[MUSIC] So all of this work started in Caltech and continued in Geneva, where Dick Epstein moved in the early 60,s was finally presented to the community at a meeting in 1963, and published in the preceding of this meeting in 1964. So this is the map of T4, the genetic map of T4, degenerated. They have here 47, sorry, I already said 46, 47 genes that are numbered totally arbitrarily and that are put on the map. And for some of these gene they have some idea of the genetic distance and those are in black. So many, many genes. The previously known genes are E for Lizozime, R1, R3, R2, and that's about it. Those were the morphological or the R2 genes identified before, nothing else. Okay, so as such, it's not very interesting, right? We just have a collection of mutants in essential genes. But, what they did, and what they report in that paper, was the properties, the morphological properties of infection with these mutants under non permissive condition, in equal IV. I'm just going to discuss with you a few of the genes, not all of them. A few of the genes because they have different properties. Gene 32. Infection by a mutant in gene 32, makes no lysils, no DNA, no serum blocking antigen, no particles, no heads, no tails, nothing. Infection by gene 32 is extremely abortive. And this gene was later called a D0 because there's no DNA made, no synthesis of DNA, no replication. The next gene is a bit, but very significantly different. The next gene 33, makes DNA but nothing else. No particles, no heads, not tail, no fibers, nothing, just replication. This, was called later on, a maturation defective and it's interesting that the pressure, the intellectual pressure, of the operon model, enough regulation of gene expression by negative regulators, was so strong, that the authors did not realize that they had one of the first example of positive regulation. So, it's interesting in terms of the history of ideas that they could have made this very important contribution in gene regulation and they just didn't see it because everybody believed that gene expression was negatively regulated. Gene 34 codes for a fiber of the tail, one of the gene that codes for the fiber of the tail. So, this is tail fiber and these are all the allele, now you see amber alleles in this, the mutant alleles here. And you see that some of these are called amber AM and some are called TF. Well, Bob Edgar, when the Dick Epstein got his mutants, was a bit jealous. He confessed himself that he was a bit jealous, so can believe that he was maybe very jealous. And so, he then said, that's not fair, these guys who were looking for something completely different and they bumped into something fantastic. I want my own genes to play with. And so, Bob Edgar was at the time slightly older, he was an Assistant Professor, and he was a good friend of Norman Horowitz, who had actually characterized the first temperature sensitive mutants. The temperature sensitive mutants are capable of making an infection at 30 degree, or 25 degree, but not at 42 degree. So he isolated many TFs, and he had TF's he had identified genes, that Epstein had not seen. For instance this gene, gene 30 was has only TF mutants, no amber. But very quickly he found that all of most of these TFs mapped to gene that were identified already or had been identified already by the amber. And in act the TF and the amber are just what we call now conditional lethal mutants. They are lethal in one condition, not lethal in another condition, conditional lethal. And by definition you can isolate conditional lethal in any gene that codes for an essential function. You cannot for unessential genes. So one of the things that I point to you in this because I think it's very indicative already of their thinking in terms of morphogenesis or assembly or development, is that the amber mutants in gene 34 make DNA. The TF mutant also make DNA. The amber mutant and the TF mutant all make normal particles without the fibers, they hadn't seen the fibers in the At the time, but there was one big difference. When they look at the antigen, when they look at the protein, epithelia protein pieces that can react with antibodies, they found that all of the amber made no detectable antigen, zero. And all of the TFs made detectable antigen. So, the TFs are proteins that at high temperature are not properly folded, compacted, assembled. The amber make no protein, and we know today and we actually have discussed slightly out of order, the notion that the amber are nonsense codons that can be suppressed by suppressor tRNA. We've discussed that in the previous session. Okay. So after this, they represented the map in a slightly different way. Each gene here is the same size. And they give for each gene a phenotype, the name of the gene or the number, and the phenotype. And you can see that gene 34 makes particles but no infectious viruses. These have no fibers, these are tail fibers. Gene 33 is a maturation defective. And it's not the only gene that is maturation defective because there's another one that was found a few years later called gene 55. And then they have the genes that made heads but no tail and tail but no heads. For instance, 5, 6, 7, 8 are tail genes, they make heads. 21, 22, 23, which are here, 20, actually, it starts with 20, all of these genes make tails and no heads. So they already notice something, that this genetic map is not random. If you look at the genes that are necessary for making DNA for replication, all of these the D0 genes are here, from 41 to 45, plus gene 32, which we've discussed before. So that's what they had at the time when they finished and published this paper. They had a very crude idea about which gene is important for what, and they had many mutants at that time, between the TFs and the amber they had about a thousand different mutants. They noticed also that this map is not random. Head genes tend to be together, tail genes tend to be together. Replication genes tend to be together, fiber genes tend to be together. The genome is organized in what looks like intelligent organization. Don't take this for intelligent design please. This is a way of organizing the chromosome that has some rationale in the way the genes are going to be expressed. And, this kind of organization tends to be lost in cell genomes or in human genomes, because genes are dispersed throughout the genome and usually not all that linked. So, now we have, we have done the first part which is discussing these genes. At the end in 19, they published a review two years later 1965 in Scientific American and they had 56 genes and four morphological genes. Now, we know that they're 63 essential genes. They didn't have all of them but had a pretty high number. They knew that some of this genes necessary for making DNA code for enzymes, and at the time enzymes were proteins. They proposed that some of these gene are what we call today chaperones. Protein that help another protein to form properly. Which was not a concept not to be really identified for another 20 or 30 years. They predict that there are unknown genes in the genome because it looks like there're regions on the genome that look empty. So the amount of information that was obtained by this very, very simple method is quite amazing. And this will be used in a fantastic way by Bill Woods, who was working with Bob Edgar. Now it also tells you, the next story will tell you that these mutants were used throughout the community. They were distributed. Many, many people have them. Many, many people use them. The original paper, what Frank Stahl calls The Birth Certificate of the Amber Mutants, was never published. It was in a drawer of Dick Epstein, and the manuscript or actually many versions of this manuscript were in the drawer and when Dick Epstein died a few people decided that it was probably worthwhile to try to finish this manuscript that was been in preparation for 50 years. And the Journal of Genetics agreed to publish it, so the birth certificate was published in 2012. 50 years afterwards. That's the complete description? Not complete because some of the figure had been lost or we couldn't find them, but more or less complete. Fortunately for the people interested in history, the paper on the TF mutant was written by Edgar who is much better writer that Dick Epstein and much less hesitant about sending to the journal. And so we have the story of TF mutant. But Edgar had his mutants, he had the TF, he was associated with Epstein for all the work. And he used the amber mutants to do biochemistry because it was much easier than to do it with the TF. And that's going to be the next paper.
