. LOWENSTEIN VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides) Edited by JOEL G. HARDMAN AND BERT W. O’MALLEY VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by JOEL G. HARDMAN AND BERT W. O’MALLEY VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function) Edited by BERT W. O’MALLEY AND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B) Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C) Edited by W. A. WOOD VOLUME XLIII. Antibiotics Edited by JOHN H. HASH VOLUME XLIV. Immobilized Enzymes Edited by KLAUS MOSBACH VOLUME XLV. Proteolytic Enzymes (Part B) Edited by LASZLO LORAND VOLUME XLVI. Affinity Labeling Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XLVII. Enzyme Structure (Part E) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLVIII. Enzyme Structure (Part F) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLIX. Enzyme Structure (Part G) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME L. Complex Carbohydrates (Part C) Edited by VICTOR GINSBURG VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism Edited by PATRICIA A. HOFFEE AND MARY ELLEN JONES VOLUME LII. Biomembranes (Part C: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER

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VOLUME LIII. Biomembranes (Part D: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIV. Biomembranes (Part E: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LV. Biomembranes (Part F: Bioenergetics) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVI. Biomembranes (Part G: Bioenergetics) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVII. Bioluminescence and Chemiluminescence Edited by MARLENE A. DELUCA VOLUME LVIII. Cell Culture Edited by WILLIAM B. JAKOBY AND IRA PASTAN VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME LX. Nucleic Acids and Protein Synthesis (Part H) Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME 61. Enzyme Structure (Part H) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 62. Vitamins and Coenzymes (Part D) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods) Edited by DANIEL L. PURICH VOLUME 64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Complex Enzyme Systems) Edited by DANIEL L. PURICH VOLUME 65. Nucleic Acids (Part I) Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME 66. Vitamins and Coenzymes (Part E) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 67. Vitamins and Coenzymes (Part F) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 68. Recombinant DNA Edited by RAY WU VOLUME 69. Photosynthesis and Nitrogen Fixation (Part C) Edited by ANTHONY SAN PIETRO VOLUME 70. Immunochemical Techniques (Part A) Edited by HELEN VAN VUNAKIS AND JOHN J. LANGONE


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VOLUME 71. Lipids (Part C) Edited by JOHN M. LOWENSTEIN VOLUME 72. Lipids (Part D) Edited by JOHN M. LOWENSTEIN VOLUME 73. Immunochemical Techniques (Part B) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 74. Immunochemical Techniques (Part C) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 75. Cumulative Subject Index Volumes XXXI, XXXII, XXXIV–LX Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 76. Hemoglobins Edited by ERALDO ANTONINI, LUIGI ROSSI-BERNARDI, AND EMILIA CHIANCONE VOLUME 77. Detoxication and Drug Metabolism Edited by WILLIAM B. JAKOBY VOLUME 78. Interferons (Part A) Edited by SIDNEY PESTKA VOLUME 79. Interferons (Part B) Edited by SIDNEY PESTKA VOLUME 80. Proteolytic Enzymes (Part C) Edited by LASZLO LORAND VOLUME 81. Biomembranes (Part H: Visual Pigments and Purple Membranes, I) Edited by LESTER PACKER VOLUME 82. Structural and Contractile Proteins (Part A: Extracellular Matrix) Edited by LEON W. CUNNINGHAM AND DIXIE W. FREDERIKSEN VOLUME 83. Complex Carbohydrates (Part D) Edited by VICTOR GINSBURG VOLUME 84. Immunochemical Techniques (Part D: Selected Immunoassays) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 85. Structural and Contractile Proteins (Part B: The Contractile Apparatus and the Cytoskeleton) Edited by DIXIE W. FREDERIKSEN AND LEON W. CUNNINGHAM VOLUME 86. Prostaglandins and Arachidonate Metabolites Edited by WILLIAM E. M. LANDS AND WILLIAM L. SMITH VOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereo-chemistry, and Rate Studies) Edited by DANIEL L. PURICH VOLUME 88. Biomembranes (Part I: Visual Pigments and Purple Membranes, II) Edited by LESTER PACKER

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VOLUME 89. Carbohydrate Metabolism (Part D) Edited by WILLIS A. WOOD VOLUME 90. Carbohydrate Metabolism (Part E) Edited by WILLIS A. WOOD VOLUME 91. Enzyme Structure (Part I) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 93. Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 94. Polyamines Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME 95. Cumulative Subject Index Volumes 61–74, 76–80 Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly and Targeting (General Methods; Eukaryotes)] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processing and Recycling) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 99. Hormone Action (Part F: Protein Kinases) Edited by JACKIE D. CORBIN AND JOEL G. HARDMAN VOLUME 100. Recombinant DNA (Part B) Edited by RAY WU, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 101. Recombinant DNA (Part C) Edited by RAY WU, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 102. Hormone Action (Part G: Calmodulin and Calcium-Binding Proteins) Edited by ANTHONY R. MEANS AND BERT W. O’MALLEY VOLUME 103. Hormone Action (Part H: Neuroendocrine Peptides) Edited by P. MICHAEL CONN VOLUME 104. Enzyme Purification and Related Techniques (Part C) Edited by WILLIAM B. JAKOBY


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VOLUME 105. Oxygen Radicals in Biological Systems Edited by LESTER PACKER VOLUME 106. Posttranslational Modifications (Part A) Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 107. Posttranslational Modifications (Part B) Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 108. Immunochemical Techniques (Part G: Separation and Characterization of Lymphoid Cells) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS VOLUME 109. Hormone Action (Part I: Peptide Hormones) Edited by LUTZ BIRNBAUMER AND BERT W. O’MALLEY VOLUME 110. Steroids and Isoprenoids (Part A) Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 111. Steroids and Isoprenoids (Part B) Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 112. Drug and Enzyme Targeting (Part A) Edited by KENNETH J. WIDDER AND RALPH GREEN VOLUME 113. Glutamate, Glutamine, Glutathione, and Related Compounds Edited by ALTON MEISTER VOLUME 114. Diffraction Methods for Biological Macromolecules (Part A) Edited by HAROLD W. WYCKOFF, C. H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 115. Diffraction Methods for Biological Macromolecules (Part B) Edited by HAROLD W. WYCKOFF, C. H. W. HIRS, AND SERGE N. TIMASHEFF VOLUME 116. Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS VOLUME 117. Enzyme Structure (Part J) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 118. Plant Molecular Biology Edited by ARTHUR WEISSBACH AND HERBERT WEISSBACH VOLUME 119. Interferons (Part C) Edited by SIDNEY PESTKA VOLUME 120. Cumulative Subject Index Volumes 81–94, 96–101 VOLUME 121. Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 122. Vitamins and Coenzymes (Part G) Edited by FRANK CHYTIL AND DONALD B. MCCORMICK

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VOLUME 123. Vitamins and Coenzymes (Part H) Edited by FRANK CHYTIL AND DONALD B. MCCORMICK VOLUME 124. Hormone Action (Part J: Neuroendocrine Peptides) Edited by P. MICHAEL CONN VOLUME 125. Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 126. Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 127. Biomembranes (Part O: Protons and Water: Structure and Translocation) Edited by LESTER PACKER VOLUME 128. Plasma Lipoproteins (Part A: Preparation, Structure, and Molecular Biology) Edited by JERE P. SEGREST AND JOHN J. ALBERS VOLUME 129. Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism) Edited by JOHN J. ALBERS AND JERE P. SEGREST VOLUME 130. Enzyme Structure (Part K) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 131. Enzyme Structure (Part L) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 132. Immunochemical Techniques (Part J: Phagocytosis and Cell-Mediated Cytotoxicity) Edited by GIOVANNI DI SABATO AND JOHANNES EVERSE VOLUME 133. Bioluminescence and Chemiluminescence (Part B) Edited by MARLENE DELUCA AND WILLIAM D. MCELROY VOLUME 134. Structural and Contractile Proteins (Part C: The Contractile Apparatus and the Cytoskeleton) Edited by RICHARD B. VALLEE VOLUME 135. Immobilized Enzymes and Cells (Part B) Edited by KLAUS MOSBACH VOLUME 136. Immobilized Enzymes and Cells (Part C) Edited by KLAUS MOSBACH VOLUME 137. Immobilized Enzymes and Cells (Part D) Edited by KLAUS MOSBACH VOLUME 138. Complex Carbohydrates (Part E) Edited by VICTOR GINSBURG


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VOLUME 139. Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins) Edited by ANTHONY R. MEANS AND P. MICHAEL CONN VOLUME 140. Cumulative Subject Index Volumes 102–119, 121–134 VOLUME 141. Cellular Regulators (Part B: Calcium and Lipids) Edited by P. MICHAEL CONN AND ANTHONY R. MEANS VOLUME 142. Metabolism of Aromatic Amino Acids and Amines Edited by SEYMOUR KAUFMAN VOLUME 143. Sulfur and Sulfur Amino Acids Edited by WILLIAM B. JAKOBY AND OWEN GRIFFITH VOLUME 144. Structural and Contractile Proteins (Part D: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME 145. Structural and Contractile Proteins (Part E: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME 146. Peptide Growth Factors (Part A) Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 147. Peptide Growth Factors (Part B) Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 148. Plant Cell Membranes Edited by LESTER PACKER AND ROLAND DOUCE VOLUME 149. Drug and Enzyme Targeting (Part B) Edited by RALPH GREEN AND KENNETH J. WIDDER VOLUME 150. Immunochemical Techniques (Part K: In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors) Edited by GIOVANNI DI SABATO VOLUME 151. Molecular Genetics of Mammalian Cells Edited by MICHAEL M. GOTTESMAN VOLUME 152. Guide to Molecular Cloning Techniques Edited by SHELBY L. BERGER AND ALAN R. KIMMEL VOLUME 153. Recombinant DNA (Part D) Edited by RAY WU AND LAWRENCE GROSSMAN VOLUME 154. Recombinant DNA (Part E) Edited by RAY WU AND LAWRENCE GROSSMAN VOLUME 155. Recombinant DNA (Part F) Edited by RAY WU VOLUME 156. Biomembranes (Part P: ATP-Driven Pumps and Related Transport: The Na, K-Pump) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER

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VOLUME 157. Biomembranes (Part Q: ATP-Driven Pumps and Related Transport: Calcium, Proton, and Potassium Pumps) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 158. Metalloproteins (Part A) Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 159. Initiation and Termination of Cyclic Nucleotide Action Edited by JACKIE D. CORBIN AND ROGER A. JOHNSON VOLUME 160. Biomass (Part A: Cellulose and Hemicellulose) Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG VOLUME 161. Biomass (Part B: Lignin, Pectin, and Chitin) Edited by WILLIS A. WOOD AND SCOTT T. KELLOGG VOLUME 162. Immunochemical Techniques (Part L: Chemotaxis and Inflammation) Edited by GIOVANNI DI SABATO VOLUME 163. Immunochemical Techniques (Part M: Chemotaxis and Inflammation) Edited by GIOVANNI DI SABATO VOLUME 164. Ribosomes Edited by HARRY F. NOLLER, JR., AND KIVIE MOLDAVE VOLUME 165. Microbial Toxins: Tools for Enzymology Edited by SIDNEY HARSHMAN VOLUME 166. Branched-Chain Amino Acids Edited by ROBERT HARRIS AND JOHN R. SOKATCH VOLUME 167. Cyanobacteria Edited by LESTER PACKER AND ALEXANDER N. GLAZER VOLUME 168. Hormone Action (Part K: Neuroendocrine Peptides) Edited by P. MICHAEL CONN VOLUME 169. Platelets: Receptors, Adhesion, Secretion (Part A) Edited by JACEK HAWIGER VOLUME 170. Nucleosomes Edited by PAUL M. WASSARMAN AND ROGER D. KORNBERG VOLUME 171. Biomembranes (Part R: Transport Theory: Cells and Model Membranes) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 172. Biomembranes (Part S: Transport: Membrane Isolation and Characterization) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER


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VOLUME 173. Biomembranes [Part T: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 174. Biomembranes [Part U: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 175. Cumulative Subject Index Volumes 135–139, 141–167 VOLUME 176. Nuclear Magnetic Resonance (Part A: Spectral Techniques and Dynamics) Edited by NORMAN J. OPPENHEIMER AND THOMAS L. JAMES VOLUME 177. Nuclear Magnetic Resonance (Part B: Structure and Mechanism) Edited by NORMAN J. OPPENHEIMER AND THOMAS L. JAMES VOLUME 178. Antibodies, Antigens, and Molecular Mimicry Edited by JOHN J. LANGONE VOLUME 179. Complex Carbohydrates (Part F) Edited by VICTOR GINSBURG VOLUME 180. RNA Processing (Part A: General Methods) Edited by JAMES E. DAHLBERG AND JOHN N. ABELSON VOLUME 181. RNA Processing (Part B: Specific Methods) Edited by JAMES E. DAHLBERG AND JOHN N. ABELSON VOLUME 182. Guide to Protein Purification Edited by MURRAY P. DEUTSCHER VOLUME 183. Molecular Evolution: Computer Analysis of Protein and Nucleic Acid Sequences Edited by RUSSELL F. DOOLITTLE VOLUME 184. Avidin-Biotin Technology Edited by MEIR WILCHEK AND EDWARD A. BAYER VOLUME 185. Gene Expression Technology Edited by DAVID V. GOEDDEL VOLUME 186. Oxygen Radicals in Biological Systems (Part B: Oxygen Radicals and Antioxidants) Edited by LESTER PACKER AND ALEXANDER N. GLAZER VOLUME 187. Arachidonate Related Lipid Mediators Edited by ROBERT C. MURPHY AND FRANK A. FITZPATRICK VOLUME 188. Hydrocarbons and Methylotrophy Edited by MARY E. LIDSTROM VOLUME 189. Retinoids (Part A: Molecular and Metabolic Aspects) Edited by LESTER PACKER

