JOURNAL OF BACTERIOLOGY, May 1968, P. 1835-1843Copyright © 1968 American Society for Microbiology

Vol. 95, No. 5Printed in U.S.A.

Requirements for Macromolecular Synthesis in theEstablishment of o-Galactosidase Repression in

Zygotes1JOHN H. CASTER2 AND NORMAN E. MELECHEN

Department of Microbiology, Saint Louis University School of Medicine, Saint Louis, Missouri 63104

Received for publication 16 February 1968

Inhibitors of protein synthesis do not consistently prevent formation of the lacoperon repressor, according to several published reports, although direct evidenceindicates that the repressor is a protein. Inhibition of ribonucleic acid (RNA)synthesis has never been shown to block lactose repression. These results haveraised the possibility that repressor is synthesized in some unusual fashion. Wehave studied the effect of various inhibitors upon the establishment of repression inzygotes, utilizing conditions which minimize catabolite repression. Inhibition of pro-tein synthesis by either chloramphenicol treatment or tryptophan deprivationblocked repressor formation in our experiments. Sodium borate and 6-azauracilare compounds reported to be specific inhibitors of RNA synthesis, and their be-havior in control experiments is consistent with this specificity. Both delayed theestablishment of repression. Thymine deprivation, either by starvation of a thymineauxotroph or by treatment with 5-fluorodeoxyuridine, did not delay the onset ofrepression. We conclude that repressor formation requires RNA synthesis andprobably utilizes the usual protein-forming mechanisms.

When a lactose operon regulator gene (i+) anda 3-galactosidase structural gene (z+) are trans-ferred together by conjugation into an i-z-Escherichia coli female, the z+ character is ex-pressed immediately, even in the absence of anexogenous inducer, and j3-galactosidase is syn-thesized for 1 to 2 hr before normal repressionbecomes established. This period is thought to bethe time required to accumulate effective amountsof cytoplasmic repressor (24).A number of recent reports, especially that of

the isolation of an i gene protein by Gilbert andMuller-Hill (9), provide substantial evidence thatthe lac repressor is a protein or at least containsan essential protein component. However, theconflicting results of in vivo inhibitor studies haveraised the possibility that repressor is not formedby the usual mechanisms of protein synthesis.Pardee and Prestidge (25) originally reported thatneither chloramphenicol nor 5-methyltryptophanprevented the formation of repressor, and Syp-

1 This work constitutes part of a thesis submittedby John H. Caster in partial fulfillment of the require-ments for the Ph.D. degree.

2Present address: Department of Microbiology,The School of Medicine, University of Pennsylvania,Philadelphia, Pa. 19104.

herd and DeMoss (31) found that chlorampheni-col apparently stimulated its production. Morerecently, Barbour and Pardee (2) reported thattreatment with puromycin or 4-methyltryptophaninhibited repressor synthesis, but they were unableto come to any definite conclusion regarding theeffect of chloramphenicol. H. V. Rickenberg(Federation Proc. 26:678, 1967) found that re-pressor was apparently formed during chloram-phenicol treatment, as well as during eithertryptophan or cysteine starvation, but not duringdeprivation of methionine, arginine, isoleucine, orhistidine. Horiuchi and Ohshima (13) are alonein reporting that chloramphenicol does inhibitrepressor formation. They also found the puro-mycin treatment or methionine deprivation wascompletely inhibitory and that 5-methyltrypto-phan was partially inhibitory.There has been no direct evidence that ribo-

nucleic acid (RNA) synthesis is required forlactose repression. Horiuchi and Ohshima (13)reported that uracil deprivation did not preventthe establishment of repression, even thoughRNA synthesis was reduced to 4% of that incontrol cultures. Similarly, 5-fluorouracil treat-ment did not affect repression. Rickenberg (Fed-eration Proc. 26:678, 1967) used borate treatment

