Genetic fusions for operon analysis.

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Annu. Rev. Genet. 1978.12:193-221. Downloaded from www.annualreviews.org by New York University - Bobst Library on 02/01/15. For personal use only.

Ann. Rev. Genet. 1978. 12:193-221 Copyright © 1978 by Annual Reviews Inc. All rights reserved

GENETIC FUSIONS

+3138

FOR OPERON ANALYSISl Naomi C. Franklin Department of Biological Sciences, Stanford University,

Stanford, California 94305

CONTENTS INTRODUCTION ....................................................................................................... . FASHIONING FUSIONS ............................................................................................ Deletions .................................................................................................................... Transducing Phages ..................................................................................................

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I Definitions and Abbreviations: Operon

=

194 195 197 198 199

200

Unit of transcription; including at least a promoter

and one structural gene, but possibly several structural genes whose products may have related

functions. Operons are designated in E. coli by three-letter abbreviations, e.g. ara. gal. lac. and

a

m l. for arabinose, galactose, lactose, and maltose utilization, respectively; argo bio. his. trp

for arginine. biotin, histidine, and tryptophan biosynthesis. respectively; rpo for RNA polyme rase. Structural gene

=

Gene coding for an RNA or protein product. Generally trans -acting

and called cistron for its ability to complement, but in special cases cis -specific. Genes are

designated by an uppercase letter, as Z in lacZ, their products by gpo as in gplacZ. Regulatory site

=

Site at which operon expression is regulated; cis-dominant; designated here by a

lowercase letter preceding the operon name, as p-Iac. o-lac. The following are known classes of regulatory sites: Promoter (P) = Site of transcription initiation, also site of RNA polymerase binding, and possibly also site of initial recognition between DNA and polymerase, prior to binding. The promoter is also considered to include sites of positive regulation. i.e. binding regions for activator proteins such as the catabolite activator protein (CAP) or gpara c. Operator (0)

=

Site of negative regulation of the promoter where specific protein repressors

bind. The operator may overlap with the promoter, as in trp. or lie adjacent to it, as in lac (Figure 1). Terminator (t) = Site of transcription termination. at operon ends. and sometimes

within operons. When used to regulate biosynthetic operons such as trp and his. t is called

an attenuator (am). Leader (I) = Stretch of transcribed DNA between the promoter and the first structural gene. = Sit e in bacteriophage A for the recognition of gpN in its role as antiterminator .

N-utilization site (nut)

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FRANKLIN


FUSIONS IN ANALYSIS AND MANIPULATION OF OPERONS .... . ... . ........... Operon Units Defined ................................................................................................ Promoter and Operator Sites Defined .. .................................................................... Regu lators of the Regulator Genes .......................................................................... Transcription Termination: Sites and Means .... .................................. ....................

Termination and antitermination in A and in transcriptional polarity .......................... Regulation by termination in the trp operon .... ... ........... ........... '" .............................. Termination at operon ends ........ ....................... .................... ...... ...... .. .... ................. Maximized Function of Structural Genes ... ... ...................... ... . .... ............................ Protein Fusions .......................................................................................................... Sequencing of RNA and DNA ................................ ........... .................................. .....

FUSIONS FOR THE STUDY OF GENE REGULATION IN EUCARYOTES .. SUMMARY ....................................................................................................................

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20 1 202 204

206 206

209

210 2 10 211

2 12

2 13 2 14

INTRODUCTION The operon of procaryotes is of interest as a unit of regulation of gene expression. Originally the operon was recognized by the coordinate expres sion observed for certain sets of genes, generally closely linked (1). Study of such coordinately expressed genes has allowed formulation of regulatory mechanisms, dependent as a rule on genetic determinants outside the "structural" genes whose products are observed. Coordinate expression was critical to the analysis of regulation at the level of transcription, since coregulated genes were found to be cotranscribed from the same origin to give polycistronic messages (2-6). Insofar as single genes must also be transcribed from a defined origin and in regulated fashion, they too consti tute operons. The operon thus comes to be defined as a unit of transcription, subject to regulated expression. Each is composed of one or more cistrons of DNA coding for RNA or protein products, the "structural genes," plus elements of DNA that serve to regulate the functioning of the structural genes. This generalization can be applied to eucaryote DNA, as well as to procaryote, even though coordinate expression of eucaryote genes has been observed only rarely. The opportunities for genetic regulation, all coded in the DNA itself, are proving far more varied and subtle than was once imagined. Transcription, initiated at specific promoter sites and terminated also at specific sites by RNA polymerase, can be regulated at all stages: by RNA polymerase itself, by protein molecules able to modify polymerase or compete with it for sites of recognition, by DNA sequence at sites of recognition or at sites of actual effect, by proteins able to bind and mOdify these sites, and by the structure of the RNA transcript, its susceptibility to nucleases more or less specific and its ability to initiate and to terminate translation. The translation process involves many more components and therefore also many possibili ties for influencing gene expression. Probably the regulatory interactions observed so far are only the grossest.


GENETIC FUSIONS FOR OPERON ANALYSIS

195

New techniques will contribute greatly to the quest of understanding genetic regulation. The new capability for exact sequencing of DNA (7-9) allows genetic features to be scrutinized to the last intimate detail. The newly recognized range of specificities of "restriction" endonucleases (10 ) allows splicing of DNA in any desired union (subject to the rationality of NIH guidelines). Such splicings will augment the well developed in vivo methods for rearranging DNA considered below. In addition, the cloning of spliced or fused DNA into multicopy plasmids provides enrichment of functions as well as of DNA (11, 12). As these enlarged possibilities come into play, it is well to review the kinds of information bearing on gene function and its regulation that have proved accessible as a consequence of operon fusions. Operon fusions are a convenient contrivance, joining the regulatory elements of one operon to the structural genes of another. We consider examples of the various ways in which they have contributed both to the knowledge of gene regulators, when substituted structural genes provide ease of assay or means of selec tion, and to the knowledge of structural genes, when substituted regulators allow them to function under abnormal conditions. Examples are taken primarily from E. coli, its phages and transposons. Gene designations follow Bachman et al (13). Genetic maps are given in Figure I; abbreviations are explained in Footnote I, below. A contemporary review of the contributions made by fusions involving the lac operon is available for further developments there (13a).

