By Dahm, Ralf
Few remember the man who discovered the “molecule of life” three- quarters of a century before Watson and Crick revealed its structure On February 26, 1869, in the old university town of Tubingen in southwest Germany, the young Swiss doctor Friedrich Miescher, who had settled there only a few months earlier, completed a letter to his uncle in which he described a momentous discovery. He had found a substance that he was certain resided in the cell nucleus and which differed in chemical composition from proteins or any other compound known at the time. Without grasping the reach of his work, Miescher had started one of the greatest scientific revolutions. Years later, it would completely change the fundamental understanding of life and lead to medical breakthroughs unimaginable in Miescher’s time.
Johann Friedrich Miescher was born into a family of scientists in 1844 (he was always known as Friedrich, even in his publications later in life). Miescher’s father and his maternal uncle, Wilhelm His, were distinguished medical doctors and professors of anatomy and physiology at the University of Basel in Switzerland. A range of scientists frequently visited the home, and their lively discussions exposed the young Miescher to a variety of scientific ideas and concepts. In such surroundings, Miescher developed a keen interest in the natural sciences. At the age of 17 he started his studies of medicine in Basel and graduated in 1867 when he was only 23 years old.
At first he considered practicing medicine, like his father. However, poor hearing from an illness he had contracted in childhood would have made some parts of that job difficult for him. His fascination with the sciences suggested research as an avenue for him to pursue. Inspired by his uncle’s conviction that the “last remaining questions concerning the development of tissues could only be solved on the basis of chemistry,” Miescher decided to study biochemistry.
In the spring of 1868 he moved to Tubingen to work under the guidance of two of the most renowned scientists of the time: the organic chemist Adolf Strecker, in whose laboratory Miescher worked for one semester, and the biochemist Felix Hoppe-Seyler, one of the pioneers in a nascent branch of science referred to as “physiological chemistry.” Between 1860 and 1871, Hoppe-Seyler headed one of the first biochemical laboratories worldwide. It was located in Tubingen’s medieval castle, high above the old town and the surrounding river valleys (Hoppe-Seyler’s laboratory was in a converted laundry room, whereas Miescher’s was put in the former kitchen). Hoppe-Seyler had previously accomplished, among other things, groundbreaking experiments concerning the properties of hemoglobin-seminal work that was to influence countless subsequent studies on the structure and function of this and other proteins. In a very short span of time, his laboratory had attained a reputation that reached far beyond the boundaries of the city.
Elementary Analyses
Under Hoppe-Seyler’s guidance, Miescher set out to determine the chemical composition of cells. Lymphocytes were to serve as the source material for these studies. By studying this “most simple and independent cell type,” he hoped to understand the secrets of cellular life. But lymphocytes proved difficult to purify from lymph glands in the large quantities needed for chemical analyses. Hoppe- Seyler, who had a long-standing interest in the nature of blood, likely suggested that Miescher turn to closely related leukocytes instead. Thus the discovery of DNA made a rather unappetizing start: Miescher isolated the cells he needed from the pus on wound dressings he obtained from the surgical hospital in Tubingen. At the time, a plentiful production of pus from wounds was still widely held to be a requirement to purge the body of harmful substances. Antiseptics were not yet commonly used and purulent bandages were available in large quantities.
First of all, Miescher had to develop methods to wash the leukocytes from the surgical cloth. He tested a variety of salt solutions, always checking the outcome of his trials under a microscope. Once he had established the conditions, he set out to characterize and categorize the different proteins and lipids he isolated from the cells. Like many of his contemporaries, he hoped to discover how cells work by analyzing their proteins, so Miescher described their properties and attempted to classify them. But his work was plagued with setbacks. The diversity of proteins within a cell was too much for the relatively primitive methods and equipment of his time. During his experiments, however, Miescher detected a substance with unexpected properties. It could be precipitated by acidifying the solution and redissolved by making the solution more alkaline. Unknowingly, Miescher had, for the first time, obtained a crude precipitate of DNA.