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to inhibit RNA synthesis and found that theestablishment of repression was not affected,dcspite an absence of measurable de novo RNAsynthesis. Both groups of investigators have,therefore, suggested the possibility that the re-pressor is synthesized in an abnormal fashion,perhaps directly on the deoxyribonucleic acid(DNA) template.We have studied the effect of inhibition of

DNA, RNA, and protein synthesis upon theestablishment of p-galactosidase repression inzygotes under conditions which reduce cataboliterepression. In this paper, we present data whichdemonstrate that RNA synthesis is required forrepressor formation and that inhibition of proteinsynthesis, by either chloramphenicol treatment ortryptophan deprivation, will delay the onset ofrepression. Our data support the view that thesynthesis of repressor utilizes the usually acceptedmechanisms of protein synthesis.

MATERIALS AND METHODSBacterial strains. The following are derivatives of

E. coli K-12: x2l2, F- (Plkc) ara-2- leu- azir Tlrlacz- laci-s pro-3 T6r ade-3- trp- strr mtlh xyl-2-thi-; x434, Hfr OR5 prototroph lac+ T3r str5 (bothobtained from Roy Curtiss, III); and SL67-101, athy- derivative of X212 selected on aminopterin-agar plates (5). The X212 strain is stringent by the testof Alfoldi, Stent, and Clowes (1).

Culture media. Liquid media were based on M63medium (24) adjusted to pH 7.0 and supplementedwith 0.125 mg/ml each of: DL-alanine, L-argi-nine HCl, L-asparagine H20, L-asparatic acid, L-cysteine -HCI, L-glutamic acid, L-glutamine, glycine,L-histidine, L-isoleucine, L-leucine, L-lysine * HCl,DL-phenylalanine, DL-serine, and L-threonine, andwith 0.01 mg of thiamine per ml. Unless otherwiseindicated, this medium received the following addi-tional supplements: for overnight cultures, 0.05mg/ml each of adenine, proline, tryptophan, andthymidine (when required), and 2.0 mg of glucoseper ml; for logarithmic parental cultures, 0.05 mgof adenine per ml, 0.01 mg of proline per ml, 0.02mg of tryptophan per ml, 0.02 mg of thymidine perml (when required), and 2.0 mg of glycerol per ml.

Defined solid media were based on medium A (asused at Cold Spring Harbor Laboratory), whichcontained (milligrams per milliliter): K2HPO4, 10.5;KH2PO4, 4.5; Na citrate 5H20, 0.47; MgSO4, 0.05;(NH4)2SO4, 1.0; and agar, 15. To select Lac+ Strrrecombinants, this was supplemented with strepto-mycin, 1.0 mg/ml; lactose, 2.0 mg/ml; adenine,proline, leucine, and tryptophan, 0.02 mg/ml each;and, when necessary, thymine, 0.1 mg/ml. Tryptoneagar (Difco tryptone, 10 mg/ml; NaCl, 5 mg/ml;thiamine, 0.01 mg/ml; and agar, 15 mg/ml) was usedwhen a complete medium was required.

Mating procedure. Except where indicated, cultureswere maintained at 37 C with active aeration. Strainsfor mating were subcultured and grown to 2 X 10to 4 X 108 cells/ml in the medium described above.

They were then centrifuged and resuspended in two-fifths of the original volume in identical medium butwithout proline or glycerol. At t = 0, male and femalecells in a ratio of about 3:7 were mixed in a thin layerof medium (adjusted with 1 N HCl to pH 6.3) inthe bottom of a large flask and incubated withoutaeration for 35 min. The culture was then shakenviolently, diluted fivefold in medium to which wasadded 1.0 mg of streptomycin per ml and 0.03 mg ofsodium lauryl sulfate per ml (to inhibit the strepto-mycin-sensitive males and to prevent further mating),and then divided and treated as required. Isopropyl-fl-D-thiogalactopyranoside (5 X 10-4 M) was used toinduce ,B-galactosidase.

f3-Galactosidase assay. The assay for j3-galactosidasewas adapted from Melechen (21). To conserve ma-terials in some experiments, 0.1-ml samples werediluted in 0.9 ml of chilled reducing buffer (27) with0.01 ml of 5% sarcosyl and 0.01 ml of toluene, theincubation time was extended to allow adequatecolor development, and the optical density was readquickly at 420 miu against a reagent blank. The ab-sorbance, measured in a Gilford model 2000 spectro-photometer, was proportional to enzyme concentra-tion up to at least A4o = 2.80. No loss of enzyme ac-tivity was detected during incubation for as long as300 min. One enzyme unit is defined by the release of1.00 m,umole of o-nitrophenol per min.

