SCIENCE AT THE SHINE DOME canberra 3 - 5 may 2006

New Fellows Seminar

Wednesday, 3 May 2006

Macfarlane Burnet Lecture

Professor Jenny Graves
Research School of Biological Studies, Australian National University, Canberra

Introduction by Professor John Shine – Jenny Graves has a highly acclaimed international reputation for her work in mammalian genetics and comparative genomics on Australian marsupials and monotremes. Her research has raised profound questions about fundamental human biological and mammalian evolution. She has made extensive groundbreaking discoveries relating to the cell cycle, control of DNA replication, evolution of the mammalian genome and the function and the evolution of sex chromosomes. She graduated from Adelaide University and received a Fulbright Award to undertake her PhD in molecular biology at the University of California in Berkeley. Jenny was selected as the 2006 Laureate for the Asia Pacific Region L’Oreal–UNESCO Awards for Women in science. She is currently the Research Director at the Australian Research Council’s Centre for Kangaroo Genomics and Head of the Comparative Genomics Research Group at the Research School of Biological Science at the Australian National University (ANU).

Exploring the genomes of weird Australian mammals

Thank you very much. It’s a huge honour to receive this award and I thank the Academy for bestowing it on me. I was lucky enough to meet Macfarlane Burnet when I first returned to Australia from Berkeley. I recall we had an argument about DNA repair, a subject that neither of us knew very much about. I sometimes wonder what Macfarlane Burnet would have thought about the genome information we now have that shows how very complicated the immune system is. I think he probably would not have been very surprised. I like to think that he would have got a kick out of the work that we published a few months ago on the marsupial immune system, showing how the human system evolved.

The title of my talk, ‘Exploring the genomes of weird Australian mammals’, was actually chosen by the Academy and Hugh Tyndale-Biscoe while I was at an email free meeting in Hawaii. I think they were scared I was going to talk about sex again. So I’m going to talk about genomics and highlight the special opportunities that Australia has in making a very big contribution to the understanding of the human genome project. This work has gone on over many years and I have many people to thank, including an army of postgraduate students and postdocs, first of all at La Trobe University and then at ANU. I also want to acknowledge the fantastic support of my family, particularly my husband, John, who is here today, and my father, the legendary T J Marshall, who at 99 is still enthusiastic and encouraging.

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Well, as I’m sure you all know, the human genome is done – at a huge cost. We now know about three billion base pairs of the human genome. But this is very boring information unless we know what it does. That’s where we come in because the question that I am most often asked is: well, we’ve got the human sequence now, why do we need other mammals? Particularly, why do we need weird animals from Australia? So what I want to convince you of today is that this is going to help us find new genes and find out what they do so we can understand human genetic disease and how to treat it, and maybe even make money in developing new drugs and a better trace for our domestic animals. The thing that is dearest to my heart is that it’s going to tell us how a human genome evolved. Comparative genomics is a field that used to be a bit of a Cinderella during the human genome sequencing, but is now coming into its own.

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The first thing I want to do is show you what’s being done in sequencing genomes, and how the sequenced animals are related to each other. Genomes of a number of different placental mammal species have been sequenced, including the human genome, which has been sequenced to a very great depth (ie multiple times). We also have the sequences for chimps, mice, rats, dogs, cats, and even the elephants are now lined up for sequencing. But these animals are actually all rather closely related. They shared a common ancestor only 100 million years ago, and that isn’t enough time for the genome to have changed sufficiently for us to get the maximal information out of it. If we go to the other extreme and look at animals very distantly related – that is birds, frogs and even fish – they share a common ancestor with mammals 300 or 400 million years ago and that’s too far because now the sequence is so different it’s actually hard to line up.

Wouldn’t it be lovely if there were some animals in the middle? Well, that’s exactly where Australian animals are. Marsupials and monotremes last shared a common ancestor with humans about 200 million years ago, so they’re exactly in the right spot to give us maximal information that we need to make these comparisons. So Australia is in a fantastic position. We were instrumental in getting sequencing projects on the road and now I’m happy to tell you that the genomes of two marsupials and the platypus are going to be completely sequenced. So Australia has a fantastic opportunity to make a major contribution to the understanding of the human genome by using our own mammals.

Now, I wish I could tell you that Australia is making a fantastic contribution to international genomics. Australia has made small contributions to the cow genome project but, in fact, Australia has really not participated in a very large way in international comparative genomics. Our one standout is the kangaroo. Australia is doing half the sequencing of this and the Baylor Genome Center in Houston is doing the other half. The Australian component is not being funded by the Federal Government but by the Victorian Government who, I think, are being very far sighted here. It is the kangaroo genome project that is saving Australia’s reputation in the genomics community and giving us a seat at the genomics table, so it is here I will begin.

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There are 26 species of kangaroo and we chose to sequence the Tammar Wallaby. This is a good choice because the classic marsupial biochemistry and physiology was done on this species by the research groups headed by Hugh Tyndale-Biscoe and Marilyn Renfree. There are two centres working on this species, the ARC Centre that I direct is doing the mapping and preparing the genome for sequencing, and the Australian Genome Research Facility (AGRF) is doing the sequencing in Melbourne and Brisbane.

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The kangaroo genome is about the same size as the human genome at just over three billion base pairs (we call that gigabases or gigs). But it’s packaged very differently - into just eight enormously large chromosomes that are a dream to work with. The other good thing about marsupials is that their chromosomes are all very closely related, so if you’ve seen one genome you’ve seen them all.

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The very large kangaroo chromosomes can be cut out of a photograph of a cell, arranged in a row and assigned numbers according to their size and location of the centromere. There are two copies of each chromosome, one from the mother and one from the father. There are seven pairs of what we call autosomes – that is ordinary chromosomes – and a pair that’s different in males and females, these are the sex chromosomes X and Y. So the Tammar is great because it only has eight pairs and they’re easy to distinguish from one another, compared with humans who have 23 pairs that are all rather small and dull. In fact, Tammar Wallaby chromosomes are so beautiful that a few years ago Australia Post actually made a stamp depicting them.

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The first thing that you need to do when sequencing an animal, is make a map of the genome. There are two different kinds of maps you need to make. One is a genetic map made by mating animals with different characteristics – these can be anything, including fur colour, eye colour, or sections of DNA. The other kind is a physical map where you’re actually looking at the location of particular bits of DNA on the chromosomes under a microscope. Once you have both maps you need to put them together and that’s what our centre is doing. We already have a genetic map of the Tammar, which is essentially just lines on a piece of paper showing the relationship of these genes and how they sort out in the offspring. There are various kinds of markers that we use to map the genes. One is real live genes, another is just anonymous pieces of DNA – we don’t know what they are or what they do, but if they’re different in our parents we can map them.

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The really interesting things are the visible markers and, for instance, we could – we haven’t yet but we could – map onto this linkage map fur markers, resistance to pests like ticks or even qualities that we’re interested in, like milk. This means we can then zero in and physically get the gene that we’re interested in. At the same time we’re looking at mapping genes by a physical means and that means we clone the gene in a very large piece of kangaroo genome and we colour the DNA with different dyes. Then we hybridise it to chromosomes on a microscope slide and we can actually see spots where those genes are. There are, of course, two copies of the spots because the chromosome has already divided.

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So we can do that with many bits of DNA containing genes, and we construct a map of different markers that are on chromosome one and two and so forth. This sort of map is what we’re going to need before we start putting the sequence together.

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Our gene map also has another benefit and that is that all these genes exist in humans as well and we know where they are. So we can colour the bits of the kangaroo genome according to what parts of the human genome they represent. For instance, we might map a milk gene in the Tammar, we know exactly where that gene is going to be in the human genome and we can go in and look for it in the human genome sequence. So this comparative map is already extremely valuable to us.

What about other marsupial genomes? I mentioned to you that other marsupials have very few rearrangements and we’ve been able to show this with a beautiful technique that we call chromosome painting. What our collaborators have been able to do is to physically sort the kangaroo chromosomes out by size, then make DNA from individual chromosomes and colour them with different dyes.

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For instance, the DNA from chromosome 2 of the Tammar Wallaby was coloured green, chromosome 7 DNA was coloured pink, and the X chromosome DNA was coloured white. We then hybridised this DNA onto chromosomes of the Swamp Wallaby to home in on sequences which are homologous. We found that chromosome Y2 of the Swamp Wallaby is nothing more than DNA from chromosomes 2 and 7 jammed together and the enormous X chromosome is nothing more than DNA from chromosomes 2, 7 and X all jammed together. We’ve been able to do this with practically every marsupial species. So we know where a gene in a Swamp Wallaby will be in a Tammar Wallaby or a Tasmanian Devil.