But where did this enigmatic substance come from? When Miescher had extracted leukocytes with acids, he had noticed that prolonged exposure of the cells to diluted hydrochloric acid resulted in a cellular residue consisting of what looked like isolated nuclei. He also noticed that these nuclei could no longer be stained yellow with iodine, an indication that the proteins had been largely extracted. Very weak alkaline solutions led to a strong swelling of the nuclei, without, however, dissolving them. Based on these observations Miescher speculated that his mysterious precipitate could only belong to the nuclei.
At that time, very little was known about this organelle. Although the nucleus had been discovered as early as 1802, its function in the cell remained a matter of intense controversy and speculation. However, in 1866, three years prior to Miescher’s discovery, the influential German biologist Ernst Haeckel had proposed that, the nucleus contained the factors responsible for the transmission of hereditary traits. This suggestion led to a renewed interest in the role of the nucleus. Miescher’s serendipitous finding opened a door to gleaning more information on the nature of this mysterious organelle.
Before being able to further characterize the nuclear precipitate, however, Miescher had to develop protocols to isolate nuclei with higher purity. After many trials, he finally hit on a method. He rinsed the cells several times with fresh solutions of a diluted hydrochloric acid over a period of several weeks at “wintry temperatures,” which were important to minimize degradation of his material. This treatment broke apart the cells’ membranes and stripped most of the cytoplasm off the nuclei. He next removed the lipids by vigorously shaking the material in a combination of water and ether. When the mixture settled, Miescher observed mat the extracted nuclei sank to the bottom of the vessel as fine granules. When he added alkaline solutions to these nuclei, he found that they swelled and faded, just as he had seen with his earlier preparations. When he added acid, on the other hand, the swelling was reversed and again a white, woolly precipitate appeared. With these experiments Miescher showed that the precipitate he had previously observed had indeed come from the nuclei. As a consequence, Miescher later named it nuclein, a term still preserved in today’s name deoxyribonucleic acid.
Despite nuclein’s unusual behavior, Miescher was not yet entirely convinced that it was distinct from protein. He thus devised further experiments to learn more about the nature of this strange molecule. Chiefly, he intended to determine its elementary composition, but to do so he needed still purer nuclein. In particular, he had to remove as much of the contaminating cytoplasm as possible. Miescher decided to try a method that Wilhelm Kuhne had described only one year earlier in his textbook on physiological chemistry. Kuhne had observed that cells can be broken apart with solutions containing the digestive enzyme pepsin, which dissolves cytoplasm without attacking nuclei.
This approach was precisely what Miescher needed. Unfortunately, at the time, pepsin could not be ordered from a chemical supplier. Instead Miescher had to isolate it for himself. Thus he embarked on the second unsavory part of his scientific journey: He rinsed out pig stomachs with diluted hydrochloric acid and filtered the washed- out contents to obtain a crude solution of protein-digesting enzymes. Treating cells with this solution not only chewed apart the proteins, it also showed that nuclein was indeed not a protein.
Now Miescher finally had an optimized protocol to isolate DNA. He began by washing the leukocytes several times with warm alcohol. This broke the cells up and removed most of the cytoplasm. Moreover, it dissolved most lipids. Subsequently, he digested the extract with his pepsin solution. This treatment resulted in a fine, gray sediment. To remove residual lipids, Miescher shook the sediment in ether and again in warm alcohol. He noted that the “nuclear mass” purified in this way showed the same chemical behavior as the nuclear extracts isolated with his previous protocols. Next, Miescher washed the preparation with alkaline solutions, such as highly diluted sodium carbonate. When subsequently adding an excess of acetic or hydrochloric acid, he got a flocculent precipitate, which he could redissolve by adding alkaline solutions. This precipitate was the first comparatively dean preparation of DNA, pure enough for Miescher to finally embark on an analysis he had been planning for some time: to determine which elements make up nuclein. Elementary analyses were one of the few methods available to characterize novel molecules at the time. The procedures involved heating the substance in the presence of various chemicals that selectively reacted with the different constituent elements. The resulting reaction products were weighed to determine the amount of each element present in the substance under test. The process was laborious and time consuming, so much so that Miescher called it “factory work,” but he kept at it. So far, Miescher had established that nuclein behaved differently from proteins and lipids in his isolation procedure: Enzymes capable of breaking down proteins were unable to degrade it, and it could not be extracted by strong organic solvents. The analysis of its elementary composition held another surprise for Miescher. Besides containing the elements carbon, oxygen, hydrogen and nitrogen, which are known to be very abundant in proteins, the molecule did not contain sulfur and it did harbor large quantities of phosphorus. The latter was a very unusual finding because virtually no other organic molecules containing phosphorous were known at the time. This result finally convinced Miescher that he had discovered a fundamentally new type of cellular substance.
