Kitabı oku: «The Mysterious World of the Human Genome»

In April 1927 a young Frenchman, René Jules Dubos, arrived at the Rockefeller Institute for Medical Research, in New York, on what would appear to have been a hopeless mission. Tall, bespectacled and a recent graduate of Rutgers University, New Jersey, with a PhD in soil microbiology, Dubos had an unusually philosophical attitude to science. He had become convinced, through the work of eminent Russian soil microbiologist Sergei Winogradsky, that it was a waste of time studying bacteria in test tubes and laboratory cultures. Dubos believed that if we really wanted to understand bacteria we should go out and study them where they actually lived and interacted with one another and with life in general, in the fields and the woods – in nature.

On graduation from Rutgers Dubos had found himself unemployed. He had applied to the National Research Council Fellowship for a research grant but had been turned down because he wasn’t American, but somebody had scribbled a handwritten message on the margin of the rejection letter. Dubos would later reflect upon the fact that it was written in a female hand, almost certainly added as a kindly afterthought by the official’s secretary. ‘Why don’t you go and ask advice and help from your famous fellow countryman, Dr Alexis Carrel, at the Rockefeller Institute?’ Dubos duly wrote to Carrel, which brought him, in April 1927, to the building on York Avenue, on the bank of the East River.

Dubos knew nothing about Carrel, or indeed about the Rockefeller Institute for Medical Research, and was surprised on his arrival to discover that Carrel was a vascular surgeon. Dubos had no academic knowledge of medicine and Carrel knew nothing about the microbes that lived in soil. The outcome of their conversation was all too predictable; Carrel was unable to help the youthful microbiologist. Their conversation closed about lunchtime and Carrel did Dubos the courtesy of inviting him to have lunch with him in the Institute’s dining room, which had the attraction for a hungry Frenchman that they served freshly baked bread.

It seemed entirely by accident that Dubos found himself sitting at a table next to a small, slightly built gentleman with a domed bald head who addressed him politely in a Canadian accent. The gentleman’s name was Oswald Theodore Avery. Although Dubos later confessed that he knew as little about Avery as he did of Carrel, Professor Avery (his close associates referred to him as ‘Fess’) was eminent in his field, which was medical microbiology. It would prove to be a meeting of historic importance both to biology and to medicine.

Avery subsequently hired Dubos as a research assistant, in which role Dubos discovered the first soil-derived antibiotics. Meanwhile Avery led his small team – who were working on what he called his ‘little kitchen chemistry’ – on another quite different quest, one that would help unravel the key to heredity. Why then does society know little to nothing about this visionary scientist? To understand how this anomaly came about we need to go back in time to the man himself and the problems he faced three-quarters of a century ago.

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In 1927, when Dubos first met Avery, the principles of heredity were poorly understood. The term ‘gene’ had been introduced into the nomenclature two decades earlier by a Danish geneticist, Wilhelm Johannsen. Curiously, Johannsen had adopted a vague concept of heredity, known as ‘pangene’, that was first proposed by Charles Darwin himself. Johannsen modified it to take on board the belated discovery of the pioneering work of Gregor Mendel, which dated back to the nineteenth century.

Readers may be familiar with the story of Mendel – the cigar-smoking, Friar-Tuck-like abbot of an Augustinian monastery in Brünn, Moravia (now the Czech Republic) – who undertook some brilliantly original studies of the peas he cross-bred in the monastery vegetable garden. From these studies Mendel discovered the basis of what we now know as the laws of heredity. He found that certain characteristics of the peas were transmitted to the offspring in a predictable manner. These characteristics included tallness or dwarfishness, presence or absence of the colours yellow or green in the blossoms or axils of the leaves, and the wrinkled or smooth skin in the peas. Mendel’s breakthrough was to realise that heredity was stored within the germ cells of plants – and this would subsequently be extrapolated to all living organisms – in the form of discrete packets of information that somehow coded for specific physical characters or ‘traits’. Johannsen coined the term ‘gene’ for Mendel’s packet of hereditary information. At much the same time, a combative British researcher, William Bateson, extrapolated the term ‘gene’ to the discipline he now called ‘genetics’ to cover the study of the nature and workings of heredity.