Chemical assays. The chemical assays used todetermine the concentration of macromolecules havebeen described previously (30).

Control cultures for biochemical manipulations. Forpurposes of comparing the course of macromolecularsyntheses in mating cultures, containing not only thezygotes but the unmated males and females, culturesof the F- strain alone were treated in the same manneras mating cultures, with exogenous proline addedafter 35 min of deprivation to mimic the entrance ofthe pro+ gene into the zygotes. Such control cultures,however, tended to have lower initial levels of proteinand RNA than did zygote cultures (following theperiod of proline starvation), because male cells inthe latter continued to synthesize these macromole-cules until inhibited by streptomycin. The controlcultures may also have exhibited more synchronythan the zygote cultures, since the zygotes did notall receive the pro+ gene at the same time.

Isotope incorporation experiments. De novo proteinsynthesis in zygotes was followed in some experimentsby the incorporation of DL-tryptophan-3-14C (VolkRadiochemical Co., Burbank, Calif.) into hot acid-insoluble material. The mating culture was starvedfor tryptophan from t = 0. At 35 min, about 0.02,Ac of 14C-tryptophan and 0.02 mg of 12C-tryptophanwere added per ml of culture. At intervals, 1.0-mIsamples were precipitated with cold 0.3 M trichloro-acetic acid, heated to 90 C for 15 min, filtered (0.45-,Afilter; Millipore Corp., Bedford, Mass.), washed,dried, and counted in a windowless gas-flow counter(Nuclear-Chicago Corp., Des Plaines, Ill.).

Inhibitors and analogues. The sources of the in-hibitors were as follows: chloramphenicol, Parke,Davis & Co., Detroit, Mich.; 5-methyl-DL-tryptophan,Mann Research Laboratories, New York, N.Y.;

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5-fluorodeoxyuridine, Hoffman-La Roche, Inc., Nut-ley, N.J.; 6-azauracil, Sigma Chemical Co., St. Louis,Mo.; streptomycin, Chas. Pfizer & Co., Inc., Brook-lyn, N.Y.; aminopterin, Lederle Laboratories, PearlRiver, N.Y.

Isopropyl-jS-D-thiogalactopyranoside and o-nitro-phenyl-,f-D-galactopyranoside were obtained fromMann Research Laboratories.

RESULTS

Experimental system. The experiments reportedhere demonstrate the effects of various inhibitorson the establishment of i gene repression inzygote cultures, as reflected by changes in (-

galactosidase synthesis. Parental cells were grownin glycerol-amino acid medium and then shiftedto a medium containing amino acids but lackingglycerol, to minimize catabolite repression (2, 16).Male cells were inhibited with streptomycin, andunmated F- cells were inhibited by prolinestarvation. Since the x434 Hfr transfers thepro-3+ locus immediately before the lace locus,all lac+ zygotes are pro+ (28). Induced and con-stitutive enzyme synthesis in the presence orabsence of proline is shown in Fig. la. j3-Galacto-sidase synthesis in the absence of proline beganonly slightly later than in cultures to which pro-line was added when the mating was interrupted(35 min). Induction increased (3-galactosidaseproduction even before the formation of effectivelevels of repressor, suggesting that a low level ofinducer-specific repression occurs early in thezygotes. This phenomenon has been discussed byBarbour and Pardee (2).As a result of proline deprivation, the cell