Well, what about platypus? The platypus is even more distantly related to humans than kangaroos, and it has characteristics that seem rather reptile and bird-like, for example they lay eggs. So there is a lot of international interest in how the platypus evolved. The cover of a journal in which we published last year showed a rather quaint idea that, in fact, the platypus is the result of hybridisation between a duck and a beaver.

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The platypus genome is surprisingly small at around 2.6 gigs – making its size somewhere between a bird and a placental mammal. The platypus has 26 chromosomes – they’re rather small, they’re not that easy to work with and they are really weird. When we line the chromosomes up we find something very strange. Most of the chromosomes are perfectly normal – two copies in both males and females – but there are 10 chromosomes that don’t have a partner in males.

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These turn out to be 10 sex chromosomes (5X and 5Y) – unheard of for a vertebrate. When we look at these chromosomes at meiosis – the reduction division where the diploid number of 52 gets reduced to the haploid number of 26 – we’re able to show that these chromosomes don’t pair up. Instead they form a big, long, strange looking chain with alternating X’s and Y’s. The five X chromosomes and five Y chromosomes line up at meiosis. We have no idea how they make baby platypuses or how baby platypuses know what sex they are supposed to be. But we have indirect evidence that all five X chromosomes go into one sperm and all five Y chromosomes go into another sperm and that’s how sex is determined.

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The chain is also interesting because when we look at the genes that are on the sex chromosomes we find that the X chromosome at one end of the chain has homology to the mammalian X and Y chromosome, and the X chromosome at the other end has homology to the bird sex chromosomes. Scientists thought that mammalian and bird sex chromosomes were completely different and evolved separately, but we now think that this is wrong. We probably started off with the same system that birds and reptiles had and we evolved a new system by exchanging our sex chromosomes with other chromosomes. This has been quite a surprise in the sex chromosome world.

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I now want to talk about the special value of weird mammals, particularly weird Australian mammals. There are two reasons why they are especially important. One is that they’re very distantly related to humans, as I mentioned before, and this gives us a good opportunity to discover genes and the sequences that control them. The other is that they’re different enough to have unique traits that might be useful.

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If we look at a map showing the phylogeny of mammals we see how different they are. There are three groups of mammals and Australia has a monopoly on two of them – marsupials and monotremes. The important thing is that they shared a common ancestor so long ago that genomic differences can be seen.

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What we can do is line up the DNA sequences in particular regions and make comparisons. For example, the prion gene. We lined up the human prion gene with mouse and we looked at the per cent similarity of sequence – the more similar they are the higher the peak. We did the same thing with human and sheep, human and cow, and human and Tammar Wallaby. The comparisons with mouse, sheep and cow, have peaks everywhere, so it’s hard to spot the really important sequences. The biggest peaks are where the gene actually is, but outside the gene there are lots of smaller peaks as well. Researchers have spent a lot of time and money looking at some of these peaks to see whether they control the prion gene. But when you look at the comparison with Tammar most of these peaks disappear. Only the gene itself, a few sequences on either side and a very interesting sequence downstream of the gene are conserved. It is here that we think researchers should look for important control factors.

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It might surprise you – it always surprises people – that our lab has actually discovered 13 new genes. Not only that, they are human genes. We discovered them mostly by accident when we were looking at something else. It turns out that looking for a homologue in marsupials is actually a jolly good way of finding new genes. We first hit the headlines when we were looking at a gene that was supposedly the male determining gene.

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Students in my lab discovered that this gene was not on the Y chromosome in kangaroos and other marsupials, and it should have been if it’s the correct male determining gene. So this meant it was the wrong gene. That led my student, Andrew Sinclair in his next position in London, to isolate the SRY gene on the human Y chromosome – which is also found on the kangaroo Y chromosome – and this is the right gene. That finding led to the isolation, by another student in my lab, of a gene on the X chromosome called SOX3 – the female partner of SRY – and it turns out to be a very important gene.

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Similarly, we were looking for a gene on the human Y chromosome that is important in making sperm. We searched for a kangaroo version of it and, indeed, we found it. But the surprising thing was we found it had a partner on the X chromosome which we call RBMX, and we were able to clone the human version of this new gene,. It maps to a very interesting region which is deleted in families with mental retardation. Since then we have taken this gene in a zebra fish, knocked it down – that is we made it express less – and those zebra fish have a brain that rots away. So we seem to have accidentally discovered yet another gene that is probably required for building a brain and making it work.

