86 BioEssays Vol. 2, NO. 2 ROOTS Shall Be

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86 BioEssays Vol. 2, NO.ROOTSshall be able to decide whether thevarious procedures applied and contem-plated are really inocuous. How manyyears must elapse before one can saythat nothing unusual or unexpected hashappened to the children producedunder such unnatural conditions? Afterall, the destiny of a human being begins,but does not end, with birth. Morestrictly, it has, in fact, begun withconception. If the cases multiply, inwhich fertilization and pregnancy fol-lowed the new scientific observance, andwith Murphys Law kept in mind, it isnot far-fetched to assume that the man-or womanhandling of the embryo willprove, seldom or often, far fromharmless. A very long time will have topass before statisticians and patho-logists will be able to arrive at an

opinion. Although disappointed in thecase of chemical and radiation injuries,I much rather put my trust in thekeenness of the legal profession, whohave developed an exquisite feeling forthe possibility of malpractice suits.Society is obviously bewilderedbefore the advances of science; it hasgrown accustomed to expect them, butdoes not know how to digest them. Forthe physician, brought up to heal thesick, everybody is a patient. There aremen who want to be fathers, andca.nnot; there are women who want tobe mothers, and cannot. What morenatural than to trick nature? But eventhe physician or the scientist ought to befrightened by the irreversibility of whatthey are doing. There is no recall of aliving being except by murder. This is no

longer the exercise of the healers art; itis a Manichaean undertaking in whichthe scientist plays demiurge.I wish Zoe the best of this world. Mayshe prosper, but on condition that sheremain the one and only swallow - heone that does not make a summer. Whata simple and transparent world it waswhen we were told that it was the storkwho brought the babies into the world.He at least knew what he was doing.0 Erwin Chargaff

E R W I N C H A R G A F F , ormerly wilhthe Department of Biochemistry, theCollege of Physicians and Surgeons,Columbia Unviv ersity, is at 350 CentralPark We st, New York, New York 10025,U S A . This article is a chapter fro m a bookwhich the author is preparin g.

Molecular Basis of Gene Expression: Origins from thePajama ExperimentArthur B.PardeeSummaryThe Pajama (P ardee, Jacob, Mo nod)experiment provided a breakthrough inour understanding o the molecularmechanisms by which gene expression isregulated. Today , twenty-$ve year s lateri t provides a paradigm f o r thinking aboutproblems o gene expression, such asgrowth regulation and differentiation.From this experiment emerged entitiessuch as repressors, regulatory genes, theoperon as a group o jointly controlledgenes, and messenger R N A .BackgroundThe Pajama experiment resulted from aunion of bacterial physiology, genetics,and enzymology. The main conclusionreached from this experiment is thatgene regulation must depend upon ahitherto unsuspected regulatory mol-ecule, the repressor protein. This repres-sor provides the link between externalagents and the genetic expression pat-tern of the cell, by interacting on one ofits sites with the low molecular weightinducer and on the other with a specificregulatory gene. This Janus-likeproperty of the repressor - ooking

simultaneously in two biochemicaldirections - s very like that of enzymeswith both catalytic and regulatory sites(the allosteric property), so important inthe control of enzyme molecule activity.The Pajama experiment was carriedout entirely within a few months in thefall and winter of 1957 while I was onsabbatical with Jacques Monod at thePasteur Institute in Paris. Our broadaim was to make connections betweeenthe extracellular inducer moleculeswhich turn on specific enzyme produc-tion and bacterial genes.To understand this problem betterone should have some feeling for itsstatus in 1957. From the turn of thecentury bacteria were known to producecertain enzymes only when their sub-strates were present. This property wasregarded as benefitting the organism,and so these enzymes were calledadaptive. In contrast other enzymespresent under all conditions of growthwere named constitutive.2*A classicalexample of enzyme adaptation is /?-galactosidase which can increase10,000-fold in activity after addition oflactose, so long as a better carbon sourcesuch as glucose is ab ~ e n t . ~nother

