Genetic Engineering: Principles and Methods PDF
Preview Genetic Engineering: Principles and Methods
Genetic Engineering Principles and Methods Volume 11 GENETIC ENGINEERING Principles and Methods Advisory Board Carl W. Anderson Donald D. Brown Peter Day Donald R. Helinski Tom Maniatis Michael Smith A Continuation Order Plan is available for this series. A continuation order will bring delivery of each new volume immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact the publisher. Genetic Engineering Principles and Methods Volume 11 Edited by Jane K. Setlow Brookhaven" National Laboratory Upton, New York Plenum Press· New York and London The Library of Congress cataloged the first volume of this title as follows: Genetic engineering: principles and methods, v. 1- New York, Plenum Press [1979- v. ill. 26 cm. Editors: 1979- J. K. Setlow and A. Hollaender. Key title: Genetic engineering, ISSN 0196-3716. 1. Genetic engineering-Cellected werke. I. Setlow, Jane K. II. Hollaender, Alexandei, date. QH442.G454 575.1 79-644807 MARC-S ISBN 978-1-4615-7086-8 ISBN 978-1-4615-7084-4 (eBaak) DOl 10.1007/978-1-4615-7084-4 © 1989 Plenum Press, New York Softcover reprint of the hardcover 1s t edition 1989 A Division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013 All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher PREFACE TO VOLUME I This volume is the first of a series concerning a new technol ogy which is revolutionizing the study of biology, perhaps as pro foundly as the discovery of the gene. As pointed out in the intro ductory chapter, we look forward to the future impact of the tech nology, but we cannot see where it might take us. The purpose of these volumes is to follow closely the explosion of new techniques and information that is occurring as a result of the newly-acquired ability to make particular kinds of precise cuts in DNA molecules. Thus we are particularly committed to rapid publication. Jane K. Set low v ACKNOWLEDGMENT The Editor is most grateful to June Martino, who not only did all the final processing of the manuscripts, but also caught some of the Editor's mistakes as well as some of the authors'. vii CONTENTS DNA METHYlA.SES............................................. 1 A. Razin ADVANCES IN DIRECT GENE TRANSFER INTO CEREALS.............. 13 T.M. Klein, B.A. Roth and M.E. Fromm THE COPY NUMBER CONTROL SYSTEM OF THE CIRCLE 2~m PLASMID OF Saccharomyces cerevisiae........................ 33 B. Futcher THE APPLICATION OF ANTISENSE RNA TECHNOLOGY TO PLANTS...... 49 W.R. Hiatt, M. Kramer and R.E. Sheehy THE PATHOGENESIS-RELATED PROTEINS OF PLANTS................ 65 J.P. Carr and D.F. Klessig THE MOLECULAR GENETICS OF PLASMID PARTITION: SPECIAL VECTOR SYSTEMS FOR THE ANALYSIS OF PLASMID PARTITION....... 111 A.L. Abeles and S.J. Austin DNA-MEDIATED TRANSFORMATION OF PHYTOPATHOGENIC FUNGI....... 127 J. Wang and S.A. Leong FATE OF FOREIGN DNA INTRODUCED TO PLANT CELLS.............. 145 J. Paszkowski GENERATION OF cDNA PROBES BY REVERSE TRANSLATION OF AMINO ACID SEQUENCE..................................... 159 C.C. Lee and C.T. Caskey MOLECULAR GENETICS OF SELF-INCOMPATIBILITY IN FlOWERING PLANTS........................................ 171 P.R. Ebert, M. Altschuler and A.E. Clarke PULSED-FIELD GEL ELECTROPHORESIS........................... 183 M.V. Olson INDEX. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 229 ix DBA HETHYIASES Aharon Razin Department of Cellular Biochemistry The Hebrew University-Hadassah Medical School Jerusalem, Israel 91010 INTRODUCTION DNA methylases are widespread in nature; they all use S adenosylmethionine (SAM) as the methyl donor, but differ in their DNA subs tra te specifici ty and may be highly sequence specific. In general, DNA methylases recognize a cytosine or adenine residue in a specific sequence of the DNA and transfer the methyl group from SAM either to the 5 position in the cytosine ring or the amino group at the 6 position of adenine moieties (Figure 1). The methylase reaction is always a postreplication process. It may take place immediately after replication on the hemimethyl ated DNA (maintenance methylation) or on unmethyiated sites (de novo methylation), creating symmetrically methylated sites (Figure 2). The symmetric nature of the methylated site and the maintenance methylation allow clonal inheritance of the methyla tion pattern over many cell generations. On the other hand "demethylation" and de novo methylation provide a mechanism for controlled changes in the methylation pattern 0). Being specific for the base and the sequence in