SEQUENCE SPECIFICITY IN TRANSCRIPTION AND TRANSLATION

Proceedings of a conference from a UCLA Symposium held in

Steamboat Springs, Colorado, April, 1985

Editors

Richard Calendar Department of Molecular Biology

University of California Berkeley, California

Larry Gold Department of Molecular,

Cellular, and Developmental Biology University of Colorado

Boulder, Colorado

Alan R. Liss, Inc. · New York

Contents Contributors xv Preface xxv

I . P R O K A R Y O T I C P R O M O T E R S

Interaction of R N A Polymerase with the LAC P s Promoter In Vivo and In Vitro

James A . Borowiec and Jay D . Gralla 3 Regulation of Transcription Initiation in Escherichia coli

Will iam R. McClure and Barbara C. Hoopes 11 Promoters of the Ε. coli System: In Vivo Strength and the Contribution of Elements in the Transcribed Region

Hermann Bujard, Ulrich Deuschle, Wolfgang Kammerer, Reiner Gentz, Wilhelm Bannwarth, and Dietrich Stueber 21

SI Analysis of Plio Activity in the Ε. coli rpoBC Operon After AminoacyltRNA Limitation or Rifampicin Treatment

Brian A . Morgan and Richard S. Hay ward 31

Bacteriophage N4 Virion RNA Polymerase Promoters Lucia B. Rothman-Denes, Lynne Haynes, Peter Markiewicz, Alexandra Glucksmann, Cherie Malone, and John Chase 41

Comparison of the Bacteriophage T3 and T7 RNA Polymerases Will iam Τ. McAllister, Nancy J. McGraw, Claire E. Morris, and John F. Klement 55

Control of the Late Transcription of the B. subtilis Phage 029 DNA Rafael P. Meilado, Jose L . Carrascosa, and Margarita Salas 65

Reciprocal Transactivation by P2 and P4 Phages Richard Calendar, Gail Christie, Emily Dale, and Conrad Hailing . 75

I I . E U K A R Y O T I C P R O M O T E R S

Molecular Genetics of Yeast RNA Polymerases C. James Ingles, Howard J. Himmelfarb, Matthew Moyle, Lori A. Allison, Michael Shales, and James D. Friesen 85

Characterization of the Rhesus Monkey Ribosomal DNA Promoter S. House, R . M . Learned, and R. Tjian 95

Interaction of RNA Polymerase I Transcription Factor(s) with Mouse rDNA Promoter Elements

Ingrid Grummt, Detlev Buttgereit, and Joachim Clos I l l Sequences Conserved in the Promoter Regions of Nematode Vitellogenin Genes

Thomas Blumenthal., Erin Zucker, Karen Denison, Jerome Cane, and John Spieth 125

ix

χ Contents

I I I . T E R M I N A T O R S E L E C T I O N

Mechanisms of RHO-Dependent Transcription Termination Site Selection

David G. Bear, James A . McSwiggen, William D. Morgan, and Peter H . von Hippel 137

Characterization of a RHO-Dependent Termination Site and its Relationship to RNA Processing in Generating the Mature 3' End of E. coli Tryptophan Operon mRNA

Terry Piatt, John E. Mott , Jill L . Galloway, and Raymond A. Grant 151

Interaction of R H O Protein with λ cro and T7 D i l l Early Gene Transcripts

Marion Ceruzzi and John P. Richardson 161 Gene Expression in Coliphage λ: Proteins and Sites Involved in Transcription Antitermination

A.T. Schauer and D . I . Friedman 171 Role of Escherichia coli NusA Protein in Transcription Termination: T S Mutant and RNA Recognition

Yoshikazu Nakamura and Akiko Tsugawa 185 Regulation of Transcription Termination by the nus Gene Proteins of Escherichia coli

Jack Greenblatt, Robert Horwitz, Yukiko Goda, and Joyce L i . . . . 197

I V . T R A N S C R I P T I O N A L R E P R E S S O R S AND A C T I V A T O R S

M N T Repressor-Operator Interactions: Altered Specificity Requires N-6 Methylation of Operator DNA