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As well as discovering new genes, we can also use these comparative techniques to look at regions outside the gene to find out what controls the activity of the gene and what parts of the protein – that the gene encodes for – are critical for activity. Perhaps the best example of this work is the SRY protein.

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When we first looked at this protein and compared kangaroos, mice and humans, the strange thing we found was it was very different. The only part that was the same was a small section in the middle and it turns out that that codes for the ‘business end’ of the protein. This region is important because it binds to DNA and bends it into a particular angle and somehow makes you male.

Everyone assumed that this gene worked by turning on other genes, but its lack of conservation means instead that it may function as an inhibitor by simply getting in the way of something else. So we can find out a lot about how a gene works, just by lining up sequence between species. Similarly, we can find out a lot about other sequences that are needed to control it.

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For example, the prion gene. We looked for very small sequences outside the gene and also between the exons that are conserved. There is a lot of conservation between humans, mice, cows and sheep, but as soon as you add the Tammar Wallaby the conservation drops away. There are transcription factor binding domains that are conserved and presumably have something to do with turning on and off the prion gene. The prion protein, of course, is the one that goes wrong in mad cow disease, so there is a lot of interest in it.

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Now I want to talk about the unique traits of Australia’s weird animals, and how we might exploit these. Kangaroos – quite famously – do reproduction very well and this is one unique trait that we hope to exploit. The female can switch on and off embryonic development and they have a very sophisticated milk system that we can possibly learn from.

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At a very early stage the blastocyst can go into a quiescent state and stay there for up to 11 months before the signal to develop is turned on again. Of course, we would love to know what genes are involved in turning off and then turning on embryonic development because it would mean we could understand development more and possibly manipulate it in domestic animals and maybe even humans.

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The kangaroo is born at a very immature stage – about the size of a jellybean and with no back legs or gonads. It crawls up into the pouch, latches onto a teat and suckles milk which nurtures these early stages of development.

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Then later on while the pouch young is still in the pouch and still suckling it receives milk of a completely different constitution . The extraordinary thing is that the two completely different constitutions of milk are delivered by two teats lying next to each other like two adjacent petrol bowsers. We want to know what’s in that milk, particularly the growth factors, and understand how does the kangaroo switch from one type of milk to the other?

As reported in The Age recently, kangaroo milk contains a very powerful new antibiotic. Researchers have been studying kangaroo milk and saliva for some time because the pouch is a very dirty, grotty sort of place for a poor little pouch young who doesn’t even have an immune system. Something must be killing off the bacteria and it turns out that there are a number of antibiotics in milk. Maybe these can be harnessed for treating bacterial infections in other animals, including humans. Kangaroo milk may be a strange place to search for new products but it looks like it has delivered.

My own enthusiasm is in genome evolution and I’m particularly interested in the evolution of the sex chromosomes. We can find out a lot about where and how the sex chromosomes originated by comparing sex chromosomes from humans, kangaroos and even platypus.

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I’m sure you know that in humans, as in other mammals, females have two copies of a large gene rich X chromosome (approximately 1000 genes), while males only have one copy and a small Y chromosome. The Y chromosome is a very peculiar little chromosome because it only has 45 genes that are mostly concerned with sex determination and making sperm. That is, most of the 45 genes have a special male role. So the Y has always been known to be very peculiar so there is been a lot of interest in looking at different models of the Y.

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There are three models for the Y chromosome. The model we were all brought up with was the Y as a macho little thing because if you have a Y you’re male and that’s it. But it turns out that’s only because the Y chromosome has the SRY gene on it. The other theory is that the Y is a selfish sort of entity and it grabs genes from other parts of the genome that are handy in males. But our work on comparative mapping says that the Y is merely a wimp, a relic of the X chromosome. It started off being identical to the X but over millions of years it has been losing genes and there are hardly any left. This, of course, makes men very anxious. Will the Y chromosome disappear?

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I have indeed predicted that at the rate it’s going the Y chromosome will disappear in something like 15 million years. This doesn’t necessarily mean that males will disappear. It’s possible that we’ll evolve a new sex determining gene somewhere else in the genome.

