Unravelling the Double Helix

by Gareth Williams

  * The molecular weight of a compound is the sum of the masses of all its atoms; giving hydrogen (H) a relative weight of 1, carbon (C) has an atomic weight of 12 and oxygen (O) of 16. The simple sugar glucose has the chemical formula C6H12O6 and a molecular weight of 180, while the poliovirus is C332662H492388 N98245O131196P7500S2340 and weighs in at 8.5 million.

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  BAG OF WORMS

  Nucleic acids were not alone in failing to capture the imagination of scientists. The structure that contained them had also passed through its own cycle of discovery and neglect. When Miescher began his ‘immortal studies’ in 1868, the nucleus had been on the scene for thirty-five years but had spent most of that time buried in fine print. It was no wonder that Miescher’s plea for ‘serious study of the chemical constitution of the cell’s nucleus’ went unheeded for so long.

  Modern pictures of the cell show the nucleus sitting proudly in the middle, as conspicuous as the full moon in the night sky. In the beginning, though, it was just an ‘opaque speck’ that could have been artistic licence.

  Brownian notion

  The summer 1858 issue of the Annals and Magazine of Natural History offered up a rich haul for anyone fascinated by the living world, from the beak of a ‘rapacious bird’ to the shell-dissolving juices of the crab’s stomach. It also contained an article which rather implied that botanists had to die to be fully appreciated (‘We occupy ourselves with their lives only when they cease to exist’) and reminded a Mr Brown to get on with things before his time ran out as ‘several of his labours still await their completion’. There was unfortunate irony in these words, written some years earlier, as the piece was entitled ‘Robert Brown, Esq. Obituary Notice’.

  The deceased had followed that directive with great distinction, because this was no common or garden Mr Brown. He was Robert Brown FRS, chief botanist on the four-year voyage of HMS Investigator to Australia, and later President of the Linnean Society of London and the first Keeper of Botany at the British Museum. Being ‘a professed naturalist and a Scotchman with a cold mind’, he was ideally qualified to catalogue the 4,000 plants which Investigator brought home to England; in the process, he found over 2,000 species unknown to science. His cold Caledonian mind also saw him through the sinking of Investigator’s sister ship Porpoise and all its treasures, but allowed him to get really angry when the botanist-scoundrel Richard Salisbury published chunks of Brown’s lectures under his own name.

  Brown did his greatest work in a book-crammed house on Dean Street in Soho, London, which had been bequeathed to him by Joseph Banks, the flamboyant President of the Royal Society. His name lives on in ‘Brownian motion’, the random jiggling of tiny particles suspended in a drop of water. Brown first observed the phenomenon in 1827, while training his microscope on the minuscule granules (he called them ‘molecules’) that spilled out of rupturing pollen grains. The ‘molecules’ were not alive because similarly tiny particles of anything – even a fragment of limestone chipped off the Sphinx – performed the same perpetual dance routine.

  The microscope through which Brown tracked the antics of his ‘molecules’ looked nothing like the sleekly functional instruments that grace laboratory benches today. It was a triumph of simplicity, with just one tiny lens – a perfect sphere of glass barely a millimetre across – set in an eyepiece mounted on top of a brass pillar about a foot high. A concave mirror at the base of the pillar concentrated the light from an oil-lamp on to the specimen, which was clamped directly below the lens. The specimen could be part of a flower or a leaf, or pollen grains in a drop of water sandwiched between a thin glass slide and a coverslip of mica. The lens had an extremely short focal length (less than half a millimetre), which meant that eye, eyepiece and specimen all had to be uncomfortably close together, but the magnification was astonishing. Brown’s lenses magnified up to a thousand times – easily powerful enough to examine tissue biopsies.

  Figure 3.1 Prominent nuclei in pollen cells of the milkweed, drawn by Robert Brown in 1833. The filamentous structures are pollen tubes.