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VOLUME 190. Retinoids (Part B: Cell Differentiation and Clinical Applications) Edited by LESTER PACKER VOLUME 191. Biomembranes (Part V: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 192. Biomembranes (Part W: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 193. Mass Spectrometry Edited by JAMES A. MCCLOSKEY VOLUME 194. Guide to Yeast Genetics and Molecular Biology Edited by CHRISTINE GUTHRIE AND GERALD R. FINK VOLUME 195. Adenylyl Cyclase, G Proteins, and Guanylyl Cyclase Edited by ROGER A. JOHNSON AND JACKIE D. CORBIN VOLUME 196. Molecular Motors and the Cytoskeleton Edited by RICHARD B. VALLEE VOLUME 197. Phospholipases Edited by EDWARD A. DENNIS VOLUME 198. Peptide Growth Factors (Part C) Edited by DAVID BARNES, J. P. MATHER, AND GORDON H. SATO VOLUME 199. Cumulative Subject Index Volumes 168–174, 176–194 VOLUME 200. Protein Phosphorylation (Part A: Protein Kinases: Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression) Edited by TONY HUNTER AND BARTHOLOMEW M. SEFTON VOLUME 201. Protein Phosphorylation (Part B: Analysis of Protein Phosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases) Edited by TONY HUNTER AND BARTHOLOMEW M. SEFTON VOLUME 202. Molecular Design and Modeling: Concepts and Applications (Part A: Proteins, Peptides, and Enzymes) Edited by JOHN J. LANGONE VOLUME 203. Molecular Design and Modeling: Concepts and Applications (Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides, and Drugs) Edited by JOHN J. LANGONE VOLUME 204. Bacterial Genetic Systems Edited by JEFFREY H. MILLER VOLUME 205. Metallobiochemistry (Part B: Metallothionein and Related Molecules) Edited by JAMES F. RIORDAN AND BERT L. VALLEE


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VOLUME 206. Cytochrome P450 Edited by MICHAEL R. WATERMAN AND ERIC F. JOHNSON VOLUME 207. Ion Channels Edited by BERNARDO RUDY AND LINDA E. IVERSON VOLUME 208. Protein–DNA Interactions Edited by ROBERT T. SAUER VOLUME 209. Phospholipid Biosynthesis Edited by EDWARD A. DENNIS AND DENNIS E. VANCE VOLUME 210. Numerical Computer Methods Edited by LUDWIG BRAND AND MICHAEL L. JOHNSON VOLUME 211. DNA Structures (Part A: Synthesis and Physical Analysis of DNA) Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG VOLUME 212. DNA Structures (Part B: Chemical and Electrophoretic Analysis of DNA) Edited by DAVID M. J. LILLEY AND JAMES E. DAHLBERG VOLUME 213. Carotenoids (Part A: Chemistry, Separation, Quantitation, and Antioxidation) Edited by LESTER PACKER VOLUME 214. Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis) Edited by LESTER PACKER VOLUME 215. Platelets: Receptors, Adhesion, Secretion (Part B) Edited by JACEK J. HAWIGER VOLUME 216. Recombinant DNA (Part G) Edited by RAY WU VOLUME 217. Recombinant DNA (Part H) Edited by RAY WU VOLUME 218. Recombinant DNA (Part I) Edited by RAY WU VOLUME 219. Reconstitution of Intracellular Transport Edited by JAMES E. ROTHMAN VOLUME 220. Membrane Fusion Techniques (Part A) Edited by NEJAT DU¨ZGU¨NES, VOLUME 221. Membrane Fusion Techniques (Part B) Edited by NEJAT DU¨ZGU¨NES, VOLUME 222. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part A: Mammalian Blood Coagulation Factors and Inhibitors) Edited by LASZLO LORAND AND KENNETH G. MANN

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VOLUME 223. Proteolytic Enzymes in Coagulation, Fibrinolysis, and Complement Activation (Part B: Complement Activation, Fibrinolysis, and Nonmammalian Blood Coagulation Factors) Edited by LASZLO LORAND AND KENNETH G. MANN VOLUME 224. Molecular Evolution: Producing the Biochemical Data Edited by ELIZABETH ANNE ZIMMER, THOMAS J. WHITE, REBECCA L. CANN, AND ALLAN C. WILSON VOLUME 225. Guide to Techniques in Mouse Development Edited by PAUL M. WASSARMAN AND MELVIN L. DEPAMPHILIS VOLUME 226. Metallobiochemistry (Part C: Spectroscopic and Physical Methods for Probing Metal Ion Environments in Metalloenzymes and Metalloproteins) Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 227. Metallobiochemistry (Part D: Physical and Spectroscopic Methods for Probing Metal Ion Environments in Metalloproteins) Edited by JAMES F. RIORDAN AND BERT L. VALLEE VOLUME 228. Aqueous Two-Phase Systems Edited by HARRY WALTER AND GO¨TE JOHANSSON VOLUME 229. Cumulative Subject Index Volumes 195–198, 200–227 VOLUME 230. Guide to Techniques in Glycobiology Edited by WILLIAM J. LENNARZ AND GERALD W. HART VOLUME 231. Hemoglobins (Part B: Biochemical and Analytical Methods) Edited by JOHANNES EVERSE, KIM D. VANDEGRIFF, AND ROBERT M. WINSLOW VOLUME 232. Hemoglobins (Part C: Biophysical Methods) Edited by JOHANNES EVERSE, KIM D. VANDEGRIFF, AND ROBERT M. WINSLOW VOLUME 233. Oxygen Radicals in Biological Systems (Part C) Edited by LESTER PACKER VOLUME 234. Oxygen Radicals in Biological Systems (Part D) Edited by LESTER PACKER VOLUME 235. Bacterial Pathogenesis (Part A: Identification and Regulation of Virulence Factors) Edited by VIRGINIA L. CLARK AND PATRIK M. BAVOIL VOLUME 236. Bacterial Pathogenesis (Part B: Integration of Pathogenic Bacteria with Host Cells) Edited by VIRGINIA L. CLARK AND PATRIK M. BAVOIL VOLUME 237. Heterotrimeric G Proteins Edited by RAVI IYENGAR VOLUME 238. Heterotrimeric G-Protein Effectors Edited by RAVI IYENGAR


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VOLUME 239. Nuclear Magnetic Resonance (Part C) Edited by THOMAS L. JAMES AND NORMAN J. OPPENHEIMER VOLUME 240. Numerical Computer Methods (Part B) Edited by MICHAEL L. JOHNSON AND LUDWIG BRAND VOLUME 241. Retroviral Proteases Edited by LAWRENCE C. KUO AND JULES A. SHAFER VOLUME 242. Neoglycoconjugates (Part A) Edited by Y. C. LEE AND REIKO T. LEE VOLUME 243. Inorganic Microbial Sulfur Metabolism Edited by HARRY D. PECK, JR., AND JEAN LEGALL VOLUME 244. Proteolytic Enzymes: Serine and Cysteine Peptidases Edited by ALAN J. BARRETT VOLUME 245. Extracellular Matrix Components Edited by E. RUOSLAHTI AND E. ENGVALL VOLUME 246. Biochemical Spectroscopy Edited by KENNETH SAUER VOLUME 247. Neoglycoconjugates (Part B: Biomedical Applications) Edited by Y. C. LEE AND REIKO T. LEE VOLUME 248. Proteolytic Enzymes: Aspartic and Metallo Peptidases Edited by ALAN J. BARRETT VOLUME 249. Enzyme Kinetics and Mechanism (Part D: Developments in Enzyme Dynamics) Edited by DANIEL L. PURICH VOLUME 250. Lipid Modifications of Proteins Edited by PATRICK J. CASEY AND JANICE E. BUSS VOLUME 251. Biothiols (Part A: Monothiols and Dithiols, Protein Thiols, and Thiyl Radicals) Edited by LESTER PACKER VOLUME 252. Biothiols (Part B: Glutathione and Thioredoxin; Thiols in Signal Transduction and Gene Regulation) Edited by LESTER PACKER VOLUME 253. Adhesion of Microbial Pathogens Edited by RON J. DOYLE AND ITZHAK OFEK VOLUME 254. Oncogene Techniques Edited by PETER K. VOGT AND INDER M. VERMA VOLUME 255. Small GTPases and Their Regulators (Part A: Ras Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 256. Small GTPases and Their Regulators (Part B: Rho Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL

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VOLUME 257. Small GTPases and Their Regulators (Part C: Proteins Involved in Transport) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 258. Redox-Active Amino Acids in Biology Edited by JUDITH P. KLINMAN VOLUME 259. Energetics of Biological Macromolecules Edited by MICHAEL L. JOHNSON AND GARY K. ACKERS VOLUME 260. Mitochondrial Biogenesis and Genetics (Part A) Edited by GIUSEPPE M. ATTARDI AND ANNE CHOMYN VOLUME 261. Nuclear Magnetic Resonance and Nucleic Acids Edited by THOMAS L. JAMES VOLUME 262. DNA Replication Edited by JUDITH L. CAMPBELL VOLUME 263. Plasma Lipoproteins (Part C: Quantitation) Edited by WILLIAM A. BRADLEY, SANDRA H. GIANTURCO, AND JERE P. SEGREST VOLUME 264. Mitochondrial Biogenesis and Genetics (Part B) Edited by GIUSEPPE M. ATTARDI AND ANNE CHOMYN VOLUME 265. Cumulative Subject Index Volumes 228, 230–262 VOLUME 266. Computer Methods for Macromolecular Sequence Analysis Edited by RUSSELL F. DOOLITTLE VOLUME 267. Combinatorial Chemistry Edited by JOHN N. ABELSON VOLUME 268. Nitric Oxide (Part A: Sources and Detection of NO; NO Synthase) Edited by LESTER PACKER VOLUME 269. Nitric Oxide (Part B: Physiological and Pathological Processes) Edited by LESTER PACKER VOLUME 270. High Resolution Separation and Analysis of Biological Macromolecules (Part A: Fundamentals) Edited by BARRY L. KARGER AND WILLIAM S. HANCOCK VOLUME 271. High Resolution Separation and Analysis of Biological Macromolecules (Part B: Applications) Edited by BARRY L. KARGER AND WILLIAM S. HANCOCK VOLUME 272. Cytochrome P450 (Part B) Edited by ERIC F. JOHNSON AND MICHAEL R. WATERMAN VOLUME 273. RNA Polymerase and Associated Factors (Part A) Edited by SANKAR ADHYA VOLUME 274. RNA Polymerase and Associated Factors (Part B) Edited by SANKAR ADHYA


VOLUME 275. Viral Polymerases and Related Proteins Edited by LAWRENCE C. KUO, DAVID B. OLSEN, AND STEVEN S. CARROLL VOLUME 276. Macromolecular Crystallography (Part A) Edited by CHARLES W. CARTER, JR., AND ROBERT M. SWEET VOLUME 277. Macromolecular Crystallography (Part B) Edited by CHARLES W. CARTER, JR., AND ROBERT M. SWEET VOLUME 278. Fluorescence Spectroscopy Edited by LUDWIG BRAND AND MICHAEL L. JOHNSON VOLUME 279. Vitamins and Coenzymes (Part I) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 280. Vitamins and Coenzymes (Part J) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 281. Vitamins and Coenzymes (Part K) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 282. Vitamins and Coenzymes (Part L) Edited by DONALD B. MCCORMICK, JOHN W. SUTTIE, AND CONRAD WAGNER VOLUME 283. Cell Cycle Control Edited by WILLIAM G. DUNPHY VOLUME 284. Lipases (Part A: Biotechnology) Edited by BYRON RUBIN AND EDWARD A. DENNIS VOLUME 285. Cumulative Subject Index Volumes 263, 264, 266–284, 286–289 VOLUME 286. Lipases (Part B: Enzyme Characterization and Utilization) Edited by BYRON RUBIN AND EDWARD A. DENNIS VOLUME 287. Chemokines Edited by RICHARD HORUK VOLUME 288. Chemokine Receptors Edited by RICHARD HORUK VOLUME 289. Solid Phase Peptide Synthesis Edited by GREGG B. FIELDS VOLUME 290. Molecular Chaperones Edited by GEORGE H. LORIMER AND THOMAS BALDWIN VOLUME 291. Caged Compounds Edited by GERARD MARRIOTT VOLUME 292. ABC Transporters: Biochemical, Cellular, and Molecular Aspects Edited by SURESH V. AMBUDKAR AND MICHAEL M. GOTTESMAN VOLUME 293. Ion Channels (Part B) Edited by P. MICHAEL CONN

