About science; About building a virus

This is about science produced by the California Institute of Technology and originally broadcast by station KPCC in Pasadena California. The programs are made available to the station by national educational radio. This program is about building a virus with host Dr. Albert Hibbs and his guest Dr. Robert Edgar a professor of biology. Here now is Dr. hid. It's. About 14 years ago a long process of biological research resulted in the discovery of the structure of deoxyribonucleic acid. This extremely important molecule usually abbreviated to DNA had already been identified as the source of genetic information in the creation or the reproduction of life. The blueprint molecule which tells one creature how to reproduce the next and for that matter tells one cell how to reproduce itself or how to operate once it's in in shape and running. After this search was over and the structure of the

molecule had finally been determined a very long complicated molecule with a curious twisted structure. The next attempt was to try to understand its function and that is the way it accomplishes its task. The steps which go in between the blueprint and the final product. We have with us now. Professor Robert Edgar who has been involved with this research and specifically with a fascinating accomplishment that is the construction of a virus and Bob perhaps we ought to start out by saying what the virus is that you're working with or for that matter what is a virus to begin with to sort of lay the groundwork. What a virus. Well a virus is simply a package of genes. It's about the simplest form of life that we can study. It's just a package of genes that contain the information for building more viruses wrapped up in a coat to protect itself. So it does have a DNA molecule for example involved in it. That is right. It's just a package of

DNA. And that's why a logically active and sewn condemns things. Well it cannot do anything except find a place to function which is another cell. So a virus has to grow inside of another cell a living cell which contains the machinery for translating the information in the DNA into proteins that are needed for various types of functions. So it's sort of a parasite than in other cells. That's right it. It's a blueprint in search of a. Search of a machine of machine X right. And did you pick a particular want to work with. Just sort of random choose any that happen to be available. Well a lot of these things are accidental in nature but the virus that I work with is very convenient to work with in the laboratory its host is a bacterium and so bacteria are easy to handle in the lab rather than for instance an animal virus where one

has to work with either whole animals or tissue culture. I see so you selected it primarily on the basis of experimental convenience. That's right in Hamlet in the laboratory and do things when it's right. Sample the one you have. You pick what does it look like how big is it. Well it's. Bout A doesn't have a name by the way. Yeah it's called T4. Just means to type for office. Not a very exciting dozen compared Allwood deoxyribonucleic acid no romance and it's about I guess about one hundred thousandth of an inch or something. But for a virus it's pretty big and complicated. Many viruses are much much smaller and less complicated in structure. What does that structure consistent. Well it has had that sort of hexagonal in shape and it has a tail that's rather complicated looking at has a sort of spiral

structure on the tail and then at the tip of the tail there are six long fibers that look like tentacles that come out. And this fancy tail is really just a fancy can opener for getting the DNA that is packaged in the head into the bacterial host. So the six fibers it can attach as tentacles to the surface of the bacterium. And then this. Tail contracts much like a syringe plunging a needle into the bacterium and then the DNA that is in the head goes through the needle and enters into the bacterium. So this elaborate gadget is mainly because the bacteria has a rather tough wall and this is just a device for getting the DNA into the cell. Sounds like a very marvelous piece of machinery when you don't have a kind of bacteria doesn't in fact by the way. It's it's called Ashley Richard Cole I insist the common you know the common column bacillus that everybody has in their intestine harmless an arm that was always found.

Yeah. Well I wouldn't know when it when it finally succeeds and injecting its DNA into a so what happens to equalize at the the end of its existence was a rather sad fate I'm afraid you have the the first thing that happens is that you can detect is that the DNA of e.coli is destroyed. And this would seem to make a lot of sense after all the DNA is the blueprint for. It tells the machinery in the cell what to do in the bacterial DNA of course tells the machinery to make more bacteria and so that soon as the viral DNA gets and it destroys the bacterial DNA it sort of like performing a coup d'etat and then the viral DNA now takes over the cell and directs the proteins into sizing machinery to make viral proteins instead of bacterial pricy and so on.

As you point out the virus itself has none of this machine it is not as well scenery so it uses all of the complicated chemical capabilities of the cell. For now for its own purpose actually and how how much what sort of a reproduction factor is there how many typically how many fours come out at the end of the process. Well it takes about 30 minutes and you get 200 new viruses. Nice new complete viruses each with a set of genes in the DNA and the complicated tail and so on. It sounds like you are a fast reproductive cycle thats right but probably doubles about every two minutes. When you approach this problem what did you do and how did you how did you go looking into the machinery of this process what was a first step. Well. By training I'm a geneticist and my approach to trying to find out how the virus grows and multiplies was to approach it from a genetic point of view. The idea being that in the DNA of the virus

is the blueprint for how the virus gets built. And so like a cryptographer if I try if I can crack the code of what the various Gene functions in the DNA are I can come to understand better what this blueprint on how to read the blueprint you first have to crack the code of this particular one and find out what each of those various metal pieces of message matter. Yes in this sense by the code I mean the different gene functions how many genes are there in the key fours of DNA molecule is it just one molecule but it's just one molecule and I was right it's just one molecule just up for reference purposes how how many molecules of DNA are there in a human set of genes or sort of. Well the set of human DNA this is very hard to say. I'm nobody really no answers. About I don't remember exactly but I would think about 100000 times as

much DNA I see in the human cell as there is in this virus. Exactly how they're connected to molecules is another question but he's right it often has more material in him. That's right yeah. OK well how many so how many different genes that you figure there were in this one. Well one can only make a guess and guesses in the neighborhood of a couple of hundred. The gene is is a segment of the DNA that carries the information to make a particular protein. I see and we don't know how many different kinds of proteins the virus codes for but we know how much DNA there is and we have a reasonable idea of how much what the amount of DNA is required to make one protein. So by how you can make a calculated right out of right about up to a couple hundred perhaps. Well this seems like a rather complicated task to set out to discover the order of one or two hundred different. Genetic hundred different gene functions how I guess is a

big problem how did you go about it. Yeah well for a long time that was a big stumbling block because the usual way to do geneticist studies gene function is by getting mutants and mutants are. More mutations are errors in the blueprint errors in the DNA once and the error is made by the reproduction of the DNA. It's faithfully copied as an error. Now errors that are dilatoriness such that they result in them some change in the protein made by that gene so that the protein is nonfunctional. If that if that particular gene is essential for the growth of the virus then it will be lethal. So the we won't be able to study such mutations that seem to be quite a stumbling block in your path. That's right. For example if we were on a method of learning things as to kill the specimen before we have a

chance to examine that. Yeah and so we devise several tricks to get around this. One of them was well both of them in fact are examples of what we call conditional lethal mutations. These are mutations that affect the protein made by the gene in such a way that under some conditions the protein the mutant protein can function and in other conditions it can point to some examples of the conditions that would affect whether or not. Well the simplest one I think to visualize is temperature sensitive mutations and an example that everybody's seen I think is the Siamese cat. Which is what what is well sensitive in the cat. There's an enzyme protein that makes black pigment and in most black cats this pigment is this enzyme that makes the pigment is perfectly fine. But a mutation occurred some time

which gave rise to a modified form of this enzyme which was temperature sensitive so that it would only function this enzyme would only function in cool parts of the body. In the warm parts of the body the enzyme is inactive so that's why Siamese cats have dark ears and noses and that corals those are the cool parts of the body. And I was a ballet on the side usually stay like right. So this is an example in of a of a temperature sensitive enzyme Where did you find any temperature sensitive enzymes and T4. Yes well the idea then from this was to try and isolate mutants that showed this temperature sensitive property only not for coat color in the virus but rather for in other words mutations that would effect the virus so that it could grow in the host normally and produce progeny at a low temperature. But when the mutant was shifted to a high temperature it was incapable of producing a healthy crop of baby

viruses. How many did you find. Oh well we isolated thousands of them. And when you say it was only a couple hundred genes. That's right. Well that's the next problem in the game is to try and sort out where the mutations are and what kinds of functions they affect. And we have no way of picking them specifically. So what we do is to just isolate lots of mutants at random and then try and sort them into different genes. How do you do this. Well one way is to test whether or not the mutants independently derived from humans are defective in the same function in other words in the same gene. This can be done by taking two mutants and infecting a bacterium at the high temperature. Now if those two mutations are in different genes

the two mutants can complement each other. In other words. Mutant one has a perfectly good to Gene and mutant to has a perfectly good one gene sewn together in the cell. They can manage to make temperature stable proteins of all kinds. I see they all grow together. They'll mix their DNA in the cell. They both affect in fact the cell they will knock each other out No no got a cell but I'd rather they can grow old together in harmony whereas if the two mutations mutation one mutation two are in the same gene and we do this then they can how they can help each other because they both have the same defect. And so by doing lots of combinations of tests like distant mutants can be sorted into different genes and this is and after you are able to do this how many of your estimated want to 200 genes were you able to identify. Well I think we've got about 70 now. That's a fairly good size fraction of my sort of is it enough for your purposes.

