FUNCTION OF KINETOPLAST IN THE HEMOFLAGELLATES

本项目的总体目标是了解 寄生虫动质体内线粒体的生物发生及其作用 动质粒DNA在这一过程中起重要作用。特别强调的是不寻常的RNA 发生在线粒体中的被称为RNA编辑的处理现象 这些细胞。具体目标如下： #1.体内和体外转录和编辑的脉冲追逐动力学。 未编辑、已编辑和部分已编辑的齐聚物将用于S1和 核糖核酸酶A保护实验测定RNA的相对速率 转录和RNA编辑，并确定是否部分编辑 RNA是真正的中间体。#2.测试几个涉及假设的模型 编辑模板。#3.体外编辑类系统的分析：(1) 利用预编辑优化体外RNA编辑类体系 细胞色素b RNA。将测试各种参数，以尝试 增加内部U添加的程度，而不是3‘端U添加的程度。(2) 底物Cyb RNA的缺失分析以确定推测的 特定切割的信号，可能还有U加成的信号。(3)测试 内部TL反应中额外的预编辑和未编辑的RNA U.COIII、MURF2和MURF3 5‘末端将用作预编辑的RNA，以及 ND1、COI、ND4、ND5和细菌质粒序列将被用作 未编辑的控件。富含GC的基因间区RNA也将进行测试。(4) 体外编辑的RNA中内部U‘s的定位。他的指纹 将使用特定的核糖核酸酶消化，以及生物素-亲和素选择 标记片段以及直接克隆和测序。(5)内部是否可以使用 通过添加线粒体膜来重建添加活性 澄清的海参素萃取物的级分？如果是这样的话，这部分将是 进行了分级，并确定了重建所需的组分。 (6)肝素处理的小鼠胸腺细胞Cyb RNA切割位点的测定 体外系统。裂解是由核酸内切酶还是自身裂解引起的？ 寻找一种可以作为“修剪”酶去除的核酸外切酶 编辑站点上有额外的U。#5.线粒体TUTase的纯化 和RNA连接酶。生产特定的抗血清。#6.隔离 线粒体蛋白质由编辑后的mRNAs产生。氨基酸序列 将获得与编辑区域相对应的区域。#7.RNA的进化 在动质体内编辑。动质体泛编辑的检测 来自其他属的植物。#8.对编辑前的序列进行计算机分析。搜索 关于富含GC的基因间隔区的其他编辑基因和信号 或可能参与编辑的二级结构。

The overall goal of this project is to understand the process of mitochondrial biogenesis in the parasite kinetoplastids and the role of the kinetoplast DNA in this process. Specific emphasis is on the unusual RNA processing phenomenon termed RNA editing which occurs in the mitochondrion of these cells. The specific aims are as follows: #1. In vivo and in vitro pulse-chase kinetics of transcription and editing. Unedited, edited and partially edited oligomers will be used in Sl and RNase A protection experiments to determine the relative rates of RNA transcription and RNA editing, and to determine if the partially edited RNAs are true intermediates. #2. Test of several models involving putative editing templates. #3. Analysis of the in vitro editing-like system: (1) Optimization of the in vitro RNA editing-like system using preedited cytochrome b RNA. Various parameters will be tested in an attempt to increase the extent of internal U addition versus 3' end U addition. (2) Deletion analysis of substrate CYb RNA in order to ascertain the putative signal for specific cleavage and possibly for U addition. (3) Testing additional pre-edited and non-edited RNAs in the TL reaction for internal U. The COIII, MURF2 and MURF3 5' ends will be used as pre-edited RNAs, and the ND1, COI, ND4, ND5 and a bacterial plasmid sequence will be used as non-edited controls. GC-rich intergenic region RNA will also be tested. (4) Localization of internal U's in the in vitro edited RNA. Fingerprints of specific RNase digests will be used, as well as biotin-avidin selection of labeled fragments and direct cloning and sequencing. (5) Can the internal U addition activity be reconstituted by addition of mitochondrial membrane fractions to the clarified Triton extract? If so, this fraction will be fractionated and the components necessary for reconstitution determined. (6) Determination of sites of cleavage of heparin-treated CYb RNA in the in vitro system. Is cleavage due to an endonuclease or to self-cleavage? #4. Search for an exonuclease that could act as a "trimming" enzyme to remove extra U's at sites of editing. #5. Purification of the mitochondrial TUTase and RNA ligase enzymes. Production of specific antisera. #6. Isolation of mitochondrial proteins produced from edited mRNAs. Amino acid sequences corresponding to edited regions will be obtained. #7. Evolution of RNA editing in the kinetoplastids. Detection of pan-editing in kinetoplastids from other genera. #8. Computer analysis of pre-edited sequences. Search for additional edited genes in GC-rich intergenic regions and for signals or secondary structures that could be involved in editing.