Vol. 145, No. 1JOURNAL OF BACTERIOLOGY, Jan. 1981, p. 293-2980021-9193/81/010293-06$02.00/0

Use of Gene Fusions to Determine the Orientation of GenephoA on the Escherichia coli ChromosomeAPARNA SARTHY,t SUSAN MICHAELIS, AND JON BECKWITH*

Department ofMicrobiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts02115

We present genetic evidence which demonstrates that the phoA gene is tran-scribed in the clockwise direction on the Escherichia coli chromosome, in contrastto an earlier proposal. Our conclusion is based on analysis of various geneticfusions between the lac operon and the phoA gene.

The phoA gene, which codes for the peri-plasmic enzyme alkaline phosphatase, lies be-tween 8 and 9 min on the Escherichia colilinkage map (1). The order ofgenes in this regionof the E. coli chromosome, read in a clockwisedirection, is lacphoA proCphoR (7, 16). Suzukiand Garen (13) analyzed fragments of aLkalinephosphatase produced by strains ofE. coli carry-ing different nonsense mutations in the phoAgene. They found that protein chain-terminatingmutations located at one end of the phoA geneyielded small protein fragments related to alka-line phosphatase. It was concluded that thesewere amino-terminal fragments of the enzyme.Subsequently, Nakata et al. (7) oriented thephoA gene relative to the nearby proC gene bydoing four-factor crosses with phoA mutationslocated at opposite ends of the gene. Based onthis mapping and the previous report of Suzukiand Garen, they concluded that the phoA genewas transcribed in the direction away from theproC gene (counterclockwise).

During an analysis of genetic fusions betweenthe phoA and lacZ genes, we encountered anumber of confusing results which led us toquestion the validity of the previous conclusionsregarding the direction of transcription of thephoA gene. In this paper, we present evidencewhich shows that the direction of transcriptionofthephoA gene is indeed clockwise rather thancounterclockwise. A more detailed analysis ofthe data of Suzuki and Garen (13), in conjunc-tion with recent amino acid sequence data onalkaline phosphatase (R. Bradshaw, personalcommunication), is also consistent with this con-clusion.

MATERIALS AND METHODSMedia and chemicals. Media and chemicals used

are described in the accompanying paper (10).Bacterial and phage strains. Bacterial and phage

t Present address: Departnent of Biochemistry, Universityof Washington Medical School, Seattle, WA 98105.

strains used are listed in Table 1 and in the accom-panying paper (10). To obtain Hfr strains carryingphoA mutations, the Hfr strain CA7087 was trans-duced topro+ with P1 grown onvariousphoA mutants.proC and phoA are 95% cotranaducible. pro' trans-ductants were selected on Tris-minimal glucose-XP(5-bromo-4-chloro-3-indolylphosphate) agar. ThephoA transductants of the Hfr strain were identifiedas white colonies.

Isolation of fusions. ThephoA-lacZ fusions char-acterized in this paper have been isolated in a deriva-tive of XPh26 that is deleted for the lac region. Thisstrain is constitutive for the production of alkalinephosphatase. The technique used for the isolation offusions has been described previously (9).

Preparation of phage lysates carrying thephoA-lacZ fusions. The Casadaban technique (4)allows for isolation of A transducing phage particlescarrying gene fusions. These phages are easily ob-tained by UV induction of the strain containing thefusion.A portion of a UV-induced phage lysate from a

fusion strain was spread on a lawn of MC4100 in F topagar (2.5 ml) on Tris-minimal glucose-XG (5-bromo-4-chloro-3-indolyl-,8-D-galactoside) agar containing 10-4M inorganic phosphate. Lambda transducing phagecarrying the fusion yielded dark blue plaques on theseplates. After repurification of the plaques on the samemedium, several well-isolated independent plaqueswere obtained. Plate lysates were made from eachplaque.Mapping the amount of thephoA gene present

