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Twocomponent Signaling Systems Part C 1st Edition Melvin I Simon

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Twocomponent Signaling Systems Part C 1st Edition Melvin I Simon Twocomponent Signaling Systems Part C 1st Edition Melvin I Simon Twocomponent Signaling Systems Part C 1st Edition Melvin I Simon

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Twocomponent Signaling Systems Part C 1st Edition Melvin I Simon
Twocomponent Signaling Systems Part C 1st Edition Melvin I Simon
Methods in ENZYMOLOGY Volume 471 Two-Component Signaling Systems, Part C Edited by Melvin I. Simon, Brian R. Crane and Alexandrine Crane
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, CA 92101-4495, USA 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 32 Jamestown Road, London NW1 7BY, UK First edition 2010 Copyright # 2010, Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: permissions@ elsevier.com. Alternatively you can submit your request online by visiting the Elsevier web site at http:/ /elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made For information on all Academic Press publications visit our website at elsevierdirect.com ISBN: 978-0-12-381347-3 ISSN: 0076-6879 Printed and bound in United States of America 10 11 12 10 9 8 7 6 5 4 3 2 1
CONTRIBUTORS Adrián F. Alvarez Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, México D.F., México Babak Andi Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA Paul V. Attwood School of Biomedical, Biomolecular and Chemical Sciences (M310), The University of Western Australia, Crawley, Western Australia, Australia Paul G. Besant School of Biomedical, Biomolecular and Chemical Sciences (M310), The University of Western Australia, Crawley, Western Australia, Australia Roberto A. Bogomolni Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, USA Katherine A. Borkovich Department of Plant Pathology and Microbiology, University of California, Riverside, California, USA Robert B. Bourret Departments of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Winslow R. Briggs Department of Plant Biology, Carnegie Institution for Science, Stanford, Califor- nia, USA Edmundo Calva Departamento de Microbiologı́a Molecular, Instituto de Biotecnologı́a, Universi- dad Nacional Autónoma de México AP 510-3, Cuernavaca, Morelos, Mexico Paul F. Cook Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA xiii
Rachel L. Creager-Allen Departments of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA Nabanita De Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Jan S. Fassler Department of Biology, University of Iowa, Iowa City, Iowa, USA Marcus A. Frederickson Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, USA Sumire Fujiwara Department of Plant Cellular and Molecular Biology, Ohio State University, Columbus, Ohio, USA Dimitris Georgellis Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, México D.F., México Eric Giraud LaboratoiredesSymbiosesTropicalesetMéditerranéennes,IRD,CIRAD,AGRO-M, INRA, UM2, TA A-82/J, Campus de Baillarguet, Montpellier Cedex 5, France Zemer Gitai DepartmentofMolecularBiology,PrincetonUniversity,Princeton,NewJersey,USA Erin A. Gontang Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA Mark Goulian Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA Penelope I. Higgs Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Hans-Jörg Hippe Medizinische Klinik III, Universitätsklinikum, Universität Heidelberg, Heidelberg, Germany James A. Hoch Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA xiv Contributors
Terence Hwa Center for Theoretical Biological Physics, University of California San Diego, La Jolla, California, USA Sakthimala Jagadeesan Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany Carol A. Jones Department of Plant Pathology and Microbiology, University of California, Riverside, California, USA Alla O. Kaserer Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA Woe-Yeon Kim Department of Plant Cellular and Molecular Biology, Ohio State University, Columbus, Ohio, USA, and Division of Applied Life Science (BK21 Program) and Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, Korea Susanne Klumppw Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms- Universität, Münster, Germany Roberto Kolter Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA Michael Korth Institut für Pharmakologie für Pharmazeuten, Universitätsklinikum Hamburg- Eppendorf, Hamburg, Germany Petya V. Krasteva Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Jérôme Lavergne CEA, DSV, IBEB, Laboratoire de Bioénergétique Cellulaire, and CNRS, UMR 6191, Biologie Végétale et Microbiologie Environnementales, and Aix-Marseille Université, Saint-Paul-lez-Durance, France Bongsoo Lee Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany w Deceased June 17, 2009 Contributors xv
Jürgen U. Linder Pharmaceutical Institute, University of Tübingen, Tübingen, Germany Daniel López Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA Katrin Ludwig Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms- Universität, Münster, Germany Bryan Lunt Institute for Scientific Interchange, Viale S. Severo 65, Torino, Italy C. Robertson McClung Department of Biological Sciences, Dartmouth College, Hanover, New Hamp- shire, USA Takeshi Mizuno Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Japan Aaron M. Moore Departments of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Susumu Morigasaki Department of Microbiology, University of California, Davis, California, USA, and Graduate School of Biological Sciences, Nara Institute of Science and Tech- nology, Ikoma, Nara, Japan Marcos V. A. S. Navarro Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Jose N. Onuchic Center for Theoretical Biological Physics, University of California San Diego, La Jolla, California, USA Ricardo Oropeza Departamento de Microbiologı́a Molecular, Instituto de Biotecnologı́a, Universidad Nacional Autónoma de México AP 510-3, Cuernavaca, Morelos, Mexico Stephani C. Page Departments of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA Andrea Procaccini Institute for Scientific Interchange, Viale S. Severo 65, Torino, Italy xvi Contributors
Patrice A. Salomé Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, USA Andreas Schramm Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Alexander Schug Center for Theoretical Biological Physics, University of California San Diego, La Jolla, California, USA Joachim E. Schultz Pharmaceutical Institute, University of Tübingen, Tübingen, Germany Kazuhiro Shiozaki Department of Microbiology, University of California, Davis, California, USA Ruth E. Silversmith Departments of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Albert Siryaporn Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA David E. Somers Department of Plant Cellular and Molecular Biology, Ohio State University, Columbus, Ohio, USA Holger Sondermann Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Hendrik Szurmant Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA Stephanie A. Thomas Departments of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Tong-Seung Tseng Department of Plant Biology, Carnegie Institution for Science, Stanford, Califor- nia, USA André Verméglio CEA, DSV, IBEB, Laboratoire de Bioénergétique Cellulaire, and CNRS, UMR 6191, Biologie Végétale et Microbiologie Environnementales, and Aix-Marseille Université, Saint-Paul-lez-Durance, France Contributors xvii
Qi Wang Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Martin Weigt Institute for Scientific Interchange, Viale S. Severo 65, Torino, Italy John N. Werner DepartmentofMolecularBiology,PrincetonUniversity,Princeton,NewJersey,USA Ann H. West Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA Thomas Wieland Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Medizinische Fakultät Mannheim, Universität Heidelberg, Mannheim, Germany Takafumi Yamashino Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Japan Xiao-Bo Zhou Institut für Pharmakologie für Pharmazeuten, Universitätsklinikum Hamburg- Eppendorf, Hamburg, Germany xviii Contributors
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 xix
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 xx Methods in Enzymology
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 Methods in Enzymology xxi
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 xxii Methods in Enzymology
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 Methods in Enzymology xxiii
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 xxiv Methods in Enzymology
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 Methods in Enzymology xxv
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 xxvi Methods in Enzymology
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 Methods in Enzymology xxvii
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 xxviii Methods in Enzymology
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 Methods in Enzymology xxix
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 xxx Methods in Enzymology
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 DÜZGÜNES, VOLUME 221. Membrane Fusion Techniques (Part B) Edited by NEJAT DÜZGÜNES, 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 Methods in Enzymology xxxi
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 GÖTE 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 xxxii Methods in Enzymology
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 Methods in Enzymology xxxiii
VOLUME 256. Small GTPases and Their Regulators (Part B: Rho Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL 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 xxxiv Methods in Enzymology
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 Methods in Enzymology xxxv
VOLUME 293. Ion Channels (Part B) Edited by P. MICHAEL CONN 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 xxxvi Methods in Enzymology
VOLUME 311. Sphingolipid Metabolism and Cell Signaling (Part A) Edited by ALFRED H. MERRILL, JR., AND YUSUF A. HANNUN 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 Methods in Enzymology xxxvii
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 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 DÖTSCH, AND ULI SCHMITZ VOLUME 339. Nuclear Magnetic Resonance of Biological Macromolecules (Part B) Edited by THOMAS L. JAMES, VOLKER DÖTSCH, 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 xxxviii Methods in Enzymology
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C H A P T E R O N E Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation Albert Siryaporn and Mark Goulian Contents 1. Overview 2 2. Sources of Cross-Talk 3 3. Cross-Talk Suppression 4 4. Transcriptional Reporters 6 5. Response Regulator Localization 8 6. Phosphatase Cross-Talk 13 7. Signal Response in Cross-Talk Networks 14 8. Concluding Remarks 14 Acknowledgments 15 References 15 Abstract Cross-talk between noncognate histidine kinases and response regulators has been widely reported in vitro and, in specific mutant backgrounds and condi- tions, in vivo. However, in most cases there is little evidence supporting a physiological role of cross-talk. Indeed, histidine kinases and response regula- tors show remarkable specificity for their cognate partners. In vivo studies of cross-talk have the potential to establish mechanisms that control specificity and, if the cross-talk is observable in wild-type strains, may reveal new levels of cross-regulation. However such studies can be complicated by effects of other regulatory circuits and by the inactivation of mechanisms that would otherwise suppress cross-talk. It is thus easy to mis- or overinterpret the significance of such studies. We address potential complications associated with measuring cross-talk and discuss some methods for identifying and unmasking sources of cross-talk in cells using transcriptional reporters and in vivo DNA-binding assays. Methods in Enzymology, Volume 471 # 2010 Elsevier Inc. ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)71001-6 All rights reserved. Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA 1
1. Overview When sequence comparisons first revealed the generality of two- component signaling, it was immediately appreciated that histidine kinases could potentially phosphorylate noncognate response regulators (Nixon et al., 1986) and that such cross-talk could play a role in signal integration and processing (Stock et al., 1989) (Fig. 1.1). Early studies demonstrated the existence of cross-talk in vitro, although the cross-phosphorylation occurred with considerably slower kinetics compared with phosphorylation between cognate partners (Igo et al., 1989; Ninfa et al., 1988). Cross-talk was also observed in vivo, but only in modified strains in which various regulators were deleted or overexpressed. In the two decades since these early con- siderations, cross-talk has not emerged as a common theme in two-compo- nent signaling. Despite numerous reports in the literature, in most cases there is little evidence to support the claim that there are detectable effects from cross-talk in wild-type strains (Laub and Goulian, 2007). There are several factors that make attempts at identifying cross-talk subject to mis- or overinterpretation. In vitro studies have demonstrated that numerous histidine kinases can phosphorylate noncognate response regula- tors, with some histidine kinases showing a considerable level of promiscu- ity (Skerker et al., 2005; Yamamoto et al., 2005). However, as a rule, histidine kinases show a very strong kinetic preference for their cognate response regulator (Skerker et al., 2005). Thus, a demonstration of cross-talk in vitro, without a comparison of the kinetics of phosphotransfer between cognate and noncognate pairs, is not particularly compelling evidence for cross-talk function in vivo (Laub and Goulian, 2007; Skerker et al., 2005). There are additional complications when studying cross-talk in vivo. For most systems, it is not feasible to directly measure response regulator HK1 RR1 HK2 RR2 HK3 RR3 ? ? Figure 1.1 Cross-talk between noncognate histidine kinases (HKs) and response regulators (RRs) could potentially enable complex processing of multiple input signals. However, to date there has been relatively little evidence for cross-talk in wild-type strains. 2 Albert Siryaporn and Mark Goulian
