Proc. Nat. Acad. Sci. USAVol. 68, No. 9, pp. 2190-2194, September 1971

Establishment and Maintenance of Repression by Bacteriophage Lambda:The Role of the ci, cit, and ciII Proteins

(E. coli/lysogeny versus lysis/cy- mutants/DNA binding)

HARRISON ECHOLS AND LINDA GREEN

Department of Molecular Biology, University of California, Berkeley, Calif. 94720

Communicated by A. D. Kaiser, July 8, 1971

ABSTRACT To define the events necessary for theestablishment and maintenance of repression in a X-infected cell, we have studied the requirements for efficientsynthesis of the cI protein ("X-repressor"). Three classesof X mutants defective in the establishment of repressionare also defective in the appearance of cI protein activityat the normal time. Two of these mutational classes (cII-and cIII-) probably result from inactivation of X-specifiedproteins, but the third class (cy-) may involve a structuraldefect. We conclude that at least three regulatory elementsare likely to be required for the normal turn-on of cI pro-tein synthesis in an infected nonlysogenic cell: clI andcIII proteins and an "active" y-region of X DNA. Fromthese and other results, the complete role of cII and cIIIproteins in the establishment of repression may involve abifunctional regulatory activity: positive regulation of thecI gene and negative regulation of late genes. A possiblemolecular model for cII and cIII action is discussed. Sincethe cII and cIII genes are repressed by the cI protein underconditions of stable lysogeny, a separate mechanism isrequired for the maintenance of cI protein synthesis.After infection of a lysogen by c11- phage, the rate of in-crease of cI protein activity is substantially greater thanafter infection of a nonlysogen. From these and otherresults, the cI protein may also have a bifunctional regu-latory activity: positive regulation of the cI gene andnegative regulation of early lytic genes.

After infection by the temperature phage X, the establishmentof lysogeny requires two functionally separate events: arepression of the capacity of the phage DNA for lytic growth,and a site-specific genetic recombination event that integratesthe viral DNA into the host DNA at a specific site. Themaintenance of the lysogenic state is inherently a simplerphenomenon than its establishment; the repression of lyticcapacity must simply be maintained, and no recombinationevents are involved.The maintenance of repression for phage X is probably ac-

complished by one phage protein-the ci protein (or "X-repressor")-which acts to repress RNA synthesis from criticalearly genes required for viral DNA replication and lytic geneactivation (Fig. 1, see also refs. 1 and 2). The establishment ofrepression for phage X involves a more complex regulatory sit-uation, because at least one early viral protein (the productof the int gene) is required to catalyze the integrative recom-bination essential for stable lysogeny. Thus, the phage mustallow expression of at least some early viral genes and yetimpose repression before an irreversible commitment tolytic growth.Two general mechanisms have been considered that might

provide for the "properly timed" establishment of repression.One involves a repression of viral functions, in addition to that

provided by the cI protein; for example, an inhibition ofsynthesis of late proteins might delay the late stage of lyticdevelopment until cl-mediated repression can take over (3).Another plausible mechanism involves timing the estab-lishment of repression by the time of synthesis of cI protein;this possibility is suggested by the finding that the syn-thesis of cI protein is subject to regulation (4).

Since active clI and cII genes are essential for the effectiveestablishment of lysogeny in an infected cell, but not for themaintenance of lysogeny (5), the cII and cIII proteins arelikely to function as critical timing elements in the establish-ment of repression. As a result of previous experimental ef-forts to understand the role of the cII and cIII genes, weprovided indirect evidence that the cII and cIII proteinsperform both "activities" indicated above: an inhibition of

Head Tailint cM N cIcdHOP QRecomb Reg DNA Lysis

* A^0

FIG. 1. Genetic and functional map of X DNA. Genes withrelated function exhibit extensive clustering along the X DNA.molecule. This is indicated on the diagram by "Head" for genesconcerned with the structure of the phage head; "Tail" for genesconcerned with tail structure; "Recomb" for genes involved withgeneral and site-specific recombination; "Reg" for genes exertinga regulatory function in lytic development or lysogeny; "DNA".for genes specifying replication proteins; and "Lysis" for genesconcerned with cell lysis. Specific genes of the "regulation re-gion"-cIII, N, cI, cII-are indicated above the "X DNA", asare the integrative recombination gene int, the DNA replicationgenes OP, and the late regulator gene, Q.Approximate DNA regions transcribed and the direction of