A . K . Vershon, P. Youderian, Μ.A. Weiss, M . M . Susskind, and R.T. Sauer 209

Cross-Specificities of Functionally Identical DNA-Binding Proteins From Different Lambdoid Bacteriophages

Daniel L . Wulff and Michael E. Mahoney 219 Activation and Repression of the XPre Promoter by el l Protein

G. Gussin, J.-J. Hwang, K. Matz, M . Zuber, and D. Court 229 DNA Loop in Repression of the E. coli L-Arabinose Operon

Robert Schleif 239 Mechanism of gal Repressor Action

S. Adhya, A. Majumdar, M . Polymeropoulos, L . Orosz, and M . Irani 245

Interaction of a Virus-Coded Type I I DNA-Binding Protein Jonathan R. Greene and E. Peter Geiduschek 255

An Autoregulatory Protein is Required for P I Plasmid Replication Dhruba K. Chattoraj, Subrata K. Pal, Judith A . Swack, Rebecca J. Mason, and Ann L . Abeles 271

Contents xi

Role of an Upstream Region in Regulation of the Yeast Suc2 Gene by Glucose Repression

Laura Sarokin and Marian Carlson 281 Target Sites for Positive and Negative Regulatory Elements Controlling Expression of an Inducible Eucaryotic Gene

R.A. Sumrada and T.G. Cooper 291 Prokaryotic Promoters, Activators and Repressors: Workshop Summary

Sankar Adhya 303

V. R I B O S O M E BINDING S I T E S

Three Dimensional Organization of the E. coli 16S Ribosomal RNA Paul L . Wollenzlen and Alain Expert-Bezancon 309

Protein-RNA Interactions Direct Measurement of eIF-4A Interaction with mRNA and Nucleotides

Dixie Goss, Charles Woodley, and Albert Wahba 323 Ribosome Binding Site Sequences and Function

John Childs, Keith Villanueba, Doug Barrick, Thomas D. Schneider, Gary D. Stormo, Larry Gold, Moshe Leitner, and Marvin Caruthers 341

Rules of Translation: Studies with Altered Forms of the Yeast CYCl Gene

Steven B. Bairn, Charles T. Goodhue, Dennis F. Pietras, David C. Eustice, Michael Labhard, Linda R. Friedman, D. Michael Hampsey, John I . Stiles, and Fred Sherman 351

Human α-Interferon Genes: Control Sites and Pseudogene Expression

Arthur P. Bollon, Richard M . Torczynski, Rajinder S. Sidhu, and L . Cheryl Hendrix 363

Workshop Summary: Ribosomal RNA—Genetics & Phylogenetics Harry F. Noller 377

V I . R E G U L A T I O N O F T R A N S L A T I O N

Determination of the Coding Regions for Adeno-Associated Virus Structural Proteins Β and C : Evidence that Protein Β is Initiated by an A C G Codon

S.P. Becerra, J.A. Rose, and C.W. Anderson 383 Regulation of Translation Initiation by an Intramolecular Long Distance Interaction Within the mRNA

J. Botterman, E. De Almeida, L . Bougueleret, and M . Zabeau . . . 397 Antibiotic Resistance Mutations in 16s and 23s Ribosomal RNA Genes of Escherichia coli

Curt D. Sigmund, Mohamed Ettayebi, Susan M . Prasad, Bonnie M . Flatow, and Edward A. Morgan 409

xii Contents

Synthesis of Specialized Ribosomes in Escherichia coli Herman de Boer, Peter Ng, and Anna Hui 419

V I I . RNA P R O C E S S I N G

Processing of tRNA Precursors by an RNA Catalyst from Bacillus subtilis

T.L. Marsh, B. Pace, C. Reich, K. Gardiner, and N R . Pace . . . . 441 Analysis of mRNA Degradation in Escherichia coli

Sidney R. Kushner, Cecilia M . Arraiano, Stephanie D. Yancey, and William P. Donovan 451

Sequences and Factors Involved in Pre-mRNA Splicing In Vitro Adrian Krainer, Robin Reed and Tom Maniatis 461

Studies on the Mechanism of mRNA Splicing In Vitro Richard A . Padgett, Maria M . Konarska, Paula J. Grabowski, Markus Aebi, Charles Weissmann, and Phillip A. Sharp 475

