Proc. Natl. Acad. Sci. USA Vol. 80, pp. 4775-4779, August 1983 Genetics

Cyclic AMP-dependent constitutive expression of gal : Use of titration to isolate operator mutations (promoters/plasmid/Escheriehia coli) M. IRANI*, L. OROSZt, S. BUSBYt, T. TANIGUCHIO, AND S. ADHYAS Lahoratorv of , National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205 Communicated by Bernard D. Davis, April 1, 1983

ABSTRACT When the gal operator region is present in a mul- described here showed constitutive synthesis of gal ticopy plasmid it binds to all ("titrates") the gal repressor and "in- only in cya+ cells and not in cya- cells. duces" the chromosomal gal operon. To make operator mutations (Oa) with reduced affinity toward the repressor, plasmid DNA was MATERIALS AND METHODS irradiated with UV light and mutant derivatives were isolated that were unable to release the chromosomal gal from repres- The E. coli K-12 strains used in this study are C600 (F- thi- sion. Then with such an Oa plasmid operator revertants were iso- thr- leu- lacY- suII), SA1293 (Hfr H thi- galRss), SA1293C lated that had reacquired the ability to release repression. Both (SA1293 ilv-2: :TnlO cya-1039), and Ml101 [F- his- str relAl sets of mutations have been localized by DNA sequence analysis. lac- lacPuv5 A(gal-bio) A(galR-lysA)]. All other strains used, When the Oa mutations were transferred from the plasmid to the including those described in Table 1, were derived from Ml101 chromosome by recombination these mutant operators were found by a single or successive steps of P1 phage transduction with to make gal expression constitutive (independent of repressor) but appropriate markers. The ilv-2:TnlO and cya-854 alleles pres- still dependent on cAMP, whereas the previously reported gal op- ent in some of the strains originated from BM4468 of A. Camp- erator mutants (0C) are constitutive both in the presence and in bell and CA8306 of J. Beckwith, respectively. Phage strains the absence of cAMP. The titration method of isolating mutants AcI857 A(attL-int)313 A(gal)165, AcI857lySA galR,8, and Plkc enables the isolation of strains with operator mutations that also are from our collection. The AcI857 gal8dc transducing phage affect normal activity, and it provides an easy way to was obtained from D. Court and is described in Fig. 2. isolate revertants of operator mutations. The media composition are from Miller (9). Ampicillin and tetracycline were used at' 50 and 15 ,ug/ml, respectively. AgalOa The initial controlling step of a negatively regulated operon is phage, described in Results, were distinguished from AgalO+ the modulation of initiation from its promoter by by spotting on methyl f-D-thiogalactoside/ minimal binding of specific repressor molecules with the operator site agar plates, seeded with a Agal (Ak) strain. On this plate cells (1).'The operator is usually identified by mutations (QC) that constitutively making gal enzymes grow but those that are in- make the operon constitutive but do not affect the promoter. ducible do not (8). However, when the operator and promoter loci overlap, con- Restriction enzymes, RNA polymerase, phage T4 DNA li- ventional selection would preclude the isolation of strains with gase, and pBR322 DNA were from Bethesda Research Labo- operator mutations located in the overlap region. In this paper ratories or New England BioLabs and were used as recom- we describe a method for isolating the full range of gal operator mended by them. Calf intestinal alkaline phosphatase was from mutations with a reduced affinity for gal repressor. Boehringer, polynucleotide kinase from P-L Biochemicals, and The gal operon can be transcribed from either of its two hydrazine, dimethyl sulfate, and piperidine from Eastman-Ko- overlapping promoters, PG1 or PG2 (Fig. 1; refs. 3, 6, and 7; dak. Kinase and epimerase assay reagents were from Sigma. unpublished data). On gal DNA, pGi-promoted transcription The radioisotopes [ y-32P]ATP and [a-32P]UTP were supplied starts at +1 position (S1), and PG2 at -5 (S2). cAMP and its by New England Nuclear and ['4C]galactose by Amersham. The receptor protein (CRP) regulate the activities of PGi and PG2 in CRP was a gift from J. Krakow. opposite ways, stimulating transcription from PG1 and inhib- The plasmid DNAs were prepared by lysing the cells with iting transcription at PG2. Thus, synthesis beginning at Si pre- Triton X-100 followed by chromatography on Sepharose 4B dominates in wild-type (cya+) cells, and synthesis beginning at (Pharmacia) (10). DNA fragments for RNA transcription, DNA S2 predominates in adenylate cyclase mutants (cya-). Analysis sequence analysis, or protection against DNase digestion were of promoter mutations and other studies have established that separated on polyacrylamide gels and extracted by the methods cAMP-CRP binds at a single target site (cat), located around described by Maxam and Gilbert (11). For ligation, the DNA position -35. Binding at this site is responsible for both stim- ulation of transcription at PGI and inhibition at PG2 (2, 4, 5). The Abbreviations: CRP, cAMP receptor protein; bp, base pair(s); Gal', ga- gal repressor protein, the product of the galR , regulates lactose-utilizing; Gal-, galactose-negative; TMGs, sensitive to methyl both the promoters (6, 8). The operator defined by several Oc ,B3D-thiogalactoside; AmpR, ampicillin resistant. mutations and galR- mutations derepress the synthesis of the * Present address: Dept. of Biochemistry, University of Washington, gal enzymes and they do it in both cya+ and cya- cells-i.e., Seattle, WA 98195. they make both and PG2 constitutive. t Present address: Dept. of Genetics, Attila Jozsef -University, Szeged, PGI Hungary. Unlike the previously reported OC mutants, the 03 mutants t Present address: Institut Pasteur, Dept. Biologie Moleculaire, Paris, France. The publication costs of this article were defrayed in part by page charge § Present address: Dept. of Biochemistry, Kouchi Medical College, payment. This article must therefore be hereby marked "advertise- Nangoku-Shi, Kouchi, Japan. ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. ¶To whom reprint requests should be addressed. 4775 Downloaded by guest on September 27, 2021 4776 Genetics: lrani et al. Proc. Natl. Acad. Sci. USA 80 (1983)

