Woman of Science
an interview with Cardy Raper
Her autobiography, Woman of Science, is in print again, too.
Leon Shernoff: Tell us how you got started in mycology. What early experiences led you along this path?
Cardy Raper: Well, I knew from the third grade that I wanted to be a scientist. And of course that wasn’t what a girl was supposed to do back then – you were supposed to be a nurse or a secretary. But I went ahead with it. My first college experience was very disappointing – science was taught as a litany of facts that had to be memorized, not what I understood science to be! But then I transferred to the University of Chicago, where I got to study with John Raper.
Now he was already known as one of the foremost scientists in the world who worked with fungal sexuality and genetics. He needed a graduate assistant, so we had a talk and he decided that I was the one. Before that, I hadn’t really thought much about the fungi – I thought I would just become some kind of general biologist. But through him, I got to know the fungi well and all the really fascinating and different ways they behave sexually, so that became my career path. And as you know, we also got to know each other really well; we fell in love, married and had children.
Schizophyllum, the fungus of a thousand sexes
LS: Now, the work of yours that will be most familiar to my readers is on Schizophyllum. Your work with John is what established that it has all these 23,000+ different genders.
CR: Yes, well, when I went to work for John – he, being very redheaded, preferred to be called Red, so I called him Red – he was working with Achlya, a “water mold” (Oomycete). It was then considered a fungus, but has since been placed in the new kingdom of Chromista, which includes some other parasites like Phytophthora, and also the brown algae.
Achlya is a very interesting organism. Red found that it emitted specific sexual hormones that direct growth of a pair of individuals towards one another in mating. This had never been demonstrated in such organisms before. Anyway, while we were working on Achlya, John’s graduate student, Haig Papazian (I was Red’s first graduate student at U. of C., and I think Haig was his third) started working on Schizophyllum for his dissertation. Papazian succeeded in showing that Schizophyllum has at least three genetic loci regulating mating type: a so-called A locus and two linked B loci. Papazian then moved on to something else and, Red picked up where Papazian left off showing that the A locus has two parts as well as the B and beginning to enumerate Schizophyllum’s many sexes.
LS: Now, what was that like? I mean, surely he didn’t go into that enterprise expecting to find so many. And when they just kept coming and coming and coming…
CR: Well, it wasn’t so strange, actually. He started collecting specimens from all over the world and mating them in all combinations. Meanwhile, I completed my Masters work on Achlya and took a job at Argonne National Laboratory where I studied the comparative mutagenic effects of nitrogen mustard versus X-rays on Paramecium. It was only after we married, had children, and moved from the University of Chicago to Harvard University, that I started working with Red on Schizophyllum. Red, his technicians and graduate students continued to define the extent and nature of the mating system while I concentrated on generating mating-type mutants.
LS: So you were irradiating the fungus?
CR: Yes, although the first mating-type mutation was obtained by graduate student Yair Parag using nitrogen mustard.
LS: Why do all this?
CR: You can find out all sorts of things from simple little organisms like this, using reliable methods. The complexity comes with what some people call the “higher” animals.
And it’s fascinating that the fungi really are so much more like animals than they are like plants. The understanding of mating and sexual processes in simpler organisms translates all the way up to comparable processes in higher animals.
LS: I saw on your website (www.cardyraper.com) the beautiful culture of Schizophyllum in a Petri dish.
CR: Yes, although the mushroom of Schizophyllum, unlike portabellas, is too small to hold in your hand for making a pretty picture on a book cover, it grows well on simple media in a Petri dish and makes lovely mushrooms when viewed under the microscope. One of its advantages as a model organism for biological studies is the ease in which it can be cultivated and experimented with in the laboratory.
Once More into the Breach
LS: I saw in one of your interviews that after John’s death, you went back to school and got your doctorate.
CR: Well, I never really left! (laughs) As I put it, I’ve never really been weaned from academia.
LS: And then did you go to work at the University of Vermont directly?
