Yeast Cells

METHODS IN CELL BIOLOGY VOLUME XI1 Yeast Cells Contributors to This Volume W. BANDLOW J. MARMUR P. BAUER JOAN H. CAULTON MARJORIECRANDALL D. R. CRYER JULIANDAVIES JOHN H. DUFFUS R. ECCLESHALL D. J. FENNELL B. J. KILBEY BIRTEKRAMHBFT ANTHONY W. LINNANE H. B. LUKINS C. V. LUSENA H. PONTA U. PONTA JOHN R. PRINGLE ANTHONY H . ROSE GERALDM. RUBIN DANIEL SCHINDLER P. R. STEWART ANDRESWIEMKEN D. WILKIE D. H. WILLIAMSON E. WINTERSBERGER L. J. ZEMAN ERIKZEUTHEN Methods in Cell Biology Edited 6y DAVID M. PRESCOTT DEPARTMENT OF MOLECULAR, CELLULAR AND DEVELOPMENTAL BIOLOGY UNIVERSITY OF COLORADO BOULDER, COLORADO VOLUME XI1 Yeast Cells 1975 ACADEMIC PRESS New York San Francisco London A Subsidiary of Harcourt Brace Jovanovich, Publishers COPYRIGHT 0 1975, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY A N Y MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER. ACADEMIC PRESS, INC. 111 Fifth Avenue, New York. New York 10003 United Kingdom Edition published by ACADEMIC PRESS. INC. (LONDON) LTD. . 24/28 Oval Road, London NWI LIBRARY OF CONGRESS CATALOG CARD NUMBER:64-14220 ISBN 0- 12-5641 12-5 PRINTED IN THE UNITED STATES OF AMERICA CONTENTS xi LIST OF CONTRIBUTORS xiii PREFACE 1. Growth and Handling of Yeasts Anthony H. Rose I. 11. 111. IV. 2. Introduction Isolation and Maintenance of Yeasts Growth of Yeasts Procedures Used in Handling Yeast Populations References 1 3 10 14 15 Inhibitors of Macromolecular Synthesis in Yeast Daniel Schindler and Julian Davies I. Introduction 11. Inhibitors 111. Effects of Inhibitors on Yeast Metabolism N. Conclusion References 17 18 27 33 33 3. Isolation of Yeast DNA D. R. Cryer, R. Eccleshall, and J. Marmur 39 I. Introduction 11. Isolation Procedure References 4. 40 44 Preparation of RNA and Ribosomes from Yeast Gerald M. Rubin 45 I. Introduction 11. Cell Growth and Disruption 111. Radioactive Labeling IV. Preparation of Ribosomes V. Preparation of Ribosomal Subunits Y 46 46 47 49 vi CONTENTS VI. Preparation of Polyribosomes VII. Preparation of High-Molecular-Weight rRNAs VIII. Preparation of Low-Molecular-Weight RNAs IX. Polyacrylamide Gel Electrophoresis of Low-Molecular-Weight RNAs X. High-Molecular Weight rRNA Precursors XI. mRNA References 50 51 51 54 61 61 63 5 . DNA-Dependent RNA Polymerases from Yeasts H . Ponta, U.Ponta. and E. Wintersberger I. Introduction 11. Purification Procedures 111. Characterization of the RNA Polymerases of Yeasts 65 66 72 IV. Preparation of Yeast DNA Active as Template for Yeast RNA Polymerase V. Discussion References 73 74 75 6 . The Isolation of Yeast Nuclei and Methodr to Study Their Properties John H. Duffus I. Introduction 11. Isolation of Nuclei from Yeast Protoplasts 111. Isolation of Nuclei from Normal Yeast Cells N. Selection of a Method for the Isolation of Yeast Nuclei V. Preparation of a Dense Crescent (Nucleolar) Fraction from Yeast Nuclei VI. Electron Microscopy of Isolated Yeast Nuclei VII. Isolation and Characterization of Chromatin from Yeast Nuclei VIII. Isolation and Characterization of DNA from Yeast Nuclei Ix. Isolation and Characterization of RNA from Yeast Nuclei X. Isolation and Characterization of Nuclear Proteins XI. Yeast Nuclear Enzymes XII. Concluding Remarks References 71 78