SEQUENCE-SPECIFIC NUCLEOSOMAL DNA-BINDING PROTEINS

由于序列特异性DNA结合蛋白控制着基因在 细菌，并可能在高等生物体中发挥同样重要的作用， 应在分子水平上(除其他外)研究调节，以 了解导致差异化和发展的信号。 解决这些问题的一种方法是鉴定真核生物 序列特异的DNA结合蛋白。 果蝇核小体杂交图谱显示 富含AT的异染色质序列所特有的蛋白质(D1)。这种方法 将继续使用低丰度序列的杂交探针， 探讨D_1与富含AT的非卫星蛋白结合的普遍性 序列，并寻找更高的序列特异性DNA结合 低拷贝数基因的蛋白质。 重组杂交作图--一种新的分离方法 重叠的核蛋白和DNA物种，应该明确地识别 在二维图案中获得了“神秘”的次要粒子 来自未分离的核小体。将使用DNA-纤维素层析法 为了提纯蛋白质，这些蛋白质将与盐剥离的核小体混合， 涂抹在二维凝胶上，并通过杂交和 将DNA和蛋白质模式联系起来。 蛋白质-蛋白质交联应揭示D1之间的相互作用 分子、组蛋白和核结构的其他元素。 交联剂还可以稳定蛋白质，否则蛋白质可能不会稳定。 在核小体之间重新分配。分子的动力学和结构效应 将使用核蛋白研究d1与DNA的体外结合。 电泳法和保护(足迹)实验。 这些研究将揭示核小体d1如何具有序列特异性 DNA结合蛋白的最普遍的品种，识别DNA，以及如何 它广泛地分布在黑腹果蝇的基因组中。

Since sequence-specific DNA-binding proteins control gene expression in bacteria, and may serve the same crucial function in higher organisms, regulation should be studied at the molecular level (among others) to understand the signals responsible for differentiation and development. One way to approach these problems is to identify eukaryotic sequence-specific DNA-binding proteins. Hybridization mapping of Drosophila melanogaster nucleosomes has revealed a protein (D1) specific for AT-rich heterochromatic sequences. This approach will be continued with hybridization probes for lower abundance sequences, to investigate the generality of D1 binding to AT-rich, nonsatellite sequences, and to search for more highly sequence-specific DNA-binding proteins of lower copy number genes. Reconstitution hybridization mapping, a new approach designed to separate overlapping nucleoprotein and DNA species, should unambiguously identify minor particles which are "cryptic" in a two-dimensional pattern obtained from unfractionated nucleosomes. DNA-cellulose chromatography will be used to purify the proteins, which will be mixed with salt-stripped nucleosomes, spread on two-dimensional gels, and analyzed by hybridization and by correlating DNA and protein patterns. Protein-protein cross-linking should reveal the interactions between D1 molecules, the histones, and other elements of nuclear structure. Cross-linking will also stabilize proteins which might otherwise redistribute between nucleosomes. The kinetics and structural effects of D1 binding to DNA in vitro will be investigated using nucleoprotein electrophoretic methods and protection (footprinting) experiments. These studies will reveal how D1, a nucleosomal, sequence-specific DNA-binding protein of the most general variety, recognizes DNA, and how widespread is its distribution throughout the D. melanogaster genome.