Wearing Two Hats

G. Semenza and A.J. Turner (Eds.) Selected Topics in the History of Biochemistry: Personal Recollections. IX (Comprehensive Biochemistry Vol. 44) q 2005 Elsevier B.V. DOI: 10.1016/S0069-8032(05)44003-6

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Wearing Two Hats GUY DIRHEIMER Institut de Biologie Mole´culaire et Cellulaire du CNRS, Strasbourg, France E-mail: [email protected]

Abstract Born on July 14, 1931 I passed my childhood and the war in Strasbourg. In 1944 my parents, my brother and I were arrested by the Gestapo and sent to a SS camp in Germany. We were liberated by the American army in 1945. After studying pharmacy, physiology and biochemistry at Strasbourg University I became a researcher in 1955 at the Centre National de la Recherche Scientifique (CNRS) in J.-P. Ebel’s laboratory. I first worked on the structure and function of linear polyphosphates in yeast and bacteria. This was interrupted by my 28 months military service at a research laboratory of the health service in Lyon where I isolated and studied a soluble substrate of lysozyme. I got a PhD in pharmacy in 1961 and in sciences in 1964 and became assistant professor in 1964, then full professor (1969) at the Faculty of Pharmacy of which I was dean in 1969–1970. There I lectured biochemistry, molecular biology and toxicology. After a post-doc in R.W. Holley’s laboratory in Ithaca (USA) in 1965, I turned to the study of tRNAs, which became my main research topic for 35 years. I was concerned with their isolation, primary structure and their rare nucleotides. I discovered their specific

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cleavage by lead, and studied several aminoacyl-tRNA synthetases. In parallel, I also did research in toxicology and discovered the mode of action of ricin and of ochratoxin A on protein synthesis. My last research was concerned with the genotoxicity of mycotoxins and the formation of DNA adducts. This was possible, thanks to a very active laboratory and many gifted and hard working co-workers as well as many international collaborations. I worked at the CNRS Institute of Molecular and Cellular Biology of which I became director after the retirement of J.-P. Ebel. I was involved in many scientific organisations: President of the French Biochemical Society and of the French Toxicological Society (1979–1980), I was Secretary general of FEBS (1984–1989) and President of FEBS (1999–2003). I was also president of EUROTOX (1990–1992). I am member of the French National Academy of Medicine since 1988.

I was born, as chance would have it, in Basel on July 14, 1931. It is since that time that I have a taste for exoticism. In fact, as far as I can trace back in my genealogy, i.e., the 30 years war (1618–1648), all my ancestors were born in Alsace. However, my father, Charles Dirheimer, after his doctorate in pharmacy in Strasbourg, had taken an appointment in a pharmacy in Basel. Thus, I was born in Switzerland. Three years later my father bought the Pharmacie du Doˆme in Strasbourg and I passed my childhood in the shade of its wonderful cathedral.

The War Years, 1939–1945 Like many people of my generation, it was the war that most marked my youth. The whole population of Strasbourg was evacuated in July 1939, due to its proximity to the German border. With my mother and my brother, we took refuge in

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Quimper (Brittany), while my father was in the army. In Quimper I went to the elementary school associated with the Lyce´e Saint Yves. When we came back to Strasbourg in July 1940, the Nazis had already started their Germanisation of the population. First, everybody had to use German names. My Christian name being Guy, which has no German translation, had to be changed to Guido. Guido is not German either but was acceptable as the Italians were allied with the Germans. However, this alliance only lasted until 1943 when Italy signed an armistice with the Allies. Then I had to change my name again and take my second Christian name which is Pierre; thus I became Peter! Before the war I went to a private school, but the Nazis did not condone private schools. I had to go to the Volksschule (school of the people). It corresponded to elementary school and was called Scho¨pflinschule. It was forbidden to speak French. I did not know either German or the Alsatian dialect, as we only spoke French at home. Therefore, I had to learn German at school and Alsacian with my school friends, who came from the workingclass part of town and almost all spoke the dialect. The situation became even more complicated as we could not write in Latin letters but had to write in Su¨tterlinschrift, a horrible lettering that resembles a zigzag line. As a final complication the books were not printed in roman but in gothic characters! Fortunately, I was 9 years old and at that age learning is rapid and memory exceptional. In 6 months I managed this Babel task. We had very intimidating teachers who came from Germany and asked us if we spoke German at home. This happened to my brother Yves, renamed Karl, who was 6 years old. As I mentioned, speaking French was punishable by imprisonment in the concentration camp at Schirmeck in the Vosges. When I was 11, I went to the first class of the high school previously called Lyce´e Fustel de Coulanges, which was renamed Erwin von Steinbach Schule. There I started learning English as studying French was forbidden. It was a strange situation since the Germans were still fighting with the British,

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Fig. 1. Guy Dirheimer and his wife Marguerite (at the 28th Congress of the European Society of Toxicology, 1987).

but France had been defeated and was occupied by the German army. I had a sadistic English professor who was German. He thought my accent was French when I pronounced English words because the inflexion at the end of my words rose while the English inflexion goes down. In addition, like most Frenchmen, I did not pronounce the correctly. He came to me almost every day and pulled my hair at the temple until I pronounced the English words correctly. Finally, I spoke with a German accent when

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speaking English. Very often when I am in England or the USA, people think that I am German! The teachers who taught history, geography, German and music were Germans from the fatherland. The history teacher came from Northern Germany and finished almost all of his sentences with nich which means approximately OK. The Nazis had also invented new names for the musical notes, instead of do, re´, mi etc. they became bi, gu, la etc., therefore we had named our music teacher Bigula. However, we were very impressed when he sang the Erlko¨nig from Schubert while accompanying himself on the piano. When the professors entered the classroom we had to stand up, raise our right hand and shout Heil Hitler. We had to do the same when they left at the end of each lecture. The secondary school was very close to my home. I just had to go around the cathedral to reach it. Unfortunately, there was always a terrible wind blowing around the cathedral and sometimes the temperature dropped to minus 15–20 8C during the wintertime. The Nazis had forbidden catholic masses in the cathedral during the war, but the cathedral remained open. Therefore, I walked through it every day as a short cut! On January 2, 1942, it became obligatory for all the boys older than 10 to become members of the HJ (Hitlerjugend, Hitler Youth Organisation) in Alsace-Lorraine. As I was 10 and a half years old, I had to go to the HJ every Wednesday and Saturday afternoon, the periods we did not have school. The meeting was at my secondary school. We had mostly theoretical lectures about the Nazi party doctrine, the German army, the life of Hitler, the “Bolchevismus” (Bolshevism) being a danger for the world etc. It was boring. When we were not attentive enough we had to go to the school’s square and do Hinlegen-Aufstehen meaning one had to run, then when Hinlegen was shouted to lie down in the dust and gravel, then when they shouted Aufstehen, to stand up and run again. In general, the HJ-Fu¨hrer did not speak but shouted. Since that time I cannot bear to hear people shouting. This was a radical change for me from the cub scouts to which I belonged in

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1939/1940 in Quimper. I still had my beret from the cub scouts, but the Nazis had forbidden the Alsatians to wear berets! The HJ also had to parade almost every Sunday around Place Kle´ber, the central square of the town, which had been renamed Karl Ross Platz by the Nazis. Many Nazi flags adorned the town. I remember the Avenue de la Paix, which is a large avenue in Strasbourg, “decorated” with triumphal arches. I did not know how to march and I often stepped on the heel of the fellow in front of me. This caused him to step out of the column when this happened and he would be very angry with me. After the war I met him once in a Strasbourg jazz club, where he was part of the orchestra playing drums. We laughed a lot about this story. Fortunately, I did not belong to the HJ for very long as the Nazis, very proud of music, decided that everybody playing a musical instrument could be exempted from the HJ. Immediately, I had a vocation for the accordion and took lessons to learn how to play this instrument. But my vocation did no last very long and I have not played the accordion since that time. I read a lot during the war. This took my mind off our unbearable situation. I read mostly books by Karl May, which described adventures of Indians or Bedouins. There was a competition in my class for the person who could read the greatest number of Karl May’s books. I won it having read more than 40 books! I had also a private tutor for German and Latin. At the end of each lesson my tutor, Mr. Andre´, read me a chapter of the Odyssey. This was my first discovery of Greece. We had a lot of free time because we had only 20 h of class a week at school, divided between 4 days with 5 h each morning. During the afternoons we often went with our bicycles to the forests along the Rhine river, which at that time, were like a jungle with islands surrounded by water. We built huts there. These forests now have been mostly destroyed and transformed into industrial properties. Another favourite pastime was rollerskating. The cars in the city had been replaced by horses. Thus, we could skate quickly downhill from the cathedral. In the

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wintertime we skated on the lake at the “Orangerie” park which was frozen, as the winters were very cold during the war. The situation for the adults was much worse than that of children who adapt more easily to bad circumstances. They had to find food and clothing for their children while dealing with the Nazis and the bombings of the town by the allies. Fortunately, after 1942, we had each evening a good news programme from the BBC called Les Franc¸ais parlent aux Franc¸ais or French people speak to French people. Again, to be caught listening to this program was cause for imprisonment in Schirmeck. We lived at this time above my father’s pharmacy, which was destroyed on August 11 by American bombs dropped in error on Strasbourg city. Part of the cathedral and of the Palais Rohan were destroyed the same day. Fortunately, we were not present that day in Strasbourg. However, we were arrested by the Gestapo (Geheime Statspolizei) on August 18 and sent to an SS camp Lager in Schelklingen, near Ulm, in the German province of Wurtenberg because we were considered as Reichsfeindlich (enemies of the Empire) by the Nazis. We were in the category of people called abgesiedelt (banished). In Schelklingen we were first in a room of the camp with 38 other Alsatians, women, men and children sleeping on bunk beds. Most of them were parents of young Alsatian boys who had refused to become German soldiers. The other prisoners in the camp were mainly Polish citizens or Slovenians. The memory of the Appell every morning, the fear of the SS, the revolting food and the bugs which devoured me (I looked as if I had measles) are engraved forever on my memory. Fortunately, after several months, my father managed to get an appointment in a pharmacy in Ulm and we four were allowed to live near the camp in a small garret of 10 square meters, with no running water. We had to steal wood in the forest to warm our small room during the bitterly cold winter of 1944–1945, but it was like being in Paradise after the camp. We however were still dependant on the SS. I had to go to a college (Urspringschule) where the sons of Nazi dignitaries and officers were studying, far away from the cities which were bombed by the allies. They were

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well aware of the desperate situation of the German armies (this was in October 1944) and were generally not aggressive towards me, except for one day when the boys stole my school bag and put it high up in a tree. However, one of the schoolboys, Gerhard Zaeschmar, climbed the tree and brought my bag home to me. I still have contact with Gerhard who lives in Northern Germany and one of my sons made an exchange visit with one of his sons some years ago. The people who lent us their garret, family Kreutle, were also very kind to us. Thus, very young, I discovered that in every country there are good and bad people and that any generalisation concerning any group of people is wrong. I also learned to speak almost accent-less German, which was very useful later on in my scientific career. At that time I started to write my Kriegstagebuch (personal war diary), which I wrote with a secret alphabet that I had invented to ensure that the Nazis could not read it. I was naı¨ve at that time. I discovered recently that my good friend Tomi Ungerer, who is same age as I, did the same during the war [1]. It would be too long to describe in detail our stay in Schelklingen. What I remember, however, is the solidarity of all Alsatians inside or outside the camp. This time finally ended when we were liberated by the American army on April 22, 1945. I am still grateful to the American for having liberated Europe from the Nazi dictatorship. A good friend of mine, Marie-Louise Roth-Zimmerman, who was in the same camp as we were, has written a book about it [2].

Secondary School and University Coming back to Strasbourg we had to learn French again, having forgotten how to write it after 5 years of German occupation. The Alsatians had special “transitional classes” in order to be trained in French. At the Lyce´e Fustel de Coulanges I continued to learn English also and in 1948 made an exchange visit with an English boy living in Hemel-Hamstead. There, I attended an English secondary school for two weeks, experimenting with

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cricket. I also attended the Olympic games, which were in London that summer and went to see “Oklahoma” a very successful American musical. At that time I hesitated about my future occupation. I was attracted by natural sciences and particularly botany, my father having taught me to recognise the wonderful mountain flowers when we were hiking in the Vosges mountains near Strasbourg. Thus I did the Baccalaure´at (A-level) Sciences experimentales, in June 1949, but I was also attracted by my uncle’s job. Maurice Fougerolle was head of a large civil engineering company. So I prepared myself for the Baccalaure´at Mathe´matiques e´le´mentaires during the summer vacation, behind closed shutters, in order not to be tempted to go to the swimming pool, and passed it in September. However, I did not like the Mathe´matiques supe´rieures classes with 20 h of mathematics a week and although I had been admitted into Mathe´matiques spe´ciales, the preparatory class for the Grandes e´coles, I fell ill (it was certainly a psychosomatic illness!) and decided to abandon it. Mathematics still governs many careers in France. I turned to study pharmacy, the job of my father and grandfather. At that time pharmaceutical studies started with a practical training period in a pharmacy followed by 4 years of faculty. This, in my opinion, was a good thing because before starting University, you knew what your future job would entail. I made a nice herbarium and decorated the windows of my father’s pharmacy with scientific objects. I was very interested in the many different fields that were taught during the 4 years at the Faculty of pharmacy, everything from organic chemistry, bacteriology and immunology to botany, physiology and pharmacognosy. As we had some free time during the pharmacy studies the professor of analytical chemistry, Michel Hasselmann, had asked me to do some research in his laboratory. This I did from the third year on. During my pharmaceutical studies I was a member of the student council of the association Amicale des Etudiants en Pharmacie de Strasbourg, called H2S, and a delegate to the

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Student Council of the Association Fe´de´rative Ge´ne´rale des Etudiants de Strasbourg. The biggest political problem at that time was the French war in Indochina, then the war in Algeria. They were not popular with the university students. I was to meet the same problem 10 years later when I was at Cornell University, but the Americans had relieved the French in Vietnam. As I was one of the few pharmacy students who that spoke three languages fluently, this designated me as the delegate of the French pharmaceutical students to the 5th Congress of the “International Pharmaceutical Students’ Federation” in September 1957 in Ireland. This started my career as a committee member and gave me a taste for European affairs. In fact, my father was also a committed European and he supported for years the charity of the Nobel Peace prize winner (1958), Father Pire in favour of displaced persons. During the summer holidays I travelled with my brother Yves, first by bicycle through the provinces of Provence and Coˆte d’azur, from Orange to Menton, then through Switzerland to Italy where we took the train to Rome and Naples and then the boat to Palermo. We cycled the entire tour of Sicily, Giro di Sicilia, camping along the way. The third year I bought a Vespa scooter with the money I had earned doing temporary work in pharmacies, and travelled through Denmark and Norway to the North Cape. It was the first going to Hammerfest with a Vespa. In 1953 there was no pavement on the roads of Norway, which had suffered badly during the war, but people were very friendly to Frenchmen, remembering Narvik where French troops helped them against the Germans. I came back through Finland and Sweden, having lost 7 kg, but happy after this exciting trip. I practiced several sports. Twice a week I trained in Judo, followed by a bridge game with my friends in a Strasbourg wine pub or Winstub. I had got a taste for ski-mountaineering either during the weekends in the Vosges mountains or in the Chamonix valley during the Easter vacations. We slept in the Lognan refuge, above Argentie`res, and from there climbed to the different passes or mountain huts. The marvellous view of the

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Aiguilles Courtes, Aiguilles Droites and Aiguille Verte visible from the Refuge du Tour Noir is engraved forever in my memory. Unfortunately, in 1954 the refusal of a car driver to give me right of way caused me a complicated fracture of the radius which necessitated a bone transplant and prevented me from skiing and travelling for 2 years. I tried to make up for my disappointment by preparing a great project: the crossing of Africa from North to South by car. Starting Research at the CNRS in J.-P. Ebel’s Lab It was in my last year of pharmacy that chance decided my future career. Jean-Pierre Ebel, after a 2 year post-doc in Paris at the Colle`ge de France in the laboratory of E. Faure´-Fre´miet, came back to the Faculty of Pharmacy in Strasbourg, where he had done his pharmacy studies and his PhD. He had just gotten the Agre´gation the highest level competitive examination for recruiting Faculty of Pharmacy professors. He was lecturing in introductory chemistry and biochemistry. It was during his lectures that I discovered biochemistry. Prior to that, biochemistry had been taught by an older professor who was not a biochemist His course was disrespectfully called Pipime´trie by the students. After one of his lectures J.-P. Ebel explained that he wanted to start to built up a research laboratory in biochemistry and that he was looking for a young pharmacist who could start a PhD project with him. Of the two candidates he choose me, maybe because I had already worked in a laboratory or because I had been a good student (I had been awarded the Lobstein prize for the best average mark during pharmacy studies). We had another thing in common: Ebel had been arrested by the Gestapo in June 1943, imprisoned in Clermont-Ferrand, Moulins and Compie`gne and deported to the concentration camps of Buchenwald and Dora. He presented my candidacy to the CNRS (Centre National de la Recherche Scientifique) in September 1955 and it was accepted. This might seem very strange today where one needs at least a PhD with one or two postdocs to enter the CNRS.

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However, at that time we were not civil servants, as the CNRS researchers are today, and our fellowship had to be renewed every year after evaluation of a research report. We were Stagiaire de Recherche (researcher trainees) with a salary of 47,750 francs, equivalent to 830 euros, during preparation of a PhD and usually, after 2 years, we became Attache´ de Recherche. Thus, I started my research in professor J.-P. Ebel’s laboratory in October 1955. Here I would like to remember J.-P. Ebel. During 36 years of collaboration with him we never had a falling out. He was full of enthusiasm, full of “joie de vivre” and had plenty of energy to spare. He was entirely dedicated to biochemistry in general, but also to French and European biochemistry. He created in his laboratory a harmonious and peaceful atmosphere. He was a great democrat and all decisions were taken after democratic discussions. In addition, he was extremely open-minded and interested in both music and politics. J.-P. Ebel had a major impact on my scientific thinking. He was not only my mentor, but also like a second father. In fact, he took the career of his co-workers to heart and, although he was my boss, he was also a faithful friend. I even became the godfather of his third daughter Christine. In the lab we called him le pe`re Ebel between ourselves. I did not know all these qualities when I first started to work in his laboratory, but they rapidly became evident to me. At that time, when somebody was hired by the CNRS, a scientific godfather was also designated to follow up his research independently of his research director. Mine was Paul Mandel, Professor of Biochemistry at the Faculty of Medicine of Strasbourg since 1954. I visited him once a year to show him my results. He received me in a very friendly way and I was always impressed by his knowledge of biology and his vivid imagination. He created in 1965 the Centre de Neurochimie du CNRS. Biochemistry in Strasbourg owes him a great deal. When he died in 1992, I was chosen to deliver his obituary before the National Academy of Medicine in Paris and my speech paying homage to his numerous talents has been published [3].

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J.-P. Ebel asked me to work on inorganic linear polyphosphates (PolyP). These are energy-rich polymers of orthophosphate residues linked by high-energy phosphoanhydride bonds. In 1955 they had been characterised in algae, mushrooms, yeast and bacteria. In yeast they may represent 10–20% of the cellular dry weight. J.-P. Ebel, using the bidimensional paper chromatography system he had developed during his PhD thesis, had shown that they were comprised of polymers of different length from tripolyphosphate to long chain polyphosphates. They can work as an energy reservoir for ATP formation by phosphorylating ADP as had been described by O. Hoffmann-Ostenhof et al. in yeast and by S.R. Kornberg and A. Kornberg in E. coli. We wondered whether inorganic polyphosphates would be able to replace ATP in a direct phosphorylation reaction. We chose two reactions normally using ATP, hexokinase that catalyses glucose phosphorylation and phosphohexokinase that phosphorylates fructose 6-phosphate to yield fructose 1,6-diphosphate. When I started my experiments in 1955 these enzymes were not commercially available. The catalogue of Boehringer Mannheim showed only one product: NADP! Thus Methods in Enzymology Volume 1 published by S.P. Colowick and N.O. Kaplan in 1955 became my bedside book! In fact is was the only book in the field we had in the lab. In order to get brewers yeast I went to Kronenbourg brewery and got 40 L of yeast suspension. This had to be treated with toluene in order to lyse the cells. However, we had no container large enough for this experiment. Thus, I did it in the cellar of my father’s pharmacy where he had huge demijohns. The experiment went well. However, the whole pharmacy started to stink of beer and the customers sniffed suspiciously. They certainly thought that drinking binges were taking place in the back shop! Phosphofructokinase was isolated from rabbit muscle. I prepared different pure polyphosphates: pyrophosphate, tripolyphosphate etc. up to octapolyphosphate, by column chromatography according to N. Busch and J.-P. Ebel (1956). The hexokinase assay method is based on the fact that for each mole of phosphate transferred from ATP to glucose one acid equivalent is liberated.

