The lac-cistrons
. h t t p : / / W W W . S C I E N C E - P R O J E C T S . C O M / . . . . . . .

Controlling a Gene - 1
The Parts (Cistrons) of the Lac-Operon


Controlling a Gene - 2


..... What is development? What is adaptation? How do we - creatures both great and small - cope with our environment which might be changing by the minute or by the season? Life is full of changes that require adaptation. A great part of this is accomplished by our being able to turn genes on and off. Of what do these genetic switches consist? How do they work? These are some of the 'molecular biology' things we are going to consider in this unit.

..... We are going to again use that famous bacterium E. coli as our tool not only because it is cheap and easy to grow, but because it is simple and not complicated by too many extraneous factors such as arm and leg genes. Also E. coli has a lot of 'fast' genes appropriate for a short lab period. We are going to look at its gene which codes for the machinery that takes the sugar lactose (milk sugar) from its environment and prepares it for entry into the glycolysis pathway which you have studied earlier in the course.

..... Philosophically we have already asked the question of why E. coli would ever have a gene that was sometimes 'on' and sometimes 'off.' It is all a matter of metabolic economics. If there is no lactose around, for example, why should the bacterium waste its time and energy making unnecessary lactose-processing machinery. Yet, if the bacterium comes across some lactose, it would be well for the bacterium to use it. So the genes are turned on, the machinery is made, and the food source is used.

..... In general, the experiment we are going to do today will start with our taking a culture of E. coli that has been growing for a long time in the absence of lactose. We will then switch on the gene, and then watch how fast the lactose processing machinery is made. We will do this by monitoring how much of the lactose-splitting enzyme -galactosidase (-gal'ase) is produced as time goes by. We will have to use a couple of tricks to do this: remember that our chemist friends have provided us with a sneaky compound, IPTG, that looks a lot like lactose and will switch the gene 'on', but which itself cannot be split by -gal'ase); and we have another compound, ONPG, which also looks like lactose and is split-able. When the -gal'ase enzyme splits this second analog a yellow color develops. We can thus follow the rise of yellow color as an indication of how much -gal'ase is present, which, in turn, tells us whether or not the lac-gene has been turned on.

..... This simple experiment is one of the most profound in the annals of molecular biology. While it led directly to several peoples' getting Nobel prizes long ago, its fundamentals are in great use even today as this marvelous switch can be inserted by gene-splicing in front of other genes we want to study. Just add a little lactose or IPTG, and the new gene is turned on for us to see what it does!

..... This 'lac-operon induction' experiment has been done thousands of times before by students far and wide. Let's do something more. A next step, as it were. Real science: exploration. Let's invent some of our own tricks to see if we can crack into how does this lactose switch works. Let's tinker with the E. coli system with various treatments that damage the cell in very specific ways. If the damage does not affect the induction of lac, then we can safely say that that particular part of the cell is not involved. But if damage does affect the induction, well! Let's get started.

..... Each group in the lab will be assigned a different treatment. You will be getting these assignments from the TA's.

A Qualitative Analysis of the Lac-Operon

.....Before we begin trying to quantitatively measure the rate of induction of the lac-operon, we should check our various cultures in some sort of fast qualitative manner just to make sure we have something worthy of our spending a lot of time on it. Our 'tool'-organisms are listed in Table I:



TABLE I
Bacterial Strain List
Escherichia coli K-12
.....
(Note the characteristics listed below do not necessarily match the strain. It is our problem to properly match them!)
#StrainCharacteristics
1 W3110 or K-12+ lac+
2 C600 lacY-
3 See your supplier lacZ-
4 M7032 lacc



.....You shall be shown three petri plates upon which have been plated the types of bacteria listed in the above Table I. The plates are denoted as to whether they contain medium with the sugar glucose, or with lactose, and perhaps without any sugar. You could see how each of the strains react to each of the diferent media. Next you will have to ascertain which of the 'negatively testing' strains might have -galactosidase inside. To do this, will make 'ONPG' tests of each strain growing in similar media made without dyes. Here is how the simple qualitative 'ONPG' goes:

  1. TA's will have grown the 4 strains of bacteria on glucose and lactose plates the previous day.
  2. They will then label 8 test tubes G1, G2, G3, G4, L1, L2, L3 and L4. (G = glucose; L = lactose)
  3. Next they'll put 1 ml of "Z-Buffer" into each. Little polyethylene droppers are used. (Qualitative work usually does not require much precision.)
  4. Using a fresh toothpick each time, they'll scoop up a little of the bacteria from one of the plates, and drop the toothpick into the appropriately labeled test tube. This is done with all the possible combinations of 'bug' and medium.
  5. Next, they'll vortex each of the tubes to suspend the bacteria homogeniously in the buffer.
  6. 2 drops of "SDS" and 2 drops of CHCl3 are then added to each tube.
  7. Each of the tubes is vortexed again - this time for about 10 seconds each.
  8. At time = zero, 200 l of ONPG reagent is added to each tube and swirled to mix.
  9. Note which tubes turn yellow. Roughly how long did each of those take?
  10. Go back to the table above and match as many as you can of the strains with their correct characteristics.
  11. Now consider which of the the four strains are inducible. These will be the exciting ones for the next lab period's experiments.



