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MCB 421 HOMEWORK #9 ANSWERS 1. FALL 2008 Given a putA point mutant and a P22 generalized transducing lysate grown on a random pool of Tn10dCam (chloramphenicol resistant) transposon insertions: (Tn10dcam is a derivative of Tn10 that has the cat gene encoding resistance to chloramphenicol.) a. How could you isolate a Tn10dCam near (but not in) the putA- gene? [Hint: this will require several steps.] Draw a diagram showing how you would do the experiment and indicate the medium you would use for each selection or screen. The phenotypes of the WT and putA mutant are shown in the table below, where “+” indicates growth and “-“ indicates no growth. putA putA+ Min + Glucose + NH4 + + Min + Glucose + Proline + - ANSWER: 1) Isolate a Tn10dCam linked to the putA point mutation: Random Pool Tn10 putA+ putA- X Select CamR put+ on Min + Glucose + Cam + Proline medium. putA+ Tn10 2) Make P22 HT lysate of above strain and transduce into CamS putA-. Select on Min + Glucose + Cam + NH4  putA- will grow Replica plate onto Min + Glucose + Cam + Proline  putA- will not grow putA+ Tn10 X 3) Backcross to recipient strain and select as described above. All should be putA-. b. How could you use a Tn10dCam insertion linked to the putA+ gene to isolate point mutations in the putA gene? [Draw a figure indicating the donor and recipient and any selection or screen you would use.] ANSWER: Localized mutagenesis of phage, transduce into CamR, screen putA-. 2. Chromosomal duplications of the nadC gene were constructed in S. typhimurium. The resulting merodiploids have one mutant nadC allele in copy #1 and a different mutant nadC allele in copy #2 with a mini-Mud at the join point, as shown in the figure below. a. How could you select for maintenance of this chromosomal duplication? ANSWER: Maintain the duplication in medium with Amp. b. If one of the copies of nadC had a Tn10dCam insertion mutation (i.e. nadC::Tn10dCam), two types of segregrants can be obtained. Draw a diagram showing the two classes of recombination events that would result in segregration of the chromosomal duplication. [Clearly label the genes on the diagram, indicate the position of any cross-overs required, and show the two types of segregrants obtained.] ANSWER: leu+ nadC proA' Mud(Amp, lac) 'leu nadC ::Tn10 dCam proA+ 'leu nadC ::Tn10 dCam Mud proA' Mud leu+ nadC leu+ nadC proA' proA+ 'leu nadC ::Tn10 dCam proA+ 'leu nadC ::Tn10 dCam proA+ leu+ nadC ::Tn10 dCam proA+ leu+ nadC+ proA+ Questions to Ponder (Don’t hand in answers) Homework assignment #9 is due at the beginning of class on Wednesday, November 5. Please note the “rules” for homework in the course syllabus. You must show your work for credit. If you have any questions please come see us during office hours or discussion section. The following questions will not be graded but will give you additional practice solving problems like those you may encounter on exams. The answers to these additional problems can be found under “sample problems” in the course website. The best way to benefit from the additional problems is to try to solve them on your own, then to consult the answers on the web to check your answers. If you are unsure of any terminology, please see the Microbial Genetics glossary on the course web site. More sample problems and solutions are available on the course web site. 1. Transposon Tn5 has inverted repeats at each end of the transposon. The same inverted repeats are found at the end of every Tn5 insertion. a. Briefly describe the important features of these inverted repeats and their function. ANSWER: The inverted repeats encode transposase and the ends of the inverted repeats provide binding sites for transposase that are essential for transposition. b. Short direct repeats are observed flanking each Tn5 insertion. The sequence of these direct repeats is different for each Tn5 insertion. Briefly explain how these direct repeats are formed and why they are different for each Tn5 insertion. ANSWER: The direct repeats are host DNA from the transposition target site that are generated by staggered cuts which are subsequently filled in by DNA replication. 2. Transposon Tn10 encodes resistance to tetracycline. This is a complete transposon that encodes its own transposase. Briefly describe two ways that you could deliver Tn10 to recipient cells that would select for transposition events. ANSWER: There are many ways this question could be answered. Two are listed below. (1) Bring the transposon into a recipient cell on a defective phage that cannot replicate in the host or lysogenize the host — select for TetR. (2) Bring the transposon into a recipient cell on a suicide plasmid that cannot replicate in the host — select for TetR. 3. The araBAD operon is required for growth on arabinose as a sole carbon source. A random collection of Tn10 insertions was isolated in an araBAD- mutant background. Given a lysate of phage P1 grown on this random pool of Tn10 insertions and an ara+ recipient, how could you isolate a Tn10 insertion linked to the ara- mutation? Draw a figure showing the crosses you would do and indicate how you would select or screen for the desired mutants. ANSWER: A generalized transducing lysate with random Tn10 insertions will include insertions at many chromosomal sites, including insertions adjacent to the ara operon ( ara- Tn10 ) Transduce selecting for Tn 10 encoded Tet R ( ara- Tn10 ) Donor ara- ara Recipient ara+ Screen for Ara - on medium with arabinose as the sole carbon source. ara- Tn10 The results should be confirmed by backcrossing the Ara Tet transductants against the ara- recipient. + R 4). Given a Tn10 insertion that is cotransducible with the wild-type araBAD. a. How could you take advantage of this insertion to isolate point mutations in araBAD? Draw a figure indicating the donor and recipient strains and any selection or screen you would use. ANSWER: ara+ Tn10 Grow transducing phage lysate P1 ( ara+ Tn10 ) Mutagenize in vitro with hydroxylamine P1 ( arax Tn10 ) Transduce selecting TetR ( arax ara+ Tn10 ) Screen for Ara(Failure to grow on arabinose as sole carbon source) arax Tn10 Grow transducing phage and backcross vs ara + parent to confirm that mutation is in ara c. How would you demonstrate the mutants are in the araBAD operon? Briefly describe the donor and recipient strains and any selection or screen you would use. ANSWER: Backcross as described in the final arrow above and shown below. ( ara- Tn10 ) Backcross ara + parent selecting for Tn10 encoded TetR ( ara- Tn10 ) Donor ara- ara Recipient ara+ Screen for Ara- on medium with arabinose as the sole carbon source. ara- Tn10 5). There are two mechanisms for transposition used by bacterial transposable elements: replicative (Tn3) and non-replicative (Tn5 and Tn10). Compare and contrast the two mechanisms with respect to: a. Host DNA sequences adjacent to the ends of the element: b. Formation of co-integrants: c. Resolvase enzyme: d. Requirement for DNA synthesis: e. Structure of DNA in intermediates of the reaction: