Maxi-circles, glycosomes, gene transposition, expression sites,
transsplicing, transferrin receptors and base J.


By Piet Borst, The Netherlands Cancer Institute,

Plesmanlaan 121, 1066 CX Amsterdam,The Netherlands (



My entry into the trypanosome field was accidental, and this is typical for most of the research topics that I worked on in my life. When I came back from my postdoctoral period in New York University in the laboratory of Severo Ochoa, I had started to work on mitochondrial nucleic acids. That was a suitable niche for somebody who had done a Ph.D. on mitochondrial metabolism and oxidative phosphorylation in Amsterdam and then switched to RNA phage replication in E. coli as a post-doc in New York. I soon found that animal mitochondrial DNAs consist of relatively small 10-kb circles and that all mitochondrial circles in a single cell had the same sequence 1. This established that the mitochondrial DNA could only encode a very small fraction of the total number of proteins present in mitochondria. This was a good start and 5 years later I had reached the status of an expert in the (small) field of mitochondrial biogenesis 2,3.

          In 1971 (I think, I cannot find back the file) Maurice Steinert organized a workshop on kinetoplast DNA (kDNA) in Rhode St. Genèse. It was a small workshop with only around 20 people invited, which represented the world population of scientists working on kDNA in 1971. Maurice Steinert thought that it might be useful to ask an expert on mitochondrial biogenesis to attend the conference, to provide an outsider helicopter view. Since Amsterdam is only 200 kilometers from Rhode St. Genèse, I was the cheapest expert available and this is how I entered the trypanosome field.

          Once I saw some of the mysteries of the Kinetoplastida, I got hooked. I was also fairly arrogant in that period of my life (fortunately, this trait disappeared in later years) and I thought that the kDNA field was one to which I could contribute even with a single graduate student. Obviously, I did not think that the competition in that period was overwhelmingly competent. So I went back to Amsterdam, recruited a graduate student, Koen Kleisen, and started working on the structure of the kDNA of Crithidia 4. Even then, I considered Trypanosoma brucei more interesting, but I was not yet aware of the existence of T. brucei strains that were not infectious to graduate students. Knowing my students, I was convinced that they would manage to infect themselves, if given the opportunity.

          From the start I was convinced that the mini-circles, then known as the only component of kDNA, could not have sufficient genetic information to encode the usual set of mitochondrial proteins. This conviction remained unshaken, even when Koen Kleisen found by restriction enzyme digestion (at that time really new technology) that the mini-circles were micro-heterogeneous 5,6. So we started looking for kDNA components with more genetic information content and found the larger circles that I called maxi-circles –rather uningeniously, I admit- to distinguish them from the mini-circles 5,7. These maxi-circles were independently discovered by Steinert and Van Assel 8. The late Maurice Steinert and I became good friends through collaborations on trypanosomes, but he would later admit that inviting me to into the trypanosome field had not been such a good idea, because this generated more competition than he needed.

For about 10 years we continued work on various aspects of kDNA. In this period I collaborated with several people in the field, who helped me to get started on Kinetoplastida. Especially enjoyable was a collaboration with Maurice Steinert and Bruce Newton (Moltino Institute, Cambridge, England), in which we tried to use cytological hybridization with mini-circles to distinguish strains of T. brucei infective to humans from those that are not 9. Our final conclusion was that the distinction was very subtle, and that the human-infective strains might only differ from the non-infective strains in one or very few genes 10-15. That conclusion has been confirmed in recent work by others.

          There were several other interesting findings during our 10-year period of intensive kDNA analysis for which I refer to published papers 16-28. I was especially pleased with a collaboration with Ian Eperon (MRC, Cambridge, UK), which unambiguously showed that the 9 and 12 S RNAs encoded in the maxi-circle are exceptionally small ribosomal RNAs 29,30. I also liked the work done by Jan Hoeijmakers and Peter Weijers, who analyzed the segregation of kDNA networks, and shot some very elegant EM pictures 31. Their conclusions were confirmed and greatly extended by Paul Englund’s group later. I continued to do some work on kDNA with my trypanosome technician Francesca Fase-Fowler (FFF for insiders), in part in collaboration with (former) post-doc Wendy Gibson, but this was not a major effort and only added a few missing pieces to the puzzle 32. However, the most important discovery in trypanosome mitochondrial biogenesis remained to be made and that was RNA editing.

          Around 1981 Rob Benne, an expert in eukaryotic protein synthesis, joined my department as a senior collaborator to set up an independent group. He became intrigued by trypanosomes and took over our project on mitochondrial biogenesis 33. It was Rob Benne who discovered RNA editing. I had nothing to do with it, but of course the great pleasure of seeing this discovery being made under my nose. It is hard to understand with our present knowledge for the younger generation how difficult the right conclusion was. When Benne’s paper was submitted to Cell, two of the reviewers actually called me to ask whether this Benne was serious or a nut. Once RNA editing was discovered by Benne, several groups joined the hunt for the editing mechanism and its physiological significance. That story has been engagingly told by Benne and other contributors to this series. My only involvement in RNA editing was accidental: post-doc Ted White found a terminal uridylate transferase (TUTase) activity in trypanosome extracts, which allowed him to end-label RNAs. As there was also RNA ligase activity in the extract, Ted was able to make labeled rRNA circles 34. This was a remarkable artifact, but not the nuclear RNA polymerase we were looking for. Ted abandoned the labeling and sorted out the interesting structure of the RNA of the large sub-unit of the cytoplasmic ribosomes, which is fragmented35. Later work showed that Ted probably saw the first two enzymic activities of the complex editing machinery.


The glycosome

          In my years as a graduate student I had become interested in the reoxidation of glycolytic NADH by the mitochondria in animal cells. With some luck I even discovered a new pathway for this reoxidation, the malate-aspartate shuttle, known for some time as the Borst cycle 36,37. It is therefore not surprising that after 3 years of kDNA I became intrigued by the unusual metabolism of bloodstream form T. brucei. Others had established that T. brucei reoxidized glycolytic NADH by another substrate cycle than the malate-aspartate shuttle, the glycerol-phosphate cycle. As a student I had studied this substrate cycle in Ehrlich ascites tumor cells 38 and, notwithstanding my preoccupation with mtDNA and kDNA in the seventies, I was no stranger to metabolism. What made the glycerol-phosphate cycle in trypanosomes so intriguing was that the oxidase involved was inhibited by salicylhydroxamic acid, which had little effect on animal cells. Here was an interesting target for potential chemotherapy 39,40. Even more intriguing was that Clarkson had reported 41 that the glycerol-phosphate oxidase of trypanosomes was located in microbodies and not in the mitochondria, as it is in mammalian cells. I therefore recruited a new student, Fred Opperdoes, to look at this oxidase. Fred had done work on mitochondrial metabolism as an undergraduate student and was willing to work with me on a metabolic problem rather than on DNA.