Procedure for Assaying -Galactosidase

  1. Pardee, A. B., F. Jacob, and J. Monod. 1959. The Genetic Control and Cytoplasimic Expression of "Inducibility" in the Synthesis of -Galactosidase by E. coli. J. Mol. Biol. 1: 165.
  2. Zabin, I., and A. Fowler. 1970. -Galactosidase and Thiogalactoside Transacetylase. The Lactose Operon, p. 27. Cold Spring Harbor Laboratory.
  3. Miller, J. H. 1972. Experiments in Molecular Genetics. pp: 352-355. Cold Spring Harbor Lab.


Supplies:


METHOD:

  1. Add 1 ml of "Z-buffer" to a small test tube
  2. Add 2 drops of chloroform (CHCl3), and make sure that the CHCl3 sinks to the bottom of the "Z-buffer")
  3. Add 4 drops of the 0.1% SDS
  4. Add 6 drops of ONPG
  5. Add 0.5 ml of culture (or a scraping of cells from the surface of a petri plate using a loop or sterile toothpick)
  6. Violently vortex mix this for 10 seconds.
  7. Set aside at room temperature (or 37C for faster reaction) until yellow color developes in the "positive" control.
  8. Yellow color indicates that -galactosidase was formed and that the cell's lac-Z cistron was "on."


.....THE THINKING PART. Can you think of any questions prompted by this data. Let us consider one of those which you should have thought of: at first there was no -gal'ase, and then after a few minutes, there was some. Where did the -gal'ase come from? Why the delay in its production? Perhaps we can run an experiment that will answer the question. But first, we must speculate on alternatives. You know, often scientists spend more time talking to each other than they do on running experiments. What they do is speculate on alternative possible answers, and then try to devise a crucial experiment that selects between all the alternatives. Happy is the day when one alternative is cleanly indicated! (Oddly: Great is the day when no alternative is indicated because that means there is still much to be understood. A scientist with no problems to investigate is a hungry scientist!)

..... Let's start our own speculations by first looking at another common and perhaps related phenomenon. We know that our blood will clot if it is let out into the air. What is the clot made of? How does a clot form? In a clot, our blood cells are all entrapped in a web of a protein called fibrin. But where did the fibrin come from? Was it an induced gene-product? Not directly! Long before the blood clotted, the fibrin was already in the blood - but in a masked state. Whatever it is that triggers the clotting to occur actually causes a chemical reaction in which the starting compound called fibrinogen is modified and suddenly begins to polymerize into long ropes of fibrin that tangle together to form the web that makes the clot. Thus we see that one of our alternatives is that our -gal'ase was already in the cell but in a form not detectible by our ONPG reaction. If this be the case, then no protein has to be made in the induction process, and the presence of a protein-synthesis inhibitor such as the antibiotic streptomycin would not prevent the induction. This we might call the pre-existing or sequestered precursor model.

..... In another alternative, we might consider that no -gal'ase in any pre-existing form was present and that it had to be newly made. If this be the case, then protein synthesis must occur at some point after the inducer is added, and the presence of streptomycin would prevent the induction. Furthermore, if a protein is to be made, dogma has it that messenger-RNA is also needed to be made. mRNA synthesis can be prevented by the presence of another antibiotic - chloramphenicol. These are the mechanics for studying the genetic induction model.

..... Discussing further, we come to a very important aspect of any scientific experiment: employing proper controls. Of course, a "run" without any antibiotic would be one sort of control - called the 'normal.' Another sort of control might try to take into account the fact that both streptomycin and chloramphenicol are known killers of E. coli. The assumption we had been operating under is that not-quite-dead-yet bacterial cells might be able to do some reactions such as turn on some genes. But our STR and CHL are suspected of being involved with the pathway of transferring information from DNA through mRNA to protein. We need to use another antibiotic as a control. Maybe we could use ampicillin, which interfers with the growth of the bacterial cell envelope.

..... Thus we can penetrate the mere, yet profound, concept of the induction of a particular gene, to ask how induction is generally done in all genes. We need only to make a few parallel, but disturbed, "runs" of the simple, unadulterated induction experiment. And note, too, another beauty of this experiment as it is of the black/white or all-or-none type. The STR will either work or it won't - no partials. At least in theory! Ahem!!!


Well, now! What Does All This Mean?!

.....We have seen how the lac-operon is turned on. Presumably it can be turned off by taking away the lactose or its analog, IPTG. (That's true!) We now should see spread before us a lot of implications.

  1. In our human development, what might the factors be that turn on and off genes?
  2. In cancer, might unintentional analogs play a roll?
  3. Could we break a genetic switch in the 'off' position? Ramifications? Cancer? Aging?
  4. Conversely, could we break a genetic switch in the 'on' position? Ramifications?
  5. Genetic translocations of genes or parts of genes (moving genes to other positions on the chromosome): ramifications?
  6. Insertion of foreign genes (e.g.: viral genes) near switches: ramifications? AIDS?
  7. Genetic therapy could cure some diseases. Any ideas of how this might be possible?

..... Now do you see why Nobel Prizes were given for this rather simple idea of genetic switches? The ramifications pervade all of biology - all of LIFE.



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