transcription during the different stages of lytic growth are also.shown: v represents immediate early RNA synthesis, per-formed solely by the host transcription machinery; -_ rep-resents delayed early RNA synthesis, dependent on N protein;- - - -s. represents late RNA synthesis, dependent on Q protein (seerefs. 23, 33, 34 for recent detailed reviews). The cI protein blocksimmediate early RNA synthesis; this provides for completerepression of lytic genes, because delayed early and late RNA.synthesis is dependent upon N protein (see refs. 1, 2 for recentdetailed reviews). Mutations affecting the establishment ofrepression have been located in the cHI and clII genes and in the"y-region" of X DNA between the cI and clI genes (5, 13)-seeResults section.

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Proc. Nat. Acad. Sci. USA 68 (1971)

synthesis of late viral proteins and an activation of syn-thesis of cI protein (6). This report provides much more sub-stantial evidence for the activation of cI protein synthesis bydirect assay of the levels of cI protein in extracts from infectedcells. The results reported here also suggest that the site ofaction of the clI and cIII proteins may be between the cI andcli genes. Similar results and conclusions with a different assayfor cI protein are reported in the accompanying paper byReichardt and Kaiser (7). Conclusions similar to ours havebeen derived from other types of experiments by Eisen andPereira da Silva (personal communication), and by Kourilsky(9).

Since the cII and cIII genes are repressed under conditionsof stable lysogeny (10), the maintenance of cI protein syn-thesis would be expected to occur by a different mechanism.Our results suggest that the cI protein activates its own syn-thesis in a lysogen. This conclusion agrees with that derivedfrom RNA synthesis experiments (11, 12) and from othermeasurements of cI protein (7).

MATERIALS AND METHODSBacteriophage and bacteria

The Escherichia coli strains used were W3102su- and W3104.The X mutations were the following: the ci Amber-mutationc114, the clI mutations cII68 and cII28(Amber), the cIII mu-tations c11167 and c111611 (Amber), and the cy mutations cy42and cy2001. For the genetic characterization of the cI, clI,clII, and cy mutations, see refs. (5) and (13).Phage growth

Phage stocks were prepared by lytic growth in W3104bacteria; the growth medium was T-broth liquid (per liter:10 g of Difco Tryptone, 5 g NaCl) or T-broth agar (T-brothcontaining 1.2% Difco Bacto-agar for the underlayer and0.7% agar for the soft-agar overlayer). Phage were con-centrated and partially purified by precipitation with poly-ethylene glycol (10% polyethylene glycol-0.5 Ml NaCl), andphage stocks were stored in "adsorption buffer" (0.01 MMIgSO4-0.01 M Tris HCl, pH 7.4) containing 0.01% gelatin.

In experiments to measure the synthesis of cI protein,W3102 cells were grown at 370C in "supplemented T-broth"(T-broth + 0.2% maltose + 0.01% yeast extract) to an A59oof about 2, centrifuged, and resuspended in adsorption bufferat an A590 of 4. To this cell suspension, phage were added, at amultiplicity of 5 or 10, for an adsorption period of 20 minat room temperature. The infected cells were then diluted10-fold into supplemented T-broth (prewarmed to 370C)and aerated at 370C until the time at which the level of cIprotein was to be measured; the culture was then chilled andextracts were prepared.Assay of cI protein by DNA-binding activity

The cI protein binds tightly and specifically to X DNA (14).This DNA-binding activity can be made the basis for a simple"'membrane filter" assay for the cI protein; the DNA-proteincomplex adheres to a membrane filter, but free DNA doesnot. Excess nonspecific DNA can be added to compete forthe binding of proteins that associate with DNA, but lackthe specificity of the cI protein for X DNA. The membrane-filter assay has been used extensively in studies of the lacrepressor (15, 16) and has been used as an assay to guide thepurification of the cI protein (17, 18). In the experiments re-ported here, we have used the membrane-filter assay to

C3

>

0z 2z

z

0

cam

L

0oMINUTES AFTER INFECTION

FIG. 2. Kinetics of production of active cI protein. Infectionwas at a multiplicity of 5 phage per bacterium. At thespecified times cells were chilled, centrifuged, and extracts wereprepared. DNA-binding activity was measured by the retentionof 32P-labeled X (or Ximm434) DNA on a nitrocellulose filter inthe presence of excess unlabeled "chicken-blood DNA". DNA-binding activity is expressed as DNA-binding units/109 cells.-0-0- represents cI + infection, assayed with XDNA; -0-0- repre-sents cI- infection, assayed with XDNA; -A-A- represents cI+infection, assayed with Ximm434 DNA.

measure the level of cI protein in crude extracts. The criticalcontrol experiments are presented in Fig. 2 (Results).

