Preface The modern biologist takes almost for granted the rich repertoire of tools currently available for manipulating virtually any gene or protein of interest. Paramount among these operations is the construction of fusions. The tactic of generating gene fusions to facilitate analysis of gene expression has its origins in the work of Jacob and Monod more than 35 years ago. The fact that gene fusions can create functional chimeric proteins was demonstrated shortly thereafter. Since that time, the number of tricks for splicing or inserting into a gene product various markers, tags, antigenic epitopes, structural probes, and other elements has increased explosively. Hence, when we undertook assembling a volume on the applications of chimeric genes and hybrid proteins in modern biological research, we con- sidered the job a daunting task. To assist us with producing a coherent work, we first enlisted the aid of an Advisory Committee, consisting of Joe Falke, Stan Fields, Brian Seed, Tom Silhavy, and Roger Tsien. We benefited enormously from their ideas, suggestions, and breadth of knowledge. We are grateful to them all for their willingness to participate at the planning stage and for contributing excellent and highly pertinent articles. A large measure of the success of this project is due to the enthusiastic responses we received from nearly all of the prospective authors we ap- proached. Many contributors made additional suggestions, and quite a number contributed more than one article. Hence, it became clear early on that given the huge number of applications of gene fusion and hybrid protein technology for studies of the regulation of gene expression, for lineage tracing, for protein purification and detection, for analysis of protein localization and dynamic movement, and a plethora of other uses it would not be possible for us to cover this subject comprehensively in a single volume, but in the resulting three volumes, 326, 327, and 328. Volume 326 is devoted to methods useful for monitoring gene expres- sion, for facilitating protein purification, and for generating novel antigens and antibodies. Also in this volume is an introductory article describing the genesis of the concept of gene fusions and the early foundations of this whole approach. We would like to express our special appreciation to Jon Beckwith for preparing this historical overview. Jon's description is particularly illuminating because he was among the first to exploit gene and protein fusions. Moreover, over the years, he and his colleagues have xiii xiv PREFACE continued to develop the methodology that has propelled the use of fusion- based techniques from bacteria to eukaryotic organisms. Volume 327 is focused on procedures for tagging proteins for immunodetection, for using chimeric proteins for cytological purposes, especially the analysis of mem- brane proteins and intracellular protein trafficking, and for monitoring and manipulating various aspects of cell signaling and cell physiology. Included in this volume is a rather extensive section on the green fluorescent protein (GFP) that deals with applications not covered in Volume 302. Volume 328 describes protocols for using hybrid genes and proteins to identify and analyze protein-protein and protein-nucleic interactions, for mapping molecular recognition domains, for directed molecular evolution, and for functional genomics. We want to take this opportunity to thank again all the authors who generously contributed and whose conscientious efforts to maintain the high standards of the Methods in Enzymology series will make these volumes of practical use to a broad spectrum of investigators for many years to come. We have to admit, however, that, despite our best efforts, we could not include each and every method that involves the use of a gene fusion or a hybrid protein. In part, our task was a bit like trying to bottle smoke because brilliant new methods that exploit the fundamental strategy of using a chimeric gene or protein are being devised and published daily. We hope, however, that we have been able to capture many of the most salient and generally applicable procedures. Nonetheless, we take full responsibility for any oversights or omissions, and apologize to any researcher whose method was overlooked. Finally, we would especially like to acknowledge the expert assistance of Joyce Kato at