Well the unknown ones play a role that is vital to you. We the unknown ones are unknown. We don't know how I would die. There is really just question that there are number of genes that are not essential for the virus for the viruses growth and such. And if that's the case then we wouldn't no matter how long we kept trying we wouldn't get any Taisha of the kind I described never see a lethality. That's right take place. Just be silent regions of the DNA and we can mutate them. Well having not having identified some of the genes and the mutations what's the next step and do you now write down the code and reproduce the blueprint of what else. What do you do next. Well the next step is to find out what each of these genes does that is to try to identify the protein that is responsible for it to some degree yes. And the way this is done is simply to take a mutant.

From a particular gene and in fact bacteria for instance at high temperature for this gene will not function properly. And then ask what does happen after infection. Can the virus do anything. Presumably all of the genes have the potential of functioning in the infected bacterium except that one that is mute. And so by comparing what happens under these defective conditions with different mutants defective in different genes we can try and get an idea of what really is wrong with that particular mutant And so in for what the function of the gene is. So in this way you can then begin actually to reproduce the coded blueprint or understand it and begin to understand the particular gene functions you know. For example what if in some cases we would find for instance that after infection there would be no. Virus DNA synthesis and this would then suggest that that particular gene is

is needed for the reproduction of the virus DNA. In other cases the model saying that the fundamental reproductive set. That's right. And another mutant we might find that that the viral DNA is reproduced normally in the infected cell and the infected cell bursts open at the normal time and instead of liberating virus particles it might only liberate for instance tails and no heads and thus we would suspect that this mutant is defective in making the head and that affected gene and is a gene that is needed for building the head. And so in this way we can identify at least to some degree of certainty what the various genes are doing in myself and what their function is right for then starting from this could you then sort of correct the process in any way or other than the temperature method how did you how did you go about accomplishing what.

What I mentioned we started with that is building the virus. So far you've told us how you destroyed them in various diabolical ways how do you put it back together once right. Well. We were quite surprised to find an enormous number of genes while about 45 of the 70 that we identified appeared to be concerned with the building of the virus particle itself we suspected most of the genes would be concerned with making the DNA reproducing the DNA. But apparently a large number of them were actually built concerned with assembling the virus itself. And they suggested that the assembly of the virus the putting together the head and the tail and so on was a pretty complicated process. So we decided to look into this more closely and when Bill would join the faculty Kel-Tec we decided to see if we could. Study this more directly by trying to build a virus outside the cell. And we took advantage of the mutants as. A reservoir of

parts. In other words I mentioned a particular mutant would produce for instance tails and no heads and so such a mutant infected cell is then a source of tales. Now another mutant would make heads but no tail so that's the source of head. How long can you keep please once you've made a batch mixed up a batch of Iris tails and put them in the refrigerator. Well whatever keeps them in a deep freeze and then keep for awhile they haven't really studied how at least I'm sure that all their hard pressed for four instantaneous experiments one media after the other you have some time. We have a little time that right. Yeah so what after after getting a few of the parts I made Incidentally how many different parts would you manage to break the T4 down into bits before you began assembly procedure. Well it isn't quite it isn't so easy to really answer that question in these mutant infected cells in the electron microscope you can see particular parts of the virus for in so you

can see the head and you can see the tail. You can see these fibers that have the sole right and a few of the other intermediate parts of the tail. But the fact that there were so many genes concerned with assembling the virus suggested that there are lots of little. Little bit so you could think of nuts and bolts and so on which would be maybe one just one protein molecule say that hooks the tail onto the head of one protein molecule attaches a fiber to each of the fibers to that to tip of the tail and so on. And these of course we can't see. So the problem is really to use these extracts really of mutant infected cells to sort out what was going on. So then having having a set of parts some of which you could see in the electron microscope and some of which you could only guess at. How did you manage to get them back together again outside a cell.