Extracts for assay of cI protein were prepared as follows.Infected cells (about 6 X 1010) were collected by centrifuga-tion and suspended in 3 ml of "lysis buffer" [0.05 M TristHCl (pH 7.9)-2 mM EDTA-0.45 M NH4Cl-14%(w/w)sucrose]. The resultant cell suspension was centrifuged, andthe pellet was frozen and stored at -20°C. For the prepara-tion of lysates, the frozen cells were suspended in 1.5 ml oflysis buffer and lysozyme was added to a concentration of 330,jg/ml. After incubation for 5 min at 370C, the suspensionwas chilled, and MgCl2 and 2-mercaptoethanol were added to50 mM and 14 mM, respectively. Lysis was completed by theaddition of the nonionic detergent Brij-58 to a concentrationof 0.5%. After 30 min at 0WC, the NH4Cl concentration wasincreased to 1.0 M and the lysate was centrifuged for 2 hrat 100,000 X g. The supernatant fraction was dialyzedinto a buffer containing 0.01 M Tris HCl (pH 7.4)-0.01 AIMgCl1-0.02 M NH4CI--0.1 mM dithiotreitol. The dialyzedextract was clarified by centrifugation for 15 min at 7500rpm, and the supernatant fraction ("crude extract") wasstored at 4VC. The DNA-binding activity of the cI proteinwas quite stable in extracts prepared and stored as describedabove-generally less than 50% of the activity was lost afterseveral weeks of storage.The DNA-binding assay was essentially that described

previously (18). An aliquot of crude extract was mixed with:0.25 ml of "binding buffer" (0.01 M Tris HCl (pH 7.4)-0.01 1\I magnesium acetate-0.02 M KCl-0.25 mM EDTA-14 mM 2-mercaptoethanol), 40,gg of "chiclen-blood DNA"(CalBiochem), and 0.5 jig of 32P-labeled X DNA, in a finalvolume of 0.35 ml. After incubation for 5 min at 0WC, 0.1 ml

Phage Lambda Repression 2191

2192 Biochemistry: Echols and Green

of the mixture was added to a membrane filter (Schleicherand Schuell B6, 25 mm); the filter was washed with 0.4 mlof binding buffer, dried, and the retained radioactivity wasdetermined by liquid scintillation counting. One unit of DNA-binding activity is defined as the quantity sufficient to retain0.5 ,ug of X DNA on the filter. The retention of X DNA onthe filter was approximately proportional to added extractin the range from 0.1 to 0.5 DNA-binding units, and assayswere routinely performed in this range.

RESULTSKinetics of appearance of cI protein activity ininfected cells

As a first step in exploring the establishment of cl-mediatedrepression in infected cells, we performed experiments de-signed to measure the kinetics of synthesis of cI protein. Theassay for ci protein was specific binding to X DNA, as judgedby retention of X DNA to a membrane filter. Fig. 2 shows theresults of DNA-binding experiments for extracts prepared atdifferent times after infection. The appearance of X-specificDNA-binding activity begins about 6-9 min after infectionwith normal X (cI+cII+cIII+) phage. We attribute this DNA-binding activity to the cI protein because of two control ex-periments, also shown in Fig. 2. First, substantial DNA-bind-ing activity is not found after infection by a cI- mutant.Second, the X-specific DNA-binding activity found aftercI+ infection is not demonstrable if X imm434 DNA is usedfor the assay instead of X DNA; X imm434 DNA lacks thebinding sites for the X cI protein (14) (see Fig. 1) and there-fore should not be retained on the filter in the presence of theXcI protein.The experiments presented in Fig. 2 thus show that the

DNA-binding assay can be used to measure cI protein ac-tivity in extracts prepared from infected cells. The deTayed-appearance of cI protein activity is consistent with the con-

