Chapter 8: Walk This Way

Sex determination, how an embryo decides to become male or female, is such a fundamental process that it comes as a surprise that we had no clue how it worked until a little over half a century ago; in 1959, in a great example of scientific serendipity, the beginnings of the answer were born out of the boredom of a 24-year-old researcher in Edinburgh, Pat Jacobs.

Pat’s expertise was in cytogenetics, the analysis of chromosomes, and in the wake of the 1950s nuclear arms race, she was hired by a small MRC-funded group to compare the chromosomes from patients with radiation-induced leukaemias with those with normal leukaemia. It was a laudable project, but unfortunately, Pat couldn’t find a single example of a radiation-induced leukaemia and had to mark time making a larger and larger control sample of normal leukaemic chromosome spreads. Therefore, when her boss Michael Court Brown introduced her to John Strong, an endocrinologist who wanted some chromosome spreads performed from one of his patients, she jumped at the chance to break the monotony. Strong’s patient had been classified as suffering from Klinefelter’s syndrome, a sex-reversal disorder then held to result from a female with two X chromosomes somehow becoming a male. However, Pat noticed something rather odd when she looked at the chromosome spreads; there were definitely two X chromosomes, but instead of the normal human count of 46 chromosomes, there were 47. Even more weirdly, the interloper was small, and to Pat’s practised eye, looked an awful lot like a Y chromosome. Because the Y chromosome was popularly held to be a piece of functionless junk, this seemed odd, but analysis of other Klinefelter’s patients yielded the same result—all had two X chromosomes and an additional Y.

Her youth and relative naivety meant that it wasn’t until Pat showed the data to Michael Court Brown that she had any idea of its huge significance, but Court Brown realised immediately that what he was looking at was going to overturn all notions regarding how human sex was determined. Jacobs and Strong’s paper, published in Nature in 1959, was the first demonstration of the binary nature of sex determination in mammals—if there is a Y chromosome, you make boys, and if there isn’t, you make girls. However, it would take until 1990 until it was finally known what it was on the Y chromosome that was responsible for making the decision. It would fall to another researcher who had wandered into the field whilst marking time waiting for reagents, to make the discovery. This time, however, the path to success had many more twists and turns than ever encountered by Jacobs and Strong, and the competition proved to be unhealthily intense.

In the 30-odd years between the Y being fingered as the source of the Testis Determining Factor, or TDF (developed testes being the physiological criteria for maleness), and the discovery of what exactly on the Y was responsible, science had to cover a lot of ground. In 1959, genetics, the study of how characteristics are inherited, had accumulated a venerable history, but the gap between the statistically derived maps of linked genetic loci, based on the frequency with which two loci were inherited together or separately, and the physical reality of the chromosomes, where the genes corresponding to the loci were actually located, was enormous. How did one superimpose the abstract linkage maps onto the DNA? Those working in the field of human genetics had an additional problem, because they were unable to set up the breeding experiments that were the lifeblood of genetic research in lower organisms, and could only make very basic linkage maps based on a few family pedigrees.

Fortunately for the human geneticists, in 1960, a group in France made a discovery described by J.B.S. Haldane as a substitute for sex, possibly the first and only time that such an idea has been viewed with unbridled enthusiasm. The idea of mammalian somatic cell or parasexual genetics, as it was more prosaically called, was not new, having been proposed by Guido Pontecorvo in the 1950s. Pontecorvo was one of the first microbial geneticists, hunting for fungal genes by mutation and recombination, and had noticed a rare spontaneous event in his favourite fungus Aspergillus nidulans, in which nonsex (somatic) cells fused together and generated offspring bearing characteristics of both parent cells, circumventing the need for mating. Ponte, as he was universally known, realised that if somatic animal cells could be made to fuse in this way, vast genetic opportunities would open up; unfortunately, Ponte does not occupy the place in history he deserves for this fundamental insight, because he did not succeed in getting mammalian cell fusion to work. However, this had now been remedied. Ponte must have been very unlucky, because the paper published by Serge Sorieul, Francine Cornefert, and Georges Barski was apparently very straightforward.

Guido Pontecorvo.

(Photograph courtesy of CRUK London Research Institute Archives.)

Sorieul, Cornefert, and Barski’s method for somatic cell fusion was based on tissue culture. They reported that if they mixed two different sorts of cells together, the resulting mixed cultures contained cells with characteristics of both parental types. With Boris Ephrussi, Sorieul showed in 1962 that the cells had in effect “mated”—their chromosomes had mixed together and then resegregated to create offspring carrying a genetic contribution from each parent. Most excitingly for geneticists, in 1967, Mary Weiss and Howard Green found that if mouse cells were mixed with human cells, the battle of which chromosomes were retained by the hybrid interspecies offspring was almost invariably won by the mouse chromosomes.

With the advent of somatic cell hybrids, as they were called, it was possible to derive cell lines whose chromosome complement included just one human chromosome, with all the rest being mouse. These could then be used to map loci to a particular chromosome, as long as one had an assay for the locus of interest. Panels of cell lines carrying different human chromosomes could be screened for the presence or absence of a human gene product, and hence its parental gene. Experiments were relatively simple: Set up your assay, whatever it was, on the 20 or so cell lines, each with their single human chromosome, and see which line comes up positive. Check which chromosome is in that cell line, and bingo! You’ve mapped another gene. Combined with the fact that breeding was no longer necessary, experiments took days rather than years, and in the decade following, somatic cell hybrids were used to map a few hundred genes with known and assayable gene products onto specific human chromosomes, including the X.

Sadly, for those interested in the Y chromosome, the pickings were still very thin, even using somatic cell genetics. The problem lay in the fact that, unlike the X, which was positively bursting with genes, the Y chromosome seemed to be an echoing wasteland—none of the known genes seemed to map there at all. This emptiness, however, fitted very neatly with the prevailing hypothesis regarding sex determination, which held that the default fate of an embryo was to become female, and this could be diverted into maleness by the expression of just one gene. Such a big decision, it was argued, should be a simple on/off switch, a binary decision, to leave as little room as possible for mistakes. An interesting, if completely wrong, idea arose suggesting that maybe the Y only held one gene, the testis-determining TDF, and that therefore, the only criterion for being TDF was to be Y-linked. A large number of adherents of this idea, who should probably have known better, hared off after the H-Y antigen, the only protein known to be associated with the presence of a Y chromosome, and it was acclaimed as TDF for some 10 years, until the theory was comprehensively demolished in 1984 by the British embryologist Anne McLaren and her colleagues, who showed that the presence or absence in mice of H-Y antigen didn’t make a blind bit of difference to whether mice turned out male or female.

After this and other equally misguided attempts to find a gene, any gene, on the Y, the more enlightened amongst the field realised that to identify the genes on the Y chromosome and to find which of them was TDF, was clearly going to need a different technique, one that was independent of what the genes encoded; in other words, the genes would have to be mapped simply by their presence on the Y, not what they did. And, at the end of the 1970s, a suitable mapping technique appeared, provided, inevitably, by the fertile minds of molecular biology.