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I determined the rate of acid formation by direct titration using a very sensitive Radiometer pH meter. This was placed under an oven in a small 3 m!3 m lab which was Ebel’s office, located in the old Faculty of Pharmacy built in 1904–1906. In front of Ebel was the huge refrigerated MSE centrifuge I used to spin down my yeast. These were the only specific apparatus we had, together with a fraction collector from which we could not take our eyes off because it only collected fractions correctly when fancy took it. However, my comings and goings did not disturb J.-P. Ebel in the numerous phone calls that already occupied him at that time. However, none of the condensed phosphates could replace ATP in the enzymatic reactions. Moreover, the linear polyphosphates had a strong inhibitory action on the reactions in the presence of ATP, whereas cyclic polyphosphates, called metaphosphates, had none. The longer the chain of the linear polyphosphates, the stronger was the inhibition. I showed that this inhibition was due to their complexing power towards MgCC ions which are the cofactor of these reactions and which increases with their chain length. This research lead to my first publication, published in November 1956 with J.-P. Ebel in the Bulletin de la Socie´te´ de Chimie Biologique [4]. Of course, it was written in French, but with a German and English summary. In fact, I wrote my first paper in English only in 1967 for Acta Biochemica Polonica! It was followed by my second paper in English in 1968 in Nature. The consequences of this French writing was that French biochemistry was often overlooked by Anglo-American scientists.

Crossing Africa But let’s come back to 1956. It had cost me 11 months salary to buy a small 2 horse-power Citroe¨n van to start my trip through Africa. On Friday July 13 (I am not superstitious), I took the Pierre Loti, a ship of the Messageries Maritimes Line from Marseilles that went to Madagascar. I was lucky to pass through the Suez Canal just a few weeks before Nasser nationalised it.

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Along with some passengers, I took a taxi in Port Said and visited Cairo during the time our liner crossed the canal. The numerous police controls (6 times in 150 Km), 10 m high portraits of Nasser and triumphal arches decorated with guns, canons and tanks were signs pointing to an imminent war. We were happy to get back on our ship in Suez. For reasons of economy I had taken a third class ticket. The only cool place in this class during the voyage down the Red Sea (42 8C in the shade) was the bar. Together with Belgians returning to the Belgian Congo we tested all the tax-free cocktails and played bridge. After a 2 week trip I arrived in Mombasa (Kenya) and drove to Nairobi where I met my brother Yves who had taken a plane to join me. Describing my 13,000 km trip through Eastern Africa to the Cape of Good Hope would be too long here. The highlights of it were the visits to several National Parks: Royal Nairobi National Park in Kenya, Elisabeth Park in Uganda, Albert National Park in the Belgian Congo and an excursion to a Pygmy village. We made a 5 day hike on Ruwenzori mountain, up to the limit of the glaciers (4200 m), and were filled with wonder at the Victoria falls between Northern and Southern Rhodesia. All these countries have become independent since that time and have changed their names. What has happened in the wonderful hills of RuandaUrundi? My brother Yves who had to take some exams at the Faculty of Medicine in Strasbourg where he was studying, left me in Johannesburg (South Africa) and I continued alone visiting the Kruger National Park, Swaziland, and the coasts of the Indian Ocean passing through Durban, a Zulu Native Reserve, Port Elisabeth and East London to the Cape of Good Hope. On September 14 I took the R.M.M.V. “Winchester Castle,” a Union-Castle Line Mail vessel, which brought me back to Southampton after 6000 nautical miles sailing with only one port of call for a few hours at Las Palmas (Canary Islands). I had made a 90 min long film of our trip and was asked to present it several times once back in Strasbourg. Let us recall that there was very little television at that time. As it was a silent film I had to

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comment on it each time and this was a good training for future oral presentations of my scientific results.

Inorganic Polyphosphates When I returned to the laboratory I started to work on another problem posed by PolyP in yeast. At the beginning of the year 1956, S.R. Kornberg had described the presence of a cyclic PolyP, called trimetaphosphate in yeast extracts. Thus, we wanted to know whether other metaphosphates like tetrametaphosphate, pentametaphosphate etc. existed in yeast. I cultivated yeast on a glucose-(32P)phosphate containing medium and isolated the PolyP by three different techniques: 90 8C hot water, cold water or cold 5% trichloroacetic acid. They were analysed either by bidimensional paper chromatography, elution and quantitation of the spots with a Geiger-Muller counter, or by ion exchange column chromatography. The results were quite different according to the extraction procedure. Trimetaphosphate was found after hot water extraction whereas none was found after cold water or trichloroacetic acid extraction. These results showed that trimetaphosphate did not pre-exist in yeast, but was formed during hot water extraction. Using synthetic long chain polyphosphates I could confirm that metaphosphates were formed by hydrolytic degradation of PolyP at neutral or alkaline pH at 90 8C [5]. When I started to work in J.-P. Ebel’s lab there were only three people, Norbert Busch, an assistant at the Faculty of Pharmacy, who was working on the separation of PolyP, a technician, Monique Blanck (who worked with me until she retired in 2000) and myself. Thereafter the number of researchers in the laboratory increased regularly. Simone Felter was recruited in 1956 and Jacques-Henry Weil in 1957, both as Stagiaires de Recherche at the CNRS. I was in charge of teaching them the know-how of the laboratory. I remember a poster (in English!) I had put in the lab “Better ask twice than do wrong once.”

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The material also increased; we bought a sewing machine because our separations were on ascending paper chromatography. The paper was rolled in a cylinder, sewn and put on a Petri dish in the solvent under a bell shaped microscopic cower. But we still had no spectrophotometer. Georges Cohen, working at that time in Gif-sur-Yvette, proposed to give us his old Beckmann. I went with my small car to Gif to pick it up. The spectrophotometer was a very heavy parallelepiped about 1 m long, functioning with a car battery. Georges Cohen, who was the head of a well-known laboratory, helped the young student I was to carry it to my car. He was a great scientist and a very kind person. In 1957 and 1958 I also obtained two science degrees, one in biochemistry and one in animal physiology. This was necessary if a pharmacist or a physician wanted to prepare a PhD in science. Pierre Chambon, an MD working at the biochemistry laboratory of the Faculty of Medicine, also passed his science degrees the same year as I did. Finally, the year 1957 was also a happy year for me because I met Marguerite (called Titi) Mangin who was studying at Ecole des Beaux-Arts (art college) in Nancy. She became art teacher at the high school in Saint Die´ (Vosges). We became engaged at Christmas 1957 and married on December 20, 1958 in Thionville (Moselle). In the third year of my thesis I started, with an Egyptian student Michel Yacoub, to work on the problem of the association of RNA and polyphosphates. These were found to contaminate one another after extraction from the cells. A.N. Belozerski and J.S. Koulaiev had postulated that there existed a covalent binding between the two components which would give ATP-like properties to this “combination.” With J.-P. Ebel and M. Yacoub we presented the problem at the IVth International Congress of Biochemistry in August 1958 in Vienna [6]. This was the first of a long series of international congresses I have attended. I solved the problem only later, after my return from obligatory military service. It is interesting to point out that this was the beginning of

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the numerous studies on RNAs, which were performed for years in Ebel’s laboratory and still continue at the “Institut de Biologie Mole´culaire et Cellulaire” (IBMC) of the CNRS in Strasbourg.

Military Service and Lysozyme The military service normally had to be done at the age of 18. However, for students the age was 25, and for medical and pharmaceutical students it was 27, in order that they could be helpful in the army health service. We had our recruitment training in Bar-le-Duc. I prefer not to dwell on the lack of hygiene of the kitchen and the stupidity of some junior officers who threw their weight around. After 2 months I went to the reserve officer’s school in Bordeaux, I worked hard there because at the end a competitive examination was given. According to our marks we got to choose the region where we would continue our military service. At that time France was at war in Algeria, which was hypocritically called Maintien de l’ordre or Pacification, and nobody wanted to go there. Fortunately, I got high marks and was appointed in a research laboratory of the health service called STRESSA (Section Technique de Recherches et d’Etudes du Service des Sante´ des Arme´es), with the rank of officer cadet. The head of the laboratory was an army medical major, named Louis Colobert (we called him CocoZCommandant Colobert!). He had done a PhD in 1955 in the laboratory of Professor Claude Fromageot, a leading biochemist in France, at Boulevard Raspail in Paris. The STRESSA laboratory was located in a special building at the military hospital Des Genettes in Lyon. The atmosphere was nothing but military and several young pharmacists and physicians, having worked before in different laboratories, did their military service there. They have remained my friends and have since become directors of different laboratories in France: Bernard Montagnon, Director of Virology Production at Me´rieux (Lyon), Andre´ Kirn, Professor of Virology at the Faculty of Medicine of Strasbourg, Ge´rard Rebel, Director

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of Research in Strasbourg. L. Colobert asked me to isolate a soluble substrate of lysozyme. Since the discovery of this enzyme by A. Fleming in 1922, it had been well studied, but in 1958 its mechanism of action was not really known as all the authors followed its activity on a suspension of Micrococcus lysodeikticus which was clarified by the enzyme. I cultivated large amounts of bacteria in 200 bottles carpeted with a solid culture medium. As an Alsatian I was not too much disturbed by their sauerkraut odor. The bacteria were treated in sealed ampoules by 0.5 NaOH for 2 weeks at 56 8C and after several precipitations and purifications I lyophilized the compound obtaining a nice white, cornflakes-like, compound. Unfortunately, this turned out to be composed of two products on moving boundary electrophoresis (a method difficult to set up, as it did not use a solid support and which has completely disappeared since the discovery of polyacrylamide gel electrophoresis). Thus, I separated them by column chromatography on DEAE cellulose. The question was to desalt completely the substrate. Fortunately, we had obtained a 10 g sample of a compound called Sephadex produced by an unknown Swedish company called Pharmacia. The properties of Sephadex had not yet been published (J. Porath and P. Flodin published their first results in 1959), but in the advertising leaflet it was was stated that Sephadex permitted the column chromatographic separation of compounds according to their molecular weight. Using Sephadex G25 this desalting was easy. Our separation was thus one of the first obtained with Sephadex. Then I studied the composition of the compound by the classical colorimetric techniques showing the presence of amino acids and amino sugars, but I could not see the compounds. Like St Thomas, I only believed what I saw! I proposed to L. Colobert to separate them by paper chromatography but he refused stating that this technique would not work. Therefore, I did the experiments secretly in the cellar of the institute, with materials I had smuggled in from Strasbourg. When, after 1 month I showed L. Colobert, the wonderful separation of the amino-acids first and then the separation of glucosamine and muramic acid from the

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hydrolysate of my peptidoglycan, he did not send me to courtmartial and began to believe in chromatography and in my ideas. In fact, L. Colobert was very enthusiastic about science. He went often to Paris where he discussed with Pierre Jolle`s, the lysozyme specialist, and Pierre Douzou. Each time he came back he had new ideas and I had to fight at length with him to keep on my subject and not to change it every week. I finally studied the action of lysozyme on my substrate. I started doing that with a rotating microviscosimeter (another apparatus that has disappeared from our laboratories), but the result was not specific. Then I measured the release of reducing sugars by lysozyme. The then recently published book “The Enzymes” by M. Dixon and E.C. Web became my bedside book and I translated into French the chapters on enzyme kinetics (I still have this translation!). Initial velocity, Michaelis constant, all kind of inhibition mechanisms, effect of pH, of ions etc. had no more secrets for me! This study allowed me to explain the paradoxical inhibition of lysis by high lysozyme concentrations, which was due to the formation of a complex between the degradation product of the substrate and the enzyme. This could be dissociated by sodium chloride, explaining the activation of the reaction by salts. I remember that I did all of my kinetic experiments with the same 0.2 mL pipette, used about every 20 s to take a sample out of the reaction mixture with my “chief” the captain–pharmacist, Olivier Creach, noting the seconds of the stopwatch. As officer cadet I was paid about 180 euros a month together with a piece of soap and 10 packets of cigarettes. Many young men started to smoke during their military service because of these free cigarettes. The culpability of the state herein is evident. Fortunately, my wife got a position as art teacher at a secondary school in Lyon and with the help of my parents and my savings we lived decently in Lyon. We liked this lively city, attended the films of Ingmar Bergmann in the avant-garde cinemas and even went skiing in the Alps. Our first son Florent was born on August 15, 1960 in Lyon. After 18 months of military service I was retained

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in service for 10 additional months. However, this was with the salary of a lieutenant, which was higher than my salary at the CNRS. My wife stopped working in order to take care of our first child. As our second child Bertrand was born on October 18, 1961, after we had returned to Strasbourg she did not continue teaching. She began to paint again and to exhibit her beautiful paintings in 1988 after our three children (Pascale was born in 1966) had left our home. I presented my results at a meeting in Geneva where one of the participants asked me several questions. I defended my proposition without hesitation. After the session L. Colobert was very excited when he came to see me “Do you know who asked you the questions?,” “No, I have no idea,” “It was P. Desnuelle” at that time considered the pope of Biochemistry in France (he has also written his memoirs in this book series in 1983). I published my results with L. Colobert in three papers which appeared in 1960, 1961 (my first one in BBA, still in French) and 1962 [7–9]. In addition, I presented my work on lysozyme as the manuscript for a Doctor of Pharmacy (Doctorat d’Etat en Pharmacie) in October 1961. At that time, 150 copies of the thesis had to be printed. This cost a lot of money, and when my wife asked me to buy armchairs for our new home I answered: “Let’s sit on my theses.” Unfortunately, my sense of humour was not always appreciated! My doctorate permitted me to become Charge´ de Recherche at the CNRS with a better salary and the armchairs finally found their way into our living room.

Polyphosphates and RNAs When I returned in J.-P. Ebel’s lab at the Faculty of Pharmacy it had moved into a new building on the Rue de l’Argonne, beside the old Faculty. There was a great hustle and bustle in the lab because everybody was preparing for the Colloque International sur les Acides Ribonucle´iques et les Polyphosphates – Structure, Synthe`se et Fonctions to be held in Strasbourg July 6–12, 1961

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organised by J.-P. Ebel and Marianne Grunberg-Manago at the Centre of Research on Macromolecules (CRM) in Strasbourg. This was a great success. Many participants were already, or have become in the meantime, leading scientists around the world. When I look at the photograph (Figure 2) of the about 80 participants I recognise P. Berg, G. Bernardi, P. Chambon, H. Fraenkel-Conrat, F. Gros, L. Hirth, C. Lie´becq, U. Littauer, V. Luzatti, P. Mandel, R. Monier, J. Montreuil, E.J. Ofengand, C. Sadron, G. Schramm, M. Staehelin, H. Zachau, J.-P. Zalta, etc., in addition to the researchers of the lab: G. Aubel-Sadron, G. Beck, S. Muller-Felter, A. Stahl, J.-H. Weil, and myself. This meeting had a great influence on the development of our laboratory. The importance of research on RNAs increased; we had now contact with all the leading persons in the field and our contribution was recognised. A congress book was published by the CNRS. In 1961 I came back to my research on the biological role of PolyP. Using Sephadex G200 I separated the polyphosphates from RNA showing that they were not linked[8]. This led me to try to separate the ribosomal RNA from the so-called soluble RNA with J.-H. Weil [10]. This worked very well, but the soluble RNA gave a shouldered peak that corresponded to the tRNAs preceded by the 5S-RNA, as I was told by R. Monier who had just discovered this RNA. We had now clearly shown that sRNA was a mixture of tRNAs and 5S-RNA. However, we did not continue to work on 5S-RNA. Having learned to work with bacteria in Lyon I started looking whether PolyP could replace ATP in these organisms, the reaction I had failed to observe in yeast. M. Szymona had succeeded in it in 1956–1957 with Mycobacterium phlei. I tried with Corynebacterium xerosis using long chain PolyP (Graham salt) and different sugars as substrates. In the presence of PolyP both glucose and glucosamine were phosphorylated in position six whereas in the presence of ATP mannose and fructose were also phosphorylated. This excluded an indirect mechanism, i.e., a phosphorylation of contaminating ADP to ATP by the PolyP, followed by the phosphorylation of the sugars by ATP. This was confirmed by

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Fig. 2. Participants at the “Colloque International sur les Acides Riborucleiques et les Polyphosphates— Structure, Synthe`se et Functions”. Strasbourg, July 6–12, 1961.

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the fact that the reaction was not inhibited by apyrase, which hydrolyses ATP, nor was it inhibited by eliminating the contaminant nucleotides by filtration of the enzyme on a anion exchanger. Thus, I had demonstrated a PolyP glucokinase in C. xerosis [11]. Continuing with this bacterium I also characterised a PolyP-AMP phosphotransferase which converted AMP to ADP [12]. Since ADP can be converted to ATP by adenylate kinase, PolyP constituted a reservoir of energy in these bacteria. Since 1963 several authors have described and studied these reactions in several bacteria. In 1963 a new subject was started in J.-P. Ebel’ lab. He had shown that the action of bromine on phosphorous acid (PO3H3) in the absence of water gave a mixture of polyphosphoric acids. He asked me to check whether this reaction could be applied to AMP in order to obtain adenosine polyphosphates. With my student, Hubert Wittersheim, who was assistant at the Faculty of Pharmacy, we obtained both ADP and adenosine-2 0 (3 0 ), 5 0 -diphosphate with this reaction in DMF. Starting from isopropyliden adenosine, where only the 5 0 of the ribose is free, we obtained AMP, ADP and ATP. H. Wittersheim was my first graduate student and obtained a PhD in 1966. He became director of a medical analysis laboratory near Strasbourg. In 1962 the Professor of Biochemistry at the Science Faculty in Strasbourg retired and J.-P. Ebel, who had been Professor of Biochemistry at the Faculty of Pharmacy since 1958, succeeded him. This was a much better position as the research credits were higher at the Science Faculty. In addition, the Science Faculty decided to build a new Physiology and Biochemistry Institute where a whole floor was reserved for Ebel’s laboratory. J.-P. Ebel’s lab remained at the Faculty of Pharmacy until 1965 when the construction of this institute was finished. Professor Pierre Me´tais succeeded J.-P. Ebel as Professor of Biochemistry at the Faculty of Pharmacy. I asked for the former position of P. Me´tais and was temporarily appointed as Maıˆtre de Conferences, which is equivalent to assistant professor in the USA and is now called Second Class Professor in France. I had to teach toxicology and

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part of the biochemistry course. I asked the CNRS for a leave of one year for lecturing and in order to prepare my agre´gation. The personnel officer of the CNRS answered that she had registered my resignation. If I had failed in the agregation I would have become redundant! Times were hard at the CNRS in 1962!