Caltech, whose administrative skills were essential in organizing these books. JEREMY THORNER Scott D. EMR JOHN N. ABELSON Contributors to Volume 326 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. JON BECKWITH (1), Department of Microbiol- ogy and Molecular Genetics, Harvard Med- ical School, Boston, Massachusetts 02115 JOSHUA A. BORNHORST (16), Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215 LISA BREISTER (22), Stratagene Cloning Sys- tems, La Jolla, California 92037 IRENA BRONSTEIN (13), Tropix, Inc., PE BiD- systems, Bedford, Massachusetts 01730 CLAYTON BULLOCK (14), Department of Phar- macology, College of Medicine, University of California, Irvine, California 92697 ANDREW CAMILLI (5), Department of Molecu- lar Biology and Microbiology, Tufts Uni- versity School of Medicine, Boston, Massa- chusetts 02111 CHARLES R. CANTOR (19), Center for Ad- vanced Biotechnology and Departments of Biomedical Engineering and Pharmacology and Experimental Therapeutics, Boston University, Boston, Massachusetts 02215 and Sequenom, Inc., San Diego, Califor- nia 92121 JOHN M. CHIRGWIN (20), Research Service, Audie L. Murphy Memorial Veterans Ad- ministration Medical Center and Depart- ments of Medicine and Biochemistry, Uni- versity of Texas Health Science Center at San Antonio, Texas 78229-3900 SHAORONG CHONG (24), New England BiD- labs, Inc., Beverly, Massachusetts 01915 R. JOHN COLLIER (33), Department of Micro- biology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115 LISA A. COLLINS-RACIE (21), Genetics Insti- tute, Cambridge, Massachusetts 02140 JOHN E. CRONAN, JR. (27), Departments of Microbiology and Biochemistry, University of Illinois, Urbana, Illinois 61801 MI LLARD G. CULL (26), Avidity, L.L. C, Elea- nor Roosevelt Institute, Denver, Colorado 8O2O6 BRYAN R. CULLEN (11), Howard Hughes Medical Institute and Department of Genet- ics, Duke University Medical Center, Dur- ham, North Carolina 27710 BRIAN D'EoN (13), Tropix, Inc., PE Biosys- tems, Bedford, Massachusetts 01730 SALVATORE DEMARTIS (29), Institute of Phar- maceutical Sciences, Department of Applied BioSciences, Swiss Federal Institute of Technology Zurich, CH-8057 Zurich, Swit- zerland ELIZABETH A. D1BLAS10-SMITH (21), Genetics Institute, Cambridge, Massachusetts 02140 Roy H. Dol (25), Section of Molecular and Cellular Biology, University of California, Davis, California 95616 CHARLES F. EARHART (30), Section of Molec- ular Genetics and Microbiology, The Uni- versity of Texas at Austin, Austin, Texas 78712-1095 DOLPH ELLEFSON (31), Department of Molec- ular Microbiology and Immunology, Ore- gon Health Sciences University, Portland, Oregon 97201 JOSEPH J. FALKE (16), Department of Chemis- try and Biochemistry, University of Colo- rado, Boulder, Colorado 80309-0215 CATHERINE FAYOLLE (32), Unit~ de Biologie des R~gulations Immunitaires, CNRS URA 2185, Institut Pasteur, Paris, Cedex 15, France CORNELIA GORMAN (14), DNA Bridges, Inc., San Francisco, California 94117 ix X CONTRIBUTORS TO VOLUME 326 PIERRE GUERMONPREZ (32), Unitd de Biolo- gie des R~gulations Immunitaires, CNRS URA 2185, Institut Pasteur, Paris, Cedex 15, France NICHOLAS J. HAND (2), Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 FRED HEFFRON (6, 31), Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Port- land, Oregon 97201 DANNY Q. HOANG (22), Stratagene Cloning Systems, La Jolla, California 92037 PHILIPP HOLLIGER (28), MRC Laboratory of Molecular Biology, Cambridge CB2 2QH United Kingdom JOE HORECKA (7), Department of Molecular Biology, NIBH, Tsukuba, Ibaraki 305- 8566 Japan ADRIAN HUBER (29), Institute of Pharmaceu- tical Sciences, Department of Applied Bio- Sciences, Swiss Federal Institute of Technol- ogy Zurich, CH-8057 Zurich, Switzerland SATOSHI INOUYE (12), Yokohama Research Center, Chisso Corporation, Yokohama 236-8605 Japan RAY JUDWARE (13), Tropix, Inc., PE Biosys- terns, Bedford, Massachusetts 01730 GOUZEL KARIMOVA (32), Unitd de Biochimie Cellulaire, CNRS URA 2185, Institut Pasteur, Paris, Cedex 15, France CHRIST1AAN KARREMAN (9), Institute of On- cological Chemistry, Heinrich Heine Uni- versity, 40225 Duesseldorf,, Germany DANIEL LADANT (32), Unit~ de Biochimie Cellulaire, CNRS URA 2185, Institut Pas- teur, Paris, Cedex 15, France EDWARD R. LAVALLIE (21), Genetics Insti- tute, Cambridge, Massachusetts 02140 CLAUDE LECLERC (32), Unit~ de Biologie des Rdgulations Immunitaires, CNRS