pMl3

EcoRi I Hind III I I I I AN galE K 1L gaIT 1L gal i

I -_

,' OPERATOR CAT '9 GT - _ _ '9'9 ~~~~CA '9 ~~~~~~~~0'33,3 -137 -80 -70 - 60 -50 -40 -30 -20 -10 +1 +10 +45 CCAA---GGCTAAATTCTTGTGTAAACGATTCCACTAATTTATTCCATGTCACACTTTTCGCATCTTTGTITATG TATGGTTATTTCATACCATAAG ---CCG GGTT---CCGATTTAAGAACACATTTGCTAA>GATTAAATAAGGTACAGTGTSiAAA AGCGTAGAAACAATAC T ACCI TAAAGTATGGTATTC---GGC B1 °38°034°S°4 S2 ACT A T A TGA T A T SI 01e3 0e3o5 G T C A FIG. 1. Structure of the plasmid pMI3 as described in ref. 2 and the gal operon ofEscherichia coli. The gal controlling region covers the two transcription starts S1 (+ 1) and S2 (-5) and their respective promoters, including the Pribnow sequences (boxed regions) (3), the cat site around the -35 region (4), and the operator segment around -58 (5). The coordinates referto the start point ofthe cAMP CRP-dependentgal transcription, S1, taken to be + 1. The operator is amplified and shows the sequence changes associated with the three oa mutations, Ol, Oa, and O', described in this paper along with seven O' mutations, including O°1, described before (5, 6). The plasmid pMI3 carries the regulatory region extending from +45 to -187, between the HindMI and EcoRI sites of pBR322. The BstEII restriction site present at +42 has been used to reconstitute the intact gal operon from pMI3 as described in the text and Fig. 2. Small square indicates base protected by cAMP CRP from methylation by dimethyl sulfate. Wavy underlinings indicate protection against DNase digestion by cAMP*CRP at the 3' side of the indicated base (4). fragments were isolated from agarose gels by the perchlorate that reduces the binding of the repressor to the plasmid gal method (12). Transformations were performed with host cells operators, it would fail to derepress the chromosomal gal genes. that were.aged for 3-24 hr in 75 mM CaCl2 on ice. To perform the current study, we inserted a small fragment of For DNA sequence analysis, the plasmid DNA was opened gal DNA containing all known control sites into plasmid pBR322. with HindIll and the 5' ends were labeled with polynucleotide The resultant plasmid, pMI3, harbors a 270-bp piece of gal DNA kinase. After digestion with EcoRI, the 270-base-pair (bp) frag- that contains the operator, two promoters (including the two ment was isolated from a 5% (wt/vol) polyacrylamide gel and transcription start sites), and the coding sequence for the first its sequence was determined by the method of Maxam and Gil- six amino acids of the galE cistron (Fig. 1). When pMI3 (galO+) bert (11). is introduced into a strain (SA1293) carrying a gal super-re- For UV light mutagenesis, host cells were irradiated with 13 pressor mutation, galRS, it changes the cell phenotype from J/m2 and were transformed immediately with DNA samples galactose-negative (Gal-) to galactose-utilizing (Gal'). Thus, previously.irradiated with a dose of 540 J/m2. For isolation of SA1293/pMI3 grows on galactose minimal agar plates and forms revertants, cells were grown overnight in the presence of N- red colonies on MacConkey galactose agar plates. SA1293 with- methyl-N'-nitro-N-nitrosoguanidine at 100 pug/ml in LB me- out the plasmid does not grow on minimal galactose and forms dium. white colonies on MacConkey galactose agar plates, because Epimerase and kinase assay conditions have been described the super-repressor binds to the operator even in the presence previously (13). Units of are nmol of product formed of galactose. Also, pMI3 enables an inducible galR+ strain (C600) per min per ml of cells. to grow on minimal galactose agar in the presence of the gra- tuitous anti--methyl p-D-thiogalactoside. C600, without RESULTS the plasmid, does not grow on methyl thiogalactoside/galac- tose. These