general selection for specific dna-binding activities

Copyright 0 1986 by the Genetics Society of America

GENERAL SELECTION FOR SPECIFIC DNA-BINDING ACTIVITIES

NICHOLAS BENSON, PAUL SUGIONO, STEVEN BASS, LYNN V. MENDELMAN AND PHILIP YOUDERIAN

Department of Biologzcal Sciences, University of Southern Calijornia, Los Angeles, Calzfornia 90089-1481

Manuscript received October 1, 1985 Revised copy accepted May 7, 1986

ABSTRACT

We present a general strategy for the selection of bacterial clones that express DNA-binding activities corresponding to particular DNA recognition sites. T h e selection uses a “challenge phage” vector, P22 K n 9 arc-amH1605, into which is substituted a synthetic DNA-binding site for a site that controls transcription of the P22 antirepressor (ant ) gene. Constitutive synthesis of antirepressor channels a challenge phage into lytic development and efficiently kills an infected host, unless the substituted site is bound by a specific protein; in this case, the challenge phage prefers lysogenic development, and the host survives and acquires an antibiotic-resistance phenotype. Infections with challenge phages carrying the E. coli Lac operator, phage XOL, operator, or synthetic, “idealized” E. coli Trp and TnlO Tet operators select clones that express each of the corresponding binding activities. T h e use of challenge phage vectors may be extended to select clones that express eukaryotic DNA-binding activities.

ENE expression is often regulated by transcription factors that bind to G specific, short sequences of DNA. These cis-acting regulatory sites are normally situated close to a regulated structural gene; often, they overlap the promoter (RNA polymerase binding site) for the structural gene. Presumably, many eukaryotic transcription factors act as their prokaryotic counterparts, repressors and activators, by binding specific sites to inhibit or stimulate the initiation of transcription.

Motivated by the idea that some eukaryotic transcription factors, when expressed in prokaryotic cells, will retain their DNA-binding activities, we have developed a selection for isolating clones that express binding proteins corresponding to specific DNA sites. I t is our hope that, by extending this selection to eukaryotic regulatory sites, we shall be able to select clones that express corresponding transcription factors from plasmid cDNA libraries.

Our selection involves a derivative of temperate Salmonella phage P22 called a “challenge phage.” Challenge phages exploit the unique genetic properties of the P22 imml region (Figure 1). The primary feature of the imml region is the ant gene. The product of ant, antirepressor, can inactivate the primary prophage repressor, c 2 protein (LEVINE et al. 1975; BOTSTEIN et al. 1975;

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2 N. RENSON E T AL.

a.

b.

-35

Pant

arc * mnf -

Pmnr

-10 +I +34 c. ~~GATAGAAGCACTCTAC~V~~T~~TCAAT[PGGTCCACGGTGGACC$TATTGTGAGGTGAA~

Panr Omnr arc

FIGURE 1 .-The P22 imml region. a , Organization of the P22 prophage genome, showing the pattern of early transcription (arrows). The imml region (thick line) is embedded within the late operon, in the right prophage arm. b, Transcriptional organization of the imml region. The a n t promoter and Mnt operator are represented by a thick line; genes are indicated by open boxes. c , Partial sequence of the sense DNA strand of the an t promoter and Mnt operator, showing the position of the operator with respect to the most highly conserved promoter sequences and the initiation codon of the arc gene (boxed).

SUSSKIND and BOTsTEIN 1975). P22 c2 repressor, like X cI repressor, is directly responsible for preventing the transcription of the phage early, vegetative functions (BRONSON and LEVINE 1971; POTEETE et al. 1980; POTEETE and PTASHNE 1982). Constitutive synthesis of antirepressor prevents the action of c2 repressor and, thus, prevents lysogeny.

P22 must produce two repressors to maintain the lysogenic state: c2 repressor, to prevent the synthesis of early vegetative functions, and Mnt (maige- nance) repressor, to prevent the synthesis of antirepressor. Mnt protein acts by binding a single operator site located at the startpoint of transcription of the ant promoter I O prevent the binding of RNA polymerase (SAUER et al. 1983; \ r ~ ~ ~ ~ ~ ~ et al. 1985a; D. HAWLEY and W. R. MCCLURE, unpublished results).

