CHAPTER FIVE 5—3 In a genetics lab, Kim and Maria infected a sample from an E. coli culture with a virulent bacteriophage. They noticed that most of the cells were lysed, but a few survived. The survival in their sample was about 1 x 10-4. Kim was sure the bacteriophage induced the resistance in the cells, while Maria thought that resistant mutants probably already existed in the sample of cells they used. Earlier, for a different experiment, they had spread a dilute suspension of E. coli onto solid medium in a large Petri dish, and, after seeing that about 105 colonies were growing up, they replica plated that plate onto three other plates. Kim and Maria decided to use these plates to test their hypotheses. They pipette a suspension of the bacteriophage onto each of the three replica plates. What should they see if Kim is right? What should they see if Maria is right? 5—4 Evaluate the following statement: “No two cells in your body have the identical nucleotide sequence.” Do you agree or disagree? Explain your reasoning. 5—15 Look carefully at the structures of the molecules in Figure 5—5. A. What would you expect if dideoxycytidine triphosphate (ddCTP) were added to a DNA replication reaction in large excess over the concentration of deoxycytidine triphosphate (dCTP)? B. What would happen if ddCTP were added at 10% the concentration of dCTP? C. What effects would you expect if dideoxycytidine monophosphate (ddCMP) were added to a DNA replication reaction in large excess, or at 10% the concentration of dCTP? 5—16* How would of the absence of a 3’-to-5’ proofreading exonuclease activity of DNA polymerase in E. coli to affect the rate of DNA synthesis? Explain your reasoning. 5—25 Conditional lethal mutations are very useful in genetic and biochemical analyses of complex processes such as DNA replication. Temperature- sensitive (ts) mutations, which are one form of conditional lethal mutation, allow growth at one temperature (for example, 30°C) but not at a higher temperature (for example, 42°C). A large number of temperature-sensitive replication mutants have been isolated in E. coli. These mutant bacteria are defective in DNA replication at 42°C but not at 30°C. If the temperature of the medium is raised from 30°C to 42 C, these mutants stop making DNA in one of two characteristic ways. The ‘quick-stop’ mutants halt DNA synthesis immediately, whereas the ‘slow-stop’ mutants stop DNA synthesis only after many minutes. A. Predict which of the following proteins, if temperature sensitive, would display a quick-stop phenotype and which would display a slow-stop phenotype. In each case explain your prediction. 1. DNA topoisomerase I 2. A replication initiator protein 3. Single-strand binding protein 4. DNA helicase 5. DNA primase 6. DNA ligase 5—27 DNA repair enzymes preferentially repair mismatched bases on the newly synthesized DNA strand, using the old DNA strand as a template. If mismatches were simply repaired without regard for which strand served as template, would mismatch repair reduce replication errors? Would such an indiscriminate mismatch repair be better than, worse than, or the same as no repair at all? Explain your answers. 5—29 The different ways in which DNA synthesis occurs on the leading and lagging strands raises the question as to whether synthesis occurs with equal fidelity on the two strands. One clever approach used reversion of specific mutations in the E. coli lacZ gene to address this question. E. coli is a good choice for such a study because the same polymerase (DNA poll III) synthesizes both the leading and the lagging strands. The lacZ CC 106 allele can regain its function (revert) by converting the mutant AT base pair to the normal GC base pair. This allele was inserted in both orientations on one side of the normal origin of replication (Figure 5-12A). As shown in Figure 5—12B, misincorporation of C opposite T on one strand, or of C opposite A on the other strand, could lead to reversion. Previous studies had shown that C is only very rarely misincorporated opposite A, and when it is, DNA polymerase is inefficient at extending the chain. Thus, the most common source of reversion is from misincorporation of G opposite T. To eliminate the complicating effects of mismatch repair, the experiments were done in two mutant strains of bacteria. One was defective for mismatch repair, which eliminates it from consideration; the other was defective in the proofreading exonuclease, and introduces so many mismatches that it over whelms the mismatch-repair machinery. Accurate frequencies of lacZ reversion were measured in the two strains, along with the frequencies of mutation at the rif gene, whose orientation in the chromosome was constant (Table 5- 2). A. On which strand, leading or lagging, does DNA synthesis appear to be more accurate? Explain your reasoning. B. Can you suggest a reason why DNA synthesis might be more accurate on the strand you have chosen? 5—35 The laboratory you joined is studying the life cycle of an animal virus that uses a circular, double-stranded DNA as its genome. Your project is to define the location of the origin(s) of replication and to determine whether replication proceeds in one or both directions away from an origin (unidirectional or bidirectional replication). To accomplish your goal, you isolated replicating molecules, cleaved them with a restriction nuclease that cuts the viral genome at one site to produce a linear molecule from the circle, and examined the resulting molecules in the electron microscope. Some of the molecules you observed are illustrated schematically in Figure 5—14. (Note that it is impossible to tell one end of a DNA molecule from the other in the electron microscope.) You must present your conclusions to the rest of the lab tomorrow. How will you answer the questions that your advisor has posed for you? (1) Is there a single, unique origin of replication or several origins? (2) Is replication unidirectional or bidirectional? 5—41 Which one of the following statements about the newly synthesized strand of a human chromosome is correct? A. It was synthesized from a single origin solely by continuous DNA synthesis. B. It was synthesized from a single origin solely by discontinuous DNA synthesis. C. It was synthesized from a single origin by a mixture of continuous and discontinuous DNA synthesis. D. It was synthesized from multiple origins solely by continuous DNA synthesis. E. It was synthesized from multiple origins solely by discontinuous DNA synthesis. F. It was synthesized from multiple origins by a mixture of continuous and discontinuous DNA synthesis. G. It was synthesized from multiple origins by either continuous or discontinuous DNA synthesis, depending on which specific daughter chromosome is being examined. 5—51 You have recently purified and partially sequenced a protein from a ciliated protozoan that seems to be the catalytic subunit of telomerase. You identify the homologous gene in fission yeast, which makes it possible to perform genetic studies that are impossible in the protozoan. You make a targeted deletion of the gene in a diploid strain and then induce sporulation to produce haploid organisms. All four spores germinate perfectly, and you are able to grow colonies on nutrient agar plates. Every 3 days, you re-streak colonies onto fresh plates. After four such serial transfers, the descendants of two of the original four spores grow poorly, if at all. You take cells from the 3-, 6-, and 9-day master plates, prepare DNA from them, and cleave the samples at a site about 35 nucleotides away from the start of the telomere repeats on two of the three chromosomes. You separate the fragments by gel electrophoresis, and hybridize them to a radioactive telomere-specific probe (Figure 5—26). A. What is the average length of telomeres in fission yeast? B. Do the data support the idea that you have identified yeast telomerase? If so, which spores lack telomerase? C. Assuming that the generation time of this yeast is about 6 hours when growing on plates, by how much do the chromosomes shorten each generation in the absence of telomerase? D. If you were to examine the yeast cells that stop dividing, do you suppose they would be longer, shorter, or about the same size as normal yeast cells?
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