If one were to stretch out the chromosome as an untangled circle, one could imagine about 7,000 genes spread out along its length.     (There are about 1000-times as many genes packed into each one of almost all of your complicated cells!     Can you name the exception?) Pictured here is a diagram of E. coli's chromosome, and on it are depicted only a very few of its scattered genes.

        Among the 'background' of incidental genes, we have chosen the R-genes and LAC both because they represent extremes and because we know a great deal about them both. R-genes code for the making of r-RNA, a necessary function that is always 'on' in a growing cell.     But LAC is a gene that is only turned 'on' when the cell encounters the sugar lactose in its environment; otherwise, the gene is turned 'off.' .. In this way, the highly generalized scheme of relaying the genetic code from DNA to protein is regulated.

        Let us first take a look at the seven nearly identical R-genes as a group.     These seven short stretches of DNA are responsible for the production of nearly 85% of all the RNA produced in bacterial cells.     Indeed this quantity of ribosomal-RNA (rRNA) constitutes up to 30% of the non-water stuff in the cell!     These seven genes are really very 'hot', as some molecular biologists like to say. (rRNA + rProteins -> ribosomes, which are the workbenches upon which all the different proteins are synthesized.)

        The section of a gene that is called the 'promoter', P, might be thought of as hailing RNA-polymerase (alias:     transcriptase) to begin the process of reading the gene and converting (transcribing) code from that of a DNA sequence into one of an RNA sequence in the form of what we call messenger-RNA (mRNA).     While normal genes will have only one starting point for transcriptase, each of the seven r-genes has three.     No self-respecting transcriptase could bypass that siren call!     Thus it is a rare moment indeed that there is not a line of transcriptases waiting to begin decoding an R-gene.     This hyperactivity is, of course, needed to provide all the rRNA molecules that are needed.     Oh, one more point:     the R-gene is unusual in another way - it doesn't lead to the making of m-RNA.     Instead, it codes [mainly] for RNA's that become integral structural components of ribosomes - the workbenches upon which proteins are made from codes derived from the m-RNA products of other genes (translation).     Even the remainder of the gene is decoded into amino acid transfer RNA's (tRNA) which grab specific amino acids and help the ribosome/mRNA complex line them up properly to be linked into forming a protein.

        And not to overlook that group of molecular biologists who ominously call themselves the 'Terminators,' because they have interest in what kind of code ends the genes, we see that r-genes end with some sort of 'stop' signal.     Presumably, the transcriptase reaches the 'stop' and is de-railed and falls off the DNA so that it does not continue to read onwards into the next gene.     It is then available to go back and start again.

        Now let us turn to a rather normal sort of gene - one that is needed by the cell to feed on an energy-packed sugar called lactose (milk sugar).

The 'lac'-gene can be turned 'on' (induced) leading to the production of two enzyme tools - a membrane component called permease that grabs passing molecules of lactose and shoves them inside the cell, where the other tool, an enzyme called ß-galactosidase, snips the lactose in two yielding glucose and galactose.     Another gene elsewhere on the chromosome codes for the enzyme galactose/glucose epimerase that converts the galactose into glucose, and then the glucoses move on to be fodder for glycolysis.

        Now let's see how all this is accomplished by a genetic system.     We need to account for not only the production of permease and ß-galactosidase, but also for the on/off switch of induction and repression. In the following diagram of the gene, we see a few familiar friends: we see P, some loci (called 'cistrons') that result in products, and a 'stop.'     But there are also a couple new-comers - a detached sequence of DNA that is called "R" (repressor locus), and another sequence called "O" (operator locus).     If the lac-gene did not have the R and O, then it would act somewhat like a cooled-down r-gene:     it would be 'on' all the time - somewhat slowly since it has only one "P".     But that would be a waste of time and energy for the cell if it didn't happen to have any lactose around to chew on - and that is most of the time as lactose is rather uncommon in the earth's environment.

    So what's this R and O all about?     Well, R is a rather normal type of DNA sequence as it is transcribed into mRNA, which, in turn, is translated into a protein.     This particular protein is called 'repressor.'     Repressor has a great fondness for binding to a very specific DNA sequence - just the one found in and only in "O".     Thus repressor wraps around O and overlaps into adjacent sequences, one of which is the important P region.     If P is not exposed to attract transcriptase, no reading of the gene occurs:     de facto making the gene 'off.'

