Dr. George Wolf has recently come to Switzerland in search of documentation for an account of the discovery of DNA. The undertaking started off as a wild-goose chase. Hoping to find Miescher's laboratory notebooks he went to Basel and visited the "F. Miescher Institute", which is part of the pharmaceutical company "Novartis". There was almost nothing to be found there. At the Basel university library, he dug up a postcard from 1920, which stated that the notebooks had been received at the Institute of Physiology and were being stored "with the platinum". He rushed there, only to be told by the director that there was no platinum, that the notebooks had either been thrown out or were given to members of Miescher's family. He then visited a grand-niece of Miescher, who lives in Basel above the Rhine river. She fed him a delicious strawberry pie, but had no clue. In the meantime, he has been able to piece together the missing experimental details from other sources.
The historical account of the discovery of DNA in 1869 includes a brief biography of Miescher and a description of the historical context and of the emergence of research schools. It concentrates on the experiments Miescher carried out for the isolation, analysis and characterization of DNA.
You can also download the full manuscript as a 65 kB PDF file: Friedrich Miescher, the Man who Discovered DNA
George Wolf , Adjunct Professor
Dept. of Nutritional Sciences and Toxicology
University of California, Berkeley, CA 94720-3104
FRIEDRICH MIESCHER, THE MAN WHO DISCOVERED DNA
A historical account of the discovery of DNA in 1869, including a brief biography of Miescher and a description of the historical context and of the emergence of research schools. It concentrates on the experiments Miescher carried out for the isolation, analysis and characterization of DNA.
“Who discovered DNA ?” “Watson and Crick, of course !” most students will answer. However, DNA was isolated, analyzed and recognized as a unique macromolecule in 1869 by Friedrich Miescher, an eminent physiological chemist from Basel, Switzerland. His life and contribution to science have been repeatedly described in chapters of books on the history of nucleic acids, for instance by Portugal and Cohen, and recently by Lagerkvist, by Fruton and by Gehring. The present preoccupation with DNA in so many walks of life, unprecedented for a substance of physiological importance, suggests an occasion for a look at the way it was discovered and the man who discovered it.
Friedrich Miescher was born in Basel in 1844, the eldest of 5 sons of Friedrich Miescher-His, professor of pathologic anatomy at Basel University, and a successful practicing gynecologist. Miescher’s mother, Antonie, was of the His family, Basel aristocracy if such had existed. Her brother, Wilhelm His, who lived in the same house as the Mieschers, was professor of anatomy and physiology, distinguished for his work in embryology and histology. He had a life-long influence on his nephew Friedrich. The household was of that academic, haut-bourgeois kind well-known at that time, comfortable, spacious, hospitable, filled with music, books and intellectual pursuits, mainly, of course, centering on biology and medicine. In the Miescher-His house gathered a wide circle of friends, old and young, from diverse social classes. Even decades later, friends of the Miescher sons who had been treated like members of the family, felt grateful for the intellectual stimulus they had received in that house. Wilhelm His, in a monumental work, describes Miescher's life and appends a collection of Miescher's correspondence with family and colleagues, as well as a selection of his scientific papers.
The young Miescher, though shy and withdrawn - no doubt because he was hard-of-hearing - graduated at the top of his high school (Gymnasium)) class. He began his medical studies in Basel in 1862, attending lectures by both his father and uncle. In the summer of 1865, he worked in the chemical laboratory of F. Wohler in Gottingen, but came down with typhoid fever and could not resume his studies until 1866. He graduated in 1868, presenting an outstanding thesis.
At the time of his graduation, he wrote a long letter to his father, discussing his career plans. He wanted to be a practicing physician, but because of his hearing difficulties, he chose ophthalmology or otology, where listening through a stethoscope would not be needed. On the other hand, he had a great desire to work in basic research: ”It was only in the lectures on physiology that the entire splendor of research on organic matters became apparent to me “. He therefore proposed a compromise: he would practice ophthalmology and in his spare time do research. Miescher’s father showed this letter to Miescher’s uncle, Wilhelm His, who instantly saw that the compromise solution would not work. He proposed that “in view of the considerable mental talents which Fritz has “, he should enter the career he found most appealing, that of research in physiology. His thought that someone so eminently theoretical in nature as Miescher would find satisfaction only in scientific research. Miescher, who idolized his uncle, followed his advice and his father agreed. So, in 1868, the young physician decided to join the laboratory of physiological chemistry of the Felix Hoppe-Seyler, "the rising new star", as he was called by Lagerkvist. He had established the first faculty of natural science in Germany. Miescher wanted to investigate the chemistry of the cell, following the conviction of his uncle that “the ultimate solution of the problem of tissue development will be found in chemistry”.
Young Miescher, with his characteristically methodical approach to research, decided, before starting what we would now call the post-doctoral job in physiological chemistry, to spend a semester in the chemistry laboratory of A. Strecker in Tübingen, to become familiar with organic-chemical techniques. In the fall of 1868 he began his work in Hoppe-Seyler’s laboratory, also in Tübingen. His mentor was interested in the chemistry of body fluids, particularly of blood. He had recently discovered the binding of oxygen to hemoglobin in erythrocytes. Now he put Miescher to work on other blood cells, the lymphocytes. These were difficult to obtain in quantity, so Miescher isolated leukocytes, similar to lymphocytes and thought to be derived from them. He prepared leukocytes from pus. From these, he then succeeded in isolating pure nuclei. He extracted an acid-insoluble, alkali-soluble, high-phosphorus containing substance from the nuclei and characterized what he recognized as a new class of substances. By December 1869, a year after beginning his research, he submitted a manuscript to Hoppe-Seyler, describing the new substance, which he called “nuclein”, later called “nucleic acid” by R. Altmann and now known as DNA (Miescher’s name for it, nuclein, will be used in this article).