Synthesis of antirepressor is also repressed during lytic development by the product of the first gene in the ant operon, arc (for antirepressor control) (SUSSKIND 1980). Upon infection of a sensitive host by P22 arc- phage, the state of occupancy of the M n t operator is a critical determinant of the developmental course of the infection. I f the Mnt operator is free of Mnt repressor, the ant gene is expressed at high constitutive levels, antirepressor inactivates c2 repressor, and the infection proceeds lytically. If, on the other hand, active M n t repressor preexists in cells infected by P22 arc- phage, Mnt binds its operator, ant is not transcribed and antirepressor does not interfere with the establishment of lysogen),.

Challenge phages are derivatives of P22 arc- phage that carry a substitution of another DNA-binding site for the Mnt operator (YOUDERIAN et al. 1983). Since the state of occupancy of the h ln t operator governs the developmental decision between lysis and lysogeny for P22 arc- phage, the state of occupancy

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CHALLENGE PHAGE SELECTION 3

of the substituted site governs this decision for a challenge phage. Challenge phages carry an additional mutation, Kn9, the substitution of an antibiotic- resistance determinant for the mnt repressor gene, so that lysogens surviving a challenge infection may be recognized by a selectable phenotype. The challenge phage arc gene is conditionally inactivated by an amber mutation to facilitate growth under permissive (suppressed) conditions (SUSSKIND 1980). These three mutations, arc-amber, an operator substitution and Kn9, conspire to enable a challenge phage to selectively lysogenize, and therefore spare from death, a host cell that produces a specific DNA-binding activity corresponding to its substituted site.

In previous reports, we demonstrated that infection with challenge phages carrying symmetric, mutant Mnt operators could be used to select mutant Mnt repressors with altered specificities of DNA binding (YOUDERIAN et al. 1983; VERSHON et al. 1985b). In this report, we extend this selection to show that challenge phages with any of several different binding sites of prokaryotic origin can be used to select clones that produce specific, corresponding DNA- binding activities.

MATERIALS AND METHODS

Bacterial and phage strains: Bacterial strains are derivatives of S. typhimurium LT2 or E. coli K12. Salmonella strains DB7000 (leuA-am414 sup"; SUSSKIND, WRIGHT and BOTSTEIN 1971) and its supE derivative MS1363 (SUSSKIND 1980), used for the permissive growth of P22 arc-am phages, have been described. Otherwise isogenic hosts, MS1868 (sup") and MS1883 (supE), also carry a mutation ( r - ) that inactivates one of several Salmonella restriction systems preventing efficient transformation ( G R A ~ ~ A , YOUDERIAN and SUSSKIND 1985). MS1367 is a derivative of MS1363 that carries a P22 c2+ mnt- prophage deleted for the imml region (SUSSKIND 1980). MS1582 is an immune (c2+ m d ) lysogen of prophage P22 16-amH1455 sieA44 Ap68tpfr49 in MS1363, used for the isolation of virulent mutants of P22 ( G R A ~ ~ A , YOUDERIAN and SUSSKIND 1985). Plasmids were constructed in E. coli strains C600 (APPLEYARD 1954) or MM294 (ME- SELSON and YUAN 1968) before transformation into Salmonella (LEDERBERG and COHEN 1974). F'lac episomes were introduced into MS1868 by conju al transfer from E. coli E5014 (F'lac+; SIGNER and WEIL 1968) and E. coli X90/F'lacl~1 (AMANN, BROSIUS and PTASHNE 1983).

Media, enzymes, and chemicals: Media and general phage techniques have been described (LEVINE 1957; LEVINE and CURTIS 1961; SIGNER and WEIL 1968; SUSSKIND, WRIGHT and BOTSTEIN 1971; BOTSTEIN, CHAN and WADDELL 1972; WEINSTOCK, SUSSKIND and BOTSTEIN 1979; YOUDERIAN et al. 1983). Antibiotics were obtained from Sigma Chemical Company. Restriction endonucleases, E. coli DNA polymerase I large fragment, T 4 polynucleotide kinase and T 4 DNA ligase were purchased from New England Biolabs. Deoxyribonucleoside triphosphates and ATP were purchased from PL Biochemicals. C Y - ~ ~ P - ~ A T P (700 Ci/mmol), used in DNA sequence analyses, was from ICN Biomedicals, Inc. Synthetic XbaI (5' CTCTAGAG 3') and XhoI (5' CCTCGAGG 3') linkers were purchased from New England Biolabs. Synthetic Lac operator DNA (see Figure 3) was a generous gift from PONZY Lu. Synthetic XO,, DNA was a generous gift from ROBERT SAUER. Synthetic consensus, symmetric T r p and Tet operator DNAs were synthesized on a Systec Microsyn P1450A Automated DNA Synthesizer, using phosphoramidite substrates from ABN and Baker HPLC grade solvents (CARUTHERS 1982).

