7.03 Genetics: Solutions to Problem Set 3 (2001)

Solutions to 7.03 Problem Set 3 1. a. In order to suppress TAG and TAA nonsense mutations, it requires two different tRNA’s that have anti-codons mutated to CUA and UUA. Having two mutations in two different tRNA genes at the same time is as extremely unlikely as it is to get two back mutations at the same time. b. The possible mutations are: G � A C � T Amber mutation is TAG. tRNA has to have the anti-codon CUA. Gene that encodes this tRNA must have the sequence CTA. This sequence, given the possible mutations, could have mutated from: CTG � CTA CCA � CTA Glutamine and Tryptophan tRNA’s. Wild-type Glutamine tRNA: 3’ tRNA: 5’ GUC CAG mRNA: 5’ 3’ Coding sequence of DNA for the tRNA: 5’ 3’ CTG GAC 3’ 5’ Wild-type Tryptophan: 3’ tRNA: 5’ ACC UGG mRNA: 5’ 3’ Coding sequence of DNA for the tRNA: 5’ 3’ CCA GGT 3’ 5’ Mutant: 3’ tRNA: 5’ AUC UAG mRNA: 5’ 3’ Coding sequence of DNA for the tRNA: 5’ 3’ CTA GAT 3’ 5’ c. The possible mutations are: A � T T � A Ochre mutation is TAA. tRNA has to have the anti-codon UUA. Gene that encodes this tRNA must have the sequence TTA. This sequence, given the possible mutations, could have mutated from: ATA � TTA TAA � TTA TTT � TTA Tyrosine, Leucine, Lysine tRNA’s. Wild-type Tyrosine: 3’ tRNA: 5’ AUA UAU mRNA: 5’ 3’ Coding sequence of DNA for the tRNA: 5’ 3’ ATA TAT 3’ 5’ Wild-type Leucine: 3’ tRNA: 5’ AAU UUA mRNA: 5’ 3’ Coding sequence of DNA for the tRNA: 5’ 3’ TAA ATT 3’ 5’ Wild-type Lysine: 3’ tRNA: 5’ UUU AAA mRNA: 5’ 3’ Coding sequence of DNA for the tRNA: 5’ 3’ T TT AAA 3’ 5’ Mutant: 3’ tRNA: 5’ AUU UAA mRNA: 5’ 3’ Coding sequence of DNA for the tRNA: 5’ 3’ TTA AAT 3’ 5’ 2. a. In order to suppress the nonsense mutation with another mutation in the same gene but not in the nonsense codon, you would need a frameshift mutation before the nonsense codon and another frameshift mutation that puts the reading frame back in the original reading frame. This is a highly unlikely event, because EMS generally does not cause frameshift mutations. b. Because P1 was grown on his+ Kanr strain, cotransductants selected for Kanr are his+. cotransduction frequency = # of cotransductants = 40/100 = 40% total transductants c. If the revertant were a back mutation, 40% of the transductants would have been his-, because his- cotransduces with Kanr 40% of the time. If the revertant were an extragenic suppressor, then all the transductants would be his+, because the extragenic suppressor would suppress the his- mutation. Therefore, it must be an extragenic suppressor. d. The revertant must have been a back mutation. The cotransduction frequency is consistent. The only difference is that Kanr is cotransducing with his- allele instead of his+. e. Because his1-1am and his1-2am are both mutations in His1 gene, Tn5 can’t be between these two mutations. Otherwise, His1 gene would be disrupted by Tn5. Experiment 1 Tn5 1am + + + 2am Experiment 2 Tn5 + 2am + 1am + Possible Transductant Genotypes Tn5 1am + All cotransduce: Tn5 + 2am Only Tn5: Tn5 1am 2am 1am cotransduce: Tn5 + + 2am contranscuce: Tn5 + 2am Tn5 1am + Tn5 + + Tn5 1am 2am P1 Grown on: P1 Infects: Count 12 2 Since the locus of the mutations don’t change between the experiments, each type of recombination should occur at the same frequency. In a quadruple crossover event, “All contransduce” and “Only Tn5” classes are not possibilities. Therefore, Tn5 and 2am cotransductants must be the quadruple crossover class. We find that gene order is: Tn5 his1-1am his1-2am 3. a. In cells that have restriction enzyme but no modifying enzyme, DNA is digested, therefore the cells would not survive. In cells that have no restriction enzyme, DNA is not digested, therefore the cells can survive on Ampr given that the plasmid transforms. In cells that have both restriction enzyme and modifying enzyme, it’s a race between these two enzymes recognizing the same sequence and either protecting or digesting it. Therefore, some of the cells with the plasmid will survive and some will not. This follows that ORF1 encodes for the restriction enzyme and ORF2 encodes for the modifying enzyme. b. λ grown on wild-type E. coli plated on E. coli carrying clone 1. DNA not modified, no restriction enzyme � high efficiency λ grown on wild-type E. coli plated on E. coli carrying clone 3. DNA not modified, restriction enzyme � low efficiency λ grown on E. coli carrying clone 1 plated on E. coli carrying clone 1. DNA modified, no restriction enzyme � high efficiency λ grown on E. coli carrying clone 1 plated on E. coli carrying clone 3. DNA modified, restriction enzyme � high efficiency λ grown on E. coli carrying clone 3 plated on E. coli carrying clone 1. DNA modified, no restriction enzyme � high efficiency λ grown on E. coli carrying clone 3 plated on E. coli carrying clone 3. DNA modified, restriction enzyme � high efficiency