The Science

 Since most readers are not biologists, I thought it might be helpful to provide a short summary of Monod’s and Jacob’s critical observations, discoveries, and ideas.  This information is divided into: i) what was known about genes, DNA, and proteins as they began their work; ii) Monod’s and Jacob’s discoveries that are covered in the book; and iii) important subsequent, related discoveries. (This is also in the book's Appendix)

Genes, DNA and proteins

Chromosomes and genes
From the pioneering work by Thomas Hunt Morgan (Nobel Prize 1933), it was known that chromosomes are the physical entities responsible for heredity, and that specific genes reside at specific positions along chromosomes. Oswald Avery’s studies (1944-1946) demonstrated that DNA is the chemical component of the chromosome responsible for inheritance. Each chromosome contains a long molecule of DNA.

DNA
James D. Watson and Francis Crick deciphered the structure of deoxyribonucleic acid (DNA) in 1953. DNA molecules in cells are made of two strands of four distinct bases. These chemical building blocks are denoted by the single letters A, C, G, and T. The strands of DNA are held together by strong chemical bonds between pairs of bases that lie on opposite strands – A always pairs with T, C always pairs with G.

Solving the structure of DNA immediately revealed how the two fundamental processes of inheritance and mutation worked at a molecular level.  That is, a DNA sequence could be faithfully copied and passed on because the base present at each position on one strand determined its complement on the other strand. Mutations result from errors in this copying process where the wrong base or an extra base(s) gets inserted, or a base(s) may be deleted, generating a change in the DNA sequence. 

Proteins 
Proteins are the molecules that do all of the work in cells, breaking down nutrients, assembling cellular components, copying DNA etc. Proteins are made up of chains of building blocks called amino acids. There are twenty different amino acids. The chemical properties of these amino acids, when assembled into long chains averaging about 400 amino acids in length, determine the unique activity of each protein. Enzymes are proteins that catalyze specific chemical reactions.

At the time of Monod’s and Jacob’s seminal work, the relationship between the sequence of bases in DNA and the sequence of amino acids in proteins was not understood. Crick asserted that the main function of DNA was to encode proteins, but how that information was decoded and the nature of the “genetic code” were unknown (Crick’s Black Box) until the early 1960’s. 

Monod’s and Jacob’s discoveries

Diauxy
The observations that started Monod on the path to fundamental insights and the Nobel Prize concerned the growth of bacteria in the presence of simple sugars. When bacteria are grown in the presence of a single sugar such as glucose, they grow exponentially until the sugar is exhausted. But, Monod noticed that when bacteria were grown in the presence of certain combinations of two sugars, glucose and lactose for example, they grew exponentially, then paused briefly before resuming exponential growth.  He called this phenomenon “double growth” or “diauxy.” By shifting the relative ratios of the sugars, he found that he could shift the relative length of each part of the double growth curve. From that observation, he deduced that the bacteria were using up one sugar before utilizing the second, less -preferred sugar.

Enzyme adaptation and enzyme induction
The time lag in bacterial growth on a less-preferred sugar was interpreted as an example of “enzyme adaptation”, in which growth is delayed briefly until the enzyme required to break down a nutrient appears. 

Monod specifically focused on the control of lactose metabolism. Lactose is a disaccharide made up of the two monosaccharides glucose and galactose.  Lactose itself cannot be used as an energy source in E. coli bacteria, it must be broken down into glucose and galactose, which is the function of the enzyme β- galactosidase. Importantly, the enzyme is normally not produced in the absence of lactose, but appears when lactose is the sole energy source provided to the bacteria (a case of enzyme adaptation). Since the appearance of the enzyme occurred in the presence of the sugar it broke down, Monod and others renamed the phenomenon “enzyme induction,” and substances that were able to elicit the enzyme were dubbed “inducers.” 

The lactose operon
How a simple bacterium “knew” which sugar to use, and when to produce β-galactosidase – were the crux of the mystery Monod set out to solve. 

