Jacob and Monod: From Operons to EvoDevo
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Current Biology 20, R718R723, September 14, 2010 2010 Elsevier Ltd All rig
Pasteur 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, rising
a lysogen is immune to further infection by another lambda:the repression that keeps the prophage silent also silencesany new incoming phage as well.identity had been revealed to the Gestapo, he was forcedjoined his lab. Indeed, Jacob himself speculated that it wasCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
perhaps the good mood engendered by this discovery thatE-mail: firstname.lastname@example.org a prominent leadership role. Following concern that histo move out of his apartment and steer clear of the Sorbonne.He was given temporary bench space in Andre Lwoffs lab
signal, 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 JacobWhile the prophage within a lysogen is almost infinitelystable if left unmolested, it can, upon receipt of a suitableJacob and Monod:From Operons to EvoDevo
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 Monodsoffice 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. Jacobs 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 Monods 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 thehts reserved DOI 10.1016/j.cub.2010.06.027
at the Pasteur Institute, and never left; by 1970 he was theInstitutes director.
Workingon theE.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 adaptationinvoked 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;Lwoffs 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,
ing anti-sera raisedecial substrate thatnzyme. In addition,ually both lactose)rivatives that eitherdidnt serve as its
pounds deriveda long line of Ameri-ated manipulation ofained rudimentary.
entered 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 Wollmans 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 carriedknown, and easily assayed both usagainst the protein, and by use of a spchanged colour when cleaved by the ethe roles of inducer and substrate (uscould be separated using chemical deinduced synthesis of the enzyme butsubstrate, or vice versa. These comlargely by Mel Cohen, one of the first incans to visit the lab allowed sophisticthe system, even while its genetics rem
The PaJaMa experimentFigure 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 years literature winner, MikhailSholokhov, stands slightly apart. (Photo re-produced with permission of Getty Images.)
weakened Lwoffs resistance to hisjoining the lab. Lwoffs 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 course
Special IssueR719So 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 lambdas discoverer, Esther). Bacte-rial mating doesnt 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 Wollmanused 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 wasnt, two strikingobservations were made. First, the lambda prophageout in 1956.In late 1957 a year before Jacobs 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
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 didnt 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 wouldnt act in trans).In a perfect example of the synergy between the systems,Jacob realized that mutants with exactly these characteris-
Current Biology Vol 20 No 17R720tics 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 cells immunity). And those mutations are dominantin cis: only genes on the same phage chromosome as...