FASHIONING FUSIONS DNA fusion is a consequence of "illegitimate recombination" events (141 6), that is, joinings between DNA segments not previously associated and with no appreciable nucleotide sequency homology (IS). In E. coli, such events (deletions, insertions, inversions, duplications, etc) are haphazard and not expedited by the recombination (rec) functions that facilitate recombination between homologues. Inefficient rec-promoted recombina tion between barely homologous regions may also create fusions. Since fusions are observed only rarely (10-5 to 10-10 per bacterial cell), their discovery depends upon selective methods. The frequency of fusions is increased to 10-3 to 10-6 per bacterial cell when brought about by transpo sons, that is, units of DNA such as temperate bacteriophages, F episomes, drug resistance episomes, and insertion elements, all of which can integrate themselves more or less frequently into a host chromosome at locations ranging from specific to random ( 17). Current interpretation of transposons leads to the expectation that their insertions may depend on special site specific recombination functions coded by the transposons themselves, -

196

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arginine

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,E , PP,C

a)

B

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bfe

H,

"

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tryptophan = , C ,

o

..... P,', E , 0

b)

80 and colicins V and B, can be selected against by exposing bacteria to T h c/>80, colicins, or a mixture of these (42, 43). Since tonB lies adjacent to the tryptophan (trp) operon, selection against it has been used to generate deletions that terminate throughout the trp operon (44) or that fuse trp to genes of a prophage integrated at the adjacent attachment site specific for phage c/>SO (att80) (45). If the prophage has been constructed to include bacterial operons, fusion of trp to those operons is also possible by tonB deletion (46, 47). Other transpositions (below) of genes to the vicinity of tonB make those genes also subject to fusion with the trp operon by deletion of the intervening tonB gene. An other convenient selection for deletions uses a temperature-inducible pro...

fusing gal to

bio to be found. (d) Fusion between the ora operon (1.3 min) and the transposed lac operon. Selection against an intervening Mu prophage (not shown) allows deletions fusing lac to araC to be found. (e) Major regulatory regions of bacteriophage A, with various fusions of E. coli trp to the A N operon indicated by a set of DNA lines below the A genome. Lambda structural genes are on the DNA line, and regulatory loci are above the line, except for

oLpL and oRpR, the major leftward (L) and rightward (R) operator-promoter regions. (I) Enlargement of the regulatory region at the start of the trp operon, showing overlapping promoter and operator regions, the leader region and the tip of the first structural gene

trpE. The leader is the transcribed region preceding trpE, whose initial part is probably translated into a labile polypeptide (see text), and whose distal part includes a transcription

termination site or attenuator (atn ) preceded by two G· C-rich regions of overlapping symme

try, indicated by the dotted lines.


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phage as antagonist. A A prophage with a mutation, c Its, making its immunity function heat-labile (48), is lethal to its host upon heat induction. When A is located at its attachment site of preference between the galactose (gal) and biotin (bio) operons, deletions of A that fuse gal to bio have been found among survivors of heat induction (49). This selection for deletions is enhanced by using a A mutant unable to excise itself from the bacterial chromosome, so that prophage removal depends entirely upon deletion events. The original opus of operon fusion (50) made use of the trans dominant lactose operon (lac) repressor mutation is, a mutation coding for a super-repressor rendering the lac operon noninducible and thereby non functional. When is is present in diploid, only mutations or deletions of the lac operator (o-lac) can restore lac function. Thus selection for lac function allowed recovery of a set of deletions covering o-lac and extending into lac Z on the one hand and into adjacent operons on the other hand; when the deletion extended into the purine E operon (purE), lacY function was found to be repressed by adenine, showing dependence of lacY on the operator-promoter for purE. Since lac and purE are separated by genes essential to E. coli, this fusion could be obtained only in a strain where an F' episome provided diploidy for that region of the chromosome, as well as for is. Analogous selections to overcome cis-dominant promoter or polar mutations can also result in deletion of the mutation with possible fusion to adjacent genes or operons (51-55, 169). Transducing Phages

The ability of temperate phages such as A to generate specialized transduc ing derivatives is an important tool in operon analysis, because it enables limited stretches of DNA to be extracted from a bacterial chromosome and perpetuated in a replicon with less than 1% as much DNA. Lambda integrates its circularized DNA into the E. coli host chromosome by a reciprocal exchange at a specific attachment site (att) with an element common to host and phage, using a phage-coded site-specific "integration" enzyme (14, 19, 56-58a). Prophage excision is normally the efficient reversal of this process, but exceptional excisions bring different lengths of bacterial DNA adjacent to the prophage into the phage genome as permanent, cova lently linked parts of the phage chromosome. Each such exceptional phage carries with it a particular block of bacterial DNA which it introduces into subsequent hosts, a process called high frequency transduction. Since pack aging of A DNA depends on cuts at specific A DNA ends (59, 60, 60a), packaging of adjacent bacterial DNA requires that these ends be intact, and that the DNA total fall between 72% and 107% of a normal A genome to make a stable package able to inject its DNA. A ·substantial range of bacterial DNA can be represented among A particles abnormally excised,



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since up to 30% of A DNA can be substituted without affecting vegetative phage development, and since even defective particles with about 95% substitution of A (61, 62) can be propagated in the presence of helper phage. Thus pieces of bacterial DNA up to about the length of A (49.6 kilobases, or kb), can be incorporated into a reproducible phage particle if only the specific phage ends are retained. This potential of phage for packaging reproducible lengths of host DNA has been expanded to genes not normally adjacent to A: (a) by finding alternative attachment sites for A when its preferred att site is deleted (63-65a); (b) by using temperate phages related to A in life-style, but having different specific att sites (66-67a); (c) by transposing bacterial DNA into range of such prophages at their different sites of integration (see below) (43); (d) by transposing A to different bac terial locations using homology of already incorporated bacterial genes (68); and (e) by the use of A with an insertion of a piece of the promiscuous bacteriophage Mu (see below) (55). The possibilities are even further ex panded by in vitro splicing of E. coli (or other) DNA into A DNA (34-37, 69,70) followed by transfection of the DNA into new host cells (71) or in vitro packaging of the DNA into complete phage particles able to infect normally (60a, 72, 73). In this way virtually all of E. coli DNA becomes subject to division into units of 1 to 50 genes, reproducible as part of a temperate phage genome, therefore available in quantity, readily purifiable, and able to reinfect new hosts and reintegrate into the bacterial chromo some, using either the att-specific functions of the phage or the DNA homology provided by the bacterial DNA. This ability to fuse convenient units of E. coli DNA to temperate phage genomes is not greatly different from the use of bacterial plasmids as cloning vehicles (30, 32, 33, 74). For study of operon fusion the A vehicle has the advantage of offering possibili ties of fusion to well-characterized A operons, of easy transfer between different cell lines, and of reinsertion into the bacterial chromosome. Operon Transpositions