Hoppe-Seyler delayed publication for 2 years, in order to repeat the experiments himself. He confirmed Miescher’s results in every respect and published the paper of Miescher together with his own confirmation, in addition to a paper demonstrating the presence of nuclein in the nucleated erythrocytes of birds and snakes.
Miescher was still not satisfied with his research training and sought more experience in physiological research in the laboratory of C. Ludwig in Leipzig, where he worked on nerve pathways in the spinal cord (1869-70). Though conscientious as usual, he was not enthusiastic about this topic. The international character of the research school there, with scientists from 6 nations, including an Egyptian of Islamic faith, was most unusual at that time, and he there made many good friends for life.
He returned to Basel in 1870 and in 1871 became Privatdozent upon completing his Habilitation , a senior thesis, which dealt with the regulation of breathing. His experiments proved that it was not lack of oxygen, but increased carbon dioxide in the lung alveoli that acted as the respiratory stimulus.
Miescher began his research career in Basel under the tutelage of his uncle, Wilhelm His, famous for his studies on the origin of tissues in the developing embryo. He influenced Miescher to investigate the small white spheres present in the yolk of hens’ eggs, which His thought may be the cells that form the early embryo upon fertilization. Miescher concluded from some experiments that they contained nuclein, but later came to doubt this. His was also interested in the development of fish eggs and Miescher began to study them chemically with a view to isolating nuclein from their nuclei. However, he soon realized that fish sperm cells might be a more rewarding source for that purpose, because they were simple independent cells, known from previous histologic studies to consist almost entirely of nuclei. Moreover, on the practical side, the salmon migration up the Rhine and the consequent large-scale salmon fisheries made this material easily available in large quantities.
At that time (1872) his uncle and mentor, Wilhelm His, became professor of anatomy and embryology in Leipzig and Miescher was appointed to the newly-created chair of professor of physiology in Basel at the exceptionally young age of 28, after receiving the highest recommendations from both Hoppe-Seyler and Ludwig. Nonetheless, there was talk in the university of the “triumph of family politics”. Miescher started with very poor facilities, his laboratory consisting of a converted corridor and helped only by a quarter-time technician. His old friend, A. Jaquet, after Miescher’s death, commented that, when Miescher took over the professorship in Basel, aware of his great new responsibilities, he tried to make up for his youth and inexperience by redoubling his research and teaching efforts. He overtaxed his strength and the consequences of overwork began to affect his health.
Miescher succeeded in isolating nuclein from salmon spermatozoa, similar in properties to the nuclein of leukocytes from pus, except that it was combined in salt-like form with a basic protein he called “protamin”, still known by that name to-day. He later isolated nuclein from the sperm of carp, frog and bull, apparently a constant component of nuclei of spermatozoa (1871-73).
The concept of the existence of a separate substance, characteristic of the cell nucleus and different from protein was vigorously contested by scientists in Germany, France and England. Miescher, perhaps discouraged by this vociferous opposition to his discoveries, ceased to investigate sperm nuclein, at least as far as the public record goes, though some notes show that even in 1876 he still carried out chemical studies on this topic.
Instead, he turned his attention to the physiology of the maturation of the salmon’s reproductive organs. The spawning salmon, while traveling up the Rhine, does not feed, yet the male continues to produce sperm and the ovaries of the female grow to a quarter of its body weight. Miescher showed that the trunk muscles of the fish degenerated, while the products of muscle breakdown were transported by the blood to build up the germ cells. He termed the process “liquidation”. The results of this study were published in 1880 and consist of an enormous number of careful measurements and statistical data on the migration and spawning of salmon in the Rhine, on changes in their body weight at different periods, their trunk muscles, ovaries and spleen .
Upon a request from the government, Miescher in the 1880’s prepared an extensive report on the nutritional requirement of prisoners, a task he described as “thankless and tedious”. Similarly he felt it a distraction from his passionate devotion to research to be asked to write a comprehensive paper on the nutrition of the Swiss population in light of recent scientific findings and complained bitterly of “becoming the guardian of the stomachs of 3 million of his countrymen”.
A new institute of physiology was opened in Basel in 1885, constructed according to Miescher’s plans and under his supervision, called “Vesalianum”, named by him to honor the Renaissance anatomist Andreas Vesalius, who published the first scientifically accurate work on anatomy and physiology in Basel in 1543. Miescher organized the first ever congress of physiology in Basel in 1889, attended by 50 scientists. In 1878, he married Marie Ann Rusch; they had three children.
At the Vesalianum, Miescher continued his work on respiration that he had begun in 1871 for his Habilitation thesis. This led on to his studies on the effect of high altitude on the composition of blood, which, however, were never completed.
In 1885, Miescher contracted pleurisy, perhaps because of the long hours spent in his ice-cold laboratory, when he neglected his ill health to the extent of not taking any nourishment for long periods. In 1894, he was diagnosed as suffering from tuberculosis. He died of the disease in a sanatorium in Davos in 1885, at age 51. As quoted by Portugal and Cohen, his former teacher, C. Ludwig, wrote to him shortly before his death: ”I know what it is to give up well-loved, hopeful work. Sad as it is, there remains for you the satisfaction of having completed immortal studies in which the main point has been the knowledge of the nucleus; and so, as men work on the cell in the course of the following centuries, your name will be gratefully remembered as the pioneer of this field.” Prophetic words, indeed !
Miescher left behind a mass of unpublished work as laboratory notes and manuscripts. These were compiled by is old friend from Leipzig days, O. Schmiedeberg, then professor of pharmacology at Strasbourg, and published in 1896 .