          Initially, Fred got nowhere. He was unable to unambiguously locate the glycerol phosphate oxidase in a microbody. A major problem in our cell fractionation experiments was the fact that we had no suitable marker enzyme for the mitochondrial fraction. In bloodstream form trypanosomes mitochondrial biogenesis is highly repressed and the usual mitochondrial enzymes (cytochrome c oxidase or Krebs cycle enzymes) were not available. Fortunately, I had experience with the repressed mitochondria of anaerobic yeast and I knew that these had retained an activity typical of the mitochondrial ATP generating machinery, the oligomycin-sensitive ATPase. When Fred used this enzyme activity in his cell fractionations, the result was unambiguous: the glycerol phosphate oxidase was mitochondrial 42,43. We had been led astray by Clarkson, and especially by the extensive discussion of his results in an authoritive review in the Annual Review of Microbiology by Miklos Müller 44. We had another unfortunate experience with Clarkson: When we published that T.brucei bloodstream form trypanosomes were killed by a combination of salicylhydroxamic acid and glycerol 45, Clarkson accused us of duplicating his published data. With lab books in hand this could be shown to be without any ground. It was the only time in my life that I was accused of scientific theft and it was an unpleasant experience.

          With the glycerolphosphate oxidase firmly located in mitochondria, another remarkable result remained to be explained: the second enzyme

involved in the glycerol phosphate cycle, the glycerol phosphate dehydrogenase (GPD),

an enzyme found in the cytosol in animal cells, seemed particle-bound in T. brucei 43. This particle was clearly not the mitochondrion, nor the lysosome. We concluded that it was a microbody 46. This created a specific problem for me: at that time most scientists believed that microbodies were freely permeable to substrates and co-factors, because they were permeable when isolated. I was convinced, however, that this was an isolation artifact and that microbodies were just as impermeable in vivo as other cellular organelles. Hence, I did not understand how the trypanosomal GPD could work, if placed in a microbody. How could the substrates of the enzyme reach their target behind an impermeable membrane and how could the products of the reaction leave the organelle again? This question 46led to one of the most exciting weeks in my scientific life, as I was drawing different versions of the glycolytic system and Fred Opperdoes was testing enzymes.

          I have once described this story of the birth of the glycosome in detail elsewhere 47 and I leave it to Fred Opperdoes to paint an accurate picture. Let me only add that I still have mixed feelings about the name glycosome that I coined for our new organelle 48. Even in 1977, we had already good evidence that the glycosome was a highly modified microbody 46 and giving a microbody a new name, just because it has adopted a new set of enzymes, is an action that can be criticized with hindsight. However, the name glycosome was an instant hit and it has stuck.

          When Fred Opperdoes left my lab to take up an independent position in the Institute of Cellular Pathology (ICP) in Brussels, he wanted to take the glycosome with him. I let him take it, after a student who had joined me from another department, Nico Visser, had finished 49. I have often done that in my career and I have no regrets. I lost interesting lines of research, but I was forced to tackle new problems, which turned out to be  just as interesting.  Changing topic creates problems, because grant-giving bodies do not like it, especially in small countries like The Netherlands. I had a reputation for working on mitochondrial biogenesis in yeast and then I switched to glycosomes and antigenic variation in trypanosomes. My Dutch colleagues were aghast: they preferred me to stick to what I was good at, instead of taking the risk of starting something new. It can be done, however, and I survived. Switching topics also has clear advantages. Entering a new field unburdened by established dogma’s, may allow you to take a successful route considered blocked by insiders. Sometimes interesting discoveries can be made between fields and if you never peer over the edge of your chosen field, you may miss golden opportunities. My regular switching of topics (the 7 year itch) has also helped me in my side jobs on advisory committees and prize juries. It has proven to be very useful to have first-handknowledge of a number of very different fields of biomedical research on such committees.

          Looking back from a 28-year distance at the glycosome, I think that this discovery contains a few lessons for young investigators. First and foremost, it shows the advantage of a broad background. If I had not had extensive experience with cell fractionation in animal cells, with metabolic pathways, with yeast mitochondria, we would not have found the glycosome. Specialization gets you into a tenure track fast, but may get you stuck later in your career. Secondly, it shows the importance of not believing everything you read, even in the Ann. Rev. of Microbiology. Once, we disbelieved and went back to the original data, hidden in a thesis, we immediately saw the elementary error that Clarkson had made. Finally, of course, it shows the importance of having good graduate students. It took a Fred Opperdoes to get there.


Routing enzymes into glycosomes

          Since Fred was more interested in metabolic aspects of glycosomes than in glycosome biogenesis, I retained the biogenesis part of the project. I wanted to find out what the targeting signal was for getting enzymes into glycosomes. At the time we started this project in 1980, peroxisomal targeting sequences had not yet been identified.

We began cloning and sequencing the genes for glycolytic enzymes in collaboration with Paul Michels, Fred Opperdoes and Wim Hol 50-53. Our strategy was simple: Opperdoes and I had already shown that there are two versions of several glycolytic enzymes, a glycosomal and a cytosolic one. We hoped that the two versions would be similar and that the differences would help us to define the glycosomal targeting sequences. We focused on the phosphoglycerate kinase isoenzymes. Trypanosomatids have 3 genes, which we called A, B, C. Gene B encodes the cytosolic enzyme, C the major glycosomal enzyme50, A a minor glycosomal species 54. The genes are kept similar by gene conversion 55 and differential expression is controlled post-transcriptionally. We found two major differences between the deduced aminoacid sequences of genes B and C,: enzyme C had clusters of basic aminoacids, and a C-terminal tail of aminoacids missing in B 50. Since all the glycosomal glycolytic enzymes analyzed had the basic clusters, we proposed that two basic hotspots 40 Å apart on the surface of these proteins, were the glycosomal targeting sequences 56. We were carried away by the enthusiasm of Wim Hol, but by the time this paper was submitted, I had doubts and I should have stopped its publication. We subsequently found that the cytosolic and glycosomal phosphoglycerate kinases of Crithidia are virtually identical, but for  the C-terminal tail in the glycosomal enzyme 57. No basic hotspots whatsoever. We came as far as a C-terminal targeting sequence, but at that time (1988) we were still unable to do transfection experiments with T.brucei and we could therefore not test whether our suggestion was correct. Later research in other systems brought the C-terminal tripeptide targeting sequence of microbodies in general to light and research by other labs confirmed that this is also one of the targeting sequences for glycosomes. By that time I had left the glycosome biogenesis field, however, because yeast molecular biologists managed to find conditions to induce the biosynthesis of peroxysomes (microbodies) in yeast. As I had worked myself with yeast I knew the formidable power of yeast molecular genetics and cell biology to solve basic problems in eukaryotic biology, and I did not want to confirm in trypanosomes what had been found before in yeast. Indeed, in a very short period most of the riddles of microbody (peroxysome) biosynthesis were solved in yeast and later work in trypanosomes has shown that the targeting and import use similar principle in trypanosomes and in yeast, although Paul Michels has recently come up with some interesting innovations specific for tryps.

