Current Biology 20, R718–R723, September 14, 2010 ª2010 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2010.06.027

From Operons to EvoDevo

Guest EditorialJacob and Monod:

Alexander Gann

‘‘The game was that of continually inventing a possibleworld, or a piece of a possible world, and then ofcomparing it with the real world...What matteredmore than the answers were the questions and howthey were formulated.’’

Francois Jacob, The Statue Within.

As Francois Jacob tells it, one afternoon in September 1958,just back from New York, he walked into Jacque Monod’soffice at the Pasteur Institute in Paris; he believed he hadsomething exciting to discuss. But he found an unimpressedMonod brusquely dismissive. Tired from his flight, Jacobquickly gave up and went home to bed. The next day hereturned re-energized and found an altogether more receptiveMonod. Although the two had worked at the Pasteur for anumber of years, their conversation that day launched anintense period of collaboration that resulted in one of the trueintellectual and experimental triumphs of molecular biology.

Jacob had been in New York to deliver the Harvey Lectureat Rockefeller University and in the weeks leading up to thisprestigious event, while pondering what he might say, some-thing rather remarkable had occurred to him: perhaps thetwo unrelated biological systems studied at the Pasteurwere controlled in the same way. The two systems in ques-tion were lysogeny by bacteriophage lambda, and the abilityof Escherichia coli to make an enzyme that digests lactoseonly when the cell encounters that sugar. Jacob’s idea wasthat the mechanisms underlying the regulation of these twootherwise distinct phenomena were identical.

Below I give an overview of how Jacob and Monod cameto work together, and of the historic experiments they com-pleted 50 years ago this year. This is not for mere arcaneamusement on this important anniversary, but because theirwork remains relevant. Many observations reported sincein studies of gene regulation and developmental biologyare in essence re-runs of their experiments, in differentways and in a variety of systems. They also provide thecontext for what follows in this special issue on the evolutionof gene expression. Jacob and Monod’s model of gene regu-lation is essential for an understanding of the emerging fieldsof EvoDevo and the evolution of gene circuitry.

Before the Collaboration BeganMonod was the elder of the two men by 10 years — andwould have been 100 this year were he still alive. A keenrock climber, accomplished cellist and sailor, he joined thePasteur after the liberation of France in 1944. During thewar he had worked in a lab at the Sorbonne while also takingpart in the guerilla activities of the French Resistance, risingto a prominent leadership role. Following concern that hisidentity had been revealed to the Gestapo, he was forcedto move out of his apartment and steer clear of the Sorbonne.He was given temporary bench space in Andre Lwoff’s lab

Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.

E-mail: ganna@cshl.edu

at the Pasteur Institute, and never left; by 1970 he was theInstitute’s director.

Workingon the E.coli lacsystem, Monod andhis colleagueshad developed a range of biochemical tools with which theycould show that the presence of lactose in the growth mediuminduced synthesis of the enzyme, b-galactosidase, that wasresponsible for digesting lactose. This was already a majorinsight: previously the favored model of ‘enzyme adaptation’invoked a precursor enzyme taking on the ability to digestlactose when it encountered that sugar — the enzyme, itwas suggested in the most extreme version of this model,used lactose as a template on which to fold itself into therequired shape to digest that substrate. When another sugarpresented itself, the enzyme would change shape again andbecome active on that new substrate instead. (This, it shouldbe remembered, was at a time before it was widely acceptedthat a given protein had a defined three-dimensional shape,even a consistent composition.)