phosphorylation in cells. Instead, phosphorylation is inferred from transcrip- tion measurements, e.g., with transcriptional reporters. However, it is often difficult to disentangle the effects of other regulatory factors that may act at the promoter of interest. In addition, studies in mutant strains may identify cross-talk that is irrelevant in the context of wild-type cells. Mechanisms have been identified that suppress cross-talk between noncognate pairs in vivo (Groban et al., 2009; Siryaporn and Goulian, 2008). When these mechanisms are eliminated by mutation, significant cross-talk may emerge (Kim et al., 1996; Silva et al., 1998; Siryaporn and Goulian, 2008), which can give the false impression that cross-talk functions in wild-type strains. Here we describe some of the issues one should take into account when looking for sources or effects of cross-talk. These methods can also be used to explore various mechanisms that limit cross-talk and to explore the molecular determinants of specificity among histidine kinases and response regulators. This chapter covers potential sources of cross-talk, cross-talk suppression mechanisms, and methods for measuring cross-talk in cells. In particular, it provides specific details on how to measure the association of response regulator-fluorescent protein fusions to DNA in vivo, a method which can distinguish cross-talk from the effects of other regulatory path- ways. We also briefly discuss methods for characterizing phosphatase cross- talk and the effects of signal response on cross-talk networks. 2. Sources of Cross-Talk A response regulator can potentially be phosphorylated by a cognate histidine kinase, noncognate histidine kinases, and small molecule phos- phodonors. It is generally assumed that when genes for a response regulator and a histidine kinase are in the same operon, the encoded proteins are cognate pairs. Situations in which more than one histidine kinase or response regulator is encoded in an operon, however, may be more com- plex. It is also not unusual to encounter ‘‘orphan’’ histidine kinases and response regulators for which no clear partner has been identified. Many microbial genomes appear to have unequal numbers of histidine kinases and response regulators (Alm et al., 2006), which suggests that some response regulators may have more than one source of phosphorylation and similarly some histidine kinases may phosphorylate multiple response regulators. In some systems, the histidine kinase is bifunctional and mediates response regulator phosphorylation and dephosphorylation. In this case, there could in principle be cross-talk from either the phosphotransfer or phosphatase activities. However, we are unaware of any reports of phos- phatase cross-talk, even in mutant strains where phosphotransfer cross-talk has been observed. For example, the histidine kinase CpxA phosphorylates Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation 3
and dephosphorylates its cognate response regulator CpxR. Cross-talk from CpxA to OmpR can be detected in strains deleted for envZ and cpxR, however, dephosphorylation of OmpR-P by CpxA is not detected (Siryaporn and Goulian, 2008). Kinetic modeling of the histidine kinase– response regulator interaction suggests that phosphatase activity may emerge for sufficiently strong interaction between a bifunctional histidine kinase and response regulator (Siryaporn et al., in preparation). Thus, the difficulty in observing phosphatase cross-talk may reflect the relatively weak interac- tions between noncognate partners. A number of response regulators are phosphorylated by the small mole- cule phosphodonor acetyl phosphate (McCleary and Stock, 1994; Wanner and Wilmes-Riesenberg, 1992; Wolfe, 2005). The cellular concentration of this high-energy source of phosphoryl groups is strongly dependent on growth conditions, such as carbon source (Wolfe, 2005). Concentrations can be quite high in some cases, which raises the possibility that acetyl phosphate may integrate metabolic status into some two-component systems (Fredericks et al., 2006; Klein et al., 2007; Wanner, 1992). For most two-component systems, however, a role for acetyl phosphate has not been established. There is also the possibility that other small molecule phosphodonors (Lukat et al., 1992) could function to phosphorylate response regulators in some contexts in vivo. The ability of a phosphodonor to affect the steady-state phosphorylation level of a particular response regulator depends on the rate of the phosphory- lation reaction relative to the rates of other sources of phosphorylation and dephosphorylation. While phosphorylation rates of cytoplasmic fragments of histidine kinases are readily measured in vitro using purified components (Laub et al., 2007), the effects of individual phosphorylation sources on levels of phosphorylated response regulator in vivo can be quite sensitive to the strain background and can be difficult to measure. For example, a bifunctional histidine kinase that has high rates of phosphorylation and dephosphorylation can give levels of phosphorylated response regulator that are comparable to those that would arise from a histidine kinase that has weak phosphorylation activity but lacks a mechanism for response regulator dephosphorylation (Fig. 1.2A). However, through a careful and systematic analysis using tran- scriptional and translational reporter fusions, the contributions from different phosphorylation sources can in some cases be disentangled. 3. Cross-Talk Suppression Studies have shown that cross-talk can be suppressed by cognate histidine kinases and response regulators (Fig. 1.2B and C) (Groban et al., 2009; Kim et al., 1996; Silva et al., 1998; Siryaporn and Goulian, 2008). 4 Albert Siryaporn and Mark Goulian
Mutations that relieve these suppression mechanisms could produce signals of cross-talk that would otherwise be squelched in the wild-type strain. Toavoid over-or misinterpretingthe resultsfromanalysisof mutant strains, it is important to take into account the potential effects of these mechanisms. HK1 A B RR1 RR1 HK1 P P HK2 RR1 RR1 P P HK2 RR1 RR2 HK1 RR1 HK2 X X Histidine kinase buffering Response regulator inhibition C Figure 1.2 (A) Two different scenarios in which very different rates of phosphoryla- tion can give the same steady state level of phosphorylated response regulator (RR-P). (Left) A bifunctional histidine kinase phosphorylates and dephosphorylates a response regulator with very high rates such that, under moderate stimulation, a moderate level of RR-P is obtained at steady state. (Right) A histidine kinase that phosphorylates a response regulator very weakly may, in the absence of any phosphatase activity, also produce moderate levels of RR-P. (B, C) Suppression of cross-talk from a histidine kinase HK2 to response regulator RR1 by cognate partners (Groban et al., 2009; Siryaporn and Goulian, 2008). (B) The high flux of phosphorylation and dephosphory- lation of RR1 by the bifunctional histidine kinase HK1 can suppress or buffer against cross-talk from HK2. (C) Suppression of cross-talk by a cognate response regulator. The cognate response regulator RR2 outcompetes the noncognate regulator RR1 for interaction with the histidine kinase HK2. By removing RR2, significant cross-talk from HK2 to RR1 may be detected, which would be otherwise absent in the wild-type strain. Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation 5
Bifunctional histidine kinases set the level of phosphorylated response regulator through a balance of phosphorylation and dephosphorylation. This cycle can effectively act as a buffer to suppress the effects of weaker phosphorylation sources (Fig. 1.2B) (Groban et al., 2009; Kim et al., 1996; Silva et al., 1998; Siryaporn and Goulian, 2008). Therefore, deletion of a bifunctional histidine kinase could reveal phosphorylation of the response regulator from a noncognate histidine kinase or other phosphodonor (e.g., Batchelor et al., 2005; Danese and Silhavy, 1998; Hutchings et al., 2006; Wolfe et al., 2008). However, this alternate source may give a much lower flux of phosphoryl groups and may thus be physiologically irrelevant in wild-type strains. A response regulator can also prevent its cognate histidine kinase from participating in cross-talk (Fig. 1.2C). For example, cross-talk from CpxA to OmpR is suppressed by CpxR (Siryaporn and Goulian, 2008). This is likely to occur through competitive inhibition, in which the cognate response regulator outcompetes noncognate response regulators for inter- action with the histidine kinase. Thus, deletion or inactivation of a response regulator could potentially produce cross-talk from the cognate histidine kinase to some other response regulator. Significant overexpression of the histidine kinase could also have a similar result. 4. Transcriptional Reporters In most two-component systems that have been studied, the phos- phorylated response regulator controls transcription of effecter genes. Therefore, transcriptional reporter fusions have been a standard tool for measuring the output of signal transduction systems. A reporter gene such as gfp or lacZ is placed under control of a promoter of interest and expression of the reporter is measured through fluorescence or enzyme assays. Cross-talk can be measured using transcriptional reporter fusions. Chromosomal fusions are preferable to constructs on multicopy plasmids since they avoid potential effects on reporter gene expression from titrating out response regulator and changes in plasmid copy number. It is always a good idea to assessthe dynamic range of reporter expression sincethis gives an indication of the sensitivity to changes in output under various conditions. The basal expression level can be determined from measurements in a strain deleted for the response regulator of interest. The range of activation can be determined by making measurements for different levels of input stimulus, if the stimulus is known, or by varying expression of a constitutively active version of the histidine kinase, if one is available. For experiments in which a fluorescent reporter such as gfp is used, it is preferable to grow cultures using a growth medium with a low level of autofluorescence such as a 6 Albert Siryaporn and Mark Goulian
minimal salts medium (Miller, 1992), as this generally increases the sensitivity of measurements. Rich media often give moderate levels of autofluores- cence, which could reduce the sensitivity of fluorescence measurements. Evidence for the presence and absence of cross-talk can be followed by monitoring changes in transcriptional reporter expression for different genetic backgrounds and conditions. Depending on the application, it may be necessary to remove cross-talk suppression mechanisms described above by deleting appropriate histidine kinases and response regulators before cross-talk can be detected. The observation of cross-talk in this case should not be used to conclude that there is cross-talk in the wild-type strain. Specific histidine kinases that are potential sources of cross-talk can be tested by changing expression levels or stimulation with signal. However, their effects on cross-talk will depend on details of the histidine kinase and its interaction with the noncognate response regulator, as discussed below. A histidine kinase sets the level of phosphorylated response regulator through phosphorylation alone (monofunctional behavior) or through a balance of phosphorylation and dephosphorylation (bifunctional behavior). For histidine kinases displaying monofunctional behavior, the level of phosphorylated response regulator is expected to be quite sensitive to histidine kinase expression level (E. Batchelor and M. Goulian, unpublished observations; Miyashiro and Goulian, 2008). On the other hand, when the histidine kinase has bifunctional behavior, modeling and experiments sug- gest the level of phosphorylated response regulator can be relatively insen- sitive to histidine kinase expression levels (Batchelor and Goulian, 2003; Miyashiro and Goulian, 2008; Shinar et al., 2007). Thus, cross-talk may be observed in a context of histidine kinase overexpression even though it appears that cognate response regulator phosphorylation is unchanged. In addition, in at least some cases, a histidine kinase can exhibit bifunctional behavior against its cognate partner while displaying monofunctional behavior against a noncognate partner (Siryaporn and Goulian, 2008). The choice of carbon source may have a significant effect on the level of acetyl phosphate in the cell (McCleary and Stock, 1994) and thus the level of response regulator phosphorylation. The desired level of acetyl phosphate may depend on the experimental condition being tested. In some cases, significant levels of acetyl phosphate may provide a means of increasing the basal level of response regulator phosphorylation and hence increase the sensitivity to cross-talk by other phosphodonors. In other cases, one may want to eliminate acetyl phosphate or keep its level to a minimum in order to distinguish its effects from other sources of response regulator phosphor- ylation. For Escherichia coli, growth using carbon sources such as glucose, pyruvate, and acetate produce high levels of acetyl phosphate. Low levels can be achieved by growing on glycerol. One can also modulate acetyl phosphate levels by deleting one or both of pta and ackA, depending on the flux of acetate through the Pta (phosphotransacetylase)–AckA (acetate Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation 7
kinase) pathway (Wolfe et al., 2008). Deletion of pta ackA abolishes production of acetyl phosphate. However, care should be taken in altering the Pta–AckA pathway as this may have additional indirect effects on some two-component systems (Wolfe et al., 2008). While transcriptional reporter fusions can provide effective measure- ments of two-component system output, they are also subject to the effects of other regulatory factors. There are many examples of genes whose expression is controlled by multiple regulatory proteins and small RNAs. Even many relatively well-characterized systems may have additional unidentified regulators whose effects could give the false impression of cross-talk. In some cases, promoters may also be controlled by multiple response regulators (e.g., Batchelor et al., 2005; Mouslim and Groisman, 2003). To distinguish between cross-talk and alternate regulatory pathways, one may need to use additional methods to follow response regulator phosphorylation or binding to promoters. 5. Response Regulator Localization If a functional fluorescent protein (FP) fusion to the response regulator of interest is available then one can use a fluorescence localization assay to provide further support for cross-talk in vivo (Batchelor and Goulian, 2006; Siryaporn and Goulian, 2008). The method takes advantage of the tendency of plasmids with partitioning systems to form clusters in the cell (Pogliano, 2002). In this assay, cells express the response regulator-FP fusion and also contain a plasmid that has one or more binding sites for the response regulator. Plasmid clustering provides a high local density of response regulator binding sites in the cell (Fig. 1.3A). Significant response regula- tor-FP binding to DNA results in intense localized regions of fluorescence, which appear as bright foci or spots under a fluorescence microscope and can be easily quantified by image analysis (Fig. 1.3B–D). Assuming DNA binding is modulated by response regulator phosphorylation, changes in response regulator-FP colocalization with the plasmid can be used to infer changes in response regulator phosphorylation. We have been able to construct functional fluorescent protein fusions to the C-termini of several different response regulators in E. coli, although in all of the cases that we tested the fusions showed decreased activity. We are unaware of any general rules regarding the choice of C- or N-termini for the fusion, or whether or not to include a flexible linker. It is probably best to try several different constructs. For the plasmid, we have used constructs based on pRS415 (Batchelor and Goulian, 2006; Simons et al., 1987), which is derived from pBR322. Many other plasmids should work as well although they may show differing levels of clustering. Plasmid clustering can 8 Albert Siryaporn and Mark Goulian