  Brown’s ‘peculiar taste in botany’ centred on the sex life of orchids, which is leisurely and sticky and may involve foreplay with other species.* While studying the intimate details of the process under his microscope, he noticed that every cell in the skin of orchid leaves contained a single circular ‘areola’. The cells of irises, lilies and other plants also showed an areola, always one per cell and usually lying centrally (Figure 3.1). Brown steadily built up a detailed picture of the areola: ‘exactly round’, granular and ‘somewhat opake’. Remarkably, he managed to dissect the areola out of the cells that form the hairs in the flowers of spiderwort; the extracted areola, squeezed out with the tip of a fine needle like a surgeon winkling out a cataract, was lentil-shaped when viewed from the side and apparently wrapped in ‘an enveloping membrane’.

  The areola had already been drawn by the botanical artist Franz Bauer in some of his diagrams of orchids, but he gave it ‘little importance’. Now, Brown identified the areola as a constant finding in a wide variety of plant cells. Apart from speculating that it produced the pollen tube to fertilise the ovum, he had no idea what it might do.

  We unconsciously remember Brown today because he rechristened the areola in his landmark paper on reproduction in orchids (1833). Using the Latin word for the kernel of a nut, he referred to the ‘nucleus of the cell, as perhaps it might be termed’. And the new name stuck.

  Nuclear proliferation

  Within a few years of Brown’s discovery, the nucleus was recognised as an obligatory feature of almost all animal and plant cells. Some nuclei are relatively dainty, whereas the lymphocytes that pack the thymus gland (a classic source of DNA) are almost all nucleus, with just a thin rim of cytoplasm. Most nuclei are spherical or lens-shaped, but the white blood cells that Miescher isolated from pus have a multi-lobed affair that looks like a rubber glove filled with water.

  There are rare exceptions to the ‘one cell, one nucleus’ rule, including the red blood cells of mammals, from which the nucleus is popped out during maturation in the bone marrow. By contrast, the red cells of birds and reptiles hang on to their nuclei – and so provided the nuclein that allowed Hoppe-Seyler’s student Plósz to verify Miescher’s unbelievable discovery.

  By the mid-1850s, it was generally accepted that cells multiplied by splitting in two, and that the nucleus also divided and miraculously reappeared in each of the two daughter cells. Most biologists assumed that the nucleus was essential to the life of the cell, because cells from which it was removed experimentally soon died. Others, however, believed that the nucleus was just a fellow-traveller, swept along by more important components of cellular machinery. The biggest name on the anti-nuclear bandwagon was Thomas Huxley, President of the Royal Society and ‘Darwin’s Bulldog’, who famously savaged the evolution denialist Samuel Wilberforce during a debate at the Oxford Union. Huxley insisted that nuclei (and even cells) were artefacts of microscopy – and that a strange jelly dredged off the bottom of the North Atlantic in 1857 was a revolutionary nucleus-free life form. The jelly had no microscopic structure and did absolutely nothing, but Huxley named it Bathybius (‘life from the deep’) haeckelii, after Ernst Haeckel, a German polymath and self-publicist who at the time also thought the nucleus irrelevant. Huxley continued to believe in Bathybius for over twenty years after the jelly was shown to be a chemical artefact.

  By then, the mercurial Haeckel had changed his mind and joined the nuclear family. This was because the nucleus had come in from the cold and, despite its infuriating habit of disappearing just as things were getting interesting, was beginning to share its secrets. And the new findings were pointing in an intriguing direction. In 1866, as though it had been blindingly obvious all along, Haeckel wrote that ‘the nucleus provides for the transmission of hereditary characters’.

  It took another twenty years for the evidence to catch up with Haeckel’s statement of fact. This was thanks to advances in optics an
d ‘histology’, the study of tissues under the microscope. Brown’s glorified magnifying glass evolved into the ‘compound’ microscopes familiar today, with separate lenses in the objective (immediately above the specimen) and the eyepiece. These produced a much clearer, brighter image and could be trained on living cells or on very fine slices of tissue that had been permeated with paraffin wax to preserve the internal structures. The slices were thin (a stack of 200 sections would be only a millimetre high) and transparent, which allowed cellular features to be highlighted with synthetic dyes. These histological stains transformed the monochrome landscape of microscopy. They react with specific components such as proteins, fats or nucleic acids and light them up with colours that could have been stolen from an artist’s palette. The first stains included methyl green, eosin (deep pink, and named after the Greek goddess of dawn) and toluidine blue, which picks out the nucleus in a rich shade of ultramarine. Friedrich Miescher could have been a trailblazer in this new field of ‘histochemistry’. In 1874, he found that a clear solution of nuclein turned a beautiful blue-green on adding methyl green; but he had no wish ‘to join the Guild of Dye-Makers’, and abandoned the observation for someone else to rediscover.