Various tricks have been devised to transpose bacterial operons, placing them adjacent to other operons, to sites of prophage attachment or to convenient deletion targets such as tonB. A major instrument of such in vivo transpositions is the mutant episome Fts-lac, whose replication is temperature sensitive as a consequence of mutation within the episome itself. Unable to maintain itself in E. coli at high temperature, Fts-lac occasionally becomes integrated into a lac-deleted bacterial chromosome, resulting in stable lac+ colonies, selectable at 41°C (75). In the absence of lac homology, the integration events are more or less random, depending at least in part, as we now know, upon transposon sequences common to F-lac and to the bacterial chromosome (76). For example, integration of


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F-Iac on either side of att80 made possible the generation of two classes of c/>80 lac transducing phages, each transcribing lac from opposite DNA strands (77). The temperature-sensitive quality of Fts-lac can be recombined into other F' episomes, such as F-ara (carrying genes for arabinose utiliza tion), allowing alternative sets of host genes to be transposed when F is forced into host dependency at high temperatures (78). Recombination of Fts can be facilitated by translocatable drug-resistance elements (29). A selection similar to that using Fts forces fusion between two incompatible episomes in a recA-host, selecting an essential function of each episome. When operons of the first episome are joined with temperate phage attach ment sites on the second, it becomes possible to generate new transducing phage combinations (79, 80).

Phage Mu Insertions

The art of in vivo fusions reaches its zenith in the constructions of Casada ban (47, 81), combining fusion elements generated through the years in the Beckwith laboratory. In addition to many of the above contrivances, these constructions invoke phage Mu to generate homology within operons slated for fusion (82). Mu is a temperate phage of E coli able to insert itself into the bacterial chromosome at virtually any location. Insertions within struc tural genes negate the functions not only of those genes but also of down stream genes in the same operon, because of Mu-generated polarity (20, 83, 84). The objective, for example, of fusing structural genes of thelac operon to regulatory operator-promoter sites of the ara operon (which directs catabolism of arabinose) was achieved initially by the following steps (47): 1. Strain A was derived from a strain of E coli with a deletion of the natural ara operon. Transducing phage c/>80 d a ra (78) established a trans position of ara by lysogenizing at att80, near trp and tonB, with ara in the same orientation as trp and downstream from it. Insertion of Mu into araB, first structural gene of the ara operon, then negated all ara func tions. 2. Strain B was derived from a strain of E. coli with a deletion of the naturallac operon. Transducing phage c/>80 dlac (46) established a trans position oflac by lysogenizing at att80, withlac in the same orientation as trp. Deletion of tonB together with p-lac fusedlacZYA to the end of the trp operon, under regulation of p- tr p (54). Insertions of Mu into trpE, the first structural gene of trp, negated trp function directly, and also greatly reducedlac function as a consequence of polarity. 3. By making strain A into a male donor, it was then possible to recom bine strains A and B, selecting for Trp+ and screening for low levellacZ function. Such recombinants result from homology between the two Mu inserts, when these occur in the same orientation.


201

4. Finally, deletions of the recombinant Mu were found which fused

lac Z to p-ara. Such fusion strains make 500-fold more ,8 -galactosidase in

the presence of inducing arabinose than in its absence.

(81) makes use of A p( /ac Mu), a A derivative lacZYA fused to a piece of Mu, replacing the integration


A simpler construction that includes

,

function of A. This phage can be made to use its Mu DNA for integration,

by homologous recombination with a Mu prophage. Since the Mu prophage can be located in or near any gene chosen for study, the proximity of

lacZ to that gene can be brought about by A p(lac, Mu) integration.

Deletion of the Mu prophage with adjacent DNA can then effect fusion of

lac Z to the regulatory regions of any E. coli gene. Finally, this fused

segment can be immortalized on a A transducing phage, by abnormal excision of the adjacent A.

Although these schemes have broad potential, they are cumbersome to

achieve. For this reason they are likely to be superseded by in vitro splicing of DNA (11, 12). Splicings, however, generally make use of those endonu cleases with greater site specificity (sequences of 5 to 6 nucleotides), and therefore generate fusions between relatively large blocks of DNA (1 to 50 kb long). The fine tuning of in vivo fusions may be needed in conjunction with DNA splicing to achieve desirable variety. Or more sophisticated splicings (38, 85) may be made to serve.

FUSIONS IN ANALYSIS AND MANIPULATION OF OPERONS

Operon Units Defined

Operon fusion can contribute to the definition of unknown operons. Were a recognizable gene fused to a strong and inducible promoter, the new inducibility of that gene in coordination with contiguous genes would show how many of those genes belonged to the same transcriptional unit. The

,8

and

,8'

subunits of RNA polymerase, coded by contiguous genes and

fluctuating in parallel under different growth conditions, were shown defini tively to be products of the same operon when a deletion entering

rpoB rpoC (coding ,8') to the adjacent arginine operon, placing rpoC under regulation by arginine repression (Figure 1a) (86). Unexpected relationships were discovered among genes for ribosomal (coding

,8)

was found to fuse

proteins, for cofactors in protein synthesis, and for subunits of RNA poly merase by incorporating large blocks of genes into A transducing phages (62). In these studies operon groupings were actually determined by tran

script analysis and by polarity due to insertions, rather than by fusion to a A operon. (In fact, the antitermination feature of major A operons, dis

cussed below, would make such operon analysis unfeasible in the A context.)