important example is penicillinase;appearing when penicillin is provided, itprotects bacteria by degrading thisdrug.5Around 1950, emphasis turned fromconsidering this process primarily interms as an adaptive response beneficialto the bacteria towards inquiring intothe mechanism.6 The terminology wasexplicitly changed from adaptiveenzyme formation to enzyme induc-tion, to emphasize mechanism and tostress the need to understand its mol-ecular basis. These two viewpoints areadmirably contrasted in the introductoryarticle on teleonomic advantage to thecell and in the summarizing article onmolecular mechanisms of the firstSymposium on Cellular RegulatoryMechanisms.The problem of enzyme inductionwas to discover the connections thatexist between induced enzymes, lowmolecular weight inducers, and the bac-terial genetic and biochemical machi-nery. It had been shown that inducersare not necessarily substrates or inhibi-tors of /?-galactosidase, and vice versa.Inducers could therefore not functionby directly interacting with the enzyme.*


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Enzyme synthesis by constitutive mu-tants occurred in the absence ofinducers.This gave rise to the hypothesis that aconstitutive cell produces an inducingcompound within itself. This idea,reflected today in the autocrine hypo-thesis of malignancy, was consistentwith data on sequential induction ofenzymes in metabolic pathways, asworked out primarily by Roger Stanier.3Each substrate as it was formed in themetabolic pathway was a hypotheticalinducer of the enzyme for its ownmetabolism.The one gene-one enzyme hypothesisof Beadle and Tatum states that theformation of each enzyme ultimatelydepends on its structural gene. Mutantstherefore should exist that are altered inability to produce inducible enzymes;such mutants were indeed f0 u n d . l ~~Induction was, however, far too rapid tobe accounted for by selective outgrowthof a mutant with much higher intrinsicenzyme forming capacity than cells ofthe general population, a hypothesisthen held by some. Direct assays afterinduction showed increases of enzymeactivity within minutes.*v5 Of greatimportance for this story, a novel classof mutants were constitutive; theyproduced B-galactosidase at high levelsin the absence of inducer. Genescontrolling induction itself evidentlymust exist, having been mutated, inaddition to the structural genes.The biochemistry of induction wasrecognized as requiring synthesis of newenzyme protein molecule^.^ But this wasnot very helpful since the biochemistryof protein synthesis was just beingworked out. Ribosomes and tRNAswere known, but not mRNA nor thegenetic code linking base sequences ingenes (DNA) to amino acid sequencesof proteins. There was no clue as to whatmolecule interacts with inducers.Much insightful research on enzymeinduction4 by Jacques Monod attractedme to his laboratory. My interests overthe preceding eight years were inprocesses used by cells to control theirgrowth and metabolism, such as feed-back control of enzyme activity (see theprior 'Roots' articlee),and also enzymeforming controls such as induction, andalso derepression which is the increasedactivity of enzymes when a relatedbiosynthetic metabolite is in short

The advent of a novel genetic tech-nique was crucial for performing thePajama experiment. It had been foundthat genetic material could be transfer-red from a donor to a recipient bac-terium, where it was eventually stably

supply.5, o

expressed. Thus after the gene forP-galactosidase (z') was transferredinto a galactosidase-deficient(2-) ecipi-ent, it allowed the recipient cell to growinto a colony on a plate containinglactose as the sole carbon source.*

Monod and I planned to study theinduction of P-galactosidase after selec-tively destroying the /3-galactosidasegene. Elizabeth McFall, Gunther Stentand I had earlier demonstrated thatrandom damage to bacterial DNA stopsenzyme synthesis. This damage wasaccomplished by decay of 32P ncor-porated into all DNA. We concludedthat DNA integrity is essential forenzyme induction." We planned totarget the /3-galactosidase gene moreselectively for damage by incorporating32P into a donor bacterium, and thentransfer this radioactive gene by matinginto a galactosidase-negative mutantcell which was not radioactively labeled.Decay in this cell could occur only in thepiece of DNA carrying the radioactivegene, and one could study the conse-quences for enzyme induction. Thisresearch combines three techniques:labeling a gene with 32P,ransfer of thisunstable gene into a stable environmentby mating, and directly assaying loss ofP-galactosidase activity. Previously thetransferred gene's activity could only bemeasured after a day by countinglactose-positive colonies. These 32P x-periments, done later by Monica Riley,gave the hoped for result of enzymedependence on integrity of thegene.12Our preliminary experiments, as isoften the case, led us down a path moreinteresting than the original one. Thiswork, the Pajama experiment,' is des-cribed here as I remember it. It has oftenbeen discussed from other points ofview -m ~ l e c u l a r , ~ ~ist~rical, '~andeven philosophical. 5The Pajama Experiment