CR: No, I got my doctorate at Harvard, and then I worked as a postdoc in the Netherlands with Jos Wessels. He was very biochemically oriented. It was a good experience. He and one of his students had developed a new technique for removing Schizophyllum’s cell walls and manipulating the organism as viable single-celled protoplasts. You can do all sorts of things with protoplasts. Generating protoplasts is a necessary first step in applying molecular genetic techniques to fungi—DNA preps will not penetrate the fungal cell wall.
So I learned how to make protoplasts in Wessels’s lab, then got a paying job – at Wellesley college. Now of course, that’s an undergraduate school, and somewhat limited in terms of how much research you can do. But I came to like it very much: working with undergrads.
I went from there to the University of Vermont, with an interlude at the University of California at Davis, where I worked with Bill Timberlake, a very good geneticist. He was developing the use of molecular techniques with his fungus, Aspergillus nidulans, and that’s where I learned – or began to learn – the techniques of molecular genetics for application to Schizophyllum.
LS: And by that you mean things like sequencing?
CR: Well, things like how you isolate and separate DNA from RNA, and how you identify genes, or the section of the genome that you’re interested in: the very basics of molecular genetics possible in the 1980s. From that time, of course, the techniques have developed tremendously. That’s one of the most fascinating things about this whole field of genetics, it’s always evolving into something new, and revealing surprises.
LS: Now, I always get Wellesley mixed up with Wesleyan…
CR: Wellesley is an all-girls college west of Boston.
LS: And was that something that was valuable to you – to encourage women in science?
CR: Oh, absolutely. That was of course… well, let’s see, when do we usually say the feminine revolution starts? Around the sixties, right? But that was clearly on everybody’s mind when I was there, between ‘78 and ’84. And yes, it was another issue that interested me. I think I helped a little.
LS: So you went to UC Davis, you learned more of these techniques there, and then you went to Vermont. Have you had any particular focus within genetics since then?
CR: My whole research career, as PI (principal investigator) has been here at the University of Vermont, and I’ve been concentrating on the mating-type genes of Schizophyllum. I started in collaboration with a former graduate student of my husband’s, Bob Ullrich, and his buddy, Charles Novotny, who was in the department I joined here. And they each had graduate students, so it was like a little colony of us studying the genetics of mating in Schizophyllum.
Here, at the University of Vermont, Ullrich and Novotny were working on the A mating type system, and found that it involves genes encoding something called homeodomain proteins that are DNA transcription regulators similar to a host of comparable genes used for developmental processes throughout the animal kingdom. So his group was working on that, and my lab was working on the B system which we, along with Drs. Erika Kothe and Marjatta Raudaskovsky, found involves genes for pheromones and pheromone receptors. The neat thing we found at these loci is that they’re full of genes; The two B loci, B alpha and B beta, each contain several pheromone genes and one pheromone receptor gene.
It’s like a system of locks and keys, where the keys are the pheromones, and the lock, which is a seven-trans protein that weaves seven times through the cell membrane, is the receptor molecule. An unlocked receptor sets off a whole cascade of events leading to meiosis, or fertilization.
A locked receptor in an unmated individual just sits there. Along comes a compatible pheromone, which is a very small molecule; it lands on the receptor and titillates it. Like a fitting key, it opens up that receptor, with all its membrane domains, and then the altered receptor interacts with another protein, called the G protein, the programming protein, which sets off a whole cascade of genetic events leading to actual fertilization, diploidy, and meiosis.
The A genes kick in, somewhere along the line; we don’t know just when and how, but they’re collaborative, we do know that…
Nowadays, the guy who’s really carrying on with Red Raper’s work, from my lineage, from my lab, is Tom Fowler at Southern Illinois University, Edwardsville. In addition to doping out which pheromones fit which receptors, Tom and a technician in our lab identified a transposon that is key to a commonly recurring mutation that changes hyphal morphology in our organism.
LS: Umm, so could you just tell me a little bit about what transposons do?