URA 2185, lnstitut Pasteur, Paris, Cedex 15, France BETTY LIU (13), Tropix, Inc., PE Biosystems, Bedford, Massachusetts 01730 ZHIJIAN LU (21), Genetics Institute, Cam- bridge, Massachusetts 02140 COLIN MANOIL (3), Department of Genetics, University of Washington, Seattle, Washing- ton 98195 CHRIS MARTIN (13), Millennium Predictive Medicine, Cambridge, Massachusetts 02139 DINA MARTIN (13), Tropix, Inc., PE Biosys- terns, Bedford, Massachusetts 01730 ROBERT A. MASTICO (34), Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom MARK McCORMICK (23), Novagen, Inc., Mad- ison, Wisconsin 53711 JOHN M. McCoY (21), Biogen, Inc., Cam- bridge, Massachusetts 02142 ROBERT C. MIERENDORF (23), Novagen, Inc., Madison, Wisconsin 53711 DARIO NERI (29), Institute of Pharmaceutical Sciences, Department of Applied Bio- Sciences, Swiss Federal Institute of Technol- ogy Zurich, CH-8057 Zurich, Switzerland FREDRIK NILSSON (29), Institute of Pharma- ceutical Sciences, Department of Applied BioSciences, Swiss Federal Institute of Technology Zurich, CH-8057 Zurich, Swit- zerland CORINNE E. M. OLESEN (13), Tropix, Inc., PE Biosystems, Bedford, Massachusetts 01730 JAE-SEoN PARK (25), Sampyo Foods Co., Ltd., Seoul 132-040, Korea DAVID PARKER (31), Department of Molecu- lar Microbiology and Immunology, Oregon Health Sciences University, Portland, Ore- gon 97201 HENRY PAULUS (24), Boston Biomedical Re- search Institute, Watertown, Massachusetts 02472-2829 RONALD T. RA1NES (23), Departments of Biochemistry and Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706 LAL1TA RAMAKRISHNAN (4), Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5124 KELYNNE E. REED (27), Department of Biol- ogy, Austin College, Sherman, Texas 75090 CONTRIBUTORS TO VOLUME 326 xi DEEPALI SACHDEV (20), University of Minne- sota Cancer Center, Minneapolis, Minne- sota 55455 SOFIIE REDA SALAMA (8), Microbia, Inc., Cambridge, Massachusetts 02139 TAKESHI SANO (19), Center for Molecular Im- aging Diagnosis and Therapy and Basic Sci- ence Laboratory, Department of Radiology, Beth Israel Deaconess Medical Center, Har- vard Medical School, Boston, Massachu- setts 02215 PETER J. SCHATZ (26), Affymax Research In- stitute, Palo Alto, California 94304 THOMAS G. M. SCHM1DT (18), Institut far Bioanalytik GmbH, D-37079 GOttingen, Germany HAlE-SuN SHIN (25), Sampyo Foods Co., Ltd., Seoul 132-040, Korea THOMAS J. SILHAVY (2), Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 ARNE SKERRA (18), Lehrstuhlfiir Biologische Chemie, Technische Universitdt Mfinchen, D-85350 Freising- Weihenstephan, Germany JAMES M. SLAUCH (5), Department of Micro- biology, University of Illinois, Urbana, Illi- nois 61801 STlEPHlEN SMALL (10), Department of Biology, New York University, New York, New York 10003 DONALD B. SMITH (17), Garden Cottage, Clerkington, Haddington, East Lothian, Scotland, United Kingdom GEORGE F. SPRAGUE, JR. (7), Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403 MICHAEL N. STARNBACH (33), Department of Microbiology and Molecular Genetics, Har- vard Medical School, Boston, Massachu- setts 02115 PETER G. STOCKLIEV (34), Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom IAN TOMLINSON (28), MRC Laboratory of Molecular Biology, Cambridge CB2 2QH United Kingdom A~yEs ULLMANN (32), Unit~ de Biochimie Cellulaire, CNRS URA 2185, Institut Pasteur, Paris, Cedex 15, France PETER VA1LLANCOURT (22), Stratagene Clon- ing Systems, La Jolla, California 92037 RAPHAEL H. VALDIVIA (4), Department of Molecular and Cell Biology, University of California, Berkeley, California 94702 ADR1ANUS W. M. VAN DlER VELDIEN (31), De- partment of Molecular Microbiology and Immunology, Oregon Health Sciences Uni- versity, Portland, Oregon 97201 THOMAS R. VAN OOSBRIEIE (23), Novagen, Inc., Madison, Wisconsin 53711 FRANCESCA V1TI (29), Institute of Pharmaceu- tical Sciences, Department of Applied Bio- Sciences, Swiss Federal Institute of Technol- ogy Zurich, CH-8057 Zurich, Switzerland JOHN C. VOVTA (13), Tropix, Inc., PE Biosys- tems, Bedford, Massachusetts 01730 MICAH J. WORLIEY (6), Department of Molec- ular Microbiology and Immunology, Ore- gon Health Sciences University, Portland, Oregon 97201 MING-QuN Xu (24), New England Biolabs, Inc., Beverly, Massachusetts 01915 Yu-XIN YAN (13), Tropix, Inc., PE Biosys- tems, Bedford, Massachusetts 01730 CHRISTOPHER C. ZAROZINSKI (33), Depart- ment of Microbiology and Molecular