Conservation of Structure in Intermediate Filament Proteins and Their Genes

Elaine Fuchs, Douglas Marchuk, Angela L . Tyner, Amlan RayChaudhury, Marjorie Lindhurst, Sean McCrohon, and Margaret J. Eich man 487

V I I I . E N H A N C E R S

Functional Elements of the Yeast rRNA Transcription Unit Elaine A . Elion and Jonathan R. Warner 501

The Strong Enhancer Element in the Immediate Early Region of the Human Cytomegalovirus Genome

Michael Boshart, Frank Weber, Rüdiger Rüger, Karoline Dorsch-Häsler, Gerhard Jahn, Jay Stoerker, Walter Schaffner, and Bernhard Fleckenstein 511

Regulation of Enhancer-Promoter Interactions by SV40 T-Antigen Paul Robbins and Michael Botchan 521

Preliminary Analyses of the Glucocorticoid Receptor Gene and its Expression: A DNA-Binding Protein Essential for Hormone-Dependent Transcriptional Enhancement

Roger Miesfeld, Sandro Rusconi, Sam Okret, Ann-Charlotte Wikström, Jan-Äke Gustafsson, and Keith R. Yamamoto 535

Workshop Summary: Eukaryotic Promoters and Enhancers Karen U . Sprague 547

I X . C O M P L E X R E G U L A T O R Y L O O P S

Site Directed In Vitro Mutagenesis in the Study of the Molecular Mechanism of Attentuation in the Threonine Operon of Escherichia coli

A l i Roghani, Richard I . Gumport, and Jeffrey F. Gardner 553

Contents xüi

Post-Transcriptional Enhancement of Bacterial Gene Expression by a Retroregulatory Element

Hing C. Wong and Shing Chang 565 Translational Control of the Positive Regulator of Amino Acid Biosynthetic Genes in Yeast

Alan G. Hinnebusch 585 Regulation of the Phosphatase Multi-Gene Family in S. cerevisiae

A. Tait-Kamradt, J. Lemire, R. Koren, and K . A . Bostian 595 RNA Metabolism During Sporulation

Stephen Kurtz, Edys Gordon, and Susan Lindquist 611 Regulation of Mitochondrial Gene Expression in Trypanosoma brucei

K. Stuart, J.E. Feagin, and D.P. Jasmer 621 Coupled Circuits of Gene Regulation

Michael A . Savageau 633

X . T H E H E A T S H O C K R E S P O N S E

Induction of the Bacterial Heat-Shock Response Frederick C. Neidhardt, Philip M . Kelley, and Ruth A . VanBogelen 645

Expression of the 5. cerevisiae Hsp70 Multigene Family Elizabeth A. Craig, Michael R. Slater, Will iam R. Boorstein and Karen Palter 659

Translational Regulation in the Drosophila Heat Shock Response Eileen R. Sirkin and Susan Lindquist 669

A Nucleolar Function for Mammalian and Drosophila 70kD Heat Shock Proteins

Hugh Pelham, Michael Lewis, and Sean Munro 681 Index 689

Sequence Specificity in Transcription and Translation, pages 511-520 © 1985 Alan R. Liss, Inc.

THE STRONG ENHANCER ELEMENT IN THE IMMEDIATE EARLY REGION OF THE HUMAN CYTOMEGALOVIRUS

GENOME1

Michael B o s h a r t 2 , Frank Weber^, Rüdiger Rüger 2, Ka r o l i n e Dorsch-Häsler-^, Gerhard Jahn 2, Jay S t o e r k e r 2 ,

Walter Schaffner^, and Bernhard F l e c k e n s t e i n 2

I n s t i t u t für K l i n i s c h e V i r o l o g i e der Universität Erlangen-Nürnberg, D-8520 Erlangen;

^ I n s t i t u t für Mol e k u l a r b i o l o g i e I I der Universität, CH-8093 Zürich