Miescher’s personality was described by one of his students , F. Suter: he appeared to be insecure, restless and introverted, no doubt because he was hard-of-hearing and myopic. The impression he gave was of a person completely taken up by his inner mental activity, without contact with the outer world. However, when called upon, he was always ready to help others. As a lecturer, he was difficult to understand, fidgety, in constant motion, with little contact with his audience. He assumed much greater knowledge and interest amongst his listeners than they really had, even though he spent days and often weeks preparing for his lectures. Yet he was extremely hospitable and invited all students who attended his courses to his house once a semester.
Two traits of his personality were most striking: his passion for his scientific goals that he pursued without rest, often working in the laboratory from dawn to midnight, neglecting his family and his health - not even noticing the beginning of the sickness of which he died prematurely. And his extreme modesty and self-criticism. He hesitated before publishing any work he would consider to be incomplete. This was the reason for his very sparse publication record and he even reproached himself for having published some results too soon. He wrote: ”Physiological chemistry is made up of such a great number of interdependent facts that it makes little sense to add still more to the confusion. That being so, I have deprived myself of the great joy experienced by publishing a few scattered discoveries. But I can do nothing else”.
Although the expression “cell” was used as early as the 17th century by R. Hooke and M. Malpighi, it was not until the 19th century, with the development of microscopy, that the cell was observed in plant and animal tissues. As a microscopist, R. Brown discovered the cell nucleus in plants in 1831 without realizing its nature, and J.E. Purkinje described (and named) the cell’s protoplasm as a ground substance in the embryo in 1839.
It was T. Schwann who first came to regard the cell as the basic element in the structure of animal and plant tissues. In his “Microscopic Investigations” (1839), he gives a clear description of cells and of their nuclei in lymphocytes and epithelia. His theory of cell formation involved aggregation of extracellular substances to form cells and it was not until 1852 that R. Remak demonstrated that cells originated by cell division. In 1858 R. Virchow wrote: “Wherever a cell originates, in that place there must have been a cell before, just as an animal can only originate from an animal and a plant from a plant”.
At the time of Miescher’s first investigations, the protoplasm of the cell was regarded as paramount. However, already in the 1850’s was the nucleus considered to be of importance and it was realized that cell division was accompanied by nuclear division. E. Haeckel, in his great work on physiology and anatomy, published in 1866, clearly distinguished between nucleus and cytoplasm. He ascribed to the nucleus the property of inheritance of the cell’s inheritable characteristics and a role in the cell’s reproduction, whereas the cytoplasm served the adaptation of the cell to its environment and the nourishment of the cell. He thought both cytoplasm and nucleus consisted of protein, albeit of different chemical properties, proteins being made up of combinations of carbon, hydrogen, oxygen, nitrogen, sulfur and often phosphorus. He stated that the proteins were extremely labile and that the slightest stimulus could transform or destroy their complicated atomic structure. T.H. Huxley remarked in 1868 “all organisms, both animal and vegetable, are fundamentally one... their action, form and elementary composition are basically proteinaceous”.
At that period, physiologists like Wilhelm His adhered to the view that chemistry would explain the function and development of cells, in opposition to the prevailing reliance on microscopy and the staining techniques with the many newly-discovered organic dyes. His and Miescher distrusted the staining procedures , because the interaction of tissue components with dyes lacked exact chemical definition. Miescher’s aim was to go beyond staining methods and to attempt the chemical isolation and analysis of cell components (“I must again defend myself against the guild of the dyers...”). As Olby points out, Miescher, though he isolated pure nuclei from pus cells, he so distrusted staining procedures that he never used the popular carmine stain specific for nuclei.
The application of chemistry to physiology goes back to H. Boerhaave, whose textbook on physiology (1708) taught that all physiological functions obey chemical and physical laws. Justus von Liebig was doubtless the father of modern biochemistry. He developed the basic method for carbon and hydrogen analysis of organic compounds later used by Miescher and, in the 1830’s, among a host of discoveries in organic and biochemistry, he studied the metabolism of protein to urea and uric acid, and isolated lactic acid from muscle. He established the significance of protein, carbohydrate and fat in animal and human nutrition in the 1840’s. His view that catalysis consisted of molecular movement that sets up sympathetic vibrations in other molecules found an echo much later in Miescher’s ideas on the process of fertilization.
The towering figure in biochemistry, with whom Miescher worked 1868-9, was Felix Hoppe-Seyler. His overarching view was that all life processes must be chemical. As a young physician, Hoppe-Seyler entered Virchow’s research institute in Berlin in 1854. In 1861, he was appointed to the chair of applied chemistry in Tübingen, where he instituted the first independent laboratory of physiological chemistry. His achievements in biochemistry were epoch-making. Not only did he write the most influential textbook for many years on physiologic and pathologic chemical analysis for physicians and research students, he also founded the famous Zeitschrift fur physiologische Chemie.. Following on from Liebig’s analytical methods, he established reliable analytical procedures for biochemistry, including spectral analysis, gas analysis and colorimetry. He isolated and distinguished between different proteins - serum and egg albumin, globulin, fibrin, myosin. He purified and established the composition of lecithin, isolated cholesterol and recognized the binding of oxygen to erythrocytes as a function of hemoglobin. One of Hoppe-Seyler’s students, A. Kossel, followed up Miescher’s work and, starting in 1879, identified the hydrolytic split products of yeast nuclein (nucleic acid) as uracil, thymine and cytosine, as well as purine and pyrimidine bases from animal tissues .
