An Interview with Werner Arber

Interview The Inventiveness of Nature: An Interview with Werner Arber

Jane Gitschier* Departments of Medicine and Pediatrics and Institute for Human Genetics, University of California San Francisco, San Francisco, California, United States of America

Image 1. Werner Arber stands outside the Biozentrum at the University of Basel, Switzerland. doi:10.1371/journal.pgen.1004879.g001

I admit to having a soft spot for the years, restriction and modification was not spawned extremely useful technology. In bacterial phenomenon of host-specific only a remarkable story in itself, but also past issues, I have interviewed Hamilton modification and restriction. I vividly remember first learning about this mech- Citation: Gitschier J (2014) The Inventiveness of Nature: An Interview with Werner Arber. PLoS Genet 10(12): anism to stave off marauding DNA as a e1004879. doi:10.1371/journal.pgen.1004879 beginning graduate student in the mid- Published December 18, 2014 1970s. Preceding the discovery of its Copyright: ß 2014 Jane Gitschier. This is an open-access article distributed under the terms of the Creative modern-day defensive counterpart, Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, CRISPR (clustered regularly interspaced provided the original author and source are credited. short palindromic repeats) by about 50 * Email: [email protected]

PLOS Genetics | www.plosgenetics.org 1 December 2014 | Volume 10 | Issue 12 | e1004879 Smith, who isolated the first type II Edouard Kellenberger, who ran the La- new preparation techniques. You could restriction enzyme, as well as Herb boratoire de Biophysique in the basement spread the lysate out on the film and see Boyer, whose production of recombinant of the Institute of Physics there. It housed and count phage-related structures. In DNA in collaboration with Stan Cohen electron microscopy as well as radiation nonmutant strains of lambda, of course, launched the biotech industry. For this studies. I went there within the next few there were the nice phages with heads and interview, I was able to go back even days, and I was satisfied and accepted the tails, whereas for some of the mutants, farther into the tale and hear first-hand job. there were heads but no tails attached, and how restriction and modification came to I have to tell you the beginnings of this. sometimes the tail was missing. In some be understood at the molecular level. The Institute of Physics had been headed other cases the heads were empty. This came at the tuition of Werner by another Swiss professor, Jean Weigle. Gitschier: Where did all these mutants Arber (Image 1), who received the Nobel Weigle had agreed to help a Swiss come from? Prize together with Smith and the late company specialized in vacuum systems Arber: From the lab itself, and we got Dan Nathans. Arber remains active in to develop an electrostatic electron micro- some from the Pasteur Institute. Then one science; he heads the Pontifical Academy scope. It was given to the Institute in the day, Weigle brought me the defective of Sciences and has a keen interest in 1940s, free of charge. lambda transducing prophage that had understanding evolution’s molecular driv- Weigle then had some health problems been isolated in the Lederbergs’ lab by ers, one of which—horizontal gene trans- and later quit the directorship of Physics. Larry Morse. He asked whether lambda fer—is a direct descendent of his work on He was very interested in biology, and he could also transduce, like P22, and it did! phage transduction. In early June, after a moved to Caltech in California in 1949. But it transduced only galactose markers. quick jaunt to Paris (to watch Rafael But he still had an apartment in the old And that was only upon induction of Nadal win the French Open for the ninth town of Geneva, and he came to Geneva phage production by lysogenic bacteria, time), I popped over to Basel to reunite every summer for three months. At not after lytic infection. Now we know that with old friends and to interview Arber. Caltech, he worked with Max Delbru¨ck; the attachment site for the lambda pro- Despite that Monday being a Swiss he was a part of that relatively small, at phage is near the galactose markers… holiday, Arber suggested meeting with that moment, phage group. Gitschier: … in the E. coli genome. me in his