In Vitro Transc r ip t ion and Trans la t ion Pro tocols

Methods in Molecular Bio logy John M. Walker, SERIES EDITOR

37. In Vitro Transcription and Translation Protocols, edited by Martin J. Tymms, 1995

36. Peptide Analysis Protocols, edited by Michael W. Pennington and Ben M. Dunn, 1994

35. Peptide Synthesis Protocols, edited by Ben M. Dunn and Michael W. Pennington, 1994

34. Immunocytochemical Methods and Protocols, edited by Lorette C. Javois, 1994

33. In Si tu Hybridization Protocols, edited by K. H. Andy Choo, 1994 32. Basic Protein and Peptide Protocols, edited by John M. Walker, 1994 31. Protocols for Gene Analysis, edited byAdr/an J. Harwood, 1994 30. DNA-Protein Interactions, edited by G. GeoffKneale, 1994 29. Chromosome Analysis Protocols, edited by John R. Gosden, 1994 28. Protocols for Nucleic Acid Analysis by Nonradioactive Probes, edited by

Peter G. Isaac, 1994 27. Biomembrane Protocols: I1. Architecture and Function, edited by

John M. Graham and Joan A. Higgins, 1994 26. Protocols for OHgonucleotide Conjugates, edited by Sudhir Agrawal, 1994 25. Computer Analysis of Sequence Data: Part II, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 24. Computer Analysis of Sequence Data: Part I, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 23. DNA Sequencing Protocols, edited by Hugh G. Griffin

and Annette M. Griffin, 1993 22. Optical Spectroscopy, Microscopy, and Macroscopic Techniques,

edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas, 1994

21. Protocols in Molecular Parasitology, edited by John E. Hyde, 1993 20. Protocols for Oligonucleotides and Analogs, edited by

Sudhir Agrawal, 1993 19. Biomembrane Protocols: I. Isolation and Analysis, ec~ted by

John M. Graham and Joan A. Higgins, 1993 18. Transgenesis Techniques, edited by David Murphy

and David A. Carter, 1993 17. Spectroscopic Methods and Analyses, edited by Christopher Jones,

Barbara Mulloy, and Adrian H. Thomas, 1993 16. Enzymes of Molecular Biology, edited by Michael M. Burrell, 1993 15. PCR Protocols, edited by Bruce A. White, 1993 14. Glycoprotein Analysis in Biomedicine, edited by Elizabeth F. Hounseli,

1993 13. Protocols in Molecular Neurobiology, edited by Alan Longstaf f

and Patricia Revest, 1992 12. Pulsed-Field Gel Electrophoresis, edited by Margit Burmeister

and Levy Ulanovsky, 1992 11. Practical Protein Chromatography, edited by Andrew Kenney

and Susan Fowell, 1992 10. Immunochemical Protocols, edited by Margaret M. Manson, 1992

Earl ier volumes are stil l available. Contact Humana for details.

r •

Methods in Molecu la r Biology * 37

In Vi t ro T r a n s c r i p t i o n

a n d T r a n s l a t i o n P r o t o c o l s

Edited by

Mart in J. Tymms Monash University

Clayton, Victoria, Australia

Humana Press ~ Totowa, New Jersey

© 1995 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

All rights reserved.

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Library of Congress Cataloging in Publication Data

Main entry under title:

Methods in molecular biology.

In vitro transcription and translation protocols/edited by Martin J. Tymms p. cm.--(Methods in molecular biology; 37)

Includes index. ISBN 0-89603-288-4 1. Molecular genetics--Laboratory manuals.

3. Genetic translation--Laboratory manuals. molecular biology (Totowa, NJ); 37 QH442.I557 1995 574.87'3223---dc20

2. Genetic transcription--Laboratory manuals. I. Tymms, Martin J. I. Series: Methods in

94-34486 CIP

P r e f a c e

Most laboratories conducting studies that use molecular biology techniques employ in vitro transcription and translation systems as a routine part of their day-to-day research. The commercial availability of purified bacterial RNA polymerase and the availability of robust trans- lation systems has made in vitro systems attractive not only as an alter- native to the in vivo expression of genes, but also as good model systems for studying specific aspects of transcription and translation. Although fairly efficient eukaryotic translation systems have been established for a number of years, reconstitution of transcription in vitro has proved to be more difficult. Recent improvements in fractionation techniques and the cloning of proteins involved in transcription have made this a fast moving area of research. Considerable progress has also been made in recent years in developing in vitro systems to study transcription and translation in chloroplasts and mitochondria, together with systems for the study of protein import.