When Miescher’s uncle persuaded the young man to forsake the profession of a practicing physician and to take up a research career instead, Miescher chose first to train in chemical research methods with A. Strecker in Tübingen. The concept of “learning” research was relatively new. Strecker had been a pupil of Liebig’s, one of the pioneers in the establishment of research schools. Liebig’s began in the 1840’s. Prior to that time, scientific research was carried out by rich private individuals as an avocation rather than a profession, as for instance by A.-L. Lavoisier, or by amateurs gathered together under the auspices of an academy or society, as for instance the Royal Society in London. Viewed from to-day’s point of view, it is amusing to read in a novel by I. S. Turgenev (“Fathers and Children”, 1862) of the hero who brings his chemical apparatus with him in his overnight bag on a visit to the country house of a friend and there pursues his “chemical research”.
The earliest of research schools was developed by the Czech anatomist and physiologist J.E. Purkinje in Breslau (then in Germany), where he was appointed professor of physiology in 1823. His reforms of the medical school curriculum were based on the teachings of the Swiss educational reformer J.H. Pestalozzi (1746-1827), ultimately reflecting the ideas of the Enlightenment and the writings of J.J. Rousseau. Pestalozzi was primarily concerned with the education of children, “not with what a child learns, but how it is learned” and that “genuine education is self-instruction, activity that permits...the child to build his or her own view of the world - knowledge is a self-generated product”. Purkinje at first introduced “experimental courses” in which the professor and assistants carried out experiments watched by students, later allowed students to take part in these demonstrations. Later still, starting in 1828, by application of the Pestalozzian principles of learning-by-doing, he was able gradually to establish the modern research institute, with students conducting chemical and microscopic investigations under his direction. As to-day, the professor was in charge of the research agenda, the doctoral candidate or medical student received advanced training, including in the use of elaborate apparatus and instruments, and was rewarded by publication of his results (no women participated), and ultimately was helped by the professor to obtain a research position. An important achievement of Purkinje was to maintain the research institute within the purviews of the university, where previously only lectures had served for the education of the students.
At about the same time (1824), the young Justus von Liebig began the establishment of a research school in Giessen (Germany) that became the model for such institutions and trained some outstanding scientists. He was mainly responsible for the professionalization of scientific research, through the reshaping of university curricula, experimental training in a new institute, specialist qualifications (the Ph.D.), with emphasis on original work, a written thesis and specialized publications. The foundation on which Liebig’s laboratory was built was a reliable, repeatable method for the analysis of organic compounds, a combustion apparatus for quantitative organic analysis. Its development was completed by 1830, and it was used by Hoppe-Seyler and Miescher amongst many others. As pointed out by Morrell:”The use of a set of relatively simple, fast and reliable experimental methods allowed those students who were less than brilliant to do and to publish competent work. When these techniques were deployed on a large scale, a knowledge factory was the likely result”. Publication was in Liebig’s own journal, Annalen der Chemie . Soon, Liebig established a great reputation, which ensured a supply of increasingly gifted students as manpower (again, no women) and institutional support. The similarity of the research schools of the 1840’s to our present system, at least in the American universities, is unmistakable.
Of course, there were (and are) disadvantages. Membership of a school could inhibit imagination. It was the opinion of A. Kekule, who had been offered (and declined) an assistantship by Liebig, that it is essential “for doing something of your own...to get rid of preconceptions due to early training”.
The research schools attended by Miescher were those of Strecker and Hoppe-Seyler in Tübingen and of C. Ludwig in Leipzig. Even though Miescher was professof of physiology with his own laboratory and later his own institute, the Vesalianum, he never founded a research school. He had few students and clearly lacked charisma. He wrote to his friend, A. Jaquet, at the end of his life (1894): ”If I could instill into my students the idea that everything in physiology is not only interesting but also easily acquired, I would have great success as a teacher. However I regard such teaching as a deception”.
At the time Miescher began his research in Hoppe-Seyler’s laboratory in the fall of 1868, proteins, lipids (including lecithin and cholesterol) and carbohydrates were known as chemical components of tissues. Also, mainly through histochemistry, different cell types in tissues and components of cells (protoplasm, nucleus, nucleolus, plasma membrane) were known.
As a new post-doctoral assistant, Miescher had his own ideas, greatly influenced by his uncle, of the importance of investigating the chemical properties of cell components. In practical terms, this meant finding a material for study and then developing chemical methods for such investigations. He had, of course, to fit into the current Hoppe-Seyler research program. A letter from Miescher to His clearly shows that he brought his own ideas into complete harmony (Einklang) with those of his research director’s, to study the chemical composition of lymph cells : “I was captivated by the thought to trace the general conditions of the life of cells by means of the simplest and most independent forms of animal cells”. Lymph cells were difficult to obtain in quantity from blood or lymph glands. Hoppe-Seyler had already attempted to isolate cells from pus and suggested to Miescher to use this material. W. Kuhne in 1868 had proposed that white blood cells, lymphocytes, were the precursors of the leukocytes of pus. Besides, Hoppe-Seyler was interested in understanding the formation of pus.
Few methods were then available for identification, isolation in chemically pure form and characterization of substances from animal tissues. In many cases, the isolation procedure itself served as a method of identification, depending on solubilities in different solvents or, for proteins, in aqueous solutions of salts at different pH’s. Only a small number proteins was known, so a newly-discovered protein was assumed to be identical to one already known, if their solubility properties were the same. Color reactions were: for protein, an early type of Biuret reaction (violet color with copper sulfate and alkali); xanthoprotein reaction (yellow color with nitric acid); Millon’s reaction (red color with mercuric nitrate and nitric acid); for purines (uric acid, xanthine, guanine), the murexide reaction (purple color with nitric acid followed by ammonia).
The basic method for identification and characterization was the elementary analysis. Miescher used elementary analysis data not only to compare different isolates for identity or difference, but also to demonstrate purity by purification to constant analysis. In his publications, Miescher does not state which was his method for carbon and hydrogen analysis, most likely it was that of Liebig , by heating the substance with copper oxide and absorbing the water formed in calcium chloride and the carbon dioxide in alkali, both of which were weighed. He mentions the method of Varrentrapp and Will for determination of nitrogen, in which the substance was heated with sodium and calcium hydroxide and the evolved ammonia trapped in hydrochloric acid. The ammonium chloride formed was precipitated by excess platinum chloride as the chloroplatinate and weighed.