Professor of Biochemistry 1964 was a labour intensive year for me. I had to prepare my new lectures, but I also wrote my doctoral thesis in Science (Doctorat e`s Sciences) about my 6 years research on PolyP. At that time the PhD preparation in France normally took 6–7 years! I presented my PhD in Science on March 20, 1964. After all that, I was still only temporary Assistant Professor. In order to obtain the full position, I had to succeed in a competitive biochemistry exam, the Concours d’agre´gation. This took place in Paris. It consisted of presenting a 1 h lecture in biochemistry after 8 h preparation in the library, and a 45 min lecture in Pharmaceutical Chemistry after 6 h of preparation. I trained for this competition for a whole year in Strasbourg. J.-P. Ebel gave me a subject every week and I had to prepare the lecture and present it under the same conditions as the agre´gation. One of the main objectives was that the lecture took exactly 1 h. At the lecture presentation the alarm clock rang after 1 h. Those who finished their lecture early had to wait in front of the jury saying nothing; those who did not finish on time were brutally interrupted and had to leave, never mind the wonderful conclusion they had planned. This was also good training for my future congress presentations. For the concours I drew as subjects, respectively, glutamic acid, and natural and synthetic anticoagulants. We also had to do a practical. I got chromatographic separation of amino-acids. Finally, we had to present our CV. Everything went well, although I was very stressed, and I got the best rank, which allowed me to choose the professorship in Strasbourg. In fact, I was still not a civil servant, but trainee assistant professor

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(Maıˆtre de confe´rences agre´ge´ stagiaire). Things were complicated at that time! The departure of Ebel to the new Institute of Physiology and Biochemistry at the Science Faculty in 1965 posed a problem. As professor at the Faculty of Pharmacy I had to have a research laboratory there, but I had started to look after several students preparing their thesis in Science, who preferred to move. Thus, I started to have two laboratories. I worked and lectured in the morning at the Faculty of Pharmacy and in the afternoon at Ebel’s lab. Starting with tRNAs in Holley’s Lab At that time when doing a PhD one had, in addition to the experimental work, to write what was called a second thesis, a bibliographical review. For my thesis in Pharmacy in 1961 my subject was “The problem of the sequence of nucleotides in RNAs.” The only thing that was known in 1960 was that the sRNAs (now called tRNAs) all started with pGp. at the 5 0 terminus and were terminated by .pCpCpA at the 3 0 terminus. In 1961 P. Berg et al. had shown a great heterogeneity starting before this sequence of three nucleotides. Concerning viral RNAs H. Fraenkel-Conrat et al. had shown in 1961 that the TMV-RNA had an adenine at both the 5 0 and 3 0 end positions. This aroused my interest in the domain of RNA sequencing. But in 1964 the problem had much evolved and several laboratories were already working on the structure of purified tRNAs. The studies in J.-P. Ebel’s laboratory had changed to the comprehension of protein synthesis both at the ribosomal level and that of aminoacyl-tRNA synthetases. As such, it had become urgent to isolate pure tRNAs and to determine their structure. We thought that the first step of this isolation would be to do a countercurrent distribution of bulk yeast tRNA produced in gram quantities by Boehringer Mannheim. J.-P. Ebel obtained the funds to buy a countercurrent machine from the DGRST (De´le´gation Ge´ne´rale a` la Recherche Scientifique et Technique) early in 1965. But which

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one should we buy? Three models were on the market; the EC apparatus used by R.W. Holley at Cornell University in Ithaca (N.Y.) and H. Zachau in Cologne (Germany), another one used by I. Smith at King’s College (London) and yet another one located in New York. So I went to visit the different laboratories. I went to Cologne to discuss with Hans Zachau and Horst Feldmann, who were sequencing serine-tRNAs, and was very well informed. I went to London too, and finally to Ithaca for 2 months. R.W. Holley, who had just finished the sequencing of yeast alanine-tRNA, the first nucleic acid sequenced, for which he got the Nobel Prize in 1968, had very kindly agreed to show me the countercurrent distribution and the tRNA sequencing techniques used in his laboratory. This was to be my first stay in the USA. The cheapest company to fly there was Icelandic airlines. I took a prop-jet plane in Luxembourg on April 14, 1965 to New-York via Reykjavik. On our arrival in Iceland the pilots decided to go on strike! This allowed me to visit the capital of Iceland waiting for a solution. Finally after 14 h we got a PanAm plane jet coming from Berlin, almost empty, and flying twice as rapidly as our Icelandic airlines prop-jet. From New-York a very small Mohawk airlines plane brought me to Ithaca. I was very surprised upon arrival because it was snowing whereas I had left Strasbourg in Spring. The laboratory of R.W. Holley was at 20 min walking distance from Telluride house, where he had found a room for me. I walked with flat shoes through the snow. R.W. Holley was a very nice, but shy man. He spent almost the whole day in his office, so I was trained mainly by an English PhD student John R. Penswick and a Hungarian technician. I did not like the EC countercurrent apparatus they used because it was fed on one side with tRNA, but all the fractions coming out at the other side of the apparatus were thrown away. This was because Holley was only interested in alanine-tRNA which, with the lowest solubility in this organic phase, remained in the apparatus when it was perfused with the organic phase. In Europe after the war we were thrifty and I was not happy about this waste of precious material. I also learned to test the purified fractions for their amino-acid accepting activity

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with E. Keller and the fractionation of the oligonucleotides, obtained after the hydrolysis of the tRNA, by different ribonucleases on a 2.5 m high narrow diameter column which was very difficult to fill with DEAE-cellulose (about a day’s work!). During my stay in Ithaca I was invited twice by Holley, once at home with L.A. Heppel and once at the Faculty Club with B. Magasanik, where they served the same Alsatian wine I drink at home (Lorentz from Bergheim)! I also became more familiar with the American student way of life. As Ithaca was a very small city, all their activities were localised on the campus. This was very different from Strasbourg where all faculties were in the centre of the city. The libraries were open until midnight (a dream in France!). The war in Vietnam was in the centre of all discussions and many meetings and demonstrations took place. I remember particularly the celebration of the 100th anniversary of the charter of Cornell University. The Governor of New York, Nelson Rockefeller, had come for this big feast held in a great hall and attended by about 4000 people, half of them being parents of students or former students of the University. The rector had just finished his speech and Rockefeller stood up to give his talk when a long procession of about 200 students with posters, “Cornell feasts while one bombs Indochina,” came in, shouting “End the war in Vietnam, end the war in Vietnam..” This took at least 5 min of general consternation. Rockefeller remained standing and waited until they had left. Then he said “This proves that we live in a free country.” Having no car to travel in the region of the Finger Lakes I went several times to New York for the weekends, visiting all the marvellous museums of the city. I even discovered in the early Gothic Hall of the Cloisters, the Virgin from the 13th century choir screen from the cathedral in Strasbourg! Its a pity that it is not in the Muse´e de l’Oeuvre Notre Dame in Strasbourg where the original sculptures of the cathedral are shown. To be fair I must add that it was purchased legally by the Americans for 89 pounds and 5 shillings in 1903!

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I also went to visit the factory of “Post Scientific Instruments” where they were building a countercurrent apparatus. I liked it very much and ordered this machine, composed of 500 tubes, each with 20 mL capacity. In this factory everybody spoke German and I was invited to the home of the head of the factory for a typical German Kaffeeklatsch. Thus, I discovered another aspect of American Society. Finally, I stayed for two weekends with my friend Tomi Ungerer who had settled near Central Park in 1957. He had just published “The Underground Sketchbook” which was very appreciated. “Its a must for anyone contemplating mankind in all its delicate glory” said Ronald Searle about this book. But his children’s books were also great successes. I went with him, his second wife and little girl to Long Island where he had a magnificent villa. He invited me to a party where he had also invited Gu¨nter Grass, who later got the Nobel Price for literature. As I had to give a seminar in Holley’s lab the next day, I returned to Ithaca on Sunday afternoon. I regret it a little now, but I console myself by saying that I was not in the States for society life! Returning to Strasbourg was another adventure. Icelandic Airlines were still on strike, thus we were put on a plane of a Dutch company. After the stop-over in Reykjavik the pilot decided to land in Amsterdam, instead of Luxembourg where our families were waiting for us. This created a revolution in the plane. We wrote a petition, which was signed by the passengers. Finally, the company hired a plane to take us to Luxembourg where my wife had caught a severe cold waiting for me. Since that time she never waits for me at airports and she is right given the constant delays of the planes, every day for another reason. Countercurrent Distribution of tRNAs The countercurrent apparatus, however, arrived in Strasbourg by plane without problems and a technician from the Post Factory in New-York built it up in the 24 8C thermostated room I had installed in Ebel’s lab. I did the first distribution of 1.5 g tRNAs in

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1966 and obtained, after 1100 transfers, a wonderful result with seven distinct peaks of A260 nm. With my technician, Olga Blasi, we determined the accepting activity for 17 amino-acids. Alanine and phenylalanine tRNAs were almost pure, the others often 7–15 times enriched. The isoacceptors were clearly located in different peaks. We repeated the experiment. It completely failed! This was a great disappointment. However, we found out that the machine had been contaminated with a ribonuclease. We washed it completely with a sulfochromic solution and did all experiments with gloves and the third experiment again gave a very nice result. I published these results with J.-P. Ebel in 1967 [13]. Now we were ready to purify the countercurrent fractions in order to obtain several pure tRNAs. We used different column chromatographies, hydroxyapatite with a technique I had devised in the meantime, DEAE cellulose, and later reverse phase chromatography. With Georgette Sabeur in Ebel’s lab I isolated aspartate tRNA in 1967 [14]. I had also started to set up a group on tRNA sequencing at the Faculty of Pharmacy. My first co-worker was a pharmacist, Bernard Kuntzel, who came to my lab in 1966 and was engaged by the CNRS in 1967. He isolated one of the isoacceptors of argininetRNA, we had called tRNAArg3. This was published in Nature in 1968 [15]. In 1967, Jacques Bonnet, a chemical engineer, came to our laboratory where he first worked on the purification of yeast tRNAVal[16]. I used the countercurrent distribution of tRNAs until 1990. Every 2 years a technician came from New York to look after the machine and to change the defective parts. It was a wonderful machine, running like clockwork day and night, preparing purified fractions of tRNAs for the whole laboratory. My technician, Monique SchIegel ran the machine. In fact, with the years, the demand for purified tRNAs increased, first for the sequencing of several tRNAs in my own group, then for the crystallisation of tRNAs by Richard Giege´ and for their structural determination by X-ray diffraction by the group of Dino Moras and Jean-Claude Thierry. It was the pillar of the development of

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Ebel’s and my laboratories. Now it is waiting in a cellar until it is requested by a museum of scientific equipment. In 1965 I started a collaboration with a specialist of circular dichroism and thermodynamics, J. Brahms, working at the Centre of Molecular Biophysics that had been created in Orle´ans by Charles Sadron after he left the CRM in Strasbourg. AnneMarie Aubertin during her senior diploma (masters equivalent) isolated several trinucleotides. Marianne Grunberg-Manago at the Institut de Biologie physicochimique in Paris prepared other trinucleotides by enzymatic synthesis. A comparison of circular dichroism and thermodynamic properties studied under different conditions of ionic strength, concentration and temperature allowed us to detect the conformational characteristics of the polynucleotide chains. This was my first publication with Marianne Grunberg-Manago [17]. Anne-Marie Aubertin was later recruited by INSERM (Institut National de la Sante´ et de la Recherche Me´dicale). This National Institute for Medical Research functions similarly to the CNRS with Committees recruiting researchers and judging the laboratories. She became, after the retirement of Andre´ Kirn in 1997, the director of an INSERM/University unit “Pathogenicity of Persistent Viral Infections.”

Starting Toxicology with Ricin But let’s go back to 1963 and the title of this chapter. As I mentioned above, I taught toxicology at the Faculty of Pharmacy and I believe that a University Professor should do research in the field in which he teaches. I set up the Certificat d’e´tudes supe´rieures of toxicology, with lectures and practicals, which had not previously existed at the Faculty of Pharmacy. This attracted every year two to three students from France or other countries. Some of them prepared a thesis in toxicology at my laboratory afterwards. I also set up a research group in toxicology. P. Me´tais suggested to me that I work on ricin, the toxic

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glycoprotein of castor bean that had been discovered as early as 1889 by H. Stillmark. Its mechanism of action was unknown. This interested me and, with a pharmacist, Francis Haas, using Sephadex G 100 filtration and hydroxylapatite chromatography we prepared microelectrophoretically pure ricin [18]. One microgram was enough to kill a mouse in 48 h, but the symptoms appeared only after 30 h. Examining the different constituents of blood of rats intoxicated with ricin, I found a decrease of serum albumin [19]. This led me to the curious hypothesis that ricin could inhibit protein synthesis in liver. I purified the microsomes of a rat 6 h after ricin intoxication, when the rat did not show any symptoms of intoxication and added a 105,000 g supernatant from healthy control animals to assay the protein synthesis. and it worked! In one experiment there was a 45% inhibition, in the other a 71% inhibition. Using the 105,000 g supernatant of intoxicated animals with microsomes of healthy animals I did not find any inhibition. I had discovered that ricin inhibits protein synthesis at the ribosomal level. I published this result in a congress book (in French) in 1968 [20]. This discovery was completely overlooked! This effect was rediscovered in intact cells in 1971 by J.-Y. Lin et al., and in vitro by S. Olsnes and A. Pihl in 1972, then by L. Montanaro et al. in 1973. Between 1966 and 2003, 2474 papers have been published on ricin (according to PubMed). Proteins acting in a similar way have been discovered in many organisms and are now called “ribosome inactivating proteins” (RIP). Fortunately, the French toxicologists had been interested in my presentation, particularly Professor Michel Gaultier, the head of the first French Poison Centre at Hopital Fernand Widal in Paris. This encouraged me to carry on my research in toxicology. I now had the hat of a toxicologist! In fact I called the hat “Molecular Toxicology” because I was interested in the mechanism of action of toxic compounds at the molecular level. I must note, however, that doing toxicological research in France was very difficult. There was no financial support for it. Only researchers working on drugs or in hospitals were funded and were given positions. Thus, I worked for years with only one

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or two researchers. Following F. Haas was Alain Lugnier who started working in the lab in 1964 (without being paid until 1966). Then he went off for 16 months to do his military service. When he returned in 1968 he was recruited by INSERM.

Analogues of Nucleotides The recruitment of Ebel’s team had much increased after he moved to the Faculty of Sciences. He hired Pierre Re´my who was a chemical engineer from the Ecole supe´rieure de Chimie in Strasbourg. With him we continued to work on the synthesis of ATP analogues. The idea was to replace the tripolyphosphate chain of ATP with a chain containing phosphorous atoms of a lower oxidation state. In 1965, Pierre Re´my synthesized adenosine 5 0 -phosphohypophosphate (AMPOPP) [21] and later guanosine 5 0 -phosphohypophosphate (GMPOPP). AMPOPP was tested in the reactions catalysed by hexokinase and valyl-tRNA synthetase. It could not replace ATP, but behaved as a competitive inhibitor in the two reactions. GMPOPP was tested on protein synthesis which was also inhibited [22]. This research was done with Michel Revel, who worked at that time in Franc¸ois Gros’ laboratory in Paris and who is now at the Weizmann Institute in Rehovot (Israel). In the binding of fMet-tRNA to E. coli ribosomes GMPOPP could replace GTP, but with a much lower efficiency than GTP and another analogue of GTP guanylyl-5 0 -methylenediphosphonate (GMPOPCP) synthesised by J.W.B. Hershey and R.E. Monro in 1966. Since that time this latter analogue has been extensively used. An analogue of ADP adenosine-5 0 -hypophosphate was also synthesised and tested on adenylate kinase and pyruvate kinase. In this second reaction it was phosphorylated into adenosine hypophosphophosphate a new analogue of ATP [23,24] which was able to phosphorylate glucose and fructose 6-phosphate. In this work we were helped by Jacques Setondji, a PhD student from Benin (Africa), who defended his thesis in Cotonou in December 1972. As this was the

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first thesis presented in that country, where they had only one professor at the University, J.-P. Ebel and I went there. I remember the sweltering temperature in the overcrowded lecture room. Ebel and I were in briefs under our silken gowns. I also remember the enthusiasm of the audience when we granted the PhD at the end of the defence. The female African students were dancing in the aisles of the amphitheatre. J. Setondji became Professor of Biochemistry the year after and Director of the Enseignement Technique. Our laboratory still has excellent scientific relations with the Biochemical Laboratory in Cotonou now headed by Professor Ambel Sanni.

CNRS Commissions In 1967 I was elected member of the Commission de Biochimie du CNRS. Here I have to explain briefly the functioning of the CNRS and its system of evaluation. The CNRS has its own laboratories and institutes, built and owned by the CNRS, where researchers and technicians belonging to the CNRS, but also from universities, are doing research. University laboratories can also be associated with the CNRS and get part of their support money from the CNRS. As universities have much less money for research, this type of association with the CNRS (or INSERM) is much sought after. Ebel’s laboratory was associated with the CNRS beginning in 1965. For the evaluations, several specialised commissions exist at the CNRS (35 in 1967); Biochemistry, Cell Biology, Physiology, Organic Chemistry, Mathematics etc. In Biochemistry the CNRS recruits every year 5–15 full time researchers depending on the government budget for research. The candidates are first selected by their laboratories, then they present a written report with their CV and projects, which they defend orally before the commission. Then, after discussion with two referees, members of the commission, the commission votes by secret ballot. The highest ranking candidates are proposed for recruitment to the Council of the Department for Life Sciences

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which normally accepts, but has the prerogative to change the classification. Finally, the Director of the Department, the Scientific Council and the General Director of the CNRS have to agree. This seems very complicated, but the proposals of the commission are rarely changed, at least for the top of the list. The commission has a great importance both for the recruitment of researchers, their career advancement, and the development of laboratories. Half of the members of this commission are appointed by the Director of the CNRS the other half being elected. The commission has also to propose promotions, for example from Charge´ de recherche to Directeur de recherche which is a major change in the responsibilities of a researcher. Finally, the commission evaluates the activities of all the laboratories and institutes working in biochemistry in France. These were evaluated every 2 years (now every 4 years) and compared with the other laboratories. The tasks of the commission are very important and represent at least 3–4 weeks of full time work a year. The first time I was in this commission I was much impressed by the personalities I encountered there. Jacques Monod, particularly, had a great influence and when once, in one of my reports, I spoke of tRNAsynthetases instead of aminoacyl-tRNA synthetases he pointed out that a tRNA synthetase is an enzyme synthesizing a tRNA and not what I was speaking of! This was extremely embarrassing and a hole to crawl into would have been a welcome solace. Other well-known biochemists belonged to the 1967 commission, Paul Boulanger, Jean-Emile Courtois, Jean Jacques, Pierre Joliot, Serge Lissitzki, Marianne Grunberg-Manago, Jean Lavorel, Jean Roche, Jeannine Yon, Georges Cohen, Jean Coursaget, Raymond Dedonder, Roger Monier and Maurice Vigneron. We were elected or appointed for 3–4 years. Over my career years, I have been elected four times to this commission (1967–1970, 1971–1975, 1980–1983, 1991–1995). This way I could follow the evolution of French biochemistry and became acquainted with its principal actors. The name of the commission has changed several times. In 1991 it became Biomolecules: structure–function relationships.

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As member of the national commissions, I also became member of the evaluation committees of several CNRS Institutes in Marseilles, Bordeaux and Paris.

The Events of May 1968 France, with General Ch. de Gaulle as President and Georges Pompidou as Prime Minister, was in early 1968 a prosperous and politically very stable country. However, the situation in French universities was bad. In Strasbourg, the number of students had increased from 5,340 in 1950 to 21,530 in 1967/1968. This increase was particularly important in literature, whereas in pharmacy the number had only increased from 500 to 862. This was due to the typical French problem. In France everybody having obtained the baccalaureat at the end of secondary school is accepted into the university without any additional selection. However, the number of teachers and jobs offered to the holders of university diplomas had not increased in proportion. In 1968 there was only one University in Strasbourg, which was divided into seven Faculties each headed by a Dean. The seven Deans met several times a year with the Rector, Maurice Bayen, who was appointed by the government, and the Mayor of the city of Strasbourg who was at that time Pierre Pflimlin. The Deans were elected by the council of their respective faculties (comprising only full professors). The Professors sans chaire and Maitres de confe´rences were sitting in the second row of the Council. Equivalent grade researchers from CNRS and INSERM were not members of the Council. The events of May 1968 did not directly touch the Faculty of Pharmacy except for the exam of the Certificat d’Etudes Spe´ciales (CES) of biochemistry (a national exam held in all French Faculties at the same moment, a requisite to open a laboratory of medical biochemistry). On May 11 the examination room was invaded by students, mainly from the Faculty of Protestant Theology. The pharmaceutical students who, after

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1 year’s preparation, wanted to get their exam, fought against them. Having been appointed Professeur sans chaire the January 1, 1968, I was in charge of this exam and had difficulty separating them. At the Faculty of Literature the students installed a tramp in the Dean’s office and the black flag of the anarchists was flying over the Faculty. After this, the students of the Faculty of Pharmacy decided to occupy the Faculty in order to protect it against the revolutionary students who might destroy the scientific equipment. All the exams were postponed until September. The events of May 1968 in Strasbourg are well described in the Feuerstein’s book [25]. After the elections of June 23 and 30, 1968 the new Minister of National Education, Edgar Faure, prepared the new Law of Orientation of University Education (Loi d’orientation de l’Enseignement supe´rieur). The situation at the Faculty of Pharmacy was confused because the Dean, Michel Hasselmann, refused to discuss with the students and even the diplomas were not signed. However, as the youngest professor I had good relations with them, but also with M. Hasselmann. He even proposed to transform his professorship of Analytical Chemistry and Toxicology into Analytical Chemistry and Bromatology, and to ask for the creation, for me, of a professorship in Toxicology. This request was accepted by the Council of Professors on January 24, 1969 and I was appointed full professor by decree of the president, Georges Pompidou, on October 8, 1969. In France, becoming Professor was a serious affair!

Dean of the Faculty of Pharmacy According to the Orientation Law we had to elect a council comprising representatives of all the categories of people working at the Faculty from professors to students including researchers, technicians and administrative personnel. This council of 41 persons met for the first time on February 20, 1969. I was elected President by 36 votes. Gilbert Laustriat was elected first

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Vice-President and Charles Stirnweiss, a student, second Vice-President. The Dean, M. Hasselmann, resigned the day after at the assembly of professors. On February 26 the assembly of delegates was transformed into the Conseil transitoire de gestion (Transitory Management Council) of which I was elected President. I was Charge´ des fonctions de doyen (in charge of the functions of Dean) on April 11, 1969. Our council was initially a constitutive council as we had to elaborate the statutes of the new teaching and research unit, UER (Unite´ d’Enseignement et de Recherche) of Pharmaceutical Sciences, which was replacing the Faculty of Pharmacy. We had many discussions, often extending late into the night, but we managed to elaborate them in time, as well as the by-laws. A scientific committee, an executive committee and several working groups were created. An option Gestion des enterprises was realised in collaboration with the Institute of Business Economy (Institut d’e´conomie applique´e aux affaires). Two new CES options, one of Therapeutic Chemistry the other of Industrial Galenic Pharmacy were set up. I obtained from the Education Ministry the creation of three new professorships, Organic Chemistry, Analytical Chemistry and Physiology, which allowed the development of new fields of teaching; phyto- and zoo-pharmacy, dermopharmacy, homeopathy, environment. I also developed language teaching, English and the Alsatian dialect. Alsatian dialect had not been taught before at the University, although I thought it was important for pharmacists working outside the big cities where many Alsatians, particularly elderly people, continued to speak the dialect. We also worked a lot on a Reform Bill for Pharmaceutical Studies. C. Stirnweiss and several other students from Strasbourg defended it before other Faculties but the majority of them rejected it. Since May 1969 we had also been very much occupied with the creation of several new Universities of Strasbourg replacing the unique one we had before. We wanted to have a large scientific university in Strasbourg, but for political reasons many professors were hesitant. Some wanted to make a University of Law

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and Pharmacy! We had to lobby a lot to convince the former Faculty of Medicine to join the Faculty of Sciences, which was considered to be leftist oriented. In May 1969 the different Deans met almost twice a week with the Rector! Fortunately, we were served a whisky after each meeting! Finally, the decision to create the University of Strasbourg 1, named Universite´ Louis Pasteur (ULP) was voted. I was delegated on April 1970, with C. Stirnweiss, to the Constituent Assembly Committee in charge of elaborating the statutes of the ULP. After many meetings its statutes were adopted in June 1970. We finally needed to convince an eminent scientist to become the first President of the University. I remember I went to see Guy Ourisson at his house outside Strasbourg to convince him to be a candidate for the first President of the University Louis Pasteur, which he became. How many Committees did I attend in these feverish years? I counted 29 Councils at the Faculty of Pharmacy in 1970. In order not to lose too much time I often scheduled them at 11.00. When people become hungry they do not speak unnecessarily. Some of the meetings were held in the evening at 20.30 and ended at 0.30. There was much less television in these days! The old Faculty inside the city being too small for the increasing number of students and for the development of research, we were already lobbying at the time when M. Hasselmann was Dean, for the building of a new Faculty on the campus of Illkirch, 6 km outside the town. In 1970 it was placed as a priority on the 6th Economical Development Plan of the government. I met Andre´ Bord, an Alsatian minister in G. Pompidou’s government, who promised me to support our request for financing. However, it took years of lobbying by the Deans who succeeded me, Gilbert Laustriat and Pierre Me´tais, before the Faculty was built in 1979! In the new statutes of the UER we had written that the Deans could not be re-elected. Thus, Gilbert Laustriat was elected in January 1971 as director of the UER of Pharmaceutical Sciences and the council offered me the title of Honorary Dean, being apparently happy with the work I had done. I remained President

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of the Scientific Council which we had set up in 1969 and remained an elected member of the Faculty Council for years.

Sequencing tRNAs During these feverish years, research did not stop in my two groups. In the morning I was at the Faculty of Pharmacy doing my job of Dean and running my own lab and in the afternoon at the Faculty of Sciences in Ebel’s lab. There I now had two new co-workers, Ge´rard Keith and Jean Gangloff who had done a Masters in Biochemistry. We were also particularly aided by a skilful and hard working technician Marie-Louise Gangloff who remained in our group from 1964 to 2003. Together they started to sequence brewer’s yeast tRNAAsp. This took two full years. They improved the purification of tRNAAsp by using column chromatography on benzoylated DEAE-cellulose (BD cellulose) [26], a system elaborated by I.M. Gillam et al. in 1967 [27]. Several grams of pure tRNA were necessary for the sequencing. J. Gangloff and G. Keith also developeded a quick bidimensional electrophoresis method for the fractionation of the mixtures of oligonucleotides obtained by either pancreatic or T1 hydrolysis of the tRNA as well as a method for the determination of their nucleotide sequences by spectrophotometric analysis [28]. The structure of tRNAAsp was published in French in 1970 [29] and in English in 1971 in Nature [30]. From that time on almost all my publications were in English. I presented the structure of brewers yeast tRNAAsp in June 1970 in Riga at a tRNA Meeting. It was the first time I had visited the USSR and I had to obtain in Moscow the authorisation to go to Riga. The police phoned to the hotel I was supposed to stay in Riga to check if what I told them was true. Unfortunately, the organisers of the congress had changed my accommodation in Riga and I became a suspect! It took about 2 h of phone calls before I was allowed to fly to Riga. I have another story about Riga. I was staying in the town whereas J.-P. Ebel and M. Grunberg-Manago where in a hotel at a seaside resort.

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I went with them in the afternoon and returned to Riga in the evening by train. I bought a ticket at the railway station, as I do at home, and took the train. In the wagon I spoke with a man (in German) who asked me: “Are you alone?,” “Yes,” I said. “But where is your guide?” He had never seen a foreign visitor without a guide! The tRNAAsp was a jackpot for several laboratories at IBMC as, after having isolated the aspartyl-tRNA synthetase in 1973, the complex was crystallized and its structure resolved by X-ray diffraction by M. Ruff et al. in the laboratory of Dino Moras. Now that the techniques of sequencing tRNAs were well established, determination of tRNA structure was much more rapid. The chromatographic methods for their isolation were also continuously improved. Reversed phase column chromatography permitted J. Bonnet, to isolate brewers yeast tRNAval2 in 1969 and to sequence it in 1971. This was my first paper in FEBS Letters [31]. However, this tRNA differed in four positions from the one published by A.A. Bayev et al. [32] for bakers yeast tRNAVal1. After discussion with A.A. Bayev we decided to reinvestigate their structure with the tRNAVal1 they supplied and found that it was the same as ours. I decided to publish this corrected structure together with Bayev and his co-authors [33], which he very much appreciated. We remained good friends and met often at French-Soviet meetings on structure and function of nucleic acids which for a long time were organised one year in France and the alternate year in USSR. A.A. Bayev has written “The Paths of my Life” in Volume IV of these Personal Recollections (1995). Jacques Bonnet is now Professor of Biochemistry at the Faculty of Sciences in Bordeaux. My co-workers at the Faculty of Pharmacy had also some excellent job at the end of the 1960s. Bernard Kuntzel, aided by a pharmacist whom I had recruited into the CNRS in 1969, Jean Weissenbach, had succeeded in the determination of the structure of tRNAArg3 [34]. The other major isoacceptor in brewers yeast tRNAArg2 was sequenced by J. Weissenbach and Robert Martin, a Masters of Science student, who had joined my group in 1971 [35].

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Robert Martin also reinvestigated, together with J. Penswick, the structure of yeast tRNAAla which had been sequenced by R.W. Holley et al. [36]. We suspected this sequence to present an error in the D-arm and loop because it did not conform to the general rules governing the tRNA structures deduced from the 32 tRNA structures known at that time [37]. We showed [38] that a G–C sequence had to be deleted in the D-stem of the structure given by R.W. Holley et al. The interest of isolating tRNAs and determining their primary structure is also to be able to study their interaction with their corresponding aminoacyl-tRNA synthetases. Therefore, I asked J. Gangloff, who wanted to stop sequencing tRNAs, to isolate yeast aspartyl-tRNA synthetase (AspRS). He set up a quick isolation technique for pure enzyme after only two-column chromatographic steps [39]. This enzyme was thereafter studied for years by J. Gangloff and his co-workers. While purifying AspRS, J. Gangloff found that it forms a complex not only with yeast tRNAAsp, but also with yeast tRNAArg [39,40]. This discovery led to many further studies, particularly in Richard Giege´’s group.

Ricin Toxic Peptides During this time in another field of research A. Lugnier had continued to work on ricin. This protein was known to be very resistant to proteolytic enzymes. We showed that under certain conditions it could be hydrolysed by trypsin giving rise to toxic peptides. A. Lugnier, who had become Charge´ de recherches in INSERM, started to isolate these peptides in 1968 [41]. He also elaborated a new method to iolate ricin using two successive steps of column chromatography [42]. Its purity was controlled by polyacrylamide gel electrophoresis, a method just published by C.R. Parish and J.J. Marchalonis in 1969. He also proved that ricin was different from two phytohemagglutinins which had been isolated from castor bean [43]. With Hans Ku¨ntzel from the

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Max Planck-Institut fu¨r Experimental Medizin we showed that ricin inhibited Neurospora crassa and yeast mitochondrial protein synthesis which was surprising given that ricin did not inhibit E. coli protein synthesis [44]. Aminoacyl-tRNA Synthetases B. Kuntzel also started the purification of arginyl-tRNA synthetase. After he left in 1973 to set up a medical analysis laboratory in Metz, J. Gangloff took over this problem. With Annie Schutz he isolated the enzyme and studied its properties [45]. In J.-P. Ebel’s group, Franco Fasiolo, Nicole Be´fort and Yves Boulanger isolated phenylalanyl-tRNA synthetase and Daniel Kern valyl-tRNA synthetase. With all these pure enzymes and their cognate and non-cognate tRNAs available, J. Bonnet, D. Kern, R. Giege´ and F. Fasiolo could study the specificity of aminoacyl-tRNA synthetases. An important contribution of our laboratories was to show that this specificity is essentially based on the kinetic properties of the aminoacylation reaction: an aminoacyl-tRNA synthetase recognises numerous tRNAs with comparable affinities, but the Vmax of the charging of the cognate tRNA is about 2000–10,000 times higher than that of the non-cognate tRNA [46]. Arginyl-tRNA synthetase is interesting because it does not form an aminoacyl adenylate complex that can be isolated, nor does it catalyse the pyrophosphate-exchange reaction in the absence of tRNA. In 1966, J. Gangloff went to Cambridge (UK) to study with Alan R. Fersht the reaction pathway and rate determinating step in the aminoacylation of tRNAArg using quenched-flow methods [47]. These studies encouraged J. Gangloff to construct with F. Ritter a new apparatus for rapid kinetics which allowed one to measure, in a unique stroke (in a range of 5–450 ms), enough data (8–11 points) to establish the kinetics of a fast reaction, and in particular, the pre-steady-state of an enzymatic reaction [48]. A patent was taken out on this apparatus. Four copies of it were built in a workshop at our University for laboratories in China, Germany and France.

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Specific Modifications of tRNAs: Lead and Carcinogens With Bernard Krebs, a PhD student, and Christiane Werner, who became assistant in toxicology at the Faculty of Pharmacy in October 1970, I wanted to link the toxicological studies to our research on tRNAs. The publications of H. Borsok et al. [49] showing that lead inhibits protein synthesis and that of W.R. Farkas [50] showing that lead hydrolyses a mixture of unfractionated tRNAs gave me the idea to test the action of lead acetate on pure tRNAs and to study its degradation products. We started with brewer’s yeast tRNAPhe which we prepared in large quantities by countercurrent distribution. We renatured the tRNA in the presence of 0.9 M NaCl so it was in physiological conditions, and treated it by 1 mM lead acetate (Pb2C). The hydrolysis was followed by polyacrylamide gel electrophoresis. This showed only one major cleavage site even after 2 h at 37 8C. The analysis of the two isolated fragments showed that the tRNA had been specifically cut after the hU in position 17. We published these results in November 1972 in the Journal Europe´en de Toxicology [51]. In the presence of 0.5 M NaCl there were cleavages after hUp16 and hUp17 as well as a partial one after pGp1. We also characterised the cleavage points of pure tRNAVal and tRNAAsp. The results were different from those obtained with tRNAPhe, tRNAAsp. being essentially cleaved in the anticodon loop [52]. The tRNA most sensitive to Pb2C cleavage was tRNAVal, which was still cleaved with a concentration of Pb2C of 5!10K6 M in 0.15 M NaCl. The specific cleavage of tRNAs with Pb2C had great success. It was the first well-characterized example of a metal-promoted site-specific cleavage reaction involving RNA. We did not discover the mechanism of action of lead, but I discussed the problem with Aron Klug who was very interested. With R.S. Brown et al. [53] on the basis of crystallographic studies, he was able to elaborate a detailed mechanism of cleavage in which Pb(OH)C is positioned by co-ordinating to U59 and C60 and abstracts a proton from the

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2 0 -OH group of D17 to facilitate phosphodiester hydrolysis via a cyclic 2 0 ,3 0 -phosphomonoester intermediate. The discovery of ribozymes (T.R. Cech, 1987; S. Altmann 1989) placed this seemingly anomalous reaction in a broader mechanistic context. Thus, we had discovered the first ribozyme, but without having understood what we had found! A whole family of self-cleaving RNAs at a specific phosphodiester bond in the presence of lead has now been discovered and named leadzymes by T. Pan and O.C. Uhlenbeck [54]. Pb2C ions cleavage has been used as a sensitive structural probe for the folding of RNAs, first in the group of J.-P. Ebel and R. Giege´ [55] then by many others, and also as a footprinting agent for the study of complexes between RNAs and proteins. B. Krebs and C. Werner left my laboratory after completed their PhD and I did not continue to work on the subject. I found 5100 papers dealing with lead and RNA on March 2003 referenced by Medline. Thus, our paper of 1976 [52] should be my most cited one. Do you believe in the Citation Index? Another search for specificity at the tRNA level was done with Leilah Massouh-Rizk a pharmacist from Damascus (Syria). We studied the in vitro interaction of 7-bromomethylbenz(a)anthracene with four isolated yeast tRNAs because F. Pochon and A.M. Michelson [56] had shown that this carcinogen gives substitutions at C8 of guanine and adenine in polynucleotides. With G. Keith we localised the site of substitution in the dihydroU loop of tRNAPhe whereas in tRNAAsp G69, located close to the 3 0 -C–C–A end of the molecule was modified [57]. This result showed a very specific modification depending on the tertiary structure of the tRNAs. L. Massouh-Rizk received her PhD in 1975 and is now Professor of Toxicology in Damascus. I was invited three times to lecture to graduate students in this fascinating city where one can still see the place on the city wall where Saul of Tarsus (St Paul) was lowered in a basket to escape from his persecutors. It was not always easy to lecture because electrical failures stopped the slide projector. Thanks to Leilah and her husband, Professor Hani Rizk, who became good friends of mine, I visited the most interesting places of Syria, Bosra and its famous Roman theatre,

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Ma’alula where the inhabitants still speak Aramaic, the language of Christ, the oasis of Palmyra with the impressive ruins of Queen Zenobia’s city, and the medieval citadel built by the crusaders called the Crack des chevaliers. However, everything was not paradisiac in Syria. During my first stay a professor of law disappeared arrested by the secret police of Hafez al-Assad, and nobody knew where he was. Between 1978 and 1984 I also noticed the evolution of the society, the increase in the number of tchadors amongst the students marking the progress of fundamentalism.

Building the IBMC Since 1966 J.-P. Ebel and Le´on Hirth were thinking about the possibility of joining all laboratories working in Strasbourg on the structure, functioning and regulation of the molecules involved in gene expression. In addition, Ebel’s laboratory at the Institute of Physiology and Biochemistry had become overcrowded. Furthermore, we had no technical services. Thus, he and Le´on Hirth convinced the CNRS to built the Institut de Biologie mole´culaire et cellulaire (IMBM) next to the Institute of Physiology and Biochemistry. They drew up a general file and in 1968 the University gave 3500 m2 to the CNRS on which to build a CNRS Institute. This building was started in 1971 and finished on July 18, 1973. It took much longer to get the money to build the greenhouse and animal house on an additional 800 m2. They were finished only in 1979. In the meantime P. Me´tais had problems in building up his laboratory at the Faculty of Pharmacy because I occupied the main laboratory of biochemistry. Therefore, J.-P. Ebel suggested that I set up my group from the Faculty of Pharmacy in the new IBMC. In 1973 I had the task of supervising the inner installations of the institute with the architect, who had never built a scientific institute before, and the different tradesmen. This was a new experience for me, particularly when I found out, among other curiosities, that water and gas mains coming down

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from the ceiling, were running in front of the bench drawers preventing them from opening! On the other hand, I appreciated that the architect had thought of left-handed people. They are not forced to use their right hands at the IBMC as all the cupboards open on the left! My two groups were now fused into the Laboratory of Biochemistry 2 of IBMC, a group of twelve people. J.-P. Ebel headed the Laboratory of Biochemistry 1 and J.-H. Weil the Laboratory of Biochemistry 3. Athough scientifically independent, our three groups of biochemistry put all their funding together, both the money obtained from the CNRS, INSERM and the University, but also the private research grants coming from Ligue Nationale Franc¸aise contre le Cancer, Association pour la Recherche sur le Cancer, Fondation pour la Recherche Me´dicale Franc¸aise, Commissariat a` l’Energie Atomique (CEA), industry etc. As every group had good and bad years concerning the different grants this system gave an average, which remained almost constant. It permitted the development of research topics which, at first, were not a` la mode and were not financed by grants. At the beginning of each year all the researchers of the three groups met in a great “happening” to decide what large instruments we would buy. We also decided who would go to the different international congresses and workshops. In my opinion, this system developed co-operation and democracy; however it only works if every group asks for all possible grants and this policy does not work if some rely on the others in obtaining funding. At the IBMC there were, in addition to the three biochemistry laboratories, a laboratory of Biophysics, which came from the CRM, now called Institute Charles Sadron, in Strasbourg, and was headed by Michel Daune, a laboratory of Plant Virology headed by Le´on Hirth and a laboratory of Physiological Genetics, headed by Franc¸ois Lacroute, who came from Piotr Slonimski’s group in Gif-sur-Yvette. In addition, the IBMC had different workshops with technicians in carpentry, mechanics, electronics, painting, photography and electron microscopy, as well as its own animal house and greenhouse. At the beginning

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in 1975, 104 researchers were working at the IBMC, 40 from the CNRS, 28 from the University, 1 from INSERM and 35 nonpermanent (doctoral students or post-docs). Additionally, we had 53 technicians and administrative personnel of which 43 were from the CNRS. In 1977 a group of Immunology, directed by Marc van Regenmortel was established in virology. In 1979 a laboratory of Biological Crystallography headed by Dino Moras was created. In 1983 the number of people working at the IBMC was 227, an increase of 44% in 10 years. I have written the history of IBMC which can be found on its home page http://www-ibmc.u-strasbg.fr.

Mitochondrial tRNAs After having corrected the sequence of Saccharomyces cerevisiae alanine-tRNA [38], Robert Martin started to study the mitochondrial tRNAs (mt-tRNAs) of the yeast S. cerevisiae. This was first done in collaboration with Andre´ Stahl’s laboratory at the Faculty of Pharmacy, where B. Accoceberry had developed the isolation of yeast mitochondria. With J.-M. Schneller he showed that mt-tRNAPhe, which hybridises with mitochondrial (mt) DNA, is devoid of the rare base Y, otherwise found in the cytoplasmic tRNAPhe [58]. This result allowed us to exclude the possibility that cytoplasmic tRNAPhe took part in mitochondrial protein synthesis. By preparative chromatography, the mt-tRNAs corresponding to 11 amino acids could be separated and the existence of two isoaccepting species for methionine, tRNAMet/m and tRNAMet/f, both encoded by mt-DNA was shown [59]. The modified nucleoside composition from highly purified yeast mitochondria showed that out of 21 modified nucleosides found in cytoplasmic tRNAs, only eight existed in mt-tRNAs [60]. Using the twodimensional polyacrylamide gel electrophoresis system developed by A. Fradin, H. Gru¨hl and H. Feldmann [61] 27 tRNA spots were separated, all but one hybridizing to mt-DNA. This allowed us to state that the yeast mt-DNA encodes a full set of mt-tRNAs

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sufficient for mitochondrial protein synthesis by the mt-tRNAs alone [62]. This was a very original result. In addition, it was found that one nuclear-encoded tRNA was partially associated with the mitochondrial matrix. This imported tRNA was later characterized as a lysine-accepting tRNA (anticodon CUU) [63]. This was the first clear demonstration of the mitochondrial import of a cytosolic tRNA, although import of tRNA had earlier been suggested by the lab of Y. Suyama to exist in Tetrahymena pyriformis mitochondria [64]. In the meantime, tRNA import has also been demonstrated to occur in mitochondria of a variety of organisms including protozoans, fungi, lower and higher plants and even mammals (for recent reviews see 65–67). The discovery of an imported tRNALys in yeast mitochondria lead to many projects in our laboratory concerning the role of this tRNA, the mechanism of its import, and the possibility to exploit the tRNA import pathway to cure human neurodegenerative diseases due to pathogenic mutations in mitochondrial tRNA genes. These studies are being continued in Robert Martin’s lab.