Soon after arriving at the InstitutPasteur I set up a direct system formeasuring the transfer of the /?-galacto-sidase gene. In these first matingexperiments (Figure 1) the donor wasB-galactosidase positive (z+) whereasthe recipient was negative (z-). Further-more, the recipient was chosen to beresistant to streptomycin (Sm.), a drugpreviously used to prevent growth ofsensitive (SmS) donor cells on lactose-containing plates8I found that enzymicexpression of z+ in the SmS donor wasalso prevented by the drug. Thus onlythe recipient cells that had acquired z+by transfer were capable of producing

the enzyme since the original recipientslacked activity (being z-).We already knew from experimentsby Francois Jacob and Eli Wollman thatwhen this mating was interrupted atintervals after mixing parental strains,the P-galactosidase gene was stablytransferred a t 17 minutes, as determinedby plating and emergence of lactose-positive colonies.8Direct measurementsof enzyme activity, done with FrancoisJacob, showed that the gene allowedenzyme synthesisat this time of transfer.This result is consistent with the modelof sequential gene transfer duringmating.8 We found, furthermore, thatthe Z+ gene functioned at maximal ratewithin a few minutes of its transfer.Having the experimental techniquesunder control, we turned to the questionof how the i gene determines inducibilityvs. constitutivity. In the most interestingexperiment the donor was inducible (i+)and z+, as before. The recipient cell nowwas constitutive (i-), and it was z- SOthat it could not produce /3-galacto-sidase. When mating was carried out wefound something novel and very ex-citing, namely that high activities ofP-galactosidase rapidly appeared in theabsence of any inducer (Table 1). The Z+gene from the donor became activewhen it entered the constitutive (i-)recipient. This was of course consistentwith the idea that the i- recipientcontains an intracellular inducer mol-ecule, perhaps some endogenously pro-duced /3-galactoside.Even more striking was our obser-vation that this constitutive synthesisdisappeared within about 2 hours.Apparently the i+ gene that was intro-duced along with the z+ gene neededthese 2 hours to act, but then becamedominant over the resident i- gene,

r I 1 I I 125 t

;!-0 P I& I J I5 t d-0 40 60 80

Time (min)Fig. 1. p-Galactosidase induction by mated bac-teria. Donor z+ i4 Sm 8 E. coli were mated wirhrecipient z- I+ Sm' bac teria in the pres ence ofinducer and Sm . Samples were assayed at intervals(0-0). A control mating with heavily UVirradiated recipients is shown (-a) demon-strates that mated cells are responsible for theactivity.


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88 BioEssays Vol. 2, NO.2ROOTSTA B LE I . Loss of constitutivity

Time (min)Inducer 0 60 120 180

p-galactosidase- 8 36 60 61+ - 39 113 139Donor z+ i + SmS E . coli were mated withrecipient z- i- Sm' bacteria. A t 10 minutes Smwas added, and inducer to one culture but not to asecond. Samples were assayed for 8-galactosidaseactivity at intervals.