          With hindsight, I admit that our ambitious glycosomal targeting project yielded no major discovery, even though it generated a lot of interesting and useful results. I wrote some highly quoted reviews on peroxisome biogenesis in general 58-60 and came up with a new hypothesis on why inborn errors in peroxisome synthesis are lethal in humans 58. Armtwisted by a friend who was professor of Pediatrics, I even helped to develop the first prenatal test for an inborn error in human peroxisome synthesis, the Zellweger syndrome 61. But we missed the big discovery, the identification of peroxisomal targeting sequences.


A brief excursion into Toxo.

          In 1982 Prosper Overdulve, a seasoned parasitologist who contributed substantially to the delineation of the life cycle and intermediate hosts of Toxoplasma (Isopora) gondii, asked me whether he could work for a sabbatical year in my lab to learn some molecular biology. For long the classical parasitologists had hoped that molecular parasitology was an insignificant fad that would pass their door. By 1982 it had become clear, however, that molecular biology was there to stay and Overdulve had the courage to go back to the bench to learn some of the tricks. What could we do with Toxo? Uningeniously, I suggested that we try to isolate DNA circles from Toxo, a trick that I had done before. Soon we had 12-micron circles, but these were unusual in that the covalently closed circles had a short tail of 0.4 micron 62. Using standard circle technology we worked out that the circles contained a 1.7-μM palindrome, like chloroplast DNA, which contains two copies of the ribosomal DNA genes in head-to-tail configuration. In 1984 nobody could yet imagine that Toxo would contain a degenerated chloroplast, the apicoplast, and we settled for a mitochondrial origin of our circles. Unable to pinpoint their cellular location by in situ hybridization, we had to leave the issue open and Overdulve returned to his sick cats in Utrecht.


The mechanism of antigenic variation in trypanosomes

          As we were studying the glycosome on the one hand and the function of kDNA on the other, antigenic variation was a recurrent topic of discussion in the lab. This was obviously a major problem to be solved and it also looked solvable: After George Cross had discovered in 1975 that the trypanosome coat is essentially made up of a single variant surface glycoprotein (VSG), which can be repeatedly replaced, it looked as if the mechanism of antigenic variation would be amenable to an attack with the available molecular biological tools of the day. Nevertheless, it remained a daunting problem to tackle for an inexperienced lab. So we did nothing until Jan Hoeijmakers took up the challenge.

          Jan worked on kDNA and in three years he had generated sufficient material for a nice thesis. With one year left in his contract (which is four years for Dutch students) he decided to take a crack at antigenic variation. We were unequipped for this project, and I therefore approached George Cross whether he was interested in a collaborative effort. This proved a fortunate choice. George knew everything about the biology of antigenic variation and about VSGs, but he had not been trained in DNA work and had therefore hesitated to start with the molecular genetics of antigenic variation on his own. With our complementary expertise, and a rather similar approach to science, we made a good combination and our collaborative project got rapidly underway.

          The year was 1979 and unfortunately the Dutch had become thoroughly worried about the risks of recombinant DNA experiments. Of course, without permission for cloning genes, we could not isolate VSG genes to find out what happens when T.brucei switches its surface coat. Fortunately, the Swiss were less apprehensive than the Dutch, and my friend Charles Weissmann had been allowed to set up a recombinant DNA lab in his department in Zurich. This is where Jan Hoeijmakers went to clone the VSG genes expressed in four different variants from the Cross collection, 117, 118, 121 and 221. The strategy was a classical one: subtractive hybridization, i.e. cloning a cDNA highly expressed in one variant and not in the others. This approach worked like a charm, and after a few months of very hard work and cloning several pieces of contaminating yeast DNA, Jan Hoeijmakers returned to Amsterdam triumphantly with segments of his four VSG genes cloned 63.

           Once we had VSG genes, we could start to look what happened when the trypanosomes switch from coat A to coat B. From the start I had hoped that trypanosomes would combat our immune system with a strategy similar to the one our body uses to tackle parasites: the re-assortment of gene segments to make an infinite number of antibodies and T-cell receptor variants from a limited set of genes. This is why we started to look from day one for DNA rearrangements accompanying the switch to synthesis of a new VSG.

          Our initial Southern blots were black, however. This was interesting in itself, because it showed that the trypanosome is not stingy when it comes to reserving genome capacity for coat synthesis. Obviously, there were large gene families with lots of homology and our VSG gene probes cross-hybridized to many members of these large gene families 64. Since the N-terminal aminoacid sequence of VSGs was known to be relatively unique through the work of George Cross, we started to use smaller probes and stringent hybridization conditions. To our great joy this led to a considerable simplification of our blots 65. Clearest results were obtained with the 118 VSG gene for which we only obtained a single band on the Southern blot when we used the 5’-half of the gene. The most striking result came, however, when we looked at variant 118, which expressed the 118 gene. This had an extra band on the blot, an Expression Linked Copy, an ELC, as Jan Hoeijmakers found together with a new student, André Bernards, and a postdoc from Argentina, Carlos Frasch. Similar results were obtained for two other VSG genes, 117 and 121, but the results were not as clean. These genes belong to a small family of highly related genes and it was difficult to avoid cross-hybridization to the other family members.

          The only gene that did not behave at all was the VSG 221 gene. When this gene was activated, we did not see an ELC. Jan Hoeijmakers was very upset about this and he wanted to sort out how the 221 gene is activated before anything was published. That was scientifically sound, but nevertheless I did not agree. I was very pleased that we had found something new and important: We had shown that expression of at least three VSG genes was accompanied by the appearance of an extra copy of the gene in a new environment. This strongly suggested that the gene to be activated had to move to an expression site by a duplicative transposition. Even though we had not found the reassortment of gene segments that I had hoped for, the gene transposition mechanism was also cute, and I was convinced that our evidence for this mechanism was unambiguous. If the 221 VSG gene did not behave, we would have to sort this out later. I have to admit that the existence of strong, competing groups (I knew at least 3 others) was also a consideration and George Cross and I therefore decided to send a letter to Nature, which I wrote rapidly, notwithstanding the reluctance of Jan Hoeijmakers. Our paper impressed even Nature and, as far as I remember, it was accepted without any haggling 66. With hindsight I think that our decision to publish was correct. Everything in that Nature letter has been substantiated by later experiments and it has taken us a long time to sort out the activation of the 221 gene, which later proved to be by in situ activation 67.

          What followed was one of the most exciting periods in my scientific life. Both George Cross and I managed to assemble teams of outstanding collaborators, allowing us to make rapid progress. A combination of incidental factors helped me to attract good students. The most important one was probably the presence of Dick Flavell in the lab. Dick had initially worked for 2 years in my group as a post-doc and he had returned as an independent project leader after 2 years in Zürich with Charles Weissmann. Dick gave practical courses to the undergraduate students in biology and he had an exceptional knack to bring out the attractive aspects of science. He would, for instance, challenge students to find new restriction enzymes in bacteria that had never been used before to make restriction enzymes. This was a refreshing change in a curriculum filled with dusty courses.

          Dick also did fabulous research. With his post-doc Alec Jeffreys (Sir Alec of later DNA fingerprint fame) he had found the intron in the ß-globin gene and developed the first methods to map mammalian genes by Southern blotting. He was using this methodology to map the large gene deletions in inborn hemoglobinopathies, such as the thalassemias. This attracted outstanding undergraduate students for a 9-month stint in the lab and some of these students got interested in our trypanosome work. In this way André Bernards, Titia de Lange and Lex van der Ploeg ended up working on trypanosomes instead of globin genes. Good postdocs joined: Carlos Frasch  had come earlier from Argentina, Paul Michels came from The Netherlands, Alvin Liu from the USA. Also George Cross, who had moved from the Molteno Institute in Cambridge to a laboratory in the pharmaceutical industry, found very good people there, notably John Boothroyd.

          Progress was rapid; already in 1982 George Cross and I wrote a review for Cell with the confident title: “The molecular basis for trypanosome antigenic variation” 68. Indeed, in a few years the molecular genetics of antigenic variation in trypanosomes were basically solved in outline 30,50-58,60-118. This is not to suggest that we did it all, far from that. Competition was fierce, there were several other competent investigators studying antigenic variation, notably Etienne Pays and Maurice Steinert in Belgium, Harvey Eisen in Paris, Nina Agabian in Chicago, John Donelson in Iowa and Dick Williams in the ILRAD laboratory in Kenia, to mention only the most prominent competitors. Although we had many enjoyable and friendly moments with these colleagues, not everybody was up to the pressure. I vividly remember how I tried to get cDNAs for other VSG genes from competitors. PCR did not exist yet and cDNAs were precious commodities. George Cross and I had provided all our materials freely to other investigators, as soon as they were in the public domain, as we have always done. However, getting something back proved difficult: One investigator did not only fail to answer my letters (no email yet), but other investigators in the field sent me copies of the telegrams that he had sent to them: “Under no circumstances can you provide my cDNA to Piet Borst.” Another investigator wrote back: “How wonderful that you want to start a collaboration with us. I have always wanted that.” Of course, the cDNA that was in the public domain was not sent.

          I have discussed the research of these productive early years of antigenic variation in a review in Annual Reviews of Biochemistry 100 and I am not going to repeat all of it here. I only lift out a few highlights:


The telomeric location of VSG gene expression sites.

This was established in a classical experiment by Titia de Lange in which she showed that restriction fragments containing the expression-linked VSG gene copy were preferentially lost when large DNA was digested with an exonuclease (in this case Bal31 nuclease), before the restriction digestion 76. The blot with the telomeric fragments getting shorter and shorter and shorter was long one of my favorite slides in trypanosome seminars.

Telomeric growth and contraction.

We had noted at an early stage that the telomeres adjacent to the ELC varied in size in different trypanosome clones. This suggested that the number of telomeric repeats of a given telomere might vary widely during the life of a trypanosome and its progeny. I considered this an interesting problem, but my brilliant collaborators thought differently and nobody wanted to work on this. The standard comment in our highly democratized lab was: “Why don’t you do these experiments yourself, Piet?” Finally, I found a medical student, totally naïve, Carsten Lincke, who had not been brainwashed yet by my collaborators into thinking that this would not be an interesting experiment. So he did it.           The results uncovered the spectacular growth and contraction of trypanosome telomeres 78,86, later shown to be a general property of all eukaryotic chromosomes, albeit not as exuberantly as in trypanosomes. (This is what I remembered, but Paul Michels has convinced me that I have grossly embellished and simplified this story. In fact, Paul had already isolated the trypanosome populations required before Carsten Lincke entered the lab and it was André Bernards who did most of the work. The moral is clear: mature scientists should not rely too much on the accuracy of their memory).

The sequence of the telomeric repeats of trypanosomes.

Lex van de Ploeg managed to clone the telomeric repeats of T. brucei and found the sequence (GGGTTA)n at the same time as the group of Liz Blackburn. The papers were published back to back in Cell 86, long before it was known that the same sequence is present at mammalian telomeres.

Fractionation of chromosome-sized DNA molecules by pulsed field gradient gel electrophoresis (PFGE).

There were several problems around 1983 that required size-fractionation of large DNA molecules. There were indications that trypanosomes contained a set of mini-chromosomes 81, and we were interested in the question whether the duplicative transposition of VSG genes could occur between different chromosomes, as we anticipated. Trypanosomes do not condense their chromosomes and there was no way of studying these questions in 1983. I then happened to give a talk at a meeting where David Baltimore was present. He is an old friend of mine, because we both worked on phage replication in New York, and he told me about a guy in the laboratory of Charles Cantor in Columbia University in New York, David Schwartz, who seemed to have developed a new electrophoretic technique to separate intact yeast chromosome-sized DNA. Since we had shown that T. brucei contains only twice the amount of nuclear DNA as yeast 119, David Baltimore perceptively deduced that trypanosomes might also have chromosomes separable by the new technique. I knew Charles Cantor, because we were both members of the Scientific Advisory Committee of the EMBL in Heidelberg. (Yes, knowing people helps in science). I first wrote to him and, when he did not respond, I called. Charles was less than enthusiastic. David Schwartz still had not proven that he was really separating yeast chromosome-sized DNA molecules with his PFGE technique and he thought that trypanosomes would be a distraction. So I had to go to New York to convince Schwartz and Cantor of the potential of their new technique for studying real problems rather than baker’s yeast, and they finally consented in having one of my star-students, Lex van der Ploeg, as a guest in their lab to test PFGE on trypanosomes lysed in agarose blocks. The visit of Lex was a great success and the first papers of chromosome-sized DNA fractionation of yeast and trypanosomes were published back to back in Cell 90. We rapidly set up PFG electrophoresis in Amsterdam, and showed the general applicability of the new technique for chromosome-sized DNA separation in other protozoa, such as Leishmania and Plasmodium 92. Today the molecular genetics of kinetoplastida is unthinkable without PFGE, but nobody refers anymore to our groundbreaking papers.