Ricin, Final Episode At the IBMC A. Lugnier, helped by a pharmacist from Togo (Africa), Edmond E. Creppy (who came to my laboratory in 1975 to complete the Certificat d’Etudes Supe´rieures de Toxicologie and then was appointed assistant of toxicology after the departure of C. Werner in 1976), had set up trypsin hydrolysis conditions for ricin and had fractionated several peptides. They were still toxic, and showed, like the native ricin, a strong inhibitory action on in vitro protein synthesis in a cell-free eucaryotic system, but they were without any action on a procaryotic cell-free system [68]. However, an unfractionated limited trypsin ricin hydrolysate inhibited E. coli protein synthesis [69]. The peptides responsible for this inhibition have still to be isolated. A. Lugnier also studied the kinetics of action of ricin on eucaryotic protein synthesis and particularly the reasons for the lag time which was always

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observed [70]. Studies with eucaryotic cell-free protein synthesis systems were carried out with Anne-Marie Le Meur and Pierre Gerlinger, who is now Dean of the Faculty of Medicine of Strasbourg. We also performed a comparative study of ricin on hepatoma cells and isolated hepatocytes. The action of ricin was followed by scanning electron microscopy in collaboration with E. Petzinger and M. Frimmer in Giessen (Germany). The hepatoma cells developed numerous protrusions on their surface whereas the hepatocytes were an order of magnitude less sensitive to ricin [71]. However, the mechanism of action of ricin was still unknown. We suggested that ricin might act as a specific nuclease on ribosomal RNAs or as a specific protease on ribosomal proteins. With Jean-Jacques Madjar of the laboratory of Professor JeanPaul Reboud in Lyon and Julian Gordon and Guy A. Howard at the Friedrich Miescher Institute in Basel, we studied the ribosomal proteins of a rabbit reticulocytes cell-free protein synthesizing system that had been inhibited by ricin. However, the two-dimensional electrophoretic patterns of the ribosomal proteins were identical to those of the controls [72]. Thus, ricin appeared to be without action on ribosomal proteins. Since according to S.J. Mitchel et al. [73], ricin did not cause any modification of the electrophoretic mobility of ribosomal RNAs, its mechanism of action remained obscure. However, in 1987 Y. Endo et al. discovered that ricin catalysed the cleavage of the N-glycosylic bond of the adenosine at position 4324 in the 28S rRNA [74]. This remarkably specific depurination of this single nucleotide among 7000 accounts for the irreversible inactivation of the ribosome. A. Lugnier had left my laboratory in 1978 and is now Professor of Toxicology at the Faculty of Pharmacy of Strasbourg. In 1986 E.E. Creppy did a series of experiments with H. Bingen and J.P. Gut of the laboratory of A. Kirn in order to establish by electron microscopy a chronological description of rat liver lesions after ricin administration. It appeared that the Kupffer cells were the first target of ricin-induced hepatitis [75]. It is only

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secondarily that hepatocyte injury occurs, probably due to endogenous bacterial endotoxins, which can no longer be cleared by Kupffer cells. It is likely that the death of the animals is not linked to hepatocellular deficiency, but rather to the scattered phenomena of vascular coagulation.

tRNA Modification In the domain of tRNAs I had a collaboration with Evelyne Dubois, from the University of Louvain (Belgium) working in the group of J.-H. Weil, who had already started to work on plant chloroplasts and mitochondria. The question was to know whether there were specific tRNA methylases different from the cytoplasmic methylases in these organelles. Using pure yeast tRNAAsp and tRNAAla E. Dubois showed that the organellar enzymes from bean specifically methylated A7 to m1A in tRNAAsp and G46 into m7G in tRNAAla [76]. It was the first time that organelle-specific tRNA methylases were demonstrated in plants. Following a seminar I gave in Erlangen (Germany) in June 1974 a collaboration started between Helga Kersten’s and my laboratories concerning the methylation of tRNAs by Bacillus subtilis adenine-1 methyltransferase. In collaboration with R. Raettig, a student from her laboratory, J. Weissenbach studied the specificity of this enzyme. Using 16 individual tRNAs they showed that the enzyme was highly specific for the adenosine residue at the 3 0 -terminal of the D-loop (A*). Five tRNAs from S. cerevisiae were methylated in the sequence AA*GGC, but a Torulopsis utilis tRNAIle containing this sequence was not methylated. This pointed to the role of the tertiary structure in the enzyme-tRNA recognition [77], but it also might be that there are determinants and anti-determinants on tRNAs involved in their recognition by methylases, like the identity elements recognised by the aminoacyl-tRNA synthetases. The study of the modified nucleotides of tRNAs were a focal point of our research. In tRNAArg3 we had found an unknown

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nucleotide in the wobble position of the anticodon. Its properties were similar to those of the methyl ester of 5-carboxymethyluridylate (mcm5Up) which had been previously reported by T.D. Tumaitis and B.G. Lane [78] to be released when fractionated yeast tRNA was digested with RNAse A. By comparing the unknown nucleoside with synthetic mcm5U we confirmed this hypothesis [79]. J. Weissenbach showed afterwards that this modification of a uridine prevents its wobble with G [80]. Thus, tRNAArg3, which in yeast represents more than half of the arginine isoacceptors, recognises only one of the six codons of this amino acid. This study on codon–anticodon interactions led to a fruitful collaboration with Rebecca and Ernesto Falcoff working at Institut Curie in Paris, which later was decisive for the career of J. Weissenbach. They had shown that preincubation of extracts of interferon treated cells resulted in an impairment of messenger RNA translation. We showed that this inhibition was reversed, and protein synthesis restored, when one of the four yeast leucine tRNAs was added to the protein synthesising system [81]. J. Weissenbach showed that this tRNALeu had an anticodon U–A–G [82]. Since leucine is coded by six codons, these results could not be explained by the wobble hypothesis alone and showed that this tRNA was able to translate the six codons of leucine, i.e., the codons UUA and UUG in addition to the four codons CUA, CUG, CUC and CUU. This led us to assume that pairing ambiguities (existence of G:U pairs) could take place between the first letter of a codon and the third letter of the anticodon. This was a very original result [82]. J. Weissenbach left my laboratory after his PhD in 1977 for a post-doctoral stay of one year in Michel Revel’s laboratory in Rehovot where he worked on human interferon mRNA. Then he went to Pasteur Institute in Paris where Pierre Tiollais offered him exciting possibilities of development. He is now director of the Genoscope, the French National Sequencing Centre, in Evry, near Paris, and a member of the French National Academy of Sciences.

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Continuing the Sequencing of tRNAs In the 1970s G. Keith sequenced two tRNAsTrp from brewer’s yeast [83], then the tRNAsPhe from beef, calf and rabbit liver [84]. These last two tRNAs were shown to have identical structures. This led to the hypothesis that specific tRNAs have the same structure in mammals, which has since been confirmed. G. Keith developed other techniques for sequencing tRNAs. The sequencing of in vivo 32P-labelled bacterial tRNAs was done according to F. Sanger, G.G. Brownlee and B.G. Barrell [85] and applied to tRNAPhe and tRNAVal2 from Bacillus stearothermophilus with a doctoral student Cecilia Guerrier-Takada from Henri Grosjean’s laboratory, who at that time was working at Universite´ Libre de Bruxelles [86]. Sequencing of tRNAs by our group led to numerous collaborations. Jean-Pierre Garel and Danie`le Hentzen from Lyon were interested in the tRNAs from the posterior silk gland of the silkworm Bombyx mori, known to synthesise silk fibroin which is composed of only four amino-acids. (Gly, Ala, Ser and Tyr). Using a countercurrent distribution of 1500 transfers we obtained the corresponding seven tRNAs (there are two isoacceptors for each amino acid except tyrosine) with a purity of 60–90% and studied their modified nucleotides and their anticodon structures [87]. The sequence of tRNAPhe of Bombyx mori posterior silk gland was determined in 1980 [88]. Another collaboration was with the laboratory of Julie Labouesse in Bordeaux who worked on bovine liver tryptophanyl-tRNA synthetase. With Michel Fournier from her laboratory, Ge´rard Keith and Christine Fix sequenced the tRNATrp [89]. This was a complicated task as several positions were heterogenously occupied by two different nucleotides. This pointed to the existence of several tRNATrp isoacceptors and is particularly interesting as it works as a primer for avian retrovirus reverse transcriptase. Christine Fix was a very skilful technician who worked in our group for 36 years. J. Weissenbach also determined, with a Hungarian student Istvan Kiraly, the primary

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structure of two yeast threonine tRNAs differing by two nucleotides [90]. In 1977 I was awarded the CNRS silver medal for my overall research results. I was elected president of the French Society of Toxicology in 1979. Shortly after J.-P. Ebel arrived from Paris and phoned me (at half past eleven in the evening, as he normally did) and announced to me triumphantly that I had been elected president of the French Biochemical Society. He did not understand why I was not enthusiastic until I told him that I was now president of two societies. I had to wear two hats at the same time for 2 years. In the end, I survived.

Yeast Mitochondrial tRNA Structures and the Genetic Code Continuing his work on S. cerevisiae mitochondria, Robert Martin, aided by a Master of Science student, Annie-Paule Sibler, started to sequence mitochondrial tRNAs. We first determined the sequence of yeast mitochondrial tRNAPhe. This tRNA was purified by two-dimensional polyacrylamide gel electrophoresis with a yield of about 10 mg out of 250 mg of total mt-tRNA put on the gel. Owing to the small quantities of pure material obtained, either in vivo 32P-labeled or 5 0 -32P-postlabelled material was used for sequencing [91]. Due to its very low GCC content this tRNA had a very low Tm of 28 8C and only 6 G:C out of the 21 base pairs in the stem. In addition U50 is excluded from base pairing and gives a bulge in the stem of the T-loop, misnamed in this case because it does not contain a T! We presented this result at the Cold Spring Harbor Meeting on tRNAs in August 1978. At that time, only one other mitochondrial tRNA structure was known. It was that of the initiator tRNA of N. crassa sequenced in Tom RajBhadary’s group. We continued to sequence yeast mt-tRNAs until 1986. The advantage of sequencing the tRNAs instead of their genes is the possibility of characterizing their modified nucleotides. We were also much interested in the codon reading

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patterns of these tRNAs. Every new tRNA sequence brought its share of surprises. Some yeast mt-tRNAs had a characteristic procaryotic structure, like tRNAHis which had eight base pairs in the acceptor stem, tRNAMet/f which had no base-pairing at the 5 0 -end and the presence of TJCAA in the T-loop (work done with Jean Canaday), and RNATyr which has a large variable loop. Later on it was found that tRNAMet/m and tRNALys/2 have a nucleotide bulging out of the TJC stem and that in the tRNAPro the “universal” residues U8, A21 and Py48 were replaced by the residues A8, C21 and A48 respectively (for a review see [92]). At the beginning of the 1980s it became clear that the genetic code used by mitochondria differs in a number of ways from the standard code. Yeast mitochondrial threonine-tRNA was sequenced in our laboratory [93] and shown to have an unusual anticodon loop composed of eight nucleotides instead of the seven present in almost all tRNAs. The anticodon U–A–G of this tRNA allows translation into threonine of the four C–U–N codons, which mean leucine in the universal code. A second exception to the standard genetic code is the use of U–G–A, the opal termination codon in the universal code, to specify tryptophan in mitochondria. We then sequenced yeast mt-tRNATrp which was shown to have a U*–C–A anticodon and thus was able to translate the opal codon into tryptophan [94]. With Henri Grosjean and Suzanne de Henau from Brussels, we could show that this tRNATrp, when injected into Xenopus oocytes together with rabbit globin mRNA, suppressed U–G–A termination with high efficiency, thus leading to a b-globin-related readthrough protein. This also showed that a mitochondrial tRNA was able to function in cytoplasmic protein synthesis thus interacting with elongation factors and with the 80S cytosolic ribosome, allowing transpeptidation and competing with the opal termination factor(s). However, the suppressor activity was strictly dependent on co-injection of E. coli tryptophanyl-tRNA synthetase which was needed to charge the mt-tRNATrp in the oocyte cytoplasm. The absence of a cytoplasmic enzyme capable of acylating the yeast mt-tRNATrp suggested that there was a biological barrier for the

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activity of a mt-tRNA in the cytoplasm, a security in case a tRNA exchange between the two cellular compartments occurred. These results were published in Nature in 1980 [95]. However, the genetic code used in mitochondria not only differs from the standard code, but also it varies from one organism to another. In N. crassa and Aspergillus nidulans mitochondria, A–U–A specifies isoleucine following the universal code whereas in mammalian mitochondria it codes for methionine. We therefore determined the nucleotide sequence of the S. cerevisiae mitochondrial tRNAIle, tRNAMet/f and tRNAMet/m [96,97]. These tRNAs have long stretches of almost identical nucleotide sequences in common, suggesting that they may have arisen by gene duplication from a common ancestor. The tRNAMet/m with a C–A–U anticodon was shown to decode A–U–A in addition to A–U–G. This implied that a C in the first position of the anticodon wobbles with A and G in the third position of the methionine codons. This might be due to the outloop that occurs in the TJC stem of this tRNA. Another peculiarity of the mitochondrial translation apparatus is the restricted number of mt-DNA-coded tRNAs (24 in yeast mitochondria), which is far below the minimal number of tRNAs, i.e., 32, necessary to translate all sense codons according to the wobble hypothesis. From the sequence of six N. crassa mt-tRNAs determined by the group of U.L. RajBhandary [98], the following codon recognition rules were proposed: the tRNAs recognising a four codon family have an unmodified U in the wobble position of their anticodon, whilst tRNAs which recognise a two-codon family ending in a purine have a modified U. We confirmed this rule by sequencing S. cerevisiae tRNASer/2, tRNAGly, and tRNAPro which belong to four-codon families and tRNAArg/1, tRNALys/2 and tRNALeu which belong to two-codon families ending in a purine [99]. We wanted to identify the modified U in the wobble position of the anticodon. So we isolated it from tRNALeu and tRNATrp by thin layer chromatography and HPLC. Its chromatographic, UV spectral and mass spectrometric properties where shown to be identical with those of 5-[[(carboxymethyl)amino]methyl]uridine

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(cmnm5U) [100]. This work was done with Charles Gehrke and Kenneth Kuo from the University of Missouri-Columbia and Cancer Research Center and with James A. McCloskey and Charles G. Edmonds from the University of Utah. To close the chapter of mt-tRNAs, I wrote a review with R. Martin in 1990 about “Mitochondrial tRNAs; structure, modified nucleosides and codon reading patterns” [92]. With Re´my Bordonne´, a pharmacist who joined our laboratory in 1980, R. Martin studied different problems related to yeast mitochondria. First, they showed that a temperature-sensitive mutation leading to a mitochondrial protein synthesis defect phenotype, was due to T-to-A transversion in the long extra-loop of mt-tRNATyr. This base-alteration destabilized the conformation of the tRNA and impaired its aminoacylation parameters. As a consequence, mitochondrial protein synthesis was drastically reduced at the restrictive temperature [101]. With Alexander Hu¨ttenhofer and Brigitte Weiss-Brummer from the Institute for Genetics and Microbiology in Munich, R. Martin studied another mitochondrial tRNA mutation in the serine2-tRNA (anticodon UGA), which leads to suppression of aC1 frameshift mutation in the yeast mitochondrial oxi1 gene. They showed that the suppressor effect was due to a C42 to U base transition and an undermodification of J27 to U, both in the anticodon stem. These changes had a destabilizing effect on the conformation of this anticodon stem, which might induce a structural change in the anticodon loop, enabling the tRNA to read a four-base codon, U–C–C–A, and thus restoring the wild-type reading frame in the oxi1 mRNA [102]. R. Bordonne´ and R. Martin also studied the transcription initiation and RNA processing of a cluster of 5 mt-tRNA genes. The five tRNAs were cotranscribed from a transcriptional initiation site located upstream from the tRNAAla gene [103]. The expression of the oxi1 and maturase-related RF1 genes in yeast mitochondria was also studied [104]. R. Bordonne´ is now Director of Research at the CNRS Institute of Molecular Genetics in Montpellier.

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In 1987, another pharmacist, Etienne Schwob, joined the group of Robert Martin. He started to work on the yeast mitochondrial aminoacyl-tRNA synthetases and showed first that there is no glutaminyl-tRNA synthetase in yeast mitochondria, but that the mitochondrial glutamic acid tRNA synthetase is able to aminoacylate glutamine-tRNA. This deficit in glutaminyl-tRNA synthetase is compensated for by the presence of an aminotransferase transforming Glu-tRNAGln into Gln-tRNAGln. This was a very original result. He also showed that there are two distinct threonyl-tRNA synthetases in yeast mitochondria, one specifically charging the tRNAThr translating the codons ACN, whereas the second enzyme specifically charges tRNAThr, translating the codons CUN, which in the “universal” genetic code specify leucine. It has always been my policy to give more and more autonomy to my co-workers after their thesis and post-doc in order that they take care of their PhD students, start new collaborations and develop new research themes, although remaining in the field of RNAs and protein synthesis. This was the case of Robert Martin who became Director of Research in 1988. He was thesis director of E. Schwob who, later left for post-docs in Dieter So¨ll’s (Yale, USA) and Kim Nasmyth’s (IMP Vienna, Austria) laboratories. E. Schwob is now Directeur of Research at the CNRS Institute of Molecular Genetics in Montpellier. Robert Martin left our Institute in 1992 for the Institute of Physiology and Biochemistry (located next door) where he is now Director of the CNRS laboratory Mode`les Levures de Pathologies Humaines.

Workshops and Congresses Our results on the structure of tRNAs were presented at several workshops in the 1970s and 1980s: the bilateral symposium USSR-France on “Structure and function of nucleic acids” in Puchino near Moscow (1974) followed by a wonderful trip to Armenia, in Concarneau, Britanny (1975), in Tashkent,

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Uzbekistan (1977) and in Tskhaltubo, Georgia (1982). I also attended the 9th and 10th International Congresses of Biochemistry respectively in Stockholm (1973) and Hamburg (1976) and the International conference on the “Synthesis, Structure and Chemistry of tRNAs and their components” in Dymaczewo-Poznan (1976). Many collaborations with Polish researchers ensued. It was more difficult with Soviet researchers. I officially invited several times V.D. Axelrod, from the laboratory of A.A. Bayev (Moscow), with whom we had reinvestigated the structure of yeast tRNAVal, to come for a post-doc to my lab, but his visa was always refused by the authorities. I did not understand why until T.V. Venkstern once told me confidentially not to continue to waste my time because he was Jewish! I never asked about the religion of my coworkers! Fortunately, things have changed since those days in the Soviet Union. V.D. Axelrod as well as T.V. Venkstern later went to the USA. I had more success with Ricardo Ehrlich. One day of 1974 I was contacted by a Dominican monk working for human rights. He asked me whether I could accept in my lab a medical student from Uruguay who was political refugee in Argentina. If he could not find a job in Europe he would be send back to Uruguay and put in a camp there. I approached J.-P. Ebel and told him the story. We had no money to pay him a fellowship, but I told Ebel “You and I have been in a camp during the war. Here we have the opportunity to permit somebody to escape from this situation. We must do something.” J.-P. Ebel immediately accepted and we wrote a false document telling that we could pay him a researcher’s salary and he was allowed to leave Argentina to come to our lab. There he worked with Pierre Re´my, did his PhD in sciences and finally entered the CNRS. When Uruguay became democratic he went back to his country. He is now Dean of the Faculty of sciences of Montevideo. I also set up an interest group of the French Biochemical Society called “Protein Biosynthesis” which met the first time in Strasbourg in 1978, the second time in Paris in 1979, then

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again in Strasbourg, then in Palaiseau in 1982 organised by Sylvain Blanquet. Several of us also attended the EMBO Workshop on tRNA structure and function organised by Fritz Cramer in Go¨ttingen in 1971, by U. Lagerkvist in Gothenburg (Sweden) in 1973 and by Uri Littauer in February 1975 in NofGuinossar, on the bank of the Sea of Galilee. There I learned about the organisation of a Kibbutz. The following tRNA meetings, which I faithfully attended, took place in Sonderborg, Denmark in 1976 and Poznan, Poland 1976. J.-P. Ebel and I also organised an EMBO-tRNA Workshop in Strasbourg from July 16–21, 1980. Prior to sending out invitations I had gone through the Chemical Abstracts of the last 10 years in order to invite all specialists of tRNAs and to not forget anyone. It was a very successful meeting. We went afterwards to the tRNA Meetings that were organised thereafter in Hakone (Japan) in 1983 by Susumu Nishimura, in Banz (Germany) in 1985 by Helga Kersten, in Umea (Sweden) in 1987 by Kerstin Straby, in Vancouver (Canada) by Gordon Tener in 1989 and in Rydzyna (Poland) in 1992 by M. Wiewiorowski and A.B. Legocki. In 1993 I collaborated with Sylvain Blanquet in the organisation of my last tRNA meeting in Cap d’Agde (France). I took a lot of photos at these workshops and congresses and I like to flip through my photo albums remembering all the scientists I met there.