ABSTRACT The human cytomegalovirus (HCMV), a member of the herpesvirus group, was found t o possess a strong t r a n s c r i p t i o n enhancer i n the immediate e a r l y gene region. C o - t r a n s f e c t i o n of enhancerless SV40 DNA w i t h randomly fragmen­ted HCMV DNA y i e l d e d two SV40-like recombinant v i r u s e s , each c o n t a i n i n g HCMV DNA fragments t h a t were s u b s t i t u t i n g f o r the missing SV40 enhancer. The two i n s e r t s , 341 and 262 bp i n l e n g t h , are overlapping segments of genuine v i r a l DNA r e ­presenting p a r t of the 5'f l a n k i n g region of the major immedistte e a r l y gene i n HCMV. Studies w i t h d e l e t i o n mutants showed t h a t d i f f e r e n t non-overlapping subsets of the HCMV enhancer region can s u b s t i t u t e f o r the 72 bp repeats of SV40. Transient expression assays i n d i c a t e d t h a t the HCMV enhancer i s s i g n i f i c a n t l y stronger than the SV40 element, a c t i v a t i n g c i s - l i n k e d heterologous promoters i n a wide spectrum of c u l t u r e d c e l l s . I t appears t h a t the HCMV enhancer i s p o s i t i v e l y r e g u l a t e d by v i r a l immediate e a r l y genes.

This work was supported by Deutsche Forschungsgemeinschaft, Schwerpunkt V i r u s p e r s i s t e n z , the Swiss Nat i o n a l Research Foundation, and the Kanton Zürich.

512 Boshart et al

INTRODUCTION

Human cytomegalovirus (HCMV), one of the four herpes­vi r u s e s of man, i s a pathogenic agent of widespread occur­rence. The v i r u s has c l i n i c a l importance i n p r e n a t a l i n ­f e c t i o n s , and i t causes severe pneumonitis and septicemia i n immunocompromised p a t i e n t s . The genome of HCMV i s a l i n e a r double-stranded DNA molecule o f about 235 kbp. The v i r i o n DNA co n s i s t s of a long (U ) and a sh o r t (U ) unique r e g i o n , each fl a n k e d by a p a i r of i n v e r t e d repeats ( 1 , 2 ) . Both regions, U g and U , may be o r i e n t e d i n e i t h e r d i r e c ­t i o n , r e s u l t i n g i n four isomeric forms of the v i r a l genome (3 ) . The v i r a l genes are t r a n s c r i b e d i n three phases. The pr o t e i n s of the immediate e a r l y (IE) phase seem t o have r e ­g u l a t o r y f u n c t i o n s and t o be necessary f o r i n i t i a t i o n of the second ( e a r l y ) phase o f t r a n s c r i p t i o n . This phase i s follo w e d by DNA r e p l i c a t i o n and synthesis of l a t e gene pro­ducts. Four IE genes are l o c a l i z e d i n the H i n d l l l E-frag-ment of the HCMV s t r a i n AD169 ( 4 ) . A polyadenylated 1.9 kb-t r a n s c r i p t enconding a 72 Kd phophoprotein represents the most abundant IE RNA of HCMV ( 5 ) . T r a n s c r i p t i o n of t h i s gene i s blocked during the l a t e r phases of v i r u s r e p l i c a ­t i o n . The s t r e g t h of the major IE gene promoter seemed us t o p o i n t t o a pos s i b l e r o l e o f an enhancer-like element i n the c o n t r o l of t h i s t r a n s c r i p t i o n u n i t .

A number of heterologous enhancers can s u b s t i t u t e f o r the enhancer of SV40 or Polyomavirus, r e s u l t i n g i n v i r a l DNA r e p l i c a t i o n and Τ antigen expression (6, 7, 8, 9, 10). This allowed t o d e t e c t enhancer elements i n large genomes by an "enhancer t r a p " assay, i f s h o r t randomly fragmented heterologous DNA was cotr a n s f e c t e d w i t h l i n e a r i z e d SV40 DNA la c k i n g i t s own enhancer. I n c o r p o r a t i o n of f o r e i g n DNA w i t h enhancer f u n c t i o n l e d t o l y t i c a l l y growing SV40-type recom­binants (11). Applying t h i s method, we i d e n t i f i e d an en­hancer sequence i n the IE region of HCMV which i s unusual i n s t r e g t h and f u n c t i o n a l o r g a n i s a t i o n .