The molecular biologists’ annoyingly practical habit of doing things from the bottom up, starting from the DNA, proved invaluable to the geneticists, struggling with their scientifically indigestible chromosomes. As the 1970s progressed, molecular biology, as related in Chapter 2, began to provide the tools with which to manipulate and analyse both small and large pieces of DNA. Restriction enzymes, able to cut DNA at a particular sequence, made it possible to reduce chromosomes to more manageable, smaller chunks. The fragments could be separated according to size by gel electrophoresis and then cloned and amplified by insertion into plasmid vectors. Chunks of DNA could be searched for genes, using Southern blots, and the sequence of bases of which the fragments were composed could be determined, after the development of DNA sequencing. In theory at least, it was now possible to go from a chromosome to a gene.

As geneticists and molecular biologists alike began to wield their new tools in earnest, they realised that the combination of DNA cloning and the availability of increasingly more sophisticated vectors for carrying larger and larger fragments of DNA meant that it was possible to fragment chromosomes and, indeed, genomes and clone all of the fragments into vectors: DNA libraries could be made carrying complete genomic sequences in manageable chunks. These libraries could be probed with radiolabelled DNA or RNA molecules in the same way as Southern blots, giving another way to isolate genes, but their existence also opened up an even more interesting possibility: If there was a way of ordering overlapping library clones in the same way as a library of books, the result would be a DNA analogue of the genetic maps made by linkage analysis. Finally, the prospect of mapping genes on the Y chromosome simply by their location seemed more than just a daydream, as long as someone could figure out how to order the DNA library clones into a coherent linear array.

The key to the puzzle of how to order DNA libraries was provided in 1980 by the newly appointed Director of the ICRF, the 43-year-old Walter Bodmer. Human genetics by this time was a busy anthill of scurrying activity, and although it seems somewhat implausible to liken him to any type of ant, Walter Bodmer was a central figure in the antheap. His scientific lineage was impeccable. At Cambridge, he had been one of the last students of Sir Ronald Fisher, a genius who was the father of both modern statistical analysis and population genetics, and he had then moved to Stanford to work with Josh Lederberg, one of the founders of molecular biology. He got on so well with Lederberg that he became a faculty member at Stanford, where he made his codiscovery of the Human Leucocyte Antigen (HLA) system, crucial in the body’s rejection or otherwise of organ transplants. Bodmer’s HLA work led him from his first interests, plant and bacterial genetics, into human genetics, and he returned to Oxford as Professor of Genetics in 1970, where he continued his work on HLA, becoming a world leader in the field that he had founded. Around the time of his arrival at ICRF, he and Ellen Solomon, one of the new faculty he brought with him, proposed an idea, suggested at the same time by David Botstein, Ray White, Mark Scolnick, and Ron Davis in the United States, that solved the problem of how to order DNA libraries. (As a footnote, Botstein and his colleagues received rather more recognition than Bodmer and Solomon for their idea, perhaps because the Botstein paper was entitled, “Construction of a Genetic Linkage Map in Humans Using Restriction Length Polymorphisms,” and Bodmer and Solomon’s was called “Evolution of Sickle Variant Gene.” In his defence, Walter always said he didn’t bother promoting his idea much because he thought is was blindingly obvious.)

Walter Bodmer. (Photograph courtesy of CRUK London Research Institute Archives.)

The method proposed in the two papers centred on the discovery of restriction fragment length polymorphisms, or RFLPs (“rifflips”). Sequences in even the most conserved regions of the genome tend to vary slightly between different people, and the slight variations are heritable, meaning that these polymorphisms, often a change in just one nucleotide, can be used as genetic markers. Detecting RFLPs might seem a complex challenge, but fortunately, as the acronym RFLP suggests, biologists had a way of “seeing” small snippets of the genome, namely, the recognition sequence of a restriction enzyme. For example, if in a large fragment of DNA such as those cloned in the genomic libraries, the sequence GCGGCCGC occurred twice, a restriction enzyme called NotI would be able to cleave the fragment twice because GCGGCCGC is its recognition sequence. If the cleaved fragments were then run on a Southern blot and probed with the appropriate radiolabelled DNA, three bands would be seen. (Why three? Cut a piece of string in two places and it becomes evident.) However, if the same fragment was extracted from a different person’s genome, it might be that one of the NotI sites was no longer present, owing to a mutation having caused a change, say, of one of the C bases to an A. Therefore, cleavage by NotI would result in only two bands on a Southern blot, and thus one would have a marker, an RFLP, to differentiate between the first and the second person’s DNA in that area. So now, because such markers exist in many places in the genome, not just for NotI but for many other restriction enzymes, there was a way of telling the difference between the genomes of two different people. Using RFLPs, the isolation of a locus based on its position, rather than its function, was now possible, and the process acquired a new name, positional cloning, or reverse genetics. RFLPs had huge ramifications for the cloning of disease genes because normal DNA could be compared with DNA from disease carriers, and the faulty region identified. The first locus to be successfully linked to a disease was the region of the X chromosome carrying the Duchenne Muscular Dystrophy gene, in 1982, and this was rapidly followed by many others.

Following on from the idea of using RFLPs as a way of distinguishing between different genomes, the one extra conceptual step taken by Bodmer and Botstein and their colleagues was to realise that RFLP technology could also be used within a single genome to create an ordered map. By comparing the restriction enzyme patterns made by digesting all of the clones in a DNA library made from just one genome, shared patterns, indicating overlaps between different clones, could be detected. Rather like piecing together a panoramic photograph from individual overlapping shots, clones could then be put in order. This simple but clever idea was the seed from which the Human Genome Project grew, culminating in the publication in 2001 of a working draft of the complete DNA sequence of the human genome.

But what of the Y? The advent of positional cloning techniques fired the starting gun in the real race to find TDF, and the runners and riders were an interesting bunch. Two in particular seemed to be worth betting on: in London at the ICRF, Peter Goodfellow, a former Bodmer graduate student recently returned from Stanford, had moved onto the Fifth Floor and was looking for a project with which to make his name; and in Boston, David Page, a newly qualified MD and protégé of Bodmer’s RFLP rival David Botstein, was just starting his own lab at the newly formed Whitehead Institute.

Goodfellow and Page are an interesting pair, about as different in personality and appearance as could be imagined, although their origins have some similarities: Page was born in rural Pennsylvania, Goodfellow in the depths of the East Anglian Fens, and both were the first in their families to go to university, Page to Swarthmore, and Goodfellow to Bristol. After that, things begin to diverge.

