Preface Since the mid-1980s, there has been an explosion in our understanding of the mechanisms underlying cell cycle transitions. At the core of this progress is the realization that a family of cyclin-dependent kinases (Cdks) catalyzes the events of the cell cycle by phosphorylating numerous target proteins, thereby triggering the replication and segregation of the chromo- somes. Despite this key insight, much remains to be learned about the regulation and action of the Cdks. The elucidation of the regulatory net- works impinging on the Cdks is an ongoing challenge. Moreover, the cata- loging of the substrates of Cdks and an understanding of how phosphoryla- tion of these targets results in the execution of cell cycle events remain unfulfilled goals in most experimental systems. Apart from the unraveling of the mechanisms of cell cycle transitions, the description of the many biological contexts in which cell cycle control is a key determinant is an everblossoming area of research. It is well established (or highly likely) that cell cycle regulators play critical roles in the commitment to terminal differentiation, progression to malignant transformation, triggering of apoptosis, and attainment of senescence. The study of cell cycle molecules in these contexts will be of great intellectual and practical importance. A critical feature of cell cycle research is that complementary studies of diverse organisms and experimental systems have resulted in a synergistic rate of progress in our understanding of key regulatory paradigms. The conserved nature of cell cycle control mechanisms in vertebrates, marine invertebrates, and yeast has facilitated a rapid acquisition of universally applicable principles. In recognition of this process of discovery, this volume is largely subdivided according to the major experimental systems that are widely used by current cell researchers. Specifically, sections of the volume are devoted to mammalian cells, various yeasts, and several "embryonic" systems. In addition, there is one section on techniques that generally can be applied regardless of the experimental organism. We thank the various authors who took the time to describe their experimental methods in a clear and accessible manner. It is hoped that these articles will serve as a resource for future progress in this rapidly burgeoning area of science. WILLIAM G. DUNPHY XV Contributors to Volume 283 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. PETER D. ADAMS (5), Dana-Farber Cancer Institute, Boston, Massachusetts 02115 NATALIE G. AnN (3), Howard Hughes Medi- cal Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309 SOREN S. L. ANDERSEN (2), EMBL, Cell Biology Programme, D 69117 Heidelberg, Germany CHANG BAI (11), Verna and Marrs McLean Department of Biochemistry, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030 MOHAN K. BALASUBRAMANIAN (37), Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 UDO BARON (]2), Zentrum fiir Molekulare Biologie, 69120 Heidelberg, Germany DOUGLAS E. BASSETr, JR. (10), Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, and The National Center for Biotechnology Infor- mation, National Library of Medicine, National Institutes of Health, Bethesda, Maryland 20894 J. JULIAN BLOW (41), Imperial Cancer Re- search Fund, Clare Hall Laboratories, South Mimms, Hefts. EN6 3LD, England MARK BOGUSKI (10), The National Center for Biotechnology Information, National Li- brary of Medicine, National Institutes of Health, Bethesda, Maryland 20894 ANGELIKA L. BON1N (12), BASF Bioresearch Corporation, Worcester, Massachusetts 01605-4314 LINDA L. BREEDEN (25), Fred Hutchinson Cancer Research Center, Division of Basic Sciences, Seattle, Washington 98109 HERMANN BUJARD (12), Zentrum far Molek- ulare Biologie, 69120 Heidelberg, Germany JUDITH L. CAMPBELL (30), Braun Labora- tories, California Institute of Technology, Pasadena, California 91125 ANTONY M. CARR (36), MRC Cell Mutation Unit, Sussex University, Brighton BN1 9RR, United Kingdom REY-HuEI CHEN (43), Department of Physiol- ogy, University of California, San Fran- cisco, San Francisco, California 94143-0444 YONG CH1 (28), California Institute of Tech- nology, Pasadena, California 91125 JAMES P. J. CHONG (41), Imperial Cancer Re- search Fund, Clare Hall Laboratories, South Mimms, Hefts. EN6 