  Luckily, others were more interested in the new stains and their power to reveal details inside the cell that had previously been invisible. And before long, strange shapes – beautiful but baffling – began to emerge from the granular innards of Robert Brown’s nucleus.

  Divided loyalties

  In its resting state, which occupies well over 99.99 per cent of the lifespan of most cell-types, the nucleus gives away little under the microscope. It sits quietly in the cell, as impassively as a master poker-player; then, out of the blue, it is engulfed in a flurry of activity so confusing that even the sharpest-eyed microscopists could not agree about what happened. The nucleus melts away, leaving peculiar shape-shifting structures in its place. Then the cell elongates, and two nuclei materialise at its opposite ends. Finally, the whole thing tears across the middle to produce two daughter cells, each with an intact nucleus that looks just like the original.

  Cell division is fundamental to the life, health and repair of organisms. Tissues and organs grow and expand because their constituent cells multiply by dividing in two. A few cell-types, such as certain nerve-cells (neurones) in the brain, live out their long lives without the excitement of dividing, but the rest have greater ambitions. The cells in the skin and the lining of the gut suffer much wear and tear, and therefore have to regenerate themselves more frequently to keep those surfaces intact. Even in these high-turnover tissues, cell division is a rare event; for example, it fills just the last hour of the three-day lifespan of an epithelial cell in the colon. Cells divide more frequently in the embryo, and when tissues repair themselves after injury – a vivid example being the new leg grown by a larval newt following an unhappy encounter with an experimental biologist.

  Thanks to their favourable anatomy, some species have contributed generously to the study of cell division. To the naked eye, the horse threadworm looks like 5 inches of disconcertingly mobile spaghetti; under the microscope, it is the answer to a biologist’s prayer – hermaphroditic and with see-through gonads in which the development of both eggs and sperm can be followed in a single specimen. The larvae of amphibians such as newts and salamanders are blessed with large, microscopist-friendly cells in the skin, gills and bladder. And the salivary glands of flies contain extraordinary giant chromosomes so exquisitely patterned that mutations can actually be seen.

  The first attempts to make sense of cell division were made in living cells (more accurately, slowly dying ones), without using histological stains. By the mid-1870s, various researchers had reported that short rod-like structures – which Edouard Van Beneden called ‘bâtonnets’, or ‘little sticks’ – appeared in the disturbed cytoplasm where the nucleus had last been seen. But the mysteries of what the ‘little sticks’ were made of, where they came from and what they did, remained unsolved until one man sat down at his microscope and dedicated forty years to working out what was really going on.

  Picking up the threads

  Walther Flemming was one of the few genuinely nice people in the history of DNA. He was loved by his students for his ‘cordiality and benevolence’ and by the paupers of his adopted city for giving them a quarter of his salary and teaching their children free of charge.

  When the thirty-three-year-old Flemming took up post as Professor of Anatomy in Kiel in February 1876, he was returning to his roots in northern Germany. After a happy childhood in Sachsenberg, his medical training took him on a nomadic journey from Göttingen to Rostock, via Tübingen (missing Friedrich Miescher by a couple of years) and Berlin. After graduating as a doctor in 1868, he worked in Prague where hard-line Czech nationalist students made his life hell – hence his retreat to the run-down Anatomy Institute at the Christiana Albertina University in Kiel, one of Germany’s smallest.

  Thanks to its large merchant fleet, Kiel was a prosperous city, but with deep pockets of deprivation. When Flemming arrived, the Anatomy Institute was squeezed into a once-magnificent but now faded mansion near the city centre. He had to battle against chronic shortages of money, space and corpses for dissection – not to mention the university administrators who tried to steal his earnings. But he did great things with these unpromising materials, and transformed his institute into one of the world’s top centres for studying the processes of life.