density in the cultures remained nearly constant(Fig. lb), and protein synthesis was effectivelyrestricted to the zygotes. Incorporation of 14Ctryptophan into hot acid-insoluble material byunmated F- control cultures was inhibited atleast 90% by proline deprivation. When macro-molecular synthesis was measured colorimetri-cally in a zygote culture and compared with thatin a homogeneous control culture (proline addedat 35 min), the increase in protein concentrationin the zygote culture (between 50 and 210 min aftermating) was 11% of that in the control (Table 1).This is consistent with the estimated proportionof zygotes in mating cultures, based on recom-bination frequencies and rates of induced ,B-galactosidase synthesis. The relative increase inRNA in the zygote culture was about 16% of thatin the control, and the increase in DNA wasabout 48% of that in the control. Most of theDNA in the mated culture was made before 90min. This indicates that unmated females initiallysynthesize appreciable amounts of DNA eventhough deprived of proline.

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FIG. 1. Characteristics of the mating system. x434,fr i+ z+ pro+ trp+ sir8, was mated with x212, Pzr pro-3t trpr strr, at t = 0 in the absence ofprolineglycerol. At 35 min, the mating was interrupted,

e culture was divided, and inducer or proline, orth, was added. (a) #-Galactosidase synthesis. En-Pme induced with IPTG at 35 min (@) or not inducedk). Parallel cultures to which proline was added atmin and induced (0) or not induced (A). There wasdetectable ,B-galactosidase synthesis by Hfr con-

ol cultures under these conditions. (b) Total viableUls and strr lac+ recombinants in cultures deprivedproline and induced (@) or not induced (A), astermined by dilution and plating on suitable media.ie ordinate is a logarithmic scale.

"4C-tryptophan incorporation was used in sometrly experiments to correct for inhibitor-inducedriations in the rate of overall protein synthesis,it it was discovered that the kinetics of 14C-yptophan incorporation differed from thenetics of net protein synthesis as measureddorimetrically. Induction of ,s-galactosidase did)t alter the rate of net protein synthesis, asteasured by either method, so it was possible toatermine the effect of inducer-specific repressionsimple comparisons of induced and non-

duced cultures.Mating efficiency varied in these experiments-tween 10 and 30%, with corresponding varia-xns in enzyme level. The Hfr strain was reselectedhen efficiency fell below 10%.Inhibition of protein synthesis. If the repressora norrmally synthesized protein, it should be

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TABLE 1. Net synthesis ofmacromolecules in a mated culture of x434 X x212, compared with synthesis bythe F- strainz alonie under comparable conditionsa

Zygote culture Control culture

Macromolecule Concn (jtg/ml of culture) Concn (jsg/ml of culture) culture compared toIncrease Increase controlb

Initial Final Initial Final(50 min) (210 min) (50 min) (210 min)

Protein. 34 52 53 11 61 455 11RNA....... 14 20c 43 5 18 260 i 16DNAd ....... 1.2 3.0 150 0.9 3.7 311 48

a Proline was added to the control culture after 35-min deprivation to mimic the introduction of thepro+ gene into the zygotes.

b Increase in zygote culture compared to control = (% increase in zygote culture/% increase in con-trol culture) X 100.

c RNA concentration given is maximal value, obtained at 110 min. This gradually decreased to 16,ug/ml at 210 min.

d Ratio of initial DNA concentrations (1.2 versus 0.9 ,ug/ml) is similar to ratio of initial viable cellcounts: 108 cells/ml in zygote culture versus 8 X 107 cells/ml in control.

possible to prevent or at least delay its formationby treatment with inhibitors of protein synthesis.It is surprising then, that most published reportsstate that chloramphenicol, a well-known inhibi-tor of protein synthesis (4, 32), does not stoprepressor synthesis. We have tested the behaviorof chloramphenicol in our mating system, and inaddition we have deprived zygotes of tryptophanto inhibit protein synthesis. Our results indicatethat either treatment will delay the establishmentof repression.