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VOLUME 294. Ion Channels (Part C) Edited by P. MICHAEL CONN VOLUME 295. Energetics of Biological Macromolecules (Part B) Edited by GARY K. ACKERS AND MICHAEL L. JOHNSON VOLUME 296. Neurotransmitter Transporters Edited by SUSAN G. AMARA VOLUME 297. Photosynthesis: Molecular Biology of Energy Capture Edited by LEE MCINTOSH VOLUME 298. Molecular Motors and the Cytoskeleton (Part B) Edited by RICHARD B. VALLEE VOLUME 299. Oxidants and Antioxidants (Part A) Edited by LESTER PACKER VOLUME 300. Oxidants and Antioxidants (Part B) Edited by LESTER PACKER VOLUME 301. Nitric Oxide: Biological and Antioxidant Activities (Part C) Edited by LESTER PACKER VOLUME 302. Green Fluorescent Protein Edited by P. MICHAEL CONN VOLUME 303. cDNA Preparation and Display Edited by SHERMAN M. WEISSMAN VOLUME 304. Chromatin Edited by PAUL M. WASSARMAN AND ALAN P. WOLFFE VOLUME 305. Bioluminescence and Chemiluminescence (Part C) Edited by THOMAS O. BALDWIN AND MIRIAM M. ZIEGLER VOLUME 306. Expression of Recombinant Genes in Eukaryotic Systems Edited by JOSEPH C. GLORIOSO AND MARTIN C. SCHMIDT VOLUME 307. Confocal Microscopy Edited by P. MICHAEL CONN VOLUME 308. Enzyme Kinetics and Mechanism (Part E: Energetics of Enzyme Catalysis) Edited by DANIEL L. PURICH AND VERN L. SCHRAMM VOLUME 309. Amyloid, Prions, and Other Protein Aggregates Edited by RONALD WETZEL VOLUME 310. Biofilms Edited by RON J. DOYLE VOLUME 311. Sphingolipid Metabolism and Cell Signaling (Part A) Edited by ALFRED H. MERRILL, JR., AND YUSUF A. HANNUN


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VOLUME 312. Sphingolipid Metabolism and Cell Signaling (Part B) Edited by ALFRED H. MERRILL, JR., AND YUSUF A. HANNUN VOLUME 313. Antisense Technology (Part A: General Methods, Methods of Delivery, and RNA Studies) Edited by M. IAN PHILLIPS VOLUME 314. Antisense Technology (Part B: Applications) Edited by M. IAN PHILLIPS VOLUME 315. Vertebrate Phototransduction and the Visual Cycle (Part A) Edited by KRZYSZTOF PALCZEWSKI VOLUME 316. Vertebrate Phototransduction and the Visual Cycle (Part B) Edited by KRZYSZTOF PALCZEWSKI VOLUME 317. RNA–Ligand Interactions (Part A: Structural Biology Methods) Edited by DANIEL W. CELANDER AND JOHN N. ABELSON VOLUME 318. RNA–Ligand Interactions (Part B: Molecular Biology Methods) Edited by DANIEL W. CELANDER AND JOHN N. ABELSON VOLUME 319. Singlet Oxygen, UV-A, and Ozone Edited by LESTER PACKER AND HELMUT SIES VOLUME 320. Cumulative Subject Index Volumes 290–319 VOLUME 321. Numerical Computer Methods (Part C) Edited by MICHAEL L. JOHNSON AND LUDWIG BRAND VOLUME 322. Apoptosis Edited by JOHN C. REED VOLUME 323. Energetics of Biological Macromolecules (Part C) Edited by MICHAEL L. JOHNSON AND GARY K. ACKERS VOLUME 324. Branched-Chain Amino Acids (Part B) Edited by ROBERT A. HARRIS AND JOHN R. SOKATCH VOLUME 325. Regulators and Effectors of Small GTPases (Part D: Rho Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 326. Applications of Chimeric Genes and Hybrid Proteins (Part A: Gene Expression and Protein Purification) Edited by JEREMY THORNER, SCOTT D. EMR, AND JOHN N. ABELSON VOLUME 327. Applications of Chimeric Genes and Hybrid Proteins (Part B: Cell Biology and Physiology) Edited by JEREMY THORNER, SCOTT D. EMR, AND JOHN N. ABELSON VOLUME 328. Applications of Chimeric Genes and Hybrid Proteins (Part C: Protein–Protein Interactions and Genomics) Edited by JEREMY THORNER, SCOTT D. EMR, AND JOHN N. ABELSON

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VOLUME 329. Regulators and Effectors of Small GTPases (Part E: GTPases Involved in Vesicular Traffic) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 330. Hyperthermophilic Enzymes (Part A) Edited by MICHAEL W. W. ADAMS AND ROBERT M. KELLY VOLUME 331. Hyperthermophilic Enzymes (Part B) Edited by MICHAEL W. W. ADAMS AND ROBERT M. KELLY VOLUME 332. Regulators and Effectors of Small GTPases (Part F: Ras Family I) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 333. Regulators and Effectors of Small GTPases (Part G: Ras Family II) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 334. Hyperthermophilic Enzymes (Part C) Edited by MICHAEL W. W. ADAMS AND ROBERT M. KELLY VOLUME 335. Flavonoids and Other Polyphenols Edited by LESTER PACKER VOLUME 336. Microbial Growth in Biofilms (Part A: Developmental and Molecular Biological Aspects) Edited by RON J. DOYLE VOLUME 337. Microbial Growth in Biofilms (Part B: Special Environments and Physicochemical Aspects) Edited by RON J. DOYLE VOLUME 338. Nuclear Magnetic Resonance of Biological Macromolecules (Part A) Edited by THOMAS L. JAMES, VOLKER DO¨TSCH, AND ULI SCHMITZ VOLUME 339. Nuclear Magnetic Resonance of Biological Macromolecules (Part B) Edited by THOMAS L. JAMES, VOLKER DO¨TSCH, AND ULI SCHMITZ VOLUME 340. Drug–Nucleic Acid Interactions Edited by JONATHAN B. CHAIRES AND MICHAEL J. WARING VOLUME 341. Ribonucleases (Part A) Edited by ALLEN W. NICHOLSON VOLUME 342. Ribonucleases (Part B) Edited by ALLEN W. NICHOLSON VOLUME 343. G Protein Pathways (Part A: Receptors) Edited by RAVI IYENGAR AND JOHN D. HILDEBRANDT VOLUME 344. G Protein Pathways (Part B: G Proteins and Their Regulators) Edited by RAVI IYENGAR AND JOHN D. HILDEBRANDT VOLUME 345. G Protein Pathways (Part C: Effector Mechanisms) Edited by RAVI IYENGAR AND JOHN D. HILDEBRANDT


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VOLUME 346. Gene Therapy Methods Edited by M. IAN PHILLIPS VOLUME 347. Protein Sensors and Reactive Oxygen Species (Part A: Selenoproteins and Thioredoxin) Edited by HELMUT SIES AND LESTER PACKER VOLUME 348. Protein Sensors and Reactive Oxygen Species (Part B: Thiol Enzymes and Proteins) Edited by HELMUT SIES AND LESTER PACKER VOLUME 349. Superoxide Dismutase Edited by LESTER PACKER VOLUME 350. Guide to Yeast Genetics and Molecular and Cell Biology (Part B) Edited by CHRISTINE GUTHRIE AND GERALD R. FINK VOLUME 351. Guide to Yeast Genetics and Molecular and Cell Biology (Part C) Edited by CHRISTINE GUTHRIE AND GERALD R. FINK VOLUME 352. Redox Cell Biology and Genetics (Part A) Edited by CHANDAN K. SEN AND LESTER PACKER VOLUME 353. Redox Cell Biology and Genetics (Part B) Edited by CHANDAN K. SEN AND LESTER PACKER VOLUME 354. Enzyme Kinetics and Mechanisms (Part F: Detection and Characterization of Enzyme Reaction Intermediates) Edited by DANIEL L. PURICH VOLUME 355. Cumulative Subject Index Volumes 321–354 VOLUME 356. Laser Capture Microscopy and Microdissection Edited by P. MICHAEL CONN VOLUME 357. Cytochrome P450, Part C Edited by ERIC F. JOHNSON AND MICHAEL R. WATERMAN VOLUME 358. Bacterial Pathogenesis (Part C: Identification, Regulation, and Function of Virulence Factors) Edited by VIRGINIA L. CLARK AND PATRIK M. BAVOIL VOLUME 359. Nitric Oxide (Part D) Edited by ENRIQUE CADENAS AND LESTER PACKER VOLUME 360. Biophotonics (Part A) Edited by GERARD MARRIOTT AND IAN PARKER VOLUME 361. Biophotonics (Part B) Edited by GERARD MARRIOTT AND IAN PARKER VOLUME 362. Recognition of Carbohydrates in Biological Systems (Part A) Edited by YUAN C. LEE AND REIKO T. LEE

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VOLUME 363. Recognition of Carbohydrates in Biological Systems (Part B) Edited by YUAN C. LEE AND REIKO T. LEE VOLUME 364. Nuclear Receptors Edited by DAVID W. RUSSELL AND DAVID J. MANGELSDORF VOLUME 365. Differentiation of Embryonic Stem Cells Edited by PAUL M. WASSAUMAN AND GORDON M. KELLER VOLUME 366. Protein Phosphatases Edited by SUSANNE KLUMPP AND JOSEF KRIEGLSTEIN VOLUME 367. Liposomes (Part A) Edited by NEJAT DU¨ZGU¨NES, VOLUME 368. Macromolecular Crystallography (Part C) Edited by CHARLES W. CARTER, JR., AND ROBERT M. SWEET VOLUME 369. Combinational Chemistry (Part B) Edited by GUILLERMO A. MORALES AND BARRY A. BUNIN VOLUME 370. RNA Polymerases and Associated Factors (Part C) Edited by SANKAR L. ADHYA AND SUSAN GARGES VOLUME 371. RNA Polymerases and Associated Factors (Part D) Edited by SANKAR L. ADHYA AND SUSAN GARGES VOLUME 372. Liposomes (Part B) Edited by NEJAT DU¨ZGU¨NES, VOLUME 373. Liposomes (Part C) Edited by NEJAT DU¨ZGU¨NES, VOLUME 374. Macromolecular Crystallography (Part D) Edited by CHARLES W. CARTER, JR., AND ROBERT W. SWEET VOLUME 375. Chromatin and Chromatin Remodeling Enzymes (Part A) Edited by C. DAVID ALLIS AND CARL WU VOLUME 376. Chromatin and Chromatin Remodeling Enzymes (Part B) Edited by C. DAVID ALLIS AND CARL WU VOLUME 377. Chromatin and Chromatin Remodeling Enzymes (Part C) Edited by C. DAVID ALLIS AND CARL WU VOLUME 378. Quinones and Quinone Enzymes (Part A) Edited by HELMUT SIES AND LESTER PACKER VOLUME 379. Energetics of Biological Macromolecules (Part D) Edited by JO M. HOLT, MICHAEL L. JOHNSON, AND GARY K. ACKERS VOLUME 380. Energetics of Biological Macromolecules (Part E) Edited by JO M. HOLT, MICHAEL L. JOHNSON, AND GARY K. ACKERS VOLUME 381. Oxygen Sensing Edited by CHANDAN K. SEN AND GREGG L. SEMENZA


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VOLUME 382. Quinones and Quinone Enzymes (Part B) Edited by HELMUT SIES AND LESTER PACKER VOLUME 383. Numerical Computer Methods (Part D) Edited by LUDWIG BRAND AND MICHAEL L. JOHNSON VOLUME 384. Numerical Computer Methods (Part E) Edited by LUDWIG BRAND AND MICHAEL L. JOHNSON VOLUME 385. Imaging in Biological Research (Part A) Edited by P. MICHAEL CONN VOLUME 386. Imaging in Biological Research (Part B) Edited by P. MICHAEL CONN VOLUME 387. Liposomes (Part D) Edited by NEJAT DU¨ZGU¨NES, VOLUME 388. Protein Engineering Edited by DAN E. ROBERTSON AND JOSEPH P. NOEL VOLUME 389. Regulators of G-Protein Signaling (Part A) Edited by DAVID P. SIDEROVSKI VOLUME 390. Regulators of G-Protein Signaling (Part B) Edited by DAVID P. SIDEROVSKI VOLUME 391. Liposomes (Part E) Edited by NEJAT DU¨ZGU¨NES, VOLUME 392. RNA Interference Edited by ENGELKE ROSSI VOLUME 393. Circadian Rhythms Edited by MICHAEL W. YOUNG VOLUME 394. Nuclear Magnetic Resonance of Biological Macromolecules (Part C) Edited by THOMAS L. JAMES VOLUME 395. Producing the Biochemical Data (Part B) Edited by ELIZABETH A. ZIMMER AND ERIC H. ROALSON VOLUME 396. Nitric Oxide (Part E) Edited by LESTER PACKER AND ENRIQUE CADENAS VOLUME 397. Environmental Microbiology Edited by JARED R. LEADBETTER VOLUME 398. Ubiquitin and Protein Degradation (Part A) Edited by RAYMOND J. DESHAIES VOLUME 399. Ubiquitin and Protein Degradation (Part B) Edited by RAYMOND J. DESHAIES VOLUME 400. Phase II Conjugation Enzymes and Transport Systems Edited by HELMUT SIES AND LESTER PACKER