Today, if we visit the free dictionary online, we get the following definition of a gene: ‘The basic physical unit of heredity; a linear sequence of nucleotides along a segment of DNA that provides the coded instructions for the synthesis of RNA, which, when translated into protein, leads to the expression of hereditary character.’ But Mendel had no notion of genes as such, and he certainly knew nothing about DNA. His discoveries languished in some little-read papers for forty years before they were rediscovered and their importance was more fully understood. But in time his idea about the discrete packets of heredity we now call genes helped to answer a very important medical mystery: how certain diseases come about through an aberration of heredity.

We now know that genes are the basic building blocks of heredity in much the same way that atoms are the physical units that make up the physical world. But during the early decades of the twentieth century, nobody had any real notion of what genes were made from, or how they worked. But here and there, people began to study genes in more depth by examining their physical expression during the formation of embryos or in the causation of hereditary diseases. Fruit flies became the experimental model for pioneering research in the laboratory of Chicago-based geneticist Thomas Hunt Morgan, where researchers located genes, one by one, onto structures called chromosomes, which were themselves located within the nuclei of the insect’s germ cells. The botanical geneticist Barbara McClintock confirmed that this was also the case for plants. McClintock developed techniques that allowed biologists to visualise the actual chromosomes in maize, leading to the groundbreaking discovery that, during the formation of the male and female germ cells, the matching, or ‘homologous’, chromosomes from the two parents lined up opposite each other and then the chromosomes swapped similar bits so that the offspring inherited a jumbled-up mixture of the inheritance from the two parents. This curious genetic behaviour (which is known as ‘homologous sexual recombination’) is the explanation of why siblings are different from one another.

By the early 1930s biologists and medical researchers knew that genes were actual physical entities – chemical blocks of information that were lined up like beads in a necklace along the lengths of chromosomes. In other words, the genome could be loosely compared to a library of chemical information in which the books were the chromosomes. The discrete entities known as genes could then be compared to discrete words in the books. The libraries were housed in the nuclei of the germ cells – in human terms, the ova and sperm. Humans had a total stack of 46 books, which were the summed complement of ova and sperm, in every living cell. This came about because the germ cells – the ovum and the sperm – contained 23 chromosomes, so that when a human baby was conceived the two sets of the parental chromosomes united within the fertilised ovum, passing on the full complement of 46 chromosomes to the offspring. But this initial unravelling of the ‘heredity mystery’ merely opened up a Pandora’s box of new mysteries when it came to applying genetics to the huge diversity of life on our fecund planet.

For example, did every life form, from worms to eagles, from the protozoa that crawled about in the scum of ponds to humanity itself, carry the same kinds of genes in their nuclei-bound chromosomes?

The microscopic cellular life forms, including bacteria and archaea, do not store their heredity in a nucleus. These are called the ‘prokaryotes’, which means pre-nucleates. All other life forms store their heredity in nuclei and are known collectively as ‘eukaryotes’, which means true nucleates. From the growing discoveries in fruit flies, plants and medical sciences, it was becoming rather likely – excitingly so – that some profound commonalities might be found in all nucleated life forms. But did the same genetic concepts, such as genes, apply to the prokaryotes, which reproduced asexually by budding, without the need for germ cells? At this time within the world of early bacteriology there was even a debate as to whether bacteria should be seen as life forms at all. And viruses, which were for the most part several orders of magnitude smaller than bacteria, were little understood.

Over time many researchers came to see bacteria as living organisms, classifying them according to the binomial Linnaean system; so, for example, the tuberculosis germ was labelled Mycobacterium tuberculosis and the boil-causing coccoid germ was labelled Staphylococcus aureus. Oswald Avery, with his extremely conservative nature, kept his options open, eschewing the binomial system and referring to the TB germ as the ‘tubercle bacillus’. It is instructive for our story that Dubos, who came to know Avery better than any other colleague, would observe that ‘Fess’ was similarly conservative in his approach to laboratory research. Science must adhere with a puritanical stringency to what can be logically observed and definitively proven in the laboratory.