We got fired up when Lex van der Ploeg and Alvin Liu discovered that the 5’-end of the VSG mRNA was not encoded contiguously with the gene and the DNA, but encoded in a separate mini-exon. 74,80,85 Similar results were independently obtained by John Boothroyd in the laboratory of George Cross and by the group of Nina Agabian (who called this mini-exon the spliced leader). Soon it became clear that there were many mini-exon genes in the genome and these were clustered far away from the transcribed VSG gene in the expression site 82. We fantasized about very long precursor RNAs, but these fantasies went into the dustbin, as the same mini-exon was found on all mRNAs we and others looked at 87,89. This proved that the mini-exon was linked up to the main exons of protein coding genes by transsplicing, a novel mechanism in biology at that time 93,100. We contributed to the characterization of a mini-exon derived precursor RNA 85,99,106, but the main findings in this area came from other labs.

The mechanism of the duplicated transposition (gene conversion) resulting in the replacement of the VSG gene in the expression site by another VSG gene.

At an early stage we realized that there was preciously little sequence homology between the incoming VSG gene and the resident gene in the expression site. This suggested that the cross-over between the incoming gene and the resident gene might occur in the 3’-end of the gene. By comparing the sequence of the mRNA and the corresponding basic copy gene, we found that the cross-over indeed occurred within the gene 71and we pinpointed the position of the cross-over for several independent events in which the 118 gene was activated by duplicative transposition 79,80. The only sequence homology upstream of the gene was a few 70-bp repeats 80, which had homology with the large stretches of 70-bp repeats found upstream of the ELC and later upstream of all telomeric VSG genes. Proving that the cross-over between the incoming VSG gene and the resident gene in the expression site took place in such 70-bp repeats was technically difficult, but was finally accomplished by Jan Kooter and Titia de Lange 93. Relatively short stretches of imperfect homology between the incoming gene and the resident gene are therefore responsible for the duplicative transposition of VSG genes. This raises a number of questions that I have discussed in several reviews. Our 1996 model for this duplicative transposition 120 is still, I think, a reasonable explanation for all data available.

The expression site promoter.
With transplicing established, the next challenge was to get a complete expression site (ES) cloned. BACs did not exist yet, and Jan Kooter therefore decided to chromosome-walk to the start of the transcription unit. This was a daunting task, because the twenty or so expression sites are similar and one might easily walk from one ES into the next and construct an average site rather than a unique one. With hindsight, we now know that we chose a really tough ES, as the 221 ES contains multiple duplications and even a triplication. Nevertheless, Jan Kooter constructed a plausible map 109 and, what is more, this map has been fully confirmed by the recent sequence analysis of a 221 ES BAC clone 121.
          In my talks about ES, I would always present a model showing a 60-kb primary transcript of the entire ES. I would add that we never saw such a transcript in real life, presumably because the nascent RNA was rapidly processed after synthesis. One day Phil Sharp cornered me after my talk and asked why we did not locate the promoter by UV irradiation inactivation of transcription. That is a tough experiment, but post-doc Pat Johnson managed to master the technique and unambiguously showed that the ES is a single 60-kb transcription unit

RNA Polymerase I transcribing protein-coding genes.

In 1984 Jan Kooter set up a nuclear run-on transcription system to study synthesis of nascent RNA in T. brucei 95. This allowed Jan to check the α-amanitin sensitivity of the transcription of various genes. In other eukaryotes Pol I transcribes the rRNA genes and is relatively α-amanitin insensitive; Pol II transcribes proteincoding genes and is highly sensitive; and Pol III transcribes genes for small RNAs and has intermediate sensitivity. Jan Kooter got similar results with his trypanosome nuclei with one exception: the transcription of genes in the active VSG gene expression site was as insensitive to α-amanitin as transcription of the rRNA genes. We concluded that the expression site was actually transcribed by Pol I, the first example ever of a protein-coding gene being transcribed by Pol I 95. The result made sense: by that time transsplicing had been found and the major obstacle to the use of Pol I for rRNA synthesis was therefore absent in Kinetoplastida. This obstacle is that in other eukaryotes the Pol II machinery is associated with mRNA capping and uncapped mRNAs are rapidly degraded. In trypanosomes the primary transcript does not need to be capped, because the cap is provided by the mini-exon. It also made sense to recruit Pol I for heavy duty RNA synthesis, because large amounts of mRNA have to be made from a single copy gene. Pol I may be more up to this task than Pol II.

          Our results were initially met with skepticism, if not hostility. No other lab could get the nuclear run-on system to work and I received rather unfriendly comments on the irreproducibility of our experiments. Eventually, however, other labs also learned the trick and all results of Jan Kooter were fully confirmed. It took long, however, before definitive proof was obtained for our tentative conclusion that RNA Pol I transcribes expression sites. Other labs gradually accumulated more evidence, but the final blow came only 20 years later.

          We came back to the problem twice:

First, Joost Zomerdijk verified that Pol I can indeed produce functional mRNA precursors by showing that an rRNA promoter can be used to efficiently drive mRNA synthesis 115. Gloria Rudenko also did the reverse experiment and showed that an rRNA promoter can replace the expression site promoter in an active VSG gene expression site 117,122. This modified expression site could be switched on and off at the same rate as the site with an authentic promoter 122.

          An early popular model for expression site control was that the active expression site was selected out of 20 equivalent sites by moving it into a privileged location in the nucleus. An obvious place for this location was the nucleolus, the place where ribosomal DNA genes are normally transcribed by Pol I. So, we determined where the nascent RNA was produced from the active expression site and this was clearly not in the nucleolus 123, as suggested by others.