Well the first thing we tried to do. We we were really very dubious that it would work with the first thing we tried to do was to attach the fibers on to the. Virus particle and so we then took mutants that were concerned with making the fibers and infected bacteria under conditions where the genes couldn't function. And then after the cells burst open particles were produced that lacked fibers and these were noninfectious. These then we could purify by centrifuge geisha methods and then this was a source of bodies if you like. It lacked the fibers and we took another mutant that was defective in building the head and infected cells under conditions where that Gene couldn't work. So the cell line was full of tails and fibers and nuts and bolts and all sorts of other things that we don't know about but at least there were no heads there were no virus particles. Then we take those infected bacteria and break them open. And so we just have a guck. It's

got all sorts of stuff in it everything that would be needed including the fibers to build a virus but no no go ahead. And so we mix this extract then that contains the fibers with the particles that we previously prepared. And to our great delight. The particles became active and able to infect cells and we were able to show by examining them in the electron microscope that they had acquired fibers so that once the cell had gone through the process of making all of the pieces all that was necessary then was to put them in to the mix together and they would find their way to each other and hook up the right way and become at least back even as far as you could go normal before. That would appear to be the case. And in that particular case however where the considering attachment of the fibers to the

particle we think there is more involved. See we're dealing with an impure system this extract. And in that there could be the fibers and for instance an enzyme needed to attack the fibers onto the body. Yes so even though you destroyed the cell wall some of the cell machinery might still be in there or not right. That's right. So. In this particular case it requires an further analysis of what is in the extract that is needed to attach the fibers. And in this particular case Bill Wood has been looking into this in more detail and he in fact does have some evidence that there is a an enzyme that is needed to attack the fibers on to the body of the phage which is not a part of the finished. That's Iris. That's right. It seems to be like a workman if you like but the building with the walls up but he doesn't you know the part is he would

have to have along with the parts in order to accomplish this final step of putting them together. That's right this is one of the things that interested us very much in this problem because there were had been earlier work about 15 years ago tobacco mosaic virus which is a very simple plant virus had been assembled in vitro outside the cell. In this particular case it was a case of purifying the virus and then dissociating it into its nucleic acid protein and then showing that it could spontaneously reform just by putting its all its molecules in the same that's right. And this gave rise to the notion of what's been called self assembly where the structure is is a spontaneous product of just the coming together of the bits and pieces. And we hear that at some stage. Evolution the construction of complicated structures requires more than just the spontaneous falling together pieces that

additional genetic information is needed. Little workman to go around and tighten nuts and bolts and so on. And in time. Yeah that's right. And so it looks like probably in this case there is such. So you guys you would reason from that the T4 is higher up the evolutionary ladder by a few steps than and tobacco has a right right. But I suppose the tobacco mosaic corresponds fairly closely to some of the concepts of the origin of life. I mean oh acid's by some means or other accidentally came together and began making DNA spontaneously. This is the sort of thing it's. Apparently being done in the tobacco. I was a I said I assume also that when you had finished with this particular example you just talked about that the resulting DNA from the product was characteristic of the part with the head right rather than the part of there than all the other stuff was right over all those supposedly in the extract from the cell with all the parts.

There was some of the other DNA also floating around right. But it couldn't get in because the head was already had its DNA in the right foot. What other pieces did you manage to break off the two besides fibers and put back on again could you do this with any other portions of the structure. Well yes we've been able to. Study the assembly of the virus and quite a bit more detail in just tacking on of the fibers. And we've been able to. Well first there's an exam in that first experiment really encouraged us a great deal. And so we wanted to see for example whether we could build a virus by putting the heads and tails together and so it was simply a matter of taking one cell extracted contains everything except heads and either extract it contained everything except tails and mixing together and we got lots of virus and so by taking lots of different mutants defective in different genes and making extracts of the cells under conditions

where the genes didn't function mixing in them together and seeing whether or not we got virus and comparing that to what we knew the previously about the gene functions we gets begin to get some idea of what kind of steps in the assembly could be carried out in vitro outside of the cell. And to some extent we were able to purify some of the parts for instance the head and some of the tail parts and so on and begin then to work with semi purified systems and try to work this out and what is come out of this is the. The thing that sort of pleased me is that looks from this these studies that the virus is built much like an automobile is assembled in other words it's build up in a step by sequence of some reaction mass it's a really an assembly line process and that each step in the assembly line is controlled by a

particular gene. And so when we have a particular gene not functioning just all the workmen at that particular place of being second the assembly line piles up and lead cumulate and in the infected cell the precursors parts to that particular thing. Well I suppose it's probably not a fair question to ask you but you told me that in the in the within the cell once it gets infected by T4 it takes about 30 minutes to reproduce the progeny just as a dirty Question How long does it take you. About 30 years. Bob thank you very much for joining us tonight and taught us about how you put together a virus. This was about science with host Dr. Albert Hibbs and his guest Dr. Robert Edgar. Join us again for our next program when Dr. Robert McGregor Lanham will lead a discussion about bio engineering of vital organs.

About science is produced by the California Institute of Technology and is originally broadcast by station KPCC in Pasadena California. The programs are made available to the station by national educational radio. This is the national educational radio network.