The all-important phosphorus analysis, which led to the discovery of nuclein, was described by Hoppe-Seyler, though not by Miescher in his publications, and consisted in combusting the phosphorus-containing substance with sodium nitrate and carbonate, dissolving the residue in nitric acid and precipitating with ammonium molybdate. The precipitate of ammonium phosphomolybdate was dissolved in ammonia, re-precipitated with magnesium sulfate and weighed as magnesium pyrophosphate.
All the methods used were gravimetric and therefore laborious and time-consuming. In 1876, Miescher complains in a letter to his friend Boehm about the “factory work” that is needed to carry out the “mountain of quantitative analyses” .
NUCLEIN FROM LEUKOCYTES
Miescher started with the aim to isolate and characterize leukocyte proteins. His first task was to isolate the cells from pus in a pure state. No techniques for the isolation of such cells were known. In that era, prior to the introduction of sterile surgical procedures, suppurating wounds following operations were frequent in hospitals. Miescher had bandages from the local surgical clinic sent to him daily. Those with a bad smell were discarded. To wash out the cells from the bandages, he tested various salt solutions and was successful with a 9 : 1 water : sodium sulfate solution. Centrifuges were not then available as laboratory equipment, so that he had to wait several days for the cells to settle out. He washed the cells 2 - 3 times and finally collected them by filtration, checking progressively by microscope.
The next problem he tackled was the nature of the cells’ proteins. The hypothesis of Hoppe-Seyler then current was that it probably consisted mostly of myosin. Miescher tested authentic muscle myosin by extraction of muscle with dilute alkali and precipitation upon neutralization with acid. The resulting authentic myosin was soluble in sodium chloride solution. He ultimately distinguished five “proteins” extracted from whole leukocytes: a type of albumin soluble in dilute acid; a protein coagulating at 48-49 o C, insoluble in dilute acid; a protein similar to serum albumin; a protein insoluble in sodium chloride, soluble in dilute acid; and finally a substance that appeared to be unlike a known protein, insoluble in dilute acid or sodium chloride, soluble in alkali and re-precipitable with acid. Therefore, myosin was not present in the leukocytes.
By alcohol extraction of the leukocytes, Miescher obtained a lipid he identified as lecithin by means of phosphorus analysis. In the ether-insoluble fraction of the alcohol extract, he found, upon hydrolysis, a reducing sugar (by the copper sulfate-sodium hydroxide reaction) that he thought represented “cerebrin”, similar to cerebrosides obtainable from nerve tissue.
The next task undertaken by Miescher was to determine the composition of the cell nucleus of the leukocytes. For this purpose he isolated cell nuclei, a feat never achieved before. He began by prolonged treatment of the cells with dilute acid, expecting the proteins of the protoplasm to dissolve, leaving the nuclei. This was not successful - residues of protoplasm remained adhering to the nuclei, However, after weeks of treating the cells with 1/1000 diluted hydrochloric acid in a cold room (in winter), he shook the undissolved mass with ether and water. Part of the remaining material was retained at the boundary between the two liquids. A fine powder accumulated at the bottom of the vessel, microscopically shown to be pure nuclei, with smooth contours and a distinct nucleolus. Miescher correctly deduced that the nuclei sank to the bottom because their specific gravity was greater than that of the remaining proteins. Extraction of the nuclei with sodium carbonate produced a solution from which a substance could be precipitated with acid. This substance, insoluble in water and sodium chloride, soluble in sodium hydroxide and disodium phosphate, non-coagulable by boiling, differed in properties from known proteins and appeared to be a characteristic component of the nucleus. Miescher concluded that it was the same acid-insoluble, alkali-soluble substance that he had extracted from whole leukocytes. At that point, Miescher was still convinced that he dealt with a protein, because it gave the xanthoprotein color reaction. He later realized that this reaction was caused by proteinaceous impurities contaminating the nuclear substance.
To produce greater quantities of this material, Miescher made use of the recently developed method for enzymatically dissolving protein by W. Kuhne. Miescher first removed lipids from the pus cells with warm alcohol, then digested the residue by treatment with an acid extract of pig’s stomach, which contained the proteolytic enzyme pepsin. He obtained a fine grey powder of pure nuclei as a sediment, collected it by filtration and washed it with alcohol. Again, the nuclei could be dissolved completely in caustic alkali and a precipitate formed on neutralization. This precipitate, again, dissolved in dilute sodium carbonate and was insoluble in acid, even boiling acetic acid. Miescher realized that he had isolated the same substance in three stages: first, from whole leukocytes; next, from nuclei contaminated with protein; finally, from pure nuclei. He then knew that he had found a new substance and named it “nuclein”, which we now know as DNA.
Elementary analysis of nuclein revealed about 14% nitrogen and about 3% phosphorus, with closely similar results in 9 separate analyses. This was a much higher content of phosphorus than found in protein. He rejected the hypothesis that this may be a complex of protein with lecithin (a lipid substance with a high phosphorus content, recently discovered by Hoppe-Seyler), because it was resistant to proteolysis and insoluble in alcohol. He showed that the phosphorus was organically bound by combusting the substance at high temperature. The organically-bound phosphorus escaped with the vapor and the residue contained no inorganic phosphate.
In a letter to his uncle in 1869 , Miescher wrote that preliminary observations led him to suspect that nuclein occurred also in liver, kidney, testicle and nucleated erythrocytes and suggested that it may be an acid. He remarked: ”With experiments using other tissues, it seems probable to me that a whole family of such slightly varying phosphorus-containing substances will appear, as a group of nucleins, equivalen to the proteins”. Thus, within the span of less than one year, working in Hoppe-Seyler’s laboratory, Miescher had discovered DNA, the nucleic acid from nuclei.
An unusual event then followed. On December 23rd 1869, when Miescher was already at work in Ludwig’s laboratory in Leipzig, he wrote to his parents: “On my table lies a sealed and addressed package. It is my manuscript...I am now sending it to Hoppe-Seyler in Tübingen. So the first step towards publication has been taken, provided Hoppe does not refuse.” He did refuse - at first. Miescher wanted to publish in Hoppe-Seyler’s own journal, of which the latter was editor. Hoppe-Seyler delayed and delayed - why ? He did not reject the paper of Miescher, but decided to delay publication, probably because of the sensational nature of the discovery of a new class of cell substances. He wanted to repeat Miescher’s experiments himself, to dispel the doubts that he admitted feeling about the work. He wrote: “Even though I am acquainted with the careful research methods of Dr. Miescher, I could not suppress some doubts about the correctness of his data. These are of such great physiological importance, that I have repeated that part of the work regarding the substance from nuclei that he has named nuclein”. As pointed out by Olby, Hoppe-Seyler’s doubts were perfectly reasonable and were based on the fear that the pepsin digestion of the cells may have yielded peptides, which could have combined with lecithin to yield the N/P ratio of nuclein.
Hoppe-Seyler described an exact repetition of Miescher’s experiments, finding identical solubility properties and an almost identical elementary analysis with respect to nitrogen, with slightly lower phosphorus. He also reported an almost identical analysis and chemical properties for nuclein extracted from yeast cells and from brain.
Hoppe-Seyler published Miescher’s article after a 2-year delay, in conjunction with his own article in 1871, following a series of anxious, though very respectful letters to him from Miescher. Miescher’s article carries a footnote stating that “through unforseeable circumstances” its publication had been delayed. Miescher requested that supplementary remarks be added to his article, but Hoppe-Seyler declined. These remarks explain the reason for the delay. Miescher states that he was pleased to see his results confirmed and adds that Hoppe-Seyler’s discovery of a nuclein-like substance from yeast demonstrated the existence of a new factor pertaining to the life of the lowest as well as the highest organisms, representing a basic chemical difference between nucleus and protoplasm. He suggested that the chemical degradation products that could be obtained from nuclein may be of great interest to establish its structure.
Immediately following the Miescher paper in Hoppe-Seyler’s journal is one by P. Plosz from Pesth (Hungary), which, without mention of Miescher’s methods, uses his techniques to show that nucleated erythrocytes of birds and snakes (but not non-nucleated erythrocytes from cows) contained a substance with the properties of nuclein. Plosz later described the isolation of nuclein from rabbit liver.
NUCLEIN FROM SPERMATOZOA
During a brief vacation (September-October 1869), just after completing his work in Hoppe-Seyler’s laboratory, Miescher undertook some preliminary studies with his uncle, W. His, in Basel. His had been concerned with the development of eggs into chick embryos and had discovered microscopic light-refracting white spheres in the yolk of hens’ eggs, which he assumed to be preformed cells that upon fertilization would become embryos. Since they were thought to be nucleated, Miescher began work to see whether they contained nuclein. Upon his return to Basel from Leipzig in 1870, Miescher continued these studies and isolated grains from these particles that he thought were nuclei and from which he then extracted a pepsin-resistant, acid-insoluble, alkali-soluble substance. These chemical properties were similar to those of leukocyte nuclein. Elementary analysis also showed about 14% nitrogen, but a much higher phosphorus content than pus cell nuclein. The material gave color reactions positive for protein, causing Miescher to conclude that it was a different kind of nuclein, characteristic for egg yolk, perhaps combined with a protein. Much later (1876), Miescher became doubtful of his earlier conclusion and in a letter to Hoppe-Seyler, wrote that the presence of protein even after pepsin digestion confused the whole problem. This substance has since been identified as a highly-phosphorylated, protease-resistant protein named phosvitin.
His was also interested in fish embryo development from fish eggs and persuaded Miescher to look at salmon eggs. However, these were as difficult to investigate as hens’ eggs and the step to sperm was not a big one. Just as before he had studied leukocytes from pus as separate, independent cells, possible to isolate in pure form, so he now switched his interest from fish eggs to fish spermatozoa, the heads of which were known to consist almost entirely of nuclei, as discovered by the Swiss histologist A. von Kolliker in 1841. Miescher wrote: I was, therefore, glad to find in sperm an even simpler object of study. Spermatozoa are said by histologists to consist of nuclei, or as cells with mostly nuclear mass. I have now succeeded in obtaining large amounts of pure spermatozoa from salmon”. Such nuclei might give him an opportunity to determine whether nuclein might be found in sperm cell nuclei, perhaps similar to that in pus cell nuclei, thus allowing him to expand the new class of substances to nuclei of another cell type.
Salmon sperm was a fortunate choice for two reasons: fish sperm consists almost exclusively of spermatozoa suspended in saline, in contrast to that of other species, where they are mixed with other secretions. Secondly, salmon migrated up the Rhine in the fall in vast numbers in order to spawn. Salmon fishing was an important industry in Basel and salmon sperm was plentiful and cheap.
To isolate spermatozoa, Miescher squeezed salmon testes through cheese cloth and washed with water. The suspension was made acidic with acetic acid, when the spermatozoa heads settled out as a fine powder, the tails dissolving in the acid. They were broken and lipid extracted by treatment with alcohol and ether and traces of residual protein removed by washing with water.