In 1950, the 30-year old Jacob arrived at the Pasteur — likeMonod before him, a refugee taken in by Lwoff. Havingescaped France after it fell to the Germans, Jacob foughtfor the Free French under de Gaulle and was seriouslywounded in Normandy soon after the D-day landings. Theseverity of those wounds kept him in hospital for monthsand left many pieces of shrapnel permanently buried in hisbody. After the war, he completed the medical training theconflict had interrupted but found he was unable to take upa career as a surgeon due to the effects of his injuries. Aftera number of false starts in various professions, he fell uponbiological research after reading of recent work in what wouldsoon become molecular biology. By then, Monod had his ownlab located at one end of a short corridor in the attic of thePasteur;Lwoff’s lab wasdiagonally across the hall at the otherend. Jacob first approached Monod, who directed him toLwoff. After repeated attempts to persuade Lwoff to hirehim, and despite his lack of knowledge and experience —and his age — Lwoff in the end relented when his expandinginterests in lysogeny required more helpers.

Lwoff studied lysogeny in a strain of Bacillus megaterium,but soon after Jacob arrived they switched to E. coli lyso-genic for phage lambda, recently discovered by EstherLederberg. Phage lambda infection unfolds in one of twoways: either lytically, in which the phage genome replicates,packaging proteins are made, and the cell lyses with therelease of several dozen new phage some 45 minutes later;or lysogenically, in which the phage genome integrates intothat of the host in a form called a prophage. The viral genesare almost all shut off in a lysogen, and the prophage isreplicated passively as part of the bacterial genome. Also,a lysogen is ‘immune’ to further infection by another lambda:the repression that keeps the prophage silent also silencesany new incoming phage as well.

While the prophage within a lysogen is almost infinitelystable if left unmolested, it can, upon receipt of a suitablesignal, awake from its dormant state and return to lyticgrowth (in a process called induction). Lwoff had been thefirst to show this, using UV light as inducer, just before Jacobjoined his lab. Indeed, Jacob himself speculated that it wasperhaps the good mood engendered by this discovery that

Figure 1. The Nobel Prizes, December 1965.

Monod, Lwoff and Jacob (second, third andfourth from right respectively), compare theirawards with physics winners Richard Feyn-man and Julian Schwinger. At far left, ChemistR. B. Woodward seems immersed in the deco-rative calligraphy of his own certificate. On thefar right, that year’s literature winner, MikhailSholokhov, stands slightly apart. (Photo re-produced with permission of Getty Images.)

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weakened Lwoff’s resistance to hisjoining the lab. Lwoff’s work is ratherlittle celebrated these days, though heshared in the 1965 Nobel Prize togetherwith Jacob and Monod for the work ongene regulation (Figure 1). His ownsplendid account of his work onlysogeny, in an essay entitled ‘‘TheProphage and I’’ (in Phage and theOrigins Of Molecular Biology), revealshis dry wit as well as his remarkablescience.

While Monod had largely used biochemistry to investigatethe process of enzyme induction, Jacob favored geneticswhen grappling with lysogeny. These different approachesmade sense. The products of the lambda genes encodingreplication and packaging functions were numerous andbarely characterized, and none was readily assayed. Butmutants of the phage with altered behaviors were relativelyeasy to come by. In contrast, the activity of the enzymeb-galactosidase (product of the lacZ gene) was of courseknown, and easily assayed — both using anti-sera raisedagainst the protein, and by use of a special substrate thatchanged colour when cleaved by the enzyme. In addition,the roles of inducer and substrate (usually both lactose)could be separated using chemical derivatives that eitherinduced synthesis of the enzyme but didn’t serve as itssubstrate, or vice versa. These compounds — derivedlargely by Mel Cohen, one of the first in a long line of Ameri-cans to visit the lab — allowed sophisticated manipulation ofthe system, even while its genetics remained rudimentary.

The PaJaMa experimentSo what was it that had struck Jacob while preparing for hislecture in New York? What had convinced him that lambdaand the lac genes were regulated identically? A dramaticclue had come from a pair of experiments — the racysounding ‘erotic induction’ and the rather more prim PaJaMaexperiment.

