[MUSIC] So in this first paper they have a map, a genetic map, of volutes mutants. This is the map of the chromosome. How it was defined at in 1959 essentially from Bulman and Jacobs work. You see that there are number of sites on the map, but not that many considering the disk chromosome has 4000 genes. So you have about one marker per 100 genes, or 200 genes. This is a small region of the map. You will see on the map that you can't find T1 resistance lactose and galactose that we had on the previous map last week. So they map the mutant. They map a number of i mutants and a number of z mutants, and some y mutants. This map is wrong, life, because they didn't have a very fine, they could not easily select for all the possible recombinants. And so they put the i gene between the z and the y. Now, we know that on the map, the i is here, the z is here, and the y is here. But that doesn't matter. They say that this is a tentative map. Certainly the distance between the individual marker is correct. This is all based on crosses. They cannot select for an i+ recombinant. Why? Because if you want to select for an i+ recombinant, you have to kill the i-. So you have to find the condition where the i- is dead and this one is okay. What do you need for that? Well, you need a sugar, which will kill cells that express beta galactosidase. A sugar which will give rise to a toxic product and a reaction of beta galactosidase. And this sugar should not be inducing. That's one condition. Second condition, the sugar should not induce, because if it's an inducing sugar, the i+ would be used and then it would be dead. So you want a non-inducing pre-toxic sugar. And they didn't have that at the time. Those kind of sugars will come in later, but not at the time. So the first cross, they show in the paper is a cross where they take a male and HFr. You see they use the symbol that I still use in genetic medicine or in genetics of higher organism for males and females. This male is one type for the lack region, z+, y+, i+. It is sensitive to streptomycin. The female is resistant to streptomycin, but doesn't have the zg. It's mutant for the zg. So the female cannot make beta-galactosidase because it doesn't have an intact zg. The male cannot make beta-galactosidase when there is no inducer. And cannot make beta-galactosidase when there is streptomycin. Because streptomycin would prevent synthesis of the protein. So this is an HFr cross, very similar to what we've seen last week. What we've seen last week was this part, the recombinant part, this group. But now Pardee has taken samples at 10 minutes, 20 minute, 30 minute, 40 minutes, 60 minute, 80 minute, and he has measured enzyme activity. And he says that the recombinant should not, well, that's an experimental point. But basically, both enzyme synthesis ends the potential to form recombinant appears at the same time if you do uninterrupted mating. Which suggests that the enzyme is in fact made, and this is in the presence of streptomycin, so the enzyme is made in the female. But the gene comes from the male. Because the female doesn't have a zg. It's made in the female from a gene transferred from the male. So, this is a very simple experiment. So, while they have to have a few controls, And this spent actually quite sometime doing controls. The first control is to be sure that the enzyme is not made in the male cell. So, what did they do to check and to control what's going on in the cell? The first thing is streptomycin. When you add an antibiotic to a cell, the antibiotic has to enter the cell to exert its effect. Particularly in the case of streptomycin which will inhibit ribosome. So how do you know whether streptomycin works quickly? It's not very easy but they found, they measured three elements of the response to streptomycin. One is they had cells growing exponentially. They add streptomycin. And they see how long it takes for the cell to stop growing. This is sort of a window which give you an idea about how long it takes for the streptomycin to enter the cell and bug the ribosomes. And they say it's about 2, 3 minutes at most. They also measure the incorporation of sulfate S35 sulfate into protein. And sulfur goes to 2 amino acids including methionine and then goes into protein, and that's stops within 2 minutes. And then protein measures the rate of increase of enzyme with a culture, as a function of time, and then he adds streptomycin, and he measured the arrest of synthesis of beta-ga. And he gets the same curve. So basically in two minutes it's done. So that's fine, but, we are talking about the cytoplast which is a funny word to use for bacteria which you don't have a nucleus, or morphological nucleus. So, cytoplasmic expression, so what they want to do is to see what happens when the female cell cannot grow because the female cell is deficient in one or another metabolic enzymatic reaction. The first way they do it is they use a carbon source. So the control carbon source is glucose + maltose. The deficient carbon source is maltose, and this is done in a strain which is maltose-, cannot ferment multiple. Now, under this deficient condition, the F- is Mal- and the HFr is Mal+. So what you're looking at is these two numbers compared to the control. And what you really are looking at is the percent inhibition. So you have to imagine the female cell cannot use maltose. So when you put only maltose, the female cell is starving. The male cell can eat up maltose, and be happy, and has energy, and everything is good. Now, the question to ask is how much of the product of maltosirosis which is glucose, can enter the female. And you remember the conjugation of HFr and F minus- or F+ and F- result in a bridge between the two cells, a small bridge between the two. We saw that in microscope pictures last week. 73% inhibition, so it's not perfect but it's pretty reasonable. Then they use a second system, where the F- is arg- cannot synthesize arginine, and the HFr is arg+. Under this condition, the control is with ariginine in the medium, and the deficient is without arginine in the medium. So the idea here is the following. I will draw the two cells. This is the HFR. I will draw the F- here, and then I will erase here, erase here. And make the bridge, so what they ask is this cell there's no arginine the medium. This cell is making arginine. So it has some arginine floating in the cytoplasm. Can this arginine swim through the bridge into the F- cell. That's a question. And you can see that with the arginine, the amount of inhibition is very large, 96% inhibition of enzyme formation. So from this, they conclude that in the cross, the DNA may go through, but very little else. This is tight and only the DNA will go through, to make it simple. So that's the first experiment, the series of control experiment that the system is actually working. So the next experiment is genetically a bit different. You have an HFr, which is that +i+. And you had a female, which is F-, obviously. The female is not capable of making beta-gal because it's that And it's constitutive. It's both constitutive for nothing, for not making anything. It's streptomycin resistant. The mail is streptomycin sensitive and z +i+. Now,when they do the cross, they get some anxiety being made during the first hour. If they do simply this cross the Z DNA gets in the female, the i+ DNA gets in the female, but what you make, is you make LacZ. This, question mark. And then they use streptomycin, again, and one of these sugars. TMG, this is a sugar that cannot let any use, but is not a substrate. And the curve during this part of the experiment look about the same. That's a surprise.