  Flemming spent the rest of his career unpicking the fine details of cell division. He was assisted by good microscopes, the patience of a saint, an artist’s flair for capturing the moment, and the ‘excellent’ cells of the fire salamander. This smart black and yellow newt-like amphibian is notable for being poisonous and for the large clear cells lining its gills and bladder which display its chromosomes (only six, and therefore easy to track) to great advantage. Flemming began by watching the process in unstained tissues and saw ‘threads’ appear where the nucleus had been, as its outline faded away. These corresponded to the ‘little sticks’ already reported by others, but further details were difficult to make out.

  His great leap forward was to stain dividing cells with various histological dyes. He first ‘fixed’ the samples with a lethal cocktail of metal salts and acetic acid (‘Flemming’s solution’, still used today) which caused the cellular machinery to lock solid. With the stains, a new level of understanding leaped off the microscope slide. Frozen in the instant of the cell’s death, the ‘threads’ now stood out in stunning detail, painted red with safranin or deep blue with haematoxylin. Flemming then reconstructed the whole process from the snapshots of threads caught in the act at different stages of cell division. To make sure that his findings were not unique to salamanders, he also hunted down dividing cells in irises and sea-urchins.

  He wrote up his first few years of research in three big papers and a monumental book (1882), all beautifully illustrated with his drawings of the carefully choreographed dance of the threads (Figure 3.2). They first appeared as a tangled ‘skein’ where the nucleus had been, then rearranged themselves into a radiating ‘star’ pattern which became a flat ‘plate’ in the middle of a spindle-shaped structure that formed across the cell. At that point, each thread split longitudinally up the middle. Next, the divided half-threads separated into two groups that travelled to opposite ends of the spindle; each contracted into a new skein, around which the new daughter nucleus took shape.

  Figure 3.2 Movements of the chromosomes during cell division, drawn by Walther Flemming in 1883.

  Flemming wrongly believed that the threads formed a continuous strand, which was snipped into lengths for cell division, but he got almost everything else right. He called the intensely staining material of the threads ‘chromatin’, from the Greek for ‘colour’. The term was picked up in 1888 by Wilhelm Waldeyer, who renamed Flemming’s threads ‘chromosomes’ (‘coloured bodies’). The half-threads, which gave rise to the chromosomes of the daught
er cells, were later called ‘chromatids’. Flemming’s vision of ‘threads’ survives to this day. He called the process ‘Mitose’ (from ‘thread’ in Greek), which became the modern word ‘mitosis’. His almost poetic names ‘skein and ‘star’ have been replaced by more prosaic terms, but the process of mitosis is essentially as he described it (Figure 3.3).

  Figure 3.3 Cell division (mitosis), showing the stages of the process. Compare with Figure 3.2.

  Flemming’s sharp eyes picked up other crucial details. He described the ‘centriole’, so minute as to be almost a trick of the light, which usually sat quietly beside the nucleus. Then, as the nucleus began to melt away, the centriole became a remarkably busy little body. It divided itself into two halves which migrated to opposite ends of the cell, each trailing a tail like a tiny comet. The two tails joined in the middle to form the spindle to which the chromosomes attached themselves for the final steps of their dance.

  He went on to study the formation of eggs and spermatozoa in salamanders and sea-urchins, and found that cell division did not end as in other tissues. These ‘germ cells’ ran through mitosis exactly as in the gills or bladder – but the two daughter cells then underwent a further division to yield four cells, each of which contained only half the normal number of chromosomes (e.g. three in the salamander). This observation, made in 1883, confirmed Van Beneden’s finding that the sperm and eggs of the horse threadworm contained half as many chromosomes as the cells in all their other tissues.

  Flemming described this process perfectly, but did not give it a name. That fell to J.B. Farmer and J.E.S. Moore, who called it ‘meiosis’ in a paper published in 1905. The final ‘reduction division’ is an essential step in preparing eggs and sperm for their act of union, which produces a fertilised egg containing the full complement of chromosomes, and with half the genetic material contributed by each parent.