In 1978, west of the Atlantic, Page enrolled in the joint Harvard/Massachusetts Institute of Technology medical degree programme, which encouraged a mix of research and clinical training, and for the next few years he meandered creatively between clinical medicine (spending some time at a hospital in Liberia, where he met his future wife, Elizabeth), and benchwork. Following the advice of David Baltimore, a fellow Swarthmore alumnus, who also happened to have won a Nobel prize in 1975 for his work in the burgeoning molecular biology field, Page hooked up with David Botstein. Botstein had just proposed his RFLP idea, and Page’s assignment in his lab was to begin on the genetic linkage map that would eventually encompass the entire human genome, a rather ambitious project to give to a wet-behind-the-ears medical student getting some lab experience. However, it panned out for Page because the first RFLP that he found came from a region of similarity between the X and Y chromosomes; as Page says, his entire career has been determined by a toothpick, then the weapon of choice for picking clones from the agar jelly plates on which they were grown. Towards the end of his MD, with a pleasing circularity, Botstein recommended Page back to David Baltimore, as a possible inhabitant of the newly fledged Whitehead Institute, and Baltimore hired him in 1984, aged 28, as the first Whitehead Research Fellow.

David Page, 1986.

(Photograph courtesy of Cold Spring Harbor Laboratory Archives.)

Leaving David Page for the moment, standing amongst a pile of cardboard boxes in an empty laboratory and wondering what on earth he had got himself into, we return to Peter Goodfellow, who had an equally busy run-up to his date with the Y chromosome. In 1972, after a degree in microbiology from Bristol, Peter went to do a PhD with Walter Bodmer in Oxford, partly because he’d been advised to get into human genetics because it was the coming research area, but partly to show all of the teachers at his old school that, contrary to their assertions, working-class boys from the Fens could, indeed, make it into the Oxbridge system. The Bodmer lab was absolutely flying science-wise in those years, and Bodmer was a committed and inspirational PhD supervisor, so Peter became an expert in somatic cell genetics, publishing 13 papers during his PhD, five of which were in Nature. After that, he went off to Stanford for a postdoc. Outside the lab, his social life thrived; he was spat on by Sid Vicious at the last Sex Pistols concert (realising in the process that you don’t necessarily need talent to be successful), worked for Jane Fonda and Tom Hayden during Hayden’s bid for the U.S. Senate, and hung out with his new wife Julia and their friends in Berkeley and San Francisco. Unfortunately, life in the lab was not so good. Peter’s project centred on the T/t complex, at that time believed to be the key controller of early mouse development, but as his work progressed, he realised he was in the awkward position of having to tell a lot of understandably sceptical scientists that they had got something important wrong—the T/t complex theory was a dud. Overturning the consensus in the field was unpleasant and time-consuming, although it did teach Peter how to function in adversity, a lesson that his glory days in Oxford had not required him to learn.

Peter’s arrival at ICRF was something of a shock. Scruffily dressed, rake thin, trailing a cohort of lab members whose dress sense showcased the “punk meets new romantic” vibe of the time, he was a bit of a novelty to some of the crustier members of staff, to put it mildly. The wild-looking new lab head was in addition highly driven, mostly by an intense curiosity regarding science, but also by a fierce ambition to succeed. Combined with what Peter describes as a slight social autism, this drive meant that he was a ruthless adversary in a scientific argument, taking no prisoners and never tolerating mediocrity. However, these sometimes intimidating qualities masked a person entirely lacking in malice, with an almost palpable delight in having found something he loved on which to spend his working life. His scientific persona, combined with amongst other things his penchant for poetry (good) and football (perhaps less good, given his allegiance to the Arsenal team of the 1980s), made Peter a complex, brilliant man who was a delight to his friends and a red rag to his enemies.

Peter Goodfellow, ca. 1987. (Photograph courtesy of Viesturs Simanis.)

Pleasingly, one of Peter’s new colleagues in London, who was to provide him with much help, advice, and support in his future travails, was Guido Pontecorvo, who had been one of those to kickstart the field of human genetics all those years ago. Ponte had been lured down to the ICRF from his position as Professor of Genetics in Glasgow by Michael Stoker because he was tired of administrative chores (although as his filing system for official papers comprised a wastepaper bin in his office, the chores really can’t have been that arduous). Mostly retired, he occupied a position at the ICRF best described as the Institute’s scientific godfather, a generally benevolent presence, tempering the scientific cut and thrust with kindness. As well as providing an intelligent and knowledgeable ear for new ideas, he made one more major technical contribution to the somatic cell genetics field during his time at ICRF, showing that polyethylene glycol could be used to fuse cells together. This innovation, although apparently small, turned cell fusion into a reliably efficient process and became the method of choice for many years.

Despite the substantial presence in human genetics at the ICRF, Peter, partly because he didn’t want to live forever in Walter Bodmer’s shadow, had a very different idea for his future research. His years in California had got Peter very interested in developmental biology, and he returned to England with the intention of working on mouse development. However, circumstances conspired against him because the quarantine laws of the day meant that it would have taken 18 months to import all of the mouse strains he needed from the States, by which time he would be almost a third of the way through his contract at the ICRF. Given that the conversion of that temporary, tenure-track contract into a prized permanent tenured position depended on his getting some data fast, Peter made the prosaic decision that while he was thinking what to do next, he would keep on with what he was good at, make himself useful, and get some papers out quickly. He succeeded admirably. Whilst at Stanford, Peter had used his expertise with somatic cell hybrids to bring the brand-new technique of making monoclonal antibodies to the Bay Area, and this skill was also much in demand at the ICRF. His small lab published 31 papers during the next five years, mainly joint ventures, one of which has had lasting importance. His collaboration with Mike Waterfield (described in Chapter 5), during which Peter dreamt up an innovative way of making a monoclonal antibody against the human Epidermal Growth Factor Receptor, made possible the Waterfield lab’s landmark discovery that the EGF receptor had been stolen by a virus to become the v-erb-b oncogene.

By 1984, this and his other successes meant that Peter had been given tenure, had expanded his lab, and was well on the way to becoming ICRF royalty. He had added molecular biology and the new DNA mapping techniques to his already formidable skill set, and, most satisfyingly, had found the subject that he wanted to work on: the Y chromosome.

Peter, like Pat Jacobs, and, indeed, the toothpick-wielding David Page, only became involved with Y-chromosome genetics by accident. The immunologist Andrew McMichael, a friend from his Stanford years, had asked him to characterise an antibody, 12E7, raised against a surface marker that was supposed to be found only on T cells, the white blood cells involved in recognition and clearance of infections. Far from being T-cell specific, the marker that 12E7 recognised, MIC2, turned out to be on pretty much everywhere. However, a dull project suddenly became more interesting when Peter’s lab mapped the gene encoding MIC2 to the X chromosome and then found that this gene, MIC2X, had a partner, MIC2Y, on the Y. They cloned both genes, and in 1983, MIC2Y became the first-ever published gene with a functional product to be cloned from the Y. Having landed on the Y, Peter realised that it was a wonderful unexplored landscape, perfect for someone whose skills lay in genome mapping, and that hidden within the empty landscape was a valuable prize: the gene encoding the Testis Determining Factor. Peter was hooked.