results indicate that pMI3 can derepress the chro- Isolation of Strains with Operator Mutations with Reduced mosomal gal operon by binding to all the repressor in trans. Repressor Affinity. The operator segment of the gal operon, Thus, a mutant pMI3 that fails to bind the gal repressor would when present in multiple copies in a cell, is able to bind to all be scored as Gal- in a galRs strain or as methyl thiogalactoside- ("titrate") the gal repressor in trans and derepress the chro- sensitive (TMGs) in a galR+ strain. mosomal gal genes (5, 14). Thus if a plasmid carries a mutation pMI3 DNA was irradiated with UV light as described in Ma- Downloaded by guest on September 27, 2021 Genetics: Irani et aL Proc. Natl. Acad. Sci. USA 80 (1983) 4777

terials and Methods. In experiment I, this DNA was directly occasionally obtained by ligation of HindIII/EcoRI-digested used to transform the galRs strain, and the ampicillin-resistant pBR322 DNA. (AmpR) transformants were screened for Gal- (white) colonies The phenotype of pMI30, pMI31, and pMI40 was confirmed on MacConkey galactose/ampicillin agar plates. AmpR Gal- by measuring the levels of chromosomal galactokinase, the colonies were obtained at a frequency of about 0.1%. Three of product of the galK gene, of galRS strains carrying the wild-type these were characterized further and shown to have the mu- or the mutant plasmids. pMI3-carrying cells synthesized sig- tations associated with the plasmids. One carried a mutant plas- nificant amount of kinase, whereas those carrying the mutant mid with a low copy number as judged by quantifying the plas- plasmids, as expected, did not show derepression (data not mid DNA extracts by gel electrophoresis. The other two (pMI30 shown). and pMI31) carried mutations in the 270-bp HindIII/EcoRI gal DNA Sequence Analysis of the Operator-Affinity Mutations DNA fragment as shown by reconstructing the mutant plasmid (Oa). The three affinity mutations described above are desig- by ligating the 270-bp purified fragments from mutant clones nated as Oa mutations. They were located by DNA sequence into restriction endonuclease-treated pBR322 DNA that had not analysis of the respective 270-bp gal DNA fragments, as de- been irradiated with UV light. scribed in Materials and Methods. pMI30 and pMI40 showed In experiment II, the 270-bp gal DNA fragment was excised the same transition from ANT to G-C at position -64. The mu- from the UV-irradiated pMI3 plasmid by digestion with HinduIII tations are referred to as Oa and Oa, respectively (Fig. 1). pMI31 and EcoRI, purified by gel electrophoresis, and then ligated showed a COG to APT transversion at position -53 and is as- into unirradiated pBR322 DNA to reconstruct the pMI3 struc- signed the Oa allele. Both the changes are located in the same ture. The pool of ligated DNA was used to transform the galR+ "region" defined by the previously isolated Oc mutations of the strain. The AmpR colonies were screened on minimal methyl gal operon (5, 6). thiogalactoside/galactose/ampicillin agar plates. Of 150 colo- Reversion of the (0 Mutations. pMI31 plasmid in a host with nies screened, 2 were TMGs. One of them (pMI40) was shown galdeletedwas mutagenized in LB medium containing N-methyl- by restriction analysis to carry a mutation in the 270-bp gal DNA N'-nitro-N-nitrosoguanidine. The mutagenized plasmid DNA and the other a deletion of this fragment. The latter must be was extracted and used to retransform the galRs strain SA1293. created by fusion of HindIII and EcoRI sites of pBR322-per- AmpR Gal+ revertants were obtained at a frequency of 10-6 on haps an artifact of the ligation. Similar illegitimate fusions were minimal galactose/ampicillin agar plates from the transition

Pvu II

PvuII Pst

/

/ BstE 11 \ K RI Agal8dc , PO+I A jSTEP 2 8 att A p K T E OaA AgalOa ,,~f ,- - /A- A 8 att A CHROMOSOME aroG K P0 blu X bio 165 att ISTEP 3 Aoa AC, aroG- KAPO+ blu.,M a. no K T E n ., bio---- LIMMumuburvitI-2ULnRAtl4CnR -- .-a. A A MMLUS. 104 -N F--T- I 165 A f 8 att STEP 4 K aroG T 'E __A 8bio CHROMOSOME --M. a 8 att

FIG. 2. Steps of transferring the oa mutations from an operator fragment in the plasmids to the chromosome. The plasmids and the gal operon are explained in the text. The principles of the four steps are also described in the text. The thin lines represent plasmid or phage DNA, thick and double lines represent bacterial DNA, and wavy lines represent insertion DNA. The triangles stand for various deletions. The A phage integrates at the normal attachment site. X shows the insertion of prophage A. aroG, blu, and bio are neighboring bacterial markers. Downloaded by guest on September 27, 2021 4778 Genetics: Irani et al. Proc. Natl. Acad. Sci. USA 80 (1983)

mutant O'. Presumably, the mutant plasmids have regained the Table 1. Differential rates of synthesis of UDP-galactose-4- ability to titrate the gal repressor. Two such independent re- epimerase and galactokinase in the Oa mutants vertants were found by DNA sequence analysis to contain the Epimerase Kinase at position -64, that both transition G-C to A-T demonstrating No No are true revertants. These results give credence to the partic- Strain Genotype inducer Inducer inducer Inducer ipation of Oa alleles in the gal operator function and also au- thenticate our affinity method of isolating operator mutants and 1. MI601 galO+ 2.3 45.4 1.9 21.2 their revertants in studying the interaction between operator 2. MI801 galO1 18.0 53.8 11.2 29.0 and repressor. 3. M1803 galO2 27.0 44.1 9.4 30.8 Transfer of the Oa Mutations from Plasmid to Chromo- 4. MI805 galO18 54.0 77.2 41.0 32.0 some. To study the cis effect of the Oa and Oa mutations on the 5. MI602 galO+ cya-854 4.8 55.0 0.9 9.3 gal operon, we transferred the mutations from the plasmid to 6. MI802 galOa cya-854 3.5 66.0 1.0 8.2 the chromosome as described below (Fig. 2). 7. M1804 galO2 cya-854 2.7 86.0 1.0 12.2 Step 1. Plasmids carrying an intact gal operon with the oa 8. MI806 galOsc cya-854 88.0 123.0 8.9 14.5 mutations were constructed by replacing the EcoRI/BstEII fragment from the wild-type gal plasmid pAA102 (2) with the Cells were grown in ampicillin-containing medium to OD590 = 0.2 in M56 minimal fructose/Casamino acids/biotin medium (6). Various times EcoRI/BstEII fragments derived from pMI3, pMI30, and pMI31 thereafter, samples were removed and assayed for epimerase and ki- (see Fig. 1). The corresponding reconstructed plasmids, pMI50 nase. The units ofactivity were plotted as a function ofcell density. The (Of), pMI51 (Oa), and pMI52 (Oa), when introduced into a host differential rate ofepimerase and kinase synthesis was computed from with a gal deletion (Ml101) expressed comparable levels of both the slope. Wherever necessary, a correction factor has been applied to UDP-galactose-4-epimerase, the product of the galE gene (870, compensate for the differences in copy number between wild-type and with 0.2% D-galactose added and 1,150 units at OD590 = 1.0, respectively), and ga- mutant plasmids. Cultures were induced 1,120, when OD590 was 0.2. The strains are derivatives of MI101. The rela- lactokinase (1,740, 1,727, and 2,423 units at OD590 = 1.0, re- tively reduced levels of kinase in cya- strains is because of a natural spectively), indicating that there is no defect in the promoters. polarity observed in the gal operon under conditions of cAMP defi- Step 2. The wild-type and the mutant gal operators and the ciency (15). Assay ofseparate extracts from cells ofthe same strain grown adjoining DNA regions were transferred from pMI50, pMI51, under the same conditions showed approximately the following vari- and pMI52 into a plaque-forming Agal transducing phage, abilities: kinase, ± 15%; epimerase, ±20% for high enzyme levels and Agal8dc, carrying a deletion-substitution in the galET region ±50% for low levels (6). (see Fig. 2). Agal+ recombinants were obtained at a frequency of 0.5-4%. Such recombinants arose by two cross-overs be- contrast, the synthesis of the gal enzymes remains repressed tween the plasmid and the phage regions of homology that flank in the cya- (cya-854) derivatives of the two oa mutants, as in the deletion-substitution segment. Because the cross-over in their O+ parent, and is inducible (Table 1, lines 6 and 7). Ad- the operator region could be to either side of the Oa mutations, dition of cAMP to the growth media of the cya-854 derivatives the resulting Agal+ phages could be either AgalO+ or AgalOa. restores constitutive expression of the gal enzymes in the O', An oa mutation should make the expression of the gal operon and Oa but not in the O+ strains (data not shown). Normal constitutive, an initial assumption proved true as shown below. repression of Oa and Oa in cya- cells has been con- About 20% of the Agal+ phage from pMI51 and pMI52 were firmed in another cya mutation, cya-1039 (data not shown). In gal constitutive and thus carried the galoa mutations, but none contrast, the O' mutations isolated previously showed full con- (0/18) showed constitutivity when they originated from pMI50. stitutivity in both cya+ and cya- strains, suggesting total loss Step 3. The O+, Oa, and Oa alleles, present in the Agal phages of repressor affinity (Table 1, lines 4 and 8) (6). The normal lev- isolated above, were transferred into the E. coli chromosome. els of gal enzymes in the fully induced oa mutant cells under The phages carrying a heat-sensitive A repressor mutation both cya+ and cya- conditions confirm that there is no im- (clts857) were used to lysogenize a gal deletion host (Agall65 pairment of the promoters. attA+). The structures of the prophage and the contiguous gal Binding of cAMP CRP Complex to the oa Mutants. The regions are shown in Fig. 2. Such lysogens were all Gal+ and specific binding of cAMP-CRP to wild-type and mutant gal DNA did not grow at 42°C. Temperature-resistant survivors at 42°C (from +45 to -90 region) was studied by the DNase protection have lost the prophage by recombination between homologous method (4). Fig. 3 shows the effect of cAMP CRP on DNase gal DNA sequences around the A165 marker. Temperature-re- I digestion of the lower strand of the cat segment (from -15 sistant Gal+ survivors were obtained at a frequency of 5-30% to -75) of the wild-type and O1 gal DNA. It is evident that of total survivors. More than 90% of the Gal+ survivors were the digestion pattern is very similar for the two DNAs. The constitutive-i.e., resistant to methyl thiogalactoside when the cAMP-CRP complex protects the 5' side of G42, A38, A36, and parental lysogens carried AgalOa or AgalOa, but only inducible A35 from DNase digestion and enhances it at the 5' side of A49 Gal+ colonies were obtained from a AgalO+ lysogen. Note that and C39. all of the Gal+ cells carrying O+, Oa, or Oa alleles in the chro- Transcription of the 270-bp gal DNA containing O+ or 1 mosome inherited the A8 deletion from the phage (Fig. 2). allele in a purified system in the presence of 100 utLM cAMP Step 4. An isogeneic set of cya- strains was constructed by plus CRP at 3 or 10 ,ug/ml were identical (unpublished results; introducing the cya-854 allele from.SA2345 (cya-854 ilv-2: :TnlO refs. 3 and 16). These results show that the Oa mutation does donor) by P1 transduction and selection for tetracycline resis- not alter the interaction of the cAMP CRP complex with the gal tance and screening for sorbitol utilization defect on Mac- promoter and RNA polymerase. Conkey sorbitol agar plates. Effect of the oa Mutations on the Expression of a Cognate DISCUSSION Operon. Table 1 shows the differential rate of synthesis of the chromosomal gal enzvmes in the Oa mutants constructed above. gal Operator Mutations with Reduced Affinity for Repres- Oa and O° are partially constitutive for both epimerase and ki- sor. Operator loci have been previously identified by the iso- nase and can be fully induced in a cya' strain (Table 1, lines 2 lation of strains with mutations (OC) that make the expression and 3), suggesting a partial loss of their repressor affinity. In of the cognate structural genes constitutive in the presence of Downloaded by guest on September 27, 2021 Genetics: Irani et aL Proc. Natl. Acad. Sci. USA 80 (1983) 4779

