The Role of Kinetoplast DNA-Binding Proteins in the Struc- ture and Organization of the Kinetoplast in the Trypanosome Crithidia Fasciculata

摘要9404955种动叶原生动物含有一种新形式的线粒体DNA(动体DNA或kDNA)，它由数以千计的小环和20-30个最大环连接成一个片状或网状。这些kDNA网络在体内浓缩，形成直径约1微米，厚度约0.4微米的高度有序的盘状结构。以前的研究几乎全部集中在运动基质的新的DNA成分以及它们组织成连锁网络上。这里提出的研究是关于小的组蛋白样蛋白在动泡体的结构组织中的作用。我们实验室以前的工作已经在体内发现了五种与kDNA相关的富含赖氨酸的小蛋白。其中三种基因的编码基因已经被克隆。这项拟议的研究将导致对剩余基因的克隆，并将它们用于确定这些蛋白质在动质粒结构组织中的可能作用。每一种类似组蛋白的蛋白质都将通过在细菌中表达而大量产生，并用于产生抗体和每种蛋白质与DNA相互作用的生化研究。这些研究将确定每种蛋白质的DNA结合特异性及其弯曲DNA、诱导超螺旋和凝聚kDNA网络的能力。针对这些蛋白质的抗体将为使用免疫荧光显微镜和免疫电子显微镜定位细胞内的每一种蛋白质提供必要的试剂。通过使用这一系列的实验方法，我们期望对组蛋白样蛋白在组织动泡体结构中的可能作用有一个了解。遗传信息是在活细胞中以极长的DNA链编码的，必须以某种高度浓缩和规则的方式组织或“打包”。这项研究计划旨在识别和分析在活细胞内浓缩DNA所涉及的一些因素。自然界中最高度浓缩的细胞DNA形式之一是在被称为锥虫的热带寄生生物中发现的DNA。这些生物是广泛存在于植物和动物身上的寄生虫。它们的特征之一是存在大量的DNA，称为动泡体DNA(KDNA)，位于染色体所在的细胞核之外。就像染色体的DNA一样，kDNA凝聚似乎与一组带正电的小蛋白质有关。已经开发出分离和鉴定这些蛋白质的方法。通过研究这些蛋白质与DNA的相互作用，有可能确定这些蛋白质如何与DNA结合，以及这种结合如何有助于DNA的折叠和组织成紧凑的结构。生物技术的许多发展使这些研究成为可能，包括DNA克隆和序列分析。这项研究的一个重要方面是培训这些生物技术领域的未来科学家，并协助他们获得必要的技能，以促进该国一个重要技术领域的发展。***

Abstract 9404955 Kinetoplastid protozoa contain a novel form of mitochondrial DNA (kinetoplast DNA or kDNA) consisting of thousands of minicircles and 20-30 maxicircles catenated into a single sheet or network. These kDNA networks are condensed in vivo, forming a highly organized disk-like structure about 1 um in diameter and 0.4 um thick. Previous studies have focused almost entirely on the novel DNA components of the kinetoplast and their organization into a catenated network. The research proposed here addresses the role of small histone-like proteins in the structural organization of the kinetoplast. Prior work in our laboratory has identified five small lysine-rich proteins associated with kDNA in vivo. Genes encoding three of these have already been cloned. The proposed research will lead to the cloning of the remaining genes and their use in determining the possible roles of these proteins in the structural organization of the kinetoplast. Each of these histone-like proteins will be produced in large amounts by expression in bacteria and used for producing antibodies and for biochemical studies of the interactions of each protein with DNA. These studies will determine the DNA-binding specificity of each protein and its ability to bend DNA, to induce supercoiling and to condense kDNA networks. Antibodies against these proteins will provide essential reagents for localizing each of the proteins within the cell using both immunofluorescence microscopy and immuno-electron microscopy. Through the use of this range of experimental approaches we expect to gain an understanding of the possible roles of histone-like proteins in organizing the kinetoplast structure. %%% Genetic information is encoded in living cells in extremely long strands of DNA that must be organized or "packaged" in some highly condensed and regular manner. This research program is aimed at identifying and analyzing some of the factors involved in condensing DNA within living cells. One of the m ost highly condensed forms of cellular DNA in nature is the DNA found in tropical parasitic organisms called trypanosomes. These organisms are widespread parasites of both plants and animals. One of their characteristic features is the presence of a large body of DNA called kinetoplast DNA (kDNA) which is located outside of the cell nucleus where chromosomes are found. Like the DNA of chromosomes, kDNA condensation appears to involve association with a group of small positively charged proteins. Methods have been developed for isolating and characterizing these proteins. Through the study of the interactions of these proteins with DNA, it may be possible to determine how these proteins bind to DNA and how this binding contributes to the folding and organizing of the DNA into a compact structure. These studies are made possible by the many developments in biotechnology including DNA cloning and sequence analysis. An important aspect of this research is the training of future scientists in these areas of biotechnology and assisting them to acquire the necessary skills to contribute to the growth of an important area of technology in this country. ***