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VOLUME 401. Glutathione Transferases and Gamma Glutamyl Transpeptidases Edited by HELMUT SIES AND LESTER PACKER VOLUME 402. Biological Mass Spectrometry Edited by A. L. BURLINGAME VOLUME 403. GTPases Regulating Membrane Targeting and Fusion Edited by WILLIAM E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 404. GTPases Regulating Membrane Dynamics Edited by WILLIAM E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 405. Mass Spectrometry: Modified Proteins and Glycoconjugates Edited by A. L. BURLINGAME VOLUME 406. Regulators and Effectors of Small GTPases: Rho Family Edited by WILLIAM E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 407. Regulators and Effectors of Small GTPases: Ras Family Edited by WILLIAM E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 408. DNA Repair (Part A) Edited by JUDITH L. CAMPBELL AND PAUL MODRICH VOLUME 409. DNA Repair (Part B) Edited by JUDITH L. CAMPBELL AND PAUL MODRICH VOLUME 410. DNA Microarrays (Part A: Array Platforms and Web-Bench Protocols) Edited by ALAN KIMMEL AND BRIAN OLIVER VOLUME 411. DNA Microarrays (Part B: Databases and Statistics) Edited by ALAN KIMMEL AND BRIAN OLIVER VOLUME 412. Amyloid, Prions, and Other Protein Aggregates (Part B) Edited by INDU KHETERPAL AND RONALD WETZEL VOLUME 413. Amyloid, Prions, and Other Protein Aggregates (Part C) Edited by INDU KHETERPAL AND RONALD WETZEL VOLUME 414. Measuring Biological Responses with Automated Microscopy Edited by JAMES INGLESE VOLUME 415. Glycobiology Edited by MINORU FUKUDA VOLUME 416. Glycomics Edited by MINORU FUKUDA VOLUME 417. Functional Glycomics Edited by MINORU FUKUDA VOLUME 418. Embryonic Stem Cells Edited by IRINA KLIMANSKAYA AND ROBERT LANZA


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VOLUME 419. Adult Stem Cells Edited by IRINA KLIMANSKAYA AND ROBERT LANZA VOLUME 420. Stem Cell Tools and Other Experimental Protocols Edited by IRINA KLIMANSKAYA AND ROBERT LANZA VOLUME 421. Advanced Bacterial Genetics: Use of Transposons and Phage for Genomic Engineering Edited by KELLY T. HUGHES VOLUME 422. Two-Component Signaling Systems, Part A Edited by MELVIN I. SIMON, BRIAN R. CRANE, AND ALEXANDRINE CRANE VOLUME 423. Two-Component Signaling Systems, Part B Edited by MELVIN I. SIMON, BRIAN R. CRANE, AND ALEXANDRINE CRANE VOLUME 424. RNA Editing Edited by JONATHA M. GOTT VOLUME 425. RNA Modification Edited by JONATHA M. GOTT VOLUME 426. Integrins Edited by DAVID CHERESH VOLUME 427. MicroRNA Methods Edited by JOHN J. ROSSI VOLUME 428. Osmosensing and Osmosignaling Edited by HELMUT SIES AND DIETER HAUSSINGER VOLUME 429. Translation Initiation: Extract Systems and Molecular Genetics Edited by JON LORSCH VOLUME 430. Translation Initiation: Reconstituted Systems and Biophysical Methods Edited by JON LORSCH VOLUME 431. Translation Initiation: Cell Biology, High-Throughput and Chemical-Based Approaches Edited by JON LORSCH VOLUME 432. Lipidomics and Bioactive Lipids: Mass-Spectrometry–Based Lipid Analysis Edited by H. ALEX BROWN VOLUME 433. Lipidomics and Bioactive Lipids: Specialized Analytical Methods and Lipids in Disease Edited by H. ALEX BROWN VOLUME 434. Lipidomics and Bioactive Lipids: Lipids and Cell Signaling Edited by H. ALEX BROWN

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VOLUME 435. Oxygen Biology and Hypoxia Edited by HELMUT SIES AND BERNHARD BRU¨NE VOLUME 436. Globins and Other Nitric Oxide-Reactive Protiens (Part A) Edited by ROBERT K. POOLE VOLUME 437. Globins and Other Nitric Oxide-Reactive Protiens (Part B) Edited by ROBERT K. POOLE VOLUME 438. Small GTPases in Disease (Part A) Edited by WILLIAM E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 439. Small GTPases in Disease (Part B) Edited by WILLIAM E. BALCH, CHANNING J. DER, AND ALAN HALL VOLUME 440. Nitric Oxide, Part F Oxidative and Nitrosative Stress in Redox Regulation of Cell Signaling Edited by ENRIQUE CADENAS AND LESTER PACKER VOLUME 441. Nitric Oxide, Part G Oxidative and Nitrosative Stress in Redox Regulation of Cell Signaling Edited by ENRIQUE CADENAS AND LESTER PACKER VOLUME 442. Programmed Cell Death, General Principles for Studying Cell Death (Part A) Edited by ROYA KHOSRAVI-FAR, ZAHRA ZAKERI, RICHARD A. LOCKSHIN, AND MAURO PIACENTINI VOLUME 443. Angiogenesis: In Vitro Systems Edited by DAVID A. CHERESH VOLUME 444. Angiogenesis: In Vivo Systems (Part A) Edited by DAVID A. CHERESH VOLUME 445. Angiogenesis: In Vivo Systems (Part B) Edited by DAVID A. CHERESH VOLUME 446. Programmed Cell Death, The Biology and Therapeutic Implications of Cell Death (Part B) Edited by ROYA KHOSRAVI-FAR, ZAHRA ZAKERI, RICHARD A. LOCKSHIN, AND MAURO PIACENTINI

C H A P T E R

O N E

Analysis of RNA Decay, Processing, and Polyadenylation in Escherichia coli and Other Prokaryotes Bijoy K. Mohanty, Hili Giladi, Valerie F. Maples, and Sidney R. Kushner

Contents 4 5 6 6 6 7 7 7 8 9 10 10 10 11 12 12 13 14 14 15 15 16 16 17 18

1. Introduction 2. General Considerations When Working with RNA 2.1. Importance of using isogenic strains 2.2. Inhibition of new transcription 2.3. Kits versus detergent methods for RNA isolation 3. Isolation of Total RNA from Exponentially Growing Cells 3.1. Materials 3.2. Reagents and buffers 3.3. Procedure 3.4. RNA extraction 4. Isolation of Total RNA from Stationary Phase Cells 4.1. Reagents and buffers 4.2. Procedure 5. Removal of DNA Contamination 6. RNA Yield and Purity Assessment 7. RNA Integrity Assessment 8. Isolation of Polyadenylated RNA 8.1. Procedure 9. Northern Analysis 9.1. Separation of RNA in polyacrylamide gels 9.2. Separation of RNA in agarose gels 9.3. Transferring RNA to membrane 9.4. Choice of probes 9.5. Probe labeling 9.6. Hybridization, washing, and analysis

Department of Genetics, University of Georgia, Athens, Georgia, USA Methods in Enzymology, Volume 447 ISSN 0076-6879, DOI: 10.1016/S0076-6879(08)02201-5

#

2008 Elsevier Inc. All rights reserved.

3

4

Bijoy K. Mohanty et al.

10. Analysis of RNA Polyadenylation in E. coli 10.1. Dot-blot analysis 10.2. Poly(A) sizing assay 11. Reverse Transcription PCR (RT-PCR) to Analyze 30 -ends of Specific Transcripts 11.1. Primer design for RT and PCR 11.2. Procedure 11.3. Cloning and sequencing of PCR products 11.4. RNA self-ligation for determining 30 - and 50 -ends 12. Conclusion Acknowledgments References

20 20 22 23 25 25 25 27 28 28 28

Abstract This chapter provides detailed methodologies for isolating total RNA and polyadenylated RNA from E. coli and other prokaryotes, along with the procedures necessary to analyze the processing and decay of specific transcripts and determine their 30 - and 50 -ends. The RNA isolation methods described here facilitate isolating good-quality RNA in a very cost-effective way compared to the commercially available RNA isolation kits, without employing phenol and/or alcohol precipitation. We also discuss the limits associated with polyacrylamide and agarose gels for the separation of small and large RNAs. Methods useful for the analysis of post-transcriptionally modified transcripts and the processing of very large polycistronic transcripts are also presented.

1. Introduction RNA turnover and processing have now been demonstrated to be important steps that directly affect protein synthesis and the cell’s ability to survive in nature. However, the analysis of mRNA decay and polyadenylation in Escherichia coli has long been considered technically difficult. The development over the past 15 years of methods for the isolation and characterization of both mRNA and polyadenylated species has made the study of these important pathways of RNA metabolism more straightforward (Arraiano et al., 1988; O’Hara et al., 1995; Mohanty and Kushner, 1999). By employing these techniques and using isogenic strains carrying mutations in a variety of genes encoding ribonucleases, poly(A) polymerases, and accessory proteins, it has been possible to develop a better understanding of the multiple pathways by which mRNA decay and polyadenylation take place (Arraiano et al., 1988, 1993, 1997; O’Hara et al., 1995; Hajnsdorf and Re´gnier, 2000; Mohanty and Kushner, 2000; Ow et al., 2000, 2003; Mohanty et al., 2004; Perwez and Kushner, 2006). Furthermore, the ability to examine the E. coli transcriptome in a variety of mutants deficient in the

Analysis of mRNA Decay and Polyadenylation in E. coli

5

enzymes involved in mRNA decay, processing, and polyadenylation (Lee et al., 2002; Li et al., 2003; Mohanty and Kushner, 2003, 2006; Bernstein et al., 2004) has provided a valuable new approach for identifying targets of specific enzymes. The use of reliable and reproducible methodologies is paramount to successfully investigate RNA metabolism in any organism. In the sections that follow, methods are described to allow any laboratory interested in studying RNA metabolism in any prokaryote to successfully carry out such experiments. These procedures are the results of many years of experimentation.

2. General Considerations When Working with RNA Because RNA is much less stable than DNA and is prone to hydrolysis by ubiquitous RNases, extra precautions are necessary. In particular, always wear a lab coat and powder-free gloves. Periodically change gloves and never touch an RNA sample with bare hands. All glassware for RNA work should be kept separate from everything else in the laboratory and never be used for anything else. This is particularly important, since RNase A is extremely stable and tends to stick to glassware. Glassware should be baked at 180 C for 8 h prior to use. All plastic ware, gel apparatus, and other materials that cannot be baked and may come in contact with RNA should be treated with a commercially available RNase inactivating solution (RNAaseZap, Ambion, TX, USA) or equivalent solutions from other manufacturers and thoroughly washed with RNase-free water (devoid of any RNase contamination). Never autoclave any of the material used for RNA analysis. Pipette tips and Eppendorf tubes are always used new, as these are packaged RNase- and DNase-free. Water is a major source of RNase contamination. We generate RNasefree water by purifying through a Millipore water-filtration system (Durapore CVDI Cartridge Filter) that has been retrofitted to the deionized water source in the laboratory. By using this system, we have eliminated our RNase problem and thus do not treat water with DEPC (diethyl pyrocarbonate), which was formerly our standard process for making water RNase free. In addition, RNase-free water is now available through many commercial suppliers (Ambion, TX, USA; Promega, WI, USA; and Invitrogen, CA, USA). All reagents and buffers are prepared using RNasefree water only. All appropriate safety procedures should be followed when working with toxic and carcinogenic materials, such as phenol, ethidium bromide, formaldehyde, polyacrylamide, and UV transilluminators (Sambrook et al., 1989).

6


2.1. Importance of using isogenic strains Although it might seem obvious, it is critical to carry out RNA experiments in isogenic strains. It is not clear at this point if all the enzymes involved in RNA metabolism have been identified in E. coli or, for that matter, in any prokaryote.