When Miescher treated the residue with dilute hydrochloric acid, he found he could extract a base which, with platinic chloride, precipitated as the chloroplatinate and could be crystallized as such. He wrote to his former mentor, Hoppe-Seyler: “The platinum double salt, free of phosphate, crystallized in beautiful orthorhombic prisms... the salt points to a peculiar substance, with high nitrogen content, representing a molecule in between urea and protein”. This was the base he called “protamin”. He performed elementary analyses on the platinum double salt and derived the empirical formula for the free base as C16H30N9O3, 2HCl.PtCl4 + 1/2 H2O, later confirmed by his associate, J. Piccard.
Since then, protamine has been confirmed as an important component of spermatozoa. Salmon spermatozoal protamine is indeed a “peculiar substance”, a protein with 21 arginine residues out of a total of 33 amino acids, with an empirical formula, close to that originally determined by Miescher and Piccard. During meiosis, when diploid testicular stem cells differentiate into haploid spermatozoa, histones attached to the chromosomes are replaced by protamine. The arginine residues of the protamine combine with the phosphate backbone of the DNA by salt bridges, thus preventing transcription of the mature spermatocyte DNA. It is significant in that regard that Miescher could not detect protamine in immature salmon sperm.
Here Miescher fell into an error: he detected purine bases in the protamine he had isolated by the murexide reaction, no doubt caused by contamination with adhering DNA. Later, Miescher requested that Piccard re-investigate this question. Piccard also detected purine bases in the acid extract of spermatozoa from which protamine was isolated. However, he concluded (correctly) that nuclein also contained purine bases. This confusion was not resolved until R. Altmann in 1889 separated protein (free of purine bases) from nuclein (called by him nucleic acid), containing xanthine bases.
To obtain nuclein from spermatozoa, Miescher first removed lipids with hot alcohol, then extracted protamine with dilute acid. The residue was washed with dilute acid, then treated with alkali. The dissolved nuclein was then precipitated by a 1 : 1 mixture of dilute hydrochloric acid and alcohol. The substance so prepared was free of protein, as shown by a negative Millon reaction and absence of sulfur. Miescher emphasized the necessity of working rapidly and in the cold. Frequently, he had to catch the salmon himself, with the help of his technician, from the Rhine at a spot just below the university. There were then no cold rooms, so he could only do the experiments in the winter, starting at 5 a.m.in an unheated laboratory with all windows open. No solution could be left for more than 5 minutes, no precipitate left for more than 1 hour unless covered with alcohol, all precautions no doubt to prevent degradation of nuclein (DNA) by deoxyribonucleases.
As for the chemical properties of the nuclein, Miescher realized that it had a high molecular weight, because it was non-diffusable across a parchment-like paper membrane (Pergamentpapier); and that it was a multibasic acid, as it could combine with several molecules of the basic protamine or several ions of barium or copper.
Elementary analysis of salmon sperm nuclein gave an empirical formuls: C29H49N9O22P3, with 13% nitrogen and 9% phosphorus, the result of 6 separate analyses. For comparison, a trideoxynucleotide with the bases guanine, cytidine and thymine, for instance, would have the empirical formula C29H37N10O20P3, with 14% nitrogen and 9% phosphorus.
Miescher correctly assumed that nuclein occurred in spermatozoa as a salt with protamine. He observed the physico-chemical property of a base exchange, whereby the protamine of nuclein-protamine salt in solution could be exchanged for other cations such as sodium or calcium.
Curiously, Miescher thought that nuclein formed the outer structural envelope of spermatozoa. He came to this erroneous conclusion because, when he removed the nuclein from spermatozoa with alkali, the residue that remained could be observed microscopically to be in the shape of the spermatozoon lacking the envelope and to be proteinaceous in nature.
Miescher investigated carp and frog sperm and, without actually isolating nuclein, remarked that it contained a substance with nuclein-like properties. He studied spermatozoa from bull semen and, in order to compare their nuclein with that of pus cells, he digested the bull spermatozoa with pepsin from pig’s stomach as he had done with the pus cells, and isolated the nuclein by extraction of the undigested residue with alkali, then precipitated the nuclein with acid. This nuclein had properties closely similar to salmon sperm nuclein, showing 7% phosphorus upon elementary analysis. Moreover it also closely resembled pus cell nuclein in solubilities, except that the latter had less phosphorus (3%) and showed some sulfur (2%).
Morphological studies completed Miescher’s work on salmon sperm, published in 1874. He continued to investigate salmon sperm for several years without publication. His friend, O. Schmiedeberg, compiled and published the results of these studies after Miescher’s death in 1896. There is described, in the minutest detail, the isolation, purification and analysis of nuclein from salmon sperm, though in essence identical to the original method published in 1874. Miescher there mentioned attempts at chemical degradation of nuclein and his suspicion that a product may be thymine, already observed by A. Kossel and A. Neumann. He collaborated with R. Altmann, who had developed methods for separating protein from nuclein, called nucleic acid by Altmann, on the analysis of yeast nuclein. As late as 1892, 3 years before his death, Miescher began work on “karyogen”, a supposed iron-containing protein in the sperm cell nucleus.
GENERAL IDEAS OF MIESCHER CONCERNING NUCLEIN
Miescher made the broad generalization, based on his experiments, that nuclei contained nuclein and in that way differed from protoplasm. Thus, nuclei of leukocytes from pus and of spermatozoa, as well as the “nuclei” of egg yolk granules (these later turned out to be a phosphorylated protein) all contained nuclein. Miescher remarked that, though nuclei had been discovered by histologists in a variety of tissues, even in nerve cells, histologic observations could not find a single common property amongst nuclei from different tissues. He considered that it was the chemical composition of nuclei, in particular the presence in them of nuclein, that constituted their common property. He suggested that there may be sulfur-containing nuclein (in leukocytes and egg yolk granules) and sulfur-free nuclein (in spermatozoa). No doubt, the leukocyte nuclein was contaminated with protein. As Fruton points out, “about 20 years of work was required to sort out the confusion generated by these views”. Miescher thought that a further difference between nucleins from different cells was in their salt-like combination with protamine: whereas salmon sperm cell nuclein was linked to protamine, pus cell nuclein occurred without protamine. He regarded the phosphorus moiety of nuclein as the important component and remarked “I cannot refrain from believing that we are here in presence of the most fundamental physiological form of phosphorus”.