be characterized through the use of lac operators and a fluorescent protein fusion to lac repressor, as discussed below. The ability to detect fluorescent protein localization to plasmids will depend on the binding affinity of the Low RR-P High RR-P Plasmid with binding sites for LacI and RR RR-YFP CFP-LacI A DIC CFP YFP Pixel values Pixel distance2 Extracted region B C 18 13 10 9 10 13 18 13 8 5 4 5 8 13 10 5 2 1 2 5 10 9 4 1 0 1 4 9 10 5 2 1 2 5 10 13 8 5 4 5 8 13 18 13 10 9 10 13 18 172 252 320 348 356 325 272 212 304 377 410 434 387 326 233 336 415 513 491 428 332 249 360 446 521 497 400 276 232 339 403 431 408 317 226 202 266 297 316 283 216 165 159 184 199 207 188 151 132 100 200 300 400 500 0 600 1 2 3 0 4 Pixel value Radius (pixels) RR binding sites data ( fit) Vector only data ( fit) RR binding sites Vector D Figure 1.3 Measurement of response regulator binding to DNA in vivo. (A) Schematic of the method, which uses a functional translational fusion of a fluorescent protein (e.g., YFP) to the response regulator and a plasmid containing response regulator binding sites. The plasmid also contains lac operators, which are bound by CFP-LacI. Plasmid clustering in the cell results in a high local concentration of binding sites, which is easily visualized as a bright fluorescent spot in the CFP channel. Response regulator binding to plasmid also results in a bright fluorescent spot in the YFP channel. (B) Images of cells expressing OmpR-YFP andcontaininga plasmid with OmpRbinding sites (toprow)or an empty control vector (bottom row). DIC—differential interference contrast image. (C) Example of a YFP spot, corresponding to the dashed square in the upper right image in (B), and the associated pixel values and distances from the center. (D) Gaussian fit to the profileofpixelvaluesfrom(C).Acorrespondingfittotheneighborhoodofa maximalYFP pixel in a cell containing the vector control is also shown. Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation 9
phosphorylated response regulator-FP fusion as well as the extent of phosphorylation. One can increase the sensitivity by including several binding sites in the plasmid as needed. It is generally advisable to include a means to monitor plasmid clustering independently of response regulator binding to DNA. This controls for potential changes in plasmid clustering as a result of changes in cell physiol- ogy. This is easily accomplished by cloning lac operators into the plasmid and expressing a fluorescent protein fusion to lac repressor. We routinely use a CFP-LacI fusion (Batchelor and Goulian, 2006), which can be simulta- neously imaged with a response regulator-YFP fusion. We have used an array of tandem lac operator repeats (Robinett et al., 1996) to mark the plasmid. This produces extremely bright foci, which is convenient as it provides increased sensitivity and enables shorter exposures when acquiring images. After plasmid clusters have been identified in the CFP channel, one can then quantify the YFP fluorescence in the neighborhood of this loca- tion. Under conditions of very high response regulator phosphorylation one may observe additional foci that are not associated with plasmids. In at least one case (OmpR-YFP), we have determined that these foci are due to response regulator binding to chromosomal loci (E. Libby and M. Goulian, manuscript in preparation). Fluorescence localization can be visualized using techniques adapted from general single-cell fluorescence microscopy methods (e.g., Batchelor and Goulian, 2006; Miyashiro and Goulian, 2007). 1. Grow cultures in medium with aeration at the appropriate growth temperature to saturation. To maximize the sensitivity of this assay, a culture medium that gives relatively low levels of autofluorescence should be used. A minimal salts medium is ideal in this respect and also allows for choice of carbon source. 2. Dilute cultures at least 1:1000 into the same medium and grow to early exponential phase (optical density of 0.1–0.3 at 600 nm for E. coli). 3. Shortly before cells reach the target density, preheat the microscope stage to the same temperature as that used for growing the cell cultures. Prepare 1% agarose pads made from the same growth medium and placed between a microscope slide and cover glass. For many small- scale experiments, it is sufficient to use 3 in. 1 in. 1 mm slides and 22 mm square #1.5 cover glass. Place 50–100 ml of molten agarose on the center of slide and cover immediately with coverslip. Agarose pads should be maintained at the growth temperature of the culture and may need time to dry slightly before use. Cells will not immobilize well on agarose pads that are too moist. 4. Carefully lift the cover glass off of the pad and place 5 ml of culture onto cover glass. Replace cover glass back onto agarose pad with culture between the cover glass and pad and put the slide on the 10 Albert Siryaporn and Mark Goulian
microscope stage immediately. The number of cells on the agarose pad should be such that cells are easily found in most fields but are in sufficiently low density that there is little or no contact between neigh- boring cells. Obtaining proper cell density in images is especially impor- tant for the fluorescence analysis that follows. 5. Acquire a phase contrast or differential interference contrast (DIC) image and fluorescence images of the same field on a fluorescence microscope (Fig. 1.3B). Image acquisition times should be long enough so that cell fluorescence is significantly higher than background but brief enough so that none of the pixel values is equal to the maximum allowed value. The maximum pixel values are 255, 1023, 4095, and 65355 for 8-, 10- 12-, and 16-bit images, respectively. Objective magnification and resolution of the digital camera should be sufficient so that the shortest dimension of individual cells is at least approximately 5 pixels. Images should be saved without applying any contrast enhancements or other processing. Cell fluorescence levels may be relatively weak for response regulator fusions that are expressed at wild-type levels. In addition, fluorescent foci are not always observable through the eyepiece and may photo-bleach within seconds. It is important to acquire images from fields of cells that have not been previously exposed to light from the fluorescence illumina- tor. In particular, cells should be brought into focus using phase or DIC imaging. The extent of fluorescence localization will vary from cell to cell due to fluctuations in protein expression levels, plasmid copy number, plasmid localization, or other variations in the cellular environment. A representative measure of fluorescence localization for the population can be attained by acquiring images of a large number of cells (e.g., 100–200) and quantifying the extent of localization as described below. While dramatic changes in fluorescence localization may be sufficient to support a particular conclusion, in some cases it may be necessary to distinguish smaller changes in localization that are not easily assessed by eye. The fluorescence localization of cell populations can be quantified from images using software packages such as ImageJ (National Institutes of Health, Bethesda, MD), LabVIEW (National Instruments, Austin, TX), and MATLAB (The MathWorks, Natick, MA), which provide convenient libraries of imaging tools. Modules and plug-ins provided by these packages can be stitched together through scripts or macros to process large numbers of images quickly and accurately. The basic assumption behind this analysis is that fluorescent foci can be accurately represented by point-source intensity profiles. The maximum value of the fluorescence in the cell or the maximum value in the neighbor- hood of the plasmid cluster is taken to be the peak of the fluorescence intensity profile. Pixel values surrounding this maximum are extracted, assigned pixel distances, and fitted to a Gaussian function. Numerical Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation 11
parameters derived from the best-fit intensity profiles are then used to compute an integrated fluorescence intensity, which is a measure of the extent of fluorescence localization. This method assumes that YFP fluores- cence and phase contrast or DIC images have been collected. Instructions also describe cases where the investigator has chosen to express CFP-LacI to identify plasmid clusters as described above. 1. A phase contrast or DIC image and a YFP fluorescence image should be available for analysis. If the CFP-LacI system was also used, then a CFP fluorescence image should also be on hand. 2. Determine cell boundaries using the phase or DIC image. Edge detec- tion algorithms, such as the Sobel edge filter (Castleman, 1996), can be used for identifying cell boundaries in phase contrast images. For images which contain relatively low or complex contrast, such as images from DIC microscopy, cell boundaries can be identified by thresholding pixel values (Batchelor and Goulian, 2006; Miyashiro and Goulian, 2007). 3. Once cell boundaries are determined, it is often convenient to construct a binary (black and white) mask, which defines areas where cells are present. Masks can be constructed from cell boundary images by filling in regions marked by the cell boundaries. 4. Extract pixel values from corresponding fluorescence images using the mask as a guide for cell locations. It is convenient to extract pixel values for individual cells so that each cell can be processed individually from this point on. 5. Determine the location of the plasmid cluster within the cell as follows: (a) If a CFP-LacI image marking plasmid clusters has been acquired, the brightest pixel within each cell boundary in the CFP image should be identified. If the response regulator-YFP is colocalized with the plasmid cluster, it should be observable in the vicinity of the LacI- CFP peak. Identify the location of the maximum YFP peak within the local pixel neighborhood of the CFP peak. For example, for a cell that is represented by approximately 500 pixels, one might choose to restrict the search area to be within a 3 3 pixel area. This maximum YFP value is taken to be the peak of the YFP (response regulator) intensity profile. (b) If a CFP-LacI image is not available, identify the location of the maximum pixel value within the cell boundary of the YFP (response regulator) image. This is taken to be the peak of the YFP (response regulator) intensity profile. 6. Extract pixel values surrounding the peak of the YFP intensity profile (Fig. 1.3B). The extracted area should be large enough to encompass a section representative of the spot and should not include areas outside of the cell. Assign a radial distance to each pixel value with the peak pixel value as the center (Fig. 1.3C). 12 Albert Siryaporn and Mark Goulian
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[557] Mendieta, p. 110. [558] Vol. 3, cap. 5, p. 234. [559] Herrera, dec. 2, lib. 6, p. 141. [560] Kingsborough, vol. 7, chap. 4. [561] Teutonic Mythology, vol. 3, p. 1233. [562] Ibid. [563] Fables and Rites of the Incas, Padre Christoval de Molina (Cuzco, 1570-1584), transl. by Clements R. Markham, Hakluyt Society trans., vol. 48, London, 1873, p. 48. [564] The common people wore a black llautu. See Garcilaso, Comentarios, Markham's transl., Hak. Soc., vol. 41, pp. 88, 89. [565] Ibid., p. 85. [566] Ibid., p. 89. [567] Quando vàn à sembrar las Tierras del Sol, vàn solos los Principales à trabajar, i vàn con insignias blancas, i en las espaldas unos Cordones tendidos blancos, à modo de Ministros del Altar.—Herrera, dec. 5, lib. 4, cap. 6, pp. 94-95. [568] Picart, Cérémonies et Coûtumes, etc., Amsterdam, 1735, vol. 6, p. 92. [569] Montfaucon, L'antiquité expliquée, tome 2, pt. 1, p. 33. [570] Hawkesworth, Voyages, vol. 3, p. 229. [571] Voyage to Congo, in Pinkerton's Voyages, vol. 16, p. 237. [572] Pinkerton, Voyages, vol. 16, p. 388. [573] Speke, Source of the Nile, London, 1863, p. 125. [574] London, 1877, vol. 2, p. 131. [575] Stanley, Through the Dark Continent, vol. 2, p. 330. [576] Schweinfurth, Heart of Africa, London, 1873, vol. 1, p. 154. [577] Winstanley, Abyssinia, vol. 2, p. 68. [578] This cord is worn about the neck. Ibid., p. 257.
[579] Ibid., vol. 1, p. 235. [580] Ibid., vol. 2, p. 132. [581] Ibid., p. 165. [582] Ibid., p. 292. [583] Malte-Brun, Universal Geography, vol. 4, p. 259, Phila., 1832. [584] Grimm, Teutonic Mythology, vol. 2, p. 640. [585] Nightingale, quoted in Madden, Shrines and Sepulchres, vol. 1, pp. 557, 558. [586] Leems, Account of Danish Lapland, in Pinkerton, Voyages, London, 1808, vol. 1, p. 471. [587] Brand, Popular Antiquities, vol. 3, p. 5. See also John Scheffer, Lapland, Oxford, 1674, p. 58. [588] Act IV, scene 1. [589] Benjamin, Persia, London, 1877, p. 99. [590] Cérémonies et Coûtumes, vol. 7, p. 320. [591] Du Halde, History of China, London, 1736, vol. 4, pp. 244, 245, and elsewhere. [592] Higgins, Anacalypsis, vol. 2, p. 218. [593] Vining, An Inglorious Columbus, p. 635. [594] Du Halde, History of China, London, 1736, vol. 1, p. 270. [595] Univ. Geog., vol. 3, book 75, p. 144, Phila., 1832. [596] Brinton, Myths of the New World, N. Y., 1868, p. 15. [597] Early History of Mankind, London, 1870, p. 156. [598] Voyages, vol. 3, p. 102. [599] Shâyast lâ-Shâyast, cap. 4, pp. 285, 286. In Sacred Books of the East, Max Müller's edition, vol. 5. [600] Monier Williams, Modern India, p. 56. [601] Ibid., pp. 179, 180.
[602] Cérémonies et Coûtumes, vol. 7, p. 28. [603] Marco Polo, Travels, in Pinkerton's Voyages, vol. 7, p. 163. [604] Picart, Cérémonies et Coûtumes, etc., vol. 6, pt. 2, p. 99. [605] Malte-Brun, Univ. Geog., vol. 2, lib. 50, p. 235, Philadelphia, 1832. [606] Dr. J. L. August Von Eye, The history of culture, in Iconographic Encyc., Philadelphia, 1886, vol. 2, p. 169. [607] Forlong, Rivers of Life, vol. 1, p. 120. [608] Ibid., pp. 240-241. [609] Forlong, Rivers of Life, vol. 1, p. 328. [610] Ibid., p. 323. [611] Dubois, People of India, p. 9. [612] Mythology of the Hindus. [613] Mythology of the Hindus, pp. 9, 10, 11. [614] Ibid., p. 92. [615] Ibid., p. 155. [616] Ibid., pp. 135, 154, 155. [617] Maurice, Indian Antiquities, London, 1801, vol. 5, p. 205. [618] Ibid., vol. 4, p. 375, where a description of the mode of weaving and twining is given. [619] Ibid., p. 376. [620] Ibid., vol. 5, p. 206. [621] Notes of Richard Johnson, Voyages of Sir Hugh Willoughby and others to the northern part of Russia and Siberia, Pinkerton's Voyages, vol. 1, p. 63. [622] Caron's account of Japan in Pinkerton's Voyages, vol. 7, p. 631. [623] Rev. Father Dandini's Voyage to Mount Libanus, in Pinkerton's Voyages, vol. 10, p. 286.
