Case 2, the young woman with a high-risk mutation in BRCA1, the commonest gene determining inherited breast cancer, shows us how the DNA revolution has transformed medical genetics – and how far we still have to go. Harmful mutations can now be detected and pinpointed with exquisite precision: for example, the young woman’s mutation is a single-letter switch affecting the 5,325th ‘base’ (a letter in the genetic code) of the BRCA1 gene, which is 125,951 bases in length and begins at base 43,044,295 of chromosome 17. As well as giving prognostic information, molecular genetics can bring hope. In some conditions, it is possible to work out how the abnormal protein generated by the mutated gene does harm, and to design new drugs to correct the defect. So far, though, that dream has been translated into therapeutic reality for only a few diseases, which do not include hereditary breast cancer.
The young woman’s predicament also draws our attention to an achievement for which conventional superlatives are inadequate: the letter-by-letter deciphering of the entire DNA sequence (genome) of Homo sapiens, which runs to 3.24 billion bases. Our DNA is chopped into different lengths and crammed into our forty-six chromosomes. This is an extraordinary feat of packing. A total length of around three metres of DNA is somehow coiled up and squashed small enough to squeeze into the nucleus of a single cell – and in a way that still allows the ever-busy units of cellular machinery to dive inside the tangle and lock on to the genes of the moment.
If the DNA is unpacked from the nucleus and all those coils are ironed out, the molecule is still left with a purposeful twist. It is a thing of beauty: two graceful spirals that track each other perfectly, always precisely the same distance apart, as they wind around an invisible long axis. This is the fabled double helix, to which the names of Watson and Crick are attached as intuitively as E = mc2 goes with Einstein, and tonic with gin.
And it can only sound like a cliché, but this structure holds the key to the whole of life and heredity.
The double helix: a brief interactive tour
The DNA molecule looks like an architecturally implausible stairway to heaven. It certainly goes up a long way. Scaled up to the width of a spiral staircase in a medieval turret – such as in the castle where it was discovered – the DNA from the nucleus of a single cell would stretch for over 3 million kilometres, or eight times the distance to the dark side of the moon.
This is too early in the book to start delving into the bowels of molecular genetics, but a gentle stroll down a short stretch of the human genome will help to set the scene. First find chromosome 17 and walk along it until you reach base number 43,044,295, then chop out the section that begins here and ends 125,951 bases further on. You may recall that this is the inherited breast cancer gene, BRCA1. Enlarge the sequence until it is as wide as a medieval spiral staircase, stand it on its end and look at how the whole thing is put together (Figure 1.1).
You will notice immediately that the two spirals running parallel to each other are graceful but unexciting. They are both made of the same two components, joined together and repeated ad infinitum: a chemical group called ‘phosphate’ because it is dominated by a phosphorus atom, and a small sugar molecule (deoxyribose) which gives DNA (deoxyribonucleic acid) its name. The monotonous structure of the spirals could not possibly be eloquent enough to make the genetic code, which somehow has to contain enough letters to write the instructions for making millions of different molecules. In fact, the spirals are purely structural, each acting as a backbone that keeps its helix in shape.
The magic of the double helix lies in the constant interval that separates the two spiral backbones. With the molecule standing vertically, you will see that the gap is bridged by horizontal steps set at regular intervals, with ten steps to each complete turn of the staircase. A careful look will show that all the steps share a common design, but that you cannot predict exactly how a particular step will be constructed. Every step is made up of two different halves, each rooted firmly on its spiral backbone, joined together in the middle. You will soon realise that there are only four different half-steps, and that two are long and two are short. To maintain a constant distance between the spiral backbones, all the steps must be the same length. This can only be achieved by making each step from one short and one long half-step; a step made of two shorts or two longs would make the elegant spirals buckle or bulge, and would wreck the beauty and functionality of the double helix.
Figure 1.1 The DNA molecule, pictured as a spiral staircase, with and without the ‘backbone’. Right: the four possible steps; A and T always go together, as do C and G.
Working your way through a larger sample of steps – as many as you care to examine – will show that the construction of each step is unpredictable but not entirely random. This is because the molecule always obeys a simple rule: each of the two short half-steps can only be joined to a specific long one. If we designate (not quite arbitrarily) the short half-steps C and T and the long ones A and G, then an A is always connected to a T, and a G to a C.
This rule means that if you can only see the half-steps attached to one of the spiral backbones, you can predict with absolute certainty the ones which are joined to the opposite backbone and form the other half of each step. For example, if the sequence of half-steps on one side was C, then A, T and finally G, then the corresponding half-steps on the other side can only have been G, T, A and C, in that order. The half-steps are the flat, geometric molecules called ‘bases’; the inviolable rule that C goes with G and A with T is therefore called ‘base-pairing’. The discovery of this phenomenon was judged significant enough to win a Nobel Prize; this seems reasonable, because it underpins the genetic mechanisms that make each of us what we are.
