STRUCTURAL AND GENETIC STUDIES OF CHROMOSOMAL PROTEINS

我们希望了解一个可诱导的 基因，作为我们的模型系统的热休克基因hsp 26， 果蝇 这种基因在正常情况下是静止的， 在几分钟内，在所有细胞类型中， 冲击或其他压力。 根据许多经过充分研究的例子， 活性/诱导型细胞的染色质结构概况 基因是近年来出现的。 几个显著特点 （一）整个活性基因，以及几个 周围的DNA，显示出替代包装，由 对DNA酶I的敏感性升高;这似乎反映了 改变的局部核小体结构和/或改变的更高顺序 核小体包装;（ii）核小体阵列显示模式 不连续性，观察到对DNA酶I超敏感的位点 5'（有时3'）的基因;和（iii）特定的NHC蛋白质 与启动子区相关 5、基因 这里提出的研究将描述 DNA结构元件和与之结合的蛋白质， 共同定义了hsp 26基因的这些特征。 的 将在体外分析染色质组装的过程， 随后在体内研究以鉴定调控特征。 具体而言，我们打算开展以下研究：1. 完成对蛋白质-DNA相互作用的分析， 野生型和突变等位基因的HSP 26的5 ′区。 2. 非组蛋白染色体的鉴定和表征 蛋白质结合到DNA的这个区域。 3.鉴定DNA 形成DNase I超敏反应所必需的元素 位点（DH位点）和有效利用启动子 vivo. 4.体外转录的继续发展 - 基于来自胚胎的核提取物的系统，以及 开发一种体外组装程序， 产生特定的DH位点和活性的其他特征， 染色质结构观察。 5.表征 “核小体”蛋白质被认为与一些 诱导型和/或活性基因。 首先，我们将专注于 推定的HMG蛋白和组蛋白H2 A的变体。 这些实验的总和应该可以让我们详细描述 hsp 26基因的染色质结构，分析 基因激活的机制，并开始分析 指导染色质组装的特定特征的机制 以产生转录活性模板。 这些 这些研究应该有助于更好地理解基因是如何 表达在真核生物中受到控制， 了解正常和异常的细胞生长。

We wish to understand the chromatin structure of an inducible gene, taking as our model system the heat shock gene hsp26 in Drosophila. This gene, while normally quiescent, can be activated within a few minutes in all cell types examined in response to the shock or other stresses. Based on many well-studied examples, a general picture of the chromatin structure of active/inducible genes has emerged in recent years. Several distinctive features are seen: (i) the entire active gene, and several kilobases of surrounding DNA, show an alternative packaging, indicated by an elevated sensitivity to DNase I; this appears to reflect both an altered local nucleosome structure and/or altered higher order nucleosome packaging; (ii) the nucleosome array shows a pattern of discontinuities, sites hypersensitive to DNase I being observed 5' (and sometimes 3') to the gene; and (iii) specific NHC proteins required for transcription are associated with the promoter region 5' to the gene. The research proposed here will characterize the DNA structural elements and the proteins that bind to them that together define these characteristics of the hsp26 gene. The process of chromatin assembly will be analyzed in vitro and subsequently studied in vivo to identify regulatory features. Specifically, we intend to carry out the following studies: 1. Completion of an analysis of the protein-DNA interactions in the region 5' of hsp 26 for wild-type and mutant alleles. 2. Identification and characterization of nonhistone chromosomal proteins binding to this region of DNA. 3. Identification of DNA elements essential for formation of the DNase I hypersensitive sites (DH sites) and for effective utilization of the promoter in vivo. 4. Continued development of an in vitro transcription system based on nuclear extracts from the embryo, and development of an in vitro assembly procedure capable of generating the specific DH sites and other features of active chromatin structure observed. 5. Characterization of "nucleosomal" proteins thought to be associated with some inducible and/or active genes. Initially we will focus on a putative HMG protein and on a variant of histone H2A. The sum of these experiments should allow us to describe in detail the chromatin structure of the hsp 26 gene, to analyze the mechanism of gene activation, and to begin to analyze the mechanisms that direct specific features of chromatin assembly to generate a transcriptionally competent template. These studies should lead to a better understanding of how gene expression is controlled in eukaryotes, basic formation for understanding normal and abnormal cell growth.