|
| Status |
Public on Feb 01, 2007 |
| Title |
Hot Spots_9_03_04_sir2_REV_312 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Spo11-ZZp
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Precipitated Spo11-ZZ with covalently-attached DNA
|
| Growth protocol |
In most of our experiments, the strain was grown in 200 ml of pre-sporulation medium (SPS; Nag and Petes, 1993) in a two liter flask to a cell concentration measured as OD600= 1.2. The cells were harvested by centrifugation, washed once with 1% potassium acetate, and then incubated at 220 C. for 24 hours in 200 ml of 1% potassium acetate.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The diploids JG169 and MD311 strains are homozygous for a gene encoding an epitope-tagged version of Spo11p (SPO11-ZZ::K.1.URA3) and rad50S. In rad50S strains, Spo11p is covalently attached to the DNA ends produced by the DSBs that initiate recombination (Keeney et al., 1997). As described above, strains were sporulated for 24 hours. The protocol for immunoprecipitation was that described by the Koshland lab (http://www.ciwemb.edu/labs/koshland/Protocols/Yeast/chipmod.html) with the following alterations: 1) formaldehyde was omitted, 2) the SDS lysis buffer contained 1M NaCl, 3) DNA fragments resulting from sonication were about 1.5 kb in size, 4) 5 ml of protein extract derived from 100 ml of sporulating cells were used for the immunoprecipitation, and 5) the Spo11-ZZp-DNA complexes were precipitated with IgG Sepharose beads. Precipitates were washed two times with TSE-1000 buffer (TSE buffer with 1M NaCl), followed by one wash with TSE-500 buffer. The resulting precipitate was then washed once with Li/Detergent buffer and once with TE. All washing steps were done at room temperature. About half of the cell extract from the 200 ml culture was used as input for the immunoprecipitation.
|
| Label |
Cy3
|
| Label protocol |
Both the reference genomic DNA and the experimental samples (Spo11p-associated) were used as templates for three rounds of PCR amplification, and the last round utilized Cy5-dUTP as described in the Website for the Cold Spring Harbor Microarray Course (http://www.bio.unc.edu/faculty/lieb/labpages/PDFs/2003_CSH_Array_Course_Protocols_FINAL.pdf).
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| |
|
| Channel 2 |
| Source name |
reference DNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Total genomic DNA was isolated from the S. cerevisiae JG169 strain grown in rich growth medium (YPD). This DNA was sheared by sonication to a size of about 1-2 kb.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from diploid cells of the strain JG169 using the Qiagen Genomic Extraction Kit according to the provided protocol.
|
| Label |
Cy5
|
| Label protocol |
Both the reference genomic DNA and the experimental samples (Spo11p-associated) were used as templates for three rounds of PCR amplification, and the last round utilized Cy3-dUTP as described in the Website for the Cold Spring Harbor Microarray Course (http://www.bio.unc.edu/faculty/lieb/labpages/PDFs/2003_CSH_Array_Course_Protocols_FINAL.pdf).
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed in 4XSSC, 0.2% SDS and 15 ug of poly A DNA.
|
| Scan protocol |
Images were acquired using a GenePix 4000B scanner and Genepix software (Axon Instruments)
|
| Description |
In eukaryotes, meiotic recombination events are distributed non-randomly in the genome with certain regions having high levels of recombination (hotspots) and others having low levels (coldspots). Species with similar DNA sequences (for example, chimps and humans) can have strikingly different patterns of hotspots and coldspots and it has been suggested that the sequences that locally regulate recombination activity evolve very rapidly. Below, using a microarray analysis that allows us to measure the recombination activities of all 6000 yeast genes, we show that elimination of a histone deacetylase encoded by the SIR2 gene significantly changes the recombination activity of 12% of yeast genes, elevating the recombination activity of 5% and reducing the recombination activity of 7%. We also show that many of the genes with suppressed recombination in the sir2 strain are located in large (50-100 kb) sub-telomeric regions and some of the genes with elevated recombination (for example, the ribosomal RNA gene cluster) are targets of Sir2p deacetylation in the wild-type strain. We suggest that changes in the level of chromatin-modifying proteins may be responsible for altered patterns of recombination in closely-related species.
|
| Data processing |
The normalized median log2 ratios (ch2/ch1) were download from UNC microarray database. Dye swap values were inverted to (ch1/ch2).
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| |
|
| Submission date |
Jan 15, 2007 |
| Last update date |
Jan 23, 2007 |
| Contact name |
Jason D. Lieb |
| E-mail(s) |
jlieb@bio.unc.edu
|
| Phone |
919-843-3228
|
| Organization name |
Univeristy of North Carolina at Chapel Hill
|
| Department |
Biology
|
| Lab |
Lieb
|
| Street address |
CB 3280
|
| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
| |
|
| Platform ID |
GPL4414 |
| Series (1) |
| GSE6245 |
Loss of a histone deacetylase dramatically alters the genomic distribution of Spo11p-catalyzed DNA breaks in yeast |
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