Publishing Woes
In the autumn of 1869 Miescher finished his initial analyses of nuclein and returned to Basel for a short holiday. During this time he began writing his first scientific publication on his analysis of the biochemical composition of leukocytes, including his discovery of nuclein. In his manuscript, Miescher was confident about the importance of his findings and stated that the new substance he had discovered would prove to be of equal stature to proteins. Following his holiday, Miescher returned to the laboratory. However, he did not go back to Tubingen but rather to the University of Leipzig. To broaden his scientific education, he had decided to turn to other topics and thus joined the laboratory of the noted physiologist Carl Ludwig to investigate, for instance, the nerve tracts in the spinal cord that transmit pain. Although Miescher tackled the new tasks with his characteristic conscientiousness, he did not develop the same enthusiasm he had felt for his project in Tubingen.
During his first months in Leipzig, Miescher also finalized the draft of his first publication. Shortly before Christmas of 1869, he was done, and he prepared to send the manuscript to HoppeSeyler for his approval. On December 23, he wrote in a letter to his parents: “On my table lies a sealed and addressed packet. It is my manuscript, for the shipment of which I have already made all necessary arrangements. I will now send it to Hoppe-Seyler in Tubingen. So, the first step into the public is done, given that Hoppe-Seyler does not refuse it.”
Hoppe-Seyler didn’t reject Miescher’s paper. But his former mentor was suspicious of the unusual results and wanted to verify them for himself before publication. This attitude was none too surprising given that not long before HoppeSeyler’s laboratory had been the site of a protracted argument over whether a putative phosphate-containing molecule from brain tissue actually existed. In this context, Hoppe-Seyler would of course view skeptically a junior scientist’s claim of having discovered a fundamentally new molecule. Moreover, Miescher’s manuscript was to be included in the Medicinisch-chemische Untersuchungen (Medical-chemical Investigations), a journal Hoppe-Seyler himself published. Hoppe- Seyler would have been especially stringent about which papers he accepted for publication there.
Thus, Miescher had to resign himself to months of anxious waiting for Hoppe-Seyler to validate his findings. Although overall Hoppe- Seyler was positive about Miescher’s work, his initial analyses of nudein’s elemental composition differed from Miescher’s. He cautioned that these differences might not be meaningful, but it was dear that this would delay things further. Hoppe-Seyler offered to submit the manuscript to another journal if Miescher wanted, but the younger scientist preferred to wait for the verification of his results and to see his work appear in his former mentor’s journal.
Matters were made worse by the outbreak of the Franco-Prussian war. Starting in July 1870, a federation of German states was embroiled in a bitter conflict with France, which diverted both resources and attention away from academic science. Over time, Miescher grew increasingly worried about the delay in publishing his manuscript. He wanted to submit his habilitation (a kind of postdoctoral thesis) at the University of Basel so he would be able to be appointed professor there. Moreover, he feared that other scientists might discover nuclein also and publish on it before him.
He repeatedly wrote to Hoppe-Seyler, gently trying to speed things up. Despairing over the long delay, he even contemplated sending his work to another journal and asked Hoppe-Seyler to return his manuscript. But after a year of suspense, in October 1870, Miescher received Hoppe-Seyler’s reply to his letters. Hoppe-Seyler reported that he had been able to corroborate Miescher’s results on nuclein and that he intended to publish Miescher’s manuscript in the next issue of his journal. The letter also included Hoppe-Seyler’s findings on the topic for Miescher’s comments.