Back in Boston, David Page was having a bit of a crisis of self-confidence. His position as a Research Fellow of the Whitehead Institute was entirely novel, as was the Institute itself. Jack Whitehead, its benefactor, had got together with Nobelist David Baltimore with the intention of producing a kind of artists’ colony for scientists. They wanted to attract the best possible people and give them the resources and intellectual freedom to be as wildly creative as they wished. They were clearly onto a winner because they managed to recruit the five founding members of the Whitehead from the top echelons of molecular biology and genetics. Plunged into this boiling intellectual soup, Page, a newly qualified MD with only a couple of years’ lab experience, was simultaneously exhilarated by the exalted company and terrified that they would dismiss him as an intellectual pygmy. To his further alarm, his expectations of being a kind of glorified postdoctoral fellow with Rudolf Jaenisch, a world leader in the new field of making genetically modified mice, were confounded when it became clear that he was expected to be working independently. Page had to perform a hasty mental regrouping and decided, rather like Peter Goodfellow a few years before, to stick with what he knew and was good at. In Botstein’s lab, he had started a collaboration with Finnish geneticist Albert de la Chapelle—who in 1964 had described the first cases of sex-reversed XX males—to explore how RFLP probes could be used to study such disorders. It seemed natural to carry on with the collaboration and, in keeping with the ethos of the Whitehead, to aim high. Page set about finding a research assistant with a capacity for infinite hard work; he too, had decided to clone the Testis Determining Factor.

The Y chromosome looks a little like a skittle, with a short arm (called Yp), and a long arm (Yq) separated by a constriction called the centromere, the point at which the chromosome attaches to the cell spindle during cell division. From work from Pat Jacobs’s lab in the 1960s, it was possible to infer the rough location of TDF, on the short arm Yp, because if this region gets accidentally stuck onto an X chromosome, it causes the development of XX males. Slightly narrowing the search area, TDF could not be at the very tip of Yp, because this was the pseudo-autosomal region, the part of the Y chromosome alike enough to the X to pair with it; genes located there could not be unique to the Y, as TDF had to be. This narrowed the region down a bit, but the leap from a piece of DNA some 10 million base pairs (10 Mb) long to finding a gene, perhaps a few thousand base pairs in length at most, was still huge. It would be a formidable technical challenge that perhaps only Page and Goodfellow, and one other lab, that of Jean Weissenbach in Paris, had the expertise, muscle, and ambition to contemplate realistically.

To make a map of something, the first things required are the mapping tools, and in the case of the Y chromosome, these were Y-specific DNA probes, able to recognise sequences on the Y when used in Southern blotting experiments. All of the labs working on the Y chromosome spent the early 1980s mining DNA libraries in various ingenious ways to amass such probes, so that by 1984, a fair number of Y-specific ones were known. The Weissenbach and Page labs, together with their clinical collaborators Bernard Noël and Albert de la Chapelle, joined forces to produce a low-resolution map of the Y, by seeing which of a set of Y-specific probes were recognised by DNA from sex-reversed patients. The principle, known as deletion mapping, was as follows: XX males were likely to be males because they were carrying chunks of Y-specific DNA in their genomes. Therefore, some or all of the Y-specific probes should see this on Southern blots of restriction-enzyme-digested genomic DNA. The places that the probes were binding could then be roughly mapped along the Y chromosome by the panoramic photo principle, by looking for their presence in the individual snapshots coming from each of the XX males. For example, if one restriction fragment from an XX male was recognised by probes 1 and 3, and a second fragment from another patient was seen by probes 2 and 3, the two fragments must be next to each other, with the overlap defined by probe 3. Of course, in these XX male patients, each snapshot, whether of a large chunk of the Y panorama, or a tiny part, must carry the part of the Y chromosome containing TDF; otherwise, the patients would not be male. Therefore, identifying the part of the panorama that always showed up regardless of which patient’s snapshot was on view would give a rough location for TDF. In molecular terms, this simply meant that the probes that lit up DNA from the greatest number of XX males were closest to TDF.

In 1986, the two labs published the first deletion map of part of the Y chromosome, showing that TDF had to be in a region of the short arm of the Y that they called Interval 1. Interval 1 fulfilled all of the criteria above—it was there in all of the XX males with additional Y DNA, and furthermore, it was within a larger region missing in an XY female. Interval 1, however, was not small; it still covered ∼3 Mb.

Peter Goodfellow’s lab had also been mapping busily, but using different methods. Rather than relying on patient material, which was very hard to come by in London for various annoying nonscientific reasons related to the egos of the clinicians involved, they had gone for a combination of traditional and cutting-edge molecular biology techniques, based around the MIC2Y gene. MIC2Y and its partner MIC2X lie in the pseudo-autosomal regions of the sex chromosomes, meaning that MIC2Y was at the tip of Yp, in the region next door to where TDF would be found. By meiotic mapping, looking at how often pseudo-autosomal genes were able to switch between the X and Y chromosomes during the chromosomal do-se-do of meiotic recombination, the Goodfellow lab realised that MIC2Y hardly ever switched, in contrast to the rest of their pseudo-autosomal-specific markers, which changed chromosomes with happy abandon. This reluctance to switch placed MIC2Y very close to the boundary between the pseudo-autosomal region and the Y-specific region next door, and therefore made it a very good point of departure from which to start mapping the rest of Yp, and TDF.

Armed with the knowledge of MIC2Y’s location, Peter’s graduate student and postdoc, Catrin Pritchard and Paul Goodfellow (a Canadian, unrelated to Peter), made a long-range restriction map of the short arm of the Y, using a new technique, pulse field gel electrophoresis, which is able to separate enormously long pieces of DNA on agarose gels. As a source of male DNA, they used a human cell line called Oxen, which had managed to acquire four Y chromosomes, making the task of seeing faint fragments on Southern blots much easier. The strategy was to use many different restriction enzymes to perform multiple digests of Oxen DNA, separate out the bands of digested DNA on gels, and then probe all of the different digests with four probes known to span the whole region of the Y short arm from MIC2Y right up to the far side of Page and Weissenbach’s Interval 1. A panorama of Yp could then be assembled from the individual snapshots. This worked very well, and the study was published in Nature in 1987. Its appearance in such a high-profile journal was partly due to an additional observation made by the trio; at one particular point in Yp, there was a cluster of recognition sites for restriction enzymes whose cut sequences all contained the dinucleotide CG. Such clusters (called CpG islands) were the new hot item in molecular biology, because they had been shown by Adrian Bird and colleagues in Edinburgh to frequently mark the beginnings of genes. Could the Yp CpG island be marking the position of the gene for TDF, 250,000 bases from MIC2Y?

What do you do when you have a long way to go and no form of ready transport? You walk. By late 1985, the Page and Goodfellow labs both knew where they needed to be, but to get there was going to be a terrible slog. The routine of making DNA, digesting it, running it on gels, blotting the gels onto the membranes needed for Southern blots, making probes, hybridising the probes to the blots, and then interpreting the results is as boring as it sounds, and technically hard; getting the data clean enough to make any sense is a real challenge. However, the mapping performed in both labs up to now looked like chickenfeed when compared with what was to come, as now, they had to walk from the fixed points they knew into unknown territory. The Yp panoramic photos to date were complete, and correct, but they were like looking at the Earth from space; the resolution was far too low. What had to happen now was the zooming in, the molecular equivalent of Google Earth, to find the one spot occupied by TDF.