office on the top floor of the Gitschier: Presumably, he knew Del- Arber: Yes, and at frequencies of 1025, Biozentrum. It was exceptionally hot, a bru¨ck through the physics connection. the lambda genome crosses out imprecise- record-breaking 98uF (37uC) outside, and Arber: Absolutely. He brought to ly from its attachment site to form lambda- possibly even hotter indoors. Over three Geneva the connection to the phage gal, but this hadn9t been known yet at that hours, we worked our way through a large group, as well as microbial strains. He time. So I induced the defective lysogen bottle of water, which his wife, Antonia, normally stopped over in Paris at the that we received from Weigle with UV had thoughtfully suggested he bring along. Pasteur Institute where [Franc¸ois] Jacob irradiation, and the cells lysed but I Neither of us flagged as he reminisced and [Jacques] Monod were working, and couldn9t see any phage-related structures about the field and his work in 1950s and we had, therefore, a very good connection in the lysate. 960s. with these microbial geneticists. Gitschier: No phage particles? Arber developed his interest in natural Weigle also had bacteriophage lambda, Arber: Indeed, no phage particles, no science as a child growing up on a farm which originally had been discovered by empty heads, no tails! near Aarau, south of the Jura Mountain Esther Lederberg as prophage in E coli At that moment, I decided to start range of Switzerland. After attending the K12 bacteria. genetic experiments. There were a certain local gymnasium, he studied at the Swiss The problem was that everybody had number of already mapped mutants avail- Polytechnical School in Zurich to become K12, which was lysogenic, but they didn9t able—for head, tail, and other properties. a science teacher, rather than pursuing his have lambda-sensitive, nonimmune From induction of double lysogens— mother’s hopes for his priesthood in the strains. And Weigle brought us both from having one lambda-gal and one active Protestant church. He majored in exper- Esther—the lysogenic and the sensitive phage—you get both active and transduc- imental physics and completed his under- strains. In fact, that was not easy because ing particles. In a series of genetic graduate degree in 1953, just after the Esther said she wanted to do some experiments, I could show that in the structure of DNA was discovered, and experiments with that phage herself. But lambda-gal genome, a few head and a few went on to the University of Geneva for Weigle said, ‘‘We have an electron micro- tail genes were just missing. Then we his PhD. We pick up the conversation at scope and we can look at it,’’ and realized that instead of having these head this transition. everybody was interested to see what it and tail genes, they must carry gal genes. Gitschier: You finished your teaching looked like! And that was what Kellenber- That was one of the early clear proofs that degree, but you were thinking about doing ger did—he pleased the involved scientists some viruses can serve as vectors for host basic research? with a nice electron micrograph of phage DNA integrated into the viral genome. Arber: Yes. Most of the physicists did lambda. This is now called specialized transduc- not take the broad curriculum I had, Gitschier: And were there other types tion. I reported our findings at the including biology and so on. So Paul of bacteriophage that they looked at? Assembly of the Swiss Society for Micro- Scherrer [the prominent Swiss physicist Arber: Of course, the T-evens and a biology. A few people may have under- who was Arber’s advisor] suggested I go to number of other phages. One of my jobs stood it. the University of Geneva where they had in the lab was to look at mutant bacterio- Gitschier: What year? an electron microscope, which needed phages; there were about ten different Arber: That was in 1957. With the daily care by a physicist. There they mutants of lambda available. These were impulsion of Edouard, I was invited to a looked at a variety of biological objects: prophages, so I could induce by UV conference of microbial geneticists at bacterial extracts, viruses, bacteriophages, irradiation and then look at the lysate. Royaumont near Paris, to present my and so on. Right away he phoned Kellenberger was very good at inventing work, and that had a really big echo.