FEBS Officer I have been involved in FEBS activities since J.-P. Ebel had organised the 10th FEBS Meeting in Paris in 1975 where the whole laboratory helped him in different matters. When the FEBS Executive Committee decided to set up FEBS Fellowships in 1978, J.-P. Ebel proposed me as FEBS Fellowships Officer. This was accepted by the FEBS Council in Dresden (July 1978). The FEBS Fellowships programme started in 1979, originally aimed at supporting short-term visits (up to three months) by members

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of any FEBS Constituent Societies to laboratories in another FEBS countries for the purpose of carrying out experiments with special techniques or other forms of scientific collaboration. This programme rapidly became very successful. In the course of 5 years I got 298 applications of which 200 were granted. Most fellows were young scientists with a PhD degree and they have been very appreciative in their reports and grateful for the opportunity that their fellowship gave them. As Fellowships Officer I became a member of the Executive Committee of FEBS and attended its meetings twice a year. Thus I participated in all FEBS Meetings between 1978 and 1983 (Dubrownik-Cavtat 1979, Jerusalem 1980, Edinburgh 1981, Athens 1982, Brussels 1983). In 1983, I was elected FEBS Secretary General replacing Moritz Yomtov from Sofia (Bulgaria) who had been in charge of this office since 1978. This Secretariat gave me more work than the Fellowships office. Fortunately, I was helped by Danie`le Werling the very competent executive secretary of J.-P. Ebel, who, in addition to her full time work at IBMC, attended all the meetings of the Executive Committee and typed all the reports. Thus, we went to the FEBS Meetings in Moscow 1984, Albufeira (Algarve, Portugal) in 1985, Berlin in 1986 (Figure 3), Ljubljana in 1987 and Rome in 1989. The highlights of these meetings have been nicely described by Horst Feldmann in his book “Forty Years of FEBS, 1964 to 2003, A Memoir” [105]. As Secretary General I also attended the meetings of the different FEBS Committees: Publication Committee, Advanced Course Committee and Fellowships Committee. Many changes occurred while I was Secretary General. The Fellowships Programme, under the chairmanship of Carlos Gancedo, enlarged with the creation of Summer Fellowships in 1985. In 1986 we had to replace Prakash Datta who had managed FEBS Letters with single-minded dedication, enthusiasm and hard work since 1968. FEBS Letters would not have become so successful so quickly but for his contribution. Fortunately, we found a highly competent successor for P. Datta in the person of Giorgio Semenza who, with the help of his wife and that of Dr J. Weber, ran the journal in a very successful way

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Fig. 3. Guy Dirheimer with Marianne Grunberg-Manago and J.-P. Ebel (at the FEBS Meeting in Berlin, 1986).

until 2000. We also had to replace Claude Lie´becq who had been the very dedicated chairman of the Editorial Board of the European Journal of Biochemistry since its inception in 1967. I was also very glad that Horst Feldmann, whom I knew since 1974, accepted the important office of Chairman of the Advanced Course Committee, replacing Giorgio Bernardi in 1987. As Secretary General I was also in close contact with all the members of the Executive Committee, particularly with Prakash Datta our very efficient treasurer and the FEBS presidents Yuri Ovchinnikov (1984–1986), Karl Decker (1986–1987), Vito Turk (1987–1989) and Doriano Cavallini (1989–1990) who have remained my good friends ever since. I really very much enjoyed these 11 years of work for FEBS, but I shall come back to that later on.

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DNA Methylation I presented the results of bromomethylbenz(a)anthracene reaction on tRNAs at the Erwin Riesch Symposium on modification of Nucleic Acids in Erlangen in July 1976. There I heard results about a DNA methylation at the C5 of cytosines and also about the recent results of J.K. Christman et al. showing that inhibition of DNA methylation might promote the differentiation and expression of globin genes in Friend’s erythroleukemia cells [106]. This gave me the idea to test the effect of DNA modification by a carcinogen on the enzymatic DNA methylation of this DNA. In 1977 Carlos E. Salas from Chile, who had demonstrated the presence of high levels of tRNA-(adenine-1) methyltransferases in brain tissues from young rats, came to my laboratory to study the site specificity requirements of this enzyme. Using yeast tRNAAsp as substrate we showed that it was the A14 in the D-loop which was methylated [107]. I obtained from the Ligue Nationale Franc¸aise Contre le Cancer a fellowship to support a young pharmacist Annie Pfohl-Leszkowicz to test with C.E. Salas the action of carcinogens bound to DNA upon DNA methylation. They prepared rat brain DNA (cytosine-5) methyltransferase and determined the optimal conditions for the in vitro methylation of DNA by S-adenosyl-L-methyl 3H-methionine (SAM). At the same time in our Institute Robert Fuchs and Michel Daune were working on the action of N-acetoxy-N-2-acetylaminofluorene (AAF) on DNA. They had previously shown that AAF bound on the C8 of guanine induced a rotation of the substituted bases and the accomodation of the fluorene ring between the adjacent bases of the double helix, leading to its local destabilisation. The requirements were therefore fulfilled for a successful projet which, in addition, brought together toxicology and biochemistry. An inverse relationship between levels of DNA methylation and the percentage of AAF substituted guanines in DNA was found. We published this result in Nature in 1979 [108]. It was, to my knowledge, the first time that it was shown that a chemical carcinogen inhibited enzymatic DNA methylation.

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The mechanism underlying this inhibition by the bound AAF residues was studied in detail [109] using rat liver DNA methylase. We showed that the substituted DNA not only was less methylated, but also inhibited the methylation of native DNA. The inhibition was irreversible, but only after a lag time. We proposed a model in which the methylase “walks” along the DNA strands to methylate cytosine residues and is blocked at the site substituted by AAF. With another carcinogen Glu-P-3 fixed on DNA, a similar inhibition of DNA methylation was observed, but the modified DNA did not inhibit the DNA methylation of native DNA because the enzyme had a lower affinity for the modified DNA than for native DNA [110]. Due to the role of DNA methylation in gene activity, cellular differentiation and oncogenesis, our observations were very interesting and were well received when I presented them at several meetings, particularly at the Second International Congress of Toxicology in Brussels (1980). I asked A. Pfohl-Leszkowicz, who had become assistant in toxicology in the meantime, to study systematically the methylation of modified DNAs. The first modification was done in collaboration with Serge Boiteux and Jacques Laval from the Institut Gustave Roussy in Villejuif (France). We showed that the treatment of DNA with dimethylsulfate, which yields mostly 7-methylguanine (m7G) and 3-methyladenine (m3A) did not affect its in vitro enzymatic methylation. Similarly, the presence in DNA of 7-formamidopyrimidine or the conversion of the DNA B-form to its left-handed Z-form did not change its methylation. However, the alkylation of DNA by methylnitrosourea (MNU) which yields in addition to m7G and m3A, methylphosphotriesters and O6-methylguanine, decreased its methylation [111]. Thereafter we modified DNA with 4-acetoxyaminoquinoline-1-oxide, the ultimate carcinogen of 4-nitroquinoline-1-oxide. This was done in collaboration with M.H. Loucheux-Lefe`bre et al. from Institut de Recherche sur le Cancer in Lille (France). Surprisingly, both the initial velocity and the overall methylation of this modified DNA was increased as compared to native DNA.

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This was due to an increase in the Vmax [112]. The same result was obtained with DNA substituted with N-2-aminofluorene [113]. Thus, these substitutions might induce a structure favouring the “walking” of the DNA-methylase along the DNA. In conclusion, the consequences of DNA modifications by carcinogens are not uniform and have to be determined for each carcinogen. However, both hypomethylation and hypermethylation of DNA can have biological consequences as both the expression of oncogenes and the inhibition of the expression of tumour suppressor genes can contribute to cancer development. In addition, our results were obtained in vitro and need to be validated in vivo for each carcinogen. We investigated N-nitrosomorpholine which inhibits DNA methylation in vitro. We showed, in collaboration with Karl Walter Bock et al. from the University of Tu¨bingen, that in liver nodules obtained by N-nitrosomorpholine treatment of rats, the first intron of c-myc DNA was hypomethylated. This could be responsible for persistently increased c-myc expression in liver nodules [114]. This research on DNA methylation led me also to become a friend of Dusan Drahovsky, Professor of Biochemistry in Frankfurt, who organised informal workshops on DNA methylation. The last one we attended was in the village of Montroc near Chamonix where we alternated skiing and science. Unfortunately D. Drahovsky died from a melanoma three months later. These studies increased our interest in DNA-methylase. We tested its activity in various rat tissues after administration of MNU. Both total and specific activities of the DNA methylases of the brain, where tumour induction by MNU is important, were increased. Liver DNA methylase activity did not change which correlated with the fact that this organ was not susceptible to MNU-induced cancers. We argued that there is a relationship between the effects of MNU and DNA methylase activity [115]. We also found that DNA-methylases of several organs were stimulated by divalent cobalt ions [116], particularly the spleen enzyme activity. This led us to analyse the influence of vitamin B12, methylcobalamin and coenzyme B12 on de novo DNA

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methylation. In the presence of SAM these compounds significantly enhanced methylation at concentrations up to 1 mM. At higher concentrations methylcobalamin behaved as a competitive inhibitor of SAM. The use of radioactive methylcobalamin showed that it can be used as a methyl donor in DNA methylation reactions [117]. Thus, two DNA methylation pathways could exist, one involving methylation from SAM and a second one involving methylation from methylcobalamin. This result, although original and confirmed by Russian researchers (published in Russian) has not been explored by other authors up to now. It may well be rediscovered several years hence, as was the case, as we have seen, with some of our other findings. On the other hand, our discovery concerning the effect on DNA methylation of carcinogen modified DNAs has propagated like the loaves and the fishes.

Applied Research Although we did essentially basic research, I did not refuse to do applied research when I was asked for help. Maurice Vigneron introduced me to Dr Deckers and Dr Pollmann from BoehringerIngelheim who were searching for antiviral drugs. J.-P. Ebel et al. (1968) and P. Louisot et al. (1968) had previously shown that chemically modified RNAs inhibited the multiplication of some viruses in cell cultures. In 1975 I engaged Genevie`ve Pixa who was financed by Boehringer-Ingelheim. In collaboration with Pierre Louisot, Professor of Biochemistry at the Faculty of Medicine in Lyon, we measured Sindbis virus multiplication in mice previously i.p. injected with pure tRNAs. A 100-fold inhibition was obtained with 10 mg of purified tRNA per mouse. A 22 nucleotide long fragment of yeast tRNAThr comprising its T–J–C loop was as effective as the whole tRNA. We proposed that this might compete with strategic region in viral RNA. This work led to a patent but not to a useful drug because nasal administration was without effect. Thereafter, G. Pixa sequenced

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with G. Keith tRNAPro2, tRNAIle (anticodon IAU) and two tRNAsHis from brewers yeast [118,119] and also tRNALeu from Bacillus stearothermophilus [120]. Another collaboration with the pharmaceutical industry started in 1981. It concerned trenbolone, a semi-synthetic androgenic anabolic steroid which is implanted in calves. I was approached by J.C. Bouffault from Roussel-Uclaf who asked me whether trenbolone might have a hepatocarcinogenic activity by binding covalently to DNA. It was decided to compare it to two androgenic steroid hormones, 17b-oestradiol and testosterone, which belong respectively to the oestrogen and androgen groups, to another exogenous oestrogenic anabolic agent, zeranol. The principle of the experiment, which had been carefully studied by W.K. Lutz in Zurich [121], was simple. We injected radiolabeled hormones into rats, and after a given time, killed the animals, extracted the DNA from their liver and counted its radioactivity. The first difficulty was the use of very radioactive hormones, about 50 Curies/mM, in order to be able to detect very low DNA binding. The other problem was to obtain very pure DNA, not contaminated with RNAs or proteins which are also able to fix the hormones. As the purification procedures found in the literature were not thorough enough, we had to elaborate a new one using hydroxylapatite column chromatography and centrifugation in CsCl gradients. Be´atrice Barraud was engaged by Roussel-Uclaf and performed the experiments in my laboratory. We determined what is called the covalent binding index (CBI) which is the number of mmol of product bound per mole of nucleotide of DNA divided by the number of mmole of the product administered per kilogram of animal [121]. Thus the CBI is independent of the amount of drug administered. CBI values for various carcinogens span six orders of magnitude and a direct correlation between the CBI and hepatocarcinogenicity has been demonstrated in the rat. We found, using male rats, a low but significative CBI of 7.8 for trenbolone 16 h after i.p. administration. With 17b-oestradiol we obtained a CBI of 11.4, with testosterone 4.8 and with zeranol 1.6. As a comparison, N-hydroxylaminofluorene, a proven

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hepatocarcinogen, gave a CBI of 262. Thus, trenbolone had weak genotoxic activity but the CBI decreased rapidly with time. Four days after administration it was only 1.1, emphasizing the rapid repair of DNA [122]. This study introduced us to the domain of genotoxicity testing which, as we shall see, was very useful in the study of ochratoxin A. Another applied study was carried out with Bernard Montagnon. At Institut Me´rieux in Lyon they were preparing inactivated poliomyelitis vaccines from infected Vero cells grown in microcarrier culture. However, this continuous cell line showed abnormal cytological and genetic characteristics. The purification of the vaccines must, therefore, eliminate contaminant cellular DNA. The World Health Organisation Expert Committee on Biological Standardisation asked for a reduced level by a factor of 108 of this DNA from that of the initial virus harvest. As no conventional technique was sensitive enough to assay picogram amounts of DNA, B. Montagnon asked me to develop such a method. Be´atrice Barraud-Hadidane took over this problem and with R. Martin we developped three different DNA–DNA hybridisation techniques. The results deduced for the final vaccine preparation, taking into account the dilution factor and DNA recovery, ranged from 0.025 to 1.94 pg/ml for 10 of the batches and between 2.5 and 6.3 pg/ml for four batches [123]. This technique permitted Me´rieux to obtain the approval of the Food and Drug Administration for their polio vaccine, and later on for their anti-rabies vaccine. It was always my policy to help industry, but not to do systematic determinations for it afterwards. Once the Me´rieux laboratories learned our technique we did not continue this work.

EST Congress in Strasbourg and EUROTOX I presented the trenbolone results in 1982 in Zurich at an Interdisciplinary Conference on Food Toxicology and was asked whether I would be willing to organise the 28th Congress of the

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European Society of Toxicology (EST) in Strasbourg in 1987. I accepted and became member of the Executive Committee of EST. I attended the EST congresses in 1984 in Budapest, Kuopio in 1985 and Harrogate in 1986. EST was a society with individual members, but many national societies had developed in the 1970s and the need was felt for a representative organisation for these societies. Consequently, the Federation of European Societies of Toxicology (FEST) was founded in Kuopio in 1985 and the Congress I organised in Strasbourg was an EST/FEST congress. In fact, it was organised in a “home-made” way, i.e., we did everything ourselves in the lab except the hotel reservations. I was particularly helped by our devoted secretaries Gaby Issler and Danie`le Werling but also by Monique Schlegel, Ge´rard Keith and Claudine Bollack. Even my daughter Pascale was engaged for the reception desk. The congress took place in the Humanities University that had a lecture hall for 600 people and was situated just opposite our institute. The posters were in the corridor leading to the lecture hall and the exhibition around the lecture hall which the 11 exhibitors appreciated. The rent was very cheap, about 1000 euros, so I decided to pay a painter to give the toilets a badly needed face-lift! We rented a large tent, which was set up beside the university for lunch. Fortunately, as the congress took place in September, we had wonderful weather. As EST had many members from pharmaceutical and chemical companies I was helped financially by 20 of them and could offer a cheap registration fee of 1100 francs (about 170 euros), meals included. There were three symposia, Phototoxicity, Photoallergy, Photomutagenicity and Photocarcinogenicity, Nephrotoxicity and Peroxisome proliferation, and a workshop on Adverse effects on thyroid gland, its relation to species. In choosing the dates of the congress I had carefully asked about the dates of the meetings of the Council of Europe in order not to overlap, because at that time the number of hotel rooms in Strasbourg was limited. Everything was running smoothly until 2 months before the congress when the Council of Europe decided to shift its session to the week I was running my congress.

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All the hotels with which I had written agreements for a certain number of rooms wrote me indicating that they had an order of the pre´fet (!) giving priority to the Council of Europe and that they now would give me 10% of the rooms I had reserved. I almost had a heart attack and scrambled to find rooms outside the city, even across the Rhine in Kehl (Germany), and to provide the participants of the congress with buses bringing them to the congress site. I vowed it was the last time I would organised a congress in Strasbourg and I kept my word and organised the 1999 FEBS Meeting in Nice! Except for the accomodation problem, the congress was a success with 500 participants attending from 30 different countries. The social programme was important with a reception at the Town Hall followed by an organ and trumpet recital in the Cathedral on the first evening, an excursion to Riquewihr on the evening of the second day (Figure 1) with a reception followed by wine and folkloric dances and dinner in different restaurants. Finally, on the third evening we had the congress banquet at the Restaurant du Kochersberg in Landersheim outside Strasbourg. As I like jazz I had also hired a good orchestra for dancing. The proceedings of the Meeting called; The Target Organ and the Toxic Process, were published by Philip Chambers, Claire Chambers and myself as a Supplement of Archives in Toxicology. Philippe and Claire became good friends and I was always happy to meet them at the numerous meetings which I attended thereafter. In the meantime it became evident that the activities of EST and FEST were overlapping, and efforts were taken to merge them into only one organisation called EUROTOX. As a member of the Executive Committee of EST, I was pushing for this merging having seen how successful it was in biochemistry with FEBS. EUROTOX was founded in 1989 and Christian Hodel (Basel), the last president of EST became the first President of EUROTOX for 1 year. I followed him as President of EUROTOX in 1990 until 1992 and was member of its Executive Committee until 1995. I became an Honorary Member of EUROTOX on June 27, 1999 in Oslo.

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Cloning and Sequencing of Aminoacyl-tRNA Synthetases Genes Cloning, site directed mutagenesis, and DNA sequencing was set up in our laboratory by Jean Gangloff. Continuing his research on yeast AspRS with Gilles Pre´vost and M. Sellami he succeeded in 1985 in the isolation and characterisation of the gene encoding AspRS [124]. They also established its nucleotide sequence as well as those of its 1600- and 600-nucleotide-long upstream and downstream flanking regions [125]. Screening for functional domains was realised by site directed and deletion mutagenesis followed by determination of the kinetic properties of the mutated enzyme. The domains involved in aspartic acid activation and in tRNA aminoacylation were characterised [126,127]. This was done with Gilbert Eriani a Masters in Biochemistry student who joined our group in 1984. He also studied the dimerisation of yeast AspRS and established its functional importance. He showed that an invariant proline in the structural motif I of this enzyme was a key amino acid for dimerisation [128]. Furthermore, he made a major contribution to the finding that aminoacyl-tRNA synthetases may be partitioned into two classes according to the structure of their active sites [129]. The specificity of recognition of E. coli AspRS with tRNAAsp was studied by Franck Martin during his thesis preparation in order to compare it with the results we had obtained with the yeast system. He used a tRNAAsp mutated in the anticodon (CUA instead of GUC), which was inactive and selected by a genetic screen, ten AspRS mutants active with this tRNA. By localisation of the mutated residues in the AspRS [130] mutants, he could validate the structural model of the AspRS-tRNAAsp complex elaborated in Dino Moras’ laboratory, particularly the role of the anticodon in the specific recognition of AspRS. In another genetic selection he isolated AspRS molecules that had changed their specificity, i.e., were able to recognise an amber suppressor tRNAAsnCUA. These mutants showed deletions in the N-terminal

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domain suggesting that this region of the native enzyme had antideterminant elements directed against tRNAAsn [131]. As I have already mentioned, the other aminoacyl-tRNA synthetase in which we were most interested was ArgRS. G. Eriani isolated and determined the primary structure of its gene from E. coli in 1989. This also permitted him to develop a highly efficient method for purification of its 100-fold overproduced expression product [132]. Two other aminoacyl-tRNA synthetases from E. coli were cloned and characterised in 1990, cysteinyl-tRNA synthetase and aspartyl-tRNA synthetase [133, 134]. The latter one showed important homologies of its translated amino acid sequence with asparaginyl- and lysyltRNA synthetases showing more than 25% identity. Finishing my research on tRNAs I wrote a review with G. Keith, P. Dumas and E. Westhof about primary, secondary and tertiary structures of tRNAs [135]. Teaching and Committees In France, University professors had to lecture for 80 h a year until the Savary law of 1984 which increased it to 128 h. At the beginning I lectured mainly in biochemistry and toxicology to pharmaceutical students. I also introduced teaching in genetics, which was afterwards taken up by J. Weissenbach. After a new professorship in Molecular Biology was created I lectured in general toxicology (absorption, distribution, metabolism and excretion of toxicants and general mechanisms of toxicity) and genetic toxicology at the Faculty of Pharmacy, but also at the Ecole supe´rieure de Biotechnologie, at the Science Faculty in the Master of Science and Techniques option, environmental sciences, at the Faculty of Medicine in the Diploˆme Universitaire of clinical toxicology, and in the national DEA (masters) of toxicology in Paris and, finally, in the DEA of environmental toxicology I had joined in 1989. This was organised with Professor Paule Vasseur and Professor J.-M. Jouanny between the Universities of Metz, Rouen and Strasbourg. This, together

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with all the exams to correct, that, I once calculated, took more time than lecturing, occupied also a non-negligible part of my days. However, I liked to teach and explain and tried to follow the formulae “What is clearly understood can be clearly expressed.” As biochemist I became member of EMBO in 1974 and as toxicologist member of many committees: Conseil supe´rieur des Universite´s, Comite´ national de toxicovigilance, a specialised committee of INSERM, Vice-President of the Committee of Grants for Training and Research in Toxicology of the European Science Foundation, member of the Senatorial Commission for Food Contamination (Senatskommission zur Beurteilung der gesundheitlichen Unbedenklichkeit von Lebensmitteln) at the German National Research Organisation (Deutsche Forschungsgemeinschaft) in Bonn, where all the sessions were held in German. Fortunately, I was not a member of all these committes at the same time! I was also responsible for the Action Toxicologie of the Ministery of Research of and Technology (1991–1992). I remember an extraordinary lunch with the Minister Hubert Curien at the Ministery, rue Descartes (lobster, bass fillets and oyster mushrooms a` l’Anglaise!). However, I did not systematically accept all proposals made to me. For example, I refused, to the surprise of many colleagues, to accept the proposal of Claude Paoletti, the Director of the CNRS Life Sciences, to become his Assistant-Director. This would have retained me at least 3 days a week in Paris at the expense of my laboratory and my family in Strasbourg. I am also not sure how harmonious a couple we would have been. I became corresponding member of the French National Academy of Medicine in 1983 and was elected as a full member in 1988. There, I am involved in the committee Nutrition et alimentation. Reorganisation of the IBMC An important reorganisation of IBMC took place in 1987 subsequent to the building by the CNRS (nearby to our institute)

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of the Institute of Plant Molecular Biology (IBMP) where the groups of J.-H. Weil and of L. Hirth had emigrated. In addition, a group of J.-P. Ebel’s laboratory headed by P. Re´my who was interested in developmental biology had moved next door to the Institute of Physiology and Biochemistry. In addition, Franc¸ois Lacroute had left to reintegrate the Molecular Genetics Institute in Gif-sur-Yvette and Michel Daune went to Orle´ans. Robert Fuchs had succeeded him as head of the Laboratory of Carcinogenesis and Molecular and Structural Mutagenesis. Two groups of Daune’s laboratory, those of Manfred Schnarr, who worked on the regulation of the SOS system and that of Gilbert de Murcia working on poly(ADP-ribose) polymerase, were transferred to my laboratory. They had complete scientific autonomy although I followed their very interesting research. Everybody also attended our weekly internal seminars. Thus, in 1989 my laboratory counted about 30 researchers and technicians and many researchers from Germany, Canada, Poland and Tunisia stayed for various periods with us (18 between 1987 and 1989!). I had by then given complete autonomy to the groups of G. Keith, J. Gangloff and R. Martin who were Directors of Research in CNRS and group leaders. I followed most closely the research dealing with toxicology. In addition, I was Assistant-Director of IBMC, when J.-P. Ebel was Director (Figure 4).