In fact, these were essentially the same experiment, butcarried out in each of the two systems. Jacob, togetherwith Elie Wollman, had established conjugation as a conve-nient way of mapping bacterial genes. Sex in bacteria wasdiscovered in 1946 by a graduate student at Yale, JoshuaLederberg (husband of lambda’s discoverer, Esther). Bacte-rial mating doesn’t involve the fusing of male and female cellsto create a diploid zygote, as it does in eukaryotes; rather,the chromosome of the male is threaded into the female,more male genes entering the female over time. And thisprocess — conjugation as it was named — can be cut offprematurely, leaving partial diploids (Jacob and Wollman

used a Waring blender for this experimental procedure,quickly and unsurprisingly christened coitus interruptus).By experimentally disrupting mating pairs of cells at differenttimes after mixing, and assaying which genes had by thenentered the female, genetic maps of the E. coli chromosomewere established, and a new experimental tool was born.

When a male cell lysogenic for lambda (i.e. carrying aprophage) mated with a female that wasn’t, two strikingobservations were made. First, the lambda prophageentered the female at a precise time after mating, just likeany bacterial gene. This showed that the phage genomewas associated with the bacterial chromosome at a definedlocation. But second, and more remarkably, upon entry intothe non-lysogenic female, the prophage at once induced —hence the term ‘erotic induction’ (renamed ‘zygotic induc-tion’ for publication; ‘‘more decorous’’ in Wollman’s words).This explosive result revealed that the prophage in a lysogenwas normally kept silent by a cytoplasmic factor whosepresence inhibited expression of the phage genes: thenon-lysogenic recipient female lacked this factor, and sothe prophage was expressed. This experiment was carriedout in 1956.

In late 1957 — a year before Jacob’s Harvey Lecture —Jacob and Monod decided to employ conjugation to lookat the lac genes. This was to be their first collaboration. Infact, the experiment was carried out by Art Pardee fromBerkeley, who was spending a sabbatical year at the Pas-teur. Jacob and Monod had collected mutants in lacZ thatcould not make b-galactosidase, and others, which theycalled lacI–, that rendered expression of b-galactosidaseconstitutive (no longer inducible, the genes were expressedall the time, irrespective of whether lactose was present). Soin what became known as the PaJaMa experiment, Pardee,Jacob and Monod set out to test whether inducibility orconstitutive expression was dominant. To do this, theymated a lacZ+ lacI+ male with a lacZ– lacI– female (in theabsence of inducer). They saw maximal (and constitutive)expression of b-galactosidase within minutes of the lacZgene entering the female, after which expression fell and

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once again became inducible. The main conclusion from thisexperiment was that lacI encoded a trans-acting repressormolecule which inhibited expression of lacZ. The alternativeexpectation (held by Monod) had been that the mutant cellsproduced an inducer that drove constitutive expression; hisscenario would have predicted that constitutive expressionwould be dominant. A preliminary report of the PaJaMaexperiment was published in May 1958.

The similarity between zygotic induction and the PaJaMaexperiment was striking. In both cases, genes (be theylambda lytic genes or lacZ) were released from repressionwhen passed into the repressor-free cytoplasm of therecipient female. For lambda, this led to induction of thephage and death of the cell. In the PaJaMa experiment, itmeant constitutive expression of lacZ — at least untilsynthesis of repressor from the closely linked lacI gene,also brought in by mating, reached a level sufficient toonce again repress (and make inducible) lacZ expression.

But it was only in June of that year, while starting toprepare for the Harvey lecture, that Jacob saw just howfar — and how usefully — this analogy could be extended.By assuming the two systems operated in the same wayone could pool resources — the strength of lambda geneticscould be coupled with the biochemical sophistication of thelac system, for instance. And if any feature found in onesystem was mirrored in the other, this would distinguishwhat was fundamental to gene regulation from mere eccen-tricities of either system. Jacob was about to leave Paris onan extended trip and didn’t get to discuss this with Monodright away. And, as quoted by Judson in The Eighth Day ofCreation, the one time Jacob was back in Paris before finallyreturning that September after the Harvey lecture, Monodwas ‘‘in his boat somewhere’’.