which the base appears implied that a large number of different DNA methylases exists. In fact, a variety of DNA methylases has been isolated; in some cases several different methylases were found in the same cell. The ubiquity of DNA methylases in nature suggested that DNA methylation is of major importance to the biology of the cell. Indeed, many biological functions have been attributed to DNA methylation, some of them are now experimentally verified, others are being investigated and await experimental proof. Methyla tion, being site specific, provides the DNA with additional structural identity which is superimposed on the information read in the nucleotide sequence of the DNA. It is generally accepted 2 A. RAZIN that this additional information is used for specific DNA protein interactions. It is well established that this is the case in restriction modification (RM) systems in bacteria (2). In many such systems the restriction enzyme recognizes specific DNA sequences and the state of methylation of these sites determines whether a counterpart nuclease will bind to and act upon the given site. Other cardinal biological processes that were demonstrated to be associated with DNA methylation include DNA replication (3-5), DNA repair (6), transposition (7-9) and gene ac tivi ty (10). I twas, therefore, of major interest to understand the enzymatic characteristics of the various DNA methy1ases. It was a1 so impor tan t, from a prac tical poin t of view, to have a t hand purified DNA methy1ases in large quantities. These enzymes besides being a useful tool in biochemistry, molecular biology and molecular genetics studies may serve, in the not so remote future, to solve problems in medicine and agricu1 ture. To achieve this goal a substantial number of genes coding for DNA methy1ases have recently been cloned and the methy1ases produced in large quanti ties by recombinant DNA techniques. The avail abil i ty of the cloned genes provides new information on the biochemical features of the DNA methy1ases. This new information and its implications on our understanding of the biochemistry and biology of DNA methylation and related processes will be dis cussed here. PROKARYOTIC DNA METHYLASES Modification Enzymes The largest group of DNA methy1ases in prokaryotic organisms that has been thoroughly studied is that of bacterial modifica tion enzymes which are part of restriction modification systems. These enzymes are classified according to their catalytic fea tures (2). Type I me thy1ases are coded by genes 10ca ted on the bacterial chromosome (11). The recognition function of this restriction modification system is on the hsd S protein. Modification requires the hsdS gene product and the methylase, the hsdM product. Transcripts of the hsdM or hsdM-hsdS as well as the po1ycistronic transcript of hsdR-hsdS-hsdM were detected (12). Type II me thy1ases are coded by plasmid-borne genes or chromosomal genes (13). These enzymes are the simplest of all modification enzymes acting as monomers and require no additional factors for their activity. Type III methy1ases are coded by genes located on a phage, a plasmid or the chromosome (11). The methylase, a product of the mod gene, can methylate by itself. However, the restric tion enzyme, the produc t of the res gene, requires the methylase to restrict. All modification methy1ases are capable of methylating the DNA independent of restriction. DNA METHYLASES 3 ~H2 C CH3 N{~';-C/ C12 61C1 ' H rI ,,~/ I H Hi METHYlAoENIH£ 5 METHYl CYTOSINE Figure 1. Structure of S-adenosylmethione (SAM), the universal donor of methyl groups in transmethylation reactions. Structure of N6 methyladenine and 5-methylcytosine, the common methylated bases in DNA from various organisms. m m m 5' 3' 3' lt 5' m m m Replication Maintenance Methylation Active Denovo m m m 5' 3' 3' ! 5' Replication Demethylation Methylation 5' 3' 3' 5' Figure 2. Interconversions between the 3 levels of methylation of DNA: methylated DNA is symmetrically methylated on both strands, hemimethylated DNA is methylated at all methylatable sites on one strand and unmethylated DNA. The methylation pattern is faithfully maintained by the maintenance methylase, which rapidly methylates hemimethylated DNA during replication. Non-methylated DNA can be methylated by a de novo methylase activity. Loss of methyl groups may occur by several rounds of replication in the absence of methylation (passive mechanism) or by an active mechanism such as replacement of 5-methylcytosine (58).
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