Transformation of Mitochondria in Kinetoplastid Parasites

描述（由申请人提供）：本项目拟开发转化动质体寄生虫线粒体的方法。动质体原生动物是世界热带和亚热带地区毁灭性疾病的病原体，包括内脏和皮肤利什曼病、昏睡病和恰加斯病。这些寄生虫的线粒体DNA（动基体DNA或kDNA）由数千个称为微环和20 - 30个大环的小DNA组成，其编码参与氧化代谢的蛋白质和线粒体核糖体RNA的基因。kDNA的复制和大环基因的表达涉及必需序列，例如用于启动小环和大环复制的复制起点，以及用于蛋白质表达、核糖体RNA合成和用于编辑大环转录物所需的指导RNA合成的启动子序列。为了分析这些DNA元件的功能，有必要能够在体外操纵这些序列，并能够将这些序列引入线粒体。通过将DNA电穿孔到细胞核中来转化动质体寄生虫的细胞核，在分析染色体基因的调控和表达方面已经取得了巨大成功。这种方法无法转化细胞的线粒体，这阻碍了对其他真核生物和动质体中线粒体基因的类似研究。两个最近的发展提供了一个有前途的方法来转化动质体的线粒体。首先，一个粒子枪的方法引入DNA的酵母和衣原体，一个单细胞的线粒体的发展，应适用于转化的动质体的线粒体。第二，在利什曼原虫大环中编码细胞色素c突变形式的基因已被证明赋予抗霉素A药物的抗性。该突变体是通过L. tarentolae在药物的存在下。我们建议开发含有该突变基因的质粒和/或线性DNA片段，用于转化野生型L。用涂有DNA的钨球进行粒子轰击，DNA构建体将被创建用于使用小环或大环复制起点作为线粒体附加体复制，或通过重组插入大环作为整合的DNA片段复制。赋予抗霉素抗性的基因的表达水平将通过包含其它启动子序列而增加，所述其它启动子序列包括12S核糖体RNA的强启动子。这项拟议的研究将开发改变世界热带和亚热带地区常见寄生虫线粒体DNA序列的方法。线粒体DNA携带着寄生虫代谢和能量产生机制的遗传信息。了解这些寄生虫产生能量的分子和生化基础将有助于开发治疗寄生虫病的新药。

DESCRIPTION (provided by applicant): This project proposes to develop methods for transformation of the mitochondria of kinetoplastid parasites. The kinetoplastid protozoa are the causative agents of devastating diseases in tropical and sub-tropical parts of the world and include visceral and cutaneous leishmaniasis, sleeping sickness and Chagas disease. The mitochondrial DNA (kinetoplast DNA or kDNA) of these parasites consists of thousands of small DNAs termed minicircles and 20-30 maxicircles, which encode proteins involved in oxidative metabolism and genes for mitochondrial ribosomal RNAs. Replication of kDNA and expression of the maxicircle genes involves essential sequences such as replication origins for initiation of minicircle and maxicircle replication, and promoter sequences for protein expression, ribosomal RNA synthesis and for synthesis of guide RNAs required for editing of maxicircle transcripts. To analyze the function of such DNA elements it is necessary to be able to manipulate these sequences in vitro and to be able to introduce the sequences back into the mitochondria. Transformation of the nucleus of kinetoplastid parasites by electroporation of DNA into the nucleus of the cell has been enormously successful in analyzing the regulation and expression of chromosomal genes. The inability to transform the cell's mitochondria by this method has been an impediment to similar studies of mitochondrial genes in other eukaryotes as well as in kinetoplastids. Two recent developments offer a promising approach to transforming mitochondria of kinetoplastids. First, the development of a particle gun method for introducing DNA into the mitochondria of yeast and Chlamydomonas, a unicellular alga, should be applicable to transformation of the mitochondria of kinetoplastids. Second, a gene in the maxicircle of Leishmania tarentolae that encodes a mutant form of cytochrome c has been shown to confer resistance to the drug antimycin A. This mutation was selected by prolonged growth of L. tarentolae in the presence of the drug. We are proposing to develop plasmids and/or linear DNA fragments containing this mutant gene for transformation of wild- type L. tarentolae by particle bombardment with tungsten spheres coated with the DNA. DNA constructs would be created for replication as a mitochondrial episome using a minicircle or maxicircle replication origin or as an integrated DNA segment by re-combinational insertion into the maxicircle. The level of expression of the gene conferring antimycin resistance would be increased by the inclusion of other promoter sequences including the strong promoter for the 12S ribosomal RNA. The proposed research will develop methