[MUSIC] So the first paper is not really a paper. It's barely a letter. It is actually a claim. It's the claim that bacteria have sex, which was quite a shock for many, many, many people. The title is Sober Genetic Recombination in Bacteria E coli. And it's signed first by Lederberg and second by Tatum. Published in October, 1946. So, they say that their results strongly suggest the occurrence of a sexual process in the bacterium Escherichia Coli. What they do in this paper, in this note is an outline of what will be discussed in the real paper which came out in 47. What they claim is that when they mix two population of bacteria that have genetic characteristics, they can isolate new type of bacteria that have some characteristics from the first type, some characteristics from the second type. So, new type. They are aware in that paper that the process of mutation that give rise from a normal y type strain that can grow without Biotin. You can obtain a mutant that requires biotin. But from that bacteria that requires biotin there are lots of different ways you can go back to the original state y-type of no longer not recombined by FC. And one of these ways of changing the type is to undergo reversion. And we've seen reversion in the context of the Benzer paper, in the R2 mutants. We've discussed extensively mutation and reversion. And the fact that same mutants revert, and most mutants actually revert, and occasionally in the case of R-2 it's about 10% of the mutants do not revert their deletions. So the reverting is a big problem. Because the revertant give you a background. So they realized the problem due to reversion. But Tatum, in his lab, had isolated many strains of bacteria that were requiring not only one substance, like biotin, but two substances or three substances. Now, if you have a strain, how can you get rid, you don't get rid of reversion, but you bypass the problem of reversion. How do you do that? Well, you start with a string that requires a number of elements, a number of molecules. B minus, P minus. This string can revert to B plus at the low frequency, one in a million. It can revert to P plus at a low frequency. One to one million. But the chances, the probability that this strain will revert to y type, i e B+ P+, is a product of the two probability. So it's 10 to the minus 12. That's very, very, very low. If you have another strain that is M- T- for instance, this strain can also revert to M+ T+ at the same frequency of 10 to minus 12. So low, that you don't really have to worry about reversion. Now what happens if you mix these two strains together? And that was the experiment. They mixed a B-P-, we use the word cross, with an M-T-, and they asked can I isolate a y-type, that is somebody who is B+ P+ M+ T+? That's the question. And it worked. And the answer to this question is a blatant yes. The frequency was low in this experiment, on the order of 10 minus 6, one in a million. You see this is low, so if you didn't have two characters, you couldn't pick, you couldn't distinguish the revertants from the recombinants. Now these recombinants are stable and homogeneous. And they will use the phage resistance of a sensitive phenotype as a second, unselected marker. The way they write about this is these types can most reasonably be interpreted as instances of the assortment of genes in a new combination. This is a purely Mendelian wording. Classical genetics wording. We can do Mendelians genetics with bacteria. Now so far, that was a brilliant discovery. Now, they are aware, and we'll see this, of the experiments of Avery on transformation. And they do an experiment to exclude transformation. Or they believe could exclude transformation. Then they have this, and it's interesting to see how the facts are the facts and then how you interpret them is not biased but influenced by the general picture. So they say, well if we have reassortments of genes, we must have a self fusion. We make a zygote, they use actually, the word, zygote in the other, yeah in the letter. Hypothetical zygote, which has not been seen by microscopy. Which is not unreasonable, because if it's one in a million, how many cells do you have to look at before you can see one? And so they claimed that there was a cell fusion and then something like a meiosis to go back to the recombinant. That turns out to be wrong. But it was a reasonable proposal considering everything that was known about sexual processes in living organisms. So the Reid paper was published in 1947, so Lederberg is 22 years old. He's very bright, but he doesn't have the deep knowledge of the field that Tatum had. And actually the paper has a lot of discussion about other people's result, previous results. And the paper was written by Tatum and hence Tatum is the first author. And not Lederberg. Most likely Lederberg wrote the note, the claim. In nature, but the complete paper was quite certainly written by Tatum. Okay. So the first thing they discussed in that paper, very early on, is the experiment of pneumococcus. And they say, there are two different way of doing genetics, what we would call today, genetics. One, is to look a the occurrence of mutations. That's what Lorien Delbrook did, in their paper. And the other one is, here, that's a very careful wording, the hereditary interaction of one bacteria with another. They don't talk about sexual process. They talk about hereditary interaction. Something that requires a bacteria to be together and there is out of this a new bacteria with new genetic properties. Don't discuss the mechanism. But they say that a sexual stage may be inferred. And in fact, people have used, for instance, in this context, the word of male and female. The sexual aspect has remained an important part of this. Then they discuss, after discriminating these two different approaches, they discuss the fact that bacteria are now believed, we are 1947, to have genes. So this is the paper of Luria and Delbruck 1943. The founding paper of bacterial genetics. Other papers on isolations of auxotrophs by Tatum and by another group Isolation of auxotroph by somebody we will meet again later. Andre was working on lysogeny, but also working on auxotrophy. And by the phage group, who isolated bacteria that were resistant from one phage but not from another. Resistant to T1, resistant to T2, resistant to T4, etc., etc. So all of these is evidence for genes and bacteria. Genes do not exist unless you can mutate them and have a phenotype. So if you want to make it in shortcut, the p of Mendel had only seven genes because there were only seven characters. Of course Mendel was difficult because he didn't write about it, but certainly Morgan was aware that there were genes that had not yet been identified by mutations that existed. But the one that you could name were the one that had mutants with a different phenotype. So, the fact that bacteria have genes or microbes have genes does not mean that they have a sexual life or sexual. It could have genes without sex. Of course, Tatum was totally aware of the fact the neurospora can exist as a haploid or as a diploid organism. And you can isolate neutrons in the haploid. So he was aware of that. Then they discussed previous experiment that suggests that possibly that you can do recombination bacteria. And those were older experiments in the 20s, in the 30s, and they were not very convincing. And they were heavily criticized. Then he says people have done the experiments of taking a string that is for instance P minus and crossing gate with a string that is M minus and recovering wt. But in all these crosses they could not quantitatively discriminate between the background noise of reversion, and true recombination. They had no way of distinguishing. Because if there is an enormous noise in a concert, and you sneeze, and a little kid sneezes in the way back, you don't hear it because all the orchestra is playing. This is a concept of the background noise. So this could not be interpreted. They discuss the transformation first done by Griffith, then by Avery, MacLeod and McCarty. And they are aware that the condition necessary to get transformation are extremely tricky. Everybody knew that and they spell it in the text. So there is not much serious evidence before this paper. So they're going to start by taking strains of E Coli, which they describe and I show you the table not because we have to remember the table, because the table is actually extremely interesting. They discussed the different E.coli. And you can see that all but these four are derived from E.coli K12. Whereas these four are derived E.coli B. They say how the mutagenic treatment was used to isolate a biotin minus, a threonine minus, a biotin methionine minus, etc etc etc. The two strains that will be most useful at the end will be a Y24 and Y10. Y stands for Yale because those strains were isolated at Yale. And Tatum, unlike other people, did not give the name of his initial to the strain, he gave the name of the place. So all the Y strains of e coli resonated by phatum Atelier. These two strains are threonine minus leucine minus thiamine vitamin b1 minus for one. In biotin, phenylalanine, cysteine, minus for the other. UV or X-ray. Out of these strains, most of these strains are, actually, all of these strains are T1 sensitive. Sensitive to phage T1. But from all these strains they have a, not all but most of these strains they have isolated T1 resistance by the procedure developed by Luria and Delbruck. So they have two closely related strains. For exams, Y10 T1 sensitive and Y10 T1 resistant. Okay. Now, this will become important because some of the crosses will work, and some of the crosses will not work.