Q1. An E. coli strain that is rifampicin resistant (rif r ) and auxotrophic for arginine, histidine, proline, and tryptophan biosynthesis (arg- his- pro- trp-) is mixed with an Hfr donor strain that is wild-type for al of these loci (arg+ his+ pro+ trp+ rif s ). Transconjugants that recombined the arg, his, and trp genes were individualy selected and then screned for inheritance of the other loci. The results of this experiment are shown in the table below. % Recombinant for Each Alele Selected marker arg his pro rif trp arg 10 28 31 89 12 his 7 10 43 6 1 trp 29 33 30 31 10 Based on these results, draw a circular map of the E. coli chromosome showing the relative positions of the arg, his, pro, trp and rif loci, as wel as the position and orientation of the Hfr origin of transfer (oriT). (Position the oriT at "12:0" on your map.) Q2. A strain of E. coli contains a Tn10 insertion within the mogA gene. This transposon caries a gene that confers resistance to tetracycline (Tet r ). Two other genes, yaH and htgA, are located near mogA and encode protein products that are easily selected and/or identified by biochemical asay. The E. coli strain carying the mogA:Tn10 insertion is yaH+ and htgA-. A P1 transducing lysate is made from this strain, then used to infect another strain of E. coli that is mogA+, htgA+, and yaH-. Transductant cels that were recombinant for a specific donated locus were then screned for the cotransduction of a second locus. The results of this experiment are shown in the table below. Selected phenotype Recombinants screned Number of recombinants obtained htgA- 75 Tet r htgA+ 25 htgA- 96 YaaH+ htgA+ 4 yaH+ 15 Tet r yaH- 85 Based on these results, draw a map showing the relative positions of the thre genes. Indicate the cotansduction frequency for each pair of genes cotransduced. Q3. The figure below represents the chromosome map of an F- E. coli strain carying mutations in genes required for lactose catabolism (lac), tryptophan biosynthesis (trp), tyrosine biosynthesis (tyr), and isoleucine biosynthesis (ilv). The strain is also resistant to streptomycin due to a mutation in the rpsL gene. Below this are maps of several different Hfr strains, each showing the location and orientation of its origin of transfer. (Remember that the E. coli map is actualy circular, although it's represented in linear fashion here.) Each of the Hfr strains is wild- type for al of the loci indicated on the map (i.e., lac+, trp+, tyr+, Sr s , and ilv+). Each Hfr strain was individualy mated with the F- recipient strain and cels were plated on minimal glucose (MinGlu) agar containing tyrosine, isoleucine, and streptomycin. A. What donor marker or markers are being selected in this experiment? B. Rank the five Hfr donor strains to indicate the relative numbers of transconjugants, from most to fewest, expected under this selection. C. What medium would you use to select for transconjugants that had received both the lac and tyr markers from the donor cel? Which of the Hfr strains would be expected to give the highest number of transconjugants under this selection? Q4. A P1 transducing lysate was grown on a wild-type (prototropihc) E. coli strain and used to infect a strain of E. coli that is auxotrophic for synthesis of the amino acids glutamate, proline, tyrosine, and valine (glu-, pro-, tyr-, val-). Samples of the transduced culture were spread on minimal glucose agar containing various amino acid suplements; the numbers of recombinant transductants from each of these selections is shown in the table below. A. For each plate (1-9), list the aleles that are being selected. B. Calculate the percent cotransduction frequency for each of the folowing pairs of aleles: glu + pro glu + val pro + tyr pro + val tyr + val lac trp tyr rpsL ilv Hfr 1 Hfr 2 Hfr 3 Hfr 4 Hfr 5 0 100 80 70 60 50 90 40 30 20 10 C. Draw a map showing the relative position of the four genes based on their cotransduction. Indicate on your map the cotransduction frequencies for each adjacent pair of genes. D. Based on your map, what is the expected cotransduction frequency for the glu and tyr genes? Amino acid suplements present: Plate #: Glu Pro Tyr Val Number of recombinants recovered 1 + + + - 1,00 2 + + - + 1,00 3 + - + + 1,00 4 - + + + 1,00 5 + - + - 0 6 + - - + 50 7 + + - - 0 8 - - + + 50 9 - + + - 10 For the next question, use this list that describes the characteristics of the comon amino acid side chains. Hydrophobic (nonpolar): Gly, Ala, Val, Leu, Ile, Met, Phe, Trp, Cys, Pro Polar: Ser, Thr, Asn, Gln, Tyr Charged (Basic/Acidic): Lys, Arg, His / Glu, Asp Q5. The signal sequence of the periplasmic protein ?-lactamase (the enzyme responsible for resistance to Ampicilin) is shown below: Met1 Ser2 Ile3 Gln4 His5 Phe6 Arg7 Val8 Ala9 Leu10 Ile1 Pro12 Phe13 Phe14 Ala15 Ala16 Phe17 Cys18 Leu19 Pro20 Val21 Phe2 Ala23 Part A: If this sequence was part of a translational fusion to ?