Miescher was overjoyed by the prospect of his paper finally seeing me light of day and promptly sent his comments back to Hoppe- Seyler. A few weeks later he received the proofs of his first publication. In the accompanying letter, Hoppe-Seyler pointed out that they were full of typographical errors because the printers found it difficult to decipher Miescher’s handwriting.
At long last, in early 1871, Miescher’s manuscript was published as the first paper in an issue of Hoppe-Seyler’s journal, which contained two additional articles on nuclein: one by another student of Hoppe-Seyler’s demonstrating the presence of the molecule in the nucleated erythrocytes of birds and snakes, and Hoppe-Seyler’s own article in which he reported that he had confirmed Miescher’s findings on nuclein.
Return to Basel
After his stay in Leipzig, Miescher had been offered the prospect of a professorship at the University of Basel, and so in 1870 he returned to his hometown. His scientific achievements abroad had established his reputation as a dedicated and resourceful researcher. In 1871 he submitted his habilitation, and in the following year, at the age of only 28, he was offered the Chair of Physiology at the university, the same post both his father and his uncle had once held. Miescher worked exceptionally hard in his new position, often to the point of exhaustion. In addition to his passion for science, he was driven not least by the desire to dispel any suggestions that he might have been appointed because of his family ties rather than his accomplishments.
In Basel, Miescher also resumed his work on nuclein, which had all but ceased during his stay in Leipzig. He was spurred on in some part because Hoppe-Seyler wished to continue research on nuclein but agreed to limit his work, as long as Miescher’s own efforts picked up again. Miescher’s aim was to characterize nuclein in greater detail than he had done in Tubingen. But his working conditions were poor and his progress accordingly slow.
In a letter to a friend he lamented: “During the last two years, I have feverishly yearned to be back at the meat pots of the Tubingen castle laboratory. I do not really have a laboratory to speak of here, I am just tolerated in a small corner of the chemistry lab where I can hardy twitch, as it is already overly stuffed with students and on top of that, the professor of chemistry does his research here too.” He continues, “You can surely imagine what it is like to be hindered by appalling external circumstances from energetically pursuing things that may never again be placed so conveniently beneath my fingertips….”
But Miescher did not give up. Inspired by his uncle’s interest in developmental biology, he turned to studying nuclein in eggs and sperm cells. He quickly realized that sperm cells, consisting largely of nuclei, were an ideal source to isolate nuclein in large quantities and purity. Basel proved to be the perfect place for these experiments. As it was situated on the Rhine River, which at the time had a large annual upstream migration of salmon to their spawning grounds, Basel had a thriving salmon fishing industry. This gave Miescher access to an abundance of freshly caught fish. Thus, in the autumn of 1871, he converted to using salmon sperm as his source material for nuclein and developed successive, increasingly sophisticated protocols.
As during his experiments in Tubingen, Miescher used only fresh material and worked rapidly during the isolation of nuclein. Moreover, he had to handle the material in the cold to prevent degradation of the molecule. Because cold rooms were not available in those days, he could only carry out the isolation during the winter months.
Often he would get up in the middle of the night to catch salmon from the Rhine River, bring them to his laboratory and work away during the early hours of the day with the windows of his laboratory wide open to the freezing cold outside. Arduous as it was, this procedure enabled Miescher to isolate copious amounts of the purest nuclein that he had ever had at his disposal, allowing him to carry out the comprehensive, quantitative analyses that he had planned to do in Tubingen. With these new observations, he confirmed his earlier results and determined the phosphorous content of nuclein with amazing accuracy. In 1874 he published his results on the occurrence of nuclein in the sperm of various vertebrates. At the time, scientists were seeking to find out how embryonic development works and how hereditary traits are passed on. Miescher came within arm’s reach of the answer. In his article he wrote: “If one wants to hypothesize that a single substance specifically is the cause of fertilization in any way, then-without a doubt-one would have to think primarily about nuclein.” However, Miescher did not believe that a single molecule could be responsible for inheritance and scrapped the idea, mainly because he could not envisage how the same substance could lead to the diversity of different animal species whose sperm he had examined. He wrote: “There will be differences in the chemical structure of the molecule,” but continued, “though only in a limited diversity.” Too few, according to Miescher, to be responsible even for the differences observed between individuals of the same species, let alone the sometimes vast variation between different species. Instead he favored the idea that mechanical stimuli caused by the movement of the sperm and processes-as observed during the excitation of nerves and muscle fibers-are responsible for the development of the fertilized egg.
Aside from this notion, however, Miescher also developed a hypothesis to explain the transmission of hereditary information that, although incorrect in its details, came remarkably close to describing the way information is actually stored in DNA. He speculated that information might be encoded in the stereochemical state of carbon atoms, or in other words, their arrangement within molecules. Much as an alphabet of 26 letters is sufficient to express all words and concepts in a variety of different languages, molecules could be made up of different stereoisomers, or specific geometric arrangements of the constituent atoms. The vast numbers of asymmetric carbon atoms in large organic molecules, such as proteins, would allow an enormous number of stereoisomers. A molecule containing, for example, as few as 40 asymmetric carbon atoms could have 2(40), or more than one trillion, stereoisomers. This number, Miescher reasoned, would be large enough to encode the hereditary information for all the diverse forms of life. Miescher further proposed that errors in individual molecules might be prevented from manifesting themselves in the developing embryo by the fusion of information from two germ cells during fertilization. These views seem to anticipate what is now considered common knowledge: that intact alleles from one parent can compensate for defects in the allele inherited from the other.
Broadening Interests
Over time, Miescher increasingly turned to other areas of research and no longer published on nuclein. From the mid187Os onward, for instance, he studied the changes in the anatomy of salmon during their annual migrations from the ocean to their fresh- water spawning grounds in the Rhine, a trip during which the fish cease eating entirely.
Miescher spent entire winters getting up in the middle of the night to spend the early hours of the day catching salmon on the banks of the river. He carried thousands of them to his lab, measured and weighed them, examined their muscles, internal organs and blood. He was fascinated by the fact that the sexual organs of these fish undergo an immense growth, until they make nearly a quarter of the fish’s mass, at the expense of the animal’s muscles.
Based on these studies of the metabolism of salmon, in the autumn of 1876 the Swiss government requested him to prepare a report on the diet of inmates at Basel prison. Miescher was none too happy about this task, which took months to complete, but the authorities were impressed with his work and he received similar inquiries from other prisons. His uncle later wrote about this period that “Every jail wanted to have its very own menu.” But it did not end there: Educational institutions, associations for the nutrition of the people and other institutions concerned with nutrition-all sought Miescher’s advice. Finally, he had enough of this work and became disgruntled. He asserted: “I have made myself too green and now the goats are eating me. Inquiry on the diet of the Swiss people, cookbook for workers, nutrient tables for the national exhibition, controversies with the Chamer Milk Company-in brief, I’m on the best way to becoming the guardian for the stomachs of all three million Swiss.”
Eventually, Miescher turned to a new challenge. In 1885 he founded the first anatomical-physiological institute of Basel. As its first director, Miescher took his position very seriously. He encouraged an active scientific life and recruited renowned master technicians who designed and developed new machines and instruments for physiological measurements, which functioned with unrivalled precision. He investigated how blood composition varies at different altitudes and discovered that blood’s concentration of carbon dioxide, not oxygen, regulates breathing.
Yet, over time, Miescher’s increasing commitments exhausted him. His obsession with his work and his need for perfection afforded him everdiminishing rest. He slept less and less, hardly attended social functions and continued working through his holidays. Totally worn out, his body became weaker by the day and in the early 1890s he contracted tuberculosis. He had to retire from work and moved to a sanatorium in Davos in the Swiss Alps.