While digesting that, you can make a closer inspection of the BRCA1 gene. Go to the very top and stand on the highest step. If you’re bad with heights, don’t look down: the bottom is over 67 kilometres below you. Now set off down the staircase, at a steady pace of one step each second. It’s not a comfortable walk, with a drop of over 30 cm from one step to the next, and it will take about 35 hours to reach the bottom. If you start at 9 a.m., then at 45 seconds after 10.28 that morning, you will land on the 5,325th step from the top. The half-step attached to the spiral backbone on your left will be an A, because this is the version of BRCA1 that belongs to lucky people. In the case of the young woman waiting nervously to hear the verdict handed down to her in the Genetic Counselling clinic, that A was a G. That is the only difference between lucky and unlucky; every one of the other 125,950 steps is identical.
Blockbuster
The double helix was the ‘structure for deoxyribose nucleic acid’ which J.D. Watson and F.H.C. Crick of the Cavendish Laboratory, Cambridge, proposed in a brief paper published in Nature on 25 April 1953. Their claim that the structure had ‘novel features which are of considerable biological interest’ has been thoroughly vindicated. The double helix and base-pairing have revolutionised our understanding of the mechanisms of life and heredity. Their discovery epitomises the grand challenges and glorious triumphs of science, and is seen as one of the defining moments of biology.
That moment is captured, carefully posed in 1950s monochrome, in the familiar photograph of the two pioneers and their discovery (see Figure 24.3). Francis Crick, still youthful but already balding, stands on the right, pointing at their model of the double helix with a slide-rule, extended as if in mid-calculation. Seated opposite is Jim Watson, gawky and shockingly young, gazing up at their handiwork with his mouth open as though the photographer had told him to look awestruck at what they’d created. And the spidery metal contraption standing on the laboratory bench between them is what lined them up for their Nobel Prize and their places at the top table reserved for the greatest scientists of all time.
The events leading to that photograph and the Nature paper were triggered by Watson making a connection that everyone else had missed. He spotted how the two kinds of base – one short, one long – could reach across the gap between the two spir
al backbones and click together to make one of those horizontal steps. Many people would regard this stroke of genius as the greatest discovery in the history of DNA. But it is also a wonderful example of chance favouring the prepared mind and, in this case, virtually all the preparation of Watson’s precociously brilliant brain had been done by other people. Not just the person who showed him ‘Photograph 51’ with its tell-tale helical pattern, or who corrected his calculations for fitting the bases together, but all those who had worked out the basic chemistry of DNA or pursued the outrageous notion that it might play a role in heredity.
Compare that with the revelation that fell like a bolt from the blue into a mind that was totally unprepared, because this was the very beginning and, as with the Big Bang, nothing existed before this moment.
The story of DNA opens with a bright young man of around Jim Watson’s age who also happened to work in a university city with medieval buildings overlooking a picturesque river. At that point, any resemblance ends. This young man’s experimental facilities are grim, mainly because he likes it that way; in our fussier age, his laboratory would have been shut down by the European Agency for Safety and Health at Work because of multiple infringements of Directive 89/684/EEC.
And his starting material, which spawned the whole saga of DNA, is even less wholesome: heavily soiled, stinking clinical waste that nowadays would go straight into the incinerator.
2
IN THE BEGINNING
It is a bitterly cold morning in December 1868. We are in Tübingen, deep in the heart of Germany, looking out across the black waters of the River Neckar. Our vantage point is a window on the second floor of the half-timbered Alte Burse, on the edge of the Old Town. For three and a half centuries, this room was a student dormitory; now it is a surgical ward in the University Hospital. Outside, it is a hard winter, with heavy snow caked on the bare branches of the plane trees and the temperature hovering around freezing. Inside, patients are steeling themselves while bandages are peeled away from weeping flesh in preparation for the surgeon’s visit.
The surgeon is a master of his craft. On a good day, he can cut a marble-sized stone out of your bladder in less than three minutes, and can take your leg off in half that time. Speed is not just a professional selling-point. Thanks to the recent introduction of ether, you no longer have to wish you were asleep throughout your operation, but blood transfusion is still in the realm of fantasy; a couple of bungled minutes on the operating table can tip the balance between survival and death.
The surgeon inspects the exposed wounds and then turns his attention to the pus-soaked bandages that covered them. He knows how to read pus, much as an ancient soothsayer believed he could divine the future from the guts of a sacrificed animal. ‘Laudable’ pus – pale and relatively odourless – is good news; dark discolouration and a foul smell indicate that the pus is going bad, and that the patient will shortly follow.
Proficient though he is, the surgeon has no idea about what is really happening in pus. It is a battlefield, a fight to the death between invading bacteria and billions of the patient’s white blood cells. The surgeon may have heard of ‘microbes’, but the notion of infection will not take root in his mind for another twenty years. In the meantime, he will pour scorn on anyone daring to suggest that he should wash his hands between operations – or even between the post-mortem room and the operating theatre.
After the surgeon’s visit, the pus-soiled dressings would usually be washed, or burned if beyond recycling. This morning, though, the bandages are carefully put to one side for a quiet young Swiss man who had hoped to be a doctor. He will sort through them, throwing away the ones that smell particularly foul, and carry the rest up the steep hill to the turreted twelfth-century castle perched high above the Neckar. What he will do with them there is anyone’s guess.