    But now the gene is in a lurch: it is uselessly 'off' all the time!     How does it ever get turned on?     It turns out that small amounts of lactose itself are capable of inactivating repressor protein, making it unable to bind to O.     Such small debilitating amounts might leak into the cell from the surrounding environment.     Once the gene is de-repressed, permease is made and inserted in the outer surface of the cell where it increases the influx of lactose 10,000-fold.     And this lactose is attacked by ß-galactosidase and so on.     Since repression/derepression of the operator O is the switch for all the sequence that lies downstream of it, the word 'operon' was invented to mean all the contiguous sequence from O to stop.

    Jacob and Monod shared a Nobel Prize for describing the lac-operon.     If we employ for our conceptual purposes a popular video game figure as representing a protein that ordinarily would tenaciously bind with something - an O if it is a repressor or a substrate if it is an enzyme -, we can see that there are two ways that lactose might inactivate it.     The most straightforward way would be for lactose to plug up the repressor's binding site (as shown with the lactose stuck in the creature's teeth).     The repressor, with its mouth full of lactose, could no longer bind to and turn off O, and the gene would remain 'on'.     This would be called steric inhibition.     Another possible way that lactose might inactivate the repressor protein is by lactose's binding to some other second site on the repressor that handicaps the repressor (here represented by lactose's acting as a blinder keeping the repressor from finding O).     The formal name for this is 'allosteric inhibition.'

    How about a check on our comprehension of what's going on here?     One of the best ways that scientists check themselves in these matters is by tinkering with their system - in this case they would think about making some mutants.     Consider the obvious results of an experiment on a mutant that had a damaged Z-sequence:     under no circumstances could this mutant use lactose because there was no possibility of its ever making any ß-galactosidase.     In the lab, we are going to have a look at other types of mutants.     Just to get yourself into the proper frame of mind, see if you can figure out what happens in each of the following mutations:     R is damaged; O is damaged; P is damaged; Y is damaged.

.....     Now, if you think this is the whole story, it is not!     As a preview of good things to come in later courses, just consider these:

1. Why is the lac-operon 'off' when both glucose and lactose are plentifully present together in the environment?!
2. Can "Z" be mutated without causing damage to the cistron?
3. How can it be that the ß-galactosidases isolated from different strains of E. coli possess slightly different molecular weights and net charge?
4. How can it be that the DNA that makes up the lac-operon has a much higher proportion of G and C in it that does the remainder of the E. coli DNA?
5. Why is the lac-operon normally where it is in the E. coli chromosome and not in some other location in the 'circle?'
6. Why is lacA in the lac-operon? What's its function in consuming milk sugar?
7. How does ß-galactosidase in Escherichia coli differ from lactase in Homo sapiens?
8. What evidence is there that human lactase is inducible/repressible?

    It may have occurred to you that measuring the breakdown of lactose (a slightly sweet sugar) to galactose and glucose (semi-sweet sugars) is a little risky to do in labs filled with glassware that may have once contained solutions of heavy metals or other toxins. Thus chemists came to the rescue and devised a chromogenic (color producing) compound that produce intense color if lactase were to split that molecule. This chemical ANALOG of lactose is ONPG, ortho-nitro-phenyl-galactoside. Here is the reaction as performed by lactase:

    After you have read the above pages, read your textbook and maybe some other books (e.g.:     Campbell-Reece-Mitchell 5th ed: 337-341).     Get various renditions.     Make sure you really understand this material.     Scientists like Jacob, Monod, Brenner, Cuzin and others didn't get the Nobel Prize for nothing!     Get to know your scientists, too.     And where they worked - Institut Pasteur, Cambridge.     What else have these scientists done?     What indication other than the Nobel is there of the significance of these people's work?     (Hint: Citation Index.)

Lac-Operon Kinetics
Experimental Set-Ups

IMPORTANT: Before you begin pushing test-tubes, make sure that your instructor fully understands what's going on. ..Ask:     "What's the main question in 25 words or less?"

Notes: Before induction, the bacteria have been growing for a number of hours in a broth containing NO sugars.     At zero time, the additives are mixed into the culture and ONPG tests are made periodically.
    IPTG is a non-degradable inducer and is added over a thousand-fold range of concentrations to investigate its potency as an inducer.