Chromosome walking in those days was extraordinarily tedious. You began with a probe a few thousand base pairs long from the known anchor point on the chromosome, which in Goodfellow’s case was a clone from MIC2Y, and in Page’s the first clone he’d ever isolated in Botstein’s lab, which flanked the other end of Interval 1, towards the centromere. Using a library of Y-specific sequences, in which each library clone contained at most 30,000 bp of Y DNA, you’d hybridise the anchor probe to the library. Any clones lit up by the probe were picked and grown up to amplify the DNA they contained, and then the DNA was restriction-digested along with the anchor clone. You kept looking until you found a clone that had some digested bands in common with your anchor, and then, you had an overlap. After that, you started the process all over again, using as the new probe the region of your new clone that was farthest from the anchor clone. If you were lucky, you did this 50–100 times and ended up with a beautifully detailed panoramic photo of your region of interest.

The Page and Goodfellow labs were not lucky. During their previous low-resolution mapping adventures, it had become very clear that the Y chromosome was going to be a complete pain to analyse: Almost all of the probes isolated, instead of recognising one nice, clean band on a Southern blot corresponding to one locus, saw multiple bands; the Y was covered in repetitive sequences, some of which were also found on autosomal (nonsex chromosome) and X-chromosome DNA, and some of which cropped up several times on the Y itself. This was a mapping nightmare; if the overlap clone that you found was full of repetitive sequences, it was useless, because it could be from anywhere. In Peter’s words: “We spent two years doing the longest walk …: Clone, subclone, pull out a probe, find something repetitive, go back and start again, chunk by chunk by chunk. It took four to five people two years … pretty brutal work.”

In Page’s lab, things were moving equally slowly, perhaps more so, because at that point the Page laboratory comprised just two people: David himself and his research assistant, Laura Brown, with occasional help from Harvard undergraduates with research project assignments (their Y genomic library was made by one of these, Jonathan Pollack, now a professor at Stanford). They evolved a system in which Laura worked the day shift and David worked nights, so the lab was a 24-hour operation. (Fortunately for his future happiness, David’s wife-to-be was a dermatology resident in Toronto at that point, and thus remained blissfully ignorant of her boyfriend’s Stakhanovite tendencies until she moved to Boston some time later.) David and Laura’s task was made no easier by the fact that although David knew where to start, he didn’t know which direction to take, and therefore had to try both ways. His probe was in a region of the Y with homology to the X, and only when he started to find sequences not hybridising to the X would he know that he was going the right way, into Yp. In the summer of 1986, he came into the lab for his night shift and realised from looking at Laura’s data that they had finally walked into the Y-specific sequences, and knew which way to go. Before heading off for a rare holiday the next morning, he left a note for Laura, picking up on that summer’s hottest MTV hit from Aerosmith/RunDMC; in an unlikely collision of heavy metal, rap, and science, Laura came in the next day to find a piece of paper with “WALK THIS WAY!” written on it.

Not surprisingly, as the two labs crept slowly towards their destinations through 1986 and 1987, the atmosphere was pretty intense. In human genetics, although the prize of cloning an important gene is very great, both in terms of potential utility to medicine and career advancement, the stakes are equally high. There are no consolation prizes if you get beaten, and nobody remembers the runner up. At the annual Y-chromosome conferences, the occasional dark horse popped up with some interesting data on TDF, but Page and Goodfellow were the star turns, their back-to-back presentations always the hotly anticipated event of the meeting. Living in such an atmosphere of excitement tinged with alarm is a pressurized existence, but it is in those moments that labs truly come alive, catching the wave rather than paddling aimlessly. The question, however, was who would reach the shore first.

By late 1986, Page and Laura Brown had narrowed down the part of Interval 1 carrying TDF to Interval 1A, adjoining the pseudo-autosomal tip of Yp; by studying more sex-reversed XX male DNA samples, they had found that this was the smallest part of the Y chromosome that could still specify maleness. Crucially, they also had a very interesting XY female DNA sample, WHT1013. WHT1013 had the misfortune to have had her father’s Y chromosome accidentally mixed up with autosomal chromosome 22 (formally, a reciprocal Y;22 translocation), meaning that she had apparently acquired the whole of Yp except for Interval 1A2, a subregion of Interval 1A, and a region next door to it, Interval 1B, which was known from the XX males not to be necessary for maleness. Interval 1A2 was 140,000 bp long, and all of the data suggested that TDF had to be in it somewhere.

The reduction in size of the region to be searched now meant that Page could start looking for TDF by other means, as well as the laborious chromosome walk. One of the criteria for TDF was that because it was such an important gene, it was likely to be very well conserved throughout evolution; the TDF gene in humans should be extremely similar in sequence to the TDF gene in rats, cows, dogs, and other assorted mammals. Therefore, Page wanted to start doing some Noah’s Ark blots, in which male and female DNA from multiple species could be restriction-digested and tested to see whether it would stick to any of the probes derived from the chromosome walk. In early 1987, a second Harvard undergraduate, Becky Mosher, started in the lab. Despite her complete lack of laboratory experience, she turned out to have a natural aptitude for molecular biology, and so David put her onto the Noah’s Ark project. In April 1987, returning from a meeting in Oxford, Page was met by Becky in a state of high excitement—one of her probes had detected a band on the Noah’s Ark blot that was present in the male but not the female of every species on there. Once the DNA sequence of the probe was determined, it was clear that there was an open reading frame, a sequence from which protein could be translated. Even better, the protein encoded had a recognisable sequence motif, a Zinc Finger, which cropped up in transcription factors, proteins dedicated to copying the DNA template into RNA. Finally, the Page lab had found a perfect candidate for TDF and were even in a position to speculate about what it did.

The point at which you know you have something really important, but you still have to do the confirmatory experiments and then get the paper published before you get scooped, is just excruciating. Nothing can ever be done fast enough, nothing can allay the gnawing anxiety that even as you slog away, trying to be careful but trying to be quick, someone else is doing the same work, is maybe a little closer than you.… You don’t sleep properly, you don’t do anything other than go to work, you neglect your nearest and dearest, and the whole time, a little kernel of panic in your gut nags away like a physical pain. Therefore, it was hardly a surprise that the Page lab went into overdrive. Fortunately for David, the spring of 1987 had seen the arrival of his first tranche of postdocs, all attracted by his growing reputation in the field. They all set to work with a vengeance, blotting, mapping, sequencing, and checking that everything was right, that there was no possibility of error. And they got there. In November, they sent a paper to Cell entitled, “The Sex-Determining Region of the Human Y Chromosome Encodes a Finger Protein,” and on Christmas Eve, 1987, the paper was published.