Toxicology of Madagascar Plants and Mushrooms Between 1983 and 1986 three Assistant Professors from Antananarivo (Madagascar) worked in our laboratory to prepare their doctorate. They intended to do toxicology on subjects important for Madagascar. Victor Jeannoda wanted to study the toxic principle of Cnestis glabra (Connaraceae) seeds and root barks used by the natives to set poisonous baits for noxious animals, essentially for rats and stray dogs. In my laboratory in collaboration with E.E. Creppy he isolated a neurotoxic compound by a five-step fractionation procedure [136]. It gave

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Fig. 4. Christmas 1985 in the lab. From left to right: Annie Pfohl-Leszkowicz, Edmond E. Creppy, Monique Schlegel, Guy Dirheimer, Robert Martin, Wafa Arafat and Ge´rard Keith.

convulsive attacks in mice when injected i.p. We first called this compound glabrin, but as this name had already been given to another compound, we called it cnestine. Its structure was determined to be a methionine sulfoximine [137]. It was a deception for V. Jeannoda because this compound was already known, having been found in “agenised” flour. It had been shown to be a potent inhibitor of glutamine synthetase. We crystallised it with B. Chevrier from Dino Moras’ laboratory and established its absolute configuration which is the 2S,SS form [138]. However, even if this structure was already known, it had not been found before in a plant. A second study was undertaken by Charlotte Ralison on Croton mongue a Euphorbiaceae from Madagascar. It was known to have toxic stems and seeds. We purified a thermostable toxic protein from the seeds corresponding to a molecular weight of 9000.

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It inhibited protein synthesis in hepatoma tissue culture and globin synthesis in rabbit reticulocyte lysate [139]. The third study was done with a mushroom, Boletus affinis Peck (Boletaceae) by J. Louisette Razanamparany. In the Boletaceae family many species are edible and even renowned, but some others are indigestible or have toxic properties when eaten in a fresh uncooked state. In Madagascar Boletus affinis causes the death of the zebus which graze on them, but it was not considered as toxic to man. We thought that it might contain a toxic protein, which would explain why it is toxic in the raw state and not toxic when cooked. We purified a thermolabile toxic protein of Mf 22,000, which we called bolaffinine. We determined its physico-chemical and toxicological properties and showed that it also inhibited protein synthesis in hepatoma tissue culture and globin synthesis in rabbit reticulocyte lysate [140]. V. Jeannoda presented his doctorate in Strasbourg and C. Ralison and J.L. Razanamparany in Antananarivo. All three are now University Professors in Antananarivo. This collaboration gave me the opportunity to go three times to Madagascar where I lectured in toxicology (the first time this field had been lectured there). I also travelled during two weekends in this fascinating country, once to Nossi-Be and once to St. Marie Island where I dove admiring the marvellous coral fishes. After having found a toxic protein in a boletus from Madagascar, we wondered whether European boletus also contain toxic protein. We turned to the well-known Boletus satanas that causes serious gastro-enteritis in humans. With a PhD student Olivier Kretz we purified to homogeneity a monomeric glycoprotein of Mf 63,000 we called bolesatine [141]. It possesses lectinic properties with in particular a sugar binding site for galactose [142]. The disposition and toxicokinetics of 14C-bolesatine were studied. It was highly resistant to the classical proteolytic enzymes and was found intact in the urine of intoxicated rats [143]. It was also shown to be mitogenic to human lymphocytes in vitro at very low concentrations (0.1 mg/mL) [144]. At higher concentrations,

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bolesatine inhibited protein synthesis in vitro in several systems and in vivo. But bolesatine cannot be included in the group of RIP because these organelles were still active after preincubation with the lectin [145]. It fact it had a GTP hydrolysing activity with consequent inhibition of protein synthesis [146]. Finally, when tested for cell agglutination, bolesatine agglutinated both human platelets and erythrocytes. This activity was observed for a concentration of 30 nM [147]. These studies led us to collaborate with L. Barbieri and F. Stirpe from Bologna, J.-P. Cazenave and his co-workers from the blood transfusion centre in Strasbourg and again with J.-P. Reboud et al. from Lyon. Olivier Kretz presented his thesis in 1992. He is now working at the Novartis Institute for Biomedical Research in Basel. We had another collaboration on a mushroom toxin, thus time with Jean-Michel Richard from the Faculty of Pharmacy in Grenoble. Orellanine is a nephrotoxic compound extracted from the mushroom Cortinarius orellanus that is known to induce acute renal failure several days or weeks after ingestion. We evaluated its effect on DNA, RNA and protein synthesis on a cell line of renal origin (MDCK cells). The macromolecular syntheses were strongly inhibited. However, direct addition of orellanine to a cell-free system of rabbit reticulocyte lysate did not produce any inhibition of protein synthesis, whereas when orellanine was pre-incubated with an activating rat liver microsomal system, inhibition occurred. We concluded that the in vivo inhibition of protein synthesis is most likely due to a metabolite of orellanine [148].

Evolution of the IBMC In 1991 the structure of the IBMC changed again due to the departure of J.-P. Ebel who had been appointed Director of the CNRS Institute of Structural Biology in Grenoble. The CNRS department of life sciences had proposed a new structure for the IBMC which on January 1, 1992, became a GDR (Groupement de

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Recherche). I became Director of it and Robert Fuchs AssistantDirector. It consisted of five independent units (UPRZUnite´s Propres de Recherche), “Structure of Biological Macromolecules and Mechanisms of Recognition” (Director Bernard Ehresmann), “Structural Biology” (Director Robert Fuchs), “Molecular Mechanisms of Cell Division and Development” (Director Pierre Re´my) and an associated unit “Dynamism, Evolution and Expression of Microorganism Genomes” (Director Jean-Claude Hubert). In addition, the university created for 4 years a Centre of Research composed of the same laboratories, of which I became also the Director. The great difference with the former organisation was that previously the IBMC was a unique unit, associated with the university and comprising several laboratories, whereas the GDR was composed of five independent units associated with the university through a research centre. Why do something simply if you can make it complicated? J.-P. Ebel fell ill with an acute pancreatitis during the Soviet– French workshop in Roscoff (Brittany) in June 1992 and died 3 days later in Grenoble. This was a great shock for me and for all his former collaborators and friends. I hired a bus and we all went to the little chapel of La Tour-Sans-Venin above Seyssinet-Parisy near Grenoble where he owned a house and where so many of us had been invited by him and Mrs. Jacqueline Ebel. He is buried there overlooking the valley of Grenoble surrounded by the spectacular mountains of Chaıˆne de Belledonne and Grande Chartreuse. I set up a J.-P. Ebel foundation in his memory which every year gives fellowships to young biochemists permitting them to attend an international congress or workshop. Marianne Grunberg-Manago paid homage to him in a paper published in the Comptes Rendus de l’Acade´mie des Sciences. Finally, in 1994 a new reorganisation of the IBMC took place following the departure of the “Structural Biology” unit to join the IGBMC (headed by Pierre Chambon), of the “Carcinogenesis and Molecular and Structural Mutagenesis” unit to integrate the Ecole supe´rieure de Biotechnologie, and of the “Dynamism, Evolution and Expression of Microorganism Genomes” unit to

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the Botanic Institute. The GDR was transformed into an “Federative Institute of Research” which was created for 4 years. I could no longer be Director of it because I was 63 years old, and for the CNRS one must not to be more than 61 years old to become Director of a unit or institute for a 4 year period. Jules Hoffmann became Director of the institute which now comprises three independent units, “Stucture of Biological Macromolecules and Mechanisms of Recognition” (Director Bernard Ehresmann), “Immunopeptides, Autoimmunity and Immunomodulation” (Director Marc van Regenmortel, now Sylvianne Muller) and “Immune Response and Development in Insects” (Director Jules Hoffmann). This structure is still ongoinging in 2003.

Ochratoxine A I shall finish the recollections of my research with a subject of toxicology, which occupied me and my co-workers for 22 years. As we shall see it was also closely related to my favourite subject, protein synthesis. At a Meeting in Pont-a`-Mousson, near Nancy, held on May 25, 1977 dealing with mycotoxins, which I attended out of curiosity (as I was not working in the field) I met Robert Ro¨schenthaler who was Professor of Microbiology at the University of Mu¨nster (Germany). He presented (in French!), results about ochratoxin A (OTA) a mycotoxin produced by several species of the fungal genera Aspergillus and Penicillium. It is a widespread contaminant in human food and animal feed. R. Ro¨schenthaler showed that OTA inhibits protein synthesis in Bacillus subtilis and causes accumulation of the regulatory nucleotides ppGpp and pppGpp, pointing to an inhibition of tRNA charging [149]. OTA is a chlorinated isocoumarin coupled to b-phenylalanine by a peptide bond as had been shown by K.J. Van der Merve et al. in Pretoria (South Africa) [150]. This result interested me immediately, particularly when he told me that with I. Konrad he had been able to show a lower degree of tRNAPhe aminoacylation in bacteria [151]. We decided to

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collaborate on the subject and, with I. Bunge, we showed that in a polyuridylic acid-dependent peptide synthesis system of B. stearothermophilus, the synthesis of polyphenylalanine was competively inhibited by OTA. When aminoacylated phenylalanine-tRNA was included in the system, peptide synthesis was not inhibited [152]. This clearly showed an inhibition of phenylalanyl-tRNA synthetase (PheRS). I easily convinced E.E. Creppy in my laboratory to join this very interesting project. He did essentially all the research on OTA until 1989. As we had at IBMC pure PheRS and pure tRNAPhe, both from yeast, a eukaryote, we could study the kinetics of the action of OTA on the two reactions catalysed by PheRS, aminoacid activation and tRNA charging [153]. After working with yeast enzymes we studied the action of OTA on hepatoma tissue culture cells and kidney cells. These latter cells were shown to be very sensitive to OTA [154]. Let us remark that OTA is a potent nephrotoxin in most animals and most probably responsible for Balkan endemic nephropathy in man. In all reactions OTA behaved as a competitive inhibitor of phenylalanine and high amounts of phenylalanine could reverse the inhibition. This was also shown in mice where phenylalanine prevented acute intoxication by OTA and inhibition of protein synthesis in different organs [155–157]. With F.C. Størmer, who came to Strasbourg, we showed that a metabolite of OTA in animals, 4-hydroxy OTA, is just as inhibitory as its parent compound [158]. With Pieter S. Steyn and his co-workers from Pretoria we tested the analogues of OTA where the phenylalanine moiety was replaced by other amino-acids. They behaved like OTA on their corresponding aminoacyl-tRNA synthetases [159]. OTA also inhibited phenylalanine hydroxylase, but it also behaved as a substrate of this enzyme being transformed in rat liver into tyrosine-OTA [160]. It would be too long to give details on all the work we did on OTA, which has been published in 60 papers. We have written a review on the mechanism of action of OTA which can be easily consulted [161]. With Amadou Kane, a hard working PhD student from Dakar (Senegal), we studied the distribution of

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H-labelled OTA ingested by rats [162]. This permitted us to show the enteropatic circulation of this mycotoxin [163]. The changes in urinary and renal tubular enzymes caused by subchronic administration of OTA in rats were also followed. Protection by phenylalanine was again effective [164]. We also discovered that OTA and its metabolite have an immunosuppressive activity, even with very low doses (1 mg/kg in mice) [165]. I gave several seminars on OTA in different countries of Europe and convinced several colleagues to work on the subject, for example, M. Gekle and S. Silbernagl, kidney physiologists from Wu¨rzburg (Germany), who afterwards did very nice work on the nephrotoxicity of OTA. These studies led us to work with other mycotoxins like citrinine, which may occur simultaneously with OTA in mould-contaminated commodities, and is also nephrotoxic. We showed that the two toxins have a cooperative effect on DNA, RNA and protein synthesis in MDCK cells [166]. During his Master of Science Bertrand Rihn, who is now Professor of Biochemistry in Nancy, studied the mechanism of action of patulin, another mycotoxin [167]. R. Ro¨schenthaler died prematurely in 1986. E.E. Creppy and myself, who had become very good friends with him and his family, having been several times in Mu¨nster as he to Strasbourg, were terribly sad. R. Ro¨schenthaler was an eminent scientist with a passion for science, always in a good mood. It was the best foreign collaboration I have had. As he left two doctoral students who had not yet finished their thesis we took charge of them until completion of their PhD. In addition to its nephrotoxicity, OTA has been shown to induce renal adenomas and carcinomas in mice and rats (for a review see 168). In humans, Balkan endemic nephropathy is often associated with urothelial tumours. We came on the mechanism of genotoxicity of OTA in a collaboration with Charles Frayssinet and Christiane Lafarge-Frayssinet from Villejuif (France). We showed that single stranded breaks were induced in the DNA of splenic cells exposed to OTA [169]. These breaks were also found

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in vivo in kidney and liver of mice and rats having received OTA [162]. This showed for the first time that OTA was genotoxic. E.E. Creppy succeeded in the agre´gation of Toxicology in 1989 and became Professor of Toxicology at the Faculty of Pharmacy in Bordeaux. He has set up a large laboratory and is working successfully on several subjects. A. Pfohl-Leskowicz had taken up the subject of OTA on the departure of E.E. Creppy, but we continued to collaborate with him. A wide-ranging study was undertaken in three regions of France to assay the ochratoxin A content in human blood. 3070 blood samples were tested and showed an average of 14–25% contamination with more than 2 mg/L [170]. This result was presented at an International symposium on human ochratoxicosis and its pathologies organised by E.E. Creppy in 1993 in Bordeaux. With Hassen Bacha, Professor of Biochemistry in Monastir (Tunisia), several studies were performed. They showed a high contamination of some foods in Tunisia [171,172] explaining the human ochratoxicosis in this country. In order to explain the carcinogenic effect of OTA I wanted to check whether it gave rise to DNA adducts. However, it was not possible to determine the CBI because the specific activity of our radiolabelled OTA was too low. Therefore, in 1988 I asked Ge´rard Keith to go to the laboratory of Kurt Randerath in Houston (Texas) to learn his postlabelling method. After returning he set up the method in our laboratory. With A. Pfohl-Leskowicz we showed that OTA treatment induced the formation of DNA adducts in a dose- and time-dependent manner, both in mice and in rats [173]. This was found predominantly in kidney and urinary bladder [174], but also in liver and spleen. We also showed that decreasing the glutathione level in kidney led to decrease of DNA adducts suggesting that the genotoxic metabolite might be a glutathione derivative [175]. In collaboration with M. Castegnaro and H. Bartsch from the International Agency for Research on Cancer in Lyon and with I.N. Chernozemsky from Sofia (Bulgaria), we analysed for DNA adducts tumoral tissues from three kidneys and five bladders of Bulgarian

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patients from the area of Balkan endemic nephropathy who had undergone surgery for cancer [175,176]. Several adducts, with the same RF values as those obtained from mouse kidney after treatment with OTA, were detected, mainly in kidney, but also in bladder tissues from Bulgarian patients. These results provided new evidence of a possible role of OTA in the development of tumours of the urinary tract. A. Pfohl-Leszkowics left my laboratory in 1994 and became Professor of Toxicology at Ecole Nationale Supe´rieure Agronomique in Toulouse. OTA was for a long time believed to be non-mutagenic in the classical Ames Salmonella typhimurium reverse mutation test. However, using mice kidney microsomal fractions as metabolic activators my last PhD student Sophie Obrecht-Pfumio, in collaboration with Professor Daniel Marzin from Pasteur Institute in Lille, obtained reverse mutations in the presence of either NADP or arachidonic acid as cofactors, indicating that several metabolic pathways of ochratoxin A can lead to genotoxic compounds. In addition, both base pair substitutions and frameshift mutations can be caused by ochratoxin A after metabolic activation [177]. The negative results obtained by several authors before us can be explained, simply because they used liver fractions as metabolic activators whereas OTA gives kidney and urothelial cancers but not liver cancers in rats. With Professor G.H. Degen from Dortmund we also showed that OTA induced micronuclei in ovine seminal vesicle cells [178]. S. Obrecht-Pflumio also showed that pre-treatment with indomethacin, an inhibitor of prostaglandin H synthase (PGHS), dramatically reduced the DNA adduct level in kidney [174]. In view of the prospective role of PGHS, we performed in vitro experiments using kidney microsomes, incubated with DNA, OTA and arachidonic acid. Our results showed that OTA gives rise to DNA adducts in vitro. Thus, the DNA adducts we had found in vivo were not coming from secondary effects like cytotoxicity as proposed by other authors. OTA was clearly shown to be genotoxic after its activation by the peroxidase activity of PGHS. We identified the DNA nucleotide modified by

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the OTA metabolite(s) as dGMP by using different nucleotides and polynucleotides as substrates [179]. Horseradish peroxidase, which has an enzymatic action very close to the peroxidative activity of PGHS, was also able to transform OTA into a genotoxic metabolite acting on DNA and dGMP. We published these results as my last scientific paper in 2001 [180] and I summarised what was known on the genotoxicity of mycotoxins at an international symposium in Toulouse [181]. Very recently (April 2003), J. Dai, M.W. Wright and R.A. Manderville [182] showed that OTA can form a carbon-bonded C8-OTA-dG, confirming our results.

FEBS Meeting 1999 in Nice J.-P. Ebel had proposed that the French Biochemical Society (SFBBM) organize a IUBMB congress in Paris. He had presented its candidacy in London at an Executive Committee of IUBMB, but he was not successful. He asked me to propose it at the occasion of the International Congress of IUBMB in Amsterdam 1985, but I was not more successful. Subsequently, I changed our proposal to Nice for the year 2000 and presented it in Stockholm in 1993. However, our competitor was the Biochemical Society’s proposal for Birmingham that was retained. I was very disappointed and angry about the criticism of our project, for example our budget was too tight etc. In addition, I was also not happy about the gifts that had been given to the members of the Executive Committee by our competitors and considered the decision unfair. That evening in Stockholm I went to bed late. The next morning at 8 a.m. a phone call woke me at my hotel. It was Marianne Grunberg-Manago telling me that the FEBS Executive Committee would agree to give the SFBBM (and me) the organisation of a FEBS Meeting in Nice in 1999, but that I had to give my agreement immediately. After a short moment’ thought I accepted.