Ge- netics, Harvard Medical School, Boston, Massachusetts 02115 CHAO-FENG ZHENG (22), Stratagene Cloning Systems, La Jolla, California 92037 GREGOR ZLOKARNIK (15), Aurora Biosci- ences Corporation, San Diego, California 92121 [1] THE ALL PURPOSE GENE FUSION 3 [11 The All Purpose Gene Fusion By JON BECKWITH The biological revolution of recent years has derived its greatest impetus from the development and utilization of a handful of techniques and ap- proaches for manipulating DNA. These methods include, most prominently, DNA cloning, DNA sequencing, the polymerase chain reaction, and gene fusion. Given the advent of the first three technical developments only during the past 25 years, one might have thought that the use of gene fusions also appeared during this period. In fact, gene fusion as a method for studying biological problems can be traced back to the earliest days of molecular biology. Many of the principles of the gene fusion approach appear in work on one of the classical genetic systems of molecular biology, the rlI genes of the Escherichia coli bacteriophage T4. In the late 1950s and early 1960s, Seymour Benzer and colleagues charactered two adjacent but indepen- dently transcribed genes, rlIA and rlIB, which constituted the rlI region. In 1962, Champe and Benzer described an rlI mutation in which a deletion (r1589) had removed all transcription and translation punctuation signals between the two genes and, thus, fused them into a single transcriptional and translational unit. 1 The deletion covered the sequences coding for the carboxy terminus of the rlIA protein and for approximately 10% of the amino terminus of the rlIB protein. Despite the absence of a substantial portion of the B protein, the gene fusion still exhibited B activity. This property of the r1589 deletion was to provide a very important tool for understanding fundamental aspects of the genetic code. These insights were made possible by the understanding that missense mutations in the fusion that altered the A portion of the hybrid rIIA-B protein would be unlikely to affect B function, whereas mutations that caused termination of translation in the A portion would simultaneously result in loss of B function. Benzer and Champe e found a class of suppressible rlIA mutations that did have the effect of eliminating rlIB activity when introduced into the r1589 deletion. These findings were essential to the classification of these mutations (amber) as mutations that cause protein chain termination. This was the first description of such mutations and the recognition that special signals were involved in the 1 S. P. Champe and S. Benzer, J. Mol. Biol. 4, 288 (1962). 2 S. Benzer and S. P. Champe, Proc. Natl. Acad. Sci. U.S.A. 48, 1114 (1962). Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. METHODS IN ENZYMOI.OGY, VOL 326 0076-6879/00 $30.00 4 HISTORICAL OVERVIEW [ 1] chain termination process. At the same time, Crick and co-workers 3 were characterizing a class of mutations that they suspected to be frameshifts. A key step in their analysis was the demonstration that these mutations, when introduced into the rlIA region of the r1589 fusion, also eliminated rlIB activity. These experiments were important to the use of frameshift mutations to establish the triplet nature of the genetic code. Several key concepts underlying the gene fusion approach can be found in these studies. First, the idea that it is possible to remove a significant portion of a terminus of a protein (amino terminus in this case) and still retain sufficient protein function has proved to be the case with a large number of proteins. Second, the possibility of fusing two different proteins together and retaining one or both activities was not self-evident. It seemed quite reasonable to imagine that the generation of a single polypeptide chain from two chains would result in mutual interference with proper folding and functioning of each protein. Third, and most importantly, the notion of using downstream protein activity to report on what was happen- ing upstream the reporter gene concept was key to these studies. This, of course, is the key feature of the gene fusion approach. This history has been described as though it was known at the time that the rlI genes coded for protein. Extraordinarily enough, it was not shown until many years later that this was the case. Nevertheless, the genetic