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, 2007 In the spirit of the honor code, I pledge that I have neither given nor received help on this exam. 1 Signature

2 Use the following list of reagents to answer questions for this exam. 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) minimal medium without carbon source complex medium Supplementary Reagent shelf for use in questions 5, 6 and 7 canaries, dogs, cats, alligators, mice, rabbits, monkeys, goldfish, maize plants non-specific media for growing bacteria (plates) all restriction enzymes E. chrysamthemi competent E. chrysamthemi E. coli competent E. coli plasmid pql5 which possess a Mini-Tn5 transposon that encodes neo R plasmid pam22 that has one HindIII site, two EcoRV and BglII sites and encodes amp R plasmid pam23 that has one EcoRV site, three HindIII and BglII sites and encodes amp R plasmid pam24 that has one BglII site, two EcoRV and HindIII sites and encodes amp R minimal medium complex medium ampicillin, rifampicin, neomycin DNA polymerase, DNA ligase, ATP, topoisomerase all enzyme buffers pectin, collagen, lactose, cellulose, albumin (a protein) stain for pectin, stain for collagen, stain for β-galactosidase, stain for cellulose, stain for proteins

3 1. (8 points) You have the materials on the list on the separate page available to you to make media for the following experiment. You plan to grow the transducing phage P1 on a bacterium whose genotype is leu + his cyt ara + lac + bio + tet R and with it you infect a bacterium whose genotype is leu his + cyt + ara lac + bio tet S. What medium would you make to select for a bacteria with each of the following genotypes. (Be sure to specify all ingredients in the medium.) tet R his + bio + ara + 2. (11 points) You perform an interrupted mating between two E. coli strains with the following genotypes: F - :amp R, trp --, mal +, thi +, rif S Hfr: amp S, trp +, mal --, thi --, rif R What medium would be used to select transconjugants of each of the following genotypes? Medium 1: amp R Medium 2: trp + Medium 3: mal + The conjugates are plated on these media at the times indicated and the number of bacterial colonies which grow on each are listed below: Time (min) Medium Medium Medium Graph the data on the following page. Label axes and which line corresponds to which growth medium.

4 Draw a diagram below to show the gene order and relative positions in minutes. Transferred last Transferred first If you perform replica plating of colonies from medium 3 at 15 minutes onto medium 1 would most of them grow? YES or NO (circle one) If you perform replica plating of colonies from medium 2 at 30 minutes onto medium 3 would most of them grow? YES or NO (circle one) 3. (7 points) You wish to map the relative positions of three genes using transformation. You extract DNA from a bacterium which is arg -- bio + trp + and use it to transform a bacterium which is arg + bio -- trp --. You select for cells which are bio + or trp + and determine their phenotype for the other 2 genes. You obtain the following results: Gene Selected % selected colonies which are arg + bio + trp + bio trp Make a diagram showing the gene order and relative positions. Why didn t you select for cells which were arg +?

5 4. (14 points) A. Single-stranded RNA viruses which infect eukaryotic cells face the problem that these cells generally only read one protein from a piece of mrna. How has TMV solved this problem? Be specific and mention the steps and enzymes involved. Does the strategy used by TMV resemble that used by any mammalian virus we have studied? YES or NO (circle one) If yes, which virus? B. TYMV (a tymo virus) has used a slightly different strategy to solve the problem. Describe the strategy used by this virus. Again be specific and mention the steps and enzymes involved. Does the strategy used by TYMV resemble that used by any mammalian virus we have studied? YES or NO (circle one) If yes, which virus?

6 5. A. (7 points) A disease is spreading rapidly through Seattle Grace Hospital. Doctors there have been unable to isolate and identify the causative agent. Therefore, they have called Dr. House, and as the microbiologist on his team, he turns to you. Patients are suffering of sore throat, fever, cough, and nausea. The interns pet at the hospital, a cat named Socks, has also become ill. You suspect a bacterial agent is to blame, but need to do some more research before you ll know for sure. Using what is given in the reagent shelf, describe a protocol for identifying and isolating the causative agent. Be specific. B. (3 points) Name three possible types of virulence factors that would allow this pathogen to invade the respiratory tract C. (4 points) Name four built-in resistance mechanisms not involving an immune response that prevent this organism from causing infection or prevent it from spreading to other systems

7 6. (10 points) You are studying the genes involved in the production of soft rot of potatoes by the bacterium Erwinia chrysamthemi (E. ch.). This is the disease which causes the potatoes to get soft and mushy.the bacterium which causes it is a gram negative bacterium closely related to E. coli which grows on nutrient agar. You know that pectin is involved in holding plant cells together and suspect that the enzyme pectinase may be involved as a virulence factor. You select a spontaneous rifampicin-resistant mutant for your studies. This mutant is still virulent. You decide to use transposon mutagenesis to obtain pectinase mutants to determine if this enzyme is involved in producing the disease symptoms. You have available the materials shown on the reagent shelf. Fill in the missing details in the protocol you will use to obtain your mutants. 1. Grow and introduce by (technique). 2. Plate on medium containing and to obtain transposon mutants. 3. Screen for mutants which can no longer make pectinase by (be specific). You carry out this protocol and fail to obtain any pectinase mutants although you are able to obtain non-motile mutants. Suggest a possible reason for your failure. 7. (8 points) After extensive study of E. chrysamthemi you decide that it must indeed have a pectinase and you decide to find the gene by cloning it into E. coli in order to further characterize the enzyme. Since you don t have access to the E. chrysamthemi genome sequence you must clone the gene without using its sequence. From previous studies you know that HindIII cuts the E. chrysamthemi genome roughly every 4000 bases, EcoRV cuts every 2000 bases and BglII cuts every 500 bases. Fill in the blanks in the cloning protocol below. Reference the list of reagents. 1. Isolate DNA from and (process) it with to obtain 2 kilobase fragments of DNA. 2. Introduce the DNA fragment into and (process) using to fix double stranded breaks. 3. Introduce DNA from step 2 by transformation into and grow on. 4. Identify the clone which carries the pectinase by.

8 8. (9 points) Complete the table below based on the figure. Provide the mode of transmission of each of the three curves (A, B, and C), give an example of a disease that is transmitted that way, and state a method for controlling the spread of disease based on the mode of transmission. Number of Cases Reported Jan Feb Mar Apr May Jun July Aug Sept Oct Nov Dec Jan Month Feb Mar Apr May Jun July Aug Sept Oct Nov Dec Mode of Transmission Disease Example One Method for Controlling Spread of Disease A B C A B C

9 9. A. (6 points) Briefly describe the steps that lead to the inflammatory response B. (3 points) List three ways that bacteria can evade or survive phagocytosis (10 points) Please fill out the table below with in formation corresponding to each virulence factor. Toxin Cholera Site of Action (cell type or tissue) Mechanism of Action Pathology (what is the toxic effect) Botulinum