Life's Greatest Secret (20 page)

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Twice, Boris Ephrussi asked Francis Crick a question that he could not answer, and both times it was the same question. The first occasion was at Woods Hole in 1954, the second was in a Paris café a year later. Each time, Ephrussi wanted to know why Crick was so certain that the DNA sequence encoded the sequence of amino acids.
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This pointed question went to the heart of everything that had been done since Watson and Crick had discovered the double helix – they had been avoiding the central issues of what the sequence of bases did, or to put it another way, what information they contained. Everyone assumed that there was what was called a colinearity between the sequence of bases in a DNA molecule and the sequence of amino acids in the corresponding protein, but Ephrussi’s question underlined that, in the light of the available evidence, that information could equally be linked to something as mysterious as the overall form of the protein, or it could be something else altogether. As Crick later recalled:

There was no evidence, you see; there was

that a gene made a difference in the sense that you could actually determine the amino-acid sequence and show that with a mutation it was changed.

Goaded and intrigued by Ephrussi’s challenge, in the spring of 1955 Crick began searching for an example of protein variation that he could tie to genetic variation. He looked for a protein that was widely available and was from an organism that could be studied genetically. The protein he chose was an enzyme called lysozyme, which is found in chicken eggs and in human tears. Together with the German-born biochemist Vernon Ingram, Crick tried to crystallise lysozyme from the eggs of various bird species, and even used onions to make himself cry to provide a source of tears. The project was a failure – they could get lysozyme from chickens but not from other species, meaning that a comparison between species would be impossible. So they tried looking for variation in lysozymes from different individual chickens, hoping to eventually relate such variation to genetic differences. Again they found nothing. As Crick wrote to Brenner:

Attempt by Vernon Ingram and myself to find two hens with different lysozymes so far completely negative … It is all rather discouraging. Even if we find a difference we shall still have to show it’s due to amino acid composition, and also do the genetics.

But then Ingram came up with something much more dramatic, which provided the answer to Ephrussi’s question and showed the potential applications of the work that Crick and his colleagues were doing. In 1949, two articles had appeared in
Science
reporting new findings on sickle-cell anaemia, a genetic disease that predominantly affects people of African origin and can lead to debilitating weakness or death. People with sickle-cell disease have strange sickle-shaped red blood cells – hence the name. It had first been described as a genetic disease in 1917, but it was only in July 1949 that James Neel of the University of Michigan showed that the best explanation of the pattern of inheritance of sickle cells was that the trait was caused by a single gene.
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Four months later, in November 1949, Linus Pauling and his group published a study of the haemoglobin molecule found in the blood of patients with sickle-cell anaemia, with the dramatic title ‘Sickle cell anemia, a molecular disease’.
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They described two forms of haemoglobin, one associated with sickle-cell anaemia (later called the S form) and the other with normal individuals (the A form). These two forms could be distinguished by electrophoresis – when the two haemoglobins were placed in a gel and subjected to an electric field, they moved at different rates, so after a certain time they were found at different points. Pauling’s group concluded that the way the two forms moved under electrophoresis suggested that there was a difference in the shape of the two molecules, with the S form having more positive charges than the A form. For the first time, a disease had been shown to have a molecular basis.

Max Perutz’s group at Cambridge had been studying haemoglobin for years, and had begun to look at the structure of the S form. Encouraged by Crick and Perutz, in the summer of 1956 Vernon Ingram looked at some samples of the S form of haemoglobin that had been left unused by another researcher, Tony Allison. Allison’s field work in Uganda had led him to suggest why sickle-cell anaemia still exists despite having potentially lethal consequences in patients with two copies of the sickling gene: individuals who have one normal and one sickling gene have a lower load of malaria parasites.
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Having one sickling gene is actually a good thing in malaria-ridden regions.

Ingram’s aim was to detect the precise molecular difference between the S and A forms that had been described in general terms by Pauling. The haemoglobin molecule was too long for Fred Sanger’s sequencing method, which remained relatively primitive. First, Ingram had to snip the molecules into smaller bits by using an enzyme; then he divided the components on filter paper, first separating the two forms by applying an electrical current just as Pauling had done and then applying a solvent at 90° to the direction of the current. Once a chemical had been sprayed on the paper to reveal the invisible components of the haemoglobin molecule by turning them purple, the result was a two-dimensional ‘finger print’ of the molecule.
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The differences in electrical charge shown by the two forms of haemoglobin had two potential explanations. Pauling thought it was most likely that the overall shape of the molecule was the source of the differences, and that in turn was produced by the way in which the protein was folded and shaped during synthesis. As he argued in 1954:

The interesting possibility exists that the gene responsible for the sickle-cell abnormality is one that determines the nature of the folding of polypeptide chains, rather than their composition.

The other possibility was that the difference in charge was simply to do with a difference in the sequence of amino acids in the two forms.

Ingram’s ‘finger prints’ of the two forms of haemoglobin each produced about thirty marks on the filter paper; these were identical in both cases, except for one small blob that was present in the S form and absent in the A form. He therefore analysed this particular component in great detail, and was able to show that the difference between the two molecules was due to a small part of the protein. In an article in
Nature
published in 1956, Ingram concluded:

there is a difference in the amino-acid sequence in one small part of the polypeptide chains. This is particularly interesting in view of the genetic evidence that the formation of haemoglobin S is due to a mutation in a single gene. It remains to be seen how large a portion of the chains is affected and how the sequences differ.