In London on the morning of 24 December, Gerard Evan’s friend and colleague Mike Owen, still working in the ICRF unit at St. Bartholomew’s Hospital, was winding down his experiments for the Christmas break when he received a phone call from the main laboratories in Lincoln’s Inn Fields. It was a postdoc from the Goodfellow lab, and he sounded very worried. Would Mike come over straight away and talk to his friend? Mike, who had known Peter since they were both graduate students, had seen Page’s paper, and left immediately. He found Peter underneath his desk, distraught, and spent the next three hours trying to persuade him to come out. It transpired that Peter had had almost no warning of Page’s coup, because Page had simply sent him a fax of the front page of the proofs the previous evening. To make matters worse, Peter had been right on the verge of cloning the new gene, called ZFY by Page. ZFY turned out to map right next door to the CpG island on which the Goodfellow lab had recently published, and all of the lab’s efforts over the last few months had been going into cloning it.

After such an event, it takes a while for a lab to pick itself up, dust itself down, and think what to do next, and the Goodfellow lab was no exception. Peter spent the next few months half-heartedly doing experiments, reading a lot of poetry, and drinking too much coffee. His postdoc Paul Goodfellow did manage to cheer him up slightly by pointing out to him that there were an awful lot of people in the world who would like to be failures like them, sitting in a well-funded lab full of highly talented scientists, but moving on to the next thing was always going to be hard. Matters were made no easier by the world’s press; with alarming eagerness, they had latched onto the story of how one gene can make the difference between becoming male or staying female, meaning that Peter had to dole out quote after quote saying how important the work was. Even worse, scenting a great human interest story, the press reframed the whole story as a big transatlantic fight to the finish, won by the quiet American, lost by the flamboyant Englishman, completely ignoring the tedious reality that science and, indeed, life is never that simple. Early 1988 was not a good time.

Curiously, David Page was not having quite as much fun as he should have been either. With the announcement of the cloning of ZFY, he had been catapulted from being a medium-successful scientist with a growing reputation amongst his immediate circle into a media phenomenon. He was on the NBC News, the front page of The New York Times, and many other papers worldwide, and even made it into a local paper in the Caribbean, much to the bemusement of his postdoc Lizzy Fisher, taking a well-earned Christmas holiday there in the hope that she could keep ZFY out of her life for a short while. He didn’t like it at all. The nature of the attention meant that some of his scientific colleagues, whose opinions of him had been fairly neutral up to this point, felt obliged to become more extreme; although his close colleagues were delighted at his success, many others prone to jealousy began to snipe at him.

Worse still was the attention from outside science. Page’s public visibility changed instantaneously from almost zero to maximal, and it took years for the spotlight to wander away in search of other prey. In addition to receiving heart-rending letters from people hoping he could somehow solve their reproductive and sexual problems, he rapidly became a whipping boy for those on the loonier fringes of feminism, gender studies, and psychology. He was accused of sexist thinking: Why did he talk about the “dominant” Testis Determining Factor? Why was he categorising the female state as passive, and the male state as active? Why did he seek to oppress the bisexuals, the homosexuals, the sexually ambivalent, by his assertion that sexuality was bimodal and determined by a single gene? It was Page’s misfortune that what the Testis Determining Factor did was very easy to grasp and equally easy to misinterpret.

Back in the relative sanity of the laboratories, investigators in the sex determination field were reformatting their ideas to take into account Page’s new information and beginning to climb the next experimental mountain. Contrary to what the world’s press had decided, ZFY was still only a candidate for the Testis Determining Factor; in human genetics, a gene cloned by reverse genetics can only be definitively assigned to a condition when it has passed all reasonable scientific tests, and ZFY now had to take those tests. The first, that it should be in the sex-determining part of the Y chromosome, was clearly fine, and that it appeared to be the only gene in a rather empty area of DNA was also in its favour. Whilst labs around the world got hold of ZFY probes and tested them on their own sex-reversed patient samples to make sure that the equations ZFY = male, no ZFY = female held in all cases, other experiments also got under way, but this time not in humans. Sex determination as a problem of developmental biology had long been of interest, and, unsurprisingly, the mouse model people were very interested in checking out ZFY in their favourite furry organism.

At this point, a figure in London hitherto in the shadows needs to be introduced; Robin Lovell-Badge, urbane, charming, clean shaven, immaculately dressed in his trademark jeans and ironed white shirt, was the complete opposite of Peter Goodfellow, but their collaboration, brokered by Anne McLaren—nemesis of the HY antigen theory of sex determination and Robin’s boss at the MRC Developmental Biology Unit at University College London—was to prove immensely successful. Robin’s technical expertise lay in mouse embryology, specifically in working with embryonic stem (ES) cells, and in making transgenic mice by microinjecting foreign DNA into fertilised eggs, and Anne had hired him in the hope that she could persuade him to work on her own field of sex determination. After the debacle of the HY antigen and other similarly misguided ideas, mouse sex determination was in great need of some new approaches. Anne correctly realised that a partnership between an outstandingly good human molecular geneticist and an outstandingly good mouse developmental biologist would allow a synergistic exchange of ideas and technology that would be of huge mutual benefit.

Based on the idea that TDF in humans and mice was likely to be conserved, Robin and Peter decided in 1985 at the start of their collaboration that a good strategy to clone TDF might be to try to express the human gene in mice, and hope to change female mice into males. Unfortunately, the idea was so far ahead of its time that it failed; to get the mystery gene into mice involved putting enormous chunks of the human Y chromosome into ES cells, selecting the cells that were expressing the MIC2Y antigen, and then making mice with the MIC2Y-positive cells. Because virtually every single technique in the process had to be made up as they went along, there were just too many obstacles to success.

Undaunted, Robin got talking to Liz Robertson, another transgenic/ES cell pioneer, and came up with another idea, to mutate mouse Tdy (by convention, and as not detailed in the previous chapter, gene names in humans are all capitalised, and those in mice appear as proper nouns; to further confuse matters, “TDF” is referred to as “Tdy” in mice) by hopping retroviral DNA into mouse ES cell genomes. If the retrovirus hopped into the Tdy coding sequence, it would disrupt the ability of the gene to encode Tdy protein, and therefore any mouse made from the mutant ES cell would be female irrespective of its genetic makeup. Slightly amazingly, this idea actually worked, giving rise to an XY female mouse that was even able to breed, albeit with huge difficulty. Unfortunately, when the DNA from this animal was analysed, Robin and Liz were unable to find any trace of retroviral DNA, which they needed as a flag to mark the position of the Tdy gene, so they were left in the teeth-gnashingly frustrating position of having mutated the Tdy gene but being no further on towards finding out what it was.

This new annoying mouse mutant, christened Tdy m1, joined two existing sex-reversed mouse lines: Sxra, in which a small chunk of the mouse Y chromosome was stuck onto the end of the X, leading to XX male animals, and Sxrb, in which the same process had happened, but a smaller piece of the Y was transposed. Page’s paper had shown that in mice, there were two Zfy genes, and that whereas Sxra carried both, Sxrb only carried one, Zfy2. Robin and Peter both realised that despite their misery at being scooped, they would at least be able to look at mouse Zfy in these animals, and that furthermore, because an awful lot more was known regarding the earliest stages of mouse sexual development than human, they could check whether Zfy was switched on in the right places and at the right time to be involved in sex determination.

