1. What events take place when a gene is turned on? What is the ultimate result?

2. Which of the following would you expect might influence whether a gene is turned on or not?

a. Environment of cell
b. Differentiation of cell
c. Age of organism
d. Time of day

3. Connect the regulated genes and the conditions below and state whether the genes would be expected to be ON or OFF.

Regulated Genes	Condition of E. coli
1. Genes encoding lactose metabolism 2. Genes encoding DNA repair enzymes	A. In an intestine of a person who ate a cream-filled doughnut B. In an intestine of a person who drank a large glass of milk C. On a dunghill exposed to bright tropical sunlight

4. The effect of having three genes encoding enzymes involved in lactose metabolism in a single operon is:

a. The three genes are transcribed together by a single RNA polymerase enzyme
b. The three RNA transcripts are translated together by a single ribosome
c. The three genes are replicated together by a single DNA polymerase enzyme

5. Take a look at the figure in the handout concerning the lac operon. Predict whether E. coli will or will not make lactose-utilizing enzymes if it is mutated in the following way and exposed to the following conditions:

6. The activator protein in the figure of Section 11.8 acts on RNA polymerase in a qualitatively different way from the repressor in the figure of Section 11.2. How? What would be the effect of a severe mutation in the gene that encodes the activator protein?

7. You wish to transfer the enzymatic pathway leading to the chemical producing the taste of strawberry into peanuts because,... well, you can guess. The sequences of the genes encoding the enzymes are known, and you carefully isolate the genes and clone them into a plasmid in preparation to sending them into a peanut plant. Perfectionist that you are, you clone just the open-reading frames, starting with the ATG start codon and ending with a stop codon, no more, no less. The DNA is inserted at random into the DNA of a peanut cell and a plant is regenerated. The day comes and you anxiously grind up the nuts of the recombinant plant and get: Peanut Butter and... nothing. No strawberry. Not even grape. Why? What went wrong?

8. Similar to our cloning attempts in lab, you are trying to clone the smallest possible DNA fragment that contains the gfp gene and all sequences necessary for its expression. We know that the start codon for the gfp gene begins at position 2289 in pFIU1 and the stop codon is at position 3003. Explain the following cloning results.

9. Two different forms of the gfp gene are cloned into a plasmid. The gfpG gene encodes the native form of GFP, which fluoresces green under UV light. The start codon for this gene begins at position 2100 in the plasmid and the stop codon begins at position 3500. The gfpB gene encodes a mutant form of GFP that fluoresces blue under UV light. The start codon for this gene begins at position 2000 in the plasmid and the stop codon begins at position 500.

a. The plasmid is introduced into E. coli, but the colonies do not fluoresce under UV light. What could be wrong?

b. A DNA sequence containing a known bacterial promoter is cloned into the plasmid as a BamHI to BamHI fragment into a BamHI site at position 2050. The new plasmid is introduced into E. coli. Three colony types are found: some that do not fluoresce under UV light, some that fluoresce green under UV light, and some that fluoresce blue under UV light. Explain each of these colony types.

10. You are a high-scoring venture capitalist. You're sitting in your office musing on convertible debentures when in comes a guy holding a test tube who claims he has cloned a gene which, when injected into a developing fetus, will raise the kid's future IQ by 50 points. He plans on selling it to any prospective parent at 100K a pop (or 100K a mom, as the case may be). Never mind whether he looks like Albert Einstein with three Nobel prizes or Emmett Kelley with a two-day growth, what do you need to find out before you but down the money he wants? (Consider that we are nearing the end of this course, and it would be personally pleasing to me if at least part of your answer related to something you may have learned here.)

11. At the 2nd UR SYMPOSIUM ON TRANSGENIC TOBACCO, scheduled in LESS THAN TWO WEEKS, you're intrigued by a poster by someone who plans to make a tobacco plant that is a rich source of Vitamin E. Actually, tobacco already has some Vitamin E (beta-carotene), related to the carotenoids that give tobacco its occasional orange-yellow tinge (carotenoids, carrots,... get it?). You happen to be well versed in the biosynthetic pathway that produces carotenoids and recall that it contains multiple steps, each catalyzed by a separate enzyme, each encoded by a separate gene. You are thus astonished to learn upon reading the poster that the plan is to alter a gene in tobacco whose product does not participate in the biosynthetic pathway at all! Furthermore, she already put the plan into action last weekend, and it worked! How do you explain the very yellow plant she has growing next to her poster?

12. Nothing to do with cloning, just a curiosity. I heard a commercial on the radio the other day. It began with a deep voice saying (Beethoven's Ninth in the background), "Scientific research has shown that the average person blinks 20,000 times a day." I forget what they were hawking, but the idea was that if you blink, the opportunity would pass you by. Do you believe this research? Why or why not? How does that make you feel about the rest of their claims, whatever they may be?

13. No question. Use the time you would have spent answering #13 to dig deep into your project. Go out and find a sequence from GenBank. Find an article that tells you what your pathway is about. Come and see one of us to smooth over a problem.