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In fact, I had been encouraged by Michel Lazdunski who took over the Scientific Committee. We decided to involve a maximum number of biochemists in the scientific programme and asked all the FEBS constituent societies and all French biochemists for suggestions (end of 1995). The Scientific Committee met several times and selected 19 main topics, each one comprising two to five half-day sessions. Michel Lazdunski aided by his very efficient and always cheerful secretary Mrs Yvette Benhamou had a lot to do with the 227 speakers who were invited. I also tried to organise a common congress with the cellular biologists (ECBO). SFBBM and FEBS agreed, but the ECBO council refused! I retired from the university in 1997 (Figure 5) when I was 66 years old and became Professor Emeritus. I could have stayed

Fig. 5. At home with my main co-workers and their wives (1997). From the left (standing) Agathe Keith, Ge´rard Keith, Jean Weissenbach, Jean Gangloff, Marie-Louise Gangloff, Guy Dirheimer. Sitting in front: Martine Weissenbach and my wife, Marguerite Dirheimer.

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until 69 years, but the organisation of the FEBS 0 99 took all my attention and my professorship would have suffered from it. Organising a large congress is like leading an enterprise. You rapidly know how much it will cost, but you have no idea how much it will bring in. Thus I tried to convince all the French biochemists to attend and I must say that they did it as we had 1138 French participants out of a total of 2153 regular participants. I had also to find sponsors and wrote to 163 pharmaceutical and chemical companies, contacting them personally when I knew the directors. Many did not answer, but nine were very generous sponsoring whole sessions. They were listed in the final programme and in the report I published in Regard sur la Biochimie in December 1999 [183]. The donations from French Scientific Organisations (CNRS, INSERM, CEA, INRA, Ministry of Foreign Affairs) were also high. Several members of the organising Committee and Richard Giege´ president of SFBBM had to lobby a lot for obtaining them. The City of Nice, thanks to the intervention of M. Lazdunski, and the Region Provence-Alpes-Coˆte d’Azur were also very generous. All these donations raised to nearly 1 million francs (150,000 euros). I also was lobbying a lot to convince a maximum of exhibitors to come to Nice and at each FEBS Congress, which I attended between 1989 and 1998, I visited all the exhibitors. I expected 30 exhibitors in my budget and 42 came. Thus my budget was not tight at all, in spite of the IUBMB executive committee predictions in Stockholm! Since I was retired and no longer had secretarial staff, I typed almost all the letters myself, as well as the first and second announcements. These were sent not only to the members of SFBBM, but also to members of 10 related societies, Physiology, Genetics, Microbiology, Clinical Biology etc. This also might explain the large number of French participants. Hotel/excursion and registration forms were prepared by Ge´rard Keith who also helped me a lot as treasurer of the meeting. The logo of the congress and the posters were realised by my wife and me. I had to go more than 10 times to the printer in Obernai to get

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everything in time. 50,000 first announcements and 10,000 second announcements were sent out. The abstract book gave us also a lot of work. I was helped by Sylvain Blanquet in this work, and my whole lab helped me for 2 days to read, check and classify the abstracts. The abstract book took me, personally, 2 months of full time work and was published as a supplement of Biochimie the journal of SFBBM. In fact, I was aware of this kind of work as the year before I had been in charge of editing the Proceedings of the VIII International Congress of Toxicology organised by Professor Jean-Roger Claude, Re´my Glomot and Chantal Bismuth in Paris, July 5–9, 1998, where I too had been in the organising committee [184] Again, my two caps! In Nice, Professor Raymond Ne´grel helped me a lot in recruiting 40 PhD students who projected the slides, looked after the posters, organised the public relations etc. He also organised the receptions at the Congress Centre Acropolis. In parallel to the meeting a Forum des Jeunes Chercheurs was organised by young post-docs of Nice; 340 young researchers attended who also participated in the FEBS meeting. The SFBBM, the National Committee of Biochemistry and the J.-P. Ebel Foundation gave 47 bursaries for attendance. The 26th FEBS meeting in Nice, June 19–24, was a success from both a scientific and social point of view, as well as the attendance and the finances [183]. Scientifically, it could be compared to an International Congress of Biochemistry covering almost all aspects of biochemistry. 1043 posters were presented. I was particularly happy to have 46% of junior participants. Thus FEBS meetings are young and this is very important in my opinion. This was also partly due to the 94 bursaries given by FEBS. Financially, we had a positive balance of about 400,000 francs (61,000 euros) in addition to the FEBS gift of 100,000 DM which is now used to pay every year bursaries to young French biochemists attending FEBS congresses. I was also pleased with the personnel of Acropolis, their professionalism and kindness. The social events, a welcome reception, diner at Hoˆtel Palais Maeterlink with the Plenary Lecturers Stanley Prusiner,

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Mariano Barbacid, Marc van Montagu, Hartmut Michel and the Presidents of the organising committees, organised by Michel Lazdunski and sponsored by the City of Nice, the diner with the FEBS council members in Monaco and the final gala dinner at Hoˆtel Me´ridien were also much appreciated. The success of FEBS 0 99 showed that there is still place for traditional large meetings in addition to more specific ones in Europe. The day after the meeting I made a long hike along the Esterel seaside and got a sunburn. I had been for too long in front of my computer in the shade! At the end of 1999 I made a long report writing down the way I organised the meeting with comments and advice to future organisers on what to do and particularly what not to do. I also made a fine photo album about it. Bernard Ehresmann organised a semi-scientific meeting for my departure from the IBMC on November 17, 2000. Jean Weissenbach and Michel Lazdunski gave very interesting conferences and Sylvain Blanquet summarised very nicely my scientific career. Many of my former co-workers attended and I was offered a wonderful bicycle as a retirement gift. In fact, I transferred my office to home with all my books and computer but I still go to the IBMC to attend the seminars and work in the library.

President of FEBS I became president of FEBS at the meeting in Nice, theoretically for 1 year, until the next FEBS Meeting, but as there was no FEBS meeting in 2000 and as the rules of FEBS changed in the meantime, I remained President until the end of 2002. In the executive committee I found many of my old friends, Prakash Datta and Iain Mowbray (treasurer), Julio Celis (general secretary) and the chairmen of the different committees, Israel Pecht (fellowships), Karel Wirtz (advanced courses), Willy Stalmans (publications) and Joan Guinovart (meetings).

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In 1999 it became evident that new activities should be developed by FEBS. First, a “Science and Society Committee” was set up and Professor Federico Mayor, former Director of UNESCO, agreed to be the chairman. I have summarised the activities of this committee in the book of H. Felmann under the title “New FEBS activities since 1999” [105]. On the occasion of the 26th FEBS meeting in Nice a workshop called “Teaching Biochemistry in Europe” was organised and very well attended. As a consequence of this event, I proposed to the Executive Committee in Birmingham on July 14, 2000 to establish a “Working Group on Education in Biochemistry”, education being clearly mentioned at the beginning of FEBS statutes as one of the objectives of FEBS. It was proposed that this group be headed by Jean Wallach from Lyon (France). At the council meeting in Birmingham in 2000 it was also decided to increase the Executive Committees by two new members for one term of 3 years, starting January 2002, serving not only as a liaison between the Council, the Constituent societies and the Executive Committee, but also as someone who would take care of specific tasks like chairing “Career of Young Scientists” and the “Role of Women in Science” committees. This was adopted at the council meeting in Lisbon in July 2001. Marja Makarow (Finland) and Sissel Rogne (Norway) were elected chairpersons of these committees. The activities of these three working groups have been also summarised recently [105]. In March 1999, Stefan Szedlacsek, on behalf of the Romanian Society of Biochemistry and Molecular Biology, wrote an extensive report called “Necessity for improved FEBS assistance to biochemical Sciences in East-European Countries”. He warned that the biochemistry in East-European countries is in real danger due to, first of all, the lack of financial resources of the governments (e.g., in Romania only 0.36% of the National Gross Domestic Products was devoted to R&D in 1996) and second, the continuous drain of highly qualified biochemists to the WestEuropean and American laboratories. In addition, there is a serious lack of important biochemical journals in these countries.

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A “Working group for exploring ways to assist Central and Eastern European countries (WOGA)” was set up at the council in Nice and I became its Chairman. This interested me particularly as I had had excellent relations with Russian and Polish researchers. This Committee met for the first time in Bucharest (Romania) in February 2000 and a second time in Kyiv (Ukraine) in April 2002. It visited representative units in the field of biochemistry and molecular biology and had discussions with officials from the Ministries of Education and Science and from the National Academies. It was clearly shown, in addition to the above-mentioned facts, that the number of FEBS long-term fellowships attributed to Central and Eastern European biochemists represented only 15% of the total number of these fellowships in 1999. This was not the case with short-term and Summer fellowships and with the youth travel funds of which about 50% went to biochemists from these countries. The working group summarised its findings at the FEBS Council in Birmingham on July 2000 and in Istanbul in October 2002 and made several proposals: (i) to improve the flow of information in the Eastern European countries by providing internet access, (ii) to offer free subscriptions of the two FEBS journals, (iii) to organise at least two practical courses per year in Central and Eastern European countries, FEBS providing the organising institutes with funds to buy dedicated equipment and (iv) to improve collaboration with laboratories from Western European countries where the PhD students would go from timeto-time for short periods to perform experiments not feasible in their countries. The FEBS established a new type of fellowship – the Collaborative Experimental Scholarships for Central and Eastern European Countries – that are intended for students engaged in research for a doctoral thesis. This programme has become very successful. Regarding the problem of “brain drain”, this is essentially a problem of political will for the governments. If they want to have prosperous and innovative science then they will have to provide

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the funds from government resources. The scientists, particularly the younger ones, must be encouraged financially to stay and work in their countries by providing them higher salaries than they have at present, and by giving consistent financial support to those who intend to continue their research in their country or to go back after a post-doc. At the FEBS Congress in Istanbul I organised a round table discussion with all the delegates coming from Eastern and Central European countries. This meeting clearly showed the great differences in working conditions between the different countries and different solutions were proposed. Although no longer president of FEBS in 2003, I shall continue to be in charge of the WOGA. FEBS is for me the proof that European countries can cooperate thanks to the good will of the men and women who work together to make great things happen.

Epilogue Doing both biochemistry and toxicology might appear as a drudgery. On the contrary, I feel that my research in one field always enhanced the research in the other and conformed to the well-known opinion that it is on the frontiers of different disciplines that research is most fruitful. Scientific research is certainly one of the most nicest and exciting jobs one can do. Teaching science is trying to give gifted young women and men, with their life still ahead of them, the desire for knowledge, showing them that science is not static but is always evolving. Even the most “dyed in the wool” dogma are rarely definitive. How many unexpected discoveries have been made in the life sciences during the last 50 years? Science is more fascinating than the best detective novel. I was very fortunate to do research and teaching, thanks to J.-P. Ebel and all my gifted and motivated co-workers. Practically all the colleagues I met in numerous committees were giving their time and efforts in a

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disinterested manner and were fighting for scientific quality and for facilitating the younger generations’ way to independent scientific careers.

ACKNOWLEDGMENTS

I thank Dr Barbara Winsor for help with the English.

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[59] Martin, R., Schneller, J.M., Stahl, A.J.C. and Dirheimer, G. (1976) In Genetics and Biogenesis of Chloroplasts and Mitochondria (Bu¨cher, Th. et al., eds.), pp. 755–758. Amsterdam, Elsevier/NorthHolland Biomedical Press. [60] Martin, R., Schneller, J.M., Stahl, A.J.C. and Dirheimer, G. (1976) Biochem. Biophys. Res. Commun. 70, 997–1002. [61] Fradin, A., Gru¨hl, H. and Feldmann, H. (1975) FEBS Lett. 50, 185–189. [62] Martin, R., Schneller, J.M., Stahl, A.J.C. and Dirheimer, G. (1977) Nucleic Acids Res. 10, 3497–3510. [63] Martin, R., Schneller, J.M., Stahl, A.J.C. and Dirheimer, G. (1979) Biochemistry 18, 4600–4605. [64] Chiu, N., Chiu, A. and Suyama, Y. (1975) J. Mol. Biol. 99, 37–50. [65] Schneider, A. (1994) Trends Cell Biol. 4, 282–286. [66] Schneider, A. and Mare´chal-Drouard (2000) Trends Cell. Biol. 10, 509–513. [67] Entelis, N.S., Kolesnikova, O.A., Martin, R.P. and Tarassov, I.A. (2001) Adv. Drug Deliv. Rev. 49, 199–215. [68] Lugnier, A.A.J., Le Meur, M.-A., Gerlinger, P. and Dirheimer, G. (1976) Eur. J. Toxicol. 9, 323–333. [69] Haas-Kohn, L.J.M., Lugnier, A.A.J., Tiboni, O., Ciferri, O. and Dirheimer, G. (1980) Biochem. Biophys. Res. Commun. 97, 962–967. [70] Lugnier, A.A.J., Creppy, E.-E., Le Meur, M.-A., Gerlinger, P. and Dirheimer, G. (1977) FEBS Lett. 76, 166–172. [71] Creppy, E.E., Lugnier, A.A.J., Beck, G., Dirheimer, G., Petzinger, E. and Frimmer, M. (1981) Toxicol. Eur. Res. 3, 179–184. [72] Lugnier, A.A.J., Dirheimer, G., Madjar, J.J., Reboud, J.P., Gordon, J. and Howard, G.A. (1976) FEBS Lett. 67, 343–347. [73] Mitchell, S.J., Hedblom, M., Cawley, D. and Houston, L.L. (1976) Biochem. Biophys. Res. Commun. 68, 763–769. [74] Endo, Y., Mitsui, K., Motizuki, M. and Tsurugi, K. (1987) J. Biol. Chem. 262, 5908–5912. [75] Bingen, A., Creppy, E.E., Gut, J.P., Dirheimer, G. and Kirn, A. (1987) J. Submicrosc. Cyt. 19, 247–256. [76] Dubois, E., Dirheimer, G. and Weil, J.-H. (1974) Biochim, Biophys. Acta 374, 332–341. [77] Kersten, H., Raettig, R., Weissenbach, J. and Dirheimer, G. (1978) Nucleic Acids Res. 5, 3033–3042. [78] Tumaitis, T.D. and Lane, B.G. (1970) Biochim. Biophys. Acta 224, 391–403. [79] Kuntzel, B., Weissenbach, J., Wolf, R.E., Tumaitis-Kennedy, T.D., Lane, B.G. and Dirheimer, G. (1975) Biochimie 57, 61–70.

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[80] Weissenbach, J. and Dirheimer, G. (1978) Biochim. Biophys. Acta 518, 530–534. [81] Falcoff, R., Lebleu, B., Sanceau, J., Weissenbach, J., Dirheimer, G., Ebel, J.-P. and Falcoff, E. (1976) Biochem. Biophys. Res. Commun. 68, 1323–1331. [82] Weissenbach, J., Dirheimer, G., Falcoff, R., Sanceau, J. and Falcoff, E. (1977) FEBS Lett. 82, 71–76. [83] Keith, G., Roy, A., Ebel, J.-P. and Dirheimer, G. (1971) Biochimie 53, 661–669. [84] Keith, G. and Dirheimer, G. (1978) Biochim. Biophys. Acta 517, 133–149. [85] Sanger, F., Brownlee, G.G. and Barrell, B.G. (1965) J. Mol. Biol. 13, 373–398. [86] Takada-Guerrier, C., Grosjean, H., Dirheimer, G. and Keith, G. (1976) FEBS Lett. 62, 1–3. [87] Garel, J.-P., Hentzen, D., Schlegel, M. and Dirheimer, G. (1976) Biochimie 58, 1089–1100. [88] Keith, G. and Dirheimer, G. (1980) Biochem. Biophys. Res. Commun. 92, 109–115. [89] Fournier, M., Labouesse, J., Dirheimer, G., Fix, C. and Keith, G. (1978) Biochim. Biophys. Acta 521, 198–208. [90] Weissenbach, J., Kiraly, I. and Dirheimer, G. (1976) FEBS Lett. 71, 6–8. [91] Martin, R., Sibler, A.-P., Schneller, J.-M., Keith, G., Stahl, A.J.C. and Dirheimer, G. (1978) Nucleic Acids Res. 5, 4579–4592. [92] Dirheimer, G. and Martin, R. (1990) In Chromatography and Modification of Nucleosides (Gehrke, C.W. and Kuo, K.C.T., eds.), pp. B197–B264. Amsterdam, Elsevier. [93] Sibler, A.P., Dirheimer, G. and Martin, R. (1981) FEBS Lett. 132, 344–348. [94] Sibler, A.P., Bordonne´, R., Dirheimer, G. and Martin, R. (1980) Compt. Rend. Acad. Sci. Paris 290(Se´rie D), 695–698. [95] Martin, R.P., Sibler, A.-P., Dirheimer, G., de Henau, S. and Grosjean, H. (1981) Nature 293, 235–237. [96] Canaday, J., Dirheimer, G. and Martin, R.P. (1980) Nucleic Acids Res. 8, 1445–1457. [97] Sibler, A.P., Dirheimer, G. and Martin, R. (1985) Nucleic Acids Res. 13, 1341–1345. [98] Heckmann, J.E., Sarnoff, J., Alzner-Deweerd, B., Yin, S. and RajBhandary, U.L. (1980) Proc. Natl Acad. Sci. USA 77, 3159–3170. [99] Sibler, A.P., Dirheimer, G. and Martin, R. (1986) FEBS Lett. 194, 131–138.

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[100] Martin, R., Sibler, A.P., Gehrke, C.W., Kuo, K., Edmonds, C.G., McCloskey, J.A. and Dirheimer, G. (1990) Biochemistry 29, 956–959. [101] Bordonne´, R., Bandlow, W., Dirheimer, G. and Martin, R.P. (1987) Mol. Gen. Genet. 206, 498–504. [102] Hu¨ttenhofer, A., Weiss-Bru¨mmer, B., Dirheimer, G. and Martin, R.P. (1990) EMBO J. 9, 551–558. [103] Bordonne´, R., Dirheimer, G. and Martin, R.P. (1987) Nucleic Acids Res. 15, 7381–7394. [104] Bordonne´, R., Dirheimer, G. and Martin, R.P. (1988) Curr. Genet. 13, 227–233. [105] Feldmann, H. (2004) Forty Years of FEBS 1964 to 2003. A Memoir. Blackwell Publishers. [106] Christman, J.K., Price, P., Pedrinan, L. and Acs, G. (1977) Eur. J. Biochem. 81, 53–61. [107] Salas, C.E. and Dirheimer, G. (1979) Nucleic Acids Res. 6, 1123–1134. [108] Salas, C.E., Pfohl-Leszkowicz, A., Lang, M.C. and Dirheimer, G. (1979) Nature 278, 71–72. [109] Pfohl-Leszkowicz, A., Salas, C., Fuchs, R.P.P. and Dirheimer, G. (1981) Biochemistry 20, 3020–3024. [110] Pfohl-Leszkowicz, A., Hebert, E., Saint-Ruf, G., Leng, M. and Dirheimer, G. (1986) Cancer Lett. 32, 65–71. [111] Pfohl-Leszkowicz, A., Boiteux, S., Laval, J., Keith, G. and Dirheimer, G. (1983) Biochem. Biophys. Res. Commun. 116, 682–688. [112] Pfohl-Leszkowicz, A., Galie`gue-Zouitina, S., Bailleul, B., LoucheuxLefe`bvre, M.H. and Dirheimer, G. (1983) FEBS Lett. 163, 85–88. [113] Pfohl-Leszkowicz, A., Fuchs, R.P.P. and Dirheimer, G. (1984) FEBS Lett. 178, 56–60. [114] Mu¨nzel, P.A., Pfohl-Leszkowicz, A., Ro¨hrdanz, E., Keith, G. and Dirheimer, G. (1991) Biochem. Pharmacol. 42, 365–371. [115] Pfohl-Leszkowicz, A. and Dirheimer, G. (1986) Cancer Res. 46, 1110–1113. [116] Pfohl-Leszkowicz, A., Baldacini, O., Keith, G. and Dirheimer, G. (1987) Biochimie 69, 1235–1242. [117] Pfohl-Leszkowicz, A., Keith, G. and Dirheimer, G. (1991) Biochemistry 30, 8045–8051. [118] Keith, G., Pixa, G., Fix, C. and Dirheimer, G. (1983) Biochimie 65, 661–672. [119] Pixa, G., Dirheimer, G. and Keith, G. (1984) Biochem. Biophys. Res. Commun. 119, 905–912. [120] Pixa, G., Dirheimer, G. and Keith, G. (1983) Biochem. Biophys. Res. Commun. 112, 578–585. [121] Lutz, W.K. (1979) Mutat. Res. 65, 289–356.

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