in phoA-lacZ fusions. Fusion-containing strainsXPh93, XPh94, and XPh95 were mated with HfrCA7087 derivatives containing genetically character-ized phoA mutations. Matings were performed for 60min in liquid at 300C if the Hfr contained a Mu(cts)insertion, or at 370C otherwise. Dilutions of the matingmixture were spread on Tris-glucose-XP-streptomycinplates containing no inorganic phosphate (10). After 4days of incubation, phoA+ recombinants appeared aslarge blue colonies against a background of small whitecolonies. This technique is sensitive enough to detecta recombination frequency of 10-'.When fusions were carried on a A transducing phage,

the amount ofphoA material present was determinedby spotting a drop of the phage lysate onto these sameplates (without streptomycin) which had been spread

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294 SARTHY, MICHAELIS, AND BECKWITH

with MC4100 derivatives containing different phoAmutations. After a 4-day incubation period, phoA+recombinants appear as dark blue colonies.

,-Galactosidase assays. 8-Galactosidase assayswere done according to Miller (6).

RESULTSIsolation of fusions between the lac op-

TABLE 1. Bacterial strains andphages usedaBacte- Genotype or bacterial genesrium/ aredOiiphage

BacteriumCA7087 Hfr lac+ proC YA221 thi F. JacobMZ10 F- Alac(X74) phoA J. Beckwith

phoR+ trp rpsLXPh93 F- Alac-169phoB+ phoR

glpD rpsL ,(phoA-lacZ)hyb611

XPh94 F- Alac-169phoB+ phoRglD rpsL (phoA-lacZ)hyUM

XPh95 F- Alac-169phoB+ phoRglpD rpsL D(phoA-lcZ+)a

PhageApl.209 (4)480 supC (3)aXPh93 and XPh94 carry gene fusions derived from

Mu insertions M16 and M2, whereas XPh95 carries agene fusion derived from M8.

(U118)

Xpl,209

N

P phoA' M ) phoA'I I

(U1 8)~Mu(gdtyA'I

I 3el :e GENE

---DeletionOPERONFUSION

P,pho. z Y A .xD,...pho

Excision of Xl

J. BACTERIOL.

eron and the phoA gene. We have used theCasadaban technique (4) to isolate strains inwhich the genes of the lac operon are put underthe control of the regulatory elements for alka-line phosphatase. Two types of fusions are pos-sible with this technique. Operon fusions arethose in which an intact lacZ gene is placedunder the control of the phoA promoter. Genefusions result in the formation of a hybridphoA-lacZ gene which has the portion ofthe lacZ genecorresponding to the amino terminus of P-galac-tosidase replaced by a portion of thephoA genecorresponding to the amino terminus of alkalinephosphatase. The steps used to construct operonand gene fusions are s d in Fig. 1.phoA-lacZ fusions were obtained from strains

carrying each of three different Mu insertions inthe phoA gene. The order of these insertions inthe phoA gene is M2, M16, M8, proC (10). Anoperon fusion (821) was isolated from M8 andgene fusions (1611 and 242) from M16 and M2.The isolation and partial characterization of thelatter two fusions have been described previ-ously (9). That the fusions have, in fact, put lacZunder the control of the phoA regulatory ele-ments is shown by the strong repression of 18-galactosidase synthesis in the presence of inor-ganic phosphate (9; Table 2). Alkaline phospha-tase itself is normally controlled in this way (14).Which portion ofthephoA gene is deleted

in phoA-lac fusions? Deletions which are re-

phoA'

\GENEFUSION

A' ,P.a4Z4, YXAPApX+ -1 I * I'3

. phoA'

Excision of X IFIG. 1. Isolation ofgene and operon fusions by the Casadaban technique (4). "P" indicates the promoter

for thephoA gene. lacZ(U118) is an early ochre mutation in lacZ which is incorporated into theAPI29phagewhen gene fusions are being sought.