PLOS Genetics | www.plosgenetics.org 2 December 2014 | Volume 10 | Issue 12 | e1004879 On the spot I got two invitations for looking for conjugation in Salmonella develop atomic power plants in order to post-doc—one by Joe Bertani in Los strains. And at first view, it seemed that get a new energy supply. Paul Scherrer Angeles, and the other by Franc¸ois Jacob there was recombination. But then they managed to convince the Swiss Parliament at the Pasteur Institute. I thought both could show that there were no cell–cell to vote for a big financial credit just for would be interesting, but I could only contacts. In the conjugation mixture some that. And Edouard had made a request accept one; I chose the United States of the cells lysed, being lysogenic for phage that a very small part of that big credit rather than France. A few years before I P22, which turned out to be a transducing should be spent to study radiation effects: came to Bertani’s lab, he had hoped to phage. That was the discovery of trans- biosafety. He got that money, and he obtain phage lambda and appropriate duction by Zinder. asked whether I would come back to lead hosts from the Lederbergs, but this was And then Larry Morse was charged that group. So I went back to Geneva and apparently quite difficult. He therefore with investigating whether lambda also I planned to study radiation effects on decided to isolate phage strains from other does that. And it did—but only for bacteria and on phages. lysogenic bacteria himself. He called these galactose, while the Salmonella phage Gitschier: What kind of radiation? phages P1, P2, and P3. P22 gives generalized transduction, and Arber: Any radiation. Starting with Joe and his wife Betty decided to work so does phage P1. These phages transduce UV, which we used for induction of phage with P2, and they did a lot of experiments any host genes. production by lysogenic bacteria, X-ray, with P2 using various hosts. P1 is a Gitschier: So, lambda was interesting and then also I intended to incorporate transducing phage, and Joe told me I because it was different. heavy radioactivity, 32P, in the DNA. could do anything I wanted. Practically Arber: Yes. And with my observation That’s why I went to learn the ‘‘suicide’’ every day we went to the cafeteria to have of the genetics, it became clear how, in technique from Gunther Stent. lunch together, and we spoke on science. specialized transduction, some host genes Gitschier: So, you were already plan- At the end of that one-year period, I had can become part of the viral replicating ning to come back here. enough data to draft two papers. I showed unit. The defective phage lambda-gal still Arber: Right. And the other thing I them to Bertani—I had put his name as undergoes replication, but it doesn9t make had known was that Evelyn Witkin had coauthor—and he said, ‘‘These are nice functional coats and tails. In contrast, isolated radiation-resistant mutants from papers but you are the author.’’ I got so there is no trace of viral DNA in E. coli B. The strain was B/r, and it was much input from him every day, but I had transducing P1 and P22 particles. In already available in several labs. [Readers to accept his opinion. I submitted them to generalized transduction the infective may want to refer to the Witkin interview Virology; Salvador Luria was one of the transducing phage particles are just filled in 2012.] And of course, B is not a host for editors. He accepted both papers, and at with a segment of host DNA. lambda, because lambda does not adsorb that moment he offered me a second post- Gitschier: Got it! to B. doc. I also got invited by Joshua Lederberg Arber: From bacterial conjugation Gitschier: So, you had a problem to work with his wife Esther at Stanford, experiments, it had become known by because you wanted to study lambda in and I got a third offer from Edouard the late 1950s that DNA are very long the radiation-resistant strain. Kellenberger to return to Geneva! filamentous molecules carrying their ge- Arber: Right. But Esther Lederberg After one year of post-doctoral work in netic information in a linear order. As a gave me a hint; she knew that E. coli Bis Los Angeles, I left Bertani’s lab and went matter of fact, the conjugation results maltose minus, and she thought that the to Gunther Stent in Berkeley for a few suggested that the E. coli chromosome is maltose minus character is responsible for weeks, because I wanted to learn the only one circular molecule. not adsorbing lambda. ‘‘suicide’’ method that he had invented This raises the question of how can one Gitschier: Because the maltose trans- with very heavily 32P-labeled phage. You best study single genes, one by one. People porter might serve as the lambda receptor? grow the phage in 32P medium, store then proposed to sort out short genomic Arber: Right. So she suggested to them, and assay the survivors every few segments and to splice them into a natural transduce E. coli B with P1 grown on days. After that stage, I went to Stanford gene vector such as the lambda genome. K12, and to select for maltose-plus to work with Esther for one