TABLE 1. Effect -of c11, cII -, and cy mutations on cIprotein activity

X-specificDNA-binding activity

Infecting phage 12 min 18 min

c+ 2.9 5.0cM14 0.2 0.3cII68 <0.1 0.1cII28 0.1 0.1cIII67 0.2 0.6cIII611 0.3 0.3cy42 0.1 0.2cy2001 0.1 0.2

Infection was at a multiplicity of 5 phage per bacterium. Ateither 12 min or 18 min after infection, cells were chilled, centri-fuged, and extracts were prepared. DNA-binding activity wasmeasured by the retention of 32P-labeled X DNA on a nitro-cellulose filter in the presence of excess unlabeled "chicken-bloodDNA." DNA-binding activity is expressed as DNA-bindingunits/109 cells. The designation "X-specific DNA-bindingactivity" means that the binding activity of an uninfected ex-tract (0.1) has been subtracted from the binding activity of theextract prepared from infected cells. The c+, cIl4, cII68, cIII67,and cy42 infections were repeated two or more times with similar

cept that the time of rapid synthesis of cI protein is of criticalimportance in the properly timed establishment of repression.

Effect of cl-, c11l-, and cy - mutations on the appearanceof cI protein activity

From the results presented in Fig. 2, it should be possibleto investigate regulation of cI protein synthesis through a

study of the effect of various regulatory mutations on theappearance of X-specific DNA-binding activity. Besides cI-,three classes of repression-defective mutants are known:cII-, cIII-, and cy-. As judged by complementation forlysogeny, the cIl- and cIII- mutations inactivate cyto-plasmic products (5); these cytoplasmic gene products are

probably proteins since nonsense mutations exist in both thecII and cIII genes. The nature of the cy- mutations is lessclear. cy- mutations have been located between the cI andcII genes, (Fig. 1); cy- mutant phage fail to give effectivecomplementation for lysogeny after mixed infection with cI-mutants, suggesting that cy- mutations might affect a siteconcerned with cI protein synthesis (13).The effect of cII-, cIII-, and cy- mutations on the ap-

pearance of cI protein activity is shown in Table 1; all threeclasses of mutant phage are defective. We conclude that theclI and cIII proteins are probably necessary for effective syn-

thesis of the cI protein and that the "y region" of X DNAbetween the cI and cII genes may also be important for thesynthesis of cI protein.

Complementation between repression-defectivemutants-cis-dominance of cy-

In an attempt to establish more clearly the role of the v-

region of X DNA in the synthesis of cI protein, we comparedcy- and cIL- or cIII- mutants in their ability to complementwith4I- fo- the-p dtion- 4. el pretii. The- results' are

shown in Table 2. As expected for phage with mutations thatinactivate a protein gene product, cI- and cII - or cIII- mut-

ants exhibit trans complementation for the appearance of cI

protein activity (lines 3 and 4) (the most likely anticipatedlevel would be half the c+ case (line 1) because only half as

many cI+ genes are present). In contrast, cI- and cy- phage

TABLE 2. Comnplementation between cII-, cIII-, and cy-mtutants for ci protein activity

X-specificDNA-binding

Infecting phage activity

c+ 3.8cI- 0.1cl- and cII- 1. 8cl- and cIII- 1.6cI- and cy- 0.1c+andcI-cII 2.1c+ and cI-cIII- 2.2c+ and cl-cy- 2.9

Infection was at a total multiplicity of 10 phage per bacterium(5 of each phage type for mixed infections). 12 min after infection,cells were chilled, centrifuged, and extracts were prepared and as-

sayed for DNA-binding activity as in Table 1. Each extract wasalso assayed with Ximm434 DNA to provide assurance that the"complementation activity" showed the proper specificity; only0.1-0.2 units were assayable with Ximm434 DNA. The complete

results. experiment was performed twice, with similar results.

Proc. Nat. Acad. Sci. USA 68 (1971)

Proc. Nat. Acad. Sci. USA 68 (1971)

do not show effective trans complementation (line 5), eventhough cI -cy- is recessive to cI +cy+ (line 8). The cis-dominantbehavior of the cy- mutation is most simply interpreted asmutational inactivation of a site necessary for cI proteinsynthesis. We conclude that the y-region of X DNA probablyexerts an important structural role in the synthesis of cIprotein.