Crucial to his progress was his decision to tackle the problem using genetics. During the war, Monod and Alice Audureau found that strains of E. coli bacteria that were unable to utilize lactose occasionally gave rise to colonies that could grow on lactose, and that these colonies were due to genetic mutations. This was crucial evidence that the ability to metabolize lactose was genetically determined. Therefore, components of the enzyme induction process could be identified through mutations. 

In order to figure out where these mutations occurred on the E. coli chromosome, and to sort out how they affected enzyme induction Monod teamed up with Jacob in 1957. Jacob had been studying a bacterial virus, or bacteriophage, called lambda. Jacob was a pioneer in developing methods for mapping bacterial genes. He was particularly interested in the phenomenon of lysogeny, whereby a virus hides out in a bacterial host but can be induced to emerge by certain treatments. Jacob proposed that enzyme induction and virus induction were analogous – that in each case a repressor kept genes off unless an inducer was present. 

From 1957-1960, working with key collaborators such as Arthur Pardee, Monica Riley, and Sydney Brenner, Monod and Jacob identified several components involved in the control of lactose metabolism in E. coli, and coined several general terms that have remained in use to this day :

• Structural gene, that encoded the structure of a protein, such as an enzyme
• Regulatory gene, that governed the expression of a structural gene
• Repressor, that turned off enzyme production, such as the protein encoded by the i gene
• Operator, the acceptor site for the repressor on DNA
• Operon, a set of structural genes controlled by a common operator and repressor, and usually involved in the same biochemical pathway
• messenger RNA, an intermediate that carried information from genes in DNA to the ribosome for the synthesis of specific proteins.

Monod and Jacob figured out the logic of the genetic switch by deciphering what happened in certain mutants.  For example, mutations in the repressor gene i disabled repression, but not if a second normal copy of the gene was present in the cell. Mutations in the operator (o) also disabled repression, regardless of whether another operator was present. Mutations in specific structural genes blocked the production of specific enzymes.

From these observations, they were then able to construct a general picture of the process of gene regulation that looked like the following:

The diagram, a version of which appeared in their landmark paper in 1961, depicts how the operon is regulated in the absence or in the presence of an inducer, such as the sugar lactose. The key to the logic of this genetic on/off switch is the interaction of the inducer with the repressor. When no inducer is present, the repressor binds to the operator and keeps the genes of the operon turned off. When the inducer is present, it inhibits binding of the repressor to the operator, and the genes of the operon are switched on. In this way, for example, certain enzymes are made only when a certain nutrient is available.

Subsequent discoveries

The repressor protein, a critical component of the lactose regulatory system, was not isolated until 1966. Walter Gilbert and Benno Müller-Hill of Harvard University achieved that coup, and demonstrated that the repressor bound specifically to the operator sequence of the lactose operon. Moreover, the binding of the repressor to DNA was inhibited by inducers. The latter was an example of allostery, in which the binding of a molecule alters the shape and activity of a protein. 

Allostery was yet another powerful idea conceived and coined by Monod.  When he realized that the activities of some proteins, such as repressors, might be regulated by the binding of substances, he declared: “I have discovered the second secret of life!’ Indeed, allostery underlies much biological regulation, such as how hormones regulate physiology. 

The repressor for bacteriophage lambda was also isolated in 1966, by Mark Ptashne – also at Harvard. Ptashne demonstrated that lambda repressor bound specifically to the operator region of the bacteriophage DNA.  The similarities between the two systems of regulation were in fact as great as Jacob had imagined years earlier. 

The elucidation of the lactose and lambda phage regulatory systems provided critical tools for the early days of molecular genetics, in both basic and applied research. Many of the first practical advances in genetic engineering relied on knowledge developed from study of these two systems. 

The general principle revealed by study of the lactose operon and bacteriophage was that genes were switched on and off by the action of proteins that bound specifically to DNA sequences near genes (operators). While the details are a bit different in more complex organisms, the principle is the same from E.coli to elephants to humans. As Monod anticipated in 1947, and as Jacob and Monod explicitly argued in their masterful synthesis published in 1961, the development of complex creatures and the differentiation of the many kinds of cell types in animal bodies are orchestrated by the turning on and off of different sets of genes. Most of that action is controlled through the binding of regulatory proteins to specific DNA sequences around genes.