FUNCTION OF KINETOPLAST IN THE HEMOFLAGELLATES

本项目的总体目标是了解 寄生虫动质体中线粒体的生物发生以及 DNA在这个过程中。特别强调的是不寻常的RNA RNA编辑是一种发生在细胞内的加工现象， 这些细胞。具体目标如下： #1.转录和编辑的体内和体外脉冲追踪动力学。 未编辑的、编辑的和部分编辑的低聚物将用于Sl和S2中。 RNA酶A保护实验，以确定RNA相对速率 转录和RNA编辑，并确定部分编辑的 RNA是真正的中间体。#2.几个模型的检验， 编辑模板。#3.体外编辑类系统分析：（1） 利用预编辑技术优化体外RNA编辑样系统 细胞色素B RNA。将测试各种参数， 相对于3'末端U加成，增加内部U加成的程度。（二） 底物CYb RNA的缺失分析，以确定推定的 用于特异性切割和可能用于U添加信号。(3)测试 在TL反应中额外的预编辑和非编辑RNA用于内部 联合C 0 III、MURF 2和MURF 3 5'末端将用作预编辑的RNA，并且 ND 1、COI、ND 4、ND 5和细菌质粒序列将用作 非编辑控件。还将检测富含GC的基因间区RNA。（四） 体外编辑的RNA中内部U的定位。指纹图谱 将使用特定的RNase酶，以及生物素-亲和素选择， 标记片段，直接克隆测序。(5)内部U 通过添加线粒体膜来重建添加活性 馏分澄清Triton提取物？如果是这样的话，这个分数将是 分馏并确定重构所需的组分。 (6)肝素处理的CYb RNA在细胞中切割位点的测定 体外培养系统切割是由于核酸内切酶还是由于自切割？#4. 寻找一种核酸外切酶，可以作为一种“修剪”酶， 在编辑的网站上有额外的U #5.线粒体TUTase的纯化 和RNA连接酶。特异性抗血清的产生。#6.分离 从编辑的mRNA产生的线粒体蛋白质。氨基酸序列 将获得与编辑的区域相对应的图像。#7. RNA的进化 在动质体中进行编辑动质体中泛编辑的检测 其他属。#8.预编辑序列的计算机分析。搜索 对于富含GC的基因间区域中的额外编辑基因和信号 或二级结构，可能参与编辑。

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.