BIO 301, Genetics - Spring 2000

Review 2 — Translation, Mutations, Molecular Genetics

 

Provide thorough, but concise, answers to 7 of the 8 following questions.
There is no time limit to this test, but it should not take you more than 2 hours to complete.
The review is closed book.
Calculators are permitted.
All questions are worth 20 points.
If your handwriting is lousy, please type your answers.
Staple all pages together and do not put your name on any page other than this cover page.
Return completed review to me no later than 10:30am, Wednesday, April 19, 2000.

Name (please print):

Pledge and signature:

 

Answer 7 of the 8 questions

 

  1. In the laboratory, we cloned the gene encoding tetracycline resistance into the plasmid pGEM-T, transformed competent bacteria with this plasmid, and plated the resulting bacteria on agar plates containing either ampicillin or ampicillin and tetracycline. One group observed that the ampicillin plate contained 125 colonies and the ampicillin/tetracycline plate contained 37 colonies. Why were different numbers of colonies found on the two plates? How could the experiment have been altered to determine the transformation efficiency? 

    2.  

  2. Chloramphenicol acetyltransferase (CAT) is an enzyme that catalyzes the transfer of an acetyl group from acetyl-CoA to chloramphenicol. This reaction is very easy to monitor with a simple assay. As a result, plasmids containing the CAT gene can be used in eukaryotic cells to measure the level of expression that occurs. For each of the following 4 plasmids, indicate the relative level of CAT that should be produced if these plasmids were transfected into eukaryotic cells. Justify your answers.



  3. A circular plasmid is digested with a series of restriction enzymes and the resulting DNA fragments are electrophoresed on a 1% agarose gel. The following figure is a picture of the ethidium bromide stained gel. The DNAs in each lane have been treated in the following way: lane1: EcoRI; lane 2: HindIII; lane 3 :PstI; lane 4: BamHI; lane 5: EcoRI + HindIII; lane 6: BamHI + HindIII; lane 7: BamHI + EcoRI; lane 8: PstI + EcoRI; lane 9: PstI + BamHI. The sizes of the DNA fragments in kilobases are indicated on the left. Draw a map of the plasmid, indicating the locations of the restriction enzyme sites.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4. Four Hfr strains are used to map E. coli genes. Through a series of interrupted conjugation experiments, it is observed that these Hfr strains donate the following genes to the recipient cell in the orders shown:

Strain 1 - Q W D M T

Strain 2 - A X P T M

Strain 3 - B N C A X

Strain 4 - B Q W D M

What is the order of these genes on the chromosome of E. coli? What feature of the E. coli chromosome is illustrated by these results? How do Hfr bacterial strains differ from F+ strains?

 

5. For the following lac operon partial diploids, explain which genes will be expressed in the presence and absence of lactose. NOTE: A (-) next to a gene indicates that a mutation exists in that gene that precludes expression.

A. I-O+Z+Y+A+ / I+O+Z+Y+A+

B. IsO+Z-Y+A+ / I-O+Z+Y-A+

C. I+OcZ+Y-A+ / I-O+Z-Y+A+

D. I-OcZ-Y-A+ / I+O+Z+Y+A+


6. It is known that the enzyme wildcatase is abundant in the liver of humans. How would you construct a genomic DNA library and then screen this library to identify the clone containing the wildcatase gene? Provide two different means of generating a specific probe.

 

 

7. Members of your laboratory group have cloned the wildcatase cDNA and inserted it into a prokaryotic expression vector. But your advisor isn’t satisfied. She wants to be able to regulate the expression of wildcatase in transformed bacteria (in other words, be able to turn on and off expression). You are asked to design a new plasmid that will allow regulated expression of wildcatase. Describe this new plasmid. Will it work in any E. coli or will it only function properly in certain strains of E. coli? Why?

 

 

8. Describe the important genetic features of an insertion sequence. Describe the important genetic features of a transposon. In what ways are retroviruses similar to transposable elements? What evidence exists suggesting that integrated retroviruses may move within the human genome?