Name: ____________KEY___________17.03 Exam Two -- 2005Name: ______________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_______Name: ____________KEY___________21. (31 pts) You have isolated three bacterial mutants that cannot grow withoutsupplemental serine being included in the growth medium. These three mutations lie intwo genes, SerC and SerB. The SerC– mutation is a Tn5 KanR insertion in the middleof the SerC coding region. The SerB1– mutation is a nonsense mutation that producesa protein product that is 30 kDa. The SerB2– mutation is a frameshift mutation thatproduces a protein product that is 12 kDa.The first cross: You grow P1 phage on SerC– bacteria. You use the resulting phagelysate 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 KanRtransductants. All 200 of the transductants you analyze can grow on plates containingkanamycin and serine and proline, but cannot grow on plates containing kanamycin andserine (but not proline).(a, 6pts) What is the genetic distance between the SerC and the ProA loci, expressedas a cotransduction frequency?0%There are two possibilities – either A and C are linked, or they are not.The first cross: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 cotransductionfrequency. The cotransduction frequency between ProA and SerC is 0%.If A and C are unlinkedIf A and C are linkedProA–Tn-SerC–SerC+ProA+ProA–Tn-SerC–SerC+Name: ____________KEY___________3The second cross: You grow P1 phage on SerB2– bacteria. You use the resultingphage lysate to infect ProA– SerB1– bacteria. You select for transductants that cangrow 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 platescontaining serine.(b, 5pts) Are ProA and SerB definitely, maybe, or definitely not linked bycotransduction?Definitely.There are two possibilities – either A and B are linked, or they are not.The second cross: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 bycotransduction?Maybe. SerC and ProA are unlinked by cotransduction. ProA and SerB are linked bycotransduction. It may be that the map order of these genes is such that SerB is in themiddle of SerC and ProA, so SerC and ProA are far enough to be unlinked (more than10^5 basepairs), but SerC and SerB are close enough to each other to be linked (seediagram below).If A and B are unlinkedIf A and B are linkedSerB1–ProA+ProA–SerB1+SerB1–ProA+ProA–SerB2–SerB2+Name: ____________KEY___________4 SerC SerB ProAHowever it also may be that the map order of these genes is such that ProA is in the middleof SerC and SerB, so SerC and ProA are far enough to be unlinked (more than 10^5basepairs), and then SerC and SerB are also unlinked because they are even farther fromeach other than ProA and SerC (see diagram below). SerC ProA SerBThe third cross: You grow P1 phage on SerB1– bacteria. You use the resulting phagelysate to infect ProA– SerB2– bacteria. You select for transductants that can grow onplates containing serine (but not proline). Of the 400 transductants you analyze, 3 canalso grow on plates lacking serine. The other 397 can only grow on plates containingserine.(d, 9pts) In the table below, fill in the genotypes (at the ProA, SerB1, and SerB2 loci) ofthe different phenotypic classes of transductants obtained from this third cross. Be sureto list all possible genotypes in each category.Name: ____________KEY___________5 GENOTYPE:Phenotype:at the ProA locus(+ or –)at the SerB locus (be sure to includethe genotype at SerB1 and SerB2)(+ or –)Don’t requiresupplementalserine+1+ 2+Requiresupplementalserine+1+ 2–1– 2–1– 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 bacterialchromosome that is consistent with all of the data in this problem. Your map shouldshow the positions and relative order of the ProA, SerB1, and SerB2 loci. ProA SerB1 SerB2Name: ____________KEY___________6There is only one possible order for 1e. ProA cannot be in the middle, because SerB1 andSerB2 are in the same gene. SerB1 is much more likely to be in the middle because, if B1 isin the middle, you will see a higher frequency of Ser+ transductants in the second crossthan in the third cross. (If B2 is in the middle, you would have seen a higher frequency ofSer+ transductants in the third cross than in the second cross). This is because doublecrossover events are more frequent than quadruple crossover events. Below are drawn thecrossovers necessary to create Ser+ transductants. Note that you are selecting for ProA+,so ALL transductants will be ProA+. Order One – B1 in middle Order Two - B2 in middleThe second cross:The third cross:B2+B1–B1–B2+Order OneOrder TwoB1+B1+ProA +ProA +ProA–ProA–B2+B2–B2–B1+B1+Order OneOrder TwoB1–B2+ProA +ProA +ProA–ProA–B1–B2–B2–Name: ____________KEY___________72. (31 pts) You construct a plasmid that has a wild-type copy of the LacI gene from E.coli. You transform a lacIΔ E. coli strain (that is, a strain with the LacI gene deleted)with this plasmid. You observe that, whereas the original lacIΔ E. coli strain showsconstitutive expression of beta-galactosidase, the strain carrying the plasmid showsnormal
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