Use the following passage to answer questions: The fission yeast Schizosaccharomyces pombe Rec12 protein, which is the homolog of Spo11 in other organisms, initiates meiotic recombination by creating DNA double-strand breaks (DSBs) and covalently linking to the 5' DNA ends of the break. This protein-DNA linkage has previously been detected only in mutants such as rad50S, in which break repair is impeded and DSBs accumulate. In the budding yeast Saccharomyces cerevisiae, the DSB distribution in a rad50S mutant is markedly different from that in wild-type (RAD50) meiosis, and it was suggested... Show more

Use the following passage to answer questions:

The fission yeast Schizosaccharomyces pombe Rec12 protein, which is the homolog of Spo11 in other organisms, initiates meiotic recombination by creating DNA double-strand breaks (DSBs) and covalently linking to the 5' DNA ends of the break. This protein-DNA linkage has previously been detected only in mutants such as rad50S, in which break repair is impeded and DSBs accumulate. In the budding yeast Saccharomyces cerevisiae, the DSB distribution in a rad50S mutant is markedly different from that in wild-type (RAD50) meiosis, and it was suggested that this might also be true for other organisms. Researchers have shown that linkages between Rec12 and DNA can actually be detected in Sc. pombe rad50(+) cells, which are proficient for DSB repair. In fact, genome-wide microarray analysis of Rec12-DNA reveals indistinguishable meiotic DSB distributions in rad50(+) and rad50S strains of Sc. pombe, unlike in Sc. cerevisiae. These results confirm earlier findings describing the occurrence of widely spaced DSBs primarily in large intergenic regions of DNA and demonstrate the relevance and usefulness of fission yeast studies employing rad50S. The researchers have proposed that the differential behavior of rad50S strains reflects a major difference in DSB regulation between the two species—specifically, the requirement for the Rad50-containing complex for DSB formation in budding yeast but not in fission yeast. Use of rad50S and related mutations may be a useful method for DSB analysis in other species.

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MCAT Biological and Biochemical Foundations of Living Systems: Passage 5

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4 Questions

1. Detection of DNA by Southern blotting relies on the use of a nucleotide probe that is complementary to sequences in the DNA sample. What prevents RNA from being detected by Southern blotting?

DNA can be harvested under conditions that do not allow RNA recovery.Nucleotide probes do not hybridize to RNA molecules.RNA cannot be amplified by PCR.RNA cannot be separated by gel electrophoresis.

2. The figure is a Southern blot showing the DSB pattern of three strains of Sc. pombe, RAD50, rad50S, and a DSB repair double mutant, swi5∆ rhp57∆, between 0 and 6 hours (h) after the experimental initiation of meiosis.

Based on the image, what is the most appropriate conclusion about the DSB patterns in these three strains?

DSBs are not observed in rad50+.The three strains have similar DSB patterns and rates of DSB repair.The level of DSB intermediates is higher in rad50+ than in the two mutants.The three strains have similar DSB patterns, but the two mutants do not repair the breaks.

3. After DSB and the action of Rec12 (or Spo11), what is the next step that is predicted to occur in DSB repair during meiosis?

The DSBs are repaired using the homologous chromosomes as the template.The 5' end and Rec 12 (or Spo 11) dissociate, and the 5' end invades the homologous chromosome.The 5' end and Rec 12 (or Spo 11) invade the homologous chromosome.The 3' overhang invades the homologous chromosome.

4. Which of the following is NOT a necessary step in Southern blot analysis?

Separation of DNA by gel electrophoresisAmplification of DNARestriction enzyme treatment of DNADenaturation of DNA in the gel