RESULTS

1. I d e n t i f i c a t i o n of HCMV sequences w i t h enhancer a c t i v i t y

The enhancerless l i n e a r SV40 genome l a c k i n g the e n t i r e 72 bp repeat r e g i o n was excised from the vector sequence of plasmid pET-1 (11).

E T

HCMV AD 169

I I ΙΓ τττί~3 pJN 201

.Pstl-m, PCM5027 1kb

0,075 0100 0125 0,150 map units

1.9 kb 2.3kb 2.2kb 5 k b IE RNA

er ο Uü

Ol x Ε a CG

Ο x et Ε ° 1— ι \

et ο ο CD UÜ

I — I I — H I — I \ = \ »—«

et x - ^ ™ σ

ο OD χ ο

χ LUX χ Ε ο CD

ο -Ω χ

FIGURE 1. The 235 kbp genome of human cytomegalovirus s t r a i n AD169. The i n v e r t e d repeats f l a n k i n g the long (U L) and the short (U s) unique region o f the genome are boxed. H i n d i I I cleavage s i t e s are i n d i c a t e d by v e r t i c a l l i n e s . The H i n d i I I Ε-fragment spanning most of the immediate e a r l y (IE) region i s magnified t o demonstrate the map p o s i t i o n of the P s t I m-fragment, and the EcoRI s i t e s w i t h i n the IE region are i n d i c a t e d . The o r i e n ­t a t i o n s of the major IE t r a n s c r i p t s are i n d i c a t e d by arrows. The f i r s t i n t r o n i n the 1.9 kb-gene i s i n d i c a t e d .

514 Boshart et al

Cotransfections o f t h i s enhancer t r a p w i t h randomly sheared (- 300 bp) DNA fragments of HCMV s t r a i n AD169 showed, t h a t sequences from the promoter region of the do­minant 1.9 kb-IE mRNA can r e s t o r e SV40 growth (Fig. 1 ). Two r e p l i c a t i o n competent SV40-HCMV recombinants, designa­ted C2 and C4, were i s o l a t e d and analyzed by DNA sequen­ci n g . The sizes of the i n t e g r a t e d HCMV segments are 341 and 262 bp i n recombinant C2 and C4, r e s p e c t i v e l y . These sequences overlap by 196 bp. Both regions are i d e n t i c a l w i t h the genomic HCMV v i r i o n sequence. Concluding from s t r u c t u r a l s i m i l a r i t i e s between HCMV s t r a i n AD169 and Towne (12), the i n s e r t e d sequence of C2 was found t o be located between -118 and -458 upstream of the cap s i t e f o r the dominant 1.9 kb IE mRNA, and the segment contained i n recombinant C4 represented nucleotides -263 t o -524. Four repeated elements of 17 bp, 18 bp, 19 bp, and 21 bp length are found w i t h i n the HCMV-enhancer region. The repeats are imperfect i n p a r t and are separated from each other by non-r e p e t i t i v e DNA (Fig . 2 ) . The 18 bp sequence i s repeated four times and shows an 11 bp homology w i t h a core con­sensus sequence i n the 72 bp repeats of SV40 (13). The 19 bp-sequence (repeated f i v e times) froms p e r f e c t p a l i n -droms. The 21 bp repeats contain the hexanucleotide CCGCCC or the r e l a t e d m o t i f CCCGCC reminiscent of the 21 bp r e ­peats i n the e a r l y SV40 promoter and of herpes simplex v i r u s immediate e a r l y and e a r l y upstream regions (14, 15, 16). The recombinant v i r u s e s grew about as w e l l as w i l d type SV40 on CVl c e l l s . T-antigen expression a f t e r t r a n s -f e c t i o n i n t o various c e l l s such as Hela, human f i b r o ­b l a s t s , human hepatoma c e l l s , mouse f i b r o b l a s t s and f r o g kidney c e l l s i m p l i c a t e d a broad host range a t l e a s t as wide as i t i s c h a r a c t e r i s t i c f o r the SV40 enhancer.