2.2. Inhibition of new transcription The level of each transcript in an exponentially growing cell is maintained at steady state by a combination of synthesis and degradation. The half-lives of E. coli transcripts vary between less than 30 sec to more than 20 min (Kushner, 2007). Although the steady-state level of a transcript can be measured by isolating RNA at any given time, its half-life is measured after new transcription has been inhibited. In E. coli, inhibition of new transcription is accomplished by adding rifampicin to a growing culture. The first time point (time zero) is taken 75 sec after the addition of the drug to allow for equilibration within the cell.

2.3. Kits versus detergent methods for RNA isolation We have previously taken advantage of a cationic surfactant called Catrimox-14 (tetradecyltrimethylammonium oxalate), which has the ability to lyse cells and precipitate nucleic acids (Dahle and Macfarlane, 1993; Macfarlane and Dahle, 1993), to isolate RNA from E. coli (O’Hara et al., 1995; Mohanty and Kushner, 1999). This detergent was initially developed and marketed by Iowa Biotechnology Corp. (Coralville, IA). However, shortly after Qiagen acquired Iowa Biotechnology, Catrimox-14 was taken off the market. Because the detergent-based RNA-isolation method was simple and inexpensive, we subsequently determined that a similar cationic surfactant, trimethyl(tetradecyl)ammonium bromide (Sigma) was equally effective in the isolation of RNA. For simplicity, we have called this compound Catrimide. Although there are many commercially available RNA isolation kits, they can be expensive ($5-8/sample), particularly if multiple samples have to be isolated. In contrast, the detergent-based method described here is very low cost ($0.20/sample), avoids the use of phenol and/or chloroform along with alcohol precipitation, and provides many specific advantages relative to the kits. For example, the RNA yields are consistently higher with the detergent method and the samples retain low molecular weight RNAs such as tRNAs and regulatory RNAs. In contrast, most of the column-based kits do not retain these small RNAs (Table 1.1). In addition, the detergent-LiCl-based method removes >98% of the DNA contamination from the RNA (Macfarlane and Dahle, 1997), such that for many

7


Table 1.1 Comparison of commonly used RNA isolation methods/kits

RNA isolation method/kit

Catrimide-LiCl Ribopure-Bacteria Kit (Ambion) RNeasy Protect Bacteria Mini Kit (Qiagen) Purelink Micro-to-Midi Total Purification System (Invitrogen) SV Total RNA Isolation System (Promega)

Average yield (mg)/ sample (E. coli)

Exclusion of small RNAs

200* 90# 70#

No Yes Yes

50#

Partial

36#

Unknown

* Typical actual yield. # As claimed by the manufacturer.

experiments no further DNase I treatment is necessary. Furthermore, most of the commercially available kits do not provide a mechanism to stop further metabolic activity after initial sampling of the cells. This is a significant limitation when dealing with multiple samples, particularly those used for half-life determinations.

3. Isolation of Total RNA from Exponentially Growing Cells The RNA isolation procedure from exponentially growing cells is a detergent-based procedure using Catrimide that has been modified from a previously described method (Macfarlane and Dahle, 1993).

3.1. Materials Centrifuge tubes (Corex or equivalent, 15 ml), screw-cap tubes (Pyrex, 13 100 mm), 1.5-ml Eppendorf tubes, refrigerated centrifuge (Beckman Coulter, Avanti-J25 or equivalent), tabletop microcentrifuge (with at least 13,000-rpm rating), vortex mixer, water bath (37 C), cold room (optional), Savant Speed Vac concentrator (optional).

3.2. Reagents and buffers Tris-MgCl2 (TM) buffer (5): 50 mM Tris base, 25 mM MgCl2, pH 7.2. This solution is stable at room temperature (RT) for 3 months and at 4 C for 6 months.

8


Sodium azide (1 M): The solution is stored at 4 C. Stop buffer (SB): Chloramphenicol, 50 mg (dissolved in 12.5 ml of 95% ethanol); TM buffer (5), 20 ml; RNase-free water, 80 ml; sodium azide (1 M), 2.5 ml. The volume of SB prepared should be proportionally increased or decreased according to the number of samples in a given experiment. Seven ml of the SB are dispensed into baked prelabeled centrifuge tubes and placed in a –20 C freezer at least 1 hour before use. This provides enough time for the buffer to freeze. Rifampicin (prepared fresh): 33.3 mg/ml in DMSO (Sigma). Nalidixic acid (prepared fresh): 2 mg/ml in sterile water (Sigma). Note: Rifampcin and nalidixic acid are only required if the RNA samples will be used for half-life determinations. Lysis buffer (prepared fresh): 303 ml of freshly prepared lysozyme (Sigma) solution (10 mg/ml in 1 TM buffer), 33 ml of RNase-free DNase I (Roche, 10 mg/ml), 10 ml of 1 TM buffer. Store the lysis buffer in ice before use. Catrimide: A 10% solution (w/v) of Catrimide stored at RT. Other solutions: Acetic acid (20 mM), 2 M LiCl in 35% EtOH, 2 M LiCl in water, ethanol (EtOH) (70%, 95%, and 100%). Dry-ice slurry: 1–2 lbs of dry ice is crushed in an ice bucket and mixed with 100% EtOH to make the slurry.

3.3. Procedure 1. An overnight inoculum from a single colony of each bacterial strain to be studied is prepared in 5 ml of Luria broth (LB) containing appropriate antibiotics (if applicable) at the desired temperature. Faster-growing strains such as wild-type controls should be grown as standing cultures whereas slower growing strains should be shaken. 2. The next day, the overnight inoculum is diluted (2-5%) into a desired volume of LB, which is grown with shaking (always grow a few ml more than you need) (with appropriate antibiotic, if applicable) to Klett 50 (No. 42 green filter or OD600 of 0.4 or 108 cells/ml). The cultures should undergo at least two doublings when growing at either 30 C or 37 C. For temperature-sensitive mutants (e.g., rne-1 and rnpA49), the cultures are initially grown at 30 C until they reach Klett 40 and then are shifted to 44 C for up to 2 h. The cultures should be periodically diluted with prewarmed (44 C) LB to maintain their exponential growth. 3. When a culture is ready for sampling, remove a centrifuge tube containing the frozen SB from the freezer and crush the frozen buffer using a glass rod to make an icy slurry. To maintain accurate sampling times, in half-life experiments multiple tubes can be removed from the freezer if they will be used within 5 min. 4. If the RNA will be used for half-life measurements, appropriate volumes of rifampicin (500 mg/ml final concentration) and nalidixic


5. 6.

7.

8. 9.

9

acid (40 mg/ml final concentration) are added to the culture to stop new transcription. The first (0 min) sample is removed after 75 sec to permit complete blockage of new transcription by the rifampicin. Typical time points for half-life determinations of most E. coli mRNAs are 0,1,2,4,8, and 16 minutes. At each desired time point 7 ml of cells are removed and mixed thoroughly with the crushed SB using a vortex mixer. This step blocks all further in vivo metabolic activity including RNA degradation. Each sample is held in ice until the time course has been completed. At this time the lysis buffer is prepared, the dry ice–EtOH slurry is made in an ice bucket, and all the screw-cap tubes are labeled with corresponding sample numbers and are put in dry ice–EtOH slurry for prechilling. The cells are harvested by centrifuging the samples at 5000 rpm for 5 min at 4 C in a refrigerated centrifuge. The supernatants are poured off and the centrifuge tubes are inverted on a couple of layers of Kimwipes to drain the tubes thoroughly (10–20 sec). The outsides of the tubes are wiped to remove excess liquid and are then placed in ice. The cell pellets are resuspended in 400 ml of lysis buffer and transferred to the corresponding prechilled screw-cap tubes in dry ice–EtOH. The cells are lysed by repeatedly (four times) thawing the frozen cells in a 37 C water bath and refreezing the cells in the dry ice–EtOH. After the last freezing step, the cells can be stored at –70 C until ready for RNA extraction.

3.4. RNA extraction 1. To further reduce any possible RNase activity after lysis, 67 ml of 20 mM acetic acid is added to the frozen lysed cells and each sample is placed in a 37 C water bath. 2. After the lysed cell suspensions have completely melted, 400 ml of Catrimide is added and the mixture is incubated for another 2 min in the water bath. The suspension, which is cloudy at this step, is then transferred to a 1.5-ml Eppendorf tube and centrifuged for 1 min at 5000 rpm in a microcentrifuge in the cold room. 3. The supernatants are removed either with a suction apparatus or with a pipette, being careful not to suck up the pellets (containing RNA). 4. The pellets are resuspended in 1 ml of 2 M LiCl in 35% ethanol and incubated at RT for 5 min. 5. The suspensions are then centrifuged at 13,000 rpm for 6 min in the cold room. The supernatants are removed as in step 3. 6. The pellets are then resuspended in 1 ml of 2 M LiCl in water and incubated at RT for 2 min. 7. The suspensions are then centrifuged at 13,000 rpm as described in step 5. The supernatants are removed as in step 3.

10


8. The pellets are subsequently washed by adding 200 ml of 70% EtOH and centrifuging at 13,000 rpm for 5 min, followed by removal of the supernatants as described in step 3. The pellets are then dried in a Speed Vac concentrator for 5 min (or air-dried for 15 min). Note: The centrifugations in steps 5, 6, and 8 can be carried out at RT without any significant adverse effect. 9. The pellets are finally resuspended in 200 ml of RNase-free water by incubating them for 30 min at room temperature. If time permits, overnight incubation at 4 C yields maximum RNA levels. After resuspension, the samples are centrifuged for 1 min in the microcentrifuge at full speed to pellet any cell debris. The supernatants, containing the RNA, are transferred to new Eppendorf tubes and should be stored at 4 C if they are to be used immediately. Long-term storage should be at –20 C. Repeated freezing and thawing should be avoided to prevent degradation.

4. Isolation of Total RNA from Stationary Phase Cells Isolation of RNA from stationary phase cells has been found to be difficult, because of poor cell lysis, using either Catrimox-14 or Catrimide. Thus, the preceding method has been modified as follows.

4.1. Reagents and buffers TM buffer (5), sodium azide (1 M), SB, lysis buffer, acetic acid (20 mM) are as described in the section on isolation of total RNA from exponentially growing cells. SDS (sodium dodecyl sulfate, 20%), sodium acetate (3 M, pH 5.2), acid phenol equilibrated with Tris-HCl (pH 4.5-5.2) (Sambrook et al., 1989), phenol:chloroform (5:1 solution, pH 4.5, Ambion, TX, USA), and EtOH (70% and 100%).

4.2. Procedure 1. Cells are grown overnight with shaking (16–18 h) from a single colony. No more than 2 ml of culture (faster-growing strains need only 1 ml of culture) are mixed with 7 ml of crushed ice SB in a centrifuge tube and held on ice until all samples are collected. 2. The cells are collected by centrifugation at 5000 rpm for 5 min at 4 C in a refrigerated centrifuge. Each pellet is resuspended in 600 ml of ice-cold lysis buffer. 3. The suspension is transferred to a chilled (in dry ice–EtOH), baked, screw-cap tube for four cycles of freeze and thaw treatment in dry ice– EtOH and 37 C water bath.


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4. After the fourth cycle of freezing, 100 ml of 20 mM acetic acid is added to the frozen cells and each sample is transferred to a 37 C water bath. After 1 min, 30 ml of SDS (20%) is added and the suspension is incubated further for 2 min. Clearing of the suspension is indicative of good cell lysis. 5. The cell lysate is carefully transferred to a new 1.5-ml Eppendorf tube containing 600 ml of acid phenol (pH 4.5–5.2) and mixed thoroughly by vortexing for 1 min. The mixture is centrifuged at 13,000 rpm in a microcentrifuge for 5 min at RT. 6. The top aqueous layer is transferred to a new Eppendorf tube and extracted with an equal volume of acid phenol. The final extraction is carried out an additional two times using an equal volume of phenol:chloroform (5:1 solution, pH 4.5). 7. After the fourth extraction, 0.11 vol of 3 M NaOAc (pH 5.2) and 2.5 vol of 100% EtOH are added to the supernatant. 8. The RNA is precipitated at –20 C for at least 2 hr (can be left overnight). The RNA is pelleted by centrifuging for 15 min at 13,000 rpm in a microcentrifuge in the cold room. The RNA pellet is then washed with 1 ml of cold 70% ethanol and dried either at RT (15 min) or in a Speed Vac Concentrator (5 min maximum). The RNA pellets are dissolved in 200 ml of RNase-free water and stored as described previously.

5. Removal of DNA Contamination DNA contamination can be a major problem, particularly if the RNA will be used for either dot-blot analysis or cDNA synthesis. RNA isolated from exponentially growing cells using the Catrimide method is relatively free from DNA contamination (containing 5%) of residual DNA contamination (Fig. 1.1, lanes 4–5) despite a DNase I step during the initial isolation. This contamination is due primarily to a higher cell concentration in stationary phase cultures compared to the log phase cultures and can be minimized by using smaller cell volumes. We routinely use the DNA-free kit (Ambion, Austin, TX) as per manufacturer’s instructions to remove DNA from total RNA. This is a quick process and avoids use of phenol and chloroform to remove the DNase I from the reaction mixtures.