          Later, Inez Chaves, and Gloria Rudenko went back to the problem of expression site control and asked the simple question whether two expression sites can be simultaneously fully active. They put different drug resistance markers in two expression sites and selected for double resistant trypanosomes. These were obtained, but rather than having two fully active expression sites, the trypanosomes were found to rapidly switch between the two selected expression sites 124. Interestingly, the two sites were close together in the nucleus, as if they needed to be in a privileged site to be fully active 124. Our attempts to further characterize this site were unsuccessful, but later Miguel Navarro in the laboratory of Keith Gull defined a structure containing the active expression site and called this the expression site body (ESB). This raises as many questions as it answers 125, but it does support the notion that activation of an expression site involves positive selection and possibly a privileged location in the nucleus.



          Genomics is not my cup of tea, but we provided some of the background for the genome project, a reliable estimate of the genetic complexity 12 and the nuclear DNA content 119 of T.brucei. We also determined the sequence of some of the major satellites of T.brucei, the telomeric repeats 126 the 70-bp repeats 80; the 177-bp repeats 127 that make up most of the mini-chromosomes; and the large block of 50-bp repeats that marks the border between VSG gene expression sites and chromosome-internal sequences 113. My interest in repeats probably comes from deep down, the time when I started on mitochondrial DNA in 1965. At that time one of the lead characters in molecular biology, Julius Marmur, claimed that the satellite DNAs found as separate bands in CsCl equilibrium gradients (this actually gave them their name) were mitochondrial DNAs. One of my first experiments in my new field was a cell fractionation in which I showed Marmur to be dead wrong: satellites were nuclear 128

          The fact that so much junk DNA is derived from transposons / retroviruses or from expansion of simple repeats through stuttering/slippage of the DNA replication machinery, has led to a general loss of interest in the function and origin of more complex “satellites”, such as the 50-bp and 177-bp repeats. Nevertheless, I hope that somebody will find out what these repeats do in tryps and how they are maintained.


Transferrin receptor variations

          After we determined the location of the 221 ES promoter, Joost Zomerdijk wanted to check whether activation and inactivation of the expression site promoter was associated with any change in DNA sequence. This required cloning of the 221 promoter from a variant in which it was active, and independently from a variant in which it was inactive. This was easier said than done, because expression site promoters all look the same. Joost found, however, that the promoter-proximal gene in expression sites, the ESAG7 gene, contains a hypervariable region that can be used to distinguish the genes from different expression sites, which are more than 97% identical. The hypervariable region contained a high frequency of first and second codon base changes suggesting that hypervariability was actively being selected for 113. Joost Zomerdijk used this useful property of the ESAG7 gene to clone his promoter fragment and to show that activation of the promoter was not associated with DNA rearrangements close to the promoter 113. I remained intrigued, however, by the hypervariable region. What could this gene code for? Which trypanosome function could require 20 versions of a protein differing only marginally?

          I was therefore delighted when the Overath lab reported that ESAG6 encoded the transferrin receptor (Tf-R) of trypanosomes. This receptor was already known to be located in the flagellar pocket of the trypanosome, a place where it would be confronted in principle with host antibodies. Because the receptor must see the outside world to bind Tf, the receptor surface should be a target for the host immune response. In fact, Hobbs and Boothroyd had already shown that the serum of rabbits, chronically infected with trypanosomes, contained antibodies against the ESAG6 gene product. I therefore concluded  129 that the shrewd trypanosomes had invented antigenic variation of the Tf-R and that this was the evolutionary driving force behind the small differences between the ESAG6 copies, discovered by Joost Zomerdijk.

          Soon I had recruited a new post-doc, Marjolijn Ligtenberg, who started experiments to test the hypothesis. Her attempts to get the ESAG6 Tf-R to work in insect-form trypanosomes failed, however. The ESAG6 protein expressed nicely, it was routed to the surface and spread over the entire surface, but it bound no Tf. I contacted Peter Overath and we decided to join forces to clarify the negative result. After a frustrating period, we finally found that the original identification of the Tf-R was incomplete: the receptor did not consist of ESAG6 only, but of a heterodimer of ESAG6 and 7. Peter Overath (who is scrupulous and honest) wrote a correction for the EMBO Journal, in which he kindly included Marjolijn Ligtenberg and me as co-authors, but this went one step too far for me. We had made a real contribution to the solving of the problem, but we had no part in the original mistake. Soon Marjolijn Ligtenberg had managed to reconstruct a functional Tf-R on the surface of insect-form trypanosomes 130. Overath obtained similar results in the baculovirus system and Etienne Pays in Xenopus eggs.

          This opened the way to a critical test of the hypothesis that the trypanosome has the ability to make 20 different versions of the Tf-R to avoid clogging of the receptor by host antibodies. Our initial experiments were disappointing. Antibodies that clearly bound to the top of the receptor were unable to prevent multiplication of the trypanosomes and we soon found out why: Tf bound so tightly to its receptor that antibodies just could not compete at all at the high Tf concentrations that prevail in mammalian serum.

          This would probably have been the end of this project, if Wilbert Bitter, the post-doc who had taken over from Marjolein Ligtenberg, had not known about Tf. He knew that the aminoacid sequence of Tfs has rapidly changed in mammalian evolution. Pigs and humans are both infected by trypanosomes and pig and human Tfs differ as much as 35% in sequence. How could a single receptor bind Tfs that are so different both with high affinity? Even if the Tf-R of the 221-expression site had very high affinity for bovine Tf, there might be other Tfs that this receptor would bind with much lower affinity. This was easily tested in competition experiments. Wilbert rapidly found that cold murine Tf competed efficiently with uptake of radioactive bovine Tf, but that human Tf competed poorly and dog Tf not at all 131.

          How could we find an expression site that produced a Tf-R that was able to bind canine Tf? Wilbert solved this problem with a delightfully simple experiment that I did not expect to work. Fortunately, Wilbert ignored my advice and put our 221 trypanosomes in dog serum. Initially the cells hardly grew, but after 2 weeks they suddenly picked up. To our joy the trypanosomes that grew out had switched expression site and were now producing a Tf-R able to bind dog Tf 131.

          In the years after this discovery Herlinde Gerrits, Rainer Mußmann, Rudo Kieft and Henri van Luenen refined the picture considerably 132-135. If trypanosomes are switched from calf serum-based medium to dog serum based medium, they immediately increase the expression of ESAG6 and 7. We can detect upregulation after a few hours, whereas it takes several divisions to exhaust the iron stores of the trypanosome. We infer that the trypanosome can sense the level of iron coming in from the outside, but how remains to be determined.