Plasmids: General procedures used in the construction, selection, and purification of plasmids have been described (YOUDERIAN, BOUVIER and SUSSKIND 1982). All plasmids

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4 N. BENSON E T AL.

p PY 230 pMSlOO

ant‘

At1 Hlnf I I

t x b o 1 l inkers 1

0 FIGURE 2.-Construction of pPY 112. P22 DNA is indicated by thin lines; pBK322 DNA by

wavy lines. P22 genes, or portions of genes (’) are indicated by thick lines. P, PstI; R, EcoKI; F, H i n f f ; X , XbaI; 0, HpaI/PvuII junction. Only the relevant Hinfl site is indicated.

are derivatives of pBR322 (BOLIVAR et al. 1977). pPY23O is a subclone of the Sau3AI a n t promoter fragment from P22 sieA44 mnt-tsl P,,,R1204 arc-amH1605 in the unique BamH 1 restriction site of plasmid plcIS99, oriented clockwise (YOUDERIAN, BOUVIER and SUSSKIND 1982). T h e insert, flanked by EcoRI sites, was inverted by cleavage and religation to give pPY230. pMSlOO carries an AvaII-HpaI fragment of P22 DNA subcloned into the EcoRI-PvuII “backbone” of plasmid pBR322 oriented in a clockwise direction (VERSHON et al. 1985a). pPY97 carries the EcoRII-Hind111 fragment of P22 DNA carrying mnt, P,,, and the proximal two-thirds of the arc gene inserted in the EcoRI-Hind111 “backbone” of pZ150 (ZAGURSKY and BERMAN 1984), a derivative of pBR322 carrying the phage M 13 origin of replication and packaging initiation sites.

pPYl12, otherwise isogenic with pPY97, carries a substitution of a synthetic stretch of DNA including a unique XbaI site for the Mnt operator. As diagrammed in Figure 2, fragments from plasmids pPY230 and pMSlOO were used to construct the desired substitution (&,.,). Plasmid pPY230 was cleaved with Hinfl , which recognizes a new site created by the P,,,R1204 mutation; DNA was treated with DNA polymerase I large fragment in the presence of d N T P substrates to render the Hinfl ends flush; DNA was ligated with phosphorylated XbaI linkers, then cleaved with XbaI and PstI to generate a fragment carrying the R I 2 0 4 allele. In a parallel set of reactions, pMSlOO was cleaved witb EcoRI; EcoRI ends were rendered flush; DNA was ligated with phosphorylated XbaI linkers, then cleaved with XbaI and PstI t o generate a fragment carrying the wild-

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CHALLENGE PHAGE SELECTION 5 type arc gene. These two fragments were purified by electrophoresis through and elution from a polyacrylamide gel, then mixed and ligated to give pPY 1 12. Derivatives of pPY112 carrying XOLl and Oloc were transformed into MS1363 and crossed with phage P22 sieA44 Pa,,RE56 arc-amH1605 to separate the Pa,, mutation and the operator substitution (YOUDERIAN, BOUVIER and SUSSKIND 1982). Progeny phage having the genotype P22 sieA44 P,',, 0' arc-am were selected as virulent recombinants on immune host MS1582. Recombinant phages were used to transduce plasmid pMS97 from permissive host MS 1 363 to nonpermissive host MS 1868. Tetracycline-resistant transduc- tants were found to carry mutants of P,'n, plasmid pMS97 with the OLI and Olac substitutions (pPY 149 and pPY240, respectively). Plasmid pPY140 is otherwise isogenic with pPYll2, except that it carries an Xhol linker, in place of an XbaI linker, and the wild- type allele of P0,,RI2O4. pPY300 is a subclone of the Trp operator in pPY140. pPIlOOT is a subclone of the synthetic Tet operator in pPI39 (P. J. ISACKSON and K. P. BER- TRAND, unpublished results), a deletion derivative of pPY 140 lacking both the pBR322 DNA between the unique Hind111 and Sal1 sites and the M13 origin fragment. (The pBR322 deletion removes a copy of the pBR322 Tet operator.)

