Léo Ramos ChavesEnglish geneticist Peter Pearson maintains a small collection of old microscopes, a tribute to the apparatus responsible for so much of his career. Thanks to his talent for observing patterns that are invisible to the naked eye, he chose to specialize in chromosomes and discovered new aspects of the sex chromosomes, leading him to study causes of infertility. His description of a signal in the Y chromosome and its impact on determining fetal sex has been called the “Pearson body.” He developed methods for studying DNA and managed the Human Genome Project database in the USA from 1989 to 1995. He was the head of the Department of Human Genetics at the Leiden University in the Netherlands for 18 years, and helped to establish genetic research in the country.
During this journey, he met his future wife, Brazilian geneticist Carla Rosenberg, a professor at the Institute of Biosciences of the University of São Paulo (IB-USP). After retiring at the University of Utrecht, Netherlands, he moved to Brazil, where he dedicated himself more freely to science. He has taught English classes for postgraduates, established research collaborations, and participates in scientific projects and articles, mainly at the Human Genome Studies Center, one of the Research, Innovation, and Dissemination Centers (RIDCs) funded by FAPESP. He is one of the most cited researchers in the country, with an h-index of 71.
What he considers the greatest accomplishment of his 13 years in Brazil, however, is having finished the sailboat that he has spent more than 20 years building, with a little help from his students. The boat crossed the Atlantic Ocean on cargo ships five times before sailing a single millimeter on its own. Now he invites his former students on sailing trips off the coast of São Paulo.
You recently found a way to stain triple-stranded DNA. How did this come about?
It was fun, it took me back 30 years to when I found out what Eduardo Gorab, a professor at IB-USP, was doing with triple-stranded DNA. In the early 1970s I was involved in discovering how to create bands on human chromosomes [the treatment of chromosomes to reveal characteristic patterns of horizontal bands like bar codes]. Many of the compounds we examined were intercalated DNA molecules, some based on acridine orange, an organic compound that binds to certain chromosome structures. It created different levels of color and fluorescence depending on the stacking of the dye. When I found out that Eduardo was working on another dye to locate triple-stranded DNA, we got together. I like to think I made some minor contribution to Eduardo’s thinking; the results have many uses.
Field of expertise
Human genetics and cytogenetics
BS from the University of Liverpool (1962) and PhD from the University of Durham (1967), both in the United Kingdom
University of São Paulo (USP)
Around 400 scientific articles, 3 books, and 11 book chapters
Was your previous work relevant?
I looked at it from a completely different perspective. Eduardo showed me a publication that identified a strong affinity between triple-stranded DNA and dye. It reminded me of acridine orange, whose color varies depending on the compaction, quantity, and structure of the DNA. Eduardo’s triple helix seemed to be another variant of what I had already seen. It happened by chance when we crossed paths in a corridor one day.
Corridors are very important to science, aren’t they?
Coffee rooms, actually. Here in Brazil, universities are organized very differently to others around the world. I started my career in England, then went to the Netherlands, then to the United States, and back to the Netherlands. I came to Brazil after I retired, and USP is the seventh university I have worked at.
What differentiates USP from the others?
The way the university is organized makes the job very difficult. When the military dictatorship ended in 1984, USP, like the rest of the public world, celebrated the victory of democracy and created a number of committees to run the university. USP has an abundance of committees whose existence is enshrined in law, so nobody ever questions whether they are actually doing a good job. The bureaucracy and teaching load faced by professors is enormous. You don’t need a committee when two people can achieve the same thing. That said, some of the most ingenious and intelligent people are at USP. The problem is they are not in the right environment. The genetics department has 35 professors, all hired via a public exam process where the person who gave the best presentation on the day wins. But that person may not actually be the best candidate for the job in terms of how they fit into the department. The result is 35 small kingdoms. Each professor has their own laboratory, their own office, their own little group of graduate students, and their own coffee group. Nobody has coffee with other people.
This limits those chance meetings…
Exactly. They are rare, but so important. Many institutes elsewhere in the world now have central coffee areas and people are expected to leave their labs and their offices at set times for morning coffee and afternoon tea, or whatever it may be. This does not happen at USP. The large projects that FAPESP organizes, such as the RIDCs, are only half working. The people who work on these projects aren’t used to working in a group, so the coordinators have enormous difficulty fitting the pieces of the jigsaw puzzle together. In this day and age, research is no longer an individual effort. It is done by groups of talented people working in collaboration, which is very difficult to establish.
Research today is conducted by groups of talented people working in collaboration, which is very difficult to establish
Were your family scientists?