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VOL. 145, 1981

sponsible for operon and gene fusions may ex-tend into the phoA gene, thus removing phoAgenetic material (Fig. 1). Any genetic materialremoved must correspond to the portion of thephoA gene which is between thephoA promoterand Mu. Genetic sites beyond the point of theMu insertion should be unaffected by the eventswhich lead to fusions. Thus, determination ofwhat, if any, genetic material has been removedas a result of a fusion event should give anindication of the end of thephoA gene at whichthe promoter lies.To determine if any phoA material was de-

leted in the three fusion strains, crosses werecarried out with Hfr strains carrying a numberof different phoA mutations (Table 3). As ex-pected (2), in the case of the phoA-lacZ operonfusion 821, no detectable phoA DNA was de-leted. Similarly, with the gene fusion 242,phoA+recombinants were recovered with allphoA mu-

TABLE 2. P9-Galactosidase activity in MC4100lysogenized with A phage carrying phoA-lacZ

fusions.8-Galactosidase ac-

MC4100 lysogen- tivity (U/mg of pro-ized with A trans- tein) in:ducing phage con-

. phoA-lacZ L wpo-Hihu tio

fuon from stra LOW phos- phos-pae phate

XPh93 (1611) 3,962 1.5 2,641XPh94 (242) 1,092 1.2 910XPh95 (821) 1,923 2.0 962

Units of 8-galactosidase activity are defined inreference 6. The synthesis of alkaline phosphataseitself is normally increased several hundredfold inwild-type strains by phosphate starvation.

ORIENTATION OF E. COLI phoA GENE 295

tants tested except for the original Mu insertion.In contrast to the previous two cases, the

formation of the gene fusion 1611 resulted in thedeletion of phoA genetic material. Specifically,the site corresponding to M2, which is on theproC-distal side, was deleted, whereas no mate-rial in theproC-proximal portion of the gene wasmissing. This result is consistent with the sug-gestion that the phoA promoter is at the end ofthe gene farthest from the proC gene.Proper regulation of a phoA-klcZ fusion:

requirement for the proC-distal portion ofthephoA gene and nonrequirement for theproC-proximal portion. An important featureofthe Casadaban technique is that, starting withfusion strains, specialized A transducing phagescan be isolated which contain the fusion. phoA-lacZ fusions carried by A, if properly regulated,should carry the entire promoter-proximal re-gion of the phoA gene which is fused to the lacgenes. This region can be identified by crossingthe A transducing phage with various mutationsin phoA. This technique has been used previ-ously to characterize fusions of the malB operonto the lac genes (12).Lambda transducing phages for the three lac-

phoA fusions were obtained and verified as suchby showing inorganic phosphate control (14) of,-galactosidase synthesis in lysogens (Table 2).These phages were then crossed with a series ofphoA mutations. When three independently iso-lated phages carrying the phoA-lacZ operon fu-sion 821, which was obtained from M8, werecrossed with phoA mutations that map in theproC-distal portion of the phoA gene, phoA+recombinants were obtained at a frequency of10-6. However, no phoA+ recombinants were

TABLE 3. Recombination frequency obtained from crosse8 betweenphoA mutants and strains containingphoA: :Mu(cts) insertions orphoA-lacZ fu8ions'

Strains containing phoA mutations contained in Hfr CA7087Mu(cts) inphoA geneor carrying phoA-lacZ M2 M20 U12 U5 S19 E35 S10fusion

XPh95 (821) NTa NT >2 x 10-6 >2 x 10-5 NT 9 x 10-6 2 x 10-7XPh26 containing NT NT >2 x 10-5 >2 x 10-5 NT 8 X 10-6 2 x 10-7M8

XPh93 (161) <1 x 10-8 <1 x 10-8 <1 x 108 <1 x 10-8 3.5 x 10-6 6.9 x 10-6 3.5 x 10-6XPh96 containing NT NT 8 x 10-7 1.6 x 10-5 1 x 10-6 5.6 x 10-6 1.8 x 10-6M16