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Promoter and Operator Sites Defined

Regulator sites in an operon can be defined by a set of deletions or fusions that replace overlapping segments of the operon. Such sets of fusions are generated by the random quality either of deletions or of specialized trans ducing phage formation: For this purpose, these in vivo processes may generate the desired random distributions more readily than do the more prescribed junctures made by in vitro splicing. Deletion sets can be used to order existing point mutations (44, 45, 86-93). When coupled with fusion to known genes or regulators, they allow, in addition, the mapping of regulator sites in the operon under study, as shown below. Fusions to selectable genes provide opportunities to press for genetically altered regula tors. Exemplary exploitation of fusions in operon analysis was made possible for the lac operon when it was transposed to a chromosomal site adjacent to the trp operon, and in the same orientation as trp, but separated from it by the tonB locus (77). Selection against tonB allows deletions to be found that sometimes fuse the lac operon to trp, in this case submitting lac to tryptophan repression (Figure 1 b). Deletions entering the trp operon can be found if tryptophan or indole is supplied (trpB alone suffices for the biosynthesis of tryptophan from indole); deletions that leave trp intact are selected in the absence of tryptophan (44). On the lac side, deletions that spare lacZ can be recognized by the ability of its product, ,a-galactosidase, to split chromogenic galactosides during colony growth (94). Pressure can be exerted on lacZ or Y for increased lac operon function, resulting in the selection of mutations that render the promoter stronger, less dependent on CAP, or less sensitive to lac I repressor or super-repressor. Selection against lac function is possible in galactose-sensitive bacteria or by use of a thi ogalactoside whose transport into cells by gplacY is lethal; reduced lac function could result from down-promoter mutations in the lacZYA promoter, up-promoter mutations in the lac I promoter, or changes in the lacI gene rendering its product super-repressing. Selection for lacY in the absence of lacZ, by transport of melibiose as sole carbon source, is also useful (94, 95). The structure of the lac operon was clarified by deletions fusing lac to trp regulatory regions. Deletions joining lac I to trp have no effect on lacZYA genes except to make their expression constitutive, showing that lacZYA has a promoter distinct from that governing lac I; regulation of lacI is considered further below. Deletions joining p-lac or a-lac to trp do not alter the structure of ,a-galactosidase, but put it under tryptophan repression. Fusions close to lacZ may damage its ribosome binding site. Fusions into lacZ eliminate ,a-galactosidase function and put lacY and



203

lacA under tryptophan regulation. These observations confirm the indepen dence of lacI from lacZYA; and show as well the sequence and indepen dence of lac promoter, operator, and ribosome binding sites, these located upstream from the structural genes of the operon (46, 54, 96). In tum, these relationships of lacI, p, 0, and lacZ have been corroborated at the nucleo tide sequence level (92, 97). Selection for Lac function in trp-lac fusion strains has been used to find mutations affecting trp regulation. In trp-lac fusions that place lacZYA under tryptophan repression, bacteria are Lac- in the presence of trypto phan. Selection for Lac+ under these conditions could be expected to yield mutations in the trp repression system. Mutations in trpR, coding for the repressor, predominated (98, 99); new promoters internal to trp and not repressible by tryptophan were also found (99). No mutations in o-trp were found, although other work has defined that element (100-102). A similar selection, although in another construction, used trpB for leverage on the integration ( int) gene of bacteriophage A, when int was made adjacent to trpB by a rare insertion of A into trpC (64). In this conjunction, trpB was found to function at a low level from a phage pro moter, pI, close to into Selection for increased trpB function yielded cis dominant mutations that increased int as well as trpB. Some of these mapping close to pI could be up-promoter mutations (or mutations inac tivating a terminator in the int transcript). Since these mutations inactivate the excision (xis) gene adjacent to int, it is possible that p I is located within xis, assuring that transcription from pI activates int but not xis, a distinc tion important for the integration of A (90). Workings of the biotin ( bio) operon have been clarified by fusions of p-bio A to galK, allowing more convenient assay of function, as well as selective approaches (Figure I c). The bio operon, like the arginine, arabinose, and maltose operons, shows a divergent structure: Two subunits are transcribed in opposite directions from a regulatory region with possibly overlapping functions (103). The promoter for bioA was studied by fusion to galK, under conditions such that the functioning of galK determined ability of the cells to use galactose for growth. Fusion strains are Gal- at high biotin concentrations because biotin represses function of p-bioA. Even at low biotin concentrations, fusion strains are effectively Gal- because p-bioA allows only one-twentieth the normal functioning of gal genes when ex pressed from derepressed p-gal. Selection of Gal+ at low biotin facilitated the discovery of up-promoter mutations allowing increased transcription of both bioA and galK; up-promoter mutants for bioA reduce bioBFCD function two to three fold, but do not alter the ability of biotin to repress transcription in both directions. Selection against galK function in mutants with the up-promoter for bioA results occasionally in mutations caused by


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insertions that inactivate bio transcription in both directions. Selection of Gal+ at high biotin concentrations in strains with the up-promoter for bioA produced some cis-dominant bio-linked mutations affecting o-bioA as well as o-bioBFCD: Transcription became constitutive for both bio sub units, showing loss of response to biotin but no loss of promoter capacity. . These findings demonstrate interactions between the regulators of leftward and rightward transcription in the bio operon (49). More recent fusions of p-bioA to laeZ test Lac- at high biotin concentra tions, apparently due to repression at o-bioA, but Lac+ at low biotin, despite the low efficiency of p-bioA. Selection for growth on lactose in the presence of high concentrations of biotin or biotin precursors produced a variety of mutations diminishing biotin repression, including loss of biotin transport or retention, reduced conversion of precursors to biotin, loss of repressor affinity at o-bioA, or loss of corepressor biosynthesis (104).

Regulators of the Regulator Genes

Regulation of the structural genes for regulator proteins presents a special problem, because regulator proteins cannot be easily assayed, and because they are usually small and fragile proteins, present in low amounts. Fusion of a regulator gene to laeZ, for example, allows ,B -galactosidase to stand in for the regulator protein, providing easy assay and the potential of selective lac technology (94, 95). This format has been used in the study of araC, the structural gene for both positive and negative regulators of the araBAD operon: gparaC by itself acts as repressor of araBAD, but its interaction with arabinose con verts it to an essential positive activator of araBAD (l 05). araC lies adja cent to araBAD, but is transcribed in the opposite direction from a common regulator region (Figure 1d) (106). Since the protein product of araC is unstable and hard to assay (107, lOS), fusion of laeZ to araC (SI) provided the means for study of araC regulation. Like araBAD, araC function was stimulated by the presence of catabolite activator protein (CAP) and repressible by gparaC itself (provided in trans by an episome); unlike araBAD, araC was not activated by a gparaC-arabinose complex (55). The genetic region between araBAD and araC has been probed by mutations and deletions, as well as by in vitro studies of RNA polymerase binding and transcription initiation. The I or Initiation region seems to be the promoter for araBAD, the site at which the gparaC-arabinose complex and the CAP-cAMP complex interact with DNA so as to engage RNA polymerase in transcription initiation; the 0 region behaves as traditional operator for araBAD in its interaction with gparaC as repressor (91, 105, 109, 110). The same 0 region may also serve as an operator for araC, since it, too, is repressed by gparaC (55). On the other hand, the stimulating