Combining lac and LambdaOnce he began thinking of the two systems as one, Jacobrealized that in both lac and lambda, a set of genes is keptoff by a single repressor. For lambda, the set of genes waslarge (about 50) and Jacob found it hard to envisage thesingle lambda repressor inhibiting — specifically, and indi-vidually — protein synthesis of so many different genes. Hereasoned it was more likely to impose control at the levelof DNA, where all the genes were on a single molecule. Heimagined repressor acting as a master switch at that firststep of expression. And if this was true in lambda, it wouldbe true for lac as well.

Once Monod embraced this approach, further ideas werediscussed on a daily basis. Predictions and experimentscame thick and fast. Thus, for example, if lac repressorworked at a specific location on DNA, it should be possibleto get mutations in that site that would no longer bindrepressor. Such mutations should render lacZ expressionconstitutive, just as mutations eliminating repressor itselfdid. But unlike lacI mutations, the lacOc mutations (as theybecame known) should not be complemented by a wild-type copy of the operator in trans (because, unlike thecytoplasmic repressor, the DNA site wouldn’t act in trans).In a perfect example of the synergy between the systems,Jacob realized that mutants with exactly these characteris-tics had already been described in the lambda system (butnot interpreted in this way). Thus, so-called Virulent (vir)mutations render a phage insensitive to the actions ofrepressor — they can grow on lysogenic cells (uninhibitedby that cell’s ‘‘immunity’’). And those mutations are dominant

in cis: only genes on the same phage chromosome as the virmutation (i.e., genes in cis to it) are expressed when lysogensare infected by a mix of lambda vir and lambda wild-typephage.

So Jacob and Monod set out to isolate the proposed lacOc

mutations. This — and several other experiments — requiredE. coli strains stably diploid for the lac genes (diploids of thesort obtained in the PaJaMa experiment were only transient).The problem was solved with the generation of a derivativeF-plasmid that carried the lac genes (so-called F0lacplasmid). The F-plasmid is what enables sex betweenE. coli, and having the F-plasmid is what makes a cellmale. This plasmid encodes the proteins needed to con-struct the physical connection with a female cell and feedthe plasmid into her. In the experiments described thus far,those in which the bacterial chromosome is fed into thefemale recipient cell, a so-called Hfr strain was used: inthat case, the F-plasmid has inserted into the bacterial chro-mosome, so when it passes into the female it drags the chro-mosomal DNA along with it. The lac genes carried on theF0lac derivative Jacob and Monod now used were pickedup by the F-plasmid upon excision from the chromosomeof an Hfr strain. Using F0lac plasmids, any alleles of the lacgenes could be put into stable diploid combinations andtheir dominance and recessive character observed. Thisset-up allowed the lacOc mutations to be distinguished asa class that imposed constitutive expression in cis.

But the assumption that both the lac and lambda systemsmust operate in the same way had to contend with someawkward findings as well — results that didn’t seem to fit.For example, there were three lac genes (in addition toLacZ, there were lacY, encoding the permease, and lacA, en-coding an acetylase of unknown function) and Monodpointed out that, if all three were controlled from a singlelocation by a master regulator, as predicted in the lambdacase, they should be coordinately expressed under allcircumstances. But, Monod revealed, induction with oneparticular kind of inducer activated synthesis of the per-mease but not b-galactosidase. A great skill in doing scienceis to know that sometimes an isolated result must be ignored(though remembered). It might be misleading. In this case,only later was the assay shown to be at fault. The proteinbeing assayed as the presumed product of lacY in this exper-iment was in fact made by another gene elsewhere on theE. coli genome, controlled by a different system sensitiveto this one inducer. Jacob and Monod also ignored the effectof glucose on their system. Again, this was necessary if theywere to uncover the basic truth in an experimentally tractableway. Glucose effected regulation of the lac genes througha different regulator in a different way.

Although the work of Jacob and Monod is today primarilyassociated with gene regulation, it also played a part in un-covering the basic process of gene expression and thediscovery of mRNA, as outlined in Box 1.