CR: They jump around! (laughs) in the genome. A transposon is a section of DNA that moves, from one place in the genome to another. There are many different kinds of transposons, and this particular transposon, which Fowler called scooter, has characteristics of the so-called hAT family transposons known in some other fungi. It is a DNA-mediated transposon that creates 8 base pair target site duplications and has short terminal inverted repeats.
Scooter movement requires a transport, which is an enzyme that will excise it and transport it to a new location; but Fowler wasn’t able to identify the transposase.
LS: And what is the effect when it moves around?
CR: It can cause mutations like the one I mentioned, and it can change the way genes function. More often than not, if it lands in a gene sequence it would disrupt the function of that gene. Transposons were discovered, of course, by Barbara McClintock, in corn. For several years, nobody believed her! (laughs). Jumping genes!? What?! What’s that?! And of course it was all ultimately proven. She got the Nobel Prize, eventually.
Now in Schizophyllum, there is a frequently occurring mutation called thin that transforms the hyphal network into a quite different-looking morphology; it also shuts down the mating system. Scooter was originally identified as an insertion in the pheromone receptor gene of one of the B loci. It caused disruption of Parag’s primary mutation that turns on B function all by itself, thus transforming that mutant individual from a partially functioning homothallic (that is, spores are fertile with spores from the same parent mycelium) to a somewhat defective heterothallic (spores need to mate with spores from a different parent mycelium). (laughs) We always wondered “What sort of substitution could do that?” and ultimately, when we were able to get to cloning genes, examining them, seeing what they encoded, how they behaved, we discovered that the primary mutation that turned on that self-fertilization part of the genome was an altered pheromone gene capable of unlocking the resident pheromone receptor, and the secondary mutation turned out to be caused by a transposon that disrupted and obliterated function of that resident receptor.
LS: Okay; so when you talk about these loci, it’s not just one gene, it’s an area that has several genes…
CR: Right. Initially, only two mating-type loci were identified, A and B, and they were called A-factor and B-factor. Then each of those factors each turned out to have two linked parts called alpha and beta. Ultimately, using molecular genetic techniques, we discovered that the alpha and beta loci of each of these factors contain a bunch of genes! (laughs)
LS: Now, when you say that the genes are linked, what does that mean?
CR: Ah, well, it means that they’re very close together… they’re on the same chromosome, and the position on the chromosome makes a difference in terms of how frequently they recombine.
LS: Okay, so the closer they are…
CR: the less recombination.
LS: Okay, so the more likely the sex cell is to have both sets of closely linked genes from the same parent.
CR: Yes. It’s almost like with humans, you know, how we have chromosomes: x and y.
LS: Aha. Like we have lots of other things on the x and y chromosomes besides gender [color vision and balding patterns, for example].
CR: Yes, especially on the x. Sex determination gets more complex in more complex organisms.
LS: Okay, now a question about pheromones: I’m remembering that in Achlya, the organisms will actually grow towards one another, based on the pheromones?
CR: Yes. Although Red actually called them “hormones”, in hindsight, they probably should have been called pheromones. Pheromones are generally thought to govern distance recognition and attraction. You have two individuals separate, and they’re attracted to one another by pheromones that get transported… in this case by liquid medium. And hormones are generally thought of as functioning within the same organism. Both are molecules that are involved in sexing and attraction to one another.
In the case of Achlya, they’re steroids. In the case of Schizophyllum, they’re called lipopeptides. They’re just a different kind of pheromone that somehow homes in to a target making some kind of connection to receptor molecules, embedded in the membrane. You understand; the receptor doesn’t move around, the pheromone does.
LS: and so Schizophyllum will also grow towards a compatible mate?
CR: You know, I tried and tried to identify that, and I couldn’t. But I’m sure it happens somehow – it does in Ustilago maydis. As you know, in the case of Schizophyllum, the hyphae of a fertilized, fully compatible dikaryon, have these little hook cells?