XPh94 (242) <1 x 10-8 2 x 10-6 1.6 x 10-6 2 x 10-5 6 x 10-6 9.6 x 10-6 2.2 x 10-6XPh26 containing NT NT 2 x 10-7 >2 x 10-6 4 x 106 3 x 10-6 1.2 x 10-6M2

MC4100 containing 1.5 x 10-6 NT NT NT NT NT NTM31

'Recombination frequency is defined as the number ofphoA+ recombinants obtained divided by the totalnumber of input Hfr. In the control experiment M31 x M2, Hfr containing M2 transferred the phoA ::Mu(cts)insertion. M2 and M20 are Mu(cts) insertions in thephoA gene. U12, U5, S19, E35, and S10 arephoA mutations.NT, Not tested.

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296 SARTHY, MICHAELIS, AND BECKWITH

obtained when the three phages were crossedwith phoA mutation SlO, which maps at theproC-proximal end ofphoA. From results in theprevious section we know that in strain XPh95,containing thephoA-lacZ operon fusion 821, thesite corresponding to the phoA mutation S10was not deleted. Thus, in order to incorporate a

functional phoA-lacZ operon fusion 821, thephage must include proC-distal regions but no

proC-proximal region of the phoA gene.Four independently isolated X phages contain-

ing the phoA-lacZ gene fusion 1611, which was

derived from M16, were crossed with strainscontaining phoA mutations that are present inthephoA gene on either side of M16. No phoA+recombinants were obtained in any of thecrosses. From the previous section we know thatthe deletion that generated the phoA-lacZ genefusion 1611 had removed the region ofphoA thatcorresponds to M2, which lies at the proC-distalend of the phoA gene. However, no proC-proxi-mal sites in phoA were deleted in the originalfusion. These results are similar to those withfusion 821.However, two phages carrying phoA-lacZ fu-

sion 1611 did recombine with phoA mutationswhich lie in the proC-proximal portion of thephoA gene. We sumpect that in the formation ofthese two phas the A phage excised in such a

way as to include chromosomal material at bothends (Fi. 1). Ths, in addition to carrying thefusion 1611, they also carryphoA DNA which isnot part of the fusion.

ITe same analyis was done with two inde-pendently isoated phages containingphoA-lacZgene fusion 242 obtained from M2. No phoA+recombinants were recovered when the fusionwas crossed with phoA mutations which lie inthe proC-proximal portion of the phoA gene.

All of the phages analyzed in this study con-

tain fusions of the lacZ gene to the phoA geneand hence must carry the promoter ofphoA andregions of phoA DNA between the phoA pro-moter and the lacZ gene. Therefore, the map-ping studies of the phoA gene contained inphoA-lacZ fusion indicate that the promoter ofthe phoA gene is located at the end of phoAdistal to the proC gene.Use of a nonsense mutation in the phoA

gene to establish the direction oftranscrip-tion of phoA. The evidence presented so farindicates that the promoter of the phoA gene isat the end of thephoA gene farthest fromproC.If this is the case, in phoA-lacZ fusions, theproC-distal portion of thephoA gene will be thephoA segment which is properly transcribedfrom its promoter. It is possible to interrupt thetranscription of distal genes of operons by intro-

J. BACTERIOL.

ducing chain-terminating mutations in an earlygene (8). If our conclusions so far are correct, theintroduction of a phoA chain-terminating mu-

tation into the proC-distal portion ofphoA in a

phoA-lacZ operon fusion should reduce theexpression of the lac genes.