No, I was the first person in my family to go to university. They came from an agricultural background and I was accepted into one of the best agricultural universities in the UK, but my family didn’t have a farm, so I would have just ended up working as a manager for someone who owned land. So I took the easy way out and studied biology at the University of Liverpool.
And how did it go?
Thanks to my interest in and love of microscopes, I discovered I was good at looking at chromosomes. In my last year, I worked with dry plants in the herbarium. The researchers there were trying to use microscopes, and they all had two left eyes—they could not do it. So I made the preparations and used the microscope for them; I found it easy to observe things quickly and find patterns. I was later accepted to do a PhD at Durham University, studying plant chromosomes. Human cytogenetics was just getting started at the time, and the techniques for culturing white blood cells, known as microcultures, required a compound called phytohemagglutinin to stimulate growth. I used a recipe to make the substance: I would leave beans soaking in salt water overnight and then mash them and remove the supernatant, which contained the phytohemagglutinin. I had no means of sterilizing it and it was full of contaminants, so I just added a lot of penicillin and streptomycin.
Did it work?
It worked brilliantly, and got me interested in studying human chromosomes. When I finished my PhD, the Medical Research Council offered me a place in the genetics unit at the University of Oxford. I stayed there for seven years and made several discoveries by looking at chromosomes, largely based on the intuition I had developed about the right combination of equipment for each situation. I had the best fluorescence microscope in Oxford, and people would come to my little room just to use it.
Is that where the Y chromosome discovery was made?
Yes, I had been collaborating with a colleague, Martin Bobrow, and we were trying to get hold of a product called quinacrine mustard. A group from Stockholm had published an article about staining the end of the long arm of the Y chromosome with fluorescence. It was difficult to get hold of, but there was a drug called quinacrine dihydrochloride (sold as Atebrine) available in pharmacies, which had been produced during World War II to combat malaria. So we bought some of these pills and ground them up. At that time it was standard to use buccal mucosa (saliva) to look for Barr bodies, which signal inactivation of one of the X chromosomes. Female cells should have just one Barr body; when there are two, something is wrong. We examined slides tinted with quinacrine and noticed a bright spot inside the nucleus in about half of them. Could it be the equivalent of the Barr body on the Y chromosome? I went home that Friday night, woke up very early on Saturday morning, and went around collecting samples from my neighbors. I coded them as male and female, but in a way that I myself did not know which was which. Then I stained them and recorded which ones had the bright spot. When I broke the code, it was perfect: the male samples had the spot and the females did not. I called Bobrow and showed him. Then we called the head of the institute, who came in, despite it being Saturday and the fact that he liked to watch rugby on television at the weekend. The final test would be to study males with two Y chromosomes, and by coincidence we had two such patients in the maximum security prison in Reading, which is 65 kilometers from Oxford. On Monday we sent a nurse to Reading, she brought back the samples, and that same afternoon we confirmed there were two bodies. On Tuesday I wrote the article and sent it to Nature, and it was published two weeks later. People wanted to call it the Pearson body, but I refused, and it became known as the Y body. That was in 1970. I published four Nature papers in a row.
Had chromosome band staining already been done?
No, that came after we started using these quinacrine dyes. There is something about the structure of the chromosomes that gives them a characteristic band pattern, and discovering the Y body got the ball rolling in this field. In September 1970, there was an international conference on human genetics in Paris and I was involved in several parts of the program because of my articles in Nature. On the last night there was a party, and all the wheeler-dealers in human genetics were there. Halfway through the evening I climbed on a chair and said: “I guess if you didn’t know me beforehand, you do by now. I’m thinking of leaving Oxford, so if anyone is interested in offering me a job, we can talk about it during the rest of the party.” I received three job offers that night.
Why did you want to leave Oxford?
Oxford was brilliant and I was on an incredible learning curve. At first I was on the British Medical Research Council, but very quickly I became what they called an Oxford tutor. Each tutor has four or five students who they sit down with, choose a topic, and discuss. They came to my office on Tuesday afternoons, and so I was in the library at 7 a.m. every Tuesday trying to keep ahead of them. Then I was given a position as a junior professor. It was brilliant to start my career there, but in the middle of my career, there was a lot of politics involved in becoming a member of the right college, getting dining rights at the “high table,” all this Oxford malarkey.
And the Netherlands offered you the best prospects?