          The upregulation can result in Tf-R levels up to 10-fold wild-type and a large fraction of this additional Tf-R spills out of the flagellar pocket onto the pellicular surface where it contributes to Tf uptake, because the entire surface is internalized at enormous rate, as shown by the Overath lab. If upregulation does not suffice, the strong selection for a functional Tf-R results either in selection of trypanosomes that have switched to an expression site that produces a Tf-R with higher affinity for dog Tf in the 221 receptor, or it sets off bizarre gene rearrangements resulting in modification of the Tf-R population, which also leads to increased affinity for dog Tf 135.
          The Tf-R project has been more difficult than I initially anticipated. One reason is the availability of dog serum, a key tool in our experiments. Supply is limited, unreliable and, most unfortunately, properties of dog serum are much more variable than those of calf serum.   

          A second complication was that other investigators found it difficult to believe that trypanosomes need a receptor for Tf at all, given the very high levels of Tf in mammalian serum and the high rates of trypanosomal fluid endocytosis. I am convinced, that we are right, but the final proof, will require a field test. If a high-affinity Tf-R is really important for trypanosomes one should find in lions only T. brucei with a Tf-R that binds lion Tf well and in pigs one with high affinity for pig Tf. The experiment is feasible, but not simple.


Drug transporters

          My brief excursion into the drug transporters in Leishmania had a rather weak rationale, at least with hindsight. Like many other labs we were trying around 1985 to set up a transformation system for T. brucei and did not get anywhere. T. brucei had no usable plasmid, as far as we knew then, and we therefore looked with envy to Leishmania with its talent for amplifying DNA and its ability to retain some amplified DNAs as stable replicating circles. This is why post-doc Ted White started to analyze the H-circles of Leishmania tarentolae.

          We now know that T. brucei and L. tarentolae are probably as different as mice and frogs, but at the time these experiments were done we optimistically expected a plasmid system from Leishmania to work in T. brucei. From my work on mitochondrial circular DNAs I knew how to isolate and analyze circles. My student Cees Aaij and I actually introduced ethidium bromide agarose electrophoresis to separate topoisomers of circles 136, a technology that has become so widely used that the paper is unfortunately not quoted anymore. So Ted White isolated H-circles, and characterized the inverted repeats present in them, which led us to propose a model for the origin of these circles, which still has not been shown to be incorrect today 137. H-circles could be generated by drug selection, notably by selection with methotrexate (MTX) and by arsenite. In 1984 I had started a project on multidrug resistance in cancer cells and my lab was therefore heavily involved in trying to decipher mechanisms of drug resistance. Gradually the study of drug resistance in Leishmania became a goal in itself and not only a source of plasmids to be used in T. brucei.

          It was Marc Ouellette who had taken over the project from Ted White as a new post-doc in the lab, who did an experiment that got us going for real. Since everybody in my multidrug resistance group was working with ABC transporters, such as P-glycoprotein, Marc Ouellette decided to look for the characteristic signature of ABC transporters in the Leishmania H-circles, using mammalian probes. I did not think at the time that this experiment made great sense for two reasons: first and foremost, the P-glycoproteins were known to cause resistance to relatively hydrophobic compounds and not to MTX or arsenite. Secondly, I did not expect that mammalian probes would cross hybridize to any segment of any protein-coding gene in Leishmania, because the evolutionary distance is so large and because Leishmania also has an unusually high percentage of GC in its DNA. Once more, I was too skeptical and Marc Ouellette identified the first ABC transporter in Leishmania, which we called Pgp-A, because it resembled P-glycoprotein with its 2 ATP-binding motifs and an overall putative topology of typical ABC transporters 138,139. We were surprised at that time, however, that this ABC transporter was larger than the others described to date, but this did not deter us from calling it a P-glycoprotein. Years later Susan Cole and Roger Deeley identified the Multidrug Resistance-associated Protein (MRP) in mammals and found it sufficiently different from P-glycoproteins to give it a separate name. They also noted that Pgp-A looks more like MRP than like P-glycoprotein and indeed Pgp-A can be considered to be the founding father of the MRP family, which has proven to be one of the most widespread and numerous families of drug transporters in nature.

          Besides the Pgp-A gene there were several other ABC transporter genes on the H-circles, in addition to a gene conferring resistance to MTX. The cluster was surrounded by repeats and it was obvious that the repetitive sequences were strategically placed to allow ready amplification of this DNA in the form of linear or circular amplicons 140. We postulated that this arrangement was selected in evolution to allow rapid adaptation to adverse conditions 140,141. With hindsight the H-circles were one of the first eukaryotic examples of what later became known as contingency genes, i.e. genes readily activated under stress.

          I became very pleased with the Leishmania drug resistance project, not only because we got results, but also because for the first time I had managed to forge a bridge between my work on multidrug resistance in cancer cells and my research on kinetoplastida. The bridge did not last long. When Marc Ouellette went back to Canada after 3 post-doc years, he asked my permission to take the Leishmania project with him. It was a reasonable request. After all, it was Marc’s idea to look for Pgp genes on the H-circles and so the protozoal drug resistance moved with Marc to Canada. With hindsight it was the right decision. The project proved tough, but highly fruitful and it took years of hard work and some intensive friendly competition between the labs of Marc Ouellette and Steve Beverley to sort out the mechanisms of MTX and arsenite resistance. I would never have been able to solve these complex problems as a side project in my own lab and certainly not in competition with Steve Beverley.

          The big surprise that a P-glycoprotein gene was somehow associated with arsenite resistance was solved in mammalian cells by the demonstration that MRPs, such as Pgp-A, are organic anion transporters that can also transport complexes of GSH with arsenite or antimonite (see Borst and Oude Elferink, 2002 139). Eventually Marc Ouellette would prove that Pgp-A is located in the vacuole membrane and able to transport arsenite-trypanothione complexes into the vacuole, reducing arsenite toxicity. This is not a very good strategy to get rid of the arsenite and Pgp-A is only a weak resistance determinant.


Base J.