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effect on ara l of gparaC in conjunction with arabinose detracts from araC initiation (110) so that it cannot be said whether araC remains repressed in the presence of gparaC plus arabinose, or whether its function is reduced by competition with araBAD at the promoters. Either way, the effect is to reduce synthesis of gparaC, creating a kind of feedback to balance araC and araBAD function. Fusions of lac Z that eat deeper and deeper into araC leave lacZ under continued gparaC repression, showing that the site of that repression (or competition) precedes the gene, as would be expected (55). Analysis of transcription-starts both in vitro and in vivo places the araC promoter at 150 base-pairs from the araBAD promoter, establishing their separateness, but leaving at question the observation of their functional interaction (110). "Constitutive" araC mutations (araCC) are selected as able to activate araBAD in the absence of arabinose; that is, the conformation of gp araC becomes altered by mutation so that it binds to I even in the absence of arabinose (105). Does this indicate concomitant failure to bind at o-araBAD? Using araC fused to lac Z it could be shown that only 20% of araCC mutations dictate an araC protein no longer able to repress araC itself (55), possibly indicating the nonidentity of o-araC and o-BAD. It should be possible to select for mutations in p-araC or in o-araC , using the leverage of fused lac Z, in order to define better the rela tionships in the regulatory region common to araBAD and araC. The lac fusion should also yield information on the efficiency of p-araC relative to other promoters and to the level of gparaC in cells (55). An alternative analysis of the regulator of lac I (93) was made possible by the same system of fusions to trp that was used for lac operon analysis (54, 96). lac I, coding for the repressor of the lac operon, lies adjacent to the promoter for lac ZYA: both operons are transcribed in the same direc tion (Figure Ib) (98). When the lac operon was transposed to the region downstream from the trp operon, selection against tonB allowed deletions impairing lac I function to be found, since these cause lac ZYA to function constitutively. Deletions entering p-lac I but not lac I are distinguished by their ability to recombine with all point mutations in lac I, even those that alter as early as nucleotide No. 4 in the DNA sequence for lac I. Three fusion groups that disrupt lac I function but do not extend into that gene were distinguished on the basis of recombination with either of two mutant loci giving 1- phenotype and mapping just upstream from lac I. These mutations and deletions could define either the promoter of lac I or the ribosome binding site needed for translation (93). Putative up-promoter mutations in p- lac I (111) have not been compared. Most of the deletions impairing lac I function could be shown to put lacZ at least partially under control of ptrp, since strains with defective trp repressor (trpR-) gave more lac Z function than did trpR+ recombi-


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nants. Such fusion strains may make 150 times more gplacI than normal, showing the relative inefficiency of normal lac I expression. Despite such high levels of lac repressor, these fusion strains may show strong constitutive synthesis of gp plac Z, indicating either that the repressor made is defec tive or, more interestingly, that readthrough from ptrp can traverse lac I and continue into lac Z without encountering a transcription stop at the end of lac I (93). These possibilities should be distinguishable by a cis-trans test. If the latter is true, as had already been indicated (54), then basal constitu tive levels of ,B-galactosidase in lac I+ cells may reflect readthrough tran scription from p-lac I. There are at least 300-fold fewer molecules of lac repressor than of gp lacZ (,B-galactosidase) in wild-type cells derepressed for the lacZYA operon; it is not known whether this reflects a 300-fold less active promoter for lac I or whether other factors also affect lac I expression (112). But, since the basal constitutive level of gplacZ is about WOO-fold below its induced level, it is possible that readthrough from p-lac I contrib utes significantly to basal level function of lacZ. Such an effect has been found in the gal operon, where a weak alternative promoter, not CAP cAMP-activated, apparently accounts for basal expression of the gal genes (113). Transcription Termination: Sites and Means

The importance of transcription termination as a means of genetic regula tion is of more recent recognition than are operators and promoters (114116). Termination of nascent RNA chains occurs at discrete sites preordained by the DNA sequence: Most usually a poly U terminus is preceded by a GC-rich sequence that is part of an inverted repeat, showing that the secondary structure of RNA is critical to its own termination (117, 118). Perhaps this requirement for RNA secondary structure accounts for the general observation that transcription termination occurs only in re gions of the RNA not being translated: Ribosomes following polymerase would keep the transcript in an extended form. The ability to terminate resides in RNA polymerase itself (114, 117, 119-121). Still, in many cases termination depends, in addition, on rho, a protein factor with affinity for RNA and with ATPase activity (122). Tbe. have been exposed in four landmark situations; operon fusions have con tributed to the understanding of each of these. TERMINATION AND ANTlTERMINATlON IN A AND IN TRANSCRIP

In lambdoid bacteriophages, all major transcripts can be cut short by terminations at a series of specific sites: tLI> tL2' and tL3 in the leftward or N operon; tR] and tR2 in the rightward or cro operon; and tQ in the late operon (Figure 1 e). Two phage-coded proteins, gpN and gpo. serve as essential positive regulators of phage development TIONAL POLARITY

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207

based upon their ability to "antiterminate," that is, to prevent termination. Evidence on termination came originally from in vitro study of A mRNA: Extended transcripts were synthesized from pL and pR unless a protein factor, termed rho, was added, thereby curtailing the transcript from pL to a l2S piece and the transcript from pR to 7S. The 12S and 7S messages correlated with similar short messages found in vivo with N- mutants, so that N function was implicated in antitermination essential to A develop ment (114). The implied antitermination event has since been established by proof that the l2S RNA initiated at pL becomes extended (rather than reinitiated) beyond tL, in the presence of gpN (123). In Atrp transducing phages, genes of the trp operon are substituted for genes of the N operon, both units being transcribed in the same orientation from pL. Varying portions of the trp and N operons may be deleted by the in vivo fusion event. When the trp promoter is excluded, trp function depends entirely upon readthrough from pL, and is completely repressed by phage repressor acting at 0L. When trp is fused in or to the left of elII, as in Atrp44, trp expression is dependent on N+; trp genes fused closer to pL, as in Atrp48, function independently of gpN, though they may retain an intact N gene. Such fusions identify a target site for N function, tL" located just to the left of N (124, 125). Direct measurements of the lengths of transcripts from pL have now identified two other termination sites, tL2 and tL3' in series beyond tL, (126). Termination sites tR, (leaky) and tR2 in the ero operon were identified, respectively, on the basis of the 7S transcript length and on the basis of a small deletion, ninR5, which over comes the need for gpN in long-range rightward transcription (127). It seems that message termination provides a means for A to protect its host cell (and thus itself) from transient derepression of the quiescent prophage: Transcription initiation from pL and pR cannot be extended to functions lethal to the host unless derepression is sufficently sustained to permit gpN synthesis and thence complete A development. The role of gpN in antitermination is believed to be that of a modifier of RNA polymerase (128-130). The arguments, reviewed more extensively in Franklin & Yanofsky (119), are as follows:

1. The N function of related but heteroimmune phages cp80, 21, and P22 cannot help N- mutants of A; therefore gpN is not directly anti-rho. 2. The specificity of gpNA for its own immunity type has been shown to depend on N recognition sequences (nutL and nutR) which lie 50 to 64 and, tentatively, 244 to 258 nucleotides downstream from pL and pR, respectively (118, 131). 3.

Although gpN shows recognition specificity for these sites near its own pL promoter, or near pR, the effect of gpN is observed at target ter-

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mination signals that may be distant from those promoters. The effect of gpN is seen only at targets cis to the promoter regions recognized by


gpN (116, 128, 129). 4. There is evidence that gpN becomes physically associated with RNA polymerase in infected cells (130). The long-range effect of gpN was shown dramatically by 'A.trp phages in which trp genes fused to the N operon carried nonsense mutations known to be polar in the context of E. coli. It was found that readthrough of trp from pL in the presence of N+ eliminated all polar effects, although it did not correct the nonsense mutations (129). Similar observations were made by allowing prophage 'A. to be derepressed without excision, whereupon readthrough from pL into the adjacent bacterial gal operon overcomes polarity due to mutations in gal E (116). In the 'A.trp fusion phages it was possible to examine the transcripts of the trp genes carrying polar muta tions, as well as of adjacent genes, by hybridization to cf>80-trp phages each of which carried a particular segment of the trp operon. By varying the pulse length for 3H-uridine labeling of these transcripts, it was shown that polarity (seen from pL with N- or from p-trp) was associated with instabil ity of the transcript for the gene with the polar mutation and partial loss of downstream message. When polarity was overcome by transcription from pL with N+, the transcript for the gene with the polar mutation was normal in amount and stability, even though the gene's function was not restored. Since N-modified transcription was known to overcome transcrip tion termination, it could be inferred that polarity is a consequence of transcription termination occurring at DNA signals normally in translated regions, but blocked from translation in polarity situations by nonsense mutations that terminate nascent protein chains. The instability of the transcript for a gene carrying a polar mutation then becomes a conseq uence of transcription termination, occurring at sites near the ends of genes and exposed by blocks to translation (119). Fusions served this analysis by allowing particular genetic nonsense signals to be introduced into the N operon at points more or less distant from the pL promoter, showing that the effect of N is observable over a distance of several genes at least, although dependent on recognition near the transcript start point. A bonus was the insight gained into the nature of transcriptional polarity as a conse quence of fusing trp to a 'A. promotor subject to N modification. The role of transcription termination in polarity was surmised independently from evidence that polarity suppressor mutations are actually mutations in the structural gene for rho (132-136). The theme of regulation by antitermination in 'A. appears to extend to its late operon, transcribed rightwards from pR', just beyond gene Q (137). In



209

vitro the 6S transcript from pR' is severed, independently of rho, at tQ (138). Antitermination at tQ is effected by gpQ (137; J. Sklar and S. Weiss man, ms submitted). The synthesis of gpQ itself depends upon gpN for antitermination at tRt and especially at tR2 (139). Thus regulation by antitermination can also serve to establish a temporal sequence of events, desirable for efficient phage development. That gpQ plays an antitermination role analogous to gpN's has now been shown by the ability of Q -mediated transcription to overcome polarity in cis-located genes. When Aclts is integrated at its preferred chromosomal site, but in reverse orientation (34), transcription from pR reads through into the gal operon. At 42°C with a Q+ prophage, gal expression is greatest, since derepression activates N to activ�te Q. At 34°C, or at 42°C with a Q- prophage, gal expression is much reduced. Substitution of a polar mutation or insertion for galE+ allows it to be shown that Q+ readthrough at 42°C overcomes polarity. 6S termination does not require rho. Hence Q, like N, is acting not as anti- rho but as antiterminator (D. Forbes and I. Herskowitz, personal communication). That N-modified transcription from pR can read through tQ (139, 140), a rho-independent stop, is added evidence of N versatility. Whether Q function can equal N's performance against rho-dependent stops awaits further operon fusions. REGULATION BY TERMINATION IN THE TRP OPERON

The trp op eron (biosynthesis of tryptophan) is regulated both by negative control at an overlapping operator/promoter region (101) and by transcription termi nation at a site within the transcribed segment of the operon (mRNA leader) between the promoter and the first structural gene, trpE (Figure If) (141, 142). Termination is 80 to 90% efficient when tryptophan is abundant, but less than 10% efficient when tryptophan is scarce, befitting the cell's objective of economy in synthesis. Termination occurs at a specific site, 142 to 146 nuc1eotides into the leader and 20 nucleotides before trpE, a site marked by eight consecutive T-A base-pairs in the DNA and preceded by two overlapping regions of dyad symmetry that allow for alternative hairpin structures in either DNA or its RNA transcript (117). Regulation of this termination event by tryptophan is mediated by tRNA trp (143), probably, as it now seems, through regulation of leader translation. The beginning of the leader transcript is marked by a ribosome binding site at nuc1eotides 11 to 43, with an AUG start codon at position 27 to 29; an in-phase UGA nonsense codon at 69 to 71 sets off a length of RNA sufficient to code for a 14 -amino acid polypeptide. Although this polypeptide has never been found, either in vivo or in vitro, fusion of the trp leader upstream to lacI or to trpE proves the functionality of the leader ribosome binding site: Fusion proteins are found whose sequence shows initial amino acids