Figure 2 shows the effect of treatment withchloramphenicol (25 mig/ml) upon the establish-ment of repression in a mating of X434 x X212.The inhibitor was added at 35 min and removedat 100 min. The induced culture pretreated withchloramphenicol made only about one-third asmuch ,3-galactosidase as did the induced controlduring the same period. This was correlated witha general depression of protein synthesis afterchloramphenicol treatment. The noninduced cul-ture, on the other hand, made significantly moref-galactosidase after removal of chloramphenicolthan did the noninduced control. This proportion-ately greater constitutive synthesis after inhibitortreatment can have resulted only from a delay inthe accumulation of the i gene repressor, sinceconstitutive 3-galactosidase is at least as sensitiveto changes in catabolite repression as inducedsynthesis (6). The incorporation of 14C-trypto-phan into hot acid-insoluble material was meas-ured in this experiment, and the maximal specificactivity (the amount of enzyme made during agiven period divided by the "4C-tryptophan in-corporated) was attained between 90 and 120min for the noninduced control, and between

140 and 160 min in the noninduced chlorampheni-col-treated culture, which is consistent with adelay in the onset of repression. Similar resultswere obtained with 0.01 or 0.1 mg of chloram-phenicol per ml. We conclude that the chloram-phenicol treatment delays rather than stimulatesthe establishment of repression under these con-ditions.When tryptophan deprivation was used to in-

hibit protein synthesis, some j-galactosidase ap-peared in the inhibited culture several hours aftermating, suggesting that the tryptophan block wasincomplete. This synthesis of enzyme duringtryptophan starvation was completely inhibitedby the addition of 50 ,ug of 5-methyltryptophanper ml (22), which, therefore, was added at 35min to tryptophan-starved zygotes from matingsof x434 X x2l2. Figure 3a shows that, whentryptophan was added to starved zygotes at 80min, both the induced and noninduced culturesunderwent a period of accelerated f-galactosidasesynthesis in which the rate of enzyme synthesiswas greater than the initial rates in the corre-sponding controls. The constitutive level of s-galactosidase after tryptophan deprivation roseto about the same level as in the noninducedcontrol. The onset of repression was not delayed.When the inhibition was not reversed until 120min (Fig. 3b), the accelerated synthesis was notseen. However, while synthesis of constitutive3-galactosidase had nearly ceased at this time incontrol cultures, addition of tryptophan resultedin a rapid synthesis of constitutive enzyme in thestarved culture, even though the induced synthesiswas depressed subsequent to the inhibitor treat-ment. These data suggest that during tryptophan

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FIG. 2. Effect ofprotein inhibition by chloramphen-icol (25 ,g/ml) upon the establishment of repression.x434 was mated with x212 at t = 0 in medium withouttryptophan, proline, or glycerol. The mating was inter-rupted, and '4C-tryptophan was added at 35 min. Theculture was then divided, and one part received chloram-phenicol. The cultures were filtered at 95 and 100 minas indicated, washed, and resuspended in medium withstreptomycin, sodium lauryl sulfate, and 14C-trypto-phan, but without chloramphenicol. A portion of eachwas then induced. The medium used during and subse-quent to mating was half preconditioned (2, 26) andhalffresh, a combination found to minimize the post-chloramphenicol lag without increasing 14C-tryptophanincorporation in x212 controls. i3-Galactosidasesynthesis in the induced control, 0; the noninducedcontrol, A; the culture treated with chloramphenicoland then induced, 0; and the culture treated withchloramphenicol but not induced, A. (Points indicatedfor 50-, 70-, and 90-min assays represent duplicatesamples.)

starvation there is little accumulation of f-galactosidase repressor. Tryptophan starvationwithout addition of 5-methyltryptophan gaveidentical results until the mentioned time ofappearance of enzyme.

Since neither chloramphenicol nor tryptophandeprivation will permit the establishment ofrepression, we conclude that the usually acceptedmechanism of protein synthesis is probably in-volved in repressor formation.

