[MUSIC] Today we are going to discuss the origin of the amber mutants. The amber mutants were discovered like many things by accidents. A series of accidents. The people involved all started in Rochester, in the Rochester phage group that was led by Gus Doermann in the period going from 1952, 53 until 1960. These people were great practicer of a science called radiobiology. And Frank Stahl who was among the practitioner of that science defined radiobiology as a discipline where you hurl fully characterized regions at invisible targets and hope for interpretable responses. Which I think is a bit rough but quite accurate. Basically, what people wanted to do with radiobiology was to understand how many targets do you have for gene activation by an agent like UV ray or X-ray. And basically doing what Timofeev and Delbrück and Zimmer had done in Germany in the 30s. And among the things that they were doing, they were for instance mixing irradiated phage of viruses with an irradiated particle in trying to see whether they could rescue the irradiated, lethally irradiated phage with a white type page. And all of this kind of experiments were aim at defining the number of targets. Essential targets. The nerval targets they call that. When you hit a particle like a virus with a UV or x-ray, but most of the work was done with UV, and you look at the survival of the particle versus the dose, you find an exponential decay. And the slope is giving you some information, as well as the initial curve of the slope before you decay. And using this in other kind of experiments, they found that when you have multi-complexes, that is multiple infections, you have a number of vulnerable centers, large sub unit of the genome. There was about three. Whatever the vulnerable center were, it could be counted by deduction from the slopes at about three. Now, at that time, there were only very few morphology mutants of T4. And so it was pretty hard to identify what were those vulnerable center. And one of the things that he kept trying to have done during his thesis was to calculate the number of vulnerable center when an R2 mutant, infect a K lambda lysogen, or when an R plus phage infect a K lambda lysogen. And in those experiments, the R2 look like a vulnerable center. Now, in K lambda, the R2 genes or the R2 locus look like a vulnerable center. But in E.coli B they don't. And so in a typical theoretical fashion of handling this kind of problems Epstein postulated that they must be Anti R2 genes or mirror genes that behave in B like, that don't grow in B and grow in K lambda, the mirror image of the R2 mutants. so this was extremely vague and, of course, nobody had ever attempted to isolate these mutants. And so Stahl and Charlie Steinberg, who was a student of Delbrück at CalTech at the time. Deacon moved after his PhD to CalTech. Were discussing this over and over, and according to Steinberg, Dick brought up the R2 mirror gene hypothesis several times, and I was not enamored of it. I just found it difficult to take radiobiology that seriously. So one evening I said Dick, you don't believe that cockamamie idea any more than I do because if you did, you would have looked for these genes for a long time. And so, Dick supposed that he was taken aback by my fury and said he would do that that very night after supper. And Steinberg, having started the conversation, felt obliged to go. And of course, Dick was a very kind and generous person and he does not remember Steinberg shouting or being furious, and he just remembered that they both decided to go to the lab and try to do the experiment. And so they wanted to pick a number of phage particles that were plated on K lambda and pick them and see whether they will grow on B or not. A normal page will grow on B. A mutant in a hypothetical anti R2 would not grow on. And so since that involved picking a large number of plaques, they decided to enroll a young student named Harris Bernstein. And Bernstein was a graduate student and to convince him to come rather than to go to a movie with his girlfriend, they offer him to name the mutants after him, and his nickname was Immer Wieder Bernstein, Forever Amber, which was a novel widely read at the time, hence the amber. Now some of these people, 20, 30, 40 years later remember things slightly differently, some people thought that it was named for Bernstein's mother who was known by none of the people. None of the actor. And so that was probably wrong and but certainly the Bernstein amber connection was right and of course they identified this mutants. Mutant that would grow on a particular strain, K lambda strain, and not on B. Now, Bernstein is absolutely convinced that he did not isolate the first mutant. And that he is sort of a, I mean, the name was given, but he doesn't really deserve it, because he said, at that time, I was so cautious that I would not contaminate the plaques, that I had a little platinum wire that I would burn between each step. And I burned them so much I must have killed all the phage I ever tried to pick. Because on my plates the next day there was no phage. So he's convinced that he doesn't deserve the name but the name stayed. And so the isolated mutants and it turns out that the luck, the real piece of luck, that the K lambda strain that they used at Cal Tech at that moment was not the original K lambda strain. But was a strain derived by somebody in Appleyard. And the name of the strain was CR63 because the strain had been isolated in a lab at Chalk River which had a research lab in Canada. Hence the name and that's the 63rd strain isolated at Chalk River. And so Chalk River was a little bit like Oak Ridge in Tennessee, had a research lab next to a nuclear plant. And so, they isolated one mutant, they isolated two mutants, they isolated three mutants. And so when you have two mutants, the first you're going to ask is, are these mutant affected in the same gene or in a different gene? You do this by complementation. You infect under restrictive conditions with the two mutants and you look whether you get phage progeny or not. If you don't, then in neurons are affecting the same gene and are not allowing the production of one particular protein required for the phage infection cycle. So, two different genes, mutant into two different genes would compliment. We've seen this with the R2 locus, R2A and R2B. Mutants in the same gene will not compliment. Now, the prediction from the anti-R2, the mirror genes, was that there would be three or four anti-R2 genes. They very quickly had identified eight to 10 mutants. And each of these 10 mutants was in the separate genes. So the number of gene was already much larger than what was predicted by the radio biology hypothesis. And at that time, it's not exactly clear how and when, Dick and probably Steinberg immediately realized that they had identified a general class of mutation that could potentially affect any gene in the phage genome. And so the story goes part of that, story goes that day where they realize that Max Delbrück had organized a party at his house and had invited many people and so they arrived, Charlie Steinberg and Indy Epstein, young kids, fresh from the lab and they go to Max and say, Max, Max, we don't have the anti-R2 gene, but we have something very interesting. And Delbrück answers, that's too bad. Delbrück was carrying beverages for the 30 or 40 or 50 people that were in his garden. He didn't care about the anti, the mirror genes or the anti-R2 genes at that particular moment. Of course immediately, the day after the immediately realize everybody all the lab realized the potential. And so this pick them, pick them, pick more and more mutants and they map them by recombination and classify them by complementation to genes. And that's where they has this big collection of approximately 50, the first paper had 46 genes, 46 genes. So in a few weeks, they had 20 times more genes than had been isolated in the previous 10 years by morphological studies. They had no idea what the mutation were and how come this mutation would not manifest in the CR63 lambda strain and the lysogen strain. That would come later. But they had many, many mutants and at the same time electron microscopy was very intensively used to look at infected bacteria, viruses, biological things. And so immediately what Kellenberger and his colleagues did, Kellenberger was on a sabbatical at Cal Tech, was to cut sections of infected bacteria, bacteria infected under condition where there's no production of verions and look, what do they see inside this bacteria? Do they see bits and pieces or do they see nothing? And in some cases, they saw nothing. But with some of the mutants, they saw phage heads, or phage tails. So let me just show you here. One of the picture of the complete phage. So here you have a picture taken a few years later by Kellenberger, who was a Professor of Biophysics, or Molecular Biology, as would be called later at the University of Geneva, and this picture is a nice one. I mean, you can see the phage head, you can see the tail and you can see the fibers. On the right, you have a little cartoon that give you the physical dimension in angstrom of all these morphological features. So what Epstein and his colleague realized is that they had many mutants in many genes and they could both map these genes on the map. Put them on the map, on the chromosome, and identify what was present and what was missing. So once they realize that, they knew that they had a tool to do the genetics of development for sophisticated biological system. You can identify all the genes, and then you can see what they do by microscopy or by any other means. And the second paper in the series will be experiments done by biochemistry.