In 1882 German physician Robert Koch discovered that Mycobacterium tuberculosis was the cause of the greatest infectious killer in human history – tuberculosis. Koch constructed a code of logic that would be applied to bugs when first determining if they caused specific diseases. Known as ‘Koch’s postulates’, this was universally adhered to, and once a causative bug had been identified it was studied further under the microscope. Thus the bug was duly classified in a number of ways. If its cells were rounded in shape it was a ‘coccus’, if a sausage shape it was a ‘bacillus’, if a spiral shape it was a ‘spirochaete’. Bacteriologists methodically studied the sort of culture media in which a bug would grow best – whether in agar alone, or agar with added ox blood, and so on. They also studied the appearance of the bacterial colonies when they were grown in culture plates – their colours, the size of the colonies, whether they were rough in outline or round and smooth, raised or flat, stellate, granular or daisy-head. So the textbooks of bacteriology extended their knowledge base on a foundation of precise factual study and observation. And as understanding grew, this newfound knowledge was applied to the war against infection.

One of the useful things they learnt about disease-causing, or ‘pathogenic’, bacteria was that the behaviour of the disease, and thus of the bug itself in relation to its infected host, could be altered by various deliberate means: for example, through repeated cultures in the laboratory, or by repeatedly passing generations of the bug through a series of experimental animals. Through such manipulations it was possible to make the disease worse or less severe by making the bug either ‘more virulent’ or ‘attenuated’. Bacteriologists looked for ways to extrapolate this to medicine. In France, for example, the eminent Louis Pasteur used this principle of attenuation to develop the first vaccine to be used successfully against the otherwise universally fatal virus infection of rabies.

One fascinating observation that came out of these studies was the fact that, once a bug had been attenuated or been driven to greater virulence, the change in behaviour could be ‘passed on’ to future generations. Could it be that some factor of the bug’s own heredity had been altered to explain the change in behaviour?

Bacteriologists talked about ‘adaptation’, using the same term that was coming into vogue with evolutionary biologists when referring to evolutionary change in living organisms as they adapted to their ecology over time. While it was too early to be sure if bacterial heredity depended on genes, these scientists linked it to the physical appearance of bugs and colonies, or to the bugs’ internal chemistry, and even to their behaviour in relation to their hosts. These were measurable properties, the bacterial equivalents of what evolutionary biologists were calling the ‘phenotype’ – the physical make-up of an organism as opposed to what was determined by the hereditary make-up, or ‘genotype’.

Bacteriologists also came to recognise that the same bacterium could exist in different subtypes, which could often be distinguished from one another using antibodies. These subtypes were called ‘serotypes’. In 1921 a British bacteriologist, J. A. Arkwright, noticed that the colonies of a virulent type of dysentery bug, called Shigella, growing on the jelly-coated surfaces of culture plates, were dome-shaped with a smooth surface, whereas colonies of an attenuated, non-virulent, type of dysentery bug were irregular, rough-looking and much flatter. He introduced the terms ‘Smooth’ and ‘Rough’ (abbreviated to S and R) to describe these colonial characteristics. Arkwright recognised that the ‘R’ forms cropped up in cultures grown under artificial conditions, but not in circumstances where bacteria were taken from infected human tissues. He concluded that what he was observing was a form of Darwinian evolution at work.

In his words: ‘The human body infected with dysentery may be considered a selective environment which keeps such pathogenic bacteria in the forms in which they are usually encountered.’

Soon researchers in different countries confirmed that loss of virulence in a number of pathogenic bacteria was accompanied by the same change in colony appearance from Smooth to Rough. In 1923, Frederick Griffith, an epidemiologist working for the Ministry of Health in London, reported that pneumococci – the bugs that caused epidemic pneumonia and meningitis which were of particular interest to Oswald Avery at the Rockefeller Laboratory – formed similar patterns of S and R forms on culture plates. Griffith was known to be a diligent scientist and Avery was naturally intrigued.