because it made the cells inducible. Thiscapacity for induction was shown bysupplying inducer which still turned onP-galactosidase synthesis (in the pres-ence of streptomycin to block the Smsdonors). Our hypothesis was that the i+gene makes a dominant substance thatwe called the repressor which blocksexpression of the z+ gene, and the addedinducer inhibits the inhibition by repres-sors (Figure 2) .Further experiments supported thisrepressor model. For example deletionmutants in which the i gene was totallyremoved were shown to be constitutive,a result attributable to absence of any igene product. The reverse cross in whichi- and z- genes were introduced into aninducible i+ and z+ recipient did notpermit the initial constitutive enzymeformation, again indicating that the ifgene is dominant. Also matingcould notmix the two cytoplasms, since the resultsdiffered totally depending upon which

/ \a-galactosidaseFig. 2. Schematic of the repressor model. The topsection indicates synthesis of repre ssor proteincoded by the i+ gene, but not by the i- gene. Thecenter sect ion indicates blocking of z geneexpression by bound repressor. The bottom sectionshows added inducer ( I ) combines with repressorandreleases tfi-omz D N A to allow$-galactosidaseformation. The z- mutants cannot make the enzymeeven in the presence of inducer. Thet utants lackrepressor andso are Constitutive or ormation of theenzyme.

parent carried the i+ gene. The repressorhypothesis was of course completelyopposite to the previous one of aninternal, positive inducer. These resultspermanently changed thinking regard-ing the nature of genetic regulation.Kenneth Schaffner provides an interest-ing philosophical-historical interpreta-tion of these experiments, as seen thenand more re~ent1y .l~Developments from the PajamaExperiment

The nature of the repressor moleculeremained unknown for a few years. It isto the great credit of Walter Gilbert andBenno Miiller-Hill that they isolatedand characterized the repressor.16,7This was no mean task, owing to thescarcity of this molecule in the cell; itexists in only about ten copies pernormal cell. By ingenious mutant selec-tions they were able to increase theamount of repressor per cell, and thenthey isolated it. The repressor's identityis now well established as that of aprotein, and it has the allosteric propertyof binding both the specific DNA of theP-galactosidase operator gene and theinducer. Binding of inducer decreasesrepressor affinity for the DNA operatorsite, consistent with the kinetic evidencethat we provided in the Pajamaexperiment' and its sequel.12The Pajama experiment opened upmolecular studies of gene regulation."Ideas that evolved from it includeoperator and promoter regulatorygenes, as well as organization of genesinto co-regulated sets named 0per0ns.l~The repressor protein and feedbackregulated enzymes both have two bind-ing sites, separate molecular domainsfor function and control. These findingsled to the allosteric concept for regula-tion in numerous diverse ~hen0rnena.l~These ideas are being usefully extendedto cells of higher organisms. However,it is also the case that while 'what is trueof' E . coli is true of the elephant', asJacques Monod said, it is not necessarilyso that is what is true of the elephant istrue of E. co l i ; the eukaryotic gene-regulatory machinery has features notfound in prokaryotic cells.The Pajama experiment also helped tounify concepts about the various modesof control. Thus, in repression the lowmolecular weight repressing metabolitecauses the repressor (negative-control)protein to bind more firmly to itsoperator site. Another mode, positivecontrol was shown by EIlis Englesberg.I8A small molecule (arabinose) makes apositive-control protein bind to itsoperator (control) site more firmly, and

the consequence is activation of nearbystrutural genes for arabinose utilization.Yet another control mechanism, not yetreported, would be for binding of thesmall molecule to release a regulatoryprotein from a DNA operator site thatrequires its attachment for enzymeproduction.Constitutivity still provided a puzzle,in that the absence of a repressor did notalways allow rapid enzyme formation.Constitutive enzymes exist at manylevels of activity, and so a furthermechanism must determine the rateof constitutive gene expression. Themechanism determining constitutiveenzyme production rate involves thepromoter locus, mutations in which canalter levels of constitutive enzymeactivity.17~9The discovery of mRNA largelyoriginated from kinetics observed in thePajama experiment. The rapid full speedturn-on of enzyme synthesis,' as well asrapid disappearance of enzyme formingability upon destruction of DNAthrough incorporated 3zPdecay,I2 andalso induction and deinduction with3-minute delays in unmated cells,2oallcould be explained by postulating arapidly turning over intermediatebetween gene and e n ~ y m e . ~ ~ ~hisunstable intermediate was found to bethe novel mRNA.