One last time he tried to pull together his previous work, including his research on nuclein. But his strength failed him and, in 1895, Friedrich Miescher died, at the age of just 51. After Miescher’s death, his uncle Wilhelm His published his nephew’s collected papers. In the introduction he wrote: “The appreciation of Miescher and his works will not diminish with time, instead it will grow, and the facts he has found and the ideas he has postulated are seeds which will bear fruit in the future.” Despite his favorable appraisal, even Wilhelm His could not imagine how true his words would become.
DNA Research after Miescher
Why is Miescher’s name not widely associated with DNA today? For one, unlike many diseases, species or anatomical structures, molecules are not usually named after their discoverer. Moreover, Miescher was not good at promoting his work. He was introverted and interacted with only a limited number of his colleagues. He lacked students, many of whom were put off by his reclusive nature. In addition, despite his passionate drive for scientific research, he was insecure and a perfectionist, leading him to repeat experiments and delay publication. Even during his lifetime, Miescher felt that research on nuclein was increasingly being associated with other researchers. For example, in 1889 Richard Altmann renamed the molecule “nucleic acid,” a fact that greatly annoyed Miescher because he had always been very specific about the acidic nature of nuclein. Perhaps most critically, the gap of 75 years between Miescher’s discovery of DNA and the realization of its importance may have just been too long.
For almost 50 years following Miescher’s death, most scientists believed that DNA, composed of only four types of building blocks, was too simple a molecule to encode the information required to produce the diversity of life. Proteins, with their more complicated composition and structures, were considered far more likely candidates to bear and transmit hereditary information. Widespread interest in DNA was revived only in the 1940s, when Oswald T. Avery and his colleagues Colin MacLeod and Maclyn McCarty proved that DNA does indeed cany the genetic information. In 1952 Alfred Hershey and Martha Chase confirmed DNA as the genetic material and one year later, building on x-ray analyses by Rosalind Franklin and Maurice Wilkins, Francis Crick and James Watson famously solved its structure.
At that point, the individual pieces of the puzzle fell into place. DNA not only had a structure, but this structure could also explain how it functioned, how it can be faithfully replicated prior to each cell division and how the information contained in it can be read to produce proteins. Finally, by the mid-1960s-almost 100 years after Miescher’s experiments-the genetic code was cracked and scientists could finally read the language in which genetic information is written. These breakthroughs were the foundation for the emergence of an entirely new kind of biological research, molecular genetics. Since then, DNA has become more than just a molecule. It has been transformed into the icon of the modern life sciences. But nearly 150 years after Miescher’s first experiments, there still remains a lot to discover about DNA.
For relevant Web links, consult this issue of American Scientist Online:
http://www.americanscientist.org/ IssueTOC/issue/1101
Bibliography
Dahm, R. 2008. Discovering DNA: Friedrich Miescher and the early years of nucleic acid research. Human Genetics 122:565-581.
Dahm, R. 2005: Friedrich Miescher and the discovery of DNA. Developmental Biology 278:274-288.
Lagerkvist, U. 1998. DNA Pioneers and Their Legacy. New Haven: Yale University Press.
Miescher, F. 1871. Ueber die chemische Zusammensetzung der Eiterzellen. [On the Chemical Composition of Pus Cells.] Medicinischchemische Untersuchungen 4:441-460.
Portugal, F. H., and J. S. Cohen. 1977. A Century of DNA. Cambridge: MIT Press.
Wolf, G. 2003. Friedrich Miescher, the man who discovered DNA. Chemical Heritage 21(10-11):37-41.
Ralf Dahm is group leader at the Center for Brain Research, Medical University of Vienna. His research focuses on the genetics behind the development and diseases of the eye and brain. From 1999 to 2005 he worked at the Max Planck Institute for Developmental Biology in Tubingen, Germany, where he became interested in the early history of DNA research. Address: Center for Brain Research, Division of Neuronal Cell Biology, Medical University of Vienna, Spitalglasse 4, A-1090 Vienna, Austria. Internet: [email protected]
Copyright Sigma XI-The Scientific Research Society Jul/Aug 2008
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