3LD, England DAWN COVERLEY (40), Wellcome/CRC Institute, Cambridge CB2 1QR, United Kingdom VINCENT L. CRYNS (7), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 JAMES DEGREGORI (15), Department of Ge- netics, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710 RAYMOND J. DESHAIES (28), California Insti- tute of Technology, Pasadena, California 91125 JOHN F. X. DIFFLEY (29), Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hefts. EN6 3LD, United Kingdom WENDY J. DIXON (30), Braun Laboratories, California Institute of Technology, Pasa- dena, California 91125 WILLIAM G. DUNPHY (42), Division of Biol- ogy, Howard Hughes Medical Institute, Cal- ifornia Institute of Technology, Pasadena, California 91125 ix X CONTRIBUTORS TO VOLUME 283 B. D. DYNLACHT (17), Department of Molecu- lar and Cellular Biology, Harvard Medical School, Cambridge, Massachusetts 02138 RHIAN J. EDWARDS (36), MRC Cell Mutation Unit, Sussex University, Brighton BN1 9RR, United Kingdom S. J. ELLEDGE (11, 17, 31), Verna and Marts McLean Department of Biochemistry, De- partment of Molecular and Human Genet- ics, Baylor College of Medicine, Howard Hughes Medical Institute, Houston, Texas 77030 PETER A. FANTES (34), Institute of Cell and Molecular Biology, University of Edin- burgh, Edinburgh EH9 3JT, United Kingdom ANNE FERNANDEZ (6), Cell Biology Unit, Centre de Recherche de Biochimie Macro° moleculaire, Centre National de la Recher- che Scientifique, 34033 Montpellier Cedex, France RUSSELL R. PINCHER (39), The Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822 ROBERT P. FISHER (19), Program in Cell Biol- ogy and Genetics, Memorial Sloan-Ketter- ing Cancer Center, New York, New York 10021 ELLA FREULICH (18), Department of Biologi- cal Sciences, Columbia University, New York, New York 10027 SABINE FREUNDLIEB (12), Zentrum far Mo- lekulare Biologie, 69120 Heidelberg, Germany ANDREW M. FRY (20), Department of Molecu- lar Biology, University of Geneva, 1211 Ge- neva 4, Switzerland MARTIN FUNK (23), MediGene AG, D-82152 Martinsried/Munich, Germany DAVID M. GLOVER (47), Cancer Research Campaign, Cell Cycle Genetics Group, De- partment of Anatomy and Physiology, Med- icaI Sciences Institute, University of Dundee, Dundee DD1 4HN, Scotland MANFRED GOSSEN (12), Division of Biochem- istry and Molecular Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720 KATHLEEN L. GOULD (37), Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 WOLFF GRAULICH (23), Institut far Moleku- larbiologie und Tumorforschung (IMT), Philipps- Universititt Marburg, D-35033 Marburg, Germany STEVEN I. HAASE (24), Department of Molec- ular Biology, The Scripps Research Insti- tute, La Jolla, California 92037 E. HARLOW (17), Massachusetts GeneralHos- pital Cancer Center, Charlestown, Massa- chusetts 021292 J. W. HARPER (16, 17), Department of Bio- chemistry, Baylor College of Medicine, Houston, Texas 77030 AVRAM HERSHKO (46), Unit of Biochemistry, Rappaport Faculty of Medicine and Rappa- port Institute for Research in Medical Sci- ences, Technion-Israel Institute of Technol- ogy, Haifa 31096, Israel PHILIP HIETER (10), Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185 M1NGXIA HUANG (31), Verna and Marts McLean Department of Biochemistry, De- partment of Molecular and Human Genet- ics, Baylor College of Medicine, Howard Hughes Medical Institute, Houston, Texas 77030 LASZLO JAKOI (15), Howard Hughes Medical Institute, Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710 LATA JAYARAMAN (18), Department of Bio- logical Sciences, Columbia University, New York, New York 10027 ANTHONY L. JOHNSON (26), Division of Yeast Genetics, National Institute for Medical Research, London NW7 1AA, United Kingdom LELAND H. JOHNSTON (26), Division of Yeast Genetics, National Institute for Medical Research, London NW7 1AA, United Kingdom CONTRIBUTORS TO VOLUME 283 xi WILLIAM G. KAELIN, JR. (5), Dana-Farber Cancer Institute, Boston, Massachusetts 02115 ERIC KARSENTI (2), EMB L, Cell Biology Pro- gramme, D 69117 Heidelberg, Germany BRUCE E. KEMP (31), St. Vincent' s Institute of Medical Research, Victoria 3065, Australia SUNG-Hou KIM (9), Melvin Calvin Labora- tory, University of California, Berkeley, Berkeley, California 94720 RANDALL W. KING (7), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 MARC W. KIRSCHNER (7), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 SALLY KORNBLUTH (45), Department of Mo- lecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 AKIKO KUMAGAI (42), Division of Biology, California Institute of Technology, Pasa- dena, California 91125 NED J. C. LAMB (6), Cell Biology Unit, Centre de Recherche de Biochimie Macromolecu- laire, Centre National de la Recherche Sci- entifique, 34033 Montpellier Cedex, France XIAOHONG LENG (10, Department of Bio- chemistry, Baylor College of Medicine, Houston, Texas 77030 GUSTAVO LEONE (15), Howard Hughes Medi- cal Institute, Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710 DANIEL J. LEW (24), Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710 PETER LOPEZ (5), Cytomation, Inc., Fort Col- lins, Colorado 80525 KEVIN D. LUSTIG (7), Department of Cell Bi- ology, Harvard Medical School, Boston, Massachusetts 02115 DAVID LYDALL (32), Department of Molecu- lar and Cellular Biology, University of Ari- zona, Tucson, Arizona 85721 STUART A. MACNEILL (34), Institute of Cell and Molecular Biology, University of Edin- burgh, Edinburgh EH9 3JR, United Kingdom MARK A. MADINE (40), Wellcome/CRC Institute, Cambridge CB2 1QR, United Kingdom HIRO M. H. MAHBUBANI (41), Imperial Can- cer Research Fund, Clare Hall Labora- tories, South Mimms, Herts. EN6 3LD, England DANNELL McCOLLUM (37), Howard Hughes Medical Institute and Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 THOMAS J. MCGARRY (7), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 PAUL E. MEAD (7), Howard Hughes Medical Institute and Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115 LAURENT MEIJER (9), Centre National de la Recherche Scientifique, Station Biologique de Roscoff, 29682 Roscoff Cedex, France SEROIO MORENO (4), Instituto de Microbi- ologia Bioquimica, CSIC/Universidad de Salamanca, Edificio Departmental, Campus Miguel de Unamuno, 37007 Salamanca, Spain ROLE MOLLER (23), Institut far Molekular- biologie und Tumorforschung (1MT), Philipps- Universiti~t Marburg, D-35033 Marburg, Germany DOMINIK MUMBERG (23), Department of Pathology, The University of Chicago, Chicago, Illinois 60637 MONICA S. MURAKAMI (44), ABL-Basic Re- search Program, NCI-Frederick Cancer Re- search and Development Center, Frederick, Maryland 21702 ANDREW W. MURRAY (33, 42), Department of Physiology, University of California at San Francisco, San Francisco, California 94143-0444 KENTARO NABESHIMA (35), Department of Biophysics, Faculty of Science, Kyoto Uni- versity, Kyoto 606-01, Japan xii CONTRIBUTORS TO VOLUME 283 JOSEPH R. NEVINS (15), Howard Hughes Med- ical Institute, Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710 C. NGWU (17), Massachusetts General Hospi- tal Cancer Center, Charlestown, Massachu- setts 021292 ERICH A. NIGG (20), Department of Molecu- lar Biology, University of Geneva, 1211 Ge- neva 4, Switzerland TAKEHARU NISHIMOTO (22), Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fuku- oka 812-82, Japan CHRIS NORBURY (4), Molecular Oncology Laboratory, Institute of Molecular Medi- cine, John Radcliffe Hospital, Oxford OX3 9DU, United Kingdom STEPHEN A. OSMANI (39), The Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822 MATH-IIAS PETER (27), Swiss Institute for Ex- perimental Cancer Research, (ISREC), 1066 Epalinges, Switzerland DAWN E. PHELPS (14), Lineberger Compre- hensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280 JONATHON PINES (8), WeUcome/CRC Institute and Department of Zoology, Cambridge CB2 1QR, United Kingdom RANDY Y. C. POON (21), Department of BiD- chemistry, Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong CAROL PRIVES (18), Department of Biological Sciences, Columbia University, New York, New York 10027 KATHERYN A. RESING (3), Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309 VOLKER R(bNICKE (23), Max-Planck-lnstitut fiir klinische und physiologische Forschung, Kerckhoff-Institut, D-61231 Bad Nau- heim, Germany ALISON ROWELS (41), Imperial Cancer Re- search Fund, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, England JOAN V. RUDERMAN (46), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 PAUL RUSSELL (38), Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037 ALICIA A. RUSSO (1), Memorial Sloan-Ketter- ing Cancer Center, Cellular Biochemistry and Biophysics Program, New York, New York 