We have tested this prediction in the followingway. In the previous section, the isolation of a

A transducing phage which carries the phoA-lacZ operon fusion 821 was described. The Aphage carries the end of the phoA gene which isdistal to theproC gene. Strain MC4100 contain-ing a chain-terminating mutation, phoA(U24)(15), which is located at the proC-distal end ofthe phoA gene, was infected with the A trans-ducing phage containing the operon fusion 821.Since this phage carriesphoA DNA correspond-ing to thephoA( U24) site, A lysogens which gaverise to phoA+ recombinants could be selected.ThesephoA+ recombinants can arise by a singlereciprocal crossover event at homologous re-

gions of phoA DNA contained in the operon

fusion and present in the chromosome (Fig. 2).This recombination event also allows for inte-gration of A together with the fusion directlyinto the chromosome. However the nonsensemutationphoA(U24), which was originally pres-ent in thephoA gene on the chromosome, wouldnow be crossed by the reciprocal recombinationevent onto that part of the phoA gene which isfused to the lacZ gene in the phoA-lacZ operonfusion 821. If this mutation is located in theregion of the phoA gene which is present be-tween the phoA promoter and the fused lacZgene, then expression of the lacZ gene should bereduced.From the cross between the A phage contain-

ing operon fusion 821 and the mutant containingphoA(U24), eight phoA+ recombinants whichwere also X lysogens were isolated and purified.

N Xpl,209

it A phoA proCu24

phoA z y A Xp N phoA+ proCu24FIG. 2. Recombination of a X phage containing

phoA-lacZ operon fusion 821 into the phoA genecontaining the nonsense mutation phoA(U24). ThephoA recombinant has the phoA(U24) mutation re-combined onto the phoA-lacZ operon fusion.

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VOL. 145, 1981

Ordinarily, strains containing operon fusion 821when streaked on Tris-minimal glucose-XG agarform blue colonies, indicating high levels of,B-galactosidase. To determine the polarity effectof nonsense mutation phoA(U24) on expressionofthe lacZ gene in thephoA-lacZoperon fusion,each phoA+ recombinant was tested on thissame medium. Four out of eight phoA+ recom-binants formed pale blue colonies on this me-dium, indicating that the nonsense mutationphoA(U24) recombined onto the phoA DNAfused to the lacZ gene and reduced the expres-sion of the lacZ gene from the phoA promoter.The other fourphoA + recombinants formed bluecolonies on this medium. However, each of theserecombinants when restreaked on the same me-dium also yielded a fraction ofpale blue colonies.ThesephoA recombinants may represent doublelysogens of the fusion phage which throw offpale blue single lysogens, including the phagecontaining the nonsense mutationphoA(U24) inthe phoA-lacZ operon fusion.

If the reduction of lacZ expression in theoperon fusion is due to the polarity exerted byphoA( U24) crossed onto the phoA DNA in thephoA-lacZ operon fusion, then suppression ofphoA(U24) by nonsense suppressor mutationsupC (3) should restore expression of the lacZgene from the phoA promoter. IAmbda trans-ducing phages containing the operon fusion 821with the nonsense mutationphoA(U24) crossedonto thephoA DNA were isolated. These phagesappeared as pale blue plaques on a lawn ofMC4100 on Tris-minimal glucose-XG agar. Thephages were used to test the effect of the sup-pressor mutation supC on expression of the lacZgene. The tester strains were MZ10 and its iso-genic supC derivative. Since MZ10 is deleted forthe lac genes, phage containinga functional lacZgene when spotted on a lawn of this strain onTris-minimal glucose-XG agar under conditionsof lacZ gene expression will appear as a bluespot. Two independently isolated A phages con-taining the operon fusion 821 with phoA(U24)were spotted on a lawn of MZ1O 080 supC onTris-minimal glucose-XG agar. In the presenceofsuppressor mutation supC, a spot ofthe lysateproduced a blue zone in this test, indicating thatthe nonsense mutation phoA(U24) contained inthe phoA-lacZ operon fusion could be sup-pressed to restore ,B-galactosidase activity. Adrop of the same lystate, when spotted on MZ10on Tris-minimal glucose-XG agar, produces apale blue spot. We then isolated lysogens ofthese phages in MZ10 and its isogenic supC-containing derivative in order to assay fi-galac-tosidase. Assays of these strains showed thatsuppression of the phoA(U24) mutation when

ORIENTATION OF E. COLI phoA GENE 297

present in the 821 operon fusion allowed for a10-fold increase in synthesis of 8-galactosidase.In contrast, a missense mutation can be ex-

pected not to exhibit polarity in the operonfusion. Recombination of missense mutationphoA(S19) (11) onto thephoA region present inthe operon fusion 821 by the same approach didnot reduce t-galactosidase activity in this fusion.These results also support the conclusion thatthe promoter for phoA is proC distal tophoA(U24).