-galactosidase (LacZ), please indicate at least two single base changes that might ocur to make a mutant that would lead to a significant defect in the targeting of the bla-lacZ fusion to the Sec aparatus. (Use the codon chart in your textbok to identify apropriate changes.) Do not use nonsense mutations. Indicate the original and the final amino acids, the codon number, the starting sequence of the codon, and the mutated sequence. (Formulate your answer in the folowing fashion: Ser2Arg (AGU to AGA), meaning that the 2 nd codon in the signal sequence, coding for Ser was changed to one coding for Arg.) Part B: You make a version of the bla gene that has one of your S mutations included in it. The strain of E. coli containing this bla S mutation is no longer resistant to Ampicilin, while a bla + strain grows wel on nutrient agar + 20 µg/ml Ampicilin at 37°C. How would you isolate a mutation that would supres the targeting defect of the S*Bla mutant, without selecting for a revertant of the original S* mutation? Include what gene might contain the mutation and what your selection or scren would be. Q6. Many E. coli strains resistant to streptomycin (Str R ) contain a mutation in the gene rpsL, which codes for a structural protein of the ribosome. Strains merodiploid for rpsL, having one copy of the Str R alele and a second with the Str S alele are sensitive to streptomycin. Describe how you would isolate 50 independent Str R mutants, starting with the Str S merodiploid (rpsL + /rpsL-str R ). Is your strategy a selection or a scren? Describe two clases of mutations that you would expect to come out of your Str R isolation protocol, and what the distribution of each might be among your 50 mutants. Q7. You discover a new drug that targets the bacterial RNA polymerase, and cal it Bugicilin. Resistance to Bugicilin (Bug R ) ocurs via misense mutations in the gene for the ?? subunit of RNA pol (rpoC). You observe that Bug R strains of a species of Shigela (enteric bacteria, closely related to E. coli) canot make flagela or swim in liquid media, but that Bug S strains of the same species can swim normaly. You also know that the expresion of genes coding for flagelar proteins is largely controled by RNA pol with a dedicated ? factor, ? 28 . Propose an explanation for the interference of Bug R on flagelar expresion. ANSWERS: Q1. Q2. Q3. A) trp+ B) Most ? 5 4 3 2 1 - Least C) MinLac + Trp + Ile + Streptomycin (to select against donors) Hfr 3 Q4. A) 1: val, 2: tyr, 3: pro, 4: glu, 5: pro + val, 6: pro + tyr, 7: tyr + val, 8: glu + pro, 9: glu + val B) glu + pro (#8): 5% (i.e., 50/100), glu + val (#9): 10%, pro + tyr (#6): 5%, pro + val (#5): 0%, tyr + val (#7): 0% C) D) 0% (The glu and tyr genes are farther apart than pro and val are from each other, and pro and val can't be cotransduced.) Q5. Part A: There are a number of posible solutions to the problem, but you should focus on reducing the length of the hydrophobic core of the signal sequence by substituting residues with charged side chains for hydrophobic ones in the region after the short N-terminus (4-8 residues, usualy has 2-4 basic residues). Examples include: val glu pro tyr 10% 5% 5% mogA htgA yaH 75% 96% 15% argH rif trpA hisG pro Ala15Glu (GCA to GA), Leu10Arg (CUC to CGC), Ile1Lys (AU to AGU), etc Part B: Starting from strategies described in clas, you could propose doing a selection for growth on ampicilin-containing medium at 37° C, thereby isolating a gain-of-function prl mutation in secA, secE, or secY that gives Amp R . Any mutation that satisfies the selection stil wil generaly grow wel and secrete normal S-containing proteins to the outer membrane or periplasm, in adition to exporting the S*Bla protein. Q6. Streptomycin selection: Can take a plate of Strept S cels and inoculate 50 different cultures, each with a single colony, in nutrient medium. Folowing growth, you should plate a large number of cels (about 10 9 ) from each culture onto a plate with Strept and grow at 37° C. After incubation, you should have a few colonies on at least most of the plates: chose 1 colony/plate and characterize. There should be at least 2 clases of mutations: Clas 1 (most comon, probably 80-90%) wil be nonsense/frameshift mutations that knock-out synthesis of the Str S RpsL protein (so that the strain only expreses the Str R version of RpsL). Clas 2 should be a minority clas, leading to a change in the RpsL + protein from Str S to Str R (so that the strain wil be diploid for Str R ). This clas wil be relatively rare, as they are gain-of-function mutants. Q7. The Bug R misense mutation must change the surface of the ?? subunit of RNA pol so that the drug no longer binds and inhibits the activity of the polymerase. Simultaneous to altering the Bug binding site, the misense mutation must change the surface of the RNA pol polymerase ?? subunit so that it no longer can bind to ? 28 and initiate transcription at flagelar promoters. This change to the rpoC gene is not a lethal mutation, as the RNA pol stil can bind to other (esential) ? factors. Beth Traxler ProblemSet4Answers
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