pMC7 (CALOS 1978) carries a HincII partial fragment with the lac14 gene inserted in the unique EcoRI site of tetracycline-resistant plasmid pMB9 (RODRIGUEZ et al. 1976). The plasmid source of X repressor, pLRB, carries an operon fusion of the Pl, ,W5 promoter to both the c l gene and the pBR322 tetA gene (NELSON and SAUER 1985). The source of TrpR protein, pPY150, was constructed by digestion of plasmids pRPG47 (ARVIDSON, CAN and GUNSALUS 1985) and pZ152 (ZAGURSKY and BERMAN 1984) with EcoRI and PvuII and by ligation of the products. It carries the MI3 origin from pZ152 and a fusion of the Pl,,W5 promoter to the trpR structural gene from pRPG47. The source of TetR protein is plasmid pBI501 (ISACKSON and BERTRAND 1985), a subclone of a HinclI fragment containing the TnlO tetR structural gene fused to the P1.,W5 promoter in vector pUC8 (VIEIRA and MESSING 1982).

DNA sequence analyses: Challenge phage DNA, single-stranded plasmid DNA or double-stranded plasmid DNA was sequenced by the method of SANGER, NICKLEN and COULSON (1977), using a synthetic oligonucleotide complementary to the 5' end of the arc gene as primer (5' CGGCATTTTGCTCATTCC 3'). Single-stranded plasmid DNA was prepared by the method of ZAGURSKY and BERMAN (1 984).

Construction of challenge phages: Challenge phages were constructed by crosses between plasmids and phages (YOUDERIAN et al. 1983). The operator sequences of challenge phages were determined either directly from phage DNAs or from the plasmids used in the construction crosses.

RESULTS

Construction of challenge phages: Challenge phages carrying substituted binding sites were constructed in two steps. First, sites were synthesized and cloned into the unique restriction sites of plasmids pPY 1 12 or pPY 140, derivatives of plasmid pBR322 carrying the P22 antirepressor control region. Sec- ond, sites were exchanged for the Mnt operator o n P22 K n 9 arc-am phage by homologous recombination between the plasmid and phage genomes.

As shown in Figure 3, p P Y l l 2 and pPY140 carry a subcloned region of P22 DNA that includes the mnt gene, the ant promoter and a portion of the arc gene. In place of the Mnt operator, p P Y l l 2 and pPY140 carry synthetic stretches of DNA comprised of adjacent XbaI and EcoRI, or Xhol a n d EcoRI, restriction sites, respectively. To clone new DNA-binding sites, plasmid DNA was cleaved with XbaI or XhoI, and cohesive ends were rendered flush in the presence of E. coli DNA polymerase I large fragment and deoxyribonucleoside triphosphates. Linear plasmid DNA was ligated with short, synthetic, double-

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6

a.

N. RENSON E T AL.

- I ? + I

b. p PY 97 TATATTCTCAAT~GGTCCACGGTGGACCT

~ P Y 112 T~TTLTCTAGAGAATTCCACCT ...... XboI

p PY 140 TATATT~CTCGAGGAATTCC@ACCT ...... C.

OC plasmid ZZl--

2 L I

Kn9 0’ afc-am Phage - challenge 4 phage Kn9 0‘ afc-om

FIGURE 3.-Challenge phage construction. a , Structure of plasmid pPY97, a subclone of the wild-type mnt gene, ant promoter and Mnt operator in plasmid pZ150. Transcription of the tetA gene, indicated by the arrow, originates from the ant promoter within the EcoRII (B) to Hind111 (H) fragment of P22 DNA. b, Sequences of the wild-type promoter/operator region in pPY97, and the substitutions (boxed) within this region carried by otherwise isogenic plasmids pPY 1 12 and pPY140. Unique restriction sites within the subsitutions are designated; note that they are flanked on the right by EcoRI sites (5’ GAATTC 3’) that are not present in pPY97. The PantK1204 mutation (-10, T:A + G:C) is circled. c, Diagram of the cross between plasmids and P22 Kn9 arc-am phage to construct challenge phage [P22 DNA (thin line); Tn903 (Kn9) DNA (open box); pBR322 DNA (hatched box); operator substitution (filled box)].

stranded oligonucleotides representing the new binding sites. The circular products of these reactions include plasmids with a new site positioned so that its first base pair aligns with the first base pair of the Mnt operator in the wild-type sequence, the startpoint of transcription of the ant promoter.