evidence was considered compelling enough at the time that the conclusions of these studies gained widespread acceptance among molecular biologists. The next steps in the development of gene fusion approaches came from studies on the lac operon of E. coli. The first fusions of lac were obtained unwittingly as revertants of strong polar mutations in the lacZ gene. 4 Selection for restoration of the activity of the downstream lacY gene yielded many deletions that removed the polar mutation site, the promoter of lac, and fused the lacy gene to an upstream promoter of an unknown neighboring gene. In 1965, Jacob and co-workers 5 exploited this approach to select for fusions in which the lacY gene was put under the control of an operon involved in purine biosynthesis. This was the first report of a gene fusion in which the regulation of a reporter gene was determined by the gene to which it was fused; the Lac permease was regulated by the concentration of purines in the growth media. Subsequently, Muller-Hill and Kania 6 showed that the properties of /3-galactosidase allowed an even broader use of the gene fusion approach 3 F. H. C. Crick, L. Barnett, S. Brenner, and R. J. Watts-Tobin, Nature 192, 1227 (1961). 4 j. R. Beckwith, J. Mol. Biol. 8, 427 (1964). 5 F. Jacob, A. Ullmann, and J. Monod, J. Mol. Biol. 42, 511 (1965). 6 B. Muller-Hill and J. Kania, Nature 249, 561 (1974). [1] THE ALL PURPOSE GENE FUSION 5 in this system. Using a very early chain-terminating mutation, they found that they could restore/3-galactosidase activity by deleting the polar muta- tion site and fusing the remaining portion of the polypeptide to the upstream lacI gene product, the Lac repressor. It was even possible to obtain hybrid proteins with both repressor and/3-galactosidase activity. Generalizing the Approach In all the cases described to this point, genetic fusions were obtained between two genes that were normally located close to each other on the bacterial chromosome or on an F' factor. This feature of early gene fusion studies presented quite strict limitations on the systems that could be ana- lyzed by this approach. However, beginning first with some old-fashioned approaches to transposing the lac region to different positions on the chro- mosome, 7 we began to see that the gene fusion approach might be applied more widely. A graduate student in the author's laboratory, Malcolm Casa- daban, then developed improvements on transposition techniques that en- hanced the ability to fuse lac more generally to bacterial genes. 8 Malcolm continued these improvements in Stanley Cohen's laboratory at Stanford University and ultimately in his own laboratory at the University of Chicago. 9,1° All the approaches described so far involved generation of fusions in vivo. The arrival of recombinant DNA techniques for cloning and fusing genes in the mid-1970s provided a tremendous boost to the use of gene fusions. It became possible to fuse genes from or between any organism pretty much at will. Gene Fusions for All Seasons For many years, the gene fusion tool was considered to be one useful mainly for studying gene expression and regulation by reporter gene expres- sion. However, as the ease of generating such fusions grew, other uses became evident. In 1980, we reported the first case where fusing a reporter protein to another protein of interest allowed purification of the latter protein. I~ In this case, fl-galactosidase was fused to a portion of the cytoplasmic membrane protein, MalF. The unusually large size of 7 j. R. Beckwith, E. R. Signer, and W. Epstein, Cold Spring Harbor Syrup. Quant. BioL 31, 393 (1966). M. Casadaban, J. Mol. Biol. 104, 541 (1976). 9 M. J. Casadaban and S. N. Cohen, Proc. NatL Acad. Sci. U.S.A. 76, 4530 (1979). 10 M. J. Casadaban and J. Chou, Proc. Natl. Acad. Sci. U.S.A. 81, 535 (1984). 11 H. A. Shuman, T. J. Silhavy, and J. R. Beckwith, J. Biol. Chem. 255, 168 (1980). 