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coded by the trp leader fused to polypeptides of gplacI or of gptrpE (93, 144). Allowed, therefore, the conviction that the leader can be translated in vivo, aware that the leader peptide is unusually rich in codons for tryptophan, and recognizing that the translatable region of the leader di rectly precedes the region of overlapping symmetries, one seems justified in concluding that it is the extent of leader translation, governed by the avail ability of tryptophan or tryptophan-charged tRNA, that dictates secondary structure of the RNA transcript and thence the probability of transcription termination (117). Fusions of lac to trp provided yet another opportunity for analysis of operon regulation: termination of tran scription at operon ends. Whereas fusion of lacZ to trpA results in strong function of lac,!- under tryptophan regulation, fusion of lacZ downstream from trpA may allow no lacZ function if a terminator of the trp operon intervenes. In such fusion strains, selection for lacZ uncovered either of two interesting results: deletion of the intervening terminator, allowing its loca tion and thence its structure to be ascertained, or mutations in rho, showing the importance of rho to transcription termination at the end of the trp operon (14S). Furthermore, the trp-terminator-lac fusion, rendered Lac+ by a rho- mutation, could be subjected to search for Lac- derivatives: Among spontaneous mutations to rifR, known to occur in rpoB, 3% were found to restore Lac-. Such rifR mutants seem also to increase termination at the tryptophan attenuator as well as at A terminators. Since this mutant RNA polymerase behaves similarly with a variety of rho- mutants, and since :it renders a rhoambe; mutant viable in the absence of any suppressors, it seems to be a mutant form of RNA polymerase, capable of terminating transcription at any terminator whether or not rho is available; normal RNA polymerase requires rho at some terminators, not at others (121). TERMINATION AT OPERON ENDS

Maximized Function of Structural Genes

Where the product of a structural gene is made in quantities too low for biochemical investigation, it has proved feasible to augment gene function by increasing the frequency of transcription or translation. For lac I, struc tural gene for the repressor of the lac operon, presumed promoter muta tions increase lacI function 10- to lOO-fold (111). Using the leverage of fused structural genes, putative up-promoter mutations were found to boost bio A and A's int as discussed above (49, 90). Fusion was also used to boost lacI I SO-fold, when p-trp was substituted for p-I with deletion of the trp attenuator (93). Transfer of low-functioning genes to multicopy plasmids is another effective means for increasing their productivity.



21 1

The situation is more difficult for cI, structural gene for A's immunity substance (repressor of A's major operons), due to the complexities of cI regulation, notably repression of cI's nearby promoter, prm, by cI's own gene product (146-149). In order to substitute a strong and disinterested promoter, cI was fused to p-lac by deletion ( 150), or by ingenious splicing to two tandem p-/ac sequences, the whole cloned into a multicopy plasmid (85). Although those arrangements increased cI function significantly, the level of function was still well below maximum: lacZ positioned down stream from c I in fusion to the same promoters was expressed tenfold more strongly than c I, indicating that c I function was limited at a level beyond transcription. In fact, DNA- and amino acid sequencing had shown that the start codon for cI translation lies directly adjacent to the prm promoter, with no leader transcript available for ribosome binding (147, 148). By splicing c I close to the ribosome binding site as well as the promoter of lacZ, full expression of cI was attained, allowing gpcl to account for 2% of cell protein (85). This translational block was not encountered in the maximizing of lac I, either because that gene has its own efficient ribosome binding site ( 1 5 1), or because other possible interferences at the transla tional level (85) were lacking. The case of cI points up the possibility of gene regulation at the level of translation, a possibility that has scarcely been explored. There is clear evidence for translational differences between genes, for example in the late operon of A, where at least 1 5 structural genes located on a single polycis tronic message differ widely in the number of product molecules made. Since all of these late genes are translated from a single transcript with a unique start point and, since differences cannot be found in stability of the different segments of this transcript, it seems that translational controls must be at work (6). Yet, in a current review of interactions between ribosomes and mRNA ( 1 52), only two possible cases of mutationally altered ribosome binding sites are cited. Again the fusions of lacZ to trp regulatory genes may contribute, in that various fusions with end points between lacI and lacZ showed differences in lacZ function that could not be at tributed to causes other than efficiency of translation starts (96). Protein Fusions

All DNA fusions have the potential of fusing two protein products, pro vided that both components are translated, that they are separated by no translation-stop codons, and that they lie within the same reading frame following the fusion event. The joining of two proteins may serve to stabilize a labile partner, to coordinate biochemical reactions, or to direct the cellular location of a protein. Such developments may have evolutionary impor-


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tance (153). We have seen how fusions between the trp leader and the lacI or trpE allowed detection of a polypeptide coded by leader DNA, although the natural leader polypeptide is apparently too labile to be de tected either in vivo or during coupled in vitro synthesis (93, 144). Fusions between lacI and lacZ (found among LacZ+ "revertants" of lacZ extreme polar mutation early in the gene) may unite functional lac repressor protein to functional ,8-galactosidase if a deletion between them removes only the carboxy-terminus of gplacI and the amino-terminus of gplacZ (52). Such lac-repressor-,8-galactoside fusions have allowed detection of repressor ac tivity in the dimer state, as well as detection of a nonspecific DNA binding site at the amino-terminus of the repressor molecule (1 54). Similar fusions within the histidine operon unite gphisD with gphisG when His+ "revert ants" are selected from a frameshift mutation early in hisD. In this case the fused protein is inactive, but is cleaved in vivo to fragments able to associate into active dimers (53). Fusions between proteins of different operons have been generated for the purpose of understanding the cellular distributions of proteins: cytoplasmic, membrane-bound, or periplasmic ( 1 5 S). A series of fusions between malF of the maltose operon and lacZ had the effect of making gplacZ inducible by maltose. A particular class of such fusions was generated (as in the lacI-lacZ fusions above) by selecting for LacZ+ when an extreme-polar mutation was present early in lacZ, requiring deletion of the mutation in the fusion event. Such fusions apparently unite amino-terminal portions of gpmalF to carboxy-terminal portions of gplacZ, creating fused proteins abnormal in their heat lability and reduced .B -galactosidase activity. Of special interest is the observation that the residual ,8 -galactosidase activity is associated largely with insoluble cell components, and fractionates with known components of inner membrane. Since the different protein products of the mal operon have different cell destinations, it seems to be particularly the amino-terminal portion of gpmalF, and not any other characteristic of mal operon function, that guides gpmalF to its residence in cell membranes (155). Sequencing of RNA and

DNA

Operon fusions have proved valuable assets in the sequencing of RNA and For sequencing of RNA in regions normally not transcribed, that is, operator and promoter regions, it became possible to transcribe by read through, when the desired region was fused to an upstream promotor (97, 1 5 6). This application has been largely superseded by improved methods for direct sequencing of DNA (7-9). But even for DNA sequencing, fusions prove beneficial when known DNA can be juxtaposed to unknown. Thus

DNA.