What Robin’s lab found regarding mouse Zfy was rather unexpected. There were actually four, not two, copies of Zfy in the mouse genome, two on the Y, one on the X, and one on an autosome, making it very hard to see how the gene could be functioning as a Y-chromosome-specific sex-determining factor, unless the prevailing ideas regarding how sex was determined were radically wrong. Because this was actually perfectly possible, given how little was known regarding how Zfy might work, this did not constitute solid evidence for the prosecution, but the lab’s next finding was a bit more worrying for Zfy supporters. However hard the lab tried, they were unable to find any expression at all of Zfy2 in foetal gonads, only in adult testes, and although Zfy1 was detected, it was only on in the germ cells, which play no part in testis determination; in the crucial somatic cells of the foetal gonad, there was no sign of either gene. The clincher consigning Zfy being mouse Tdy to the bin was that in Robin and Liz’s Tdy m1 sex-reversed XY females, there was no change in any of the four ZFY-like genes—they were all there, and on at normal levels. There was absolutely no way they could be responsible for maleness.

These findings were so startling that although they got their first inkling of what was going on quite quickly after the Page Cell paper was published, Robin’s lab sat on the data for some considerable time, in order to make absolutely sure that they were correct. In the meantime, however, other doubts were starting to surface regarding ZFY. One of the earliest came from an unlikely source: a marsupial genetics lab in Australia, run by Jenny Graves. Page and Goodfellow both contacted her independently to get her to probe the marsupial genome, to check that marsupial Zfy was also on the Y chromosome. To everyone’s surprise, it was not; it lay on an autosome, which was very unexpected, because maleness in marsupials is also specified by the presence of a Y chromosome. Although the discovery made it onto the cover of Nature in late 1988, in an unlikely collaboration between all three labs, it was still not conclusive proof, because marsupial sex determination could quite feasibly just be a bit different from the mammalian mechanism. At this point, Page, certainly, was not terribly worried.

So what was going on in the Goodfellow lab amidst this hive of activity of collaborators and competitors? Peter, after his first few months of despondency, had woken up to the possibility that David Page might have got it wrong. The catalyst was a crucial conversation with Paul Burgoyne, who worked on mouse sex determination at the NIMR in Mill Hill. Burgoyne had been to a meeting at which he’d seen a poster from Marc Fellous of the Institut Pasteur in Paris, describing some interesting human sex-reversal pedigrees from North African families. He realised to his excitement that the region of the Y chromosome implicated in sex reversal in these patients was not the region containing ZFY. Instead, it had to be lying close to the pseudo-autosomal boundary, almost at the limit of the Y-specific region on Yp. Once he got back to London, he went to see Peter. Between them, Burgoyne and the data were so persuasive that Peter could not help but be convinced to take another look at the pseudo-autosomal boundary region. He was back on the case, and if Page had by any chance made a mistake, he was determined to find it.

Peter contacted Marc Fellous, an old friend who had been a postdoc with Walter Bodmer when Peter was a student, and Fellous sent him his patient DNA samples. The four test samples were positive for the Y pseudo-autosomal boundary region, which Peter’s lab had recently cloned, meaning that the sex reversal was likely caused by an abnormal exchange between the X and Y chromosomes and should therefore be due to the presence of TDF, and hence ZFY. However, as Burgoyne had foreseen, all tests to find ZFY came up negative, and further mapping of exactly which part of Yp was present in the patients showed conclusively that all four of the chromosomal breakpoints were far closer to the pseudo-autosomal tip, ∼60,000 bp away from the boundary, and nowhere near Interval 1A2, where ZFY was located. ZFY could not be TDF.

ZFY could not be TDF. Peter describes the lab’s reaction to this incredible, mind-blowing news as “pretty excited,” which may be a little bit of an understatement. Combined with the Lovell-Badge data, it meant the end of ZFY’s short time in the limelight. The back-to-back papers describing the downfall of ZFY were published in the 21 December 1989 issue of Nature, two years after Page’s Cell paper; working on sex determination seems to bring with it the hazard of ruined Christmases for one’s competitors.

Once the celebrations in London were over, Peter and Robin’s labs returned to the drudgery of sifting through the horribly convoluted maze of the Y chromosome, looking for the right gene. On their first walk through the boundary region of Yp, on their way towards the CpG island next door to ZFY, nothing particularly obvious had popped up, except that the region was stuffed full of repetitive sequences; it was clear that whatever TDY was, it was going to be fairly nondescript. Further mapping experiments using clinical samples from France narrowed the region to be searched down to the 35,000 bp abutting the pseudo-autosomal boundary, and thus the two labs decided to make probes by chopping this region into small bits. After eliminating the repeat sequences, the plan was to see whether any of the remaining probes could detect areas of homology in other organisms. Andrew Sinclair, a newly arrived Goodfellow postdoc from Jenny Graves’s marsupial lab in Melbourne, began probing Noah’s Ark blots. John Gubbay in Robin’s lab used a slightly different approach, hybridising his probe set to blots containing DNA from normal male and female mice, and also Sxrb and Tdy m1 sex-reversed females. Gubbay and Sinclair both hit pay dirt at almost exactly the same time: Sinclair picked up a sequence that was conserved in multiple mammalian species, but only in the males, and Gubbay saw a band that was present in normal male mice but absent in both normal and sex-reversed Sxrb and Tdy m1 females. Fortunately for the sanity of those concerned, the probe responsible was the same in both cases, corresponding to a region with an open reading frame able to code for a short protein that, like ZFY, had all the characteristics of a transcription factor. By what is almost certainly an enormous coincidence, a similar protein had already been shown to control mating in yeast. The human gene, which Robin and Peter called SRY (for sex-determining region Y), had been missed before because it was tiny, only 896 bp long, and had no CpG island associated with it to give away its location.

In July 1990, the two labs published their findings in two articles in Nature. Together, they had constructed an almost watertight case. The Goodfellow lab had done the human genetics, the positional cloning, and the sequencing, and had further shown that SRY was conserved in the males of multiple species, and in humans was only on in testes. Lovell-Badge’s lab had cloned the equivalent mouse gene, shown that it was male specific in mice, and that, crucially, it was expressed during development of the gonads in the cells required for testis formation.

In the same issue of Nature, the Page lab published a paper in which the reason that they had been misled by ZFY was revealed. It turned out that the genome of XY female WHT1013, on which the identification of ZFY had been based, contained one extra surprise that had not been detected; instead of one deletion removing ZFY, there were two, and the second deletion covered the region in which SRY was found. Such an occurrence was almost unprecedented, and Page and his colleagues, on the evidence they had at the time, were entirely right to have published what subsequently turned out to be incorrect—Peter always maintained that had he reached ZFY first, he would have published exactly the same paper.

Although SRY was universally agreed to be an excellent candidate for the elusive TDF, there was still one final test to be performed: An experiment was needed to show that by itself, SRY could cause sex reversal. Robin, the transgenic mouse expert, set about introducing Sry into female mice and seeing if it would be enough to turn them into males. The technical side of this was pretty much a doddle for Robin’s lab, but unfortunately, there had been an outbreak of Mouse Hepatitis Virus in the NIMR animal house (Robin had moved to NIMR from University College in 1988), and in order to eradicate it, there was a complete ban on breeding for some months. Robin and his lab kicked their heels and waited out the delay and, finally, managed to make five mice carrying multiple copies of artificially introduced transgenic Sry. One of these mice, very happily for all, had two X chromosomes and no Y, but was very clearly a boy, with all the right bits both inside and out. Randy, as he was christened, was very keen on mating with females but was infertile.