2. S t i m u l a t i o n of t r a n s c r i p t i o n of a c i s - l i n k e d hetero­logous gene

HCMV enhancer sequences were excised from both r e ­combinant viruses and were i n s e r t e d i n e i t h e r o r i e n t a t i o n downstream of a cloned r a b b i t ß-globin gene. I n p a r a l l e l the P s t I m-fragment of HCMV genomic DNA (Fig . 1) was cloned downstream of the ß-globin-DNA. Enhancement of ß-globin t r a n s c r i p t i o n was q u a n t i t a t e d by SI nuclease analysis of cytoplasmic RNA a f t e r t r a n s i e n t expression i n Hela c e l l s . The HCMV sequences from the recombinents C2 and C4 enhanced the synthesis of c o r r e c t l y i n i t i a t e d ß-

C2

17 bp repeat 18bp repeat 21 bp repeat

19bp repeat 1.9 kb mRNA

CAP

t t Bat I Bai I

1 I

t t Aha II Aha II

Τ Aha II

l •

t C Aha II

•

τ t Sst I

1

s t Sphl

-700 -600 -500 -400 -300 -200 -100 • 100

FIGURE 2. Schematic p r e s e n t a t i o n of the enhancer region extending between nucleo­t i d e s -118 and -524. Brackets i n d i c a t e the enhancer region as defined by the recombinant viru s e s C2 and C4. The CAAT-(C) and TATA-(T) consensus sequences, f i r s t s p l i c e donor s i g n a l (S) and l o c a t i o n of the capping s i t e (CAP) f o r the 1.9 kb IE-mRNA are i n d i c a t e d . The elements of the four classes of imperfect d i r e c t repeats are represented by arrows. The domains of the enhancer r e g i o n , defined by AhaII s i t e s w i t h i n the palindromic 19 bp repeat, are designated A, B, C, D, E, and F ( t a b l e 1).

516 Boshart et al

globin transcripts by at l e a s t two orders of magnitude i r ­respective of orientation. While the C2 sequence was about as active as the SV40 enhancer, the enhancer i n recombinant C4 was three to five times stronger. Insertion of the HCMV Pst I m-fragment, containing the complete enhancer promoter/ region and the tr a n s c r i p t i o n a l s t a r t s i t e of the 1.9 kb IE-mRNA, showed strong polar e f f e c t s . In a construct, where the directions of transcription of the 1.9 kb IE-mRNA and the ß-globin mRNA are opposite to each other and the en­hancer i s separated from the ß-globin gene by plasmid DNA on one side and by the HCMV IE promotor on the other side almost no enhancing a c t i v i t y was detected. Transfection into Hela c e l l s of a construct where the direction of RNA synthesis from the I E promoter of HCMV and from the ß-globin gene are i d e n t i c a l , revealed synthesis of correctly i n i t i a t e d ß-globin t r a n s c r i p t s i n simi l a r amounts as stimulated by the SV40 enhancer. The Si nuclease analysis revealed an addtitional strong band of a shortened protec­ted fragment (306 nucleotides); t h i s i s probably formed by aberrant s p l i c i n g to an acceptor sequence at position +48 of the rabbit ß-globin gene ( 17 ). Transcription seems to be i n i t i a t e d from the strong I E promoter of HCMV, and the IE-RNA leader i s spliced to the ß-globin sequences. Co-transfection of in t a c t HCMV IE-genes (HindIII-E and EcoRIJ, see Fig. 1) with ß-globin/HCMV enhancer fusion Plasmids resulted i n s i g n i f i c a n t l y increased amount of correctly i n i t i a t e d ß-globin t r a n s c r i p t s . I t indicates that the HCMV enhancer i s stimulated by gene product(s) that i s (are) encoded in the 1.9 and 2.3 kb IE-mRNA.

3. Deletion mutagenesis i n the enhancer sequence

The r e s t r i c t i o n enzyme Aha I I cuts within the palindromic 19 bp repeat sequences, defining s i x struc­t u r a l domains i n the HCMV-enhancer sequence (Fig. 2; Table 1 ) . Deletion mutants of the SV40-HCMV recombinants were constructed with p a r t i a l AhaII digestion products of recombinants C2 and C4. Transfection into CVl c e l l s re­vealed that smaller deletions had l i t t l e or no e f f e c t on v i r a l r e p l i c a t i o n and T-antigen expression. Large deletions reduced T-antigen expression and resulted i n slow virus growth. However, remarkably, each single de­leti o n mutant was viable, thus retaining a t l e a s t the mini­mum enhancer a c t i v i t y allowing r e p l i c a t i o n of v i r a l DNA.