DISCUSSIONWe have used the Casadaban technique (4) to

isolate fusions of thephoA gene to the lac gene.During characterization of the phoA-lacZ fu-sions, we have generated several lines of evi-dence which suggest that thephoA gene is tran-scribed in a clockwise direction: (i) in the con-struction ofaphoA-lacZ gene fusion, somephoADNA which is distal to the nearbyproC gene onthe chromosome has been deleted; (ii) X trans-ducing phages containing phoA-lacZ fusionsneed only carry phoA DNA which is distal tothe proC gene; and (iii) nonsense mutationphoA(U24) located at the end of thephoA genedistal to proC, when crossed onto the phoADNA contained in a phoA-iacZ operon fusion,reduces expression of the lacZ gene. A similaranalysis with malT-lacZ gene fusions was usedto show the direction of transcription of themalT operon (5).

Further support for this conclusion comesfrom a combined genetic and biochemical anal-ysis of one of the gene fusion strains. The genefusion 1611 codes for a hybrid alkuline phospha-tase- -galactosidase protein. The Mu(cts) inser-tion in phoA(M16) used in the isolation of thisgene fusion is located at the end of the phoAgene which is distal to proC. The molecularweight of the hybrid protein produced by thegene fusion strain as estimated from sodiumlauryl sulfate-polyacrylamide gels is approxi-mately 115,000; hence it should contain only afew amino acids from the amino terminus ofalkaline phosphatase. (The monomer of -galac-tosidase has a molecular weight of 116,000, andin the generation of the fusion only 19 aminoacids from the amino terminus of ,B-galactosid-ase were removed.) A determination of theamino acid sequence in this segment of the hy-brid protein revealed the presence of only fiveamino acids, presumably from the signal se-quence of the precursor of alkaline phosphataseattached to fi-galactosidase, and no amino acidresidues from wild-type alkaline phosphatase(9). To generate such a fusion from M16, mostofthephoA DNA between the Mu(cts) insertion

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298 SARTHY, MICHAELIS, AND BECKWITH

and the promoter of phoA would have to bedeleted. A genetic analysis ofthis region in strainXPh93 containing the gene fusion 1611 whichcodes for the hybrid protein (phoA-lacZ)h,b1611shows that only phoA DNA at the end of thephoA gene distal to proC has been deleted (Ta-ble 3).Our conclusion concerning the orientation of

the phoA gene on the E. coli chromosome con-tradicts a previous report (7). However, our con-clusion is also supported by a reevaluation ofearlier data on fragments of alkaline phospha-tase from strains containing nonsense mutations(amber and ochre) in the phoA gene. Fromnonsense mutations which map at the proC-proximal end of the phoA gene, peptides ofmolecular weight -4,000 (40 to 50 amino acidslong) representing portions of alkaline phospha-tase were obtained. It was inferred that theseparticular lesions causing translation termina-tion must be early (i.e., promoter proximal) inthephoA gene. Nakata et aL (7) performed four-factor crosses which oriented two phoA mutantalleles relative to proC and lac. One of thesemutations mapped in the same region as theamber and ochre mutants which Suzuki andGaren had examined (13). The genetic datataken together with the earlier findings led tothe conclusion that the direction of transcriptionof phoA was counterclockwise on the E. colichromosome (7).We have reevaluated the data of Suzuki and

Garen. We have compared the amino acid com-position of the tryptic peptides contained withintheir putative 4,000-molecular-weight amberand ochre fragments with those predicted fromthe amino acid sequence of mature alkalinephosphatase as determined by Bradshaw andco-workers (personal communication). We findthat at least two of the putative nonsense frag-ments contain amino acids which lie at positions150 to 190 of mature alkaline phosphatase. Thismakes them unlikely candidates for true amberor ochre fragments since these would necessarilycontain amino acids lying at positions 1 to 50 ofthe mature alkaline phosphatase amino acid se-quence. Thus, we believe that Suzuki and Garenwere examining short degradation products ofthe true amber and ochre fragments.