6 HISTORICAL OVERVIEW [ 11 /3-galactosidase allowed ready purification of the hybrid protein, which was then used to elicit antibody to MalF epitopes, facilitating its purification. We also showed that gene fusions of/3-galactosidase could be used to study the signals that determine subcellular protein localization. Fusion of /3-galactosidase to the MalF protein resulted in membrane localization of the former protein, 11 and fusion of/3-galactosidase to exported proteins permitted the genetic analysis of bacterial signal sequences) 2a3 Another important step in the evolution of uses of gene fusions came with the concept of signal sequence traps. The first development of this concept came out of the recognition that the bacterial enzyme alkaline phosphatase is active when it is exported to the periplasm but inactive when it is retained in the cytoplasm. TM Thus, alkaline phosphatase without its signal sequence provides an assay for export signals via gene fusion approaches, i.e., alkaline phosphatase will only be active if one attaches a region of DNA that encodes a signal sequence, thus reallowing its export. Hoffman and Wright 15 and Colin Manoil and the author 16 reported sys- tems-one plasmid, one transposon that allowed the detection of signal sequences in random libraries of DNA or in a bacterial chromosome. This approach has been extended with use of numerous other reporter genes, including, most prominently, fl-lactamase? 7 Extending beyond the differentiation of exported vs cytosolic proteins, gene fusion techniques can be evolved to determine subcellular localization of proteins more generally. Clearly, the use of GFP fusions enhances this ability. TM In addition, reporter proteins that sense specific features of organ- elle environment may provide a tool for detecting location and genetically manipulating signals for the localization process. The report of a GFP that responds to the pH of its environment may be a harbinger of things to come) 9 One might imagine GFP derivatives that respond to all sorts of cellular conditions, e.g., the redox environment. Finally, gene fusions can be used for the study of protein structure, protein-protein interactions, and protein folding. The yeast two-hybrid system described by Fields and Song 2° in 1989 has become a powerful tool for analyzing aspects of quaternary structure of proteins and for detecting 12 S. D. Emr, M. Schwartz, and T. J. Silhavy, Proc. Natl. Acad. Sci. U.S.A. 75, 5802 (1978). 13 p. Bassford and J. Beckwith, Nature 277, 538 (1979). 14 S. Michaelis, H. Inouye, D. Oliver, and J. Beckwith, J. Bacteriol. 154, 366 (1983). 15 C. Hoffman and A. Wright, Proc. Natl. Acad. Sci. U.S.A. 82, 5107 (1985). 16 C. Manoil and J. Beckwith, Proc. Natl. Acad. Sci. U.S.A. 82, 8129 (1985). 17 y. Zhang and J. K. Broome-Smith, Mol. Microbiol. 3, 1361 (1989). 18 D. S. Weiss, J. C. Chen, J. M. Ghigo, D. Boyd, and J. Beckwith, J. BacterioL 181, 508 (1999). 19 G. Miesenb6ck, D. A. DeAngelis, and J. E. Rothman, Nature 394, 192 (1998). 2o S. Fields and O. Song, Nature 340, 245 (1989). [1] THE ALL PURPOSE GENE FUSION 7 novel protein-protein interactions. Whereas the structure of soluble pro- teins is accomplished relatively easily by X-ray crystallography techniques, the structure of membrane proteins still largely resists such approaches. Gene fusion techniques have been able to contribute to understanding important features of membrane protein structure. The signal sequence trap techniques have proved invaluable in the determination of the topological structure of integral membrane proteins, 21 i.e., fusion of the reporter protein to intra- or extracytoplasmic domains of membrane proteins usually reports the location of that domain accurately. Similarly, more recent techniques for detecting interactions between transmembrane segments of such proteins should allow the elucidation of additional structural features. 22'23 Although not so widely employed, gene fusion approaches can aid in the study of protein folding. Luzzago and Cesareni 24 used a cute fusion approach to isolate mutants affecting the folding of ferritin. Other such ideas must be waiting in the wings. The realm of gene fusions has continually expanded. While this volume describes a host of different issues that can be studied with this technique, it seems certain that the expansion will continue. 2z C. Manoil and J. Beckwith, Science 233, 1403 (1986). 22 j. A. Leeds and J. Beckwith, J. Mol. Biol. 2811, 799 (1998). 23 W. P. Russ and D. M. Engelman, Proc. Natl. Acad. Sci. U.S.A. 96, 863 (1999). 24 A. Luzzago and G. Cesareni, EMBO J. 8, 569 (1989). [...]