Inhibition of RNA synthesis. Assuming thatrepressor is made by the translation of a typicalmessenger RNA template, inhibition of RNA

i60 90 120 150 180 35 60 90 120 150 180 210minutes after mating

FIG. 3. Effect of proteini inhibition by tryptophandeprivation and 5-methyltryptophan treatment uponthe establishmenit of repression. x434, Hfr i+ z+ pro+trp+ str8, was mated with x212, F- i z pro-3- trp-strr, in the absence of tryptophan, proline, or glycerol.The mating was interrupted at 35 min, 5-methyltrypto-phan (50 ,ug/ml) was added, and the culture wasdivided into four parts. Tryptophan was added at thetimes indicated to reverse the inhibition. (a) f3-galac-tosidase in cultures receiving inducer and tryptophan at35 min (0), tryptophan at 35 min, without inducer(A), induced at 35 min and tryptophan at 80 min (0),and tryptophan at 80 min, but not induced (A). Thearrow indicates the 80-min addition of tryptophan.(b) ,3-galactosidase ini cultures receiving inducer andtryptophan at 35 min (0), tryptophan at 35 min butnot induced (A), inducer at 35 min and tryptophan at120 min (0), and tryptophan at 120 min, but not in-duced (A). The arrow indicates the 120-min additionof tryptophan. Similar results were obtained when theinhibition is reversed at 140 min. In each instance,tryptophan was added to 50 ,g/ml. Different enzymelevels in these and other experiments result from varia-tions in zygote concentration.

synthesis should delay the onset of repression.We have tested this with two different inhibitors,6-azauracil (11) and sodium borate (14). Bothtreatments delay the onset of repression. Pre-liminary experiments indicated that these com-pounds meet two essential criteria for inhibitorsof transcription; that is, (i) the amount of aninducible protein made in the presence of the in-hibitor depends upon the extent of preinduction,and (ii) RNA synthesis is depressed earlier thanprotein synthesis by the inhibitor treatment.Growth conditions are known to influence

greatly the effectiveness of 6-azauracil (10, 11, 34).We obtained efficient inhibition ofRNA synthesisif the azauracil treatment was preceded by aperiod of proline deprivation, such as routinelyoccurred in the mating experiments. The cells re-

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mained fully sensitive to azauracil for at least 20min after reversal of the starvation.

Azauracil-induced inhibition was almost com-pletely reversed by addition of uracil, in agree-ment with the report of Yates and Pardee (33)that azauracil inhibits the enzymes of pyrimidinebiosynthesis.

Figure 4 shows the effect of azauracil upon theestablishment of repression in a mating of X434X X212. Azauracil (0.1 mg/ml) was added to allcultures at 35 min. Uracil was added to the"control" cultures at 35 min and to the "in-hibited" cultures at 80 min. Comparison of theamounts of f3-galactosidase made after 80 minreveals that, although the induced synthesis wasreduced 35% after inhibitor treatment, the con-stitutive synthesis was stimulated by 10%. Thus,azauracil treatment delayed the onset of repres-sion. A similar delay was demonstrated in otherexperiments when uracil was added at 115 or125 min. The addition of exogenous proline at35 min did not alter the result.

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FiG. 4. Effect ofRNA inhibition by 6-azauracil uponthe establishment of repression. x434 and x212 weremated at t = 0 in the absence ofproline or glycerol.At 35 min, the mating was interrupted, and the culturewas divided into four parts. All cultures received 0.1mg of azauracil per ml at 35 min. The figure shows,3-galactosidase synthesis in cultures receiving uracil(0.2 mg/ml) and inducer (isopropyl-,3-D-thiogalacto-pyranoside) at 35 min (0), uracil at 35 min but notinduced (A), inducer at 35 min and uracil at 80 min(0), and uracil at 80 min but not induced (A). Thearrow indicates the 80-min addition of uracil.