a the cAMPCRP complex has been observed during in vitro ° WT transcription experiments (17). Normally the higher affinity of operator and repressor keeps the promoters repressed in vivo. t' -85 In an operator with somewhat reduced affinity toward repres- sor (O; or O2), the cAMP CRP complex displaces the repressor, resulting in a partially constitutive phenotype in cya' (but not t**SI-65 B cya ) cells. (ii) The oa mutations change the binding of gal DNA to gal repressor so that the repressor effectively represses PG2 '4!-55 but not pGi. Use of the Titration Method. The trans titration method of -. isolating operator-affinity mutants described here could be use- 4 ful for several purposes: (i) It may be used to identify negative regulation of an operon. If a multicopy plasmid carrying the 35 putative operator segment of the operon increases the expres- sion of corresponding chromosomal structural genes, it strongly indicates the presence of an unlinked repressor gene. (ii) Be- cause the system utilizes the multicopy operator independently i~j25 -5 of the operon, one is able to isolate operator mutations that also decrease promoter function in a system in which operator and promoter loci overlap. Because the method of selection of the 1 2 34 56 7 8 91011 CRP operator-affinity mutants does not demand promoter function, cAMP, pM 40 4- 404 - - such oa mutations when assayed for their cis effect on the cog- nate operon may yield new information. (iii) The method pro- FIG. 3. DNase protection by cAMP-CRP on gal operator-promoter vides a very powerful tool to isolate and study second-site re- fragments. The autoradiograph shows the effect of cAMP and CRP upon vertants of Oc or Oa mutations. (iv) We believe that the titration DNase I digestion of O gal DNA (lanes 7-10) and 0! DNA (lanes 2- method may be useful in isolating DNA-site mutations for other 5). The radioactive DNA fragments used in both cases were labeled at interactions. The restraints are theHindill site. They were incubated with DNase (0.1 for 1 min site-spectfwc DNA-protein only /Ag/mI) in with no cAMP or CRP (lanes 5 and 10), no cAMP and CRP at 10 /Ag/ml that the protein should bind stably and normally be present (lanes 4 and 9), 4 pM cAMP and CRP at 10 pug/ml (lanes 3 and 8), or the cell in small amounts, in order to elicit a phenotypic re- 40 aM cAMP and CRP at 10 ,ug/ml (lanes 2 and 7). The resulting mix- sponse to its titration in trans. tures were separated on the gels, which were calibrated with the prod- ucts of the sequence analysis reactions performed with the same la- Note Added in Proof. We have recently discovered the existence of a beled fragments. The wild-type A+G reaction (lane 11) and the mutant second operator locus inside the galE structural gene (18). With the use C and A+G reactions (lanes 1 and 6, respectively) were used. The cal- of an appropriate plasmid, the affinity method would allow the isolation ibration numbers shown denote the number of bp upstream of the S1 of strains with operator mutations that also cause a GalE- phenotype. transcription start (see Fig. 1). We thank Max Gottesman and Douglas Ward for valuable discus- repressor. This approach yields not only operator mutations but sions, Ira Pastan and Don Court for critical reading of the manuscript, also mutations that create a new promoter insensitive to