I chose Leiden because they had a fantastic microscopic team. Only later did I discover that the person who helped manufacture the microscope I had been using at Oxford was Dutch. One day I received a letter from someone called Plum, and the name seemed familiar: it was engraved on my microscope! He came to visit me with another Dutchman and an Australian, and started taking filters out of his pockets and changing parts of my microscope. The specimen I had been examining while waiting for them to arrive was still there, so I took another look, and it was the best image I had ever seen. We have collaborated a lot over the years. Thanks to that, we developed a lot of in situ hybridization techniques for marking specific parts of the genome.
How did you discover the pairing of the X and Y chromosomes?
I was doing a survey. I was interested in male infertility, so I took testicular biopsies, some from infertile men and some from men who were not infertile but had biopsies done for other reasons. Using quinacrine to stain the long arm of the Y chromosome, I quickly observed that it was paired with the X chromosome. Later, with a technique that allowed us to locate the chromosome centromeres, we identified that the short arm of the Y was pairing with the short arm of the X.
And this is linked to infertility?
Yes. Male infertility often occurs when this pairing fails. In a stage called diakinesis or metaphase I, the two chromosomes are completely separated in 5% of infertile men. When this happens in a large number of cells, it is associated with sperm counts of practically zero.
You also studied female infertility?
That was 30 years later at Utrecht University. I received a letter from a gynecologist called Egbert Te Velde, who said he was interested in working with me. My most cited article, with about 900 citations, is one I wrote with him about female infertility. We wanted to find markers that would allow us to predict when a woman will become infertile. Before a woman reaches menopause, it is difficult to know. The average age of menopause is 50 years, but the average age at which a woman becomes infertile is 10 years earlier. We showed that this is based on a genetically determined Gaussian curve. When comparing sisters, or mothers and daughters, there is a correlation: if one has an early menopause, so does the other.
Has your knowledge of chromosomes made you an expert on reproduction?
A little. There have been other cases. In 1970, for example, Bob Edwards [1925–2013], who invented in vitro fertilization and received the Nobel Prize in 2010, needed to detect Y chromosomes in embryos in order to verify gamete fusion and confirm fertilization. Louise Brown, the first IVF baby, was born in 1978, so Edwards was already doing this eight years earlier. These were balls of cells, which make it difficult to see the chromosomes. The ideal is to have a flat specimen, but Bob would not let me squash the embryos. So I was never able to see the Y chromosomes, but I am fairly sure they were there in half of the embryos.
You also participated in the Human Genome Project in the United States?
I managed a huge database, the first one established for the Human Genome Project. You might ask why a biologist had this job. I knew a little about computing, but the main thing was that I knew the data. At the time, the Howard Hughes Medical Institute made me an offer that was hard to refuse, and I was having difficulties in my first marriage. So it was an opportunity to leave the Netherlands. After 18 years as head of the Human Genetics Department in Leiden, I went to Johns Hopkins University in Baltimore to create this database. It was actually quite boring. The institute was paying my salary and they realized I would probably go crazy, so they gave me a small lab with a new fluorescence microscope and let me take on two postdoctoral interns. That’s how I met my wife. One day in 1990, Stylianos Antonarakis, who was at the time a Greek postdoctoral student at Johns Hopkins, told me: “There is so much drama in Barbara Migeon’s lab; every day she argues with this Brazilian woman who keeps standing up for herself and the other postdoctoral students. I think she would be great for your lab.” Barbara Migeon was a very famous scientist at the university, which was a difficult environment for a woman. So she was tough. But I had no time to think about it; that same week, my colleagues and I received an email from Barbara saying that a Brazilian named Carla Rosenberg was leaving her laboratory, and under no circumstances was anyone else allowed to pick her up for their own lab. I picked up the telephone immediately: “Stylianos, bring me this Brazilian woman now!” The rest is history, as they say.
Young postgraduate students need to use English in the lab and when writing papers
And then you went back to the Netherlands?
I decided to leave the Genome Project after six years. It was boring—my team consisted of 22 programmers. I was director of the database, but it did not store the sequences, just the location of the genes on the chromosomes. When I left, the news broke of my availability. The Royal Academy of Sciences in the Netherlands was pressuring Utrecht University to open a department of human genetics, and I was interviewed in a hotel lobby in Montreal by the chairman of the search committee. We spoke in Dutch for an hour and he offered me the job. So I set up a new department and stayed there for 10 years, until I retired the month I turned 65—which is nonnegotiable under Dutch law. Carla was doing so well as a professor in Leiden, I thought she should stay there, but she wanted to move back to Brazil for family reasons.
You mentioned the importance of having a broad knowledge base.