As illustrated in Figure 4, we have subcloned oligonucleotides corresponding to the natural binding sites for E. coli Lac repressor and phage h cI repressor, and synthetic, “idealized” binding sites for E. coli Trp and transposon TnlO Tet repressors into the unique restriction sites of these plasmids. These recombinant plasmids were transformed into Salmonella host MSl883 and were crossed with P22 KnY arc-am phage. Recombinant challenge phages that had lost the Mnt operator and had acquired the new binding sites were selected as

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CHALLENGE PHAGE SELECTION 7 a OIOC

X0,l

’rrp”

“fer“

b. 7f.p”

f f..

0roH

fmQ

c. 7 G f #I

-1p *1 tl?. t2p ”CTAG AATTGTCAGCGGATAACAATT CTAGAGAA

-19 +I t10 t 2p

-I? t 1 ‘I? e20

~TATATTJCCTCGA ACTAGT~AACTAGT TCGAGG AA

[ T T ~ T C T A G +AT c A C C G ~ ~ AGTGGTA CTA GAG A A

*I 2 P ‘2,O &%~iiiTcGp. CTCTATCATTGATAGAG TCGAGGAA

4 t I? TCGAACTAGTTAACTAGTTCGA

TCG AACTAGTTA ACTAGTBCGC

EGIACTAG&ACTAGTGCM

-1p +! TCGIACJCTTTAFSAGTACAA

-2p -I,O

-y -3,O

Y tIp CTCTATCATTGATAGAG

FIGURE 4.-Design of operator substitutions on challenge phages. a , Positions of cloned asymmetric wild-type Lac and OLI operators, and idealized, symmetric Trp (“trp”) and Tet (“tet”) operators with respect to the ant promoter. “+1” designates the base pair corresponding to the wild- type P,,, startpoint. The conserved Pant -10 hexamer is boxed. b, Comparison of the consensus Trp operator (“trp”) with the three naturally occurring Trp operators controlling the trp, aroH and trpR operons (adapted from GUNSALUS and YANOFSKY 1980). The positions of each operator with respect to the promoters they control are indicated. Differences from the idealized sequence are underlined. c, Comparison of the consensus Tet operator with the two operators controlling the tetA promoter (see DANIELS and BERTRAND 1985).

virulent progeny able to form plaques on an immune (P22 c2+ mnt+) lysogen. To distinguish recombinant challenge phages from mutants of the parental P22 K n 9 arc-am phage that had acquired mutations in the Mnt operator ren- dering the synthesis of antirepressor constitutive, challenge phage DNA was digested with endonuclease EcoRI to verify the presence of the additional EcoRI site within the operator substitution. The precise structures of the operator substitutions were confirmed by DNA sequence analyses.

Immunity properties of a challenge phage with the Lac operator: A challenge phage carrying the Lac operator was constructed from a derivative of pPY 1 12 carrying a cloned synthetic 2 l-mer identical to the wild-type Lac operator (Figure 4). Table 1 shows that P22 Kn9 Oloc arc-am phage, as well as its P22 K n 9 arc-am parent, forms plaques with normal efficiency on the permissive supE (P22 c2+ mnt-) lysogen, MS1367, or on MSl367 carrying plasmid pMB9 (RODRIGUEZ et aE. 1976), indicating that the synthesis of antirepressor upon infection of these hosts leads to lytic development. MS1367 is immune to superinfecting ant- phage, but is sensitive to superinfecting ant+ phage; the

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8 N. BENSON E T AL.

TABLE I

Substitution of the Lac operator for the Mnt operator confers new immunity properties on P22

Bacterial host

MS1363 MSl367 MSl582 MS1367/pMB9 MS13671pMC7 Phage (none) ( C Z + ) (c2+mnt+) (c2') (cZ+lacI+)

KnY Om,, arc-am 1 .o 0.9 1 .o 1.1 Kn9 01,, arc-am 1 .o 1 .o 0.7 0.9 4 0 - 5

Phage suspensions were diluted and plated on the indicated bacterial strains. Plaques were scored after incubating for 12 hr at 37". Efficiencies of plating on each bacterial strain are the titers on each indicator divided by the titer on MS1363. All bacterial hosts carry the supE40 amber suppressor allele and produce the repressors indicated below the strain designations.

TABLE 2

Survival upon challenge phage infection depends on the amount and activity of repressor

Concentration of IPTG (M)

Bacterial host 0 I 0-5 10-4 10-3

MS 1868 -a 0-7 <lo-? MSl868/F'lacI+ 2 x 1 0 - ~ 2 x 10-5 4 0 - 7 <io+ MS1868/F'lacP1 0.5 0.02 3 x 10-7 2 x MS1868/pMC7 0.5 0.5 0.5 0.2

Cells were grown in LB medium at 37" to a density of 2 X 108/ml, diluted fourfold, grown two generations in the presence of the indicated amounts of IPTG (isopropy~-@D-galactopyrano- side) and infected with P22 KnY Ole, arc-am phage at a multiplicity of infection of 20. After 20- min adsorption at 25" , infected cells were diluted and spread on plates containing the indicated amounts of IPTG. Entries in the table are the efficiencies of survival of the hosts following challenge phage infection, defined as the titer of each infected cell culture assayed on green/kan plates divided by the titer of input cells assayed on green plates.

superinfecting phage must synthesize antirepressor and inactivate c2 repressor produced by the prophage in order to grow (BOTSTEIN et al. 1975). However, the parental phage cannot form plaques with normal efficiency on MS1582, a lysogen that produces both c2 and Mnt repressors ( G R A ~ A , YOUDERIAN and SUSSKIND 1985). Likewise, the challenge phage with the Lac operator cannot form plaques efficiently on MS1367 carrying a derivative of pMB9 that produces Lac repressor, pMC7 (CALOS 1978). These hosts are homoimmune to phages with the binding sites corresponding to the repressors they produce. Consistent with this idea, mutants of P22 Kn9 Olac arc-am phage that form plaques efficiently on the "Lac-immune" Salmonella host are found to carry constitutive mutations that inactivate the Lac operator (data not shown).

Selective recovery of cells that produce Lac repressor: When a sensitive sup" host carrying pMC7 is infected at high multiplicity with a challenge phage carrying a substituted Lac operator, about 20-50% of the cells survive infection and acquire an antibiotic-resistant phenotype (Tables 2 and 3). In contrast, an otherwise isogenic host carrying the parental plasmid, pMB9, is efficiently killed upon infection of the challenge phage. The ability of a host that pro-

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CHALLENGE PHAGE SELECTION

TABLE 3

Cells that produce specific DNA-binding activities may be recovered from mixed populations using the challenge phage selection

9

~ ~~ ~~ ~- ~~

Relative concentration

2 x 10-6 2 x IO-* 2 x 10-5

Expected survivors (520) (265) (27)

Efficiency of recovery 0.3 0.2 0.2 Observed survivors 165 57 5

Cultures of MS1868/pMC7 (lac@ and MS1868/pMB9 (lacl-) were grown to densities of 3 X 10’ cells/ml in LB/tet medium at 37”. The MS1868/pMC7 culture was serially diluted and added to aliquots of the undiluted MS1868/ pMB9 culture to give mixed cultures with the indicated relative concentration of MS1868/pMC7. Aliquots of the mixed cultures were infected with P22 Kn9 Ole, arc-am phage at a multiplicity of infection of 20, incubated at 25” for 20 min to ensure efficient adsorption and then distributed to green/kan/tet plates. Colonies representing observed challenge phage survivors were scored after incubating for 24 hr at 37”. The number of expected survivors was calculated from the titer of input MS1868/pMC7 cells. All five survivors of the most dilute mix were determined to carry pMC7 upon further analysis.

duces Lac repressor to survive challenge phage infection is dependent on both the amount and activity of the Lac repressor produced by the host. As shown in Table 2, a host carrying the high copy-number plasmid pMC7 that produces Lac repressor from the strong lacZQ promoter is lysogenized by the challenge phage even at high concentrations of inducer. In contrast, when Lac repressor is produced from a single-copy lade’ promoter carried on an F’ episome, a high concentration of inducer prevents lysogeny of the challenge phage. The amount of repressor produced from the wild-type l a d promoter in single copy (F’lac+) is sufficient to permit lysogeny with reduced efficiency in the absence of inducer.

How efficiently may the challenge phage selection be used to recover rare clones that produce Lac repressor from a mixed population of cells? A recon- struction experiment was performed to answer this question. We mixed various proportions of supo cells carrying pMB9 with otherwise isogenic cells carrying pMC7. As shown in Table 3, infection of mixed populations with challenge phages permits the selective recovery of cells carrying pMC7 ( lad+) us. cells carrying pMB9 (lacZ-) at frequencies as low as one in a million on a single plate. It follows that the challenge phage selection may be used to recover clones that produce a specific DNA-binding activity efficiently from large, mixed populations of cells; that is, from plasmid or phage expression libraries that include rare clones producing specific DNA-binding activities.

Challenge phages with other operators: T o test whether the challenge phage selection could be applied in a general way to DNA-binding protein/ site interactions, we tested whether other prokaryotic DNA-binding sites, when cloned onto challenge phages, could be used to select bacterial clones that produce their specific DNA-binding activities. As shown in Table 4, challenge phages carrying the XOLl operator and phages with the “idealized” E . coli T r p

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TABLE 4

The challenge phage selection works for a wide variety of DNA-binding proteins

Challenge phage operator

Bacterial host Repressor Mnt 0 1 1 T r p Tet

MSl868/F’ZacP’ None 4 0 - 7 4 0 - 7 < I 0-7 <io-’ MSl868/F’lacP’(pPY97) Mnt 0.2 4 0 - 7 <IO-’ < 10-7 MS 1868/F’lacP‘(pLRB) X C I <io-’ 0.2 < i o - 7 < 10-7 MSl868/F’Zac~’(pPY150) TrpR 4 0 - 7 <IO-’ 0.3 <IO-’ MS1868/F’Iac~’(pB1501) TetR 4 0 - 7 <IO-’ 4 0 - 7 0.3

Cultures of MSl 868/F’lacP1, MSl868/F’Zac~’ (pPY97), MSl 868/F’ZacP1 (pLRB), MS1868/ F’lacP’ (pPY150) and MS1868/F‘ZacPL (pB1501) were grown at 37” in LB medium to a density of 5 X 108/ni1, diluted fourfold, grown two generations in the presence of M IPTG (to induce the synthesis of the particular plasmid-coded repressor protein) and infected with a multiplicity of 20 of each challenge phage. After 20-min adsorption at 25”, infected cells were diluted and spread on green/kan plates containing M IPTG. Numbers in the table are the efficiencies of survival of host cells following challenge phage infection, defined as the titer of each infected cell culture assayed on green/kan plates divided by the titer of input cells assayed on green plates.

and TnlO Tet operators selectively lysogenize hosts that produce their respec- tive repressors. It is clear that the challenge phage selection can be applied to a wide variety of specific DNA binding site/binding protein interactions.

Moreover, synthetic, symmetric versions of the Trp and Tet operators be- have as natural operators should. Together with their cognate repressors, these “idealized” operators can effect repression of the ant operon. These findings are consistent with those of SADLER, SASMOR and BETZ (1983), who demonstrated that a symmetric version of the Lac operator binds wild-type repressor with tenfold higher affinity than does the asymmetric, wild-type operator. Re- cently, we have determined that the operator of SADLER, SASMOR and BETZ (1983), a symmetric version of the X operator, and an “idealized,” symmetric version of one of the E. coli Gal operators are sensitive to each of their cognate repressors, as determined by the challenge phage selection (data not shown). Therefore, we expect that the study of many repressor/operator interactions may be modeled using symmetric or nearly symmetric operator sites.

