Immunoglobulin Genes Edited by T. HONjO Kyoto University, Japan F.W.ALT College of Physicians and Surgeons of Columbia University, New York, USA T.H. RABBITTS MRC Laboratory of Molecular Biology, Cambridge, UK Academic Press Harcourt Brace Jovanovich, Publishers London San Diego New York Boston Sydney Tokyo Toronto ACADEMIC PRESS LIMITED 24-28 Oval Road London NW1 7DX United States edition published by ACADEMIC PRESS INC. San Diego, CA 92101 Copyright © 1989 by ACADEMIC PRESS LIMITED Second printing 1990 All rights Reserved No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers British Library Cataloguing in Publication Data Immunoglobulin genes 1. Vertebrates. Immunoglobulins 1. Honjo, T. II. Alt, F.W. III. Rabbitts, T.H. 612'. 118223 ISBN 0-12-354865-9 This book is printed on acid-free paper ( Typeset by Lasertext Limited, Stretford, Manchester, England Printed in Great Britain by St Edmundsbury Press Limited, Bury St Edmunds, Suffolk Contributors F.W. Alt Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA V. Anquez Institut Jacques Monod, CNRS-Universite, Paris 7, 2 Place Jusseu, 75251 Paris, Cedex 05, France. Present address: Basel Institute for Immunology, Grenzacherstrasse 487, CH 5005 Basel, Switzerland A.N. Barclay MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK D. Baltimore Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA J. Berman Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA M.J. Bosma Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, PA 19111, USA P.D. Burrows The Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA J.D. Capra Department of Microbiology, University of Texas Health Science Center at Dallas, Dallas, TX 75235, USA M.D. Cooper School of Medicine, Division of Developmental and Clinical Immunology, University of Alabama at Birmingham, Birmingham AL 35294, USA CM. Croce The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA A. Dahan Institut Jacques Monod, CNRS-Universite Paris 7, 2 Place Jusseu, 75251 Paris, Cedex 05, France viii CONTRIBUTORS K.A. Dennis Department of Microbiology, University of California at Los Angeles, Los Angeles, CA 90024, USA J. Durdik Rosenstiel Basic Medical Sciences Center, Brandeis University, Waltham MA 02254, USA L.R. Finger The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA G. Galli Howard Hughes Medical Institute, Duke University Medical Center, Depart- ment of Microbiology/Immunology, Durham, NC 27710, USA J.W. Guise Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA F.G. Haluska The Wistar Institute, 3601 Spruce Street, Philadelphia PA 19104, USA K. Hinds Showa University Research Institute, 10900 Roosevelt Boulevard, St. Peters- burg, FL 33716, USA T. Honjo Department of Medical Chemistry, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606, Japan F. Kokubu Showa University Research Institute, 10900 Roosevelt Boulevard, St. Peters- burg, FL 33716, USA S.J. Korsmeyer Departments of Medicine, Microbiology and Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St Louis, MO 63110, USA M.E. Koshland Department of Microbioogy and Immunology, University of California, Berkeley, CA 94720, USA G.W. Litman Showa University Research Institute, 10900 Roosevelt Boulevard, St. Peters- burg, FL 33716, USA F. Melchers Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland CONTRIBUTORS ix M.W. Moore Rosenstiel Basic Medical Sciences Center, Brandeis University, Waltham MA 02254, USA S.L. Morrison Department of Microbiology, Cancer Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA. Present address: Molecular Biology Institute, University of California, Los Angeles, California, USA J.R. Nevins Howard Hughes Medical Institute, Duke University Medical Center, Depart- ment of Microbiology/Immunology, Durham, NC 27710, USA M.M. Newkirk Department of Microbiology, University of Texas Health Science Center at Dallas, Dallas, TX 75235, USA V.T. Oi Becton-Dickinson Monoclonal Center, Mountain View, CA 94043, USA D.M. Persiani Rosenstiel Basic Medical Sciences Center, Brandeis University, Waltham, MA 02254, USA G. Rathbun Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA CA. Reynaud Institut Jacques Monod, CNRS-Universite, Paris 7, 2 Place Jusseu, 75251 Paris Cedex 05, France. Present address: Basel Institute for Immunology, Grenzacherstrasse 487, CH 5005 Basel, Switzerland E. Seising Rosenstiel Basic Medical Sciences Center, Brandeis University, Waltham, MA 02254, USA R. Sen Rosenstiel Research Center and Department of Biology, Brandeis University, Waltham, MA 02254, USA A. Shimizu Department of Medical Chemistry, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606, Japan U. Storb Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA X CONTRIBUTORS P.W. Tucker Department of Microbiology, University of Texas Southwestern Medican Center, Dallas, TX 75235, USA J.C. Weill Institut Jacques Monod, CNRS-Universite Paris 7, 2 Place Jusseu, 75251 Paris, Cedex 05, France. Present address: Basel Institute for Immunology, Grenzacherstrasse 487, CH 5005 Basel, Switzerland A.F. Williams MRC Cellular Immunology Unit, Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, UK O.N. Witte Department of Microbiology, University of California at Los Angeles, Los Angeles CA 90024, USA G. Yancopoulos Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA Y. Yaoita Department of Medical Chemistry, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto 606, Japan H.G. Zachau