When I was a postgraduate student, I went to my first international conference. I was talking to some American graduate students in a bar one night and I was just blown away by how much they knew. I thought I knew a lot, but these guys completely obliterated me. Their education system was different to the British system, where you start a PhD and go into a lab. There were no formal courses at the time, you had to teach yourself. When I got back, I decided to go to the library every Tuesday afternoon and read as much as I could to pick up this background information. A tangential piece of knowledge often allows me to recognize a problem and seek solutions in unexpected places. People ask me how I do it. The answer is that I exposed myself to a lot of information, and not just what I use in my research. At USP, I ask the postgraduate students about their research and they can tell me about their project in great detail. But they do not go a single millimeter outside of that. If I try to tell them they need a broader knowledge base, they do not understand why. I have seen this many times in my career. I think I have had to redesign myself maybe five times.
In what sense?
Changing the knowledge base. I started with chromosomes, then I got into cytochemistry and fluorochromes, then I made connections with the DNA structure and moved onto things like in situ hybridization. When I arrived in the Netherlands, they were looking to organize genetics research in the country and appointed a national committee to draft a proposal. The committee met once a month for two years and was funded by insurance companies, not the government. The head of the committee, a brilliant man called Hans Galjaard, highlighted our problem: is genetics medicine or science? If it is medicine, it belongs in a hospital. If it is science, then it belongs in the university. So we organized foundations. Each medical center had an independent foundation that chose its own staff. The ministry of health accepted, and life was great: there were no fights between universities and hospitals. Within two years, the quality of genetic healthcare in the Netherlands became the best in the world. We had the people and the knowledge, it was just a matter of getting organized. The molecular stuff came about a decade later. I was interested in looking at chromosomes, and I had this idea that it could be possible to count chromosomes using DNA hybridization, even before anyone published on it and before any such techniques existed. In 1979, a Chinese researcher called Y.W. Khan published an article on alpha thalassemia, which involves two genes on chromosome 16. He had developed an in vitro DNA hybridization method that allowed us to count how many of the genes were affected. I decided I wanted to count chromosomes like that. With a brilliant technician by my side, I assembled a plasmid library and isolated chromosomes with specific probes. We created a probe on a specific part of the X chromosome that enabled us to detect a lot of genetic variation in those chromosomes. The RFLPs, which stands for Restriction Fragment Length Polymorphisms, were sensational in comparison to what was being used at the time. I shared the findings at a conference in Utrecht and everyone was speechless. I mentioned that the point was in the middle of the short arm, probably close to the Duchenne muscular dystrophy gene, and someone I had never met approached me afterward and asked me to research muscular dystrophy. I could not do it—I was head of the department and there was no space for taking on another line of research—but he promised me funding and enough money to hire staff. When I continued to resist, he invited me to a patient’s day run by the Dutch Muscular Dystrophy Foundation the following Saturday. The queen of the Netherlands was there, as well as a popstar who was famous at the time, and [there was] a circus tent. What really got my attention were the hundreds of patients in wheelchairs, almost all males. Those from the wealthiest families had electric wheelchairs, while the others were mostly pushed by their mothers. How could I refuse? So in 1983, I started a Duchenne muscular dystrophy research group, on top of everything I was already doing in Leiden. Our work was very important. We mapped the entire gene, which had 2.5 million base pairs.
Has your work with muscular dystrophy led to any collaborations here in Brazil?
Not exactly. I wanted to study the aging of stem cells here, but I had no funding. I keep myself intellectually active. I helped write the Human Genome Studies RDIC applications, for example, and was involved in the first project selection phase at the Serrapilheira Institute.
How could research organization be improved in Brazil?
Department heads are given two-year terms. Nothing can be done in such a short amount of time, so nobody is strategically directing the course of the department. You have to think ahead. In the state of São Paulo, FAPESP could create groups for applicants to organize collaborative projects. Constructive criticism would improve the project, and there would be less duplicate research caused by more than one group wanting to study the same thing. They would have to join forces and do it together. Scientific output in Brazil is thought of in terms of number of articles. Citation levels are lower than many other countries, even others that do not speak English. I created a graph of the 400 largest universities in the world, comparing the number of citations against an English proficiency index. The correlation is a straight line, and Brazil is very poorly positioned.
Is that why you set up the English for Scientists course, which you taught to postgraduate students until recently?
Yes. They had to write an essay every week, and I limited the classes to 30 students. I could have had 100, but I was already spending all week correcting and rewriting their texts, so they could see how to do it correctly. They really appreciated it. Young people at the postgraduate level need to be encouraged to speak English every day in the laboratory, at work, and in discussion groups, as well as writing. This could improve their chances of getting funded and publishing quality articles.