DISCUSSION

The majority of prokaryotic repressors that recognize specific sequences of DNA are thought to act by binding to operator sites that overlap the RNA polymerase binding sites (promoters) they control. Presumably, when a repressor binds its operator, it sterically excludes the binding of polymerase to the promoter; that is, repressors are competitive inhibitors of RNA polymerase binding. Consistent with this idea, we have shown that five different operators can function at the same site within the P22 ant operon (the startpoint of transcription).

The efficacy of a repressor as an inhibitor depends on both its intrinsic affinity for a binding site as well as the position of the binding site with respect to the promoter it controls (REZNIKOFF et al. 1969; see also, GUSSIN et al.

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1985). We have subcloned both the Lac and Tet operators at the startpoint of ant promoter transcription, at positions analogous to those within their original operons, and have shown that these operators function when paired with the heterologous ant promoter. The locations of the T r p and XOL, operators within their wild-type operons are different from the location of these operators on challenge phages (with respect to conserved promoter sequences). In both cases, the site still functions as an operator when placed at the startpoint of transcription. The operons controlled by X and Tet repressors have reiterated operators; in both cases, we have shown that a single, minimally defined operator site suffices for repression of an operon.

In previous reports (YOUDERIAN et al. 1983; VERSHON et al. 1985b), we demonstrated that, starting with a symmetric mutant operator unable to bind wild-type P22 Mnt repressor, we can select mutant repressors that recognize this mutant operator. This simple genetic strategy for selecting mutant repressors with altered specificities of binding may be applied systematically and exhaustively to a single symmetric operator. One can synthesize all possible symmetric variants of a reference, symmetric operator; introduce this set of sites into a challenge phage vector; identify the subset of mutant operators that fail to bind wild-type repressors; and isolate mutant repressors that bind each mutant operator.

In this paper, we have established that challenge phages with idealized, symmetric versions of the T r p and Tet operators can be used to select for binding of the wild-type Trp and Tet repressors (see Figure 4). Experiments are in progress to identify the genetic determinants of each of these DNA binding interactions in a thorough manner. More recently, we have shown that idealized, symmetric versions of the X and Lac operators may also be used as starting points for the analyses of these two interactions (data not shown).

We have also set the stage for the systematic isolation of eukaryotic DNA- binding proteins from expression libraries, starting with the sites they recognize. Eukaryotic binding sites can be introduced onto challenge phages with relative ease. Provided that their corresponding binding proteins can be expressed in sufficient amounts and activities in Salmonella hosts, we can select bacterial clones that produce these proteins from large, mixed populations of cells using challenge phages.

There remain two main hurdles to overcome in extending the challenge phage selection to eukaryotic DNA-binding proteins. First, we must design efficient, high copy-number plasmid expression vectors for the production of eukaryotic proteins in prokaryotic hosts, so that we might saturate binding sites with sufficient amounts of active protein. In this respect, the challenge phage selection is biased in our favor, since the source of protein may be present in high copy number, whereas the repressed binding site on the challenge phage is present in single copy in a lysogen. Second, we have yet to understand the dependence of repression on the distance between the promoter and a cloned operator. Since the boundaries of many eukaryotic sites are imprecisely defined, it is important to know what possible range of relative positions within an operon will allow an operator to function. Experiments are in progress to

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12 N. BENSON E T AL.

determine precisely how induction ratio varies as a function of increasing distance between a promoter and an operator site placed from one to 17 base pairs 3’ relative to the transcription startpoint.

In summary, we have shown that the challenge phage selection may be applied to a wide variety of specific, prokaryotic DNA-binding interactions and have laid the foundation for extending the selection to the isolation of eukaryotic DNA-binding proteins.

We are especially indebted to ROBERT GUNSALUS for providing us with plasmid pRPG47, and to PAUL ISACKSON and KEVIN BERTRAND for providing us with pBI501 and pPIlOOT before publication. We are also grateful to RICHARD DEONIER, DAVID GALAS, PONZY Lu and ROBERT SAUER for gifts of synthetic oligonucleotides, plasmids and bacterial hosts. We thank PETER BERGET and MIRIAM SUSSKIND for their detailed criticisms of the manuscript. This research was supported by National Institutes of Health grant GM34150. P.S. WAS the recipient of a 1984 summer fellow- ship from the Student Research Associates Prograni of the California Heart Association.

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