50 Years OnFifty years on, we are struck by two things from the work ofJacob and Monod: first, the beauty of their thinking and theclarity with which they designed the experiments requiredto establish their model (I have given only a flavor here);and second, the fact that their model did indeed explainnot only how bacterial cells and phage respond to theirenvironment, but also, in essence, how development of amulti-cellular organism is controlled. That is, differentiation

Box 1

Good Friday in Brenner’s Rooms at King’s.

I have in this article focused solely on the issue of regulation of gene expression. But Jacob and Monod’s work — and in particular the

PaJaMa experiment — also influenced ideas about the process of gene expression, and in particular the role of mRNA.

It was at this time known that protein synthesis took place on ribosomes; also that ribosomes contained RNA — the likely intermediate

between DNA and protein. So it was believed that ribosomes were factories, and that each made a specific protein. But there were several

niggling problems with this model. In particular, the ribosomes seemed too stable and the RNA they contained too uniform to explain the

varying patterns of gene expression required by the cell. These problems had for a few years caused a roadblock in thinking about the genetic

code and how genes are expressed.

The insight that an unstable RNA intermediate (messenger RNA) traveled from the gene to the ribosomes, carrying the genetic message that

instructed the (otherwise dumb) ribosome what protein to make, was a revelation arrived at in Sydney Brenner’s rooms in King’s College

Cambridge on Good Friday 50 years ago. Gathered there was a small group - including Jacob, Brenner, Francis Crick, Alan Garen, perhaps

Ole Maaloe, and a few others.

Jacob came to the meeting with news from Paris — constitutive operator mutations and the latest iterations of the PaJaMa experiment.

Brenner and Crick were more interested in the genetic code than in regulation. They brought other data to the discussion, in particular the

finding of Volkin and Astrachan (and earlier of Al Hershey) that, upon infection of E. coli by phage T2, an unstable RNA species was produced

which had the same base composition as the phage DNA. It had been assumed this RNA was probably involved in phage replication rather

than gene expression.

But suddenly that afternoon it became obvious — first to Brenner and Crick, and then to the others present — that the rates of induction

(and then repression) in the PaJaMa experiment predicted an unstable intermediate in gene expression, and that the unstable RNA produced

in T2 infection could represent the comparable thing. Also, the operator constitutive mutants suggested that regulation (repression) really did

act at the genetic level controlling production of the unstable mRNA. This discussion, continued that evening at a party at Crick’s house,

led directly to the experiment by Brenner and Jacob, who, together with Matt Meselson at Caltech that summer, demonstrated the existence

of mRNA. Separately, Jim Watson, Wally Gilbert and Francois Gros arrived at a similar result through different means at Harvard.

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and cell fate in higher organisms are controlled throughregulatory mechanisms very similar to those described inbacteria. That this is true is startling, and takes on evengreater significance as it emerges that evolution itself veryoften works by fiddling — Jacob would say ‘tinkering’ —with those very regulatory mechanisms. Thus, the ideas ofJacob and Monod lie at the heart of our understanding notonly of development, but also of how evolution has modeled,for example, animal diversity.

But before considering further the implications of theirwork for gene regulation more broadly, it is worth notingwhat their model did and didn’t tell us. It uncovered thefundamental fact that genes are controlled at the level oftranscription by the products of other genes encodingregulators. These regulators act through binding sites onDNA near the genes they control, each regulator recognizinga specific site and thus controlling specific genes. Accordingto Jacob and Monod, these regulators are always repres-sors; by binding near the start of the gene they wouldblock RNA polymerase and thus inhibit transcription. Theirmodel — often known as the Operon model — also claimedthat linked groups of genes (‘operons’) are controlled assingle units. This is often true in bacteria, but unlike therest of their model, is largely irrelevant in eukaryotes (wherethe regulators typically bind to separate sites at each genethey control). In fact, even in bacteria this property is farfrom universal. In the case of lambda itself, for example,many of the genes thought by Jacob and Monod to comprisea single operon are in fact controlled independently throughthe use of terminators and anti-terminators, and additionalinternal promoters. So rather ironically, the feature thatgave the model its name was the one of least generality.