month, and Because the hybrids are replicative units, derivatives. And she was right. About then on the way back to Geneva I stopped they can be expected to replicate the one of three of the maltose-plus transduc- over at MIT to work for another month inserted gene separately from the rest of tants adsorbed lambda. with Luria. the bacterial genome. This can yield Gitschier: Wow! Gitschier: So tell me, while we are on enough material to undertake both struc- Arber: But then, my lambda prepara- the subject of Esther. Was lambda the first tural and functional analyses. tion came from K12, and it adsorbed to phage to be discovered to have this Gitschier: Wow! So people were the transductant B/r, but almost no phage property of lysogeny? thinking ahead to sequence analysis even was produced. Arber: No. Lysogeny was a known back then? Gitschier: So this is a multistep phenomenon, and actually Andre´ Lwoff Arber: Yes, right! People were thinking process. After all of this manipulation, had explored lysogeny with other types of just to break the DNA by sheering forces, only a few progeny phage came out if lysogens. but they didn9t get very far with that. So you9ve grown them before in K12. So B Gitschier: So, what was it that was so the idea of recombinant DNA was already restricts lambda that is grown in K12. special about finding lambda? there, but one didn9t know how to do it Arber: Precisely. And those few lamb- Arber: From my point of view, the best. da that come out of B can go back to B special thing was specialized transduction. Gitschier: Now I understand the without restriction. Weigle and Bertani A short background: conjugation had been significance of lambda. had already worked on host-controlled shown to work with E. coli K12. And, in Arber: I have to tell you now about the modification in both, P2 and lambda, and fact, another student of the Lederbergs offer I got to return back to Geneva. This I was fully aware of their work. This had was Norton Zinder who was charged with was in 1959. In physics, they intended to happened maybe five years before.

PLOS Genetics | www.plosgenetics.org 3 December 2014 | Volume 10 | Issue 12 | e1004879 Gitschier: I was wondering why Ber- viruses with semiconserved DNA per lysed invited to write a contribution to Annual tani and Weigle didn9t keep working on cell, whereas all the other phage genomes Review of Microbiology [published in that? would be new, without 32P. And, in fact, 1965], and there I speak about methyla- Arber: I don9t know. that was confirmed. Only parental DNA, tion as a likely basis for modification. Gitschier: So now we are at the point which was still subject to radioactive Gitschier: So, just to be clear, this host that you tried to take the lambda from decay, actually had the parental modifica- modification process was really discovered K12 and grow it in B/r mal+ and you tion. Most of these experiments were done as a phage process. And later it was realize that you have host modification with P1 restriction [phage P1 carries its understood to be a basic problem. problems. own modification and restriction system], Arber: I realized rather rapidly that Arber: Right. Grete Kellenberger had but I did it later also with B and with K12 this was not limited to phage. And, of done already some studies on radiation hosts. course, we could then show that it also effects on lambda and shown that, when Gitschier: So at this stage, you know it affects conjugation, DNA transformation, heavily UV-irradiated, DNA becomes is something about the DNA. and transduction. It works in any of these rapidly acid-solubilized, which means it is Arber: That was indeed new! One did processes. We did all these experiments to degraded; same thing with 32P heavy not expect it would be about the DNA. show that it is a general phenomenon of labeling. So they wondered if upon Gitschier: These people prior to the host. restriction, the DNA is also degraded you—Luria and Bertani—they are all Actually, the proof for methylation and solubilized. saying it’s not a mutation, it’s a modifica- came later on when some of my collabo- When I came back to Geneva, there tion. Was there anything known at the rators succeeded in isolating the modifica- was a student starting her PhD study in the time about methylation? tion enzyme. This enabled us to show that radiation field: Daisy Dussoix. She started Arber: OK, I9ll tell you. I knew the modifying enzyme is a methylation to work with me on that question, and one Gunther Stent very well, and one day enzyme. I, myself, was not feeling like of the first experiments was to see whether, Stent said they had a fellow who worked doing enzymology, but I had very good like for irradiated phage, restricted phage with nucleic acid methylation. collaborators, like the Americans Bill DNA was also degraded. And it was. Gitschier: Oh, really? Was DNA Wood and Stuart Linn, as well as Urs Gitschier: And did you do that with known to be methylated at that time? Ku¨hnlein, a Swiss PhD student. 