Institut für Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universität München, FRG Foreword When Boehringer and Kitasato discovered antibodies back in 1890, they could hardly have imagined that these factors were prime examples of both the chemical and biological bases of molecular recognition. The specificity of antibodies was soon recognized as a biological puzzle by Ehrlich, who in 1905 proposed the first (selective) theory of antibody specificity. Even so, Ehrlich considered it inconceivable that there could be specific substances ready to recognize and neutralize toxins that the animal species had never encountered before. So he produced the most ingeneous idea that the toxins were the ones which by pure coincidence were capable of recognizing "side chains", located on the surface of cells, and required for the utilization "of foodstuff's". Ehrlich therefore made the remarkable prediction of the existence of receptors on the cell surface, but could not conceive the capacity of the organisms to recognize the unknown and to learn to improve such recogni- tion. While he laid the foundations of what was going to be a major preoccupation of immunologists for a long time, namely quantitative immu- nochemistry, it was the role of Landsteiner to demonstrate that antibodies were indeed capable of recognizing substances, naturally occurring or otherwise, which the animal had never seen before, through his classical studies with haptens. This remarkable property of the immune system became a dominating intellectual challenge to basic immunologists. The additional conviction that such specific recognition was capable of further improvement through a process of maturation of the immune response demonstrated by Heidelberger and Kendall in the late 1930s, was an added complication, for which no rational explanation could be proposed at the time. At first it was proposed that antigens act as a form of template, around which the antibody is synthesized or folded. Prominent proponents of such an "instructive" hypothesis were Haurowitz and Pauling. But this soon ran into difficulties, for a number of reasons. A major one was the progress in the understanding of the molecular basis of protein structure and folding, and more generally speaking, the molecular basis of biological specifity. The new ideas and the developments of new methods to study the chemistry of biological specificity, which characterizes the birth of molecular biology, had a direct impact on immunology. The emerging techniques of protein chemistry were immediately applied to the studies of antibodies by Rodney Porter, soon after his PhD supervisor, Sänger, developed the methods which culminated with the demonstration that proteins had defined amino acid Xll FOREWORD sequences. This early inroad into the protein chemistry and early amino acid sequences of the N-terminus of antibodies, in the early 1950s, gave support to the instruction theories, in that no heterogeneity could be discovered. On the contrary, a single N-terminal sequence could be discerned in rabbit immunoglobulins, leading to calculations into the highly improbable possi- bility of different molecules having identical N-terminal sequences. This may sound strange today, when we have become so accustomed to the idea of protein families, and closely related tandem arrays of genes. However, in those days, the idea that genes could arise by gene duplication only gained acceptance in the 1960s, following Braunitzer's comparison of the a and ß chains of haemoglobin. The idea that antibodies were indeed heterogeneous was very difficult to demonstrate, and was the result of a variety of studies coming from different directions that slowly built up into an inescapable conclusion at a later date. Earliest among them was the discovery of idiotypes made by Oudin in the early 1960s. The clonal selection theory proposed by Burnett and inspired by an alternative selective theory made by Niels Jerne, provided a very sound theoretical basis to the generation of specificity through protein microheterogeneity. The common structural architecture of antibody molecules made up of two heavy and two light chains could be established in the early 1960s by Edelman and Porter, with the heterogeneous population of antibodies, because of the very fact that they represented an invariant character of all molecules. The critical element which revealed the essential character of the antibody diversity, did not come from studies of antibodies themselves, but from myeloma proteins. Myeloma proteins have been known for a very long time, so much so that what turned out to be the light chains of myeloma proteins were discovered by Bence-Jones in 1847. The relationship between human myeloma proteins and antibodies arose largely from the careful antigenic analysis performed by Henry Kunkel, and extended to the mouse counterparts from the mouse plasmacytomas discovered by Michael Potter. It was the structural analysis of such molecules which brought about a further understanding of the underlying diversity within the frame of a general common architecture. The early peptide maps of human and mouse Bence-Jones proteins performed, respectively, by Putnam and colleagues and by Dreyer and his colleagues, were quickly superseded by the demonstration in 1965 by Hilschmann and Craig that such light chains consisted of a common segment and a variable segment. The