Rapid molecular turnover, seen withmRNA and also some proteins, isextremely important for metabolic regu-lation. It permits sensitive, rapid respon-ses to environmental conditions whichalter synthetic or degradative rates. The'dynamic state of body constituents'extends very significantly o the dynamicstate of regulatory constituents. Thephenomenon of unstable regulation hascome up recently for consideration ingrowth control of normal vs. cancercells.22

Future ProspectsThe Pajama experiment turned out tobe a pivotal event in evolution of ourideas regarding biological and metabo-lic regulation and molecular biology.Building on a body of preceding ideasand techniques, and with 'chance favor-ing the prepared mind', it unexpectedlygave a new insight with a remarkablenumber of consequences. Today theseconcepts of repressor, and operator and

promoter genes bear strongly on ourthinking regarding the molecular natureof all biological regulatory processes.The Pajama experiment provides aparadigm for study of other complexbiological-biochemical-genetic pro-blems. Understanding the mechanism


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BioEssays Vol. 2, No. 2 89ROOTS

by which external factors (inducers)regulate induction developed throughseveral levels of investigation. Firs t wasthe cell biological adaptive phaseY2.3then came application ofenzyme kineticsduring the induction phase.4 This wasfollowed by genetics from which evolvedthe Pajama experiment; and in thencame biochemicaP and molecularbi~logical '~tudies on the repressor andrelevant genes. One hopes that currentresearch on growth regulation in normaland cancer cells under the influence ofextracellular growth factors will rapidlyprogress along a similar path.REFERENCES1 PARDEE, . B., JACOB,. & MONOD,.(1959). The genetic control and cytoplasmicexpression of 'inducibility' in the synthesisof ,&galactosidase by E. coli. J. Mol. Biol. 1,2 GALE,E. F. (1943). Factors influencingthe enzymatic activities of bacteria. Bac-teriol. Rev. 7 , 139-173.3 STANIER,. Y. (1953). Adaptation, evolu-tionary and physiological; or Darwinismamong the microorganisms. InAdaptation inMicroorganisms. Symp .SOC.en. Microb iol.,pp. 1-20. Cambridge University Press.4 Corn , M. (1957). Contributionsofstudieson /3-galactosidase of Escherichia coli to ourunderstanding of enzyme synthesis. Bac-teriol. Rev. 21, 140-168.5 POLLQCK,M. R. (1959). Induced for-

165-178.

mation of enzymes. In The Enzymes, 2nd ed.(ed. P. D. Boyer,H. ardy & K. Myrback),pp. 619-680. Academic Press, New York.6 MANDELSTAM,. (1956). Theories ofenzyme adaptation in microorganisms. Int.7 Cold Spring Harbor Symposia on Quanti-tative Biology. (1961). No. 26. Cellular Regu-latory Mechanisms. Cold Spring HarborLaboratory, New York.8 WOLLMAN,. L., JACOB,. & HAYES, .(1956). Conjugation and genetic recombi-nation in Escherichia coli. Cold SpringHarbor Sym p. Quant. Biol. 21, 141-162.9 PARDEE, . B. (1985). Molecular basis ofbiological regulation: origins from feedbackinhibition and allostery.BioEssays 2 , 3 740 .10 PARDEE,A. B. (1959). The control ofenzyme activity. In The Enzymes, 2 nd ed.(ed. P. D. Boyer, H. Lardy & K. Myrback),pp. 681-716. Academic Press, New York.11 MCFALL, ., PARDEE, . B. & SENT,G. S. (1958). Effects of radiophosphorus.decay on some synthetic capacities of bac-teria. Biochim. Biophys. Acta 27, 282-297.12 RILEY,M., PARDEE, . B., JACOB,. &MONOD,. (1960). On the expression of astructural gene. J. Mol. Biol. 2, 216-225.13 JACOB,. & MONOD,. (1961). Geneticregulatory mechanisms in the synthesis ofproteins. J. Mol. Biol. 3, 318-356.14 JUDSON,. F. (1979). The Eighth day ofCreation: The Makers of the Revolution inBiology. Simon and Schuster, New York.15 SCHAFFNER,. (1974). Logic of discoveryand justification n regulatory genetics. Stud.Hist. Phil. Sci. 4, 349-385.