32P labeling? Arber: There were publications on Gitschier: It was in the late 960s before Arber: Yes, but not the heavy one, just DNA methylation in eukaryotes. And the restriction enzymes were isolated. slightly labeled, as a tracer. some of these papers postulated that Arber: Yes. Matt Meselson had moved So then, I felt that we should also study DNA methylation might have something to Harvard, and Bob Yuan was one of his the restriction modification mechanisms in to do with cancer. young collaborators. In 1968, they were more detail. Now, retrospectively, we Gitschier: But not yet in bacteria. the first to isolate the K restriction know that the reasons for DNA degrada- Arber: No. But Stent wondered if enzyme, and to determine its cofactor tion are different between irradiation and bacterial modification is, in fact, DNA requirements, on which our group had restriction. But actually, the money came methylation for which methionine would been properly informed. from the nuclear power plant funding to be the methyl donor. I happened to have Gitschier: This is a type I enzyme that study radiation effects, so I had to justify methionine minus bacterial hosts. You just chops things up all over the place. But why we did that other project on host- grow them in the presence of methionine. the methylation, even there, is at specific controlled modification. These bacteria are then infected with the sites. But before I justified it, we did a very phage, or in the case of lysogenic cells, Arber: Yes! There are recognition sites. important experiment. If you look at our phage reproduction is induced by UV This was always my idea that there were first JMB [Journal of Molecular Biology] irradiation. After a few minutes of further specificity sites. I had expected that the paper on restriction and modification in incubation in the complete medium, the enzymes would generally be of type II, i.e., 1962, you9ll be surprised that I did it with cells are washed and then further incubat- specific cutting sites. the ‘‘suicide’’ technique. Of course, I had ed in medium without methionine. Under Gitschier: But you were able to show read about semiconservative DNA repli- these conditions, the majority of the that the methylation was happening in cation by Matt Meselson and Frank Stahl, produced phage was not modified, even specific places in the DNA? so I knew the principles of DNA replica- though the bacterial host was a modifying Arber: For a long time, that was a tion. strain. hypothesis. Gitschier: That was 958. Gitschier: The specific testing of Gitschier: Because I was wondering, Arber: Yeah, and my experiment was methylation doesn9t come across in your without type II restriction enzymes, them- done in 1960. I prepared a stock of 1965 paper [one of a series of JMB papers selves, to specifically cleave the DNA, how lambda, which was heavily loaded with on host specificity]. You test several can you even figure that out? 32P—‘‘suicide’’ levels. Immediately after auxotrophs, but you don9t say you are Arber: I called on John Smith, an its preparation, the phage stock was testing methionine with the others as enzymologist from England, and he spent carefully purified from the radioactive controls. At the very end of the paper about half a year with us in Geneva. He medium and then used for a one-cycle you say that the chemical nature is was quite successful in mapping the sites of growth in a nonmodifying host in nonra- unknown, but that alkylation is an attrac- methylation. They had mastered RNA dioactive medium. tive possibility. sequencing, but not yet DNA sequencing, Taking into account the semiconserva- Arber: At that time I considered the so it was hard work to determine the tive DNA replication, I expected that in experiment with the methionine-minus recognition sites. the one-cycle lysate obtained after very host as an indication, but not as a proof From today’s knowledge on type I’s, low multiplicity of infection, there would that modification occurs by DNA methyl- let’s say you have a recognition site here, be no conserved DNA, but at most, two ation. In the same time period, I was here is another one, here still another one;

PLOS Genetics | www.plosgenetics.org 4 December 2014 | Volume 10 | Issue 12 | e1004879 if unmodified DNA molecules come into were there, and many other scientists who There are a multitude of specific the restricting cell, then the enzyme had contributed to the field. This was the mechanisms at work to spontaneously identifies, first, one of the specificity sites time when recombinant DNA work had promote occasional genetic alterations. I which has no methyl group. But it does successfully started, thanks to the avail- classified them into three natural strategies not cleave right away. It starts to rope ability of restriction endonucleases. A few of genetic variation. One of them pro- through the DNA on both sides. This can American participants raised the question motes local nucleotide changes upon DNA be shown by electron microscopy. Mean- whether there were some conjectural risks replication: a nucleotide substitution, the while, another enzyme molecule identifies associated with the recombinant DNA insertion or deletion of one or a few another specificity