Human Cytomegalovirus Enhancer 517

TABLE 1 COMPOSITION OF SV40-HCMV RECOMBINANTS WITH

DELETIONS IN THE HCMV-ENHANCER REGION

Domains of the enhancer region A Β C D Ε F 64 53 83 62 124 21 (bp)

Recombinant v i r u s C2 - + + + + II 11 C4 + + + - -D e l e t i o n mutant #1 - + + + - -

II II 2 + - + + - -II II 3 + + - + - -II II 4 + + - - + It II 5 + - - - -II II 6 -J- + - - - + ii ii 1 - + - + - -II II 8 - + + - - + II II 9 + - - - - + II II 10 - + - - -

DISCUSSION

The SV40 enhancer t r a p system allowed t o i d e n t i f y a f u n c t i o n a l enhancer region upstream of the major immediate e a r l y gene of HCMV s t r a i n AD169. The region extends bet­ween nucleotides -118 and -524 upstream of the capping s i t e . The enhancer stimulates c o r r e c t l y i n i t i a t e d t r a n s ­c r i p t i o n , i f i t i s i n s e r t e d downstream of α cloned r a b b i t ß-globin gene i n e i t h e r o r i e n t a t i o n . The HCMV-enhancer r e ­gion has a host range at l e a s t as wide as the SV40 enhan­cer. Deletion mutants containing non-overlapping subsets of the enhancer can s t i l l s u b s t i t u t e f o r the SV40 72 bp repeats.

The herpesvirus group i s h i g h l y heterogenous i n the f u n c t i o n a l genome or g a n i s a t i o n . Some herpesviruses such as Herpesvirus s a i m i r i or Pseudorabies v i r u s have a s i n g l e immediate e a r l y region (18, 19). Other herpesviruses have several I E - t r a n s c r i p t i o n u n i t s , f o r example herpes simplex v i r u s (HSV), which contains f i v e IE-genes. These IE-genes of HSV are stimulated in_ trans by a s t r u c t u r a l component of the v i r u s p a r t i c l e . The re g u l a t i o n i s mediated by f a r upstream regions, and i t i s suggested t h a t these regions

518 Boshart et al

share a consensus m o t i f TAATGARATNC which plays an impor­t a n t r o l e f o r t r a n s - a c t i v a t i o n (15, 20, 21, 22, 23, 24). The sequence mediating the t r a n s - a c t i v a t i o n f o r the HSV IE-mRNA 3 has weak enhancer-like e f f e c t s , i f fused w i t h the HSV thymidine kinase gene (25, 26). Sequence compa­r i s o n of the upstream regions of the f i v e HSV IE genes w i t h the HCMV-enhancer region d i d not reveal homologies ^ 70% t o any of the repeated m o t i f s i n the HCMV-enhancer, and the TAATGARATNC m o t i f was not found i n the HCMV se­quence. The sequence d i s s i m i l a r i t i e s appear t o r e f l e c t d i f f e r e n t mechanisms i n the r e g u l a t i o n of I E - t r a n s c r i p ­t i o n i n HSV and HCMV, c o r r e l a t i n g w i t h the known funda­mental d i f f e r e n c e s i n the b i o l o g y of those herpesviruses.

I t has been shown t h a t d u p l i c a t i o n s can increase the a c t i v i t y of enhancer elements (11, 27). The HCMV enhancer region contains four groups of d i r e c t l y repeated sequences. The observation t h a t d i f f e r e n t subsets of the HCMV enhan­cer s u b s t i t u t e f o r the SV40 72 bp repeats may suggest t h a t the strong enhancer sequence of HCMV has ari s e n through a m p l i f i c a t i o n and d i v e r s i f i c a t i o n of an o r i g i n a l element. This may explain why the HCMV enhancer sequence i s r e l a ­t i v e l y long and one of the strongest of these elements known so f a r .

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