Rev. Cytol. 5, 51-87.

16 GILBERT, . & MULLER-HILL,. (1966).Isolation of the lac repressor. Proc. Natl.Acad. Sci. USA 56, 1891-1898.17 The Lactose Operon (1970). (Ed. J. R.Beckwith & D. Zipser). Cold Spring HarborLaboratory.18 ENGLESBERG,., SHEPPARD,., SQUIRES,C. & MERONK,. JR. (1969).An analysis of'revertants' of a deletion mutant in the Cgene of the L-arabinose gene complex inEscherichia coli B/r: isolation of initiationconstitutive mutants (1"). J. Mol. Biol. 43,19 PARDEE,A. B. & BECKWITH,. R.(1962). Geneticdetermination ofconstitutiveenzyme levels. Biochim. Biophys. Acta , 60,452-454.20 PARD=, A. B.& ~ E S T I D G E , L. s. 1960).The initial kinetics of enzyme induction.Biochim. Biophys. Ac ta, 49, 77-88.21 PARDEE, . B. (1958). Experiments onthe transfer of information from DNA toenzymes. Exp. Cell Res. 6, 142-151.22 CROY,R. G. & PARDEE, . B. (1983).Enhanced synthesis and stabilization of M,68,000 protein in transformed BALB/c-3T3cells: Candidate for restriction point controlof cell growth. Proc. Natl. Acad. Sci. U S A80,4699-4703.

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A R T H U R B. P A R D E E is in the Dept.of Pharmacolo2y, Hai-uard Medical Schooland is chief of the Division of Cell Growthand Regulation,Dana-Farber CancerInstitute, 44 Binney St., Boston, Mass.02115, USA

Transdisciplinary Science and the Graduate CurriculumRobert B. Lawson

SummaryIn the accompan ying article contributedby Robe r t B . Lawson, proposals arema& fo r revis ing the curr iculum fo rdoctoral students in biology in order toenhance a transdisciplinary awareness ofbiological science. Th e article is writtenmainly in the context o f Dr Lawson's rolea s a scientist and educator in the UnitedStates. BioEssays wil l welcome articlesalong similar l ines fr o m educators inother countries. These should be sent tothe St af f Edi tor , Dr Ada m S . Wilkins .Science is a human enterprise which inour universities includes the domains ofgraduate education and basic research.Science has traditionally been dividedinto many disciplines, a procedurewhich we judged made the objects of

scientific inquiry easier to understandand the institutions that conduct andsupport basic research more manage-able. Today, American universities per-form approximately one-half of the basicresearch carried ou t in the United Stateswith the federal government providingabout 6570% of university researchfunding. Approximately four-fifths ofthese federal dollars flow through threeagencies to our universities, namely,Health and Human Services, Depart-ment of Defense, and the NationalScience Foundation, respective1y.l Al-though requests for funds to theseagencies are considered somewhat intransdisciplinary terms, there are strongagency infrastructures in place tha t tendto reinforce the departmental or dis-ciplinary organization of most universi-ties. Accordingly, we find tha t American

universities as major performers ofbasic research and the federal agenciesas a major funding source promote acompartmentalization of science thattends to minimize transdisciplinaryresearch and breadth in graduateeducation.

In the biomedical and life sciences, thegraduate curriculum has been shapedprimarily by departmental and disciplin-ary requirements designed to producethe most advanced researcher within alimited area of expertise. The graduatecurriculum is generally considered as aseries of disparate programs held to-gether by a minimum number ofprogram-wide requirements. The re-quirements for the doctorate in bio-chemistry are quite different from thosefor microbiology or physiology al-though each of these programs share the

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86 BioEssays Vol. 2, NO. 2 ROOTS Shall Be