STRUCTURAL AND GENETIC STUDIES OF CHROMOSOMAL PROTEINS

我们希望了解诱导物的染色质结构。 Gene，以热休克基因hsp26作为我们的模型系统。 果蝇。这种基因虽然通常处于静止状态，但可以被激活。 在几分钟内对所有细胞类型进行检查，以响应 震惊或其他压力。基于许多经过充分研究的例子，一个 活性/诱导性染色质结构概貌 基因是近年来出现的。几个明显的特征 可以看到：(I)整个活性基因，以及几千个碱基的 在DNA周围，显示了另一种包装，由一个 对DNase I的敏感度提高；这似乎反映了 改变局部核小体结构和/或改变更高的顺序 核小体包装；(Ii)核小体排列呈现一种图案 观察到对DNase I高度敏感的不连续部位 基因的5‘(有时为3’)；和(Iii)特定的NHC蛋白 转录所需的基因与启动子区域相关 基因的5‘端。这里提出的研究将描述 DNA结构元素和与其结合的蛋白质 共同定义了hsp26基因的这些特征。这个 将在体外分析染色质组装的过程和 随后在体内进行了研究，以确定调控功能。 具体来说，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 HSP26的5‘区为野生型和突变型等位基因。2. 非组蛋白染色体的鉴定与鉴定 结合在DNA的这一区域的蛋白质。3.DNA的鉴定 形成脱氧核糖核酸酶I超敏反应的基本要素 和有效利用启动子。 活着。4.体外转录的继续发展 基于胚胎核提取物的系统，以及 一种体外组装程序的开发 生成特定的DH站点和Active的其他功能 观察到染色质结构。5.对 核小体蛋白被认为与一些 可诱导和/或活性基因。最初，我们将重点放在 推测的HMG蛋白和组蛋白H_2A的变异体上。 这些实验的总和应该使我们能够详细地描述 HSP26基因的染色质结构，以分析 基因激活的机制，并开始分析 指导染色质组装特定功能的机制 以产生转录上合格的模板。这些 研究应该有助于更好地理解基因是如何 在真核生物中表达是受控制的，基本形成 了解正常和不正常的细胞生长。

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.