Life's Greatest Secret

by Matthew Cobb

  They began planning their experiment in 1954; they decided to use a heavy isotope of nitrogen (15N; normal nitrogen has an atomic weight of 14) to distinguish between the original strand and its copy. Nitrogen is an important atomic component of DNA, so DNA that was synthesised with 15N was slightly heavier than its 14N counterpart. After three years of preliminary experiments, they grew E. coli bacteria for several generations on a medium rich in 15N, thereby creating a stock of bacteria with 15N-based DNA. The bacteria were then transferred to medium containing normal 14N and were allowed to reproduce for several cycles before their DNA was extracted. Meselson and Stahl spun the samples in an ultracentrifuge machine at 44,700 r.p.m. for 20 hours – any double helix containing 14N ended up at a different position in the tube from its 15N equivalent, simply because it was lighter.

  The results were very clear. After one cycle of reproduction, in which the bacteria had made only one copy of their DNA, two kinds of DNA were detected: a heavy band, composed of the original 15N-based DNA, and a slightly lighter band composed of a mixture of 15N and 14N-based DNA. This showed that ‘conservative’ replication was wrong – that model predicted that only 14N-based DNA would be present in the newly synthesised molecules. The next step was decisive: if the bacteria were allowed to reproduce for another cycle, then a new, lighter, band composed only of 14N-based DNA appeared. These molecules must have been created by copying from a whole strand of 14N DNA that had been created after the first round of replication. This showed that Watson and Crick were right and Delbrück was wrong: DNA replication was ‘semi-conservative’ with, most probably, the whole of each strand being copied simultaneously to make new daughter molecules.

  7. Figures from Meselson and Stahl’s 1958 paper. Left: DNA produced by two identical experiments (1 and 2), shown as a band (1) and a graph (r). At the beginning there was only 15N DNA. As the bacteria reproduced on 14N-rich culture, two lighter forms appeared, one after one generation, the other after two generations. By generation 4, most of the DNA was of the lightest type. Right: Meselson and Stahl’s interpretation. In each round of reproduction the bacterial DNA was copied, using nitrogen from the 14N medium. After one generation, each DNA double helix was composed of a new 14N strand and an old 15N DNA strand. At the second generation, some molecules were composed entirely of 14N-rich DNA, and were therefore lighter again.

  By resolving the thorny problem of DNA replication, Meselson and Stahl’s elegant and precise experiment represented the final confirmation of the significance of the double helix structure of DNA. As it closed one phase of the history of molecular biology, it opened another, showing that DNA molecules – and hence the genes they contained – could be investigated by using the latest analytical techniques.

  Three studies from the late 1950s therefore pointed the way to the future: Meselson and Stahl showed that DNA could be labelled and tracked down the generations, Benzer’s fastidious work revealed that it was possible to investigate the molecular structure of the gene by exploring its tiniest components, and Ingram’s discovery that the sickle-cell mutation in the haemoglobin gene produced a single amino acid change hinted that the nature of the genetic code itself might be within reach.

  *

  Even before Jacob and Monod finally described their view of gene function in 1961, their work had led to an important discovery. On Good Friday in 1960, a small group of researchers, including Crick and Jacob, gathered in Sydney Brenner’s rooms in King’s College, Cambridge, as a kind of ‘after’ meeting following a conference in London on the previous day. Although the Cambridge and Paris groups were on very friendly terms, they were not on precisely the same wavelength. As Brenner later recalled, ‘You see, the Paris people were interested in regulation. We essentially were interested in the code. So we had a slightly different approach.’41 But that day, the two approaches suddenly fused as Jacob explained the latest results from Paris, focusing on the puzzle of how the z+ gene that enabled bacteria to produce β-galactosidase was able to synthesise such high levels of the enzyme so soon after it was introduced into a z– cell. One of the possibilities that the Paris group had briefly considered was that the gene synthesised a handful of very efficient ribosomes, which then churned out the enzyme at a high rate. But, as Jacob explained, Pardee had recently done an experiment that suggested that the z+ gene did not produce anything stable, but only a rather transitory messenger molecule.

  ‘At this point,’ recalled Crick, ‘Brenner let out a loud yelp – he had seen the answer.’42 Jacob vividly described the following minutes:

  Francis and Sydney leaped to their feet. Began to gesticulate. To argue at top speed in great agitation. A red-faced Francis. A Sydney with bristling eyebrows. The two talked at once, all but shouting. Each trying to anticipate the other. To explain to the other what had suddenly come to mind. All this at a clip that left my English far behind.43

  In that moment, Crick and Brenner had realised that the PaJaMo messenger could explain some recent results from various groups that suggested that at certain points in the reproduction of phage, a short-lived form of RNA was produced. This RNA had the same A:G base composition as phage DNA, indicating that it had been copied from the phage, and differed from the ribosomal RNA that was found in the host cell. The two Cambridge men immediately seized on the possibility that this short-lived RNA was the mysterious messenger that the Paris group had hypothesised. This would make the ribosome an inert structure in the cell – Crick described it as a reading head, like in a tape recorder.44 Messenger RNA, as Jacob and Monod called it that autumn (this was soon abbreviated to mRNA), was a tape that copied information from the DNA and then carried that information to the ribosome, which read it off and followed the instructions to make the appropriate protein.

