Name KEY 7 03 Exam Two 2005 Name KEY Exam starts at 11 05 am and ends at 11 55 am There are 7 pages including this cover page Please write your name on each page Only writing on the FRONT of every page will be graded You may use the backs but only as scratch paper Question 1 31 pts Question 2 31 pts Question 3 38 pts TOTAL out of 100 1 Name KEY 1 31 pts You have isolated three bacterial mutants that cannot grow without supplemental serine being included in the growth medium These three mutations lie in two genes SerC and SerB The SerC mutation is a Tn5 KanR insertion in the middle of the SerC coding region The SerB1 mutation is a nonsense mutation that produces a protein product that is 30 kDa The SerB2 mutation is a frameshift mutation that produces a protein product that is 12 kDa The first cross You grow P1 phage on SerC bacteria You use the resulting phage lysate to infect ProA bacteria ProA bacteria have a disruption in the ProA gene which is required for the bacteria to synthesize their own proline You select for KanR transductants All 200 of the transductants you analyze can grow on plates containing kanamycin and serine and proline but cannot grow on plates containing kanamycin and serine but not proline a 6pts What is the genetic distance between the SerC and the ProA loci expressed as a cotransduction frequency 0 There are two possibilities either A and C are linked or they are not The first cross If A and C are unlinked If A and C are linked Tn SerC Tn SerC ProA SerC SerC ProA ProA You will never get ProA KanR from this You can get ProA KanR from this Given that you never see ProA KanR the two are unlinked This problem asked you to express a distance between ProA and SerC as a cotransduction frequency The cotransduction frequency between ProA and SerC is 0 2 Name KEY The second cross You grow P1 phage on SerB2 bacteria You use the resulting phage lysate to infect ProA SerB1 bacteria You select for transductants that can grow on plates containing serine but not proline Of the 70 transductants you analyze 3 can also grow on plates lacking serine The other 67 can only grow on plates containing serine b 5pts Are ProA and SerB definitely maybe or definitely not linked by cotransduction Definitely There are two possibilities either A and B are linked or they are not The second cross If A and B are unlinked If A and B are linked ProA ProA SerB1 ProA ProA SerB1 SerB2 SerB2 SerB1 You will never get ProA Ser from this You can get ProA SerB from this Given that you do see ProA Ser transductants A and B must be linked c 5pts Are SerC and SerB definitely maybe or definitely not linked by cotransduction Maybe SerC and ProA are unlinked by cotransduction ProA and SerB are linked by cotransduction It may be that the map order of these genes is such that SerB is in the middle of SerC and ProA so SerC and ProA are far enough to be unlinked more than 10 5 basepairs but SerC and SerB are close enough to each other to be linked see diagram below 3 Name KEY SerC SerB ProA However it also may be that the map order of these genes is such that ProA is in the middle of SerC and SerB so SerC and ProA are far enough to be unlinked more than 10 5 basepairs and then SerC and SerB are also unlinked because they are even farther from each other than ProA and SerC see diagram below SerC ProA SerB The third cross You grow P1 phage on SerB1 bacteria You use the resulting phage lysate to infect ProA SerB2 bacteria You select for transductants that can grow on plates containing serine but not proline Of the 400 transductants you analyze 3 can also grow on plates lacking serine The other 397 can only grow on plates containing serine d 9pts In the table below fill in the genotypes at the ProA SerB1 and SerB2 loci of the different phenotypic classes of transductants obtained from this third cross Be sure to list all possible genotypes in each category 4 Name KEY GENOTYPE at the ProA locus or at the SerB locus be sure to include the genotype at SerB1 and SerB2 or 1 2 1 1 1 Phenotype Don t require supplemental serine Require supplemental serine 2 2 2 Note that you are selecting for ProA so ALL transductants will be ProA Ser transductants will only result if both positions in the SerB gene are wild type e 6pts Draw all of the possibilities for a map of the region of the bacterial chromosome that is consistent with all of the data in this problem Your map should show the positions and relative order of the ProA SerB1 and SerB2 loci ProA SerB1 SerB2 5 Name KEY There is only one possible order for 1e ProA cannot be in the middle because SerB1 and SerB2 are in the same gene SerB1 is much more likely to be in the middle because if B1 is in the middle you will see a higher frequency of Ser transductants in the second cross than in the third cross If B2 is in the middle you would have seen a higher frequency of Ser transductants in the third cross than in the second cross This is because double crossover events are more frequent than quadruple crossover events Below are drawn the crossovers necessary to create Ser transductants Note that you are selecting for ProA so ALL transductants will be ProA Order One B1 in middle The second cross Order Two B2 in middle Order One ProA B1 ProA B1 Order Two B2 B2 ProA ProA B2 B2 B1 B1 The third cross Order One ProA B1 ProA B1 Order Two B2 B2 ProA B2 ProA B1 B1 B2 6 Name KEY 2 31 pts You construct a plasmid that has a wild type copy of the LacI gene from E coli You transform a lacI E coli strain that is a strain with the LacI gene deleted with this plasmid You observe that whereas the original lacI E coli strain shows constitutive expression of beta galactosidase the strain carrying the plasmid shows normal inducible expression of beta galactosidase A diagram of your plasmid is shown below For this problem we are going to focus on a highlighted region of DNA sequence present early in the LacI open reading frame 5 GAGGCCT 3 3 CTCCGGA 5 LacI open reading frame a 5pts Write out the sequence that would result from transcription of the LacI gene by RNA polymerase if the lower strand was used as a template Be sure to give the sequence corresponding to the short segement that is highlighted and label any 5 and 3 ends in your drawing 5 GAGGCCU 3 b 5pts Label the correct reading frame of this gene given that a tRNA with the …
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