HOUR EXAM II BIOLOGY 422 FALL, In the spirit of the honor code, I pledge that I have neither given nor received help on this exam.

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1 Name First Last PID Number - (Please Print) HOUR EXAM II BIOLOGY 422 FALL, 2006 In the spirit of the honor code, I pledge that I have neither given nor received help on this exam. 1 Signature

2 The following is a reagent shelf which you have available for all of the experiments on this exam. Note that for some questions you may have additional reagents, bacteria, or animals available. You may not use reagents which are not given to you. amino acids vitamins sugars antibiotics threonine (thr) biotin (bio) glucose (glu) streptomycin (sm) leucine (leu) thiamine (thi) lactose (lac) rifampicin (rif) histidine (his) maltose (mal) ampicillin (amp) tryptophan (trp) nucleic acid bases arabinose (ara) tetracycline (tet) arginine (arg) adenine (ade) neomycin (neo) cytosine (cyt) carbenicillin (carb) minimal medium without a carbon source complex medium 1. A. (6 points) Using materials from the reagent shelf above you wish to prepare bacterial growth media for an experiment. You wish to grow the generalized transducing phage P1 on a bacterium whose genotype is thr + leu - bio + ade + cyt - lac + mal - ara - rif R and use it to infect a bacterium whose genotype is thr - leu + bio - ade + cyt - lac - mal + ara - rif S What medium would you use to select bacteria of each of the following genotypes? (Remember to specify all of the ingredients of the medium). thr + lac + bio + rif R B. You wish to move a plasmid which carries the gene for amp R from a bacterium which is leu - tet R rif S to a bacterium which is leu + tet S rif S. You perform the conjugation. What should you put in the medium to select for the desired transconjugates?

3 2. (14 points) You perform an interrupted mating between two E. coli strains with the following genotypes: F : tet R, leu, trp, thi Hfr: tet S, leu +, trp +, thi + What medium would you use to select transconjugants of each of the following genotypes? Medium 1, selects for leu + Medium 2, selects for trp + Medium 3, selects for thi + The conjugates are plated on these media at the times indicated. The number of bacteria which grow on each medium are counted are as follows: Time (minutes) Medium Medium Medium Graph these data on the following page. Label the axes and indicate which line represents which medium. Use the graph to draw a diagram below to show the gene order and relative positions in minutes. Transferred last Transferred first If you were to replica plate colonies from medium 2 at 15 minutes onto medium 3 would most of them grow? YES or NO (circle one) If you were to replica plate colonies from medium 3 at 20 minutes onto medium 1 would most of them grow? YES or NO (circle one)

4 3. (6 points) You wish to map the relative positions of three genes using transformation. You extract DNA from a bacterium which is leu + bio + met - and use it to transform a bacterium which is leu - bio - met +. You select for cells which are bio + or leu + and determine their phenotype for the other 2 genes. You obtain the following results: Gene Selected % selected colonies which are leu + bio + met + leu bio Make a diagram showing the gene order and relative positions. 4. (6 points) Below are process characteristic of various methods of genetic exchange in bacteria. Identify which process(es) belong with which characteristics. A-Transduction B- Transformation C-Conjugation D-None Some items will require more than one answer. Requires phage Requires competent cells Transfer of DNA Fully diploid end product Requires a pilus Resistance transfer factors are transferred in this manner Requires cell contact Requires two different species

5 Supplementary Reagent shelf for use in questions 5, 6 and 7 Competent E. coli, competent A.westus E. coli carrying psoup, E. coli tra +, pir + carrying pcar, wild type E. coli McConkey s selective medium for E. coli, Restriction enzymes: EcoRI, XbaI, BamH1, etc. Taq DNA polymerase, DNA ligase, ATP, kits to label DNA pink and to label proteins red Photographic paper, nitrocellulose filter paper, blotting paper, India ink Plasmid ps0up DNA shown below, plasmid pcar DNA Fluorescent light, Gfp, Methylene gunkate, DNA from a clone carrying the gene homologous to degunk Canaries, dogs, cats, alligators, mice, rabbits HindIII Cm R EcoRi pcar Tn5 neo R orit Ori replication pir dependent

6 5. (12 points) You are the head of a research lab investigating a newly discovered bacterium named A. newbug. This bacterium is of particular interest because it is able to degrade the environmental pollutant methylene gunkate (which is colorless) into a harmless product, gunkium hydride (which is orange), using a novel enzyme, Degunk. In your search for the degunk gene, you believe you have found a homologous gene on a related bacterium for which the sequence is known. You ask one of your students to design a protocol for cloning the degunk gene from A. newbug. a. After growing a large culture of A. newbug and purifying DNA from it, your student decides that the next step is to digest the DNA with a restriction enzyme. He looks through the sequence of the homologous gene in the related bacterium and finds that it contains a restriction site for BamH1. Pleased with this discovery, he then digests the A. newbug DNA with BamH1. Was this a good choice for a restriction enzyme? Why or why not? b. It turns out that a postdoc in the lab has just finished a digest, using the restriction enzyme XbaI, of both A. newbug DNA and the ps0up vector, and gives an aliquot of each digest to your student. Your student then mixes the digested DNA and the digested vector and incubates them at 37 C. Analysis indicates that the vector did not incorporate the DNA. Did the student do something wrong? c. The postdoc leads the student through the next few steps, resulting in a culture of E. coli containing the vector with pieces of A. newbug DNA incorporated. The student grows the bacteria on nutrient agar plates, assuming that all the bacteria grown will be transformants. Is this true? What would be a better medium for the student to grow the bacteria on in order to select for transformants? d. Do all the transformants grown above contain the degunk gene? If not, how could the student screen for those colonies expressing the degunk gene?