To make the IPTG:

• Dissolve 1.08 gm IPTG in 60 ml water. 1 ml(A) + students' 24 ml flask → 3 mM (Distribute 1 ml samples to a number of microfuge tubes; freeze.)
• Take 30 ml of (A) and add to 270 ml water. 1 ml(B) + students' 24 ml flask → 0.3 mM (Distribute 1 ml samples to a number of microfuge tubes; freeze.)
• Take 6 ml of (B) and add to 54 ml water. 1 ml(C) + students' 24 ml flask → 0.03 mM (Distribute 1 ml samples to a number of microfuge tubes; freeze.)
• Take 6 ml of (C) and add to 54 ml water. 1 ml(D) + students' 24 ml flask → 0.003 mM (Distribute 1 ml samples to a number of microfuge tubes; freeze.)
• For LAC: 3.2 gm lactose + 20 ml water; 1 ml samples into microfuge tubes; freeze.
• For GLC: 1.6 gm glucose + 20 ml water; 1 ml samples into microfuge tubes; freeze.
• For STR: 250 mg dihydrostreptomycin + 20 ml water; 1 ml samples into microfuge tubes; freeze.
• For SDS: 50 mg SDS + 20 ml water; 1 ml samples into microfuge tubes; freeze.
• For EtOH: 5 ml 95% EtOH + 15 ml water; 1 ml samples into microfuge tubes; oops: can't freeze!
• For ONPG: per section: ..400 mg ONPG powder in microfuge tube; freeze. ..Start making 1 hour in advance of section's use: dump contents into 50 ml Z-buffer; magnetically stir for 30 minutes or until dissolved.

1. is IPTG a better inducer than is the natural lactose?
2. Does more inducer increase the 'on-ness?'
3. Which of the following prevent induction: the common detergent SDS, heat, STR and/or GLC?
   1. Get your group's assignment and have these ready:
      • You have lined up 17 little test tubes which have been labelled from "-1" to "15."
        
      • Group 3.HOT: label yours: -1 0 3 6 9 12 15 ...
        
      • Add 1 ml of Z-buffer to each tube.
        
      • Add 2 drops of SDS to each tube.
        
      • Add 2 drops of chloroform to each tube.
        
      • Get your 'additives:' IPGT, LAC, SDS, STR, or whatever you have been assigned.
        
      • Get your 24 ml of growing culture into your flask and have the person with the warmest hand continuously (a MUST) swirl it in the hollow of the hand to 'mother' (incubate) it at the right temperature.
   2. At time = -1 minute, take 1 ml from the flask and put it into tube "-1"; vortex the tube for 10 seconds; put back into rack.     KEEP the flask forever     SWIRLING! KEEP the flask forever WARM!
   3. At time = "0", simultaneously (a) take 1 ml from the flask and put it into tube "0"; vortex 10 seconds; put back into rack; and (b) add your additives.     KEEP SWIRLING! KEEP WARM!
      Group 3.HOT: Immediately put your flask in the 43°C water bath and continue to swirl it in there.
   4. Every minute that goes by take 1 ml from the flask and put it into the next tube; vortex 10 seconds; put back into rack.     SWIRL!     WARM! Group 3.HOT: You take a sample every 3 minutes instead of every minute.
   5. When you have finally taken the 15-th minute sample, send someone off to clean out the flask.     Groups that leave dirty stuff behind will be dungeoned to be later thrown to very hungry lions!) (Group 3.HOT:     Work quietly as you continue to the 45-th minute sample.))
   6. Add 4 drops of ONPG from a dropper bottle into each tube. ..Do this quickly but not sloppily.
   7. Put away all your bottles; clean out plastic droppers:     IN THE BASEMENT THERE ARE LIONS WHICH ARE HUNGRY FOR STUDENTS WHO FAIL TO CLEAN UP AFTER THEMSELVES.
   8. Meanwhile your tubes might be slowly getting yellow. Keep an eye especially on those from 10 to 15 minutes.     Send a spy out to see how other groups' experiments are progressing.     Keep your eye on the CONTROL group's tubes. How do yours compare?     Once your later tubes get fairly yellow - like those of the control group, ascertain the lowest numbered tube to show yellow.     How does it compare with the group with the CONTROL?     What does this mean?
   9. Record your findings with the instructor, who will go over the lab's total results in a few minutes.
   10. Meanwhile, clean out your tubes and invert them in your rack. (Very, VERY hungry lions!)
   11. Don't escape the lab yet.     The instructor will go over the class's data.     Be prepared to tell the significance of your group's findings within the context of the class's total findings.
   12. Get your group together and argue some of the questions asked earlier in the more theoretical part of this unit - considering such things as 'sequestered model', 'hot' promotored genes, and how pieces of the lac-operon might be used in genetic engineering.

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