Effect of cl-mediated repression on the appearance of cIprotein activity

Since synthesis of the cII and cIII proteins is repressed in alysogen, the problem remains as to how the phage providesfor continued synthesis of cI protein once cl-mediated repres-sion is- established.- There-are two-obvious- possibilities: ciprotein is synthesized at a low, constitutive rate in the ab-sence of cIl/cIlI activation-a rate sufficient to provide forcontinued iepression-or cI protein activates its own synthesis.To investigate those possibilities, we sought to compare thecli-cIIl-activated and "constitutive" rates of cI proteinsynthesis to the rate found in a lysogen.To try to estimate the constitutive rate of synthesis of

cI protein, we used infection of a nonlysogen by cI- phage.To eliminate gene-dosage effects because of replication, weused phage unable to replicate effectively because of a muta-tion in the P gene (3, 19, 20). Fig. 3A compares cI proteinactivity after infection of a nonlysogen and a lysogen byP-c+ phage. Fig. 3B makes the same comparison under con-ditions where cIl-cIlI-activation is blocked by mutation.cI protein activity after cII- infection of a nonlysogenis clearly lower than that found after infection of a lysogen.Similar results to those in Fig. 3B were obtained after in-fection of a lysogen carrying a cII- prophage instead of c+.The experiments of Fig. 3B are consistent with two possibleexplanations: the cI protein activates-its own synthesis in alysogen, or, the synthesis of cI protein after cIL- infection of anonlysogen is not in the "constitutive" rate, but is negativelyregulated by another X protein. We favor the possibility thatcI activates its own synthesis because the only known nega-tive regulator of cI function-the cro gene product-does notexert a demonstrable effect on synthesis of cI protein untilabout 20 min after infection (7) (see Discussion).The results of Fig. 3A indicate that the rate of cIl-cIMI-

activated synthesis of cI protein is substantially greater thanthe presumptive cI-activated rate found in a lysogen. Thebiological relevance of the rapid cII-cIlI-activated synthesisis presumably to allow the synthesis of early phage proteins(see Introduction) and yet to permit the synthesis of cI pro-tein to catch up with the multiple copies of the replicating viralDNA. The results of Fig. 3A also suggest that c1I-cI1-activated synthesis of cI protein ceases at later times, sincethe rate of appearance of cI protein activity slows markedly.

DISCUSSIONThe role of the cII and cIII proteins in the establishmentof repression

As noted in the Introduction, the regulatory problem facedby a X phage desirous of provoking lysogeny is to providefor an effective transition from initiated lytic development tothe cl-mediated repression of lytic genes that maintains thestable lysogenic state. The phage must provide for sufficientsynthesis of int protein to achieve a high probability of in-tegration, but it must also stop lytic development beforelysis renders synthesis of int protein irrelevant.

I- .

'1-5

4

z

0.KE 0.!u

20 0 10MINUTES AFTER INFECTION

FIG. 3. Rate of appearance of cI protein after infection oflysogenic and nonlysogenic cells. Infection by P c+ and P-cII-phage was at a multiplicity of about 10 phage per bacterium, andcI protein was assayed by DNA-binding as in Fig. 2. For thisfigure, the "cI protein activity" represents the excess of bindingto X DNA over that found for Ximm434 DNA (about 0.1 unit).-0-0- represents cI protein activity after infection of W3102;-0-0- represents cI protein activity after infection of the lyso-genicstrain W3112(X+).

The results reported here suggest that the turn-on ofsynthesis of cI protein at the "proper" time is a critical factorin the establishment of repression. We have shown that threeclasses of mutants defective in the establishment of repres-sion are also defective in the normal appearance of cI proteinactivity. Complementation tests indicate that two of thesemutational classes (cII- and cIII-) result from inactivationof a gene product, but the third class (cy-) may involve astructural defect. Thus, at least three regulatory elements arelikely to be required for the normal turn-on of cI protein syn-thesis in an infected cell: clI and cIII proteins, and an "active"y-region of X DNA.