sequence and starts to technology. So we spontaneously inserted nucleotides, or a mingling of a few rope through the DNA. Sooner or later, into our program an evening session to nucleotides. By chance, these processes the two enzyme complexes run into each talk about these potential risks. can affect an existing function either other. That stops the translocation and Gitschier: This is well before the positively or negatively. initiates the cleavage of the DNA mole- Asilomar Conference. The second strategy is a rearrangement cule. Arber: Yes. And since we also wanted of DNA segments within the genome, and Gitschier: Oh, fascinating! to include ethical aspects, we invited the it is a source for deletion, amplification, Arber: This functioning has been protestant priest who managed the meet- inversion, or translocation of a DNA shown with small DNA molecules, and it ing center to attend our debate. We did segment. These processes are usually is indeed wonderful. In the presence of the not end up with clear conclusions, but we driven by specific enzymes. Phage P1, for type I enzyme, each time you bring in a identified relevant issues and questions example, carries a gene called cin (for C- foreign DNA, it gets cut at a different deserving more profound attention. Some inversion). Under its action, the C-seg- place because the recognitions at different of the participants of this evening discus- ment of the P1 genome gets periodically specificity sites are not synchronous. So sion were later among the scientists signing inverted between two specific crossover sometimes you cut here, sometimes you a letter to Science in 1974 requesting an sites. This gives rise to a flip-flop process cut there. And that means that all the international conference on risk assess- affecting a tail fiber gene, which deter- fragments are different and you do not ment for recombinant DNA technology. mines the host range of the phage. We always kill a particular gene that might be That conference was held in February have seen that, at rare occasions, the cin useful to the receiving cell’s progeny. In 1975 in Asilomar. Its participants suggest- gene product can also use a number of type II, you always cut reproducibly at the ed distinguishing between short-term and secondary crossover sites. This kind of recognition site, and if that is a part of an long-term risks. Short-term risks are those fusion can, by chance, result in a novel important gene, you kill it by restriction! for people who work in the laboratory: a gene activity or in an alternative efficiency So, for evolution involving steps by cloned gene with yet unknown function of expression of a concerned gene. horizontal gene transfer, the type I’s are could be toxic or pathogenic, so one The third strategy of genetic variation is actually more appropriate than type II should be careful and take the same horizontal gene transfer, i.e., the acquisi- enzymes. And for me, this is fantastic. precautions as for medical microbiological tion of a short segment of genetic infor- Gitschier: How are you feeling? Do analyses when samples from patients are mation from another kind of organism. you have energy for another fifteen studied. But long-term risks could relate to This may sometimes happen upon cohab- minutes or so? the release of recombinant DNA into the itation of different kinds of organisms. Arber: No problem. I9m surprised and environment. Then, one cannot know Gitschier: I recall the initial intrigue satisfied to see how informed you are. whether it can later spread horizontally. when the human genome was sequenced, Gitschier: Thank you! So let’s briefly In the 1970s, horizontal gene transfer was that there were something like 150 genes delve into this horizontal gene transfer a well known for microorganisms, but most that were suggested to come from bacteria. bit more. eukaryotic people claimed that horizontal This hasn9t panned out, but there are Arber: OK, I can just tell you how I gene transfer does not concern higher other examples, like integration of Wolba- came into studying molecular Darwinism. organisms. chia [an intracellular bacterium] genes I was an EMBO member already in Gitschier: That’s because they didn9t into the genomes of some insects. Geneva, and after moving to the Univer- know enough. Arber: Nature is much more complex sity of Basel, I proposed to organize an Arber: Indeed. Then I, and a few other than we believe. I don9t think there is EMBO workshop near Basel, at a religious people, realized in Asilomar that the time much work so far on horizontal gene meeting place called Leuenberg. This had come to study the molecular processes transfer between higher eukaryotes and international workshop on bacterial re- in Darwinian evolution. In doing so with microorganisms. It is always true that you striction and modification was held in the microbial populations, I came to realize can get new insights if you develop new fall of 1972. Ham Smith and Herb Boyer that nature is quite inventive. methodologies. This will come.

PLOS Genetics | www.plosgenetics.org 5 December 2014 | Volume 10 | Issue 12 | e1004879