Lantermann Alexandra, Strålfors Annelie, Fagerström-Billai Fredrik, Korber Philipp, Ekwall Karl
Adolf-Butenandt-Institut, University of Munich, Schillerstr. 44, 80336 Munich, Germany.
Methods. 2009 Jul;48(3):218-25. doi: 10.1016/j.ymeth.2009.02.004. Epub 2009 Feb 20.
The majority of nuclear eukaryotic DNA is packaged into nucleosome cores where DNA is wrapped tightly around histone protein octamers. Such histone bound nucleosomal DNA is less accessible than the short linker DNA between nucleosome cores or the DNA in extended nucleosome free regions. Therefore, the positions of nucleosomes relative to a DNA sequence feature, like a transactivator binding site, a transcriptional start site or an origin of replication, can have profound effects on nuclear processes like transcription, replication, recombination and repair. Now that many DNA related processes are studied in a genome-wide manner, it is increasingly important to map the basic organization of their chromosomal DNA substrate, i.e., the positions of nucleosomes, on a genome-wide scale as well. To this end, the protection of nucleosomal DNA from digestion with micrococcal nuclease (MNase) is used as an assay for the presence of a nucleosome. The MNase protected DNA fragments, so called mononucleosomal DNA, can be mapped genome-wide by hybridization to microarrays. This method has been established for Saccharomyces cerevisiae, and we present here the adaptation of the method for Schizosaccharomyces pombe. As an independent method to validate genome-wide data for individual loci, we also include a protocol for the determination of locus specific nucleosome positioning by indirect end labeling.
大多数真核细胞核DNA被包装成核小体核心，其中DNA紧密缠绕在组蛋白八聚体周围。与核小体核心之间的短连接DNA或延伸的无核小体区域中的DNA相比，这种与组蛋白结合的核小体DNA更难接近。因此，核小体相对于DNA序列特征（如反式激活因子结合位点、转录起始位点或复制起点）的位置，可对转录、复制、重组和修复等核过程产生深远影响。鉴于现在许多与DNA相关的过程都是在全基因组范围内进行研究的，在全基因组规模上绘制其染色体DNA底物的基本组织（即核小体的位置）也变得越来越重要。为此，利用微球菌核酸酶（MNase）消化对核小体DNA的保护作用，作为检测核小体存在的一种方法。经MNase保护的DNA片段，即所谓的单核小体DNA，可以通过与微阵列杂交在全基因组范围内进行定位。这种方法已在酿酒酵母中建立，我们在此展示了该方法在粟酒裂殖酵母中的应用。作为验证单个基因座全基因组数据的独立方法，我们还包括了一个通过间接末端标记确定基因座特异性核小体定位的方案。