Photographing Randy. Robin Lovell-Badge is kneeling, centre.

(Photograph by Jérôme Collignon, courtesy of Robin Lovell-Badge.)

Robin and Peter wrote up the paper and sent it off to Nature, where it was duly accepted and scheduled for publication. Much to their delight, Robin’s photo of Randy climbing on a bar with one of his normal male companions, proudly showing off his very male private parts, was accepted as the front cover image for the 9 May 1991 issue of the magazine.

Randy. (Figure used with permission of the Medical Research Council/Photo Researchers.)

The weekend before the paper was published, Robin had a few phone calls from journalists and thought that, as for the 1990 back-to-back papers, that would be it for publicity. Nonchalantly strolling into work on the day of publication, he still thought he would be having a peaceful day, perhaps drinking a little champagne with the lab. However, the news had gone viral. There was something viscerally exciting about being able to change the sex of a mouse by switching on one small gene, and Randy and his anonymous friend were splattered all over the newspapers; the Independent gave them half the front page. As had David Page before them, Robin and Peter were deluged with interview requests, but surprisingly, they attracted far less flak than Page, and the experience was less alarming. Robin recalls the only slightly awkward part was having to explain that the Nature cover star, far from relaxing in a luxury cage in the animal house, was, in fact, the Late Randy, because to check that he was definitely male, he had had to be dissected. However, by way of consolation, one of his sex-reversed compatriots sits in the Science Museum, stuffed, beside Dolly the Sheep, and his fame persists to this day.


The paths of the three main protagonists of this story have diverged considerably in the 20-something years since Randy won the day for SRY. In the first months after ZFY’s downfall, David Page had to consider what to do next, and after much thought, decided, against the massed advice of his friends and colleagues, to carry on with the Y chromosome. His tenacity, superb bench skills, and infinite appetite for wringing the last drop of information from a complicated data set seemed to be perfectly suited to navigating the convolutions and complexity of the Y, and he refused to believe that the Y contained nothing more of interest. He was right: In 1992, his lab published a paper detailing the complete cloning of the Y chromosome, making it one of the first two chromosomes to be cloned, and signaling the renaissance of the Y. He collaborated in the sequencing of the Y chromosome in the late 1990s, finally resolving all of the hideous repeat sequences that had dogged the early chromosome walks and showing that they were there for a reason; the Y chromosome, uniquely amongst its fellows, uses the massive repeats to recombine with itself, maintaining genetic diversity. Since that time, Page has shown that the Y carries genes of paramount importance in spermatogenesis, and hence male fertility, work of great clinical significance. He is now Director of the Whitehead Institute, still in the same laboratory he first occupied as a 28-year-old novice. Looking back on the ZFY story now, Page is almost nostalgic. At the time, he says, it seemed perfectly fine to be working like a dog, maintaining his position at the front of the field by sheer determination and lack of sleep, and fighting intellectual battles with Peter Goodfellow at their regular conference encounters, because if you love something enough, that is what you do. The press attention and the trauma of the very public downfall of ZFY were horrible, but, despite all of his subsequent successes (and there have been many), the exhilarating rollercoaster of those days persists as the most exciting time of his career.

For Peter Goodfellow, now a biotech consultant after a high-pressure career in the upper echelons of the pharmaceutical industry, his years hunting down SRY also seem to have been a worthwhile adventure. The race for TDF was not just the big competition that the press liked to highlight, but was the result of many people’s desire to find the same information. Although there was competition, there was also collaboration—sharing of probes, information, and patient material. Like David Page, Peter was bruised by having to endure all of his tribulations in the public eye, but finding SRY remains his proudest achievement, his “textbook fact,” the major contribution to scientific knowledge to which every scientist aspires. He and Page, in the end, are more similar than they might appear; with Peter, as for Page, what comes out most strongly is his love of science, the vocational drive that motivated him to go for the big prize because it was what mattered most, not because it would boost his career and make him famous.

Peter Goodfellow and grandchildren, 2013.

(Photograph courtesy of Peter Goodfellow.)

After the splash of the SRY papers, Peter started to get a lot of tempting job offers (although one of these was hastily withdrawn after he gave his job seminar sitting cross-legged on a table waving a pointer around), and in the end he succumbed. In 1992, the flamboyance quotient of the ICRF was reduced to a depressingly normal level when Peter moved to the University of Cambridge to become the Balfour Professor of Genetics. He still carries the distinction of being the only head of the Cambridge University Genetics Department to have a ponytail. In addition to radically reorganising the department, and reading poetry to the undergraduates, he continued to work on SRY and sex determination. SRY proved to be a difficult protein to study. After many years of effort, we now know that it is a rather weedy transcription factor, whose sole purpose as a testis-determining factor is to switch on a second gene, SOX9, cloned by Peter’s lab in Cambridge. SOX9 then does all the rest of the work in establishing maleness. Interestingly, SOX9 is not on the sex chromosomes but is an autosomal gene, and appears to be a much more ancient specifier of maleness than SRY; it exists in multiple nonmammalian species, in contrast to SRY, which is solely mammalian.

And Robin Lovell-Badge? Robin’s encounter with the media following Randy’s appearance has morphed into a long-term relationship, and he is now a highly respected commentator and communicator on science issues, especially in the moral minefield of stem cell research. In the lab, he still works at the bench when he can, and his record of publishing high-impact work in high-profile journals is undiminished. In a recent twist to the story of SRY, the long-held dogma that femaleness was a default state, and to be male one simply had to activate SRY and SOX9, was overturned in a 2009 Cell paper, in which Robin and collaborators showed in mice that the autosomal gene FoxL2 specifies femaleness, and when present, overrides Sox9 and prevents ovaries changing into testes. Loss of FoxL2 in adult female mice up-regulates Sox9, causing reprogramming of some ovarian cell types to those found in testes. Remarkably, as in life, the Sox9/FoxL2 story shows that maleness and femaleness appear to be established by what some might view as a balancing act, and others as a war.

Web Resources

http://bsdb.satsumaweb.co.uk/2010/07/17/bsdb-newsletter-summer-2010   Profile of Robin Lovell-Badge, by his friend and collaborator Liz Robertson.

http://fds.oup.com/www.oup.co.uk/pdf/0-19-829792-0.pdf   Peter Goodfellow discusses sex and molecular biology.

www.scivee.tv/node/11701   David Page talks about the Y chromosome.

Further Reading

Cohen BL. 2007. Guido Pontecorvo (“Ponte”): A centenary memoir. Genetics 177: 1439–1444.

Sulston JE, Ferry G. 2003. The common thread: Science, politics, ethics and the human genome. Corgi Books, London.