Shortly before the article appeared, Ingram presented his findings to the meeting of the British Association in Sheffield, in August 1956. The science correspondent of
The Times
was there and immediately sniffed out the story, describing Ingram’s work in some detail and highlighting its significance:

He described how in the past six weeks he had shown for the first time how a mutation in a single gene, the unit of heredity, can modify chemical structure in a substance in the body for which that gene is responsible.

Within a year, Ingram was in the pages of
The Times
again, following publication of another article in
Nature
that this time revealed the exact cause of the peptide difference between the two forms: it was all due to a single amino acid.
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In the S form, a valine molecule replaced the glutamic acid found in the normal A form. This minor change, in the most fundamental component of a protein, caused the changes in the behaviour of the molecule that led to the debilitating disease. Although the genetic code was still a mystery, Ingram’s work had shown that there was a relation between a mutation in a gene, which was made of DNA, and a change in the amino acid sequence of a protein. Ephrussi’s question had been answered. For
The Times,
this was a discovery that was on a par with Mendel’s observations that led to the foundation of genetics. The article concluded:

Dr Ingram’s demonstration of a single, identified difference between genetically determined haemoglobins is thus the nearest that has been got to a direct view of one of Mendel’s genes in action. This is indeed a landmark.

Ingram’s discovery was a brilliant confirmation of the new understanding of gene function. Previously it had been thought that the gene shaped the protein, like a three-dimensional mould. Ingram, inspired by Crick, had now shown that a gene could alter a single amino acid in a one-dimensional protein sequence, and that in turn would alter the function of the protein. A new vision of protein synthesis was appearing, and it had consequences for how the gene and the genetic code were understood.
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If the gene contained information and a change in that information led to a change in an amino acid sequence, this suggested that genetic information corresponded to nothing more than the amino acid sequence in a protein produced by a gene.

* As Crick later recognised, from a strict cryptographical point of view, the genetic code is a cipher, not a code. Crick had in mind something like the Morse Code, which is equally not a code from a technical point of view. And as he later put it, ‘“genetic code” sounds a lot more intriguing than “genetic cipher”’ (Crick, 1988, pp. 89–90). The term genetic code was first used in print in 1958, by Geoffrey Zubay (Zubay, 1958).

–     EIGHT     –

In September 1957, Francis Crick gave a lecture at University College London. He had been invited by the Society for Experimental Biology to speak at a symposium entitled ‘The Biological Replication of Macromolecules’.
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For nearly a year, Crick had been musing about what genes actually do, thinking about the mechanisms of protein synthesis, trying to tease out the biochemical steps and their theoretical consequences. The London conference was his opportunity to present his ideas about protein synthesis. It was the most influential lecture he ever gave.

The French molecular geneticist François Jacob was in the audience and recalled his impression of Crick:

Tall, florid, with long sideburns, Crick looked like the Englishman seen in illustrations to nineteenth-century books about Phileas Fogg or the English opium eater. He talked incessantly. With evident pleasure and volubly, as if he was afraid he would not have enough time to get everything out. Going over his demonstration again to be sure it was understood. Breaking up his sentences with loud laughter. Setting off again with renewed vigour at a speed I often had trouble keeping up with. … Crick was dazzling.

Crick’s recollection was not quite so positive – ‘I ran overtime, and didn’t get it over very well’, he felt.
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Crick’s lecture led to two articles – one in
Scientific American
that appeared at the same time, and another more detailed piece that was published in the symposium collection in 1958. This second paper has been cited more than 750 times and is still cited frequently.
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The bold proposals Crick made in his lecture continue to play a fundamental role in modern debates over the nature of the genetic code and the evolutionary process.

Behind its dull title, ‘On protein synthesis’, Crick’s talk explained some of the new ideas about what genes do and how they do it, all wrapped up in an elegant, conversational style that still beguiles the reader. At every step he admitted the limits of his knowledge, and distinguished clearly between established fact and logical conjecture. It was these conjectures that proved so influential. Crick’s starting point was his assumption that the role of genes is to control protein synthesis, even though, as he put it with disarming simplicity, ‘the actual evidence for this is rather meagre’:

I shall … argue that the main function of the genetic material is to control (not necessarily directly) the synthesis of proteins. There is a little direct evidence to support this, but to my mind the psychological drive behind this hypothesis is at the moment independent of such evidence. Once the central and unique role of proteins is admitted there seems little point in genes doing anything else.

Crick claimed that the involvement of nucleic acids in protein synthesis was ‘widely believed (though not by every one)’.
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The caveat was significant – even at this date, there were still those who found it hard to accept that all genes were made of DNA. A year earlier, at a symposium on ‘The Chemical Basis of Heredity’ held in June 1956, Bentley Glass noted the reluctance of some geneticists to abandon the protein part of the old nucleoprotein theory of heredity, but he was nonetheless sure that ‘most persons’ accepted that DNA (or RNA in some viruses) was the primary genetic material.
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This obviously left open the possibility that there might be other, secondary and non-nucleic acid forms of heredity. At the same meeting, George Beadle focused on this problem in his opening address, underlining that there was no experimental evidence that DNA was the genetic material in organisms other than viruses and bacteria. In his talk, Steven Zamenhof, who had worked with Chargaff and had been an early supporter of Avery’s claim that DNA was the genetic material in bacteria, accepted that although ‘extensive evidence’ supported the argument that the pneumococcal transforming principle was DNA, and that ‘no evidence to the contrary had ever been presented’, there was nevertheless ‘no absolute proof’.
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With his fellow-scientists still haunted by doubt, Beadle argued that ‘it is assumed as a working hypothesis that the primary genetic material is DNA rather than protein.’
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What now appears evident was merely a ‘working hypothesis’ in 1956.