Jacob and Monod also realized that the regulators arethemselves controlled by signals. Thus, the specificity ofthe lac repressor comes from, on the one hand, the fact it

recognizes a site on DNA near the lac genes, and on theother, by the fact that it only binds that site in the absenceof the sugar lactose. Though I haven’t described any of thework here, lactose binds the repressor and inactivates itthrough allosteric modification. Monod (together with Jean-Pierre Changeux and Jeffries Wyman) came up with thismodel for the allosteric change to lac repressor caused byinducer. And the control of regulators by physiologicalsignals — very often through allosteric changes — is yetanother very general part of their model.

Lambda repressor is inactivated by signals that inducea lysogen. Thus, UV-irradiation causes DNA damage. DNAdamage is recognized by the RecA protein, which in responsestimulates cleavage of a bacterial repressor (LexA) whichotherwise keeps repressed various genes encoding DNArepair functions. Lambda repressor has evolved to look verylike LexA, and so it too gets cleaved, triggering the prophageto switch to lytic growth and escape the damaged cell.

What Didn’t They Know?They didn’t know what repressors were made of — protein orRNA were the two obvious candidates, and they initiallyfavored RNA (it was easier to see how RNA could recognizespecific DNA sequences; and it seemed more efficient to useRNA rather than having to translate it into protein first). Soonafterwards protein became the favored candidate — ambermutations were isolated in the lambda repressor gene.Yet a number of attempts by different labs — includingMonod’s own — failed to isolate a repressor and some beganto doubt the original model. Only in late 1966 did WallyGilbert and Mark Ptashne, both at Harvard, working closelyyet in competition, isolate the lac and lambda repressors,respectively, and show that they are proteins and that theydo indeed work by binding to the operator sequencesidentified genetically by Jacob and Monod.

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Of course, since the emergence of RNAi and miRNAs,the suggestion that the repressors are RNAs doesn’t seemas wide of the mark as once it did. RNA regulators, likesite-specific DNA-binding proteins, can indeed provide thespecificity needed to direct regulatory choices (thoughusually not at the level of transcription). In light of our discus-sion thus far, a nice example to consider is hybrid dysgen-esis — a phenomenon that looks very like zygotic inductionand the PaJaMa experiment. So, for example, when a maleDrosophila bearing P-element transposons mates with afemale that doesn’t, the P-elements start transposing in thefertilized egg. This is because, like the non-lysogenic femaleE. coli that lacks lambda repressor to keep silent theincoming prophage, the female fly egg lacks the repressorneeded to keep expression of the P-element switched off.And in this situation, repression requires small RNA mole-cules working through the RNAi pathway.

There has of late been much breathless coverage in thepopular science press of so-called ‘information beyondthe genome’ that allegedly exists to regulate gene expres-sion in eukaryotic systems. In fact, all regulatory eventsneed a protein to recognize a specific DNA sequence, or anRNA to recognize its site, to give the initial specificity. Andeither event relies very much on information within thegenome.

What else didn’t they know? Their model didn’t allow foractivators. But it didn’t actually exclude them — that is,activators could readily be accommodated by the model;they too could be trans-acting factors recognizing sites onthe DNA, just like repressors. But Monod in particular wasfor several years adamant that activators wouldn’t existbecause they are not necessary (the demands of any regula-tory system could in principle be met using repressors, orrepressors of repressors, etc., as detailed most fully in theirGeneral Conclusions piece for the 1961 Cold Spring HarborSymposium of Quantitative Biology). Monod’s characteristi-cally intense conviction made it hard for evidence of positiveregulation (initially obtained by Ellis Englesberg in the Arab-inose system) to gain real traction. But by the late 1960s itwas largely accepted that, as well as repressors, positiveregulators do indeed exist. And it soon transpired that eventhe lac and lambda systems use activation as well as repres-sion, and that activators obey the principles of the Jacob andMonod model for regulators.