6. RNA Yield and Purity Assessment The RNA samples are diluted 100-fold in RNase-free water and the optical density (OD) at 260 and 280 nm is measured in duplicate. The amount of RNA (mg/ml) in the original sample is calculated by using the formula: average OD at 260 nm 40 dilution factor (100). The ratio of A260/280 is obtained to determine if RNA is contaminated with protein. A typical RNA isolation from a wild-type strain using preceding methods yields 200 mg/sample (Table 1.1) with an A260/280 ratio of 1.8–2.0. Although any spectrophotometer is acceptable for these measurements, extremely accurate measurements of 1-ml samples are obtained using an ND-1000 instrument (Nanodrop, Thermo Fisher Scientific).

7. RNA Integrity Assessment It is very important to check the integrity of each RNA sample before carrying out any further experiments, as OD measurements do not indicate whether a sample has been degraded. We routinely check the quality of the RNA by running the samples in agarose minigels. Five hundred ng of total RNA is mixed with 3–4 ml of 1 RNA loading dye (Table 1.2) and separated in a 1% agarose minigel (5 10 cm) in 1 TAE buffer (Tris-acetate/EDTA, [Sambrook et al., 1989]) at 100 V. The gel is stained with ethidium bromide and viewed with a UV transilluminator. Visualization of intact rRNAs with a ratio of 23S to 16S rRNA of approximately 2/1 (Fig. 1.1, lanes 1–2) ensures that the RNA has not been significantly degraded (Fig. 1.1, lane 3). In many cases, we see that the amount of RNA observed in the ethidium bromide stained gel varies among the samples even though all lanes have been loaded with 500 ng of RNA based on OD measurements. These discrepancies in quantification may be due to either pipetting errors or

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Table 1.2 Polyacrylamide gel solution and loading dye for Northern analysis Gel solution (6%) with 7 M urea

Gel

Urea TBE (5) Acrylagel (30%) Bis-acrylagel (2%) Water Ammonium persulfate (APS)(10%) TEMED

14.7 g 7.0 ml 7.0 ml 5.6 ml 4.3 ml 280 ml 28 ml

Acrylagel and Bis-acrylagel solutions are obtained from National Diagnostics, GA, USA. APS is made fresh. To obtain 8.3-M urea concentration, 17.4 g of urea is added to the solution. RNA loading dye (2) Xylene cyanol Bromophenol blue Na2EDTA.2H2O Deionized formamide

30 mg 30 mg 37 mg 10 ml

Tris-acetate-EDTA (TAE) (10) Tris base Sodium acetate EDTA Adjust the pH to 7.8 with glacial acetic acid

100 mM 50 mM 5 mM

incorrect OD measurements. To distinguish these possibilities, we use a second method to normalize samples in a particular experiment. The amount of RNA loaded onto the minigel is reduced from 500 ng to less than 100 ng and stained with Vistra Green (GE Healthcare). The gel is then scanned using a PhosphorImager (GE Healthcare, Storm 480) and the rRNA bands (both 16S and 23S) are quantified using Imagequant software. The difference in the amount of rRNA bands in each sample compared to the wild-type strain is then used to normalize all the samples.

8. Isolation of Polyadenylated RNA Isolation of polyadenylated RNA from E. coli is a challenge as only 5 mM and at KCl concentrations >200 mM. However, it was possible to distinguish the activity of the archaeal proteins from the activity of RNase A. In a different approach, we used a bioinformatics prediction of the archaeal exosome to purify this protein complex from S. solfataricus. Isolation by coimmunoprecipitation revealed the presence of four orthologs of eukaryotic exosomal subunits and at least one archaea-specific subunit. We characterized the S. solfataricus exosome as a major enzyme involved in phosphorolytic RNA degradation and in RNA polyadenylation. Here we describe in detail the techniques used to achieve these results.

RNA Degrading Activities in Archaea

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1. Introduction Different types of RNA are often transcribed as precursors, which are processed by enzymes, mostly ribonucleases (RNases), into mature functional molecules. These mature RNAs can be very stable (transfer RNA, ribosomal RNA) or they can be a subject of fast turnover (most messenger RNAs) (Deutscher, 2006; Houseley et al., 2006). Small noncoding RNAs have different functions and stabilities (Matera et al., 2007; Vogel et al., 2003). The half-life of eukaryotic messenger RNAs (mRNAs) ranges from tens of minutes to hours (Raghavan et al., 2002). Bacterial mRNAs are much less stable; their half-lives are in the range of seconds to minutes, enabling quick responses to changing environmental conditions (Bernstein et al., 2002; Rauhut and Klug, 1999). Nothing was known about the halflives of archaeal mRNAs at the beginning of our studies. It is well documented that in bacteria the half-lives of specific mRNA molecules significantly vary under different growth conditions—a phenomenon known to control gene expression at the posttranscriptional level. The half-life of an mRNA depends on the presence of destabilizing elements, which are recognized by degrading enzymes, and stabilizing elements, which interfere with RNase activities (Condon, 2007; Rauhut and Klug, 1999). Known stabilizing elements include the triphosphate at the mRNA 50 -end and hairpin loops at the mRNA 50 -end or 30 -end. Cleavage sites for the endoribonucleases RNase E and RNase III, and short poly(A)-tails at the 30 -end, which serve as a loading platform for the 30 to 50 -exoribonucleases PNPase, RNase II, and RNase R, destabilize RNA. RNases are often found in complexes with other RNases and with RNA helicases like RhlB, which help to unwind secondary structures. For example, PNPase is found in E. coli together with RNase E, RhlB, and other proteins in a protein complex called the degradosome (see chapters by Carpousis et al., Mauria and Deho` and Mackie et al.), but it also forms an independent complex only with RhlB (Condon, 2007; Lin-Chao et al., 2007). Bacterial enzymes involved in mRNA degradation also participate in the processing and degradation of stable RNA molecules like rRNA and tRNA (Deutscher, 2006). The preceding mechanisms were well studied in the model organism Escherichia coli, and similar decay pathways seem to exist in other gram-negative bacteria. Different mechanisms operate in Bacillus subtilis, a gram-positive bacterium that harbors a very recently characterized 50 to 30 -exoribonuclease (Condon, 2007; see chapters by Bechhofer et al. and Condon et al.). Control of gene expression on the posttranscriptional level is seemingly more complex in Eukarya (Moore, 2005; Zamore and Haley, 2005). Eukaryotic mRNA stabilizing elements are the 50 -methylated guanosine cap at the 50 -end and the long poly(A)-tail at the 30 -end. The removal of

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Elena Evguenieva-Hackenberg et al.

poly(A) and decapping are followed by exoribonucleolytic degradation in a 50 to 30 -direction by enzymes like Xrn1 or in a 30 to 50 -direction by the exosome, a protein complex with structural similarity to PNPase. Very recently, short destabilizing poly(A)-tails were also described in Eukarya (LaCava et al., 2005; Vanacova et al., 2005). This very short and incomplete depiction of important details concerning mechanisms for mRNA degradation in Eukarya (see chapters in Maquat and Kiledjian, RNA Turnover, Part B) already outlines differences and similarities to bacterial decay pathways. Generally, the molecular mechanisms operating in Archaea show more similarities to the eukaryotic ones, despite Archaea having a more prokaryotic-like than eukaryotic-like cellular structure. Very little is known about RNA degradation in Archaea. At the begin of our studies, proteins with homology to RNase III were not found in the sequenced archaeal genomes, the existence of an RNase E–like activity in archaeal extracts was reported, and orthologs of bacterial and eukaryotic exoribonucleases were predicted (Anantaraman et al., 2002; Franzetti et al., 1998; Koonin et al., 2001; Zuo and Deutscher, 2001). The structural features of mRNA that would be important for mRNA stability were assumed to resemble those of bacteria, because archaeal mRNAs are not capped and do not carry long poly(A)-tails (Brown and Reeve, 1985). Our work focuses on RNA stability in Archaea and on the identification of archaeal ribonucleases.

2. In Vivo Studies on RNA Degrading Activities in Archaea One aim of our studies was to analyze whether there is posttranscriptional regulation of gene expression in Archaea. Therefore, we studied the half-lives of selected mRNAs in the halophilic archaeon Haloferax mediterranei under different growth conditions. Furthermore, we wanted to obtain general insight into mRNA stability in Archaea. For this purpose, we used the halophilic archaeon Halobacterium salinarum NRC-1, the complete genome sequence of which was available, in microarray studies.

2.1. In vivo labeling of RNA and inhibition of transcription with actinomycin D To understand the role of degradation of certain mRNAs in gene expression, it is necessary to follow mRNA decay in vivo under different conditions. The amount of individual RNA species in total-cell RNA represents a steady-state level, which is determined by the rates of RNA synthesis and decay. One method to follow RNA decay is to determine RNA abundance


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at different time points after efficiently blocking transcription. In Bacteria, transcription is usually blocked by the addition of rifampicin, which directly interacts with RNA polymerase, to cell cultures. The archaeal RNA polymerase shares similarity with the eukaryotic RNA polymerase and is different from the bacterial enzyme. Accordingly, the archaeal RNA polymerase is insensitive to rifampicin, like its eukaryotic counterpart (Langer et al., 1995; Prangishvilli et al., 1982). We decided to use actinomycin D, which is known to interact with DNA and, thereby, block RNA transcription in Bacteria and in Eukarya (Goldberg et al., 1962; Reich et al., 1961) to block transcription in Haloferax and Halobacterium. To measure the efficiency of the block, it is necessary to have a method to detect RNA synthesis in vivo. Incorporation of radiolabeled uracil or uridine into TCA (trichloroacetic acid) precipitable material can generally be used to determine the in vivo rate of total-cell RNA synthesis (Pato et al., 1973). To monitor the inhibition of transcription by the addition of chemicals, cultures of Haloarchaea were grown to early exponential phase (OD600nm ¼ 0.45) or stationary phase (OD600nm ¼ 2.8). Radiolabeled uridine ([5,6-3H], 32 Ci/mmol; Amersham Biosciences) was added at time point 0 to a final concentration of 100 mCi/ml when exponentially growing cultures were used or 300 mCi/ml when stationary-phase cultures were used. Samples of 20 ml were collected at several time points thereafter and mixed with 500 ml of unlabeled cells. Cold TCA was added to a final concentration of 11.5% (w/v). After incubation on ice for 5 min, samples were centrifuged, and TCA-precipitated radioactivity was quantified with a scintillation counter (LS 6500 Multi Purpose Scintillation Counter, Beckman). Optimal results were obtained when 500 ml of Rotiszint Ecoplus (Roth, Germany) cocktail was used to dissolve the pellet (Hundt et al., 2007; Ja¨ger et al., 2002). To test the effect of chemicals on transcription, the agent was added 12 min after the addition of uridine. Actinomycin D was dissolved overnight at 4 C in water to generate a stock of 5 mg/ml that was used the next day. In H. mediterranei and H. salinarum NRC1, a final concentration of 100 mg/ml of actinomycin D efficiently blocks RNA synthesis ( Ja¨ger et al., 2002). It was shown by Bini et al. (2002) that actinomycin D is stable enough at high temperatures and acidic pH to be used to block transcription in Sulfolobus solfataricus. A final actinomycin D concentration of 10 mg/ml is sufficient to block RNA synthesis efficiently in this archaeon; the stock solution of (1 mg/ ml) was prepared in 50% ethanol. In S. solfataricus, uridine is not incorporated to significant levels but uracil ([5,6-3H], 33.1 Ci/mmol) is well incorporated. For in vivo labeling, S. solfataricus was grown to early exponential phase (OD540nm ¼ 0.17). Time linear synthesis was observed when 189 mCi/108 cells were used (Bini et al., 2002). In another study, Andersson et al. (2006) used actinomycin D, which was dissolved in DMSO to a final concentration of 15 mg/ml, to block transcription in S. solfataricus and Sulfolobus acidocaldarius.

