 kb apart [39,42], similar to the distribution of hotspots in S. pombe (Figure 3). The strongest human hotspot has a crossover frequency of ∼1% , lower than that at mbs1 (5%)  and presumably than at several even stronger DSB hotspots in S. pombe (Figures 3 and S7). Human DSB hotspots are expected to be weaker than those of S. pombe, given the similar hotspot spacing and map sizes of the two organisms but greatly larger physical genome size of humans. Like S. pombe and S. cerevisiae, human hotpots are positively, but only weakly, correlated with GC content; this correlation has low predictive power . Therefore, several characteristics of meiotic DSB hotspots are common to S. pombe, S. cerevisiae, and humans, but there are also distinct differences in any pair-wise comparison of the organisms.
Further comparisons of meiotic DSB patterns in these yeasts and additional species will help understand how this process, crucial to sexual reproduction, is controlled. The analysis reported here provides a foundation for identifying the genomic features that regulate meiotic DSB formation, a problem so far unsolved for any organism.
Materials and Methods
S. pombe strains.
The genotypes of strains and the sources of published alleles are in Table S3. The rec12–201::6His-2FLAG allele, called rec12-FLAG in the text, was constructed as plasmid pJF32 (Text S1). The 1.6-kb BamHI fragment of pJF32 was used to transform strain GP5622 (rec12–171::ura4+) to fluoro-orotic acid-resistance to generate strain GP5960; the chromosomal allele was confirmed by automated nucleotide sequence analysis. The rec12–202::6His-2HA allele, called rec12-HA in the text, was similarly constructed as plasmid pJF41 and transferred to the chromosome to generate strain GP5961. Strains with these alleles are recombination proficient (Table S1). Other strains were constructed by standard meiotic crosses.
Preparation of meiotic chromatin and Rec12-FLAG covalently linked to DNA and its analysis with microarrays.
Cells were grown and induced for meiosis as described  and then harvested, washed, and stored at −20 °C. Chromatin was prepared, sonicated to yield DNA 0.5–1 kb long as determined by agarose gel electrophoresis (unpublished data), and subjected to immunoprecipitation with anti-FLAG antibody (Sigma, http://www.sigmaaldrich.com) as described . Precipitates (IP) were washed, treated with proteinase K, extracted with phenol-CHCl3, and treated with RNase; the residual DNA was purified on a QIAquick column (Qiagen, http://www1.qiagen.com), amplified in the presence of amino-allyl dUTP, and labeled with Cy5 as described . DNA from unfractionated, sonicated chromatin (WCE) was prepared and labeled with Cy3 in parallel. The samples were mixed and hybridized to an Agilent 44-K S. pombe oligonucleotide microarray and analyzed as described . The IP:WCE ratio for each probe on the array (∼41,000) was then calculated after adjustment of the IP values to equalize the IP and WCE medians. Data were analyzed with Excel (Microsoft, http://www.microsoft.com), PeakFinder , and ChIPOTle  software. Peakfinder was run using the haploid and diploid datasets after noise reduction as in Figure 1, with linear plotting, Gaussian weighting, a single round using a 13-probe window, right and left minimum smoothed delta = 100, and a threshold of 2,000. ChIPOTle was run on the haploid dataset with noise reduction as in Figure S3. The Gaussian background setting was used with a window size of 1 kb, a step size of 150 bp, and a cutoff of p < 0.001.
Dataset S1. Median-Normalized Genome-Wide IP/WCE Hybridization Ratios from Two Meiotic Inductions of Haploid Strain GP6013
Shown are experiment 2 data from before induction (0 h) and 5 h after induction (5 h) and experiment 1 data from 5 h after induction (5 h).
(6.7 MB XLS)
Dataset S2. Median-Normalized Genome-Wide IP/WCE Hybridization Ratios from a Meiotic Induction of Diploid Strain GP6203
Shown are data from before induction (0 h) and 5 h after induction (5 h).
(6.0 MB XLS)
Figure S1. Meiosis-Specific Rec12-DNA Linkage Requires Rec10 and Is Abundant