[624] Henry Charles Lea, History of the Inquisition in the Middle Ages, vol. 1, p. 92, New York, 1888. [625] Müller, Sacred Books of the East, vol. 14, Vasishtha, cap. 2, par 6. [626] Ibid., Baudhâyana, prasna 1, adhyâya 5, kandikâ 8, pars. 5- 10, p. 165. [627] Saxon Leechdoms, vol. 1, pp. xli-xliii. [628] Ibid., p. xliii. [629] Brand, Popular Antiquities, vol. 2, pp. 108,109. [630] Browne, Religio Medici, p. 392. [631] Brand, op. cit., p. 110. [632] Pliny, Nat. Hist., lib. 28, cap. 22. [633] Ibid., lib. 28, cap. 17. [634] Ibid. [635] Grimm, Teutonic Mythology, vol. 3, p. 1169. [636] Burton, Anatomy of Melancholy, London, 1827, vol. 1, p. 91; vol. 2, pp. 288, 290. [637] Burton, Anatomy of Melancholy, London, 1827, vol. 1, p. 91; vol. 2, p. 290. [638] Picart, Cérémonies et Coûtumes, etc., vol. 10, pp. 69-73. [639] Dæmonology, p. 100. [640] Brand, Pop. Ant., vol. 3, p. 299. [641] Ibid., p. 170. [642] Frommann, Tractatus de Fascinatione, Nuremberg, 1675, p. 731. [643] Markham, Bogle's mission to Tibet, London, 1876, p. 85. [644] Brand, Pop. Ant., vol. 2, p. 149. [645] Thomas Wright, Sorcery and Magic, London, 1851, vol. 2, p. 10.
[646] Brand, Pop. Ant., vol. 2, p. 143. [647] Pennant, in Pinkerton, Voyages, vol. 3, p. 382. [648] Hoffman, quoting Friend, in Jour. Am. Folk Lore, 1888, p. 134. [649] Brand, Pop. Ant., vol. 2, pp. 127 et seq. [650] Grimm, Teutonic Mythology, vol. 3, p. 1174. He also speaks of the nouer l'aiguillette, ibid., p. 1175. [651] Saxon Leechdoms, vol. 1, p. xliv. [652] Black, Folk-Medicine, London, 1883, pp. 185, 186. [653] Pliny, Nat. Hist., lib. 28, cap. 9. [654] Brand, Pop. Ant., vol. 2, p. 67. [655] Ibid., p. 170. [656] Sextus Placitus, De Medicamentis ex Animalibus, Lyons, 1537, pages not numbered, article de Puello et Puellæ Virgine. [657] Etmüller, Opera Omnia, Lyons, 1690, vol. 2, p. 279, Schroderii Dilucidati Zoologia. [658] Brand, Pop. Ant., vol. 2, p. 68, footnote. [659] Ibid., p. 67. [660] Paracelsus, Chirurgia Minora, in Opera Omnia, Geneva, 1662, vol. 2, p. 70. [661] Ibid., p. 174. [662] Beckherius, Medicus Microcosmus, London, 1660, p. 174. [663] Grimm, Teutonic Mythology, vol. 3, p. 1094, footnote. [664] Ibid., p. 1096. [665] Pliny, Nat. Hist., lib. 30, cap. 12. [666] Etmüller, Opera Omnia, Lyons, 1690, vol. 2, pp. 282, 283, Schroderii Dilucidati Zoologia. [667] Ibid., p. 278a. [668] Black, Folk-Medicine, London, 1883, p. 113.
[669] Forlong, Rivers of Life, London, 1883, vol. 2, p. 313. [670] Brand, Pop. Ant., vol. 2, p. 69. [671] Notes and Queries, 1st series, vol. 4, p. 500. [672] See also Black, Folk-Medicine, London, 1883, p. 79. [673] Grimm, Teutonic Mythology, vol. 3, p. 1233. [674] Black, Folk-Medicine, London, 1883, p. 114. [675] Herrera, dec. 6, lib. 8, cap. 1, p. 171. [676] Ibid., dec. 7, lib. 4, cap. 5, p. 70. [677] Smyth, Aborigines of Victoria, vol. 1, p. 351. See also previous references to the use of such cords by the Australians. [678] Pliny, Nat. Hist., lib. 28, cap. 27. [679] Highlands of Æthiopia, vol. 1, p. 247. [680] Through the Dark Continent, vol. 1, p. 398. [681] Pliny, Nat. Hist., lib. 28, cap. 11. [682] Notes and Queries, 4th series, vol. 5, pp. 295, 390. [683] Traité des Superstitions, tome 1, chap. 3, paragraph 8. [684] Pop. Ant., vol. 3, p. 276. [685] Black, Folk-Medicine, p. 109. [686] Anatomy of Melancholy, vol. 2, pp. 288, 290. [687] Brand, Pop. Ant., vol. 2, p. 107. [688] Brand, Pop. Ant., vol. 2, p. 78. [689] Ibid., p. 91. [690] Ibid., p. 93. [691] Picart, Cérémonies et Coûtumes, etc., vol. 1, p. 41. [692] Folk-Medicine, London, 1883, pp. 185, 186. [693] P. 41. [694] Black, Folk-Medicine, p. 186.
[695] Ibid., (after Tylor) pp. 176, 177. [696] Ibid., p. 178. [697] Pop. Ant., vol. 3, p. 276. [698] Salverte, Philosophy of Magic, vol. 1, p. 195. [699] Black, Folk-Medicine, London, 1883, p. 197. [700] Pliny, Nat. Hist., lib. 24, cap. 118. [701] Forlong, Rivers of Life, vol. 1, p. 451. [702] Pennant, quoted by Brand, Popular Antiquities, vol. 3, p. 54. [703] Ibid., p. 285. [704] Folk-Medicine, London, 1883, pp. 185, 186. [705] Folk-Medicine, London, 1883, p. 113. [706] Ibid., p. 57. [707] Ephemeridum Physico-medicarum, Leipzig, 1694, vol. 1, p. 49. [708] Black, Folk-Medicine, p. 112. [709] Black, Folk-Medicine, p. 112. [710] Brand, Pop. Ant., vol. 2, p. 86. [711] Black, Folk-Medicine, p. 38. [712] Pliny, Nat. Hist., lib. 30, cap. 38. [713] Ibid. [714] Black, Folk-Medicine, p. 111. [715] Brand, Pop. Ant., vol. 3, pp. 288, 324. [716] This fact is stated by Torquemada, Monarchia Indiana, lib. 10, cap. 33, and by Gomara, Hist. of the Conq. of Mexico, p. 446; see also Diego Duran, lib. 1, cap. 20, p. 226. [717] Herrera, dec. 3, lib. 2, p. 67.
[718] John Gilmary Shea, The Catholic Church in Colonial Days, p. 472. [719] Diego Duran, vol. 3, cap. 4, p. 217. [720] Anatomy of Melancholy, London, 1827, vol. 1, p. 337. [721] Picart, Cérémonies et Coûtumes, etc., Amsterdam, 1729, vol. 5, p. 50. [722] Lady of the Lake, canto 3, stanza 4, Sir Rhoderick Dhu, summoning Clan Alpine against the king. [723] Teatro Mexicano, vol. 3, p. 323. [724] Lib. 14, cap. 4, and lib. 16, cap. 16. [725] Lib. 1, cap. 23, pp. 251-252. [726] Ximenez, Hist. Orig. Indios, p. 211. [727] Mendieta, p. 83. [728] Ibid., p. 78. [729] Researches in South America, p. 83. [730] Monarchia Indiana, vol. 2, lib. 13, cap. 45, and elsewhere. [731] Emory, Reconnoissance, p. 88. [732] Gomara, Historia de la Conquista de Méjico, Veytia's edition, p. 299. [733] Ibid., p. 310. [734] Smithsonian Contributions, Ancient monuments of New York, vol. 2. [735] Buckingham Smith, Relacion de la Jornada de Coronado á Cibola, Coleccion de Documentos para la Historia de Florida, London, 1857, vol. 1, p. 148. [736] Ibid., vol. 1, p. 150. [737] Brinton, Myths of the New World, p. 253. [738] London, 1844, vol. 1, pp. 26, 29, 36, 93. [739] Ibid., p. 278.
[740] Ibid., vol. 2, p. 389. [741] Monarchia Indiana, lib. 6, cap. 45, p. 80. [742] Ibid., lib. 19, cap. 22, pp. 357-358. [743] Ternaux-Compans, vol. 10, p. 240. [744] London, 1843, p. 248. [745] Pimentel, Lenguas Indígenas de México, vol. 3, pp. 498, 499. [746] Brinton, Myths of the New World, pp. 285, 286. [747] Ibid., p. 264. [748] Kingsborough, vol. 8, sup., p. 249. [749] Parkman, Jesuits, introduction, p. lxxxiv.
Transcriber's Notes Obvious typographical errors have been repaired. Non- standard spellings, including those in other languages, were retained as in the original. Hyphenation and accent variants that could not be clearly resolved, were retained. The few cases of ellipses shown as asterisks were also retained. p. 579, paragraph beginning Dr. Joseph Lanzoni: both chermisinum and chermesinum occurred in the original as shown. p. 585, paragraph beginning At intervals: Three several times they is as in the original.