7 e. It turns out that the actual end product of degradation of methylene gunkate is gunkium carboxylate, which is colorless. Describe briefly how the student could screen for colonies expressing the degunk gene using the homologous (known) gene from the related species. 6. (8 points) This past week Seattle Grace hospital experienced an outbreak of what appeared to be a new disease. Patients presented with stomach cramps, mild diarrhea and intestinal bleeding. All of the patients had visited a local restaurant and ordered the prime rib. A dog of one of the patients had been fed leftovers and developed diarrhea and intestinal bleeding and a loss of appetite. A bacterial agent was suspected and as the leading microbiologist in the country you're called in to identify not only the bacteria responsible but if the disease is infectious. The bad news is that the restaurant sold out of that dish, so where do you start, how do you isolate and identify the cause of this disease?

8 7. A(7points) After identifying the bacterium responsible for causing the outbreak in Seattle as A. westus you decide to use transposon mutagenesis to determine the genes involved in virulence. During the course of treating patients you observe that A. westus is apparently resistant to carbenicillin. It's been years since you've done transposon mutagenesis and when you open your old micro notebook the pages have faded and you can only make out parts of it. Fill in the blanks using the materials provided in the reagent bank. 1. Grow and. 2. Mix the two cultures and allow them to. 3. Select the resulting culture you desire by plating on medium containing 4. Test the surviving bacteria to determine which ones have mutations in genes required for virulence by This procedure selects/ screens (circle one) for bacteria that carry the transposon and selects/ screens (circle one) for bacteria carrying mutations in genes involved in virulence. B. (3 points) In the box below draw the general structure of a wild type (not geneticallyengineered) transposon: 8. (9 points) On the previous exam you were asked how influenza and polio viruses solve the problem of making multiple virus proteins in eukaryotic cells which generally only read one protein from a piece of messenger RNA. Explain how the plant viruses belonging to the brome mosaic group solve this problem. Does brome mosaic virus make stochiometric amounts (equal molar) of each protein? YES NO circle one What is the principle advantage to the virus of this strategy (briefly)?

9 9. (9 points) Which of the following are required to respond by producing antibody to an antigen which the host has not encountered before? (Circle those that are required). Macrophages or dendrites red blood cells T killer cells T helper cells B cells Plasma cells Memory cells Which of the following is likely to be most useful in killing virus infected cells? (circle one) Macrophages or dendrites red blood cells T killer cells T helper cells B cells Plasma cells Memory cells Draw the host response to exposure to an antigen on the following graph. 1 st injection 2 nd injection Time What type of antibody is made during the primary immune response? Which cells make it? What type of antibody is made during the secondary immune response? Which cells make it?

10 10. A (2 points.) Seattle Grace hospital has a new chief surgeon, Dr. Germaphobe. He carries around a spray bottle of the potent antimicrobial agent used to disinfect ER equipment and surfaces, and sprays everything before he touches it, taking special care to be extra clean in the pediatrics unit. What s wrong with this picture? B. (2 points) Based on what you know about specialized cells in the immune system, why are bacteria with capsules such a problem when it comes to pathogenesis? C. (4 points) Fill in the following table with respect to infectious diseases you might expect to encounter frequently in a small remote village in the tropics. The two diseases must have different modes of transmission. Disease Mode of transmission Why is it likely to be a problem? What could you do to reduce the disease incidence? 11A. (8 points) A pharmaceutical company is investigating drugs to treat various bacterial diseases. Fill in the grid below with yes or no to indicate whether the drug described might be efficacious against the disease listed. Action of drug Inhibit release of acetylcholine from nerve cells Increase release of acetylcholine from nerve cells Inhibit the activity of adenylate cyclase Increase the activity of adenylate cyclase Inhibit ribosylation of EF-2 Increase ribosylation of EF-2 Inhibit ADP-ribosylating enzymes Diphtheri a Cholera Botulism Tetanus 11B. (5 points) What organs in the body are most affected by diphtheria toxin? Why? Why doesn t diphtheria toxin kill C. diphtheria?

HOUR EXAM II BIOLOGY 422 FALL, In the spirit of the honor code, I pledge that I have neither given nor received help on this exam.

Name First Last (Please Print) PID Number - HOUR EXAM II BIOLOGY 422 FALL, 2010 In the spirit of the honor code, I pledge that I have neither given nor received help on this exam. 1 Signature 2 3 4 5 6