the and Frances Herder and Annette Kuo for assistance. repressor, and it leaves out operator mutations that overlap with 1. Miller, J. H. & Reznikoff, W. S. (1978) The Operon (Cold Spring the promoter. We have developed a strategy to isolate operator Harbor Laboratory, Cold Spring Harbor, NY). mutations that eliminates these difficulties and is based on a 2. Busby, S., Irani, M. & de Crombrugghe, B. (1982)J. Mot BioL 154, stringent definition of the operator locus as the site of repressor 197-209. binding. In this strategy we have employed a multicopy plas- 3. Musso, R. E., di Lauro, R., Adhya, S. & de Crombrugghe, B. (1977) mid carrying the gal operator, and we have isolated operator Cell 12, 847-854. 4. Taniguchi, T., O'Neill, M. & de Crombrugghe, B. (1979) Proc. NatL mutants with reduced repressor affinity (O0), as well as their Acad. Sci. USA 76, 5090-5094. revertants. We have also described a method of transferring 5. di Lauro, R., Taniguchi, T., Musso, R. & de Crombrugghe, B. these mutations to the host chromosome so that their effect on (1979) Nature (London) 279, 494-500. the gal operon could be studied in cis. 6. Adhya, S. & Miller, W (1979) Nature (London) 279, 492-494. Effect of cAMP on gal Constitutivity. The study of the be- 7. Aiba, H., Adhya, S. & de Crombrugghe, B. (1981)J. BioL Chem. havior of the gal operator affinity mutations (O0) located in the 256, 11905-11910. has an upon repres- 8. Buttin, G. (1963)J. Mot Biol 7, 183-205. chromosome revealed effect of cAMP gal 9. Miller, J. H. (1972) Experiments in (Cold Spring sion. The gal repressor is normally active on wild-type gal op- Harbor Laboratory, Cold Spring Harbor, NY). erator both in the presence and in the absence of cAMP, whereas 10. Yamamoto, T., Sobel, M. E., Adams, S. L., Avvedimento, E. V., strong Oc mutations are constitutive under both conditions. di Lauro, R., Pastan, I., de Crombrugghe, B., Showalter, A., However, the two mutations (01 and 02) described here cause Pesciotta, D., Fietzek, P. & Olsen, B. (1980)J. Biol Chem. 255, partial constitutivity, and only in a cya+ strain. In a cya- strain, 2612-2615. Oa 11. A. & W Methods 499-560. 0 and have no enzymes in Maxam, Gilbert, (1980) EnzymoL 65, effect. The induced levels of 12. Chen, C. W. & Thomas, C. A., Jr. (1980) Anal Biochem. 101, 339- both cya+ and cya- strains are normal, suggesting that the ac- 341. tivities of the promoters and their interactions with cAMP-CRP 13. Wilson, D. & Hogness, D. (1966) Methods Enzymol 8, 220-240. are not affected by the mutations. The latter is also evident from 14. Willard, M. & Echols, H. (1968)J. Mot Biol 32, 37-46. the cAMP-CRP binding and the transcription experiments with 15. Ullmann, A., Joseph, E. & Danchin, A. (1979) Proc. Natl Acad. O; in vitro. Sci. USA 76, 3194-3197. We have considered the models to the be- 16. Nissley, S. P., Anderson, W. B., Gottesman, M. E., Perlman, R. following explain L. & Pastan, I. (1971)J. BioL Chem. 246, 4671-4678. havior of the oa mutants. (i) Although the gal operator site 17. Nakanishi, S., Adhya, S., Gottesman, M. & Pastan, I. (1973)J BioT (around -60) and the cat locus (around -35) are physically dis- Chem. 248, 5937-5942. tinct, a functional competition between the gal repressor and 18. Irani, M., Orosz, L. & Adhya, S. (1983) Cell 32, 783-788. Downloaded by guest on September 27, 2021