p53 (7 page)

Proteins are made of amino acids, of which there are 20 different kinds. The instructions from the gene determine which amino acids are to be used, in what order and what quantities. For this
process, the string of simple letters represented by the sequence of base pairs on the gene, the As, Ts, Gs and Cs, need to be translated into more complex ‘words’, so the ribosome
protein factory reads the linear information on the strand of mRNA in three-letter chunks. These chunks, or ‘words’, are called codons, and they identify which amino acids should be
used to make a specific protein. The amino acids are brought to the assembly point, the ribosome, from other parts of the cell by transfer RNAs (tRNA), which dock on to the appropriate codons and
deposit their cargoes of amino acids. When all the amino acids are in place they are strung together as a chain, which detaches itself from the strand of mRNA and goes off to another part of the
cell to be folded. This process is vitally important, since a protein’s function is determined not just by its amino acid components but also by the way it is folded.

The other biological process central to the story of p53 is that of DNA replication, which occurs in every cell that is about to divide. In this process once again the enzyme helicase unwinds
the DNA and unzips it – not all the way along, but a fragment at a time. Small molecules called single-strand binding proteins, or SSBs, attach temporarily to each separated strand to
stabilise them while they are being copied and to ensure that they stay separate. Then DNA polymerase travels along each separated strand attaching new nucleotides – the nano-scale Lego
blocks described earlier – to each of the existing ones, pairing up the bases in the conventional way, A with T and G with C, thus constructing a parallel strand of DNA, block by block. A
subunit of the polymerase travels along behind, ‘proofreading’ the new DNA to see that it has been faithfully copied. Then an enzyme ‘glue’ called DNA ligase seals the
fragments of copied DNA into a continuous double-sided strand that rewinds itself automatically.

In the replicated DNA, one strand of the double helix will be from the original (known as the parent strand) and the other will be the new copy (known as the daughter strand). The cell is now
ready to divide into two cells with equal shares of identical genetic material. This process, going on ceaselessly in billions of cells in our bodies as we repair and replace tissue and our hair
and nails grow, is so efficient that mutations – mistakes that escape the proofreader – occur at the rate of about one in 10
⁹
nucleotides per replication.

It’s interesting to note that this knowledge, this understanding of how the machinery of life works, is built on the foundations of Watson and Crick’s discovery of the structure of
DNA. The double helix – the spiral staircase drawn originally for their paper in
Nature
by Crick’s wife Odile – remains one of the iconic images of 20th-century science.
Yet when the paper announcing their discovery came out in 1953, it was noticed by hardly anyone beyond a small group of enormously brainy and ambitious scientists working in the same field –
some of whom had been racing to make the discovery themselves first.

Of the media, only one paper, the British
News Chronicle
, carried the story about ‘an exciting discovery about what makes YOU the sort of person you are . . .’ And for
nearly a decade, only a tiny proportion of scientists writing about DNA in professional journals mentioned the double helix. It was a beautifully elegant model, but many biochemists, intensely
preoccupied with working out how we synthesise the proteins in our cells, were sceptical that genes – still rather an abstract notion in the early 1950s – had anything to do with
it.

***

The cool reception and slow build-up of recognition for the double helix – culminating in the Nobel Prize for James Watson, Francis Crick and the biophysicist Maurice
Wilkins in 1962 – are instructive. This is how science works, says Peter Hall. ‘It’s the analogy of the man in the dark warehouse with a small pen-torch. He can only see a tiny
part of what’s there. The whole thing only becomes clear when he gets more light.’

Until they had a clone of p53, the scientists were guddling around in the dark without even a pen-torch, and in the early 1980s the race was on among a handful of individuals in labs around the
world, from the UK and US to Russia and Israel, to clone, or make identical copies of, the gene – the first, essential step to finding out what it is and how it works. The first person to
succeed was Moshe Oren, who started the process while working as a postdoc in Arnie Levine’s lab in Princeton, but completed it at the Weizmann Institute in Israel, where I went to talk to
him on a hot October afternoon some three decades later. Entering his small office on the top floor of the institute, with its views up into the wide sky, I was greeted by the scent of citrus. Oren
was seated behind his desk with a little pile of orange peel and pips in front of him – clementines picked from his own garden that morning, he told me, as he offered me a handful. We sat
eating the sweet fruit as we talked.

‘I was looking for a new project because I needed to change. Here was this interesting protein that people were beginning to look at and it was my chance to clone something,’ he
recalled. ‘In those days cloning was a major technical challenge. It was probably two and a half years from me saying, okay, I’m going to clone p53 to actually getting the thing. It
took a lot of setting up protocols and testing different approaches, some of which didn’t work. It’s kind of amazing how we’ve progressed: cloning a gene now is trivial;
it’s become probably a high-school exercise! But when we did it the tools were very limited; there were very few genes that had been cloned, and each of them was cloned by a variety of
improvised tricks . . . It was not easy at all.’

One of the hardest tasks for the would-be cloner even today is identifying the individual genes on a continuous strand of DNA – where does one gene end and the next begin? Oren’s
strategy was to look for the gene after it had been switched on, when the relevant segment of DNA had been copied, or ‘transcribed’, into ‘messenger’ RNA (mRNA), left the
nucleus and gone to the ribosome – the protein-making factory – in the body of the cell. Using antibodies tailored to recognise the p53 protein from among a mass of proteins being made
at the same time, he isolated the relevant protein factory and scraps of mRNA. Using the mRNA as a template, he synthesised ‘complementary’ DNA (cDNA) by attaching new nucleotides,
block by block, along its length, pairing up the bases as appropriate. This cDNA, he hoped, would give a faithful copy of the p53 gene – or at least that part of the gene responsible for
making the protein.