The borate ion is known to form negativelycharged complexes with cis diols (3), includingthose in ribose (29). This probably accounts forthe inhibition of RNA synthesis in vivo reportedby Hurwitz and Rosano (14). We have confirmedtheir report that, within limits, the amount of ,B-galactosidase produced in the presence of borateincreases with the time of prior induction. Borateinhibition was very rapid; no f-galactosidase wasproduced if the inhibitor was added 1 min afterinduction. Similar rapidity was observed in in-hibition of incorporation of 14C-tryptophanand 3H-adenine. Adenine incorporationwas depressed immediately upon borate addition,and tryptophan incorporation was depressedsoon thereafter. The depression of induced ,B-galactosidase synthesis was less rapid, but wassimilar to that following actinomycin D treatment(17).Figure 5 shows the effect of borate on the es-

tablishment of repression in a mating X434 xX212. The inhibitor was added to the zygoteculture at 35 min after mating and removed at97 min. The onset of repression was clearly de-layed. Induced enzyme synthesis was depressed53% by the previous borate treatment, but con-stitutive synthesis was stimulated by 25%.

Since treatment with 6-azauracil or boratedelays the establishment of repression, we con-clude that i gene repressor formation requiresRNA synthesis.

Inhibition of DNA synthesis. We would notexpect DNA synthesis to be required for repressorformation, unless repressor is made by someunusual mechanism. We have, however, lookedfor such a requirement by use of two alternatemethods of thymine deprivation: (i) starvationof a thy- female, SL67-101, and (ii) treatment ofzygotes with 5-fluorodeoxyuridine, an inhibitorof thymidylate synthetase (7). Neither techniqueresulted in a delay in the establishment of repres-sion.

Figure 6 shows that f3-galactosidase synthesiswas depressed in both induced and noninducedcultures by thymine starvation (19, 23) (fromt = 0) of a mated culture of X434 X SL67-101.(The thy+ gene in the male was not transferredto the zygotes in this mating.) There was nostimulation of constitutive enzyme synthesis inthe thymine-starved culture. In fact, up to 130min, constitutive synthesis of g-galactosidase wassomewhat more depressed by this treatment thanwas induced synthesis. The onset of repressionwas not delayed.The thymine-starved culture made 37% as

much DNA between 0 and 210 min as did theculture with thymine. However, a comparablecontrol culture of this thy- mutant, with exoge-

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41

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FIG. 5. Effect ofRNA inhibition by sodium borateon the establishment of repression. x434 and X212 weremated at t = 0 in the absence ofproline or glycerol.At 35 min, the mating was interrupted and the culturewas divided. Half was treated with borate (0.03 M,

pH 7). Both parts were filtered (Millipore, 0.45 ,u)at 97 min, washed, and resuspended in preconditionedmedium with streptomycin and sodium lauryl sulfatebut without borate. A portion ofeach was then induced.The figure shows fi-galactosidase in the induced con-trol (0), the noninduced control (A), the culture treatedwith borate and then induced (0) and the culturetreated with borate but not induced (A). (The 50-and 90-min points represent duplicate assays.) Similarresults were obtained without preconditioned medium,but the recovery was very slow.

nous proline added, made only 13% as muchDNA in the absence of thymine as in its presence.This is probably a better estimate of the degreeof DNA inhibition obtained, since measurementof DNA synthesis in mating experiments was com-plicated by the DNA synthesis in unmated cells.Recombinants decreased to 10% between 55and 215 min in the thymine-starved zygoteculture, indicating that thymineless death occur-red.A thymine deficiency was induced in a mating

of X434 X X212 by the addition of 5-fluoro-deoxyuridine (0.5 mg/ml) at 35 min (Fig. 7).Both constitutive and induced syntheses were

depressed by this treatment, but the constitutivesynthesis was again more severely depressed. Inthis instance also, thymineless death occurred.