ACKNOWLEDGMENTSThis work was supported by a grant from the American

Cancer Society (VC-13F,GH). S.M. was supported by a Public

Health Service training grant from the National Institute ofGeneral Medical Sciences.We thank R. MacGillivray and A. McIntosh for excellent

technical assistance.

LITERATURE CITED1. Bachmann, B. A., and K. B. Low. 1980. Linkage map of

Escherichia coli K-12, edition 6. Microbiol. Rev. 44:1-56.

2. Bassford, P. J., Jr., T. J. Silhavy, and J. R. Beckwith.1979. Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplam. J. Bac-teriol. 139:19-31.

3. Brenner, S., and J. R. Beckwith, 1966 Ochre mutants,a new class of suppressible nonsense mutants. J. Mol.Biol. 13:629-637.

4. Ca adaban, J. M. 1976. Transposition and fusion of thelac genes to selected promoters in Escherichia coliusing bacteriophage A and Mu. J. Mol. Biol. 104:541-555.

5. DebarboulIle, M., and M. Schwartz. 1979. The use ofgene fusion to study the expresion ofmalT, the positiveregulator gene of the maltose regulon. J. Mol. Biol. 132:521-534.

6. Miller, J. H. 1972. Experiments in molecular genetics.Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.

7. Nakata, A., G. R. Petersen, E. L. Brooks, and F.Rothman. 1971. Location and orientation of the phoAgene locus on the Escherichia coli K-12 linkage map. J.Bacteriol. 107:683-689.

8. Newton, A., J. R. Beckwith, D. Zipser, and S. Bren-ner. 1965. Nonsense mutants and polarity in the lacoperon of E. coli J. Mol. Biol. 14:290-296.

9. Sarthy, A., A. Fowler, L. Zabin, and J. Beckwith.1979. Use of gene fusions to determine a partial signalsequence of alkaline phosphatase. J. Bacteriol. 139:932-939.

10. Sarthy, A., S. Michaelis, and J. Beckwith. 1981. Dele-tion map of the Escherichia coli structural gene foralkaline phosphatase, phoA. J. Bacteriol. 145:288-292.

11. Schlesinger, M. J., A. Torriani, and C. Levinthal.1963. In vitro formation of enzymatically active hybridproteins from E. coli alkaline phosphatase CRM's. ColdSpring Harbor Symp. Quant. Biol. 28:539-542.

12. Silhavy, T. J., E. Brilckman, P. J. Bassford, Jr., M. J.Casadaban, H. A. Shuman, V. Schwartz, L Guar-ente, M. Schwartz, and J. Beckwith. 1979. Structureof the malB region in Escherichia coli K12. II. Geneticmap of the maIE,F,G operon. Mol. Gen. Genet. 174:249-259.

13. Suzuki, A., and A. Garon. 1969. Fragments of alkalinephosphatase from nonsense mutants. I. Isolation andcharacterisation of fragments from amber and ochremutants. J. Mol. Biol. 45:549-566.

14. Torriani, A. 19W). Influence of inorganic phosphate inthe formation of phosphatase of Escherichia coli.Biochim. Biophys. Acta 38:460-469.

15. Torriani, A. 1974. The alkaline phosphatase of Esche-richia coli, p. 173-181. In R C. King (ed.), Handbookof genetics, vol. 1. Plenum Publishing Corp., New York.

16. Yagil, E., M. Bracha, and N. Silberstehn. 1970. Furthergenetic mapping of the phoA-phoR region for alkalinephosphatase synthesis in Escherichia coli K12. Mol.Gen. Genet. 109:18-26.

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