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The other interesting thing about this hypothesis that the Y chromosome is a relic of the X, is that the genes on the Y are all, or mostly, evolved from genes on the X. We’ve done a lot of work on some of the special function genes, like the sex determining gene SRY, and other genes like RBMY which is critical for spermatogenesis, and we found that they all evolved from genes on the X that, oddly enough, appear to be involved in brain development and function. So it looks like intelligence genes that get marooned on the Y chromosomes have been commandeered to make them into fertility genes.

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So, the Y chromosome contains sex and spermatogenesis genes, will that save it? Probably not. We know that some genes needed for making sperm in mice have been lost from primates. If this gene can be lost in other species, why not the SRY gene itself? If it becomes inactive and then lost there is no reason to keep a Y chromosome – it can just go poof and disappear I think that this is likely to happen any time between next week and 15 million years, but it’s probably not going to be a severe worry to us.

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I hope I have answered some of the questions about why you would bother sequencing animal genomes. I’ve shown you that animal genomes can give depth and meaning to the human genome sequence; our Australian animals, are particularly good for finding new genes and the sequences that control them; they help us understand diseases and possibly lead to new treatments and even cures; they can lead to the development of new antibiotics, new drugs and better breeds of animals; and they also help us discover where our genome came from and how it has changed. In fact, comparative genomics is giving us information that can help us discover how humans are made and how humans function. It’s been called the greatest scientific adventure of our age and I’m not the only one who thinks so. Science thinks so too – naming evolutionary genomics the breakthrough of the year at the end of 2005.

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Again, I want to thank the people who have worked in my lab over the last 30 years. And I will finish with a logo for the kangaroo genome organisation. Thank you.

Questions/discussion

Dr Jim Peacock: Thank you very much, Jenny. I have a different interpretation of the evolution of the X and the Y chromosome to Jenny, but that’s what science is all about. Can we have some questions or comments for Jenny, please?

Question: Thank you very much, that was a wonderful talk. Jenny, would you like to comment about the opossum? How different is that from a kangaroo or the Australian marsupials?

Professor Jenny Graves: The opossum that you mean is probably the Brazilian short-tailed grey opossum which has been chosen by the National Institutes of Health (NIH) for full sequencing. It is as different to the kangaroo as a mouse is to a human. They shared a common ancestor at least 55 and probably more like 80 million years ago. It’s proving to be tremendously powerful to have sequences from both the opossum and the Tammar Wallaby because it enables us to do the comparisons. It tells us if something is specific to marsupials or specific to a particular marsupial.

Question: I’m fascinated by the antibiotics in the milk. Why doesn’t the bacterial flora in the pouch evolve resistance as we see for virtually every other antibiotic that’s known?

Professor Jenny Graves: I think it’s probably too early to tell that. This work is being done at the Victorian Institute of Animal Sciences in Melbourne and I’m sure they will be looking at it. But a lot of researchers are looking at pouch flora. For instance, some of my colleagues sent samples off to Washington University Genome Sequencing Center to sequence the entire flora content of the gut, poo and pouch. So we’ll be able to find out what kinds of creatures inhabit the pouch and, indeed, whether there is resistance to particular antibiotics.

Question: You were very polite about it, but your slide implied that the Australian Government has failed to support genomics of Australian mammals. Why do you think this is so?

Professor Jenny Graves: Yes, indeed. I think a lot of us are very puzzled and very disappointed that the Federal Government hasn’t seen fit to fund any big genomic projects in Australia, especially those of Australian animals. Possibly this is because the projects are so big. The human genome project cost many billions of dollars. But, of course, it’s getting cheaper and cheaper and I think the platypus genome project is going to be a mere $55 million and if we wait long enough they’re talking about $1,000 genomes. I think it’s a shame that the government has stepped away from big genomics, and they’ve done this very deliberately. We were told when we applied for a centre [the ARC Centre for Kangaroo Genomics], for instance, that it wasn’t fully funded because there was such a big amount going into sequencing and that was considered ‘infrastructure’. But the trouble is there is no place to get very large amounts of money for infrastructure that isn’t big lumps of equipment. So we’ve been very fortunate to have major funding from the Victorian Government to the tune of $4.5 million. That’s been matched by the NIH in an unprecedented gesture of solidarity with Australia. That isn’t quite enough to do a twofold genome project of the Tammar Wallaby so we’re still looking for $3 million if anybody has big pockets!

Dr Jim Peacock: Jenny, thank you for a wonderful and exciting lecture. We Y chromosome types will fight on.

Professor Jenny Graves: Thank you.