Previous experiments (6) have indicated that the cII andcMII proteins also inhibit synthesis of late phage proteins,since cI -cI - or cI-c11 - mutants commence the synthesis oflysozyme at an earlier time than cI-. Recent experimentshave shown that the cy- mutation also results in an advancedsynthesis of lysozyme (Court, unpublished). The completerole of the cII and cIII proteins in the establishment of repres-sion thus may involve a bifunctional regulatory activity:positive regulation of the cI gene and negative regulation oflate genes.Based upon our rather limited present state of knowledge,

a plausible molecular mechanism is the following (see Fig. 4).The cII and cIII proteins form a regulatory oligomer*, whichacts at a site in the y-region of X DANA to provide for activa-tion of I-strand transcription from the cI gene and for inhibi-tion of r-strand transcription in the opposite direction. Therepression of r-strand transcription from the cIIOPQ regionof X DNA might inhibit the synthesis of late proteins in twopossible ways: suboptimal synthesis of Q protein, which is

* St-rack and Ziegler (32) have also suggested that the cII andcIII proteins may act as an oligomer, based upon the existence ofmutations that probably are located in the cII gene, but whichcomplement as if cII- and.cIII-. We have isolated similar muta-tions and shown that they also complement as cII-cIII- in the.assayloriL.pratein activity (Mantei,<Court, Greenr and FxLs,7.-unpublished).

Phage Lambda Repression 2193

2194 Biochemistry: Echols and Green

40liiN-

ci xvcro li 0cM N -. cI x Y. c.CI .0 P2- strandr-strand

cI protein possible binding sitebinding sites for cIl/cl proteins

FIG. 4. Possible mechanism for the action of cII and cIIIproteins. The cIL-cLIL regulatory oligomer acts at a site in they-region of X DNA to provide for activation of i-strand trans-cription toward the cI gene (e ---- and inhibition of r-strandtranscription toward the chOP genes (-A) (see text). Once thesupply of cI protein becomes sufficient, lytic genes are completelyrepressed by the ability of the cI protein to inhibit immediateearly RNA synthesis, represented by (It) (see Fig. 1.) The bind-ing sites for the ci protein are located to the left and right of the ci

gene, very close to the initiation sites for immediate early RNA.The division of the area between the ci and cHI genes into x-

and y-regions is based upon the end of the region of nonhomologybetween phages X and 434 (imm-region); x is inside the imm-region, y is outside (19). The terms x and y therefore do not denoteA genes. The cro gene is within the x-region.

needed to activate late-gene transcription (21, 22), or fail-ure of normal rates of initiation of RNA synthesis for lategenes in the absence of transcription to the initiation site forlate RNA synthesis from the cIIOPQ genes to the left (seerefs. 12 and 23-25 for a discussion of such mechanisms).The existence of another regulatory gene-cro-complicates

the definition of events during the establishment of repression.'Physiological experiments have indicated that the product ofthe cro gene (see Fig. 4) antagonizes the synthesis or activity-of the cI protein (26-29). Reichardt and Kaiser (7) have pre-sented evidence that the cro product turns off synthesis of.cI protein at later times. The physiological function of cro

might be to prevent cII- cIII-mediated repression from chan-neling all infected cells to lysogeny. Alternatively (or inaddition), the physiological role of the cro gene might be toprovide for a rapid release of repression when the lysogeniccell is induced to lytic growth.

The role of the cI protein in the establishment andmaintenance of repression

Once the supply of active cI protein becomes sufficient for thenumber of A DNA molecules, complete repression should occur

rapidly. As noted in the Introduction, the cI protein willblock synthesis of new N protein and DNA-replication pro-teins. In addition, N and replication proteins cannot acteffectively, even if present, on a X DNA molecule to which the,cI protein is bound (23, 30, 31). From our experiments and-those of others (7, 11, 12), the maintenance of cI protein syn-thesis once repression is established appears to result from4'self-activation" by the cI protein. Thus, the cI protein islikely to have a bifunctional regulatory activity: positiveregulation of the cI gene and negative regulation of earlylytic genes.

Clearly, the patterns of regulation for even so simple a

creature as phage can be relatively complex.

We thank Joseph Ferreti, Dale Kaiser, Ethan Signer, andRene Thomas for phage stocks and Don Court, Harvey Eisen,Philippe Kourilsky, Margaret Lieb, and Lou Reichardt foruseful discussion and communication of unpublished results.

This research was supported in part by U. S. Public HealthService grant GM 17078.

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Proc. Nat. Acad. Sci. USA 68 (1971)