10021 SHIGEAKI SAITOH (35), Department of Bio- physics, Faculty of Science, Kyoto Univer- sity, Kyoto 606-01, Japan YOLANDA SANCHEZ (31), Verna and Marts McLean Department of Biochemistry, Bay- lot College of Medicine, Houston, Texas 77030 CORRADO SANTOCANALE (29), Imperial Can- cer Research Fund, Clare Hall Labora- tories, South Mimms, Hens. EN6 3LD, United Kingdom WILLIAM R. SELLERS (5), Dana-Farber Can- cer Institute, Boston, Massachusetts 02115 ROBERT J. SHEAFF (13), Division of Basic Sci- ences, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104 KAZUHIRO SHIOZAKI (38), Department of Molecular Biology, The Scripps Research Institute, La JoUa, California 92037 LILIA STEPANOVA (16), Department of Bio- chemistry, Baylor College of Medicine, Houston, Texas 77030 AARON F. STRAIGHT (33), Department of Physiology, University of California at San Francisco, San Francisco, California 94143-0444 P. TODD STUKENBERG (7), Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 VALERY SUDAKIN (46), Unit of Biochemistry, Rappaport Faculty of Medicine and Rappa- port Institute for Research in Medical Sci- ences, Technion-Israel Institute of Technol- ogy, Haifa 31096, Israel E. C. SWINDELL (17), Verna and Marts McLean Department of Biochemistry, Bay- lot College of Medicine, Houston, Texas 77030 CONTRIBUTORS TO VOLUME 283 xiii /~kLVARO TAVARES (47), Cancer Research Campaign, Cell Cycle Genetics Group, De- partment of Anatomy and Physiology, Med- ical Sciences Institute, University of Dundee, Dundee DD1 4HN, Scotland PIA THt3MMES (41), Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hefts. EN6 3LD, England NICOLE VALTZ (27), Programs in Genetics and Cell Biology, Department of Biochem- istry and Biophysics, University of Califor- nia, San Francisco, San Francisco, Califor- nia 94143-0448 GEORGE F. VANDE WOUDE (44), ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Freder- ick, Maryland 21702 RATI VERMA (28), California Institute of Technology, Pasadena, California 91125 TED WEINERT (33), Department of Molecular and Cellular Biology, University of Ari- zona, Tucson, Arizona 85721 HEIKE WILHELM (2), EMBL, Cell Biology Programme, D 69117Heidelberg, Germany J. WINSTON (17), Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030 YUE XIONG (14), Department of Biochemistry and Biophysics, Program in Molecular Bi- ology and Biotechnology, Lineberger Com- prehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280 GANG XU (39), Department of Pathology and Laboratory Medicine, University of Penn- sylvania School of Medicine, Philadelphia, Pennsylvania 19104 MITSUH1RO YANAGIDA (35), Department of Biophysics, Faculty of Science, Kyoto Uni- versity, Kyoto 606-01, Japan XIAN~ S. YE (39), The Weis Center for Re- search, Geisinger Clinic, Danville, Pennsyl- vania 17822 JUNYING YUAN (7), Department of Cell Biol- ogy, Harvard Medical School, Boston, Mas- sachusetts 02115 ZHENG ZHOC (31), Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 LEONARD I. ZON (7), Howard Hughes Medi- cal Institute and Children's Hospital, Har- vard Medical School, Boston, Massachu- setts 02115 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 0. 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. NEUEELD 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 VOLUME XVI. Fast Reactions Edited by KENNETH KUSTIN xvii xviii METHODS IN ENZYMOLOGY 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 MOLDAVZ 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 MOLDAVZ VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F) Edited by Klvm MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXXI. Biomembranes (Part A) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXII. Biomembranes (Part B) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXIII. Cumulative Subject Index Volumes I-XXX Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B) Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XXXW. Lipids (Part B) Edited by JOHN M. LOWENSTEIN METHODS IN ENZYMOLOGY xix 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 n. 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 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 XX METHODSIN ENZYMOLOGY 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 KIVlE 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 Com- plex 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 VOLUME 71. Lipids (Part C) Edited by JOHN M. LOWENSTEIN VOLUME 72. Lipids (Part D) Edited by JOHN M. LOWENSTEIN [...]