Although Miescher’s conclusion regarding the existence of nuclein was strongly contested by a number of scientists, the generality of nuclein as the characteristic nuclear substance was soon confirmed. As already mentioned, P. Plosz in 1871 identified nuclein in nuclei of bird and snake erythrocytes and in 1873 identified it in liver cells . R. Altmann isolated nuclein from yeast cell nuclei. E. Zacharias in 1881, using the methods of Miescher both for isolation and characterization, reported the presence of nuclein in nuclei of frog erythrocytes and in nuclei of protozoans; most remarkably also in the nuclei of the epidermis of leaves of plants (tradescantia and ranunculus), and even in the rod-like structures (later termed chromosomes) of dividing plant nuclei.
MIESCHER’S IDEAS CONCERNING FERTILIZATION AND HEREDITY
Miescher’s views of the function of nuclein changed over time. When he first discovered nuclein and observed its high phosphorus content, he thought it was merely a storage form for phosphorus. Later, however, his preoccupation with nuclein from sperm and, as he thought, from egg, led him to ponder the question of fertilization. Liebig proposed, as early as 1839, that chemical activity consisted in the movement of molecules ; and that chemical reactions were the transmission of this movement from one substance to another upon close contact. Liebig’s thought was extremely influential at that period and was applied to the process of fertilization by T.L.W. Bischoff and others, suggesting that the spermatozoon made close contact with the egg and thereby transmitted a stimulus initiating embryonic development. This idea was taken up by Miescher’s uncle, W. His, who was then professor of embryology at Leipzig. He insisted that fertilization was molecular stimulation by molecular motion and not a transfer of substance. Growth of the embryo began with the stimulus to the egg by the molecular motion inherent in the chemical composition of the spermatozoon. This view was initially opposed and was later superseded by the work of O. Hertwig. Investigating the fertilization of the sea-urchin, he showed that spermatozoa fused completely with the egg rather than just making contact. At the moment of fertilization two nuclei united to produce the first nucleus of the new generation.
In this controversy, Miescher, as ever strongly under the sway of his uncle, adhered to the opinion that the spermatozoon and in particular the nuclein that he had detected as being localized in its envelope, would be the ideal candidate to transmit motion and thus mediate fertilization upon contact with the egg nucleus. He wrote: “If one were to assume that the specific cause of fertilization depends on a single substance, for instance through an enzyme or in some other way, perhaps through a chemical stimulus, then one would have to think without a doubt principally of nuclein”. Miescher believed “to the death” in the chemical basis of inheritance. However, this he took to mean not the transmission of a substance from sperm to egg. He believed in the concept that “fertilization is a physical process of motion”. He argued that, as he could find no single substance in sperm or egg that could be the herediatary substance - “nuclein” from egg of hen and carp differed as did nuclein from salmon and bull sperm - he concluded that “there is no specific fertilization substance”. The continuity of form was subsumed not merely in the biological molecule, but in its constituent atomic groupings, with the chemical properties depending on the nature and intensity of atomic motions, transmitted from sperm to egg. He foresaw the necessity of postulating what we now know as the haploid nature of sperm and egg: “If in the germinal cell there is an absence of a member of the series of factors which determines normal cellular activity ... the spermatozoon reintroduces that part and restores cellular activity. Like muscle after a nerve stimulus, the egg, when it received the sperm stimulus, becomes chemically and physically different”. As late as 1895, the year of his death, Miescher wrote that the egg nucleus could have a missing component that is supplied by the sperm, perhaps by mere contact or through molecular movement. Ironically, as quoted by Fruton, Hertwig, who had observed the fusion of sperm with egg and was one of the founders of the transmission theory of fertilization, thought it was most probable that the fertilization substance and the transmitter of hereditary properties, was actually nuclein !
Miescher’s ideas about fertilization changed again shortly before his death, when he isolated what appeared to be an iron-containing, phosphorus-free protein from the core of the spermatozoon after removal of the nuclein-containing envelope. He called it karyogen. He wrote: “Fertilization consists in the three fundamental substances of the male cell, nuclein, karyogen and protoplasmic protein, reduced to the germinative state, being brought together with the corresponding female cell, also in the germinative state. They are then somehow united by a karyokinetic “dance”.
Miescher considered the interesting possibility that the “boundless multiplicity” of hereditary characteristics could be found in the vast number of steroisomers resulting from the asymmetric carbon atoms contained in the amino acids of protein. Thus he calculated that a protein molecule with 40 asymmetric carbons would have 240 or about a trillion isomers. W. Gehring pointed out that the idea of the storage and transmission of genetic information in a chemical form in a single type of molecule, through an almost infinite variability, was to become the basis of modern molecular biology.
The contact theory of fertilization died with Miescher. As Fruton points out, doubts arose in the 1890’s about the role of nuclein. Perhaps, if Miescher had not adhered so closely to his uncle’s views, he might not have missed the true nature of nuclein as the hereditary substance.
The author is grateful to Mrs. M. Samimi, Friedrich Miescher Institute, Basel, Switzerland, for photocopies of laboratory notes of Miescher and for photographs, also to Mrs. A. Gelzer-Miescher, Basel, for historical articles on Miescher. The author also thanks Dr. K. J. Carpenter for help with historical references.
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