In Vitro Transcription and Translation Protocols provides many detailed experimental procedures for prokaryotic transcription and translation systems, together with protocols for many key techniques used in the analysis of eukaryotic transcription. In keeping with the successful format of preceding volumes of the Methods in Molecular Biology series, step-by-step instructions are provided, together with extensive notes that cover troubleshooting and special tips considered important. I hope that the range of techniques, together with the detailed information provided, will encourage both the new worker and more experienced practitioner to use some of the techniques outside their areas of current expertise.

I would like to thank my colleagues for their tolerance during this project and to acknowledge the support of The Victorian Health Promotion Foundation and the National Health and Medical Research Council during the preparation of this volume.

Martin J. Tymms Z)

Content s

Preface ............................................................................................................................ v Contributors ................................................................................................................. /x CH. 1. Transcription In Vitro Using Bacteriophage RNA Polymerases,

Elaine 1". Schenborn ............................................................................. 1 CH. 2. Subtraction Hybridization eDNA Libraries,

Clifford W. Schweinfest, Peter S. Nelson, Michael W. Graber, Rita I. Demopoulos, and Takis S. Papas ................................. 13

CH. 3. Quantitative Measurement of mRNA Using the RNase Protection Assay,

Martin J. Tymms ................................................................................. 31 CH. 4. Assembly and Transcription of Chromatin Templates Using RNA

Polymerase III, Sara J. Felts ......................................................................................... 47

CH. 5. Antisense Affinity Depletion of RNP Particles: Application to Spliceosomal snRNPs,

Benjamin J. Blencowe and SUvia M. L. Barabino ........................... 67 CH. 6. In Vitro mRNA Editing Using S100 Extracts,

Jobst Greeve and James Scott ............................................................ 77 CH. 7. An In Vitro Transcription Assay for Probing Drug-DNA Interactions

During Active Transcription of DNA, Don R. Phillips and Donald M. Crothers .......................................... 89

CH. 8. In Vitro Reconstitution of Progesterone-Dependent RNA Transcription in Nuclear Extracts of Human Breast Carcinoma Cells,

Milan K. Bagchi, Sophia Y. Tsai, and Ming-Jer Tsai ................... 107 CH. 9. Plastid In Vitro Transcription,

Kai Tiller and Gerhard Link ............................................................ 121 CH. 10. Microinjection of In Vitro Transcribed RNA and Antisense

Oligonucleotides in Mouse Oocytes and Early Embryos to Study the Gain- and Loss-of-Function of Genes,

IsmaU Kola and Sony Hera Samarsono ......................................... 135 CH. 11. mRNA Translation in Xenopus Oocytes,

Aldo Ceriotti and Alan Colman ....................................................... 151

vii

viii Contents

CH. 13. The Xenopus Egg Extract Translation System, Glenn M. Matthews and Alan Colman ............................................ 199

CH. 14. In Vitro Translation Using Rabbit Reticulocyte Lysate, Gregory S. Beckler, David Thompson, and Tom Van Oosbree ..... 215

Ca. 15. An In Vitro Transcription System from BraN Cells of the Silkworm, Bombyx mori,

Hiroshi Sakurai, Susumu Izumi, and Shiro Tomino ..................... 233 Ca. 16. Translation Using a Wheat-Germ Extract,

John F. Van Herwynen and Gregory S. Beckler ........................... 245 Ca. 17. In Vitro Transcription and Translation in a Cell-Free System

from Clostridium tetani, Bettina Andersen-Beckh and Heiner Niemann .............................. 253

Ca. 18. Preparation and Use of E. coli S-30 Extracts, $cottA. Lesley ................................................................................... 265

CH. 19. Posttranslational Transport and In Thylakoido Integration of Plastid Protein,

Elisabeth Kruse and Klaus Kloppstech ........................................... 279 CH. 20. Import into Isolated Yeast Mitochondria of Radiolabeled Proteins

Synthesized In Vitro, Ruby H. P. Law and Phillip Nagley ................................................ 293

CH. 21. Structure-Function Studies Based on In Vitro Expression, Martin J. Tymms and Paul J. Hertzog ............................................ 317

Ca. 22. o-Like Plastid Transcription Factors, Kai Tiller and Gerhard Link ............................................................ 337

Ca. 23. Preparation of HeLa Nuclear Extracts, B. FranMin Pugh .............................................................................. 349

Ca. 24. Purification of the Human TATA-Binding Protein, TBP, B. Franklin Pugh .............................................................................. 359

Ca. 25. DNA-Binding Studies Using In Vitro Synthesized Myb Proteins, Robert George Ramsay ..................................................................... 369

C8.26. DNase I Footprinting Using PCR-Generated End-Labeled DNA Probes,

Rocco C. lanneUo .............................................................................. 379 Ca. 27. Gene Isolation by Screening ~,gtl 1 Expression Libraries

with DNA Binding-Site Probes, Bill Kalionis ....................................................................................... 393

Ca. 28. Transcriptional Activation Analysis by the Chloramphenicol Acetyl Transferase (CAT) Enzyme Assay,

David R. Hodge, Delores M. Thompson, Alexandria Panayiotakis, and Arun Seth .............................. 409

Index .......................................................................................................................... 423

x Contributors

R o c c o C. IANNELLO • Molecular Embryology and Birth Defects Laboratory, Institute of Reproduction and Development, Monash University, Clayton, Victoria, Australia

SUSUMU IZUMI • Department of Biology, Tokyo Metropolitan University, Tokyo, Japan

BILL KALIONIS • Department of Obstetrics and Gynaecology, School of Medicine, The Flinders University of South Australia, Adelaide, Australia

KLAUS KLOPPSSTECH • Institiit fiir Botanik, Universitiit Hannover, Germany

ISMAIL KOLA • Molecular Embryology and Birth Defects Laboratory, Institute of Reproduction and Development, Monash University, Clayton, Victoria, Australia

BRIGtTTE KRUSE • Department of Physiology, Phillips- Universitiit Marburg, Germany

ELISABETH KRUSE • Institiit fiir Botanik, Universitiit Hannover, Germany

RUBY H. P. LAW • Centre for Molecular Biology and Medicine and Department of Biochemistry, Monash University, Clayton, Victoria, Australia

SCOTT A. LESLEY • Promega Corporation, Madison, WI GERHARD LINK ) Department of Plant Cell Physiology

and Molecular Biology, University of Bochum, Germany GLEN M. MATTHEWS • School of Biochemistry, University

of Birmingham, UK NALINI N . MURDTER ) Department of Physiology, Phillips-Universitiit

Marburg, Germany PHILLIP NAGLEY • Centre for Molecular Biology and Medicine

and Department of Biochemistry, Monash University, Clayton, Victoria, Australia

PETER S. NELSON • Fred Hutchinson Cancer Research Center, Seattle, WA HEINER NIEMANN • Bundesforschungsanstaltfiir Viruskrankenheiten

der Tiere, Tiibingen, Gernmany ALEXANDRA PANAYIOTAKIS • Laboratory of Molecular Oncology,

National Cancer Institute, Frederick, MD

Contributors xi

TArdS S. PAPAS • Hollings Cancer Center, Medical University of South Carolina, Charleston, SC

DON R. PHILLIPS • Department of Biochemistry, La Trobe University, Bundoora, Victoria, Australia

B. FRANKLIN PUGH • Center for Gene Regulation, Department of Molecular and Cell Biology, The Pennsylvania State University, University Park, PA

R O B E R T GEORGE R A M S A Y • Melbourne Tumour Biology Branch, Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Parkville, Australia

HIROSHI SAKURAI • Department of Biology, Tokyo Metropolitan University, Tokyo, Japan

ELAINE T. SCHENBORN • Promega Corporation, Madison, WI CLIFFORD W . SCHWEINFEST • Hollings Cancer Center,

Medical University of South Carolina, Charleston, SC JAMES SCOTT • Department of Medicine, Royal Postgraduate Medical

School, Hammersmith Hospital, London, UK ARUN SETH • Laboratory of Molecular Oncology, National Cancer

Institute, Frederick, MD SONY HERU SUMARSONO • Molecular Embryology

and Birth Defects Laboratory, Institute of Reproduction and Development, Monash University, Clayton, Victoria, Australia

KAI TILLER • Department of Plant Cell Physiology and Molecular Biology, University of Bochum, Germany

DAVID THOMPSON • Promega Corporation, Madison, WI DELORES THOMPSON • Laboratory of Molecular Oncology,

National Cancer Institute, Frederick, MD SHIRO TOMINO • Department of Biology, Tokyo Metropolitan

University, Tokyo, Japan MING-JER TSAI • Department of Cell Biology, Baylor College

of Medicine, Houston, TX SOPHIA Y. TSAI • Department of Cell Biology, Baylor College

of Medicine, Houston, TX

Contributors xii

MARTIN J. TYMMS • Molecular Embryology and Birth Defects Unit, Institute of Reproduction and Development, Monash University, Clayton, Victoria, Australia

JOHN F. VAN HERWYNEN • Promega Corporation, Madison, WI TOM VAN OOSBREE ° Promega Corporation, Madison, WI