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2.2. RNA isolation from Archaea and Northern blot analysis Total-cell RNA from haloarchaea for use in Northern blot analysis can be isolated by one of three methods. In the standard hot phenol method, a 50-ml centrifuge tube for each sample was filled with 20 ml of ice-cold culture medium and stored on ice; 7 to 10 ml of cultured cells was added to this tube, and cells were sedimented in a Sorval centrifuge SA600 rotor (6000 rpm, 4 C) for 10 min. The cell pellet was resuspended in 0.5 ml of ice-cold culture medium and transferred to a clean, nonautoclaved 1.5-ml tube. Cells were pelleted in a tabletop centrifuge (8000 rpm, 4 C) for 5 min and used immediately for RNA isolation. Alternately, they were frozen in liquid nitrogen and stored at 80 C. Cells were resuspended in 125 ml of cold resuspension buffer (0.3 M saccharose, 0.01 M sodium acetate, pH 4.5), mixed with 125 ml 2% SDS in 0.01 M sodium acetate, pH 4.5, and incubated at 65 C with a water bath or a heat block for 90 sec. After this, 250 ml of phenol water (e.g., Roth readyto-use solution is the name of the product (Phenol saturated with water) preheated to 65 C) was added. Samples were thoroughly mixed, incubated at 65 C for 3 min, and then frozen in liquid nitrogen for at least 30 sec. After incubation for 2 min at room temperature (to warm the plastic ware, which may crack when centrifuged at very low temperatures), centrifugation was performed at 13,000 rpm for 10 min at room temperature. The aqueous phase was transferred into a new 1.5-ml tube. Phenol treatment was repeated two times; 40 ml 3 M sodium acetate, pH 4.5, was added followed by ethanol precipitation. After centrifugation (13,000 rpm, 4 C) for 20 min, the pellet was washed once with 80% ethanol, air dried, and resuspended in 180 ml of storage buffer (20 mM NaPO4, pH 6.5, 1 mM EDTA); 20 ml of 10 DNase buffer (200 mM sodium acetate, pH 4.5, 100 mM MgCl2, 100 mM NaCl) were added together with 1 ml DNase I (e.g., Life Technologies, diluted 1:5 in 1 DNase buffer), and samples were incubated at room temperature for 30 min; 20 ml of 250 mM EDTA, pH 7, was added, and the DNase was removed by two phenol chloroform extractions. After final ethanol precipitation, the RNA was resuspended in a suitable volume of storage buffer (usually 50 to 100 ml). The second method follows the protocol of Nieuwlandt et al. (1995) followed by two phenol/chloroform extraction steps. Cells were pelleted and washed as described for the hot phenol method. Then 1.25 ml lysis buffer (2% SDS in 0.01 M sodium acetate, pH 4.5) and 35 ml DEPC were added, and the cells were resuspended by shaking and the use of a vortex mixer. Samples were incubated for 15 min at 37 C and then transferred to ice for an additional 15 min; 625 ml NaCl-saturated water was added to the lysate, and samples were gently mixed several times. Do not use a vortex mixer. Samples were incubated on ice for 15 min. The SDS-DNAprotein-aggregate was sedimented in a tabletop centrifuge (13,000 rpm,


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4 C or room temperature) for 30 min; 1 ml of the supernatant was carefully transferred into a nonautoclaved 2-ml tube. Two phenol/chloroform extractions and ethanol precipitation followed. Third, total RNA was alternately prepared from harvested cells by use of the RNeasy Midi kit (Qiagen, Hilden, Germany) including a DNase I treatment according to the manufacturer’s instructions. RNA isolated by this method also proved useful for microarray analyses on mRNA decay in Haloarchaea (Hundt et al., 2007). For studies of mRNA half-lives in S. solfataricus (Bini et al., 2002), RNA was isolated by acid guanidinium thiocyanate–phenol chloroform extraction (Chomczinski and Sacchi, 1987). For Northern blot analyses the RNA was dissolved in storage buffer, 7 to 10 mg of RNA per lane are run on a 1% (w/v) agarose, 2.2 M formaldehyde gel, and transferred to nylon membrane (e.g., Pall Biodyne B) by vacuum pressure blotting according to the manufacturer’s recommendations. Specific DNA fragments were radiolabeled with a32P-dCTP with nick translation (e.g., Nick translation kit, Amersham Biosciences) and purified on microcolumns (e.g., Probe Quant G-50, Amersham Biosciences). Aliquots of 210 cpm are used per hybridization reaction. The signals were detected and quantified with a BioRad molecular imager and the Quantity One (BioRad) software. With the preceding techniques, we analyzed transcripts of the mc-gvp operons for gas vesicle formation in Haloferax mediterranei. The determined half-lives were between 4 and 80 min. The stability of gvpA mRNA or a 0.45-kb transcript population derived from the 50 -part of gvpD differed when the cultures were grown at different salt concentrations or to different densities ( Ja¨ger et al., 2002). This strongly suggests that, like in Bacteria and Eukarya, mRNA processing contributes to regulated gene expression in Archaea. We also studied mRNA degradation on a global level with DNA microarrays and RNA prepared from exponentially growing cultures of Halobacterium salinarum NRC-1 using the RNaesy Midi Kit (Qiagen). The technical details for performing microarrays and for the data analysis are not given here. The half-lives of the transcripts ranged from 5 min to more than 18 min, with an overall mean half-life of 10 min. We found some relationship between gene function and transcript stability but no correlation between transcript length and its stability, providing initial insights into mRNA turnover in a euryarchaeon (Hundt et al., 2007). Stability of mRNA was also studied in the crenarchaeota S. solfataricus and S. acidocaldarius by other research groups. In a genome-wide study, the median mRNA half-life in the two species was determined to be 5 min (Andersson et al., 2006). In contrast, Bini et al. (2002) determined an average half-life of 54 min with selected mRNAs and Northern blot analysis.

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3. In Vitro Studies on Novel RNA Degrading Activities in Archaea We aimed to identify and to characterize ribonucleases (RNases) from Archaea to understand the mechanisms for RNA degradation. In two independent attempts to purify novel RNases from the halophilic archaeon H. salinarum and from the thermoacidophilic archaeon S. solfataricus, very different proteins with similar endoribonucleolytic activity were purified. In an alternate approach, a protein complex with predicted RNase function— the exosome—was purified from S. solfataricus and was subsequently characterized. For the detection of RNase activity in protein fractions and for the characterization of recombinant proteins as RNases, tests for ribonuclease activity must be performed and evaluated. For this, it is necessary to prepare suitable substrates, to incubate them with the proteins of interest, to separate the reaction products from the substrate, and to detect them. The techniques used by our group for these purposes are described in the following.

3.1. Detection of RNA degrading activity in vitro and determination of cleavage sites 3.1.1. RNA substrates The following types of radioactively labeled RNA substrates were used in our studies: synthetic poly(A) oligoribonucleotide (30-mer, CureVac GmbH, Tu¨bingen, Germany) labeled at the 50 -end by T4 polynucleotide kinase, the same oligoribonucleotide labeled at the 30 -end by polyadenylation with poly (A)-polymerase I, and internally labeled transcripts of different lengths produced in vitro with T7 RNA polymerase (Conrad et al., 1998; EvguenievaHackenberg et al., 2002; Wagner and Klug, 2007; Walter et al., 2006). 3.1.2. Labeling and purification of substrates For 50 -end labeling, 10 pmol of the poly(A) oligoribonucleotide was incubated with T4 polynucleotide kinase (NEB or Fermentas) and 30 mCi [g-32P] ATP (3000 Ci/mmol) in a 10-ml reaction mixture containing buffer supplied by the manufacturer for 30 to 60 min at 37 C. The reaction was stopped by adding 50 ml STE buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA), and the unincorporated nucleotides were removed with ProbeQuant G-25 Micro columns (Amersham Pharmacia Biotech) or MicroSpin G-25 columns (GE Healthcare). For labeling at the 30 -end with in vitro polyadenylation, 50 pmol poly(A) oligoribonucleotide was incubated with 30 mCi [a-32P]- ATP (3000 Ci/mmol) and poly(A) polymerase I (Invitrogen) in PAPI-buffer (100 mM Tris, pH 8, 200 mM NaCl, 20 mM MgCl2, 2 mM EDTA, 2 mM DTT, and 2.5 mM MnCl2) in a 20-ml


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reaction mixture for 30 min at 37 C. After addition of 30 ml STE buffer, the unincorporated nucleotides were removed as just described for the 50 -end labeling. The labeled substrates were stored at 20 C. In vitro transcription with T7 RNA polymerase in the presence of [a-32P]UTP, and purification of the resulting labeled transcripts in denaturing gels was performed as previously described with some modifications (Conrad et al., 1998; Milligan and Uhlenbeck, 1989). When cloned DNA was the substrate for in vitro transcription, the plasmid was linearized with a suitable restriction endonuclease to enable runoff transcription, thus determining the 30 -end of the transcript. When we used pUC18 for cloning, the primer matching the sequence of the RNA substrate (sense primer) was extended by the T7 promoter sequence at the 50 -end. Alternately, we used vectors containing the T7 promoter (for example, pDrive, Qiagen). When the T7 promoter was present in the sense primer, PCR products were also directly used for in vitro transcription. In addition, we used oligonucleotides with an annealed 18-mer promoter oligonucleotide for in vitro transcription of very short RNAs, such as the N26 transcript (Conrad et al., 1998; Evguenieva-Hackenberg and Klug, 2000; Schweisguth et al., 1994; Wagner and Klug, 2007). When T7 RNA polymerase and the supplied buffer from NEB were used, reaction mixtures contained ribonucleoside triphosphates (rNTPs), where the concentration of rUTP was lower than that of the other rNTPs (final concentrations: 0.5 mM rATP, 0.5 mM rGTP, 0.5 mM rCTP, 0.1 mM rUTP) and 20 mCi [a-32P]UTP (3000 Ci/mmol). Alternately, when the T7-MEGAshortscript High Yield Transcription Kit (Ambion) was used according to the recommendations of the manufacturer, we added 20 mCi [a-32P]-UTP (3000 Ci/mmol) in a final volume of 20 ml. Each in vitro transcription reaction contained 1 to 3 mg of plasmid DNA, approximately 200 ng of PCR product or 5 pmol template oligonucleotide with annealed T7 promoter oligonucleotide (25 pmol template oligonucleotide and 30 pmol T7-oligonucleotide were heated for 5 min at 70 C and incubated for 30 min at 25 C in 50 ml 10 mM Tris-HCl, pH 8). The in vitro transcription was performed for 2 to 4 h at 37 C. Internally labeled in vitro transcripts were purified with denaturing gel electrophoresis in a 6 to 12% polyacrylamide gel (25 cm 25 cm 0.5 mm) containing 8 M urea and Tris-borate-EDTA (TBE) buffer as the running buffer. Samples were amended with formamide containing dye (80% v/v deionized formamide, 6 M urea, 1 TBE buffer, 0.1% (w/v) xylene cyanol, and 0.1% (w/v) bromphenol blue), heated for 10 min at 65 C, placed on ice, and loaded after excess urea was removed from the slots by flushing them with a syringe and TBE. If radiolabeled RNA substrate does not enter the gel because of high protein content, samples can be treated with proteinase K (Roche Diagnostics, PCRGrad) before adding the formamide loading dye. Usually, the electrophoresis was performed for 90 min at 500 V. After removing one glass plate, the gel was wrapped with cling film and exposed together with the other glass plate on

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an X-ray film for 1 to 2 min; illumination with red light was performed during this time to visualize the slots and to enable the localization of the transcript in the gel thereafter (shadowing). The desired bands were cut out and the substrates eluted from the crushed gel pieces for 1 to 2 h at room temperature in 100 ml RNA elution buffer (0.5 M sodium acetate, pH 5.0, 1 mM EDTA, pH 8.0, 2.5% v/v phenol). Freeze–thaw cycles and repeated elution maximized the yield of extraction, which was followed in a scintillation counter. After phenol/chloroform extraction, RNA was precipitated with isopropanol, dissolved in RNase-free water, and stored at 20 C. Incorporated radioactivity was determined in a scintillation counter. 3.1.3. RNA degradation assays A typical 10-ml degradation assay was performed with 1000- to 5000-cpm substrate. The used enzymes or protein fractions, substrates, buffers, incubation times, and temperatures varied and were published; many examples are given in the corresponding text that follows. In general, small reaction tubes (0.5 ml) help to minimize evaporation of water. Relatively short incubations of larger reaction volumes at low temperatures (up to 37 C) can be done in a heating block or water bath. Long incubations of small volumes and incubations at high temperatures (60 C to 80 C) were done in a heating oven or in a thermocycler. 3.1.4. Thin-layer chromatography For separation of the reaction products, denaturing gel electrophoresis in TBE buffer (see 3.1.2.) or thin-layer chromatography (TLC) was performed. TLC was used to resolve nucleoside monophosphates and nucleoside diphosphates that are produced by the hydrolytic and phosphorolytic activity of RNases, respectively. The nucleosides were separated with 0.1 mm cellulose MN 300 polyethylenimine (PolygramÒ CEL 300 PEI TLC plate; Machery & Nagel) in 0.9 M guanidinium hydrochloride, pH 6.3; 3 to 6 ml of each reaction mixture (without any loading dye) was spotted 5 to 10 mm apart. After drying, the thin layer plate was soaked for 20 min in methanol and completely dried before performing the chromatography in a closed chamber. The signals were detected and quantified with a BioRad molecular imager and Quantity one (BioRad) software. To obtain UDP and UMP or AMP and ADP standards, we incubated the substrate with a PNPase-containing fraction from E. coli and with an RNase R–containing fraction from Pseudomonas syringae, respectively, as previously described (Purusharth et al., 2005). Briefly, 20 fmol substrate (1000 to 3000 cpm) was incubated with highly active degradosome fractions containing approximately 5 ng PNPase or RNase R as determined by silver staining. The reaction mixture of 10 ml contained in addition 25 mM TrisHCl, pH 8.0, 5 mM MgCl2, 60 mM KCl, 100 mM NH4Cl, 0.5 mM dithiothreitol, 5% glycerol, and 10 units RNasin (Promega). In addition,


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10 mM inorganic phosphate was present in the reaction buffer for RNA degradation by PNPase. The incubation of the samples was performed for 10 min at 22 C. 3.1.5. Primer extension analyses The exact cleavage sites of an RNase were identified by determination of 50 -ends of the distal cleavage products by primer extension analysis. The unlabeled RNA substrate was incubated with the protein of interest under suitable conditions. Generally, these cleavage tests were performed in a way that intact substrate and cleavage products were detectable. After phenolchloroform extraction and ethanol precipitation, RNA was dissolved in water and primer extension was performed. RNA was incubated with 1 to 2 105 cpm 50 -32P–labeled primer in a total volume of 8 ml in 5 mM TrisHCl, pH 7.5, 1 mM EDTA for 5 min at 70 C. Within 10 min, the mixture was cooled down to 50 C, kept for 5 min on ice, and brought back to room temperature. Thereafter, 1.5 ml of 40 mM sodium pyrophosphate, AMV reverse transcriptase reaction buffer (1 final concentration), 8 units of AMV reverse transcriptase, 10 units of RNasin (Promega) and 1 mM of each deoxyribonucleotide triphosphate (dNTP) were added in a final volume of 20 ml. Reverse transcription was carried out for 10 min at 37 C, 1 h at 42 C, and 50 min at 50 C. The reaction was stopped by adding 30 ml of formamide loading buffer. Radioactively labeled (T7Sequencing kit, USB) reactions of the cloned DNA template were loaded on the same gel to map the positions of the cleavage sites.