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FIG. 6. Effect ofDNA inhibition by thymine starva-tion on the establishment of repression. Parallel mat-ing cultures of x434, Hfr i+ z+ pro+ trp+ thy+ str',X SL67-101, F i- z- pro-3- trp- thy- strr, in me-dium lacking proline and glycerol were deprived ofthymidine or not deprived from t = 0. The matingswere interrupted at 35 min, and a portion of each was

induced. f-Galactosidase in cultures with thymidine(18 ug/ml) and induced (0), with thymidine but notinduced (A), without thymidine and induced (0),and without thymidine but not induced (A).

Neither of these results implicate DNA syn-

thesis per se in repressor formation.

DISCUSSION

Our results suggest that the lac repressor ismade by the normal processes of protein synthe-sis, contrary to the results obtained in some simi-lar studies (13, 25, 31). Several factors, otherthan the unrestricted accumulation of the i geneproduct, may have caused the depression of con-stitutive synthesis reported in these studies. Forexample, depression of ,B-galactosidase synthesismay occur in response to changes in the overallbalance of metabolic activity, that is, by the stillpoorly understood mechanism of catabolite re-pression (20). Catabolite repression may (6) ormay not (18) be partially reversed by inducer;the more severe depression of constitutive synthe-sis seen in our DNA inhibition experiments sug-gests that it is reversed. The depression observedmay also result from the accumulation, even inthe presence of an inhibitor, of the very smallnumber of repressor molecules required to estab-lish repression (9) since no inhibitory treatmentis 100% effective. Another possible explanation

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20a)~~~~

3!5 60 90 120 150 180 210minutes after mating

FIG. 7. Effect of 5-fluorodeoxyuridine treatmentupon the establishment of repression. x434 and x212were mated at t = 0 in the absence ofproline or glyc-erol. At 35 min, the mating was interrupted and theculture was divided into four parts. if-Fluorodeoxy-uridine and uracil (both 0.5 mg/mi) were added to allfour cultures at this time. The figure shows jS-galacto-sidase in cultures which in addition received thymidine(0.1 mg/ml) and inducer at 35 min (0), thymidinealone (A), inducer alone (0), or none, i.e., no addi-tions (A).

is the accumulation of a repressor precursor dur-ing inhibitory treatments, with the subsequentrapid synthesis of the active molecule (15). Forexample, accumulation of messenger RNA hasbeen reported during various types of proteininhibition (8, 12), and such accumulation mightexplain the accelerated kinetics of (3-galactosidasesynthesis which follows short periods of trypto-phan deprivation (Fig. 3a).These factors, and others, acting singly or in

combination, could produce a misleading negativeresult. For this reason, we believe that morecredence can be given to experiments in whichthe establishment of repression was actually de-layed by inhibitory treatment. The direct demon-stration that the repressor is a protein (9) sup-ports this viewpoint. An observed delay in theestablishment of repression might also be mis-leading, however, if the action of an inhibitorwere not fully understood. To guard against thisin our own experiments, we have used two differ-ent types of inhibitors for the analysis of bothprotein and RNA. It can be anticipated that chlor-amphenicol and 5-methyltryptophan have com-pletely different effects on treated cells; the sameis undoubtedy true for the effects of borate and6-azauracil.

It might be asked whether our inhibitory treat-

ments could have blocked proline synthesis in thezygotes and thus prevented repressor formationindirectly. Since experiments in which exogenousproline was added gave very similar results, weconclude that inhibition of RNA or protein syn-thesis directly blocks repressor synthesis.

In agreement with similar findings of Horiuchiand Ohshima (13), we were unable to delay theonset of repression by inhibition of DNA synthe-sis. Although it cannot be concluded that DNAsynthesis is in no way required for repressor syn-thesis, the results are consistent with the knownproperties of the normal protein synthetic mech-anism.

ACKNOwLEDGMENTS

This investigation was supported by grant G-B3347from the National Science Foundation, and by aNational Institutes of Health Predoctoral Fellowshipto John H. Caster.We are grateful to Roy Curtiss, III, who supplied

many of the strains used in these experiments andtaught us the tricks of bacterial matchmaking.Thanks are also due S. K. Bose, T. Hudnik-Plevnik,and J. S. Trupin for their helpful advice.

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