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efforts to sequence the N gene o f A have been eased by the availability of trp fusions adjacent to N (Figure 1 e). These provide an increased range of specific endonuclease targets for generating DNA segments, and the fusion DNA fragments within these segments can be identified by difference in size as well as by their content of known trp sequences (N. Franklin, unpub lished).

FUSIONS FOR THE STUDY OF GENE REGULATION IN EUCARYOTES Cloning is now making possible the interchange of genetic material between procaryotes and eucaryotes, promising new revelations about the handling of genetically coded information affecting gene expression. A few yeast, Neurospora, Drosophila, and even mammalian genes have been expressed when cloned into E. coli ( 1 57-1 62). Conversely, bacterial and bacterio phage genes are being tested for function in mammalian cells ( 1 1 , 12, 40, 1 63). To the extent that such cloned genes are being cultivated in foreign soil, their expression may differ from the native. Synthesis of an authentic product indicates some degree of translational fidelity, but neither ribosome binding nor transcription initiation need be identical with the native situa tion for translation to occur. For example, the yeast his gene fused to the major leftward operon of bacteriophage A can be transcribed either from a A promoter or from a non-A promoter: The non-A promoter may or may not be the promoter used in yeast ( 1 58). Of independent Drosophila DNA segments cloned into plasmids of E. coli, only a small proportion induce the synthesis of new proteins, not all of which are authentic Drosophila proteins ( 1 6 1 , 1 62). This low probability of expression could retlect a rela tive rarity of promoters or ribosome binding sites coded in Drosophila DNA, or the inaccessibility of such determinants to the E. coli machinery. It will be necessary to test such DNA sequences in vitro with activating machinery from different sources, either composite or with mixed purified components, in order to determine whether fusions of disparate DNAs give any direct information about regulation in the donor cells. Current results indicating the internal splicing of eucaryotic mRNAs ( 164-1 67) raise par ticular doubts that eucaryotic genes cloned into procaryotes will be ex pressed in a manner retlective of their native situations. It will be interesting to compare the expression in procaryotes of natural eucaryote DNA and of c1;>NA, the "reverse transcript" DNA copied from functional cytoplas mic RNA. On the other hand, fusions of eucaryote DNAs into procaryotes provide potent tools for eucaryote analysis, by providing enriched sources of partie-


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ular small segments of eucaryote DNA. These can be used to titer specific eucaryote mRNAs, to probe for repeated DNA sequences using hybridiza tion techniques, or to sequence DNA changes as a consequence of mutation. These applications, however, depend only on random cloning and not on meaningful fusions between operons. Disparate cloning does make use of operon fusions when eucaryotic structural genes are made to function by fusion to the regulators of procar yotic operons. Thus bacteriophage A and E. coli plasmids (under regulation by NIH Guidelines) are being redesigned so that insertions of foreign DNA can easily be selected and so that such DNA can use procaryotic promoters for expression. A striking accomplishment of disparate cloning is the in vitro engineering of the mammalian somatostatin gene into the lac operon of E. coli. In this case strong synthesis of somatostatin in E. coli was made possible not only by fusion of a DNA sequence coding somatostatin to the strong promoter and ribosome binding site of lac, but also by fusion to the lacZ gene itself, allowing somatostatin to be synthesized as part of a large stable protein, able to be cleaved in vitro to release the somatostatin moiety (168).

SUMMARY Technically it will soon be possible to arrive at any desirable rearrangement of genetic material. We have considered here the applications of rearrange ments involving gene and operon fusions in two major areas. On the one hand, fusions have contributed to the definition of regulators of operon ftinction (promoters, operators, terminators, and regulatory proteins affect ing the functioning of such sites) and to the genetic alteration of such regulators by selective pressure in order to better understand the mecha nisms of their roles. On the other hand, fusions have facilitated maximiza tion of structural gene function, in order to provide gene products for biochemical analysis or use; the alteration of those products by fusion to other proteins may contribute to their functionality. A few caveats are in order. Different fusions between the same elements may show highly variable levels of function. This is apparently due to the generation of transcriptional polarity by the fusion itself as a consequence of reading frameshifts that lead to translation stops, thereby revealing tran scription stops (46, 54, 1 19, 169). The extent of the polar effect depends in tum upon the distance to the next translation start (170). In add�tion, fusions may interfere with ribosome binding sites, or join protein products in a way incompatible with their function. Finally, the interpretation of mutations selected on the basis of fusions is as perilous as with any other selection (1 12).

GENETIC FUSION FOR OPERON ANALYSIS

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ACKNOWLEDGMENTS

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Genetic fusions defining trp and lac operon regulatory elements.

Analysis of regulation of the ilvGMEDA operon by using leader-attenuator-galK gene fusions.

Characterization of fusions between the lac operon and the ilv gene cluster in Escherichia coli: ilvC-lac fusions.

Genetic mapping of a new promoter for the lac operon.

Formation of lambda lysogens by IS2 recombination: gal operon--lambda pR promoter fusions.

A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.

Structural, molecular, and genetic analysis of the kilA operon of broad-host-range plasmid RK2.

Genetic organization of the cellulose synthase operon in Acetobacter xylinum.

Introduction of single-copy sequences into the chromosome of Escherichia coli: application to gene and operon fusions.

Genetic analysis of the export of an extracellular DNase of Vibrio cholerae using DNase-beta-lactamase fusions.

Positive regulation of the pts operon of Escherichia coli: genetic evidence for a signal transduction mechanism.

Molecular genetic studies of a 10.9-kb operon in Escherichia coli for phosphonate uptake and biodegradation.

Genetic and molecular analysis of vgU and vgW: two dominant vg alleles associated with gene fusions in Drosophila.

Radioscapholunate fusions.

Decision making for partial carpal fusions.

Analysis of a ribosomal RNA operon in the actinomycete Frankia.

Molecular analysis of the Haemophilus ducreyi groE heat shock operon.

Microsatellites for linkage analysis of genetic traits.

Microfluidics for single-cell genetic analysis.

The regulatory region of the L-arabinose operon: a physical, genetic and physiological study.

A distinct alleles and genetic recombination of pmrCAB operon in species of Acinetobacter baumannii complex isolates.

Antibody fusions with immunomodulatory proteins for cancer therapy.

Corrigendum: Engineering and optimising deaminase fusions for genome editing.

Engineering and optimising deaminase fusions for genome editing.