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  • 6. Methods in ENZYMOLOGY Volume 471 Two-Component Signaling Systems, Part C Edited by Melvin I. Simon, Brian R. Crane and Alexandrine Crane
  • 7. 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
  • 8. Academic Press is an imprint of Elsevier 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 32 Jamestown Road, London NW1 7BY, UK First edition 2010 Copyright # 2010, Elsevier Inc. All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: permissions@ elsevier.com. Alternatively you can submit your request online by visiting the Elsevier web site at http:/ /elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made For information on all Academic Press publications visit our website at elsevierdirect.com ISBN: 978-0-12-381347-3 ISSN: 0076-6879 Printed and bound in United States of America 10 11 12 10 9 8 7 6 5 4 3 2 1
  • 9. CONTRIBUTORS Adrián F. Alvarez Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, México D.F., México Babak Andi Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA Paul V. Attwood School of Biomedical, Biomolecular and Chemical Sciences (M310), The University of Western Australia, Crawley, Western Australia, Australia Paul G. Besant School of Biomedical, Biomolecular and Chemical Sciences (M310), The University of Western Australia, Crawley, Western Australia, Australia Roberto A. Bogomolni Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, USA Katherine A. Borkovich Department of Plant Pathology and Microbiology, University of California, Riverside, California, USA Robert B. Bourret Departments of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Winslow R. Briggs Department of Plant Biology, Carnegie Institution for Science, Stanford, Califor- nia, USA Edmundo Calva Departamento de Microbiologı́a Molecular, Instituto de Biotecnologı́a, Universi- dad Nacional Autónoma de México AP 510-3, Cuernavaca, Morelos, Mexico Paul F. Cook Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA xiii
  • 10. Rachel L. Creager-Allen Departments of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA Nabanita De Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Jan S. Fassler Department of Biology, University of Iowa, Iowa City, Iowa, USA Marcus A. Frederickson Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, USA Sumire Fujiwara Department of Plant Cellular and Molecular Biology, Ohio State University, Columbus, Ohio, USA Dimitris Georgellis Departamento de Genética Molecular, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México, México D.F., México Eric Giraud LaboratoiredesSymbiosesTropicalesetMéditerranéennes,IRD,CIRAD,AGRO-M, INRA, UM2, TA A-82/J, Campus de Baillarguet, Montpellier Cedex 5, France Zemer Gitai DepartmentofMolecularBiology,PrincetonUniversity,Princeton,NewJersey,USA Erin A. Gontang Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA Mark Goulian Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA Penelope I. Higgs Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Hans-Jörg Hippe Medizinische Klinik III, Universitätsklinikum, Universität Heidelberg, Heidelberg, Germany James A. Hoch Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA xiv Contributors
  • 11. Terence Hwa Center for Theoretical Biological Physics, University of California San Diego, La Jolla, California, USA Sakthimala Jagadeesan Department of Molecular Biology, Max Planck Institute for Infection Biology, Berlin, Germany Carol A. Jones Department of Plant Pathology and Microbiology, University of California, Riverside, California, USA Alla O. Kaserer Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA Woe-Yeon Kim Department of Plant Cellular and Molecular Biology, Ohio State University, Columbus, Ohio, USA, and Division of Applied Life Science (BK21 Program) and Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju, Korea Susanne Klumppw Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms- Universität, Münster, Germany Roberto Kolter Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA Michael Korth Institut für Pharmakologie für Pharmazeuten, Universitätsklinikum Hamburg- Eppendorf, Hamburg, Germany Petya V. Krasteva Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Jérôme Lavergne CEA, DSV, IBEB, Laboratoire de Bioénergétique Cellulaire, and CNRS, UMR 6191, Biologie Végétale et Microbiologie Environnementales, and Aix-Marseille Université, Saint-Paul-lez-Durance, France Bongsoo Lee Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany w Deceased June 17, 2009 Contributors xv
  • 12. Jürgen U. Linder Pharmaceutical Institute, University of Tübingen, Tübingen, Germany Daniel López Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts, USA Katrin Ludwig Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms- Universität, Münster, Germany Bryan Lunt Institute for Scientific Interchange, Viale S. Severo 65, Torino, Italy C. Robertson McClung Department of Biological Sciences, Dartmouth College, Hanover, New Hamp- shire, USA Takeshi Mizuno Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Japan Aaron M. Moore Departments of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Susumu Morigasaki Department of Microbiology, University of California, Davis, California, USA, and Graduate School of Biological Sciences, Nara Institute of Science and Tech- nology, Ikoma, Nara, Japan Marcos V. A. S. Navarro Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Jose N. Onuchic Center for Theoretical Biological Physics, University of California San Diego, La Jolla, California, USA Ricardo Oropeza Departamento de Microbiologı́a Molecular, Instituto de Biotecnologı́a, Universidad Nacional Autónoma de México AP 510-3, Cuernavaca, Morelos, Mexico Stephani C. Page Departments of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina, USA Andrea Procaccini Institute for Scientific Interchange, Viale S. Severo 65, Torino, Italy xvi Contributors
  • 13. Patrice A. Salomé Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire, USA Andreas Schramm Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany Alexander Schug Center for Theoretical Biological Physics, University of California San Diego, La Jolla, California, USA Joachim E. Schultz Pharmaceutical Institute, University of Tübingen, Tübingen, Germany Kazuhiro Shiozaki Department of Microbiology, University of California, Davis, California, USA Ruth E. Silversmith Departments of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Albert Siryaporn Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA David E. Somers Department of Plant Cellular and Molecular Biology, Ohio State University, Columbus, Ohio, USA Holger Sondermann Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Hendrik Szurmant Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA Stephanie A. Thomas Departments of Microbiology and Immunology, University of North Carolina, Chapel Hill, North Carolina, USA Tong-Seung Tseng Department of Plant Biology, Carnegie Institution for Science, Stanford, Califor- nia, USA André Verméglio CEA, DSV, IBEB, Laboratoire de Bioénergétique Cellulaire, and CNRS, UMR 6191, Biologie Végétale et Microbiologie Environnementales, and Aix-Marseille Université, Saint-Paul-lez-Durance, France Contributors xvii
  • 14. Qi Wang Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA Martin Weigt Institute for Scientific Interchange, Viale S. Severo 65, Torino, Italy John N. Werner DepartmentofMolecularBiology,PrincetonUniversity,Princeton,NewJersey,USA Ann H. West Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma, USA Thomas Wieland Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Medizinische Fakultät Mannheim, Universität Heidelberg, Mannheim, Germany Takafumi Yamashino Laboratory of Molecular Microbiology, School of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Japan Xiao-Bo Zhou Institut für Pharmakologie für Pharmazeuten, Universitätsklinikum Hamburg- Eppendorf, Hamburg, Germany xviii Contributors
  • 15. 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 xix
  • 16. 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 xx Methods in Enzymology
  • 17. 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 Methods in Enzymology xxi
  • 18. 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 xxii Methods in Enzymology
  • 19. 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 Methods in Enzymology xxiii
  • 20. 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 xxiv Methods in Enzymology
  • 21. 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 Methods in Enzymology xxv
  • 22. 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 xxvi Methods in Enzymology
  • 23. 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 Methods in Enzymology xxvii
  • 24. 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 xxviii Methods in Enzymology
  • 25. 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 Methods in Enzymology xxix
  • 26. 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 xxx Methods in Enzymology
  • 27. 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 DÜZGÜNES, VOLUME 221. Membrane Fusion Techniques (Part B) Edited by NEJAT DÜZGÜNES, 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 Methods in Enzymology xxxi
  • 28. 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 GÖTE 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 xxxii Methods in Enzymology
  • 29. 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 Methods in Enzymology xxxiii
  • 30. VOLUME 256. Small GTPases and Their Regulators (Part B: Rho Family) Edited by W. E. BALCH, CHANNING J. DER, AND ALAN HALL 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 xxxiv Methods in Enzymology
  • 31. 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 Methods in Enzymology xxxv
  • 32. VOLUME 293. Ion Channels (Part B) Edited by P. MICHAEL CONN 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 xxxvi Methods in Enzymology
  • 33. VOLUME 311. Sphingolipid Metabolism and Cell Signaling (Part A) Edited by ALFRED H. MERRILL, JR., AND YUSUF A. HANNUN 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 Methods in Enzymology xxxvii
  • 34. 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 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 DÖTSCH, AND ULI SCHMITZ VOLUME 339. Nuclear Magnetic Resonance of Biological Macromolecules (Part B) Edited by THOMAS L. JAMES, VOLKER DÖTSCH, 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 xxxviii Methods in Enzymology
  • 35. VOLUME 345. G Protein Pathways (Part C: Effector Mechanisms) Edited by RAVI IYENGAR AND JOHN D. HILDEBRANDT 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 Methods in Enzymology xxxix
  • 36. VOLUME 362. Recognition of Carbohydrates in Biological Systems (Part A) Edited by YUAN C. LEE AND REIKO T. LEE 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 DÜZGÜNES, 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 DÜZGÜNES, VOLUME 373. Liposomes (Part C) Edited by NEJAT DÜZGÜNES, 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 xl Methods in Enzymology
  • 37. VOLUME 381. Oxygen Sensing Edited by CHANDAN K. SEN AND GREGG L. SEMENZA 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 DÜZGÜNES, 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 DÜZGÜNES, 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 Methods in Enzymology xli
  • 38. VOLUME 400. Phase II Conjugation Enzymes and Transport Systems Edited by HELMUT SIES AND LESTER PACKER 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 xlii Methods in Enzymology
  • 39. VOLUME 418. Embryonic Stem Cells Edited by IRINA KLIMANSKAYA AND ROBERT LANZA 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 Methods in Enzymology xliii
  • 40. VOLUME 434. Lipidomics and Bioactive Lipids: Lipids and Cell Signaling Edited by H. ALEX BROWN VOLUME 435. Oxygen Biology and Hypoxia Edited by HELMUT SIES AND BERNHARD BRÜNE 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 VOLUME 447. RNA Turnover in Bacteria, Archaea and Organelles Edited by LYNNE E. MAQUAT AND CECILIA M. ARRAIANO VOLUME 448. RNA Turnover in Eukaryotes: Nucleases, Pathways and Analysis of mRNA Decay Edited by LYNNE E. MAQUAT AND MEGERDITCH KILEDJIAN xliv Methods in Enzymology
  • 41. VOLUME 449. RNA Turnover in Eukaryotes: Analysis of Specialized and Quality Control RNA Decay Pathways Edited by LYNNE E. MAQUAT AND MEGERDITCH KILEDJIAN VOLUME 450. Fluorescence Spectroscopy Edited by LUDWIG BRAND AND MICHAEL L. JOHNSON VOLUME 451. Autophagy: Lower Eukaryotes and Non-Mammalian Systems (Part A) Edited by DANIEL J. KLIONSKY VOLUME 452. Autophagy in Mammalian Systems (Part B) Edited by DANIEL J. KLIONSKY VOLUME 453. Autophagy in Disease and Clinical Applications (Part C) Edited by DANIEL J. KLIONSKY VOLUME 454. Computer Methods (Part A) Edited by MICHAEL L. JOHNSON AND LUDWIG BRAND VOLUME 455. Biothermodynamics (Part A) Edited by MICHAEL L. JOHNSON, JO M. HOLT, AND GARY K. ACKERS (RETIRED) VOLUME 456. Mitochondrial Function, Part A: Mitochondrial Electron Transport Complexes and Reactive Oxygen Species Edited by WILLIAM S. ALLISON AND IMMO E. SCHEFFLER VOLUME 457. Mitochondrial Function, Part B: Mitochondrial Protein Kinases, Protein Phosphatases and Mitochondrial Diseases Edited by WILLIAM S. ALLISON AND ANNE N. MURPHY VOLUME 458. Complex Enzymes in Microbial Natural Product Biosynthesis, Part A: Overview Articles and Peptides Edited by DAVID A. HOPWOOD VOLUME 459. Complex Enzymes in Microbial Natural Product Biosynthesis, Part B: Polyketides, Aminocoumarins and Carbohydrates Edited by DAVID A. HOPWOOD VOLUME 460. Chemokines, Part A Edited by TRACY M. HANDEL AND DAMON J. HAMEL VOLUME 461. Chemokines, Part B Edited by TRACY M. HANDEL AND DAMON J. HAMEL VOLUME 462. Non-Natural Amino Acids Edited by TOM W. MUIR AND JOHN N. ABELSON VOLUME 463. Guide to Protein Purification, 2nd Edition Edited by RICHARD R. BURGESS AND MURRAY P. DEUTSCHER VOLUME 464. Liposomes, Part F Edited by NEJAT DÜZGÜNES, Methods in Enzymology xlv
  • 42. VOLUME 465. Liposomes, Part G Edited by NEJAT DÜZGÜNES, VOLUME 466. Biothermodynamics, Part B Edited by MICHAEL L. JOHNSON, GARY K. ACKERS, AND JO M. HOLT VOLUME 467. Computer Methods Part B Edited by MICHAEL L. JOHNSON AND LUDWIG BRAND VOLUME 468. Biophysical, Chemical, and Functional Probes of RNA Structure, Interactions and Folding: Part A Edited by DANIEL HERSCHLAG VOLUME 469. Biophysical, Chemical, and Functional Probes of RNA Structure, Interactions and Folding: Part B Edited by DANIEL HERSCHLAG VOLUME 470. Guide to Yeast Genetics: Functional Genomics, Proteomics, and Other Systems Analysis, 2nd Edition Edited by GERALD FINK, JONATHAN WEISSMAN, AND CHRISTINE GUTHRIE VOLUME 471. Two-Component Signaling Systems, Part C Edited by MELVIN I. SIMON, BRIAN R. CRANE, AND ALEXANDRINE CRANE xlvi Methods in Enzymology
  • 43. C H A P T E R O N E Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation Albert Siryaporn and Mark Goulian Contents 1. Overview 2 2. Sources of Cross-Talk 3 3. Cross-Talk Suppression 4 4. Transcriptional Reporters 6 5. Response Regulator Localization 8 6. Phosphatase Cross-Talk 13 7. Signal Response in Cross-Talk Networks 14 8. Concluding Remarks 14 Acknowledgments 15 References 15 Abstract Cross-talk between noncognate histidine kinases and response regulators has been widely reported in vitro and, in specific mutant backgrounds and condi- tions, in vivo. However, in most cases there is little evidence supporting a physiological role of cross-talk. Indeed, histidine kinases and response regula- tors show remarkable specificity for their cognate partners. In vivo studies of cross-talk have the potential to establish mechanisms that control specificity and, if the cross-talk is observable in wild-type strains, may reveal new levels of cross-regulation. However such studies can be complicated by effects of other regulatory circuits and by the inactivation of mechanisms that would otherwise suppress cross-talk. It is thus easy to mis- or overinterpret the significance of such studies. We address potential complications associated with measuring cross-talk and discuss some methods for identifying and unmasking sources of cross-talk in cells using transcriptional reporters and in vivo DNA-binding assays. Methods in Enzymology, Volume 471 # 2010 Elsevier Inc. ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)71001-6 All rights reserved. Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA 1