  Jacob and Brenner immediately began planning how to test the hypothesis. That evening, Crick and his wife held one of their many parties. Jacob recalled the scene clearly:

  A very British evening with the cream of Cambridge, an abundance of pretty girls, various kinds of drink, and pop music. Sydney and I, however, were much too busy and excited to take an active part in the festivities. … It was difficult to isolate ourselves at such a brilliant, lively gathering, with all the people crowding around us, talking, shouting, laughing, singing, dancing. Nevertheless, squeezed up next to a little table as though on a desert island, we went on, in the rhythm of our own excitement, discussing our new model and the preparations for experiment … A euphoric Sydney covered entire pages with calculations and diagrams. Sometimes Francis would stick his head in for a moment to explain what we had to do. From time to time, one of us would go off for drinks and sandwiches. Then our duet took off again.45

  Jacob and Brenner’s proposed experiment needed the help of Meselson and his ultracentrifuges at Caltech to determine whether phage infection led to the creation of new ribosomes or, as they predicted, to a new transient form of RNA that simply employed the old host ribosomes to turn its message into protein. After a tense month in California, endlessly fiddling with the experimental conditions, Jacob, Brenner and Meselson got the experiment to work. As they had hoped, no new ribosomes appeared; instead, RNA that had been copied from the phage DNA was associated with old ribosomes that were already present in the bacterial host. Other researchers were on the same track – that autumn, when Jim Watson heard of the first results from California, he informed the trio that his Harvard group was working on something similar; meanwhile, Martynas Yčas announced that yeast also produced an unstable RNA, with the same A:G ratios as yeast DNA, suggesting that it had been copied from the yeast chromosomes.46

  In May 1961, after some delays caused by Watson’s group, which had used a different technique but came up with similar findings and wanted to publish simultaneously, the Brenner, Jacob and Meselson paper appeared in Nature, accompanied by the paper from the Watson laboratory.47 As the title of the Brenner paper put it, they had found ‘An unstable intermediate for carrying information from genes to ribosomes f
or protein synthesis’. That intermediate was messenger RNA. The story of how genetic information got out of DNA was complete.*

  *

  In 1961 Jacob and Monod stepped into the history books with three powerful works of synthesis, summarising and developing their ideas about the nature of gene regulation with a verve, elegance and rigour that still put most scientific articles to shame. First, they published a long review in the Journal of Molecular Biology (this was submitted in December 1960, and appeared in May the following year). Then, at the June 1961 Cold Spring Harbor symposium, which was entitled ‘Cellular regulatory mechanisms’ and was primarily focused on examples of negative feedback and repression, they presented a more data-rich version of their review before Monod closed the conference with a summary of the significance of their approach.48 Together, these three papers have been cited more than 5,500 times – more than Watson and Crick on the double helix structure of DNA.

  In their 1961 review article, Jacob and Monod brought together the ideas that they had been developing in a series of unnoticed publications in French and German, in which they had separated genes into two kinds according to their function. They began with the classic structural gene that coded for a protein and then described the kind of regulatory gene that they had discovered in bacteria. Their vision of what genes actually do, of the meaning of the genetic code, was ultimately framed in terms of information and control:

  let us assume that the DNA message contained within a gene is both necessary and sufficient to describe the structure of a protein. The elective effects of agents other than the structural gene itself in promoting or suppressing the synthesis of a protein must then be described as operations which control the rate of transfer of structural information from gene to protein.49

  The article described gene action as involving control mechanisms and suggested that an inducer ‘somehow accelerates the rate of information transfer from gene to protein’. The role of repression was to inhibit enzyme synthesis, but in a very different manner from the negative feedback loops that had been described by Yates and Pardee or Novick and Szilárd. Classic negative feedback involved the end-product of a reaction curtailing that reaction through some kind of protein–protein interaction. Repression involved the direct action of the repressor on the DNA of the structural gene itself. According to Jacob and Monod, the agent that acted as the repressor was the product (RNA or protein) of another gene, which they called a regulator gene:

  A regulator gene does not contribute structural information to the proteins which it controls. The specific product of a regulator gene is a cytoplasmic substance, which inhibits information transfer from a structural gene (or genes) to protein. In contrast to the classical structural gene, a regulator gene may control the synthesis of several different proteins: the one-gene one-protein rule does not apply to it.50

  Both repression and induction were controlled by regulator genes.

  Jacob and Monod also showed that the response to viral infection was controlled by regulator genes, linking bacterial genetics with the world of the phage, and implying that there was a fundamental process at work that could be applied to other organisms. They finally provided a theoretical framework to explain how genes work together, with structural and regulator genes interacting as a physiological unit. They gave their discovery a name: ‘This genetic unit of co-ordinate expression we shall call the “operon”’.51 Jacob and Monod were arguing that the operon was composed of genes that had been selected to work together as a Darwinian adaptation.