In pursuit of the chemistry of life
Friedrich Miescher was born in August 1844 and enjoyed a near-aristocratic upbringing in the prosperous Swiss city of Basel. His family tree was well manured by hereditary wealth, and his father and uncle were both powerful professors in the medical faculty at the university, the most venerable in Switzerland. Young Fritz was particularly close to uncle Wilhelm His, the Professor of Physiological Pathology, who took it upon himself to steer his nephew’s career.
Miescher’s childhood was steeped in music, literature and intellectual discussion and was spoiled only by meeting a louse that, in its modest way, changed the course of science. The louse infected the lad with typhus, which left him profoundly deaf. He coped valiantly with the disability and came top of the class in the medical school at Basel – only to realise that his ambition to be a doctor had been dashed because he could hear nothing useful through a stethoscope. Wilhelm His, worried that his nephew’s ‘considerable mental talents’ might be wasted, pushed him towards ‘the splendours of research’ and told him to think big. The twenty-four-year-old Miescher promptly changed course for the laboratory bench and set himself the task of unravelling the chemistry of life.
There was only one place for the young hopeful to go: Tübingen, and Europe’s first physiological chemistry laboratory. This had recently been set up by Felix Hoppe-Seyler, a human dynamo in his mid-forties who made other ‘rising stars’ look decidedly lacklustre. Hoppe-Seyler was famous for a string of startling discoveries in the new field of protein chemistry; the names ‘haemoglobin’ and ‘protein’ were among his inventions.
Hoppe-Seyler’s laboratory had started life as the kitchens and laundry of Hohentübingen Castle, which the university had recently invaded. It was a production line for would-be researchers from across Europe, all desperate to learn the art and science of protein chemistry from the master himself. Hoppe-Seyler’s supervision style was hands-off; after an initial briefing, a new arrival was given a project and left to his own devices. Luckily, the field was novel and living organisms were stuffed full of proteins waiting to be discovered; the laboratory enjoyed phenomenal success.
Miescher joined this research hothouse in October 1868. He was given space in a badly converted kitchen next to his chief’s personal laboratory (formerly the laundry) and instructed to find exciting proteins in white blood cells (leucocytes), which were virgin territory for protein chemists. Hoppe-Seyler suggested that pus might be a good source of leucocytes and told his new apprentice to report back when he had succeeded.
It took Miescher weeks to work out how to extract leucocytes from pus-soaked bandages, using repeated cycles of washing and filtration. As hoped, he isolated four new proteins – and then he discovered something so peculiar that it ended his career in the fashionable specialism of protein chemistry. The eureka moment might not seem that thrilling: a fluffy grey precipitate appeared when Miescher added acid to an extract of pus, and then melted away on adding alkali. However, this was striking, because proteins did not behave like that; and for some reason, the strangeness of the material made Miescher wonder whether it came from the nucleus, the rounded structure in the centre of the cell that had recently begun to excite interest.
Without asking Hoppe-Seyler, Miescher abandoned his search for proteins and instead concentrated on the fluffy precipitate that might be found in the nuclei of pus cells. He toiled through the winter of 1868–9 to achieve something that had never been done before – removing intact nuclei from living cells. Miescher’s day began at 5 a.m. and often continued far into the night; he kept his kitchen-laboratory freezing cold by leaving all the windows open, because wintry conditions made his experiments work.
Extracting nuclei from cells is like pitting cherries less than one-thousandth of their usual size. A modern benchtop centrifuge can do the job in a couple of hours, but Miescher’s extractions took weeks. Pus cells were gently soaked out of dirty bandages, filtered through a sheet and left to settle for two weeks in cold salt solution. The cycle was repeated until it yielded a fine sediment of intact leucocytes. Miescher broke them up by rinsing in weak acid and added an extract of pig’s st
omach (rich in protein-digesting enzymes) to remove cellular debris. This left a residue that the microscope showed to consist of pure, ‘completely naked’ nuclei. These were indeed the source of the curious material that had pulled him away from proteins. On adding alkali, the nuclei slipped cleanly into a yellow solution, from which the curious fluffy grey substance could be precipitated by acid.
Miescher spent several weeks collecting enough of the precipitate to work out its composition, using a tedious method that involved burning weighed samples and measuring the products of combustion. This produced another surprise. The carbon, hydrogen, nitrogen and oxygen (C, H, N and O) found in proteins were all present but sulphur (S), another diagnostic ingredient of proteins, was absent. Instead, the material contained large amounts of phosphorus (P). This confirmed that the substance was not a protein. In fact, it was unlike any compound known to biochemistry.
In deference to its source, Miescher named it ‘nuclein’.
*
From a standing start with pus-soaked bandages in late October, Miescher had made astonishing progress, especially as he did it all himself. Hoppe-Seyler’s laboratory was next door but the chief was impossible to pin down, constantly flitting between his own projects and the other students. After their initial discussion, Miescher had not consulted him at all.
Unravelling the Double Helix Page 3