How are the basic principles of the Jacob and Monodmodel extended to answer the specific demands of partic-ular regulatory situations? A complete description of thelac genes themselves reveals one simple case. As we havealready seen, lactose lifts repression of the lac genes byinactivating lac repressor, which otherwise binds the DNAand excludes binding of RNA polymerase. But in addition,the absence of glucose (a preferred energy source) triggersactivation of those same genes by the activator CAP, which,in the absence of glucose, binds near the genes and recruitsRNA polymerase to them. Thus, the lac genes are onlyexpressed at high level in response to the simultaneouspresence of two signals (presence of lactose, absence ofglucose). This is an example of ‘signal integration’, whichcan readily be expanded to include more, and different,combinations of signals, each acting through a differentregulator. Indeed, the activator (CAP) that signals absenceof glucose to the lac genes similarly communicates thatsame condition to other genes that bear the appropriateDNA binding site; and at those genes it works in combination

with different other regulators. But each regulator works ac-cording to the rules of the French.

And the complete picture of how lambda choosesbetween lytic and lysogenic development, and how theprophage is maintained stably in the lysogenic state (aclassic case of epigenetics) or is efficiently induced, remainsperhaps the most complete and elegant picture we have ofhow simple regulators solve sophisticated developmentalproblems (work largely carried out by Mark Ptashne, andrecounted in his classic book, A Genetic Switch). Lambdarepressor is both an activator and a repressor and usescooperative binding to multiple sites on DNA, and positiveand negative auto-regulation, to heighten the specificityand efficiency of its action. Another repressor (Cro) andanother activator (CII) are also involved in regulating thephage life-style choice. Another feature of this system thatis seen generally is that different promoters and regulatorsare used to establish and to maintain expression ofrepressor. All these clever ploys allow ever more fine-tuning.Yet all the regulators are working according to the model ofJacob and Monod with simple add-ons.

Regulation of DevelopmentAnd as I have said, similar systems of regulation underpin thedevelopment of higher organisms as well. The classicgenetic screens carried out by Nusselein-Volhard andWieschaus in their search for the regulators of the criticaldecisions in early Drosophila development turned up largelysite-specific DNA-binding proteins — regulators of theJacob and Monod sort. And when the actions of some ofthese regulators were worked out in detail, what was re-vealed was a series of Jacob and Monod-like regulatorysystems — the trans activators and repressors binding toDNA sites (cis regulatory sequences) upstream of targetgenes. The regulators each communicate to the gene a signal(a physiological or developmental timing cue), and worktogether, often cooperatively, and so on. Regulation of theEve stripe 2 enhancer — required for expression of thesegmentation gene even-skipped (eve) in a specific stripeof the developing Drosophila embryo — by two activatorsand two repressors is a nice case in point (see the reviewby Mike Levine in this issue).

Even in mammalian development we see again and againthat the laws of Jacob and Monod hold firm. A nice recentexample, from the work of Robin Lovell-Badge, shows howthe DNA-binding protein Sry determines male developmentin genetically male mice. The regulatory events that governthis process resemble remarkably closely the regulatorylogic of lambda. In other cases — the classic work of HaroldWeintraub on muscle differentiation, the cell fusionexperiments of Helen Blau, and more recently the ShinyaYamanaka experiment in which expression of three (trans-activating, DNA-binding) regulators was shown to be enoughto generate induced pluripotential stem (iPS) cells — all showthat even mammalian differentiation is happening in theworld as described by Jacob and Monod.