Griffith’s experiments also produced an additional finding, one that really shook and puzzled Avery.

When Griffith injected non-virulent R-type pneumococci from the strain known as type I into experimental mice, he included an additional ingredient in the injections, a so-called ‘adjuvant’, which usually pepped up the immune response to the R pneumococci. A common adjuvant for these purposes was mucus taken from the lining of the experimental animal stomach. But for some obscure reason Griffith switched adjuvant to a suspension of S pneumococci, derived from type II, that had been deliberately killed off by heat. The experimental mice died from overwhelming infection. In the blood of these dead mice Griffith expected to find large numbers of multiplying R-type I bacteria – the type that he had injected at the start of the experiment. Why then had he actually found S-type II? How on earth could adding dead bacteria to his inoculum have changed the actual serotype of the bacterium from non-virulent R-type I to highly virulent S-type II?

Researchers, including Avery himself, had previously shown that S and R types were determined by differences in the polysaccharide capsules coating the cell bodies of the bugs. Griffith’s findings suggested that the test bacteria, initially R-type pneumococci, had changed their polysaccharide coats inside the infected bodies of the mice to that of the virulent strain. But they could not have achieved this by just flinging off the old coat and putting on the new one. The coat was determined by the bacteria’s heredity – it was an inherited characteristic. Further cultures of the recovered bacteria confirmed that the S type bred true. There appeared to be only one possible explanation: adding the dead S bacteria to the living R bacteria had induced a mutation in the heredity of the living R-type bacteria, so they literally transformed into S-type II.

In the words of Dubos: ‘[At the time] Griffith took it for granted that the changes remained within the limits of the species. He probably had not envisaged that one pneumococcus type could be transformed into another, as this was then regarded as the equivalent of transforming one species into another – a phenomenon never previously observed.’

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It is little wonder that Avery was astonished by Griffith’s findings. Like Robert Koch before him, Avery subscribed to the view that bacterial strains were immutable in terms of their heredity. The very concept of a mutation – that heredity was capable of an experimentally induced change – was a highly controversial issue within biology and medicine at this time. To understand why, we need to grasp the concept of what a mutation means.

By the late nineteenth century Darwinian theory had entered a crisis. Darwin himself had been well aware that natural selection relied on some additional mechanism, or mechanisms, capable of changing heredity, so that natural selection would have a range of ‘hereditable variation’ to choose between. Generations later, in the opening chapters of his innovative book Evolution: The Modern Synthesis, Julian Huxley put his finger on the nub of the problem. ‘The really important criticisms have fallen upon Natural Selection as an evolutionary principle and centred round the nature of inheritable variation.’ In 1900, a Dutch biologist, Hugo de Vries, put forward a novel mechanism that would be capable of providing the necessary variation: the concept of a random change in a unit of inheritance. Opportunity for change exists when genes are copied during reproduction, when a random change in the coding of a gene might arise from an error in copying the hereditary information. De Vries called this source of hereditary change a ‘mutation’. It was only with what Julian Huxley termed ‘the synthesis’ of Mendelian genetics – the potential for change in the inherited genes through mutation – and Darwinian natural selection operating on the hereditary choices presented within a species, that Darwinian theory became credible again to the great majority of scientists.

In time Griffith’s finding would be confirmed to be what Avery was now wondering about: it was a mutation. Geneticists would show that the change from the R to the S strain of pneumococcus involved the transfer of a gene from the dead S-type II bacteria to the living R-type I bacteria, which was incorporated into subsequent bacterial reproductive cycles, transforming the cells of the R-type I bacterium into the cells of the S-type II bacterium. It was indeed the bacterial equivalent of a change of species. And Griffith was proven right in inferring that Darwinian natural selection had operated even in the short time frame of the infection of a cohort of laboratory mice.