          A nut that has proven tough to crack is the function and biosynthesis of base J, formally called ß-glucosyl-hydroxymethyluracil. The first indications for the presence of an unusual base in the nuclear DNA of T. brucei were independently obtained by Etienne Pays and collaborators and by André Bernards in our lab. What we both found was that a VSG gene located in an inactive expression site is incompletely cut by certain restriction enzymes, such as Pst I, whereas it is completely cut when the expression site is activated or in insect form trypanosomes 142. This indicated the presence of a DNA modification and it even suggested that this modification was associated with the inactivation of expression sites, which made us interested. Our initial attempt to determine the nature of this base went nowhere. The base was obviously present in low amounts (we now know that it replaces about 0.5 percent of T) and it did not behave like methyl C 143. Only when Janet Gommers-Ampt took the big risk of tackling this problem for her thesis research, we started to make progress. Janet introduced a sensitive system for analysis of unusual nucleotides, devised by Randerath, post labeling followed by 2-D thin-layer chromatography. After considerable fiddling she mastered the technique and discovered a new spot on the 2-D TLC, which, to our delight, migrated to a position where no other unusual nucleotide had ever ventured 144. From that moment on we knew that we were on to something really new and this justified a major effort. Jeanet developed a column procedure to purify the new base in nucleotide form and started isolating sufficient amounts of the compound for further analysis. My friends working with mass spectrometry (MS) were optimistic that this problem would be solved in weeks.     Unfortunately, however, their optimism proved unfounded. During sample preparation, the glucose fell off base J and MS never managed to get more than the hydroxymethyluracil part, that we had identified already as a putative precursor of our new base 144,145. In the end we had to combine results from a variety of analytical techniques to get the unambiguous structure finally published in Cell 146. Read the paper to understand why I consider this one of the most ingenious and interesting things done in my lab.  This was also the period that we discovered the essential role of the MDR1 P-glycoprotein in the blood-brain barrier 147 and that the MDR3 P-glycoprotein is a dedicated phosphatidylcholine transporter essential for mammalian bile formation 148. Writing papers about sophisticated methods of nucleotide analysis 146, about the blood-brain barrier 147 and about bile formation 148, all in the same year made excessive demands on my memory (then nearly 60), but it certainly was an exhilarating experience.

          Once we knew the structure, an organic chemist, my friend the late Jacques van Boom, worked out a chemical synthesis for J-nucleotide and made oligonucleotides containing base J. We used the nucleotide to raise antibodies in rabbits, which provided a much more convenient assay for J-DNA than the 2-D TLC 149. This new tool was used by Fred van Leeuwen, a new student in the lab, to determine the distribution of J within T. brucei, where it was mainly found in telomeric repeats and in some other repetitive sequences 149-151, but only in blood-stream form trypanosomes 152. In nature J was found in all kinetoplastida analyzed, in Diplonema and in Euglena 153, but not in more distant phyla 154.

          Using the J-containing oligonucleotides post-doc Mike Cross found a J-binding protein (JBP1), present in all kinetoplastida analyzed 155. JBP1 is not an essential protein in T. brucei, but required to maintain normal J levels 156. In Leishmania it is essential however, and we are still trying to find out why.

          Unfortunately, the major questions – how is J made and what is its function? – remain unanswered. We have good reasons to think that J is made in 2 steps: in the first step specific thymines in duplex DNA are oxidized to yield hydroxylmethyluracil; in a second step glucose is attached to the new hydroxyl group 156-159. Although the putative glucosyl transferase is able to transfer glucose onto hydroxymethyluracil anywhere in DNA, we have been unable using our oligo assay to find this promiscuous enzyme in trypanosome extracts. We are now using various indirect ways to get at this enzyme because knocking out one of the genes required for J synthesis should tell us more about the function of J.



          Doing science implies making mistakes. I tried to intercept these mistakes before the paper went out, but I missed some. I mentioned already the proposal that glycosomal targeting sequences consisted of two clusters of basic aminoacids 56. A second error was our conclusion that two VSG gene expression sites can be simultaneously fully active 96. When we later found that this is not possible 124, we went back to the data of the original paper and saw that they were fine, but misinterpreted. What we had interpreted as two fully active sites, was in fact a telomere exchange. With hindsight it is really amazing that this misinterpretation was not picked up either by my smart collaborators or by the competent Cell reviewers, who were usually quite suspicious of my interpretations. I can only guess that we were unable to think of reciprocal telomeric recombination at the time, since this was only discovered (by the Pays lab) after our paper came out. Much later Gloria Rudenko found that telomere exchanges is actually a frequent mode of VSG gene switching in the 427 stock under certain conditions 160.

          Finally, I am not sure whether our claim for stable transformation of T.brucei by mini-chromosomal DNA from T. congolense 161was correct. The result has not been duplicated in other labs and in my lab we noticed after the paper had been published that added DNA is sometimes carried in trypanosome cultures for many generations. The lack of a selectable marker in the mini-chromosomes made it impossible to select for the transfectants. After Carruthers and Cross developed reliable transient transformation of T.brucei, we followed with a robust stable transformation system 162 and we never went back to minichromosomal transformation.



          Although I did some experiments with my own hands in the early day of trypanosome research under the stern supervision of my trusted technician Francesca Fase-Fowler, nearly everything discussed here was done by students, post-docs and technicians, often in close and indispensable collaboration with other groups. I have mentioned most of the names, but the reader may wonder what became of all these people. Some of them stayed with trypanosomes and set up their own labs. Those probably known best to the readers of the story are Fred Opperdoes and Paul Michels (at the Institute of Cellular Pathology in Louvain), Alan Fairlamb at the Wellcome Institute in Dundee, Wendy Gibson at the University of Bristol, Patricia Johnson at UC Berkeley and Marc Ouellette at Université Laval, Quebec, Canada. Ted White at the Seattle Research Institute stayed with uni-cellular pathogens but switched to fungi. Others became famous in a different field of basic biomedical research: Jan Hoeijmakers in DNA repair; Titia de Lange, who did some of the early work on trypanosome telomeres, became well known for her contribution to proteins binding to mammalian telomeres, and for her studies on telomere structure and function. Lex van der Ploeg rapidly rose through the academic ranks of Columbia University in New York before he switched to the pharmaceutical company Merck, where he was recently appointed as director of a splendid new large research institute in Boston, close to his old friend André Bernards in the MGH Cancer Center. Fred van Leeuwen came back in a tenure track position to the Netherlands Cancer Institute, but not with a trypanosome project, but with yeast epigenetics.


Concluding remarks

          I entered the trypanosome field by accident, but I never regretted this rash new step. My trypanosome projects have been among the most stimulating and satisfactory research projects in which I have been involved.  The main reason is, of course, that the Kinetoplastida branched off the eukaryotic tree early in evolution. This is why they use original solutions for biochemical problems that are very different from those used by yeast or mice. The results obtained in the lab would usually not resemble my predictions and this is what makes research interesting. Molecular parasitology is not mainstream molecular biology, but it contains a lot of nice, helpful and interesting people and I enjoyed being part of this community. I intend to stay around for some time, at least until I know what base J does and how it is made.



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