  • 44. 1. Overview When sequence comparisons first revealed the generality of two- component signaling, it was immediately appreciated that histidine kinases could potentially phosphorylate noncognate response regulators (Nixon et al., 1986) and that such cross-talk could play a role in signal integration and processing (Stock et al., 1989) (Fig. 1.1). Early studies demonstrated the existence of cross-talk in vitro, although the cross-phosphorylation occurred with considerably slower kinetics compared with phosphorylation between cognate partners (Igo et al., 1989; Ninfa et al., 1988). Cross-talk was also observed in vivo, but only in modified strains in which various regulators were deleted or overexpressed. In the two decades since these early con- siderations, cross-talk has not emerged as a common theme in two-compo- nent signaling. Despite numerous reports in the literature, in most cases there is little evidence to support the claim that there are detectable effects from cross-talk in wild-type strains (Laub and Goulian, 2007). There are several factors that make attempts at identifying cross-talk subject to mis- or overinterpretation. In vitro studies have demonstrated that numerous histidine kinases can phosphorylate noncognate response regula- tors, with some histidine kinases showing a considerable level of promiscu- ity (Skerker et al., 2005; Yamamoto et al., 2005). However, as a rule, histidine kinases show a very strong kinetic preference for their cognate response regulator (Skerker et al., 2005). Thus, a demonstration of cross-talk in vitro, without a comparison of the kinetics of phosphotransfer between cognate and noncognate pairs, is not particularly compelling evidence for cross-talk function in vivo (Laub and Goulian, 2007; Skerker et al., 2005). There are additional complications when studying cross-talk in vivo. For most systems, it is not feasible to directly measure response regulator HK1 RR1 HK2 RR2 HK3 RR3 ? ? Figure 1.1 Cross-talk between noncognate histidine kinases (HKs) and response regulators (RRs) could potentially enable complex processing of multiple input signals. However, to date there has been relatively little evidence for cross-talk in wild-type strains. 2 Albert Siryaporn and Mark Goulian
  • 45. phosphorylation in cells. Instead, phosphorylation is inferred from transcrip- tion measurements, e.g., with transcriptional reporters. However, it is often difficult to disentangle the effects of other regulatory factors that may act at the promoter of interest. In addition, studies in mutant strains may identify cross-talk that is irrelevant in the context of wild-type cells. Mechanisms have been identified that suppress cross-talk between noncognate pairs in vivo (Groban et al., 2009; Siryaporn and Goulian, 2008). When these mechanisms are eliminated by mutation, significant cross-talk may emerge (Kim et al., 1996; Silva et al., 1998; Siryaporn and Goulian, 2008), which can give the false impression that cross-talk functions in wild-type strains. Here we describe some of the issues one should take into account when looking for sources or effects of cross-talk. These methods can also be used to explore various mechanisms that limit cross-talk and to explore the molecular determinants of specificity among histidine kinases and response regulators. This chapter covers potential sources of cross-talk, cross-talk suppression mechanisms, and methods for measuring cross-talk in cells. In particular, it provides specific details on how to measure the association of response regulator-fluorescent protein fusions to DNA in vivo, a method which can distinguish cross-talk from the effects of other regulatory path- ways. We also briefly discuss methods for characterizing phosphatase cross- talk and the effects of signal response on cross-talk networks. 2. Sources of Cross-Talk A response regulator can potentially be phosphorylated by a cognate histidine kinase, noncognate histidine kinases, and small molecule phos- phodonors. It is generally assumed that when genes for a response regulator and a histidine kinase are in the same operon, the encoded proteins are cognate pairs. Situations in which more than one histidine kinase or response regulator is encoded in an operon, however, may be more com- plex. It is also not unusual to encounter ‘‘orphan’’ histidine kinases and response regulators for which no clear partner has been identified. Many microbial genomes appear to have unequal numbers of histidine kinases and response regulators (Alm et al., 2006), which suggests that some response regulators may have more than one source of phosphorylation and similarly some histidine kinases may phosphorylate multiple response regulators. In some systems, the histidine kinase is bifunctional and mediates response regulator phosphorylation and dephosphorylation. In this case, there could in principle be cross-talk from either the phosphotransfer or phosphatase activities. However, we are unaware of any reports of phos- phatase cross-talk, even in mutant strains where phosphotransfer cross-talk has been observed. For example, the histidine kinase CpxA phosphorylates Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation 3
  • 46. and dephosphorylates its cognate response regulator CpxR. Cross-talk from CpxA to OmpR can be detected in strains deleted for envZ and cpxR, however, dephosphorylation of OmpR-P by CpxA is not detected (Siryaporn and Goulian, 2008). Kinetic modeling of the histidine kinase– response regulator interaction suggests that phosphatase activity may emerge for sufficiently strong interaction between a bifunctional histidine kinase and response regulator (Siryaporn et al., in preparation). Thus, the difficulty in observing phosphatase cross-talk may reflect the relatively weak interac- tions between noncognate partners. A number of response regulators are phosphorylated by the small mole- cule phosphodonor acetyl phosphate (McCleary and Stock, 1994; Wanner and Wilmes-Riesenberg, 1992; Wolfe, 2005). The cellular concentration of this high-energy source of phosphoryl groups is strongly dependent on growth conditions, such as carbon source (Wolfe, 2005). Concentrations can be quite high in some cases, which raises the possibility that acetyl phosphate may integrate metabolic status into some two-component systems (Fredericks et al., 2006; Klein et al., 2007; Wanner, 1992). For most two-component systems, however, a role for acetyl phosphate has not been established. There is also the possibility that other small molecule phosphodonors (Lukat et al., 1992) could function to phosphorylate response regulators in some contexts in vivo. The ability of a phosphodonor to affect the steady-state phosphorylation level of a particular response regulator depends on the rate of the phosphory- lation reaction relative to the rates of other sources of phosphorylation and dephosphorylation. While phosphorylation rates of cytoplasmic fragments of histidine kinases are readily measured in vitro using purified components (Laub et al., 2007), the effects of individual phosphorylation sources on levels of phosphorylated response regulator in vivo can be quite sensitive to the strain background and can be difficult to measure. For example, a bifunctional histidine kinase that has high rates of phosphorylation and dephosphorylation can give levels of phosphorylated response regulator that are comparable to those that would arise from a histidine kinase that has weak phosphorylation activity but lacks a mechanism for response regulator dephosphorylation (Fig. 1.2A). However, through a careful and systematic analysis using tran- scriptional and translational reporter fusions, the contributions from different phosphorylation sources can in some cases be disentangled. 3. Cross-Talk Suppression Studies have shown that cross-talk can be suppressed by cognate histidine kinases and response regulators (Fig. 1.2B and C) (Groban et al., 2009; Kim et al., 1996; Silva et al., 1998; Siryaporn and Goulian, 2008). 4 Albert Siryaporn and Mark Goulian
  • 47. Mutations that relieve these suppression mechanisms could produce signals of cross-talk that would otherwise be squelched in the wild-type strain. Toavoid over-or misinterpretingthe resultsfromanalysisof mutant strains, it is important to take into account the potential effects of these mechanisms. HK1 A B RR1 RR1 HK1 P P HK2 RR1 RR1 P P HK2 RR1 RR2 HK1 RR1 HK2 X X Histidine kinase buffering Response regulator inhibition C Figure 1.2 (A) Two different scenarios in which very different rates of phosphoryla- tion can give the same steady state level of phosphorylated response regulator (RR-P). (Left) A bifunctional histidine kinase phosphorylates and dephosphorylates a response regulator with very high rates such that, under moderate stimulation, a moderate level of RR-P is obtained at steady state. (Right) A histidine kinase that phosphorylates a response regulator very weakly may, in the absence of any phosphatase activity, also produce moderate levels of RR-P. (B, C) Suppression of cross-talk from a histidine kinase HK2 to response regulator RR1 by cognate partners (Groban et al., 2009; Siryaporn and Goulian, 2008). (B) The high flux of phosphorylation and dephosphory- lation of RR1 by the bifunctional histidine kinase HK1 can suppress or buffer against cross-talk from HK2. (C) Suppression of cross-talk by a cognate response regulator. The cognate response regulator RR2 outcompetes the noncognate regulator RR1 for interaction with the histidine kinase HK2. By removing RR2, significant cross-talk from HK2 to RR1 may be detected, which would be otherwise absent in the wild-type strain. Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation 5
  • 48. Bifunctional histidine kinases set the level of phosphorylated response regulator through a balance of phosphorylation and dephosphorylation. This cycle can effectively act as a buffer to suppress the effects of weaker phosphorylation sources (Fig. 1.2B) (Groban et al., 2009; Kim et al., 1996; Silva et al., 1998; Siryaporn and Goulian, 2008). Therefore, deletion of a bifunctional histidine kinase could reveal phosphorylation of the response regulator from a noncognate histidine kinase or other phosphodonor (e.g., Batchelor et al., 2005; Danese and Silhavy, 1998; Hutchings et al., 2006; Wolfe et al., 2008). However, this alternate source may give a much lower flux of phosphoryl groups and may thus be physiologically irrelevant in wild-type strains. A response regulator can also prevent its cognate histidine kinase from participating in cross-talk (Fig. 1.2C). For example, cross-talk from CpxA to OmpR is suppressed by CpxR (Siryaporn and Goulian, 2008). This is likely to occur through competitive inhibition, in which the cognate response regulator outcompetes noncognate response regulators for inter- action with the histidine kinase. Thus, deletion or inactivation of a response regulator could potentially produce cross-talk from the cognate histidine kinase to some other response regulator. Significant overexpression of the histidine kinase could also have a similar result. 4. Transcriptional Reporters In most two-component systems that have been studied, the phos- phorylated response regulator controls transcription of effecter genes. Therefore, transcriptional reporter fusions have been a standard tool for measuring the output of signal transduction systems. A reporter gene such as gfp or lacZ is placed under control of a promoter of interest and expression of the reporter is measured through fluorescence or enzyme assays. Cross-talk can be measured using transcriptional reporter fusions. Chromosomal fusions are preferable to constructs on multicopy plasmids since they avoid potential effects on reporter gene expression from titrating out response regulator and changes in plasmid copy number. It is always a good idea to assessthe dynamic range of reporter expression sincethis gives an indication of the sensitivity to changes in output under various conditions. The basal expression level can be determined from measurements in a strain deleted for the response regulator of interest. The range of activation can be determined by making measurements for different levels of input stimulus, if the stimulus is known, or by varying expression of a constitutively active version of the histidine kinase, if one is available. For experiments in which a fluorescent reporter such as gfp is used, it is preferable to grow cultures using a growth medium with a low level of autofluorescence such as a 6 Albert Siryaporn and Mark Goulian
  • 49. minimal salts medium (Miller, 1992), as this generally increases the sensitivity of measurements. Rich media often give moderate levels of autofluores- cence, which could reduce the sensitivity of fluorescence measurements. Evidence for the presence and absence of cross-talk can be followed by monitoring changes in transcriptional reporter expression for different genetic backgrounds and conditions. Depending on the application, it may be necessary to remove cross-talk suppression mechanisms described above by deleting appropriate histidine kinases and response regulators before cross-talk can be detected. The observation of cross-talk in this case should not be used to conclude that there is cross-talk in the wild-type strain. Specific histidine kinases that are potential sources of cross-talk can be tested by changing expression levels or stimulation with signal. However, their effects on cross-talk will depend on details of the histidine kinase and its interaction with the noncognate response regulator, as discussed below. A histidine kinase sets the level of phosphorylated response regulator through phosphorylation alone (monofunctional behavior) or through a balance of phosphorylation and dephosphorylation (bifunctional behavior). For histidine kinases displaying monofunctional behavior, the level of phosphorylated response regulator is expected to be quite sensitive to histidine kinase expression level (E. Batchelor and M. Goulian, unpublished observations; Miyashiro and Goulian, 2008). On the other hand, when the histidine kinase has bifunctional behavior, modeling and experiments sug- gest the level of phosphorylated response regulator can be relatively insen- sitive to histidine kinase expression levels (Batchelor and Goulian, 2003; Miyashiro and Goulian, 2008; Shinar et al., 2007). Thus, cross-talk may be observed in a context of histidine kinase overexpression even though it appears that cognate response regulator phosphorylation is unchanged. In addition, in at least some cases, a histidine kinase can exhibit bifunctional behavior against its cognate partner while displaying monofunctional behavior against a noncognate partner (Siryaporn and Goulian, 2008). The choice of carbon source may have a significant effect on the level of acetyl phosphate in the cell (McCleary and Stock, 1994) and thus the level of response regulator phosphorylation. The desired level of acetyl phosphate may depend on the experimental condition being tested. In some cases, significant levels of acetyl phosphate may provide a means of increasing the basal level of response regulator phosphorylation and hence increase the sensitivity to cross-talk by other phosphodonors. In other cases, one may want to eliminate acetyl phosphate or keep its level to a minimum in order to distinguish its effects from other sources of response regulator phosphor- ylation. For Escherichia coli, growth using carbon sources such as glucose, pyruvate, and acetate produce high levels of acetyl phosphate. Low levels can be achieved by growing on glycerol. One can also modulate acetyl phosphate levels by deleting one or both of pta and ackA, depending on the flux of acetate through the Pta (phosphotransacetylase)–AckA (acetate Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation 7
