[MUSIC] So now we're going to discuss this paper on positive control enzyme synthesis by Gene C in the L-Arabinose system by Englesberg and his colleagues, Joseph Irr, Joseph Power, and Nancy Lee. This was published six years after the PaJaMo paper on the lactose system, so it's pretty close in time. But it was published four years after the big review that Jacobin Monot published that was the triumph of the repressor control system model. Okay, so what do they do in this paper? They first present the arabinose gene enzyme complex. Why do they call it a gene enzyme complex? Because some of these genes do not code for enzymes. Remember, the first session of this class was dealt with the relation between genes and enzymes. Now because some of these proteins are not enzymes, so they call it the gene enzyme complex. What do we have? This is a circular chromosome of e coli. You see that in this chromosome, you have the lack region losing an arabinose. Englesberg had isolated ara minus bacteria, bacteria that don't grow with the arabinose as a sole carbon source. And then he did complementation essay, and he identified four complementation group. And it is a mapping, and he mapped this fortune in the order D, A, B, C. Now we tend to write it the other way around, with C, B, A, D. He also found a fifth gene, hence our E, because you name the genes in the order in which you isolate them, not in the order on the map. And this araE gene was the permease, and at the time believed to be the only permease. So if we look, this is E, then we have the isomerase, RA, the kinase, B, and the epimerase D, that's the same as the picture we've seen last time. So, he describes all these mutants which are indicated here, the two alleles in D, three alleles in A, three in B, and a number in C, we'll come back to those. All of these are ara minus. The phenotype is arabinose minus. This can be scored on minimal arabinose plates or on indicator plates that tell you whether the strain is fermenting or non-fermenting arabinose. So there are a bunch of mutants. And then he describes the constitutive and how he's going to analyze the constitutive. Arabinose and lactose have differences that are helpful in one case and not helpful in the other case. The big help of the arabinose system is that the ara E gene is unlinked from the other genes. Of course, it is insist with the other gene because it's on the same DNA molecule. But it's so far away that it can be considered to be independent. It's actually on the other side of the chromosome. This is the major advantage of the arabinose system. In the lactose system, all the genes are next to each other, R gene, Z, Y, A gene. They're all next to each other. Unlinked means that you can do cis-trans test very easily. Anything that acts in trans will act on our E as well as BAD, unlinked to BAD. That's a plus for the arabinose system, a big plus. Jacques Monod did not choose to work on lactose because he had a fixation on his mother. He choose to use lactose because he could identify in the doors of the pastor chemicals that were analogs of lactose. And these chemicals were fantastic because they help him dissect, separate hydrolysis by lac-z and induction. Induction deals with lac-i, hydrolysis deals with lac-z. So he had chemicals like IPTG, which is an inducer but is not a substrate. Later on, they would get other sugars and other analogs of lacto, like ex-Gal, which is a substrate, but not an inducer. So if the strain is constitutive, the colonies are blue, if the strain is inducible, the colonies are white. These sugars do not exist in the arabinose system. The only sugar that can be used is a sugar called fucose, Which is an inhibitor of the enzymes responsible for catabolism of arabinose. So not because it inhibits the enzyme, but because it inhibits RC, fucose will bind to RC at the same place as arabinose. But instead of being a red dot on the model we've seen before, it's going to be a blue dot, a non-inducing dot. It's a non inducer. It's a competitive inhibitor of arabinose for RC. So, if you take a Ara+ strain, it will grow on minimal arabinose. But it will not grow on a minimal medium that has arabinose and fucose because it's non-induced. An Ara- Will not grow on any medium of that sort because it's Ara-. But an arabinose constitutive mutant will of course grow here, but will grow on this plate. So, you can start with an Ara+ strain. You can start with a million cells. Plate them on minimal arabinose fucose, abbreviated MAF in the paper. The colony will not grow. Or the bacteria will grow very, very slowly. But if you have an Ara C constitutive mutant, you don't know which gene it is, an Ara constitutive mutant. It will grow. So you can isolate constitutive, which is not bad. So you can isolate, with black you can use a sugar analog like phenylgalactoside, which is also a non-inducing substrate. So you can isolate constitutive. And the prediction is that these constitutive will be loss of repressor. That's what you expect, because that's what you've seen in the lac system, or with lambda. So Engelsberg and his colleagues isolate 60 plus, 61 constitutive mutants. Engelsberg's papers are characterized by a notion of overkill. You don't isolate one mutant or two mutants. You isolate a large number, and you hope that with a large number, you can convince people, which is irrational. So he isolate 61 independent mutant, and he's going to map them. So how does he do the mapping? He maps them with araC-. He actually already has some idea they have to be in araC. He discusses that in the paper. But let's do the mapping within araC. So he uses a phage, which is a transducing phage, which takes a piece of the chromosome from one bacterium and transfer it to another recipient bacterium. In his case, the araC constitutive are all in strains or in two strains that are lu+. And the mapping strains are all lu-. So, what you do is you can select a minimal or mineral glucose plates. He called them mineral in this paper, later on people tend to use minimal. In minimal glucose plates, a strain that cannot make leucine cannot grow because leucine is an essential amino acid. A strain that can synthesize leucine will grow. So you select for lu+ and you know how many transductants you have. And this will give you some value, 10 to the 3, 10 to the 4, whatever. For each transduction, this is one normalizing value. And then the cross is done between RC constitutive, that's a phage, and an RC- on the chromosome. So, the C constitutive will grow on the minimal arabinose plate, the plus will grow on the arabinose plates, the C- will not grow. And on the math plates, the C constitutive will grow, and the plus will not grow. So this is how he scores the mutants. Now there is something which is not discussed in the paper, not discussed so much in later papers and reviews, but this is a pure genetic cross. If it's a pure genetic cross, you have to account for everybody. You cannot lose one recombinant class. And in this case, you can have how many recombinants? You can have four. You can have C constitutive, +, +, C-, those are the parental type. +, +, and there is the C constitutive, C- recombinant, which is not indicated in the figure, but can be made. Now, these guys we ignore because they're C-. They don't grow on arabinose plates. Okay, so we can ignore them, this character and this character. But what is the phenotype of the C-? C constitutive, the W. If it's C-, it's okay, you've lost it. But if it grows on arabinose, and you don't know, you have absolutely no clue as to this phenotype. You cannot see it easily. So this is a big question mark. This will affect your calculation because I can call this parental type p. And we call this R1 and R2. Now you can measure the parental types which is the, You can measure the C constitutive + and +, +, and what he describes is the +, + over the leucine times 100 is a recombination frequency. The +, + are guys that don't grow on math plates and grow on arabinose plates. Now if these guys were +, +, you would double the number, and you would decrease, you would change by a factor of 2 the recombination frequency. So even if you don't know what is the phenotype of the double mutant, you know that the maximum error you can make is a factor of 2 in recombination frequency, which is not much but you have to realize it. Now, so he does that with all the mutants.