In order to multiply the little scraps of cDNA, he transferred them to a bacterium – E. coli, which, you will remember from the Asilomar story, is one of the workhorses of biotechnology
because it’s easy to manipulate, efficient at taking up new genetic material and can pump out clones at a terrific rate, given a good food supply. In order to transfer the cDNA to the
bacteria, Oren had to use a suitable vector – something that would breach the walls of the bacteria and take up residence inside without killing its host – and for this he chose a
plasmid.

Plasmids are tiny rings of DNA between 1,000 and 10,000 base pairs long that some bacteria have floating around in their cells, independent of their regular genomes. When a bacterium dies and
its body – a single cell – disintegrates, the plasmids are scattered into the environment and are often absorbed into other bacteria, which then begin to express the new traits encoded
by the plasmids. This is how recombination – the phenomenon debated at Asilomar that still excites such public controversy over biotechnology today – happens in nature, and it has
provided a marvellous tool for cloners. The scientists put the plasmids in solution with the scraps of cDNA they want copied, having used a ‘snipping’ tool, an enzyme, to cut a gap in
the plasmid ring where the new material should be inserted. With a little coaxing, the cDNA moves of its own accord into the gap, where it is glued into place by ‘repair enzymes’ added
to the solution. This plasmid is now a recombinant DNA molecule – a mixture of genetic material from different organisms. The cloner then adds it to another solution containing the E. coli,
or whatever else he or she has decided to use as a ‘clone factory’, and waits for it to find its way in among the machinery.

‘On paper it looks very simple,’ said Oren, shaking his head slowly at the memory of the months of trial and error and frustration working mostly in the realms of unseeable biology.
‘In practice, we spent a year trying things that didn’t work. Then we tried this method that worked, but we went through many false negative clones before we found one that was
real.’ The trickiest part was getting the plasmid vectors to take up their new cargo, the scraps of p53 cDNA. Oren and his technical assistants screened the plasmids 10 at a time rather than
individually to see if they had succeeded – a task of enormous tedium that swallowed nearly 18 months of their lives.

‘The procedure ended up being rather inefficient and the percentage of plasmid clones that indeed contained p53 cDNA was extremely low. We had to screen many hundreds of clones –
actually I think about 1,800 – before we hit the first positive one.’ When that finally happened, it was one of the most exciting moments in Oren’s scientific career, he told me
with a smile. He and his colleagues published their results in
Proceedings of the National Academy of Sciences
in 1983.

Meanwhile, back in Princeton, Arnie Levine had started collaborating with the pharmaceutical company Genentech and one of their expert cloners, Diane Pennica, to continue the search for a p53
clone once Oren had returned to Israel. Using different strategies and different cell types from Oren, they too were successful and they published their findings in the journal
Virology
the following year.

Others were hot on the same trail and by 1984 there were a number of p53 clones, of different cellular origin, in the scientific press and in limited circulation around the labs. Among them was
one obtained at the ICRF in London by John Jenkins, one of very few people with cloning skills in Britain, where progress in manipulating genetic material had been inhibited by the controversy
surrounding recombinant DNA in Europe and the US. Jenkins, who came late to science after finding himself a misfit at school, leaving early and taking up work as a general labourer and landscape
gardener before returning to education, remembers fierce competition to obtain a clone. ‘Obviously people are driven by their career ambitions – but at some level there’s the pure
joy of beating the opposition. I mean, it’s a competitive business! Always was; always will be.’ Like Oren, Jenkins remembers how tough a task cloning p53 was. This was the cutting edge
of science: ‘Everything at the cutting edge is a challenge at the time,’ he commented.

Just how much of a challenge cloning was in the early 1980s is hard to grasp for succeeding generations of biologists. ‘My best story about that was years later,’ said Jenkins.
‘One of the people in my lab was helping me clear out my office. We came across a bound copy of my PhD thesis and she said, “Oh! Can I take this away and read it?” I said,
“Sure, no problem. Bit of old history . . .” Later she brought it back and said, “I don’t believe it! You didn’t do anything, and you got a PhD for
that
?” I said, “Listen! You try doing that work when you have to make all your own enzymes . . . There weren’t any kits in those days, you know . . . Everything you take
for granted now was just not there, so it took a long, long time – and it was tricky.”’

Oren, Levine and Jenkins had, in fact, been beaten to the finishing tape in the highly competitive race to clone p53 by a Russian scientist, Peter Chumakov, working at the Institute of Molecular
Biology in Moscow. But Chumakov had published his results – in December 1982 – in
Proceedings of the Academy of Sciences of the USSR
, a Russian-language journal with limited
readership in the West, and for some time his triumph went unnoticed beyond the Soviet Union. Back in 1979 Chumakov had just completed his doctoral thesis on SV40 and had been looking for a project
that would reveal how large T antigen caused cancer when he heard of the discovery of p53. Excited by the idea that p53 might be the key to the transformation process, he decided to clone the gene
to provide material for further studies.

But Chumakov faced even greater challenges than his Western competitors. His lab in Moscow had shortages of some of the most basic equipment, such as test tubes and pipettes, which he cleaned
and re-used over and over again. He also had to send off to a scientist in Texas, Elizabeth Gurney, for a sample of the special antibody to p53 she had made and which he realised was vital to his
cloning endeavour. He made his request without much hope of receiving the sophisticated tool. ‘I thought that even if Elizabeth decided to send the sample, a package containing a suspicious
test tube would be stopped either on the USSR border, or during postal censorship that occurred regularly with mail coming from the West,’ he told me. But he was lucky. Visiting a colleague
in a neighbouring lab one day, he noticed a small, foreign-looking package lying on a desk and he turned it over in casual curiosity. To his surprise and delight he found it was addressed to
himself and contained a small phial of the antibody together with a letter from Gurney wishing him success.