Indeed, very often the experimental manipulations usedto reveal the workings of these systems are essentiallythe same as the experiments performed by Jacob andMonod. Stable states of differentiation turn out to be main-tained by trans-acting cytoplasmic factors, and thosefactors turn out chiefly to be DNA-binding regulators. Andsuch systems can maintain very stable states in mammaliandevelopment — just as they do when maintaining a lambda

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lysogen — and yet can be turned into something else ifmanipulated to do so (experimentally, as in the Yamanakacase). One last point: the frequency of iPS cell productioncan be increased by certain treatments — DNA demethyla-tion, for example — but it is the transcription factors thatprovide the specific instruction to become an iPS cell byactivating the correct genes. Just as, in the case of musclecell differentiation, only MyoD (or another of the myogenicregulators) activates the correct genes to make a cell amuscle cell.

Evolution by TinkeringAnd what of evolution, the topic to be covered in the reviewsthat follow? Much has been written on how gene regulationcan itself evolve, and the extent to which changes in generegulation underpin changes seen in evolution.

Much of the field of EvoDevo employs the language ofJacob and Monod when describing the causes of morphoge-netic variation between animals. Thus, there is much discus-sion about whether changes in ‘cis regulator sequences’account for most variation in animal form (as opposed tochanges in coding sequences of proteins). These are thecis regulatory sequences of Jacob and Monod. It now seemsthat much evolutionary variation does indeed come down tochanges in the regulation of genes, rather than the inventionof new enzymes. (I hasten to add that I am here talking only ofmorphological diversity in higher organisms; bacterial diver-sity is metabolic diversity, and more often requires changesin protein sequences and the acquisition of novel enzymaticactivities.) Often the changes in regulation are indeedmediated by changes in the cis regulatory sequences (asJacob and Monod’s model might lead one to expect), exam-ples being found in the work of Sean Carroll, David Kingsley,and others; similarly, change in the expression of a regulatorwas shown by John Doebley to be critical in domestication ofmaize. But also, as seen in work on the evolution of generegulation in yeast (see the review by Hao Li and AlexanderJohnson in this issue), regulatory variation can also arisethrough modest changes in the sequences of the trans-acting regulators (changes that can easily alter who toucheswhom, changing which partners bind cooperatively at whatgenes, and hence when and where those genes areexpressed).

While not the first, certainly an early and articulate pro-ponent of the idea that evolution would feed off changesin patterns of gene expression — reusing the repertoireof proteins already encoded rather than inventing newones — was Francois Jacob himself, in a 1977 paper inScience entitled ‘‘Evolution and Tinkering’’. As he noted:‘‘Biochemical changes do not seem, therefore, to bea main driving force in the diversification of living organisms.The really creative part of biochemistry must have occurredvery early.’’ Instead, he argued that: ‘‘It seems likely thatdivergence and specialization of mammals, for instance,resulted from mutations altering regulatory circuits ratherthan chemical structures. Small changes modifying thedistribution in time and space of the same structures aresufficient to affect deeply the form, the functioning, and thebehavior of the final product – the adult animal..It isalways a matter of tinkering.’’

Acknowledgements

Thanks to Sydney Brenner, Allan Campbell, Sean Carroll, Richard

Ebright, Greg Hannon, Sandy Johnson, Mike Levine, Rich Losick,

Rob Martienssen, Matt Meselson, Noreen Murray, Mark Ptashne and

Gary Ruvkun.

Further ReadingCairns J., Stent G., and Watson J.D., eds. (2007). Phage And The Origins OfMolecular Biology, Centenary Edition (New York: Cold Spring Harbor LaboratoryPress).

Carroll, S., Grenier, J.K., and Weatherbee, S.D. (2005). From DNA To Diversity,Second Edition (Oxford: Blackwell Publishing).

Davidson, E.H. (2006). The regulatory genome: gene regulatory networks. InDevelopment and Evolution (San Diego: Academic Press).

Doebley, J.F., Gaut, B.S., and Smith, B.D. (2006). The molecular genetics of cropdomestication. Cell 127, 1309–1321.

Jacob, F. (1977). Evolution and Tinkering. Science 196, 1161–1166.

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