3.2. Isolation of RNA degrading activities by fractionation of S. solfataricus cell lysates In 1999, we started our attempts to isolate an RNase III–like activity from the thermoacidophilic archaeon Sulfolobus solfataricus P2. In Bacteria and Eukarya, RNase III and the proteins belonging to the RNase III family are important double-strand (ds)–specific RNases that are involved in RNA processing and degradation (Gegenheimer and Apirion, 1981; Huntzinger et al., 2005). Archaea do not harbor proteins with sequence similarities to RNase III (Anantharaman et al., 2002), but this does not exclude the possibility that novel archaeal RNases exist, which are specific for dsRNA and are different from the already known intron endonuclease (KlemanLeyer et al., 1997). To identify endoribonucleases, which cleave dsRNA, from S. solfataricus, we monitored protein fractions for such activity with N26, a well-characterized small substrate of RNase III from E. coli derived from phage T7 R1.1 (Conrad et al., 1998; Schweisguth et al., 1994). This substrate is a mostly double-stranded RNA consisting of 46 bases. S. solfataricus was grown at 70 C in a fermenter with air supply of 10 L/ min1 for 5 days in the following medium: 1 g yeast extract, 1 g casamino

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acid, 3.1 g KH2PO4, 2.5 g (NH4)2SO4, 0.2 g MgSO4 7 H2O, 0.25 g CaCl2 2H2O, 0.1 ml of the following solutions: 1.8% MnCl2 4 H2O and 4.5% Na2B4O7 10 H2O, and 10 ml of each of the following solutions: 2.2% ZnSO4 7 H2O, 0.5% CuCl2 2 H2O, 0.3% NaMoO4 2 H2O, 0.15% CoCl2 6 H2O per 1 L. The medium was adjusted to pH of 4.2 to 4.4, autoclaved, and 10 ml sterile filtered 0.3% VOSO4 2 H2O per 1 L was added. S. solfataricus cells (60 g) were resuspended in 120 ml cold extraction buffer (Rauhut et al., 1995; 50 mM Tris-HCl, pH 7.9, 0.25 M KCl, 2 mM EDTA, 1 mM b-mercaptoethanol, 0.1 mM dithiothreitol [DTT], and 0.5 mM phenylmethylsulfonyl fluoride [PMSF]) and opened by sonication. After 45 min of ultracentrifugation at 4 C and 100,000g, 2 ml of the cytoplasmic fraction was tested for endonucleolytic activity with 3000 cpm of the radioactively labeled in vitro transcript N26 as substrate in a final volume of 10 ml in the reaction buffer TMKG (30 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 130 mM KCl, and 5% glycerol). These buffer conditions were used in our laboratory for specific in vitro processing of dsRNA by RNase III (Conrad et al., 1998). The 47-nt N26 substrate is a doublestranded RNA with a central bulge comprising of one major scissile bond; the cleavage products have sizes of 38 nt and 9 nt. The RNase III binding site in this minimal RNase III substrate is located between the bulge and the terminal loop (Schweisguth et al., 1994; Zhang and Nicholson, 1997). We analyzed the N26 substrate by mfold (Mathews et al., 1999). The same secondary structures was predicted at temperatures between 20 C and 50 C; 11 nucleotides at the 50 -end and 12 nucleotides at the 30 -end were proposed to form single-stranded regions at higher temperatures. Thus, at higher temperatures only the part representing the functional RNase III–binding site is expected to remain double stranded. We performed the RNA degradation assays for 5 min at 45 C and in parallel at 65 C. Substrate and degradation products were analyzed on 10% denaturing polyacrylamide gels (2.1). We observed two degradation products of approximately 35 nt and 19 nt. The 35-nt product may nearly arise by cleavage at the RNase III cleavage site; the product of 19 nt may be due to a cleavage in the region remaining double stranded even at high temperatures. An identical degradation pattern was observed when different incubation temperatures between 30 C and 90 C were applied, and the highest activity was detected at 80 C. Further assays were performed at 70 C, and the endoribonucleolytic activity that produced this cleavage pattern was followed in the subsequent fractionation experiments. The cytoplasmic fraction was subjected to precipitation with ammonium sulfate at 25%, 35%, 50%, 65%, 80%, and 100% saturation. The precipitated proteins were pelleted and dissolved in TEG buffer (40 mM Tris, pH 8.0, 0.1 mM EDTA, 5% glycerol), dialyzed against the same buffer, and tested for activity. The endoribonucleolytic activity precipitated between final ammonium sulfate concentrations from 35 to 65%.


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These fractions were pooled and used for further purification by chromatography after dialysis against TEG buffer. The chromatography steps were performed with the FPLC System (Pharmacia). The following columns were used in this order: HiTrap heparin column (Pharmacia), cation exchanger (BioRad Econo-Pac S-cartridge), anion exchanger (BioRad Econo-Pac Q-cartridge), and HiLoadTM Superdex 200 size exclusion chromatography column (Pharmacia). The columns were used according to the instructions of the manufacturer. The elution fractions were dialyzed if necessary and were tested for activity. In parallel, their protein content was visualized in silver-stained 10% SDS-polyacrylamide gels. The endoribonuclease activity was associated with a major peak in the elution profiles and, consequently, the corresponding protein fractions contained many proteins even after the application of the four different columns. The apparent native molecular weight of the endoribonuclease-containing fractions corresponded to a 90-kDa protein, as determined by gel filtration. After this step, the endonuclease-containing pool was applied on preparative 8% native PAGE, 0.25 cm slices were cut, and the proteins recovered by diffusion. Two major proteins were detected in the fractions with the highest RNase activity. They were transferred to Immobilon-PVDF membrane (Millipore) and identified by N-terminal sequencing. The 50-kDa band corresponded to an aspartate semialdehyde dehydrogenase (NCBI accession number AAK41162), whereas the 44-kDa band was found to represent an acyl-CoA dehydrogenase (NCBI accession number AAK42872; Evguenieva-Hackenberg et al., 2002).

3.3. Characterization of the recombinant RNA degrading dehydrogenases from S. solfataricus On the basis of the knowledge that eukaryotic dehydrogenases can bind RNA and that certain RNases share homology with dehydrogenases (Baker et al., 1998; Nagy and Rigby, 1995), we characterized the RNase properties of the two S. solfataricus dehydrogenases after their purification as His-tagged proteins from E. coli. We used the vector pQE30, and the genes of interest were cloned in frame from the second codon to the stop codon. The recombinant proteins could not cleave the N26 transcript in the TMKG buffer, but they produced a different cleavage pattern in the same buffer without MgCl2 (TKG buffer). This third type of N26-cleavage pattern (the approximate sizes of the detected products were 42 nt and 36 nt) was also produced by the original S. solfataricus proteins when MgCl2 was omitted from the reaction mixture. This type of cleavage reaction was possible at MgCl2 concentrations up to 5 mM; the optimum was achieved at 60 C, pH between 6.0 and 7.5, and at 50 mM KCl. In addition to the N26 substrate, other structured transcripts derived from bacterial pre-rRNAs were also endonucleolytically cleaved in TKG buffer by the recombinant S. solfataricus

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proteins and by the original S. solfataricus fractions. Further assays were routinely performed at 60 C. During characterization of the recombinant proteins, we observed that the RNA cleavage reaction was blocked in presence of RNasin, an RNase A inhibitor (Promega). Moreover, we found that RNase A in femtomolar concentrations produces similar RNA cleavage patterns as the recombinant dehydrogenases in nanomolar concentrations. Similar cleavage patterns were also produced by some bacterial and eukaryotic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins. Primer extension analyses demonstrated that the dehydrogenasecontaining fractions and RNase A cleave the substrates in loops and bulges between pyrimidine and A, preferentially between C and A. Thus, it was necessary to clarify whether the RNase activity of the dehydrogenases was due to contamination with RNase A or, indeed, is their intrinsic property. We observed that the activity of RNase A can be distinguished from the RNA degradation by dehydrogenases by the addition of 50 ng ml1 tRNA or single-stranded DNA (ssDNA). Whereas the presence of tRNA or ssDNA inhibits the degradation of the N26 transcript by the dehydrogenases, the RNA cleavage by RNase A was enhanced. To exclude the possibility that the excess of dehydrogenase polypeptides changes the behavior of contaminant RNase A in the reaction mixture, control reactions were performed with pure RNase A in femtomolar concentration together with dehydrogenases in nanomolar concentration. In the presence of ssDNA, RNase A still efficiently cleaved the radioactively labeled RNA substrate, strongly suggesting that the RNase activity of the dehydrogenases is similar to, but distinct from, that of RNase A. In addition, we subcloned and purified His-tagged deletion derivatives of the aspartate semialdehyde dehydrogenase from S. solfataricus. A polypeptide consisting of the 205 N-terminal amino acids, which contains a predicted Rossmann fold, showed RNase activity, whereas a polypeptide consisting of the 1440 C-terminal amino acids was not active. Further subcloning revealed that the RNase active site was located in the first 73 N-terminal amino acids, which comprise the first mononucleotide binding site of the putative Rossmann fold (Evguenieva-Hackenberg et al., 2002).

3.4. Purification of a protein with RNase activity from H. salinarum NRC-1 and its identification as aIF-5A To identify enzymes involved in RNA processing and degradation in Halobacterium, we performed a biochemical screen for such activities. As a first purification step, we chose a heparin column (Amersham Biosciences) to enrich nucleic acid binding proteins. H. salinarum NRC-1 was cultivated in ATCC medium #2185 at aerobic conditions and 37 C or 42 C. Cells from 1 L culture (10 to 15 g cells, wet weight) were harvested at early stationary growth phase (OD600nm ¼ 2.0)


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by centrifugation (20 min, 5000g, room temperature), washed two times with basal salt solution (4.3 M NaCl, 80 mM MgSO4, 10 mM trisodium citrate, 27 mM KCl, pH 7.2), and resuspended in 10 to 15 ml volumes of the same solution. The suspension was diluted fivefold in lysis buffer (50 mM Tris-HCl, pH 7.5), and the cells were opened by sonication. After ultracentrifugation for 1 h at 40,000 rpm (45Ti, Beckman) and 4 C, proteins of the supernatant were precipitated with ammonium sulfate (85% saturation) and dissolved in 50 mM Tris-HCl, pH 7.5. Dialysis against the same buffer, containing additionally 100 mM KCl and 1 mM EDTA, was performed to remove the ammonium sulfate. The proteins were then loaded onto a 5-ml heparin column (Pharmacia, HiTrap), and bound proteins were eluted with an increasing KCl gradient. RNA degrading activity of fractions was analyzed by incubating protein samples with the internally radiolabeled RNA substrate ‘‘pZBP’’ in ‘‘low salt’’ reaction buffer (200 mM KCl, 50 mM TrisHCl, pH7.2) for 3 h at 37 C. The reaction products were separated on denaturing TBE gels containing 8 M urea and 10% polyacrylamide. This long incubation time and maximal KCl concentrations of 200 mM were necessary for detection of RNase activity. We found that RNA remains stable for hours when incubated at 37 C or 42 C with cell-free extract from Halobacterium under physiologic salt conditions in buffer containing 10 mM MgCl2. Endoribonucleolytic activity was observed at KCl or NaCl concentrations

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