  With the discovery of messenger RNA, the mechanism of protein synthesis had become clearer; above all, genes were now seen not simply as producing proteins, but also as controlling the activity of other genes in a coordinated unit – the operon. It was still not known whether the repressor was made of RNA or protein (they initially leaned towards the RNA option; this was eventually revealed to be wrong in this particular case) and whether it acted directly on the DNA of the operator gene or on its RNA product.

  Jacob and Monod were quite aware of the implications of their discovery of genetic regulation. As they pointed out, one of the central mysteries of life is why cells in a body do not express all their genetic information all the time, but instead differentiate and turn into different structures with different functions. Their idea of gene regulation provided a conceptual key that we are still using and exploring today. They also realised that as well as explaining normal development, gene regulation could also provide an insight into cancer; they wrote: ‘Malignancy can adequately be described as a breakdown of one or several growth control systems, and the genetic origin of this breakdown can hardly be doubted.’52

  8. Jacob and Monod’s models of the Operon. In the top model, gene regulation occurs via RNA; in the lower version, it occurs via a protein. Taken from Jacob and Monod (1961a).

  The conclusion of their article transformed our view of what genes do, placing their complex research findings in an innovative framework that fused molecular genetics, cybernetics and computing, and which unwittingly echoed Schrödinger’s view that genes ‘are architect’s plan and builder’s craft – in one’. Jacob and Monod wrote:

  According to the strictly structural concept, the genome is considered as a mosaic of independent molecular blue-prints for the building of individual cellular constituents. In the execution of these plans, however, coordination is evidently of absolute survival value. The discovery of regulator and operator genes, and of repressing regulation of the activity of structural genes, reveals that the genome contains not only a series of blue-prints, but a coordinated program of protein synthesis and the means of controlling its execution.53

  Jacob and Monod had shown that genomes are not simply blueprints. Instead, they contain programs that determine the physical and temporal patterns of gene expression, and can also interact with the environment. This demonstration, which was the proof of Schrödinger’s theoretical insight two decades earlier, has shaped biology ever since.

  As Monod indicated at the Cold Spring Harbor meeting in June 1961, the discovery of regulator genes also held out the possibility of a radically new form of genetics:

  Adequate techniques of nuclear transfer, combined with systematic studies of possible inducing or repressing agents, and with the isolation of regulatory mutants, may conceivably open the way to the experimental analysis of differentiation at the genetic-biochemical level.54

  It was decades before this vision of genetic manipulation was realised, but it eventually transformed biology and medicine by making it possible to genetically manipulate organisms, and to understand diseases such as cancer.

  The discovery of the operon and of the idea of gene regulation was the complex outcome of chances and brilliant insights, as well as a lot of hard work. To mark the fiftieth anniversary of the operon, Jacob gave a graphic description of how he and Monod made their discovery, giving a glimpse of what it was like to be part of such a process:

  Our breakthrough was the result of ‘night science’: a stumbling, wandering exploration of the natural world that relies on intuition as much as it does on the cold, orderly logic of ‘day science’.55

  In a similar vein, in his 2013 obituary of Jacob, Mark Ptashne captured the intensity of the three years’ work that led up to Jacob and Monod’s insight, describing an experience that few scientists have been lucky enough to share:

  The repressor was not so much discovered by Jacob and colleagues as imagined – an entity that would explain disparate phenomena as analogous, connected by a similar underlying reality.56

  *

  Although the main focus of the 1961 Cold Spring Harbor symposium was gene regulation, both Monod and Brenner made passing references to the coding problem. The recent identification of messenger RNA led Brenner to be optimistic that a breakthrough would soon occur, while despite the absence of any real progress Monod confidently suggested that a demonstration of the link between a DNA sequence and a protein sequence (colinearity) would soon be
at hand.57 During these brief discussions, Gordon Tomkins of the National Institutes of Health in Maryland sat still and said nothing, although he must have been bursting. Unlike everyone else in the room, indeed virtually everyone else on the planet, Tomkins knew that the genetic code had been cracked three weeks earlier. He had been the first person in the world to hear the news and had been sworn to secrecy. No one else in the sweltering auditorium knew that the code had been cracked; in fact, no one else had even heard of the two men who had made the discovery.

  * This is the explanation given by Cohn et al. (1953b), who proposed the change in terminology. Historians have suggested that this change flowed from Monod’s highly-public opposition to Lysenko’s claim that organisms could adapt to new environments by directly altering their hereditary composition. Although this may be true, there is no direct evidence to support it.

  * Benzer later recalled that at the 1956 meeting he was criticised by a French geneticist for his choice of terms: ‘At this meeting at Johns Hopkins, I was attacked by Elie Wollman, who said these are very unfortunate names because they don’t translate well into French – cistron sounds like a lemon, and muton sounds like a sheep, recon is a dirty word. [Laughter] But I had already announced these names, so I stuck with them. [Laughter] Wollman was kind of upset about that.’ (Benzer, 1991, p. 51).

  * Despite the importance of this discovery, there was no Nobel Prize for those involved. There may simply have been too many people with an equal claim – no more than three people can share a Nobel Prize.

  –TEN–

  ENTER THE OUTSIDERS