LS: You mean clamp connections?
CR: Yes, the clamp connections. Now those definitely are being attracted to the subterminal cell, and then they fuse with the subterminal cell. This happens after every cell division. And we think this is a pheromonal attraction. It hasn’t really been pinned down yet, although Erika Khote’s lab has some evidence for it.
LS: So you think that if you could somehow interfere with the pheromone, the tube that forms the clamp connection would just grow out randomly?
CR: Oh, we know that – many, many mutations show that; and some of those mutations affect pheromone recognition. But you see the problem with the terminology: There is chemical attraction, but the distance is very small, and it’s an attraction to a different area of itself, not to another individual. So is it a pheromone or a hormone?
We have all sorts of little clues, and Erika Kothe, who is a German investigator at Jena University, has done some cell biology that shows there is some accumulation of pheromones at the tips of clamp cells. What makes it difficult, Leon, is that these molecules interact in very very low amounts, so it’s very hard to tag them and see what they’re doing.
It’s a fascinating system – hook cell formation – so well regulated, and it’s so essential to the whole fertilization process.
LS: Wow. I never knew about that. I thought that the clamps were just pre-programmed to grow in a certain shape.
CR: No, the system has a purpose. Every time the dikaryotic cell divides, the two nuclei of opposite (compatible) mating-types will divide synchronously – it’s called conjugate division – and they have to be resorted into the new cell, and this hook cell is the process by which that is done.
And it’s done very precisely and reliably by Schizophyllum, but is not precise in some other mushrooms.
LS: Oh! So they’ll end up with hyphal tip cells that have only one nucleus?
CR: Yeah, or many nuclei, as in Agaricus bisporus [the commercial button mushroom] where each cell has a varied but multiple number of nuclei.
LS: And does that… affect the organism? I mean, is that a problem?
CR: Well, it’s a reflection, actually, of their mating system. A. bisporus, for example, is homothallic, essentially hermaphroditic. Generally, fungal sexes aren’t morphologically distinct – the different sexes don’t look different in any way – but Agaricus bisporus is like a hermaphrodite in that it can fertilize itself. Whereas there is this other related mushroom, called Agaricus bitorquis, which I studied for my thesis. It is a bipolar heterothallic, and it does pretty much sort things out as a dikaryon… but not as well as Schizophyllum.
LS: Now, I think I asked you about the relative frequency of the mating types.
LS: Now, are different mating types more common in different locations?
CR: Yeah, that’s a little hard to figure out, but there is certainly a higher frequency of certain sets of linked genes within clades of the worldwide population. That’s another intriguing thing about Schizophyllum commune. It is fertile with other members of its species all over the world – a specimen from Israel will mate with a specimen from Australia… They’re all interfertile! – and yet they’re distributed all over the world.
But you do have certain clades, as they call them, where you have concentrations of certain characteristics, not only the mating types but other types of genes as well.
LS: So it’s maybe from some evolutionary pressure in that environment?
CR: You know, Schizo is found all over the world. It is a terrific outbreeder, and it makes millions of sexual spores. The one that fits best in the environment that it happens to land in is the one that’s going to be successful there. You’re always getting new genetic combinations, because it’s such a… what I call promiscuous fungus. (laughs)
By the way, there’s a guy by the name of Rytas Vilgalys, at Duke. You know him as a mycologist, but I remember when he was a population geneticist and studied fungi among other things. He used to say that his favorite fungus to study is Schizophyllum, because it’s found all over the world, wherever wood is growing – on all continents except Antarctica. It’s a very, very successful fungus.
LS: Now, I’m remembering that with Achlya they’ll not only grow towards a compatible type, but they’ll change genders in order to…
CR: Ah, yes. Alma Barkesdale followed up on some of that. In Achlya it’s a matter of relative sexuality – I think that term was coined by Hartmann – such that each individual can be one or the other, either male or female – or rather, either of the two mating types, because in Achlya there are only two mating types. But it’s a balance of genes, and that’s what we were not able to get at.