existence of allotypic markers which appeared to be localized in the V region of rabbit heavy chains, but shared by IgG, IgM and IgA (the Todd phenomenon), and my own demonstration that variable segments of kappa light chains consisted of at least three non-allelic sets of V regions in association with a single C region, K K provided experimental evidence that the variable and constant domains must FOREWORD Xlll be encoded by separate genes, as proposed by Dreyer and Bennett. The comparison between the rapidly expanding sequences of V segments of myeloma proteins disclosed the existence of the hypervariable regions, which were to be called complementarity determining regions (CDRs) by Kabat, to imply that those were the residues directly involved in the antigenic recognition. The generalized common architecture predicted by the linear array of disulphide bonds, and conceptualized by the domain structure proposal of Edelman, with the hypervariable segments predictably located at the tips of the Y-shaped molecule seen by electron micrographs, received the spectaculator confirmation of the crystallographic studies which followed the crystallizations of Fab fragments of myeloma proteins by Poljak and NisonofT. This exciting period characterized by studies of myeloma proteins using protein chemistry techniques, was to be enriched by the first glimpse of the somatic hypermutation, which resulted from the comparison of different lambda chains made by Cohn, Weigert and co-workers. All this was soon to be superceded by the application of the DNA recombinant technology, in myelomas and in the newly derived hybridomas. The spectacular confirmation of the two genes/one polypeptide made at the DNA level by Tonegawa in 1976, led within a period of less than 10 years to our present understanding of the genetic arrangement and rearrangement of the antibody genes. Further success was provided by the attack on the problem of the T-cell recognition system. The long-drawn out controversy concerning the T-cell receptor was finally solved, to close the chapter of basic understanding of the genetic nature of the origin of diversity and of the structures involved in antigen recognition. The puzzle of the participation of the major histocompatibility complex spearheaded by the observations of Zinkernagel and Doherty added the first glimpse concerning the molecular bases of cellular interactions. A completely new panorama of immunological puzzles started to emerge. The complexity of cellular interactions are slowly beginning to be uncovered through the advent of monoclonal antibodies against newly defined sets of markers of cellular differentiation, the discovery and large-scale production of lymphokines and growth and differentiation factors, the viral immortalization of cells at different stages of differentiation, and the improvement in techniques of molecular and cell biology. The last 10 years of immunology may have finally settled the major questions defined by the previous 90, but at the same time have exposed new fundamental and well-defined questions. C. MILSTEIN Preface Since Kitasato and Behring discovered antibodies in animal serum in the late 19th century, the structure, function and expression of antibodies or immunoglobulins have posed exciting and important questions in immu- nology. There is no doubt that immunoglobulins are essential molecules in the immune system since most infectious diseases can be prevented or cured by appropriate specific antibodies. Protein chemical studies on the immunoglobulin structure showed, firstly, that the light chain of an immunoglobulin molecule is composed of variable and constant regions. This discovery, however, served only as the vanguard to further questions: How can single polypeptides with variable and constant regions be synthesized? How are the immunoglobulin genes organized? How can so many variable regions be produced by a limited number of genes? We had to wait until the next major technical development, namely DNA cloning, to elucidate the basic framework of dynamic rearrangement of the immunoglobulin genes. During the period up to this discovery, a variety of models were proposed, most of which did, however, turn out to be partially correct. A new technology had to become available to solve questions which emerged from immunology, but are fundamental to molecular genetics in the eukaryote. The development of the recombinant DNA techniques allowed us to explore the above questions in a straightforward manner. During the past decade, we have accumulated an enormous amount of information on the organization, structure, rearrangement and expression of immunoglobulin genes in a variety of organisms. Studies on immunoglobulin genes have had a great impact not only on immunology but also on molecular biology in general. Such studies have provided many precedents for new concepts in eukaryotic molecular biology: exon-intron organization, differ- ential splicing, site-specific as well as region-specific recombination, gene deletion and somatic mutation are examples. This book provides up-to-date overviews of various aspects of immunoglo- bulin genes by authors who have actively participated in the accumulation of our knowledge on this subject. The editors hope that this book will serve as a prelude to further advancement and look forward to new developments in the field. TASUKU HONJO FREDERICK W. ALT TERRY H. RABBITTS