  • 50. kinase) pathway (Wolfe et al., 2008). Deletion of pta ackA abolishes production of acetyl phosphate. However, care should be taken in altering the Pta–AckA pathway as this may have additional indirect effects on some two-component systems (Wolfe et al., 2008). While transcriptional reporter fusions can provide effective measure- ments of two-component system output, they are also subject to the effects of other regulatory factors. There are many examples of genes whose expression is controlled by multiple regulatory proteins and small RNAs. Even many relatively well-characterized systems may have additional unidentified regulators whose effects could give the false impression of cross-talk. In some cases, promoters may also be controlled by multiple response regulators (e.g., Batchelor et al., 2005; Mouslim and Groisman, 2003). To distinguish between cross-talk and alternate regulatory pathways, one may need to use additional methods to follow response regulator phosphorylation or binding to promoters. 5. Response Regulator Localization If a functional fluorescent protein (FP) fusion to the response regulator of interest is available then one can use a fluorescence localization assay to provide further support for cross-talk in vivo (Batchelor and Goulian, 2006; Siryaporn and Goulian, 2008). The method takes advantage of the tendency of plasmids with partitioning systems to form clusters in the cell (Pogliano, 2002). In this assay, cells express the response regulator-FP fusion and also contain a plasmid that has one or more binding sites for the response regulator. Plasmid clustering provides a high local density of response regulator binding sites in the cell (Fig. 1.3A). Significant response regula- tor-FP binding to DNA results in intense localized regions of fluorescence, which appear as bright foci or spots under a fluorescence microscope and can be easily quantified by image analysis (Fig. 1.3B–D). Assuming DNA binding is modulated by response regulator phosphorylation, changes in response regulator-FP colocalization with the plasmid can be used to infer changes in response regulator phosphorylation. We have been able to construct functional fluorescent protein fusions to the C-termini of several different response regulators in E. coli, although in all of the cases that we tested the fusions showed decreased activity. We are unaware of any general rules regarding the choice of C- or N-termini for the fusion, or whether or not to include a flexible linker. It is probably best to try several different constructs. For the plasmid, we have used constructs based on pRS415 (Batchelor and Goulian, 2006; Simons et al., 1987), which is derived from pBR322. Many other plasmids should work as well although they may show differing levels of clustering. Plasmid clustering can 8 Albert Siryaporn and Mark Goulian
  • 51. be characterized through the use of lac operators and a fluorescent protein fusion to lac repressor, as discussed below. The ability to detect fluorescent protein localization to plasmids will depend on the binding affinity of the Low RR-P High RR-P Plasmid with binding sites for LacI and RR RR-YFP CFP-LacI A DIC CFP YFP Pixel values Pixel distance2 Extracted region B C 18 13 10 9 10 13 18 13 8 5 4 5 8 13 10 5 2 1 2 5 10 9 4 1 0 1 4 9 10 5 2 1 2 5 10 13 8 5 4 5 8 13 18 13 10 9 10 13 18 172 252 320 348 356 325 272 212 304 377 410 434 387 326 233 336 415 513 491 428 332 249 360 446 521 497 400 276 232 339 403 431 408 317 226 202 266 297 316 283 216 165 159 184 199 207 188 151 132 100 200 300 400 500 0 600 1 2 3 0 4 Pixel value Radius (pixels) RR binding sites data ( fit) Vector only data ( fit) RR binding sites Vector D Figure 1.3 Measurement of response regulator binding to DNA in vivo. (A) Schematic of the method, which uses a functional translational fusion of a fluorescent protein (e.g., YFP) to the response regulator and a plasmid containing response regulator binding sites. The plasmid also contains lac operators, which are bound by CFP-LacI. Plasmid clustering in the cell results in a high local concentration of binding sites, which is easily visualized as a bright fluorescent spot in the CFP channel. Response regulator binding to plasmid also results in a bright fluorescent spot in the YFP channel. (B) Images of cells expressing OmpR-YFP andcontaininga plasmid with OmpRbinding sites (toprow)or an empty control vector (bottom row). DIC—differential interference contrast image. (C) Example of a YFP spot, corresponding to the dashed square in the upper right image in (B), and the associated pixel values and distances from the center. (D) Gaussian fit to the profileofpixelvaluesfrom(C).Acorrespondingfittotheneighborhoodofa maximalYFP pixel in a cell containing the vector control is also shown. Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation 9
  • 52. phosphorylated response regulator-FP fusion as well as the extent of phosphorylation. One can increase the sensitivity by including several binding sites in the plasmid as needed. It is generally advisable to include a means to monitor plasmid clustering independently of response regulator binding to DNA. This controls for potential changes in plasmid clustering as a result of changes in cell physiol- ogy. This is easily accomplished by cloning lac operators into the plasmid and expressing a fluorescent protein fusion to lac repressor. We routinely use a CFP-LacI fusion (Batchelor and Goulian, 2006), which can be simulta- neously imaged with a response regulator-YFP fusion. We have used an array of tandem lac operator repeats (Robinett et al., 1996) to mark the plasmid. This produces extremely bright foci, which is convenient as it provides increased sensitivity and enables shorter exposures when acquiring images. After plasmid clusters have been identified in the CFP channel, one can then quantify the YFP fluorescence in the neighborhood of this loca- tion. Under conditions of very high response regulator phosphorylation one may observe additional foci that are not associated with plasmids. In at least one case (OmpR-YFP), we have determined that these foci are due to response regulator binding to chromosomal loci (E. Libby and M. Goulian, manuscript in preparation). Fluorescence localization can be visualized using techniques adapted from general single-cell fluorescence microscopy methods (e.g., Batchelor and Goulian, 2006; Miyashiro and Goulian, 2007). 1. Grow cultures in medium with aeration at the appropriate growth temperature to saturation. To maximize the sensitivity of this assay, a culture medium that gives relatively low levels of autofluorescence should be used. A minimal salts medium is ideal in this respect and also allows for choice of carbon source. 2. Dilute cultures at least 1:1000 into the same medium and grow to early exponential phase (optical density of 0.1–0.3 at 600 nm for E. coli). 3. Shortly before cells reach the target density, preheat the microscope stage to the same temperature as that used for growing the cell cultures. Prepare 1% agarose pads made from the same growth medium and placed between a microscope slide and cover glass. For many small- scale experiments, it is sufficient to use 3 in. 1 in. 1 mm slides and 22 mm square #1.5 cover glass. Place 50–100 ml of molten agarose on the center of slide and cover immediately with coverslip. Agarose pads should be maintained at the growth temperature of the culture and may need time to dry slightly before use. Cells will not immobilize well on agarose pads that are too moist. 4. Carefully lift the cover glass off of the pad and place 5 ml of culture onto cover glass. Replace cover glass back onto agarose pad with culture between the cover glass and pad and put the slide on the 10 Albert Siryaporn and Mark Goulian
  • 53. microscope stage immediately. The number of cells on the agarose pad should be such that cells are easily found in most fields but are in sufficiently low density that there is little or no contact between neigh- boring cells. Obtaining proper cell density in images is especially impor- tant for the fluorescence analysis that follows. 5. Acquire a phase contrast or differential interference contrast (DIC) image and fluorescence images of the same field on a fluorescence microscope (Fig. 1.3B). Image acquisition times should be long enough so that cell fluorescence is significantly higher than background but brief enough so that none of the pixel values is equal to the maximum allowed value. The maximum pixel values are 255, 1023, 4095, and 65355 for 8-, 10- 12-, and 16-bit images, respectively. Objective magnification and resolution of the digital camera should be sufficient so that the shortest dimension of individual cells is at least approximately 5 pixels. Images should be saved without applying any contrast enhancements or other processing. Cell fluorescence levels may be relatively weak for response regulator fusions that are expressed at wild-type levels. In addition, fluorescent foci are not always observable through the eyepiece and may photo-bleach within seconds. It is important to acquire images from fields of cells that have not been previously exposed to light from the fluorescence illumina- tor. In particular, cells should be brought into focus using phase or DIC imaging. The extent of fluorescence localization will vary from cell to cell due to fluctuations in protein expression levels, plasmid copy number, plasmid localization, or other variations in the cellular environment. A representative measure of fluorescence localization for the population can be attained by acquiring images of a large number of cells (e.g., 100–200) and quantifying the extent of localization as described below. While dramatic changes in fluorescence localization may be sufficient to support a particular conclusion, in some cases it may be necessary to distinguish smaller changes in localization that are not easily assessed by eye. The fluorescence localization of cell populations can be quantified from images using software packages such as ImageJ (National Institutes of Health, Bethesda, MD), LabVIEW (National Instruments, Austin, TX), and MATLAB (The MathWorks, Natick, MA), which provide convenient libraries of imaging tools. Modules and plug-ins provided by these packages can be stitched together through scripts or macros to process large numbers of images quickly and accurately. The basic assumption behind this analysis is that fluorescent foci can be accurately represented by point-source intensity profiles. The maximum value of the fluorescence in the cell or the maximum value in the neighbor- hood of the plasmid cluster is taken to be the peak of the fluorescence intensity profile. Pixel values surrounding this maximum are extracted, assigned pixel distances, and fitted to a Gaussian function. Numerical Characterizing Cross-Talk In Vivo: Avoiding Pitfalls and Overinterpretation 11
  • 54. parameters derived from the best-fit intensity profiles are then used to compute an integrated fluorescence intensity, which is a measure of the extent of fluorescence localization. This method assumes that YFP fluores- cence and phase contrast or DIC images have been collected. Instructions also describe cases where the investigator has chosen to express CFP-LacI to identify plasmid clusters as described above. 1. A phase contrast or DIC image and a YFP fluorescence image should be available for analysis. If the CFP-LacI system was also used, then a CFP fluorescence image should also be on hand. 2. Determine cell boundaries using the phase or DIC image. Edge detec- tion algorithms, such as the Sobel edge filter (Castleman, 1996), can be used for identifying cell boundaries in phase contrast images. For images which contain relatively low or complex contrast, such as images from DIC microscopy, cell boundaries can be identified by thresholding pixel values (Batchelor and Goulian, 2006; Miyashiro and Goulian, 2007). 3. Once cell boundaries are determined, it is often convenient to construct a binary (black and white) mask, which defines areas where cells are present. Masks can be constructed from cell boundary images by filling in regions marked by the cell boundaries. 4. Extract pixel values from corresponding fluorescence images using the mask as a guide for cell locations. It is convenient to extract pixel values for individual cells so that each cell can be processed individually from this point on. 5. Determine the location of the plasmid cluster within the cell as follows: (a) If a CFP-LacI image marking plasmid clusters has been acquired, the brightest pixel within each cell boundary in the CFP image should be identified. If the response regulator-YFP is colocalized with the plasmid cluster, it should be observable in the vicinity of the LacI- CFP peak. Identify the location of the maximum YFP peak within the local pixel neighborhood of the CFP peak. For example, for a cell that is represented by approximately 500 pixels, one might choose to restrict the search area to be within a 3 3 pixel area. This maximum YFP value is taken to be the peak of the YFP (response regulator) intensity profile. (b) If a CFP-LacI image is not available, identify the location of the maximum pixel value within the cell boundary of the YFP (response regulator) image. This is taken to be the peak of the YFP (response regulator) intensity profile. 6. Extract pixel values surrounding the peak of the YFP intensity profile (Fig. 1.3B). The extracted area should be large enough to encompass a section representative of the spot and should not include areas outside of the cell. Assign a radial distance to each pixel value with the peak pixel value as the center (Fig. 1.3C). 12 Albert Siryaporn and Mark Goulian
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  • 56. [557] Mendieta, p. 110. [558] Vol. 3, cap. 5, p. 234. [559] Herrera, dec. 2, lib. 6, p. 141. [560] Kingsborough, vol. 7, chap. 4. [561] Teutonic Mythology, vol. 3, p. 1233. [562] Ibid. [563] Fables and Rites of the Incas, Padre Christoval de Molina (Cuzco, 1570-1584), transl. by Clements R. Markham, Hakluyt Society trans., vol. 48, London, 1873, p. 48. [564] The common people wore a black llautu. See Garcilaso, Comentarios, Markham's transl., Hak. Soc., vol. 41, pp. 88, 89. [565] Ibid., p. 85. [566] Ibid., p. 89. [567] Quando vàn à sembrar las Tierras del Sol, vàn solos los Principales à trabajar, i vàn con insignias blancas, i en las espaldas unos Cordones tendidos blancos, à modo de Ministros del Altar.—Herrera, dec. 5, lib. 4, cap. 6, pp. 94-95. [568] Picart, Cérémonies et Coûtumes, etc., Amsterdam, 1735, vol. 6, p. 92. [569] Montfaucon, L'antiquité expliquée, tome 2, pt. 1, p. 33. [570] Hawkesworth, Voyages, vol. 3, p. 229. [571] Voyage to Congo, in Pinkerton's Voyages, vol. 16, p. 237. [572] Pinkerton, Voyages, vol. 16, p. 388. [573] Speke, Source of the Nile, London, 1863, p. 125. [574] London, 1877, vol. 2, p. 131. [575] Stanley, Through the Dark Continent, vol. 2, p. 330. [576] Schweinfurth, Heart of Africa, London, 1873, vol. 1, p. 154. [577] Winstanley, Abyssinia, vol. 2, p. 68. [578] This cord is worn about the neck. Ibid., p. 257.
  • 57. [579] Ibid., vol. 1, p. 235. [580] Ibid., vol. 2, p. 132. [581] Ibid., p. 165. [582] Ibid., p. 292. [583] Malte-Brun, Universal Geography, vol. 4, p. 259, Phila., 1832. [584] Grimm, Teutonic Mythology, vol. 2, p. 640. [585] Nightingale, quoted in Madden, Shrines and Sepulchres, vol. 1, pp. 557, 558. [586] Leems, Account of Danish Lapland, in Pinkerton, Voyages, London, 1808, vol. 1, p. 471. [587] Brand, Popular Antiquities, vol. 3, p. 5. See also John Scheffer, Lapland, Oxford, 1674, p. 58. [588] Act IV, scene 1. [589] Benjamin, Persia, London, 1877, p. 99. [590] Cérémonies et Coûtumes, vol. 7, p. 320. [591] Du Halde, History of China, London, 1736, vol. 4, pp. 244, 245, and elsewhere. [592] Higgins, Anacalypsis, vol. 2, p. 218. [593] Vining, An Inglorious Columbus, p. 635. [594] Du Halde, History of China, London, 1736, vol. 1, p. 270. [595] Univ. Geog., vol. 3, book 75, p. 144, Phila., 1832. [596] Brinton, Myths of the New World, N. Y., 1868, p. 15. [597] Early History of Mankind, London, 1870, p. 156. [598] Voyages, vol. 3, p. 102. [599] Shâyast lâ-Shâyast, cap. 4, pp. 285, 286. In Sacred Books of the East, Max Müller's edition, vol. 5. [600] Monier Williams, Modern India, p. 56. [601] Ibid., pp. 179, 180.
  • 58. [602] Cérémonies et Coûtumes, vol. 7, p. 28. [603] Marco Polo, Travels, in Pinkerton's Voyages, vol. 7, p. 163. [604] Picart, Cérémonies et Coûtumes, etc., vol. 6, pt. 2, p. 99. [605] Malte-Brun, Univ. Geog., vol. 2, lib. 50, p. 235, Philadelphia, 1832. [606] Dr. J. L. August Von Eye, The history of culture, in Iconographic Encyc., Philadelphia, 1886, vol. 2, p. 169. [607] Forlong, Rivers of Life, vol. 1, p. 120. [608] Ibid., pp. 240-241. [609] Forlong, Rivers of Life, vol. 1, p. 328. [610] Ibid., p. 323. [611] Dubois, People of India, p. 9. [612] Mythology of the Hindus. [613] Mythology of the Hindus, pp. 9, 10, 11. [614] Ibid., p. 92. [615] Ibid., p. 155. [616] Ibid., pp. 135, 154, 155. [617] Maurice, Indian Antiquities, London, 1801, vol. 5, p. 205. [618] Ibid., vol. 4, p. 375, where a description of the mode of weaving and twining is given. [619] Ibid., p. 376. [620] Ibid., vol. 5, p. 206. [621] Notes of Richard Johnson, Voyages of Sir Hugh Willoughby and others to the northern part of Russia and Siberia, Pinkerton's Voyages, vol. 1, p. 63. [622] Caron's account of Japan in Pinkerton's Voyages, vol. 7, p. 631. [623] Rev. Father Dandini's Voyage to Mount Libanus, in Pinkerton's Voyages, vol. 10, p. 286.
  • 59. [624] Henry Charles Lea, History of the Inquisition in the Middle Ages, vol. 1, p. 92, New York, 1888. [625] Müller, Sacred Books of the East, vol. 14, Vasishtha, cap. 2, par 6. [626] Ibid., Baudhâyana, prasna 1, adhyâya 5, kandikâ 8, pars. 5- 10, p. 165. [627] Saxon Leechdoms, vol. 1, pp. xli-xliii. [628] Ibid., p. xliii. [629] Brand, Popular Antiquities, vol. 2, pp. 108,109. [630] Browne, Religio Medici, p. 392. [631] Brand, op. cit., p. 110. [632] Pliny, Nat. Hist., lib. 28, cap. 22. [633] Ibid., lib. 28, cap. 17. [634] Ibid. [635] Grimm, Teutonic Mythology, vol. 3, p. 1169. [636] Burton, Anatomy of Melancholy, London, 1827, vol. 1, p. 91; vol. 2, pp. 288, 290. [637] Burton, Anatomy of Melancholy, London, 1827, vol. 1, p. 91; vol. 2, p. 290. [638] Picart, Cérémonies et Coûtumes, etc., vol. 10, pp. 69-73. [639] Dæmonology, p. 100. [640] Brand, Pop. Ant., vol. 3, p. 299. [641] Ibid., p. 170. [642] Frommann, Tractatus de Fascinatione, Nuremberg, 1675, p. 731. [643] Markham, Bogle's mission to Tibet, London, 1876, p. 85. [644] Brand, Pop. Ant., vol. 2, p. 149. [645] Thomas Wright, Sorcery and Magic, London, 1851, vol. 2, p. 10.
  • 60. [646] Brand, Pop. Ant., vol. 2, p. 143. [647] Pennant, in Pinkerton, Voyages, vol. 3, p. 382. [648] Hoffman, quoting Friend, in Jour. Am. Folk Lore, 1888, p. 134. [649] Brand, Pop. Ant., vol. 2, pp. 127 et seq. [650] Grimm, Teutonic Mythology, vol. 3, p. 1174. He also speaks of the nouer l'aiguillette, ibid., p. 1175. [651] Saxon Leechdoms, vol. 1, p. xliv. [652] Black, Folk-Medicine, London, 1883, pp. 185, 186. [653] Pliny, Nat. Hist., lib. 28, cap. 9. [654] Brand, Pop. Ant., vol. 2, p. 67. [655] Ibid., p. 170. [656] Sextus Placitus, De Medicamentis ex Animalibus, Lyons, 1537, pages not numbered, article de Puello et Puellæ Virgine. [657] Etmüller, Opera Omnia, Lyons, 1690, vol. 2, p. 279, Schroderii Dilucidati Zoologia. [658] Brand, Pop. Ant., vol. 2, p. 68, footnote. [659] Ibid., p. 67. [660] Paracelsus, Chirurgia Minora, in Opera Omnia, Geneva, 1662, vol. 2, p. 70. [661] Ibid., p. 174. [662] Beckherius, Medicus Microcosmus, London, 1660, p. 174. [663] Grimm, Teutonic Mythology, vol. 3, p. 1094, footnote. [664] Ibid., p. 1096. [665] Pliny, Nat. Hist., lib. 30, cap. 12. [666] Etmüller, Opera Omnia, Lyons, 1690, vol. 2, pp. 282, 283, Schroderii Dilucidati Zoologia. [667] Ibid., p. 278a. [668] Black, Folk-Medicine, London, 1883, p. 113.
  • 61. [669] Forlong, Rivers of Life, London, 1883, vol. 2, p. 313. [670] Brand, Pop. Ant., vol. 2, p. 69. [671] Notes and Queries, 1st series, vol. 4, p. 500. [672] See also Black, Folk-Medicine, London, 1883, p. 79. [673] Grimm, Teutonic Mythology, vol. 3, p. 1233. [674] Black, Folk-Medicine, London, 1883, p. 114. [675] Herrera, dec. 6, lib. 8, cap. 1, p. 171. [676] Ibid., dec. 7, lib. 4, cap. 5, p. 70. [677] Smyth, Aborigines of Victoria, vol. 1, p. 351. See also previous references to the use of such cords by the Australians. [678] Pliny, Nat. Hist., lib. 28, cap. 27. [679] Highlands of Æthiopia, vol. 1, p. 247. [680] Through the Dark Continent, vol. 1, p. 398. [681] Pliny, Nat. Hist., lib. 28, cap. 11. [682] Notes and Queries, 4th series, vol. 5, pp. 295, 390. [683] Traité des Superstitions, tome 1, chap. 3, paragraph 8. [684] Pop. Ant., vol. 3, p. 276. [685] Black, Folk-Medicine, p. 109. [686] Anatomy of Melancholy, vol. 2, pp. 288, 290. [687] Brand, Pop. Ant., vol. 2, p. 107. [688] Brand, Pop. Ant., vol. 2, p. 78. [689] Ibid., p. 91. [690] Ibid., p. 93. [691] Picart, Cérémonies et Coûtumes, etc., vol. 1, p. 41. [692] Folk-Medicine, London, 1883, pp. 185, 186. [693] P. 41. [694] Black, Folk-Medicine, p. 186.
  • 62. [695] Ibid., (after Tylor) pp. 176, 177. [696] Ibid., p. 178. [697] Pop. Ant., vol. 3, p. 276. [698] Salverte, Philosophy of Magic, vol. 1, p. 195. [699] Black, Folk-Medicine, London, 1883, p. 197. [700] Pliny, Nat. Hist., lib. 24, cap. 118. [701] Forlong, Rivers of Life, vol. 1, p. 451. [702] Pennant, quoted by Brand, Popular Antiquities, vol. 3, p. 54. [703] Ibid., p. 285. [704] Folk-Medicine, London, 1883, pp. 185, 186. [705] Folk-Medicine, London, 1883, p. 113. [706] Ibid., p. 57. [707] Ephemeridum Physico-medicarum, Leipzig, 1694, vol. 1, p. 49. [708] Black, Folk-Medicine, p. 112. [709] Black, Folk-Medicine, p. 112. [710] Brand, Pop. Ant., vol. 2, p. 86. [711] Black, Folk-Medicine, p. 38. [712] Pliny, Nat. Hist., lib. 30, cap. 38. [713] Ibid. [714] Black, Folk-Medicine, p. 111. [715] Brand, Pop. Ant., vol. 3, pp. 288, 324. [716] This fact is stated by Torquemada, Monarchia Indiana, lib. 10, cap. 33, and by Gomara, Hist. of the Conq. of Mexico, p. 446; see also Diego Duran, lib. 1, cap. 20, p. 226. [717] Herrera, dec. 3, lib. 2, p. 67.
  • 63. [718] John Gilmary Shea, The Catholic Church in Colonial Days, p. 472. [719] Diego Duran, vol. 3, cap. 4, p. 217. [720] Anatomy of Melancholy, London, 1827, vol. 1, p. 337. [721] Picart, Cérémonies et Coûtumes, etc., Amsterdam, 1729, vol. 5, p. 50. [722] Lady of the Lake, canto 3, stanza 4, Sir Rhoderick Dhu, summoning Clan Alpine against the king. [723] Teatro Mexicano, vol. 3, p. 323. [724] Lib. 14, cap. 4, and lib. 16, cap. 16. [725] Lib. 1, cap. 23, pp. 251-252. [726] Ximenez, Hist. Orig. Indios, p. 211. [727] Mendieta, p. 83. [728] Ibid., p. 78. [729] Researches in South America, p. 83. [730] Monarchia Indiana, vol. 2, lib. 13, cap. 45, and elsewhere. [731] Emory, Reconnoissance, p. 88. [732] Gomara, Historia de la Conquista de Méjico, Veytia's edition, p. 299. [733] Ibid., p. 310. [734] Smithsonian Contributions, Ancient monuments of New York, vol. 2. [735] Buckingham Smith, Relacion de la Jornada de Coronado á Cibola, Coleccion de Documentos para la Historia de Florida, London, 1857, vol. 1, p. 148. [736] Ibid., vol. 1, p. 150. [737] Brinton, Myths of the New World, p. 253. [738] London, 1844, vol. 1, pp. 26, 29, 36, 93. [739] Ibid., p. 278.
  • 64. [740] Ibid., vol. 2, p. 389. [741] Monarchia Indiana, lib. 6, cap. 45, p. 80. [742] Ibid., lib. 19, cap. 22, pp. 357-358. [743] Ternaux-Compans, vol. 10, p. 240. [744] London, 1843, p. 248. [745] Pimentel, Lenguas Indígenas de México, vol. 3, pp. 498, 499. [746] Brinton, Myths of the New World, pp. 285, 286. [747] Ibid., p. 264. [748] Kingsborough, vol. 8, sup., p. 249. [749] Parkman, Jesuits, introduction, p. lxxxiv.
  • 65. Transcriber's Notes Obvious typographical errors have been repaired. Non- standard spellings, including those in other languages, were retained as in the original. Hyphenation and accent variants that could not be clearly resolved, were retained. The few cases of ellipses shown as asterisks were also retained. p. 579, paragraph beginning Dr. Joseph Lanzoni: both chermisinum and chermesinum occurred in the original as shown. p. 585, paragraph beginning At intervals: Three several times they is as in the original.
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