It must be the result of a balance of multiple genes – they’re probably linked, but I don’t know – and any individual that you happen to pull out of a pond might tend to have more of the genes for what we call the female mating type, whereas another individual would have more of the male genes, and it’s the relative maleness versus femaleness when they come together that determines how they react with one another.
LS: Aha. When I first read about that, I thought maybe it’s like the human system, except that you can have xx, yy, and xy, and the xy will switch if it comes to meet a pure strain.
CR: Well, you can think of that way, but we don’t really know.
Red Raper had a strong female that he had collected in this country, and then he went to England because he couldn’t find a similarly strong male type over here, and he came up with a strong male type that he collected in northern England. (laughs) So then we could put the strong female together with the strong male, and rely on the strong female always being female and the strong male always being male with these two individuals. How the Atlantic ocean happened to separate these two extreme types is a mystery.
But again, when you have intergrades meeting one another, it depends on which one is relatively more male versus female. The system has to be governed by a whole group of genes. It’s more like a quantitative difference. If it were governed by something like human genes, it wouldn’t just be like they have xx versus xy; it would be like they also have xxy, and xxyyy, and all sorts of other combinations.
But our sex genes are all in a specific location on two different chromosomes, and in Achlya there seem to be a lot more of them, but we don’t know what genes are involved, because we couldn’t make productive crosses. They wouldn’t cooperate with us in their mating… (laughs) [back to Achlya photos]
LS: You weren’t playing the right kind of music for them.
CR: Well, interestingly enough, Leon, most of the basidiomycetes do have a sexual cycle, and they are either heterothallic or homothalli. Heterothallism tends to be predominant, but there’s a whole 10% or so that have no sex at all. However, with the genome projects now, they’re finding sex genes in these sterile species – those genes just aren’t functioning properly. So you know, a lot of fungi have this whole system. There are commonalities between the ascomycetes and the basidiomycetes, and even now reaching to the zygomycetes. So it seems these are very important genes that were present at a very early stage in fungal evolution and have been passed down to all these different groups, yet sometimes they’ve mutated to where they just don’t function anymore.
LS: So these fungi have found a way to get along without sex.
CR: Oh, yeah! (laughs) And of course there’s such a thing as mitotic recombination, but… it’s not really the same thing.
Interestingly enough, Penicillium, from which we derive penicillin, frustrated us for years. You may know that my husband’s brother, Kenneth Raper, did a lot of the early work on Penicillium in this country. Well, he always wanted to be able to breed it, in order to ramp up its production of penicillin – sex is such an efficient way of recombining different gene sets! But they couldn’t find any sexual cycle. Now, with the genome project, they have found some remnants of sex genes in it. So it may have been able to breed sexually early on, but it’s lost that ability. Though of course it makes asexual spores that spread all over the place, and it’s very successful that way.
Whither Fungal Genetics?
LS: What do you think the biggest question is that the field is facing right now?
CR: Well, in terms of genetics, since that is my central interest – I mean, I work with fungi, but genetics is my central interest – I’d say the effects of what they call short RNAs. You probably know the central dogma, that DNA from the cell nucleus gets transcribed to RNA, and transcribed RNA encodes the making of proteins which function all over the cell. Now, RNA is thought to be the original genetic mechanism for doing everything. DNA comes after it, from an evolutionary standpoint.
LS: So the DNA is just a storage mechanism?
CR: Right. It stores the genetic code from one generation to the next. But it’s the RNA that translates into proteins, which do all the job of running the cell, no matter what that organism is.
And now, with the genome project… you’ve probably read that it looked for a while like 95% of the human genome was “junk”? That it didn’t code for anything?
LS: Right. I remember seeing that a long time ago.
CR (laughs): People had all these theories like “Oh yeah, that’s raw material for making mutations, making this stuff into genes…” Well, it turns out that an awful lot of that “junk” is not junk. It is making “short RNA”, and these RNAs, which aren’t long enough to make genes or proteins, are functioning as modulators of how the genetic code is expressed – of DNA sequences, or possibly other RNA sequences.