... Transcriptional Translational Translational Translational MuA derivative ~ AplacMu52" AplacMu54 ~ " AplacMu55 " AplacMu5" AplacMul 3" AplacMul5" '~imm, phage immunity; Kan R, kanamycin resistance b Fusions designated lacZYA' include the sequences necessary for translational initiation and contain functional copies of both lacZ and lacy genes, but are truncated in lacA (and are lacA ) Fusions designated lac'ZYA'... Huisman and N Kleckner, Genetics 116, 185 (1987) [21 CONSTRUCTING l a c FUSIONS IN E c o l i 25 TABLE III A p l a c M u VECTORS FOR MAKING RANDOM FUSIONS Vector Marker ~ Fusion b Fusion type AplacMu50 a AplacMu51 d AplacMu53 d AplacMul I AplacMu3f AplacMu9 j immA imm21 immA, Kan R immA imm21 irnmA, Kan R IacZYA ' lacZYA ' lacZYA ' lac "ZYA' lac "ZYA' lac "ZYA' Transcriptional Transcriptional Transcriptional... Casadaban, J Mol Biol 104, 541 (1976) CONSXRUCTIN6 lac VUSIONS IN E coli [2] 27 lacA' lacY lacZ'trpA' A J l~l~'fJffffA A ~ S cI N ~r B RA 'trpA'lacZ Ac N cI R J lacY lacA'~ S trpA' lacZ lacY lacA' ~'Jfffff~\~ J "X AR cI N S \ Ac Y cI RA ( P x ~ x " trpA'lacZ lacY lacA'~ D placMu50 cA Px ~1~ J X E Px# X"trpA' lacZ lacY lacA' K/cfffff~N~\~N~l J AR cI N ~ S FIG 2 Adapted from Bremer et al 2 (A) Schematic... fl-galactosidase remains unrivaled as a transcriptional reporter Using appropriate media, mutations that increase or decrease the expression of an operon fusion of interest can be isolated easily Conversely, screening pools of random LacZ chromosomal insertions can identify targets of a regulator (either transcriptional or translational) Finally, fl-galactosidase remains useful in the study of translational... deleted for the lacZ sequence upstream of the SacI site and therefore carries a 3' fragment comprising roughly one-third of the lacZ gene, as well as functional lacy and lacA genes phages and differences in the analysis of transcriptional and translational fusions The number of vectors available for the creation of Lac fusions is positively bewildering, and a more comprehensive listing can be found elsewhere... fusions (lysogens) are marked with Kan R '~R W Simons, F Houman, and N Kleckner, Gene 53, 85 (1987) h AmpR, ampicillin resistance; Kan a, kanamycin resistance • Fusions designated lacZYA contain functional lacZ, lacY, and lacA genes and include the sequences necessary for translational initiation Fusions designated lac "ZYA are deleted for the translation initiation sequences The lac'ZscYA fragment on pRS308... be retained for long-term study This step takes advantage of the ability of AplacMu53 to form plaques Induction of the prophage by ultraviolet (UV) light stimulates illegitimate recombination and promotes the formation of specialized transducing phage particles that carry A sequences and a portion of flanking bacterial chromosome fused to the lac operon, lac genes carried by the prophage are situated... A ] ~.RS91 A J ~ pRS308 ~ 'tet ~ ori ,~ bla' ~l II attP ~plac I]]]]}" TI4 -UV5 )l P ~T CCC I I EcoR1 Imm21 nln5 altP ~ attP ~= bla' g Sinai*** ASClac'ZYA j AAT ~C Imm21 nl.s imm434 CJIndattP imm434clind" lacZYA = bla ASClac 'ZYA FIG 1 Adapted from Simons et aL 3 (A) Schematic diagram of plasmid vectors for constructing lac fusions All of the plasmids are based on a pBR322 backbone and carry a. .. stored at -20 ° in the dark, and plates should not be prepared far in advance of their anticipated use A second type of indicator media, much less expensive than X-Gal media and invaluable for bacterial genetics, is lactose MacConkey agar This rich medium, which was originally formulated to facilitate screening for Lac" gram-negative microorganisms, contains bile salts (to inhibit the growth of nonenteric... units of/ 3-galactosidase activity for the formation of Lac + (white) colonies Somewhat paradoxically, it is precisely this insensitivity that makes tetrazolium such a useful indicator Tetrazolium agar can be used to screen for rare mutations that decrease the/3-galactosidase activity of a fusion with high activity Differences between strains with 300 Miller units of /3-galactosidase activity and those . one-third of the lacZ gene, as well as functional lacy and lacA genes. phages and differences in the analysis of transcriptional and translational fu- sions. The number of vectors available. referred to as bla' carry a truncated 5' fragment of the bla open reading frame and are sensitive to ampicillin. Amp R lysogens have an intact bla gene and are ampicillin resistant All of the plasmids are based on a pBR322 backbone and carry a fragment of the 3' end of the tetA gene. In addition, all of the plasmids carry four copies of the rrnB transcriptional