[MUSIC] So this is the map. Losing is indicated. Just because they're going to use the losing units In this part, they indicate with combination frequencies that were measure in crosses between B and C mutants. 12%,0 .6%, 6%, etc etc. They're not really additive. This is 2.4%. 2.01, etc., etc. But this is a reasonable genetic map, certainly for the time. Okay, so we have a map and we can see that the B alleles are at a distance from the C alleles. 2.45% re-combination for B8 and C12. And then where do the C araC constitutive mutant map? The araC constitutive are here Here. And that's it. And here of course. All the mutants mapped to within the araC gene, which means that the constitutive and the minus are in the same gene. There is only one gene. The model which implies an second repressor R is not a Cabot formed by these results. They all map within araC. So, this is a major result It shows that if you were to consider the system like the lack system, where you have IS and I minus, they all mapping the same gene. AraC R minus, all mapping within the same gene. So one gene is doing the regulation. Now, the only caveat in this is that the R gene could be a gene that is essential for the bacteria life and if that gene is gene is essential to the bacteria life we cannot destroy. So you can always make that argument. There's no rational argument that can make it. The second argument, which was not made at the time, but could be made, is that, in fact, the araC gene, or the araC DNA piece Is in fact coding for two proteins. For instance you can imagine that there is a C protein that is read in one direction and a C protein which is read on the opposite strands in the other direction. They would all map to the same DNA piece, because the mapping is on a linear DNA piece but they could affect two proteins. The secretive could affect one protein and the minus could affect the other protein, that's a possible scenario. It was not so that the time because the notion of overlapping genes was certainly very far from the thinking of the people. There was another notion that you could have, is that you could have both genes on the same DNA strand, But read in different reading frames, remember the cat, cat, cat, cat reading frame of the R2B with C-A-T, C-A-T, C-A-T, C-A-T. You remove one base, A-T-C, A-T-C, you add a base, C-A-T, C-A-T, C-A-T, C-A-T. Remember that, okay, so now if you think that this piece of DNA has two reading frames that are functional. The two markers, genetic markers, mutants, mapping to the two genes would be overlapping. But that crazy hypothesis was not put forward at the time, so at the time, C constitutive C minus mapped together. Now, One of the thing you may ask is okay, these guys are completely crazy, I mean, they're overkilling, look at that, they've mapped these mutants, 1, 4, 7, 10, 66, 68, 69. All map very close to the C minus 12 mutant. Maybe all these mutants are due to a hotspot? You have many times the same isolate because this accident is a frequent accident. Same for this, in which case what you do is completely ridiculous, because you do over and over and over the same, you analyze the same mutant. Was caught with that in his analysis of the IC units, I constitutive because the 8 that were recovered 15 years later and sequenced. Were found to be all eight the same base substitution. So he could've analyzed 200 he would have gotten nowhere nearer the truth because he was always analyzing the same hotspot. So in this case however, we can try to at least where the sum of these mutants are the same or whether they are different. Because the next experiment in the paper is an expression experiment where he looked for araA which is the rays. And araB which is a kinase, araB which is a kinase, and araA which is and each of these dots is a separate culture. So he has duplicate cultures for all of this movements, and he measures both enzyme in the same culture. And he finds that the constitutive are not all the same, some are more constitutive than others. Some are very constitutive like the 8, the 6, the 67, the 65, and some are very poor constitutive like the 2, the 3, the 64, the 63, okay? They're very poorly constitutive, they just tiny bit on, and after go on the math plate but not really constitutive, so, the math plates select for things that grow grow. Whether they're fully induced, or partly induced, you don't know. But, the question was, did these guys isolate the same event over, and over, and over again? Well, if you go to the column where C3 is, there is C3 in the previous slide, and C8. C3, C6, and C8 are the same, are mapped to the same place. So 8, 6, and 3, and what else? 9, 9 is here, and what else? 11, 11 is here, and then all of these, 61 to 61, 2, 3, 4, 5, 1, 2, 3, 4, 5. 1, 3, 4, 2, 5 and 7 and 70. With all of these guide map to the same plane, they are clearly not the same units. They're not repeat isolate of the same event, because eight, well you could say eight and six, I mean, it could be the same. But 8 is clearly not 3 or 9 or 11, 67 could be the same mutant as 65, but it's clearly not the same as 64, 63, 70 and 61. So you know from this that they have more than one site. Maybe they don't have a 19 site or a 61 site, but they have more than 1 site, okay? So the coordinate expression gives you not only an information that is useful, because it is really definitive expected for constitutive, but it gives you an information also about the isolates, which they don't discuss in the paper, by the way. Now, this is as dull looking as all the other figures but this is the key figure because in this case they measure A and E the per being. They measure the expression of 2 gene that are at one part of the chromosome and 50% away from it. And these two genes are coordinately expressed by each of these consecutives. This is an amazing result, this shows that constitutive in this case Is acting in trans. So it's a very important experiment, and again the duplicate are quite reasonable maybe set with this one which is a bit weird. So they don't take this point into the draw when they draw the line, now, coordinate, yes, coordinate in trans, yes, what else do they do? This is again an overkill experiment, they look at the expression of two other genes. So these two figures are there to demonstrate that constitutive expression is specific for the erogenous proteins, and is not a general effect on the bacterium genome. So they use two genes, again, slightly excessive. In one case, they use an enzyme that is part of the central metabolism, an enzyme whose activity is fueled by the. And in this case, they find that there is no significant change in the isocitrate dehydrogenase. The exam is not totally easy to assaying so this is quite as some scatter but this scatter is not, when they do the correlation curve they found a flatline. Though there is no correlation, and now they even go to the trouble of assaying glucose permease, because glucose permease is also a permease like araE. And so they want to show that membrane proteins are unaffected by the constitutivity, this is a bit exaggerated, but quite beautiful. It's not that choice of other genes is they could have used another enzyme of the Crepsac, no, they use a permease, and an enzyme, because that's what the assay for a revenue is, and assay the enzymes, and the permease. I find this particularly elegant, even though, maybe, it's a little bit, I mean, I wouldn't have liked to be in the lab, because that's a lot, and lot, and lot of experiment. They've shown coordinate, specific, expression with the constitutive mutants, that all map within the araC region, so like the lac system, they are going to do now dominance recessive test. So they will do three tests with araC minus, araC plus, and araC constitutive, the first test is using araC plus and araC minus, C+ over C-. The C- is ara minus, the C+ is ara plus. They do the test in the presence of arabinose and they measure the enzyme. And in all cases the phenotype is C+, so C- is recessive, C- is recessive to C+. Now, this is not the result expected in the repressor system, because in the repressor system, in lac system lack IS, which is like minus, is dominant over lac plus. So, this is different from the lac, okay. This is in evidence for positive control, now, then they do an experiment with the C constitutive in the C-.