So it gets more and more complex. and you wonder. When I first learned about genes, I understood they can have two modes of expression. There were dominant alleles and recessive alleles, which can get resorted with each generation. If you have a diploid organism combining both dominant and recessive alleles of the same gene, then the dominant is the one that gets expressed.
But there are always modulations to that gene expression – I mean, genes do all sorts of things that need to be turned on and off at certain times – you know, you want this effect for a while, and then you don’t want it. So they have to be modulated in a very careful way to make the whole physiology of an organism function properly at the right time. And it’s turning out that these short RNAs that are encoded by what used to be called “junk DNA” are being revealed as modulators of gene expression: turning genes off or on, lowering their function or raising it…
So that to me is utterly, utterly fascinating. (laughs) If I were to set up a lab as a youngster now, that’s what I would go for.
LS: So the short RNAs, are they methylating the genes? Is this how they’re doing this?
CR: Well, possibly. Gene methylation was worked out pretty well with ascomycetes – with Neurospora before the modulating effect of short RNAs was known. I can’t explain just how it’s done – it’s not my field.
But investigators are beginning to realize that these short RNAs can modulate gene expression, they don’t just silence it. As is known for certain transcriptionally regulating proteins, small or short RNAs apparently can interact with DNA or transcriptionally regulating proteins in such a way that at significant times they’ll turn a gene on, turn it off or… slow it down, make it less effective, more effective, and it’s kind of a temporal thing, you know? When you really think about it, every organism needs to operate temporally.
LS: So then the question of course is what modulates the small RNA?
CR: Aha! We don’t know that! (laughs) Good question! That’s what’s wonderful about science: there’s always another question.
LS: Wow. So you’ve also answered my next question which was if you had another twenty years and unlimited funding, and I guess this is it.
CR: Yes! Though I’d also need a lot more energy than I have right now. (laughs)
LS: Well, you seem to have plenty here.
CR (laughs): Well, not as much as I used to.
LS: Is there anything else that you want to say that’s been left out?
CR: Yeah, there is something that I’d like to say, and that is that working with model organisms – and many of the model organisms are among the fungi – you know, there are protozoa, there are worms, and of course you’ve heard of Drosophila, the fruit fly.
Then, besides these organisms, there are many other systems that haven’t even been investigated yet. And these systems have their own characteristics that lend themselves to discovering something new about what makes life life – what makes this organism function.
I think that the funding – and it always has been like this, but it’s gotten more so – has been very directed and very bloated towards curing human diseases. Cancer is interesting to study, because it involves so many different life processes, primarily when the cell divides, and it won’t slow down and all that stuff, but there’s so much more to learn, things you can learn from little critters like fungi that you can bring into the laboratory and – I call it – play with, and there are so many ways to design experiments and get answers that you can’t do with human cells, you can’t do with rats, you can’t do with higher animals…
Another example is zebrafish – my son studies that, in terms of how the nervous system develops and senses. But we are directing our research dollars in such great concentration towards curing human disease that we tend to be short-sighted, I think. I think there should be much more support for obscure little organisms that ultimately can tell us a whole lot about how humans function.
If I were able, I would write a book about all of the knowledge that we have gained from studying everything from bacteria to fungi, and all these other organisms. Look at restriction enzymes, for instance – if we didn’t know about restriction enzymes, we wouldn’t have the ability even to do what we already can do with human cells. And the discovery of those enzymes that cut DNA into pieces was just serendipitous: that discovery came from a bacterial study – a virus and a bacterium, that were not even pathogens. So I think that the focus on pathogens and human disease restricts us. I’d like to see much more innovative research, in organisms that are not directly related to human disease.
LS: Well, this is wonderful. Thank you so much.
CR: Well, I had fun. I love to talk about this sort of thing.