Jack R S, Eggert H
Institut für Genetik der Universität zu Köln, FRG.
EMBO J. 1990 Aug;9(8):2603-9. doi: 10.1002/j.1460-2075.1990.tb07442.x.
Sequence specific DNA binding proteins in eukaryotic cells must efficiently locate their binding sites in chromosomes. Restriction enzymes provide a simple model system with which to investigate the factors which influence this process. We have used P element mediated transformation to introduce a DNA fragment containing a set of characterized restriction sites into the Drosophila germline. Embryonic nuclei prepared from these transgenic animals were treated with restriction enzymes to probe the accessibility of the target restriction sites. The results show that the insert is within an accessible region of the chromosome and that restriction sites within the inserted sequence can be cut. However, the rate of cutting is biphasic. At each restriction site, a fraction of the chromosomes is cut rapidly after which the remainder is refractory. Similar levels of incomplete cutting are obtained when the same P element construct is examined at a different chromosomal location, when different sequence elements are introduced into the P element vector or when the experiment is carried out on nuclei from different embryonic stages. These results are discussed in terms of how sequence specific DNA binding proteins may locate their genomic targets in vivo.
真核细胞中序列特异性DNA结合蛋白必须有效地在染色体中定位其结合位点。限制性内切酶提供了一个简单的模型系统，用以研究影响这一过程的因素。我们利用P因子介导的转化将一个含有一组特征性限制性位点的DNA片段导入果蝇种系。从这些转基因动物制备的胚胎细胞核用限制性内切酶处理，以探测目标限制性位点的可及性。结果表明，插入片段位于染色体的一个可及区域内，并且插入序列内的限制性位点可以被切割。然而，切割速率是双相的。在每个限制性位点，一部分染色体被快速切割，之后其余部分则难以切割。当在不同染色体位置检查相同的P因子构建体、将不同的序列元件引入P因子载体或在来自不同胚胎阶段的细胞核上进行实验时，会获得相似水平的不完全切割。根据序列特异性DNA结合蛋白如何在体内定位其基因组靶点对这些结果进行了讨论。