... Aspects) Edited by LESTERPACKER VOLUME190 Retinoids (Part B: Cell Differentiation and Clinical Applications) Edited by LUSTERPACKER VOLUME191 Biomembranes (Part V: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEYFLEISCHERAND BECCA FLE1SCHER VOLUME 192 Biomembranes (Part W: Cellular and Subcellular Transport: Epithelial Cells) Edited by SIDNEYFLEISCHERAND BECCA FLEISCHER VOLUME 193... Theory: Cells and Model Membranes) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME172 Biomembranes (Part S: Transport: Membrane Isolation and Characterization) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME173 Biomembranes [Part T: Cellular and Subcellular Transport: Eukaryotic (Nonepithelial) Cells] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 174 Biomembranes [Part U: Cellular... 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 G1NSBURG VOLUME 139 Cellular Regulators (Part A: Calcium- and Calmodulin-Binding Proteins)... min to swell them The swollen cells are poured into a 40-ml tissue grinder (Dounce; Wheaton, Millville, N J), ground 10 times with the "tight"fitting pestle, incubated on ice for 1 hr to allow for the release of the cell contents from the perforated cells into the buffer, and centrifuged at 30,000 g at 4 ° for 30 min in an SS34 (Sorvall) rotor to remove the nuclei and cell debris The supernatant is... induced for 18 hr at 20° The cells are harvested by centrifugation in a GS3 rotor (Sorvall) at 5000 g at 4 ° for 10 min, resuspended in 25.0 mM HEPES, 300.0 mM NaC1, 0.1 mM PMSF, 5.0 mM DTT (pH 7.5), and lysed in a cell disrupter (EmulsiFlex-C5; Avisten, Ottawa, Ontario, Canada) under a pressure of 10,000 psi The lysed cells are centrifuged at 30,000 g in an SS34 rotor to remove cell debris and insoluble... requires the binding of its cyclin subunit for partial activation 6 This is a key step responsible for much of the temporal regulation of C D K activity, as cyclin protein levels are tightly controlled in a cell cycle- dependent manner by ubiquitin-dependent degradation and by transcription 2 Cyclin binding also appears to facilitate the phosphorylation of the C D K subunit by C A K , which is involved... 2) of High 5 insect cells (Invitrogen, San Diego, CA) at 80% confluency (approximately 5 x 107 cells/plate), grown in Grace's medium (20 ml/plate; GIBCO, Grand Island, NY) with 10% (v/v) fetal bovine serum (FBS; HyClone, Logan, UT), are infected with 0.5 ml of viral supernatant [1 × 108 plaque-forming units (pfu)/ml] The infection is carried out by removing the medium from the cells and gently adding... supernatant to the plate, being careful not to disturb the cells Over the period of 1 hr, the plates are gently rocked every 15 min to ensure complete coverage of the cells with the viral supernatant After incubation with the viral supernatant, 15 ml of the Grace's medium containing 10% (v/v) FBS is added back to the plates After a 48-hr incubation at 27° the cells are harvested, using sterile technique, by... supernatant from the cell pellet, the cells are resuspended in 200 ml of hypotonic buffer containing 10.0 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris), 25.0 mM NaC1, 1.0 mM ethylenediaminetetraacetic acid (EDTA), 1.0 mM phenylmethylsulfonyl fluoride (PMSF), leupeptin (1.0/xg/ml), aprotinin (2.0/zg/ L _ Polyak, M Lee, H Erdjument-Bromage, A Koff, J M Roberts, P Tempst, and J ~K Massagu6, Cell 78, 59... 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 GOTrESMAN VOLUME 152 Guide to Molecular Cloning Techniques Edited by SHELBY L BERGER AND ALAN R KIMMEL VOLUME 153 Recombinant . biological contexts in which cell cycle control is a key determinant is an everblossoming area of research. It is well established (or highly likely) that cell cycle regulators play critical. [Part T: Cellular and Subcellular Transport: Eukary- otic (Nonepithelial) Cells] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 174. Biomembranes [Part U: Cellular and Subcellular. mechanisms underlying cell cycle transitions. At the core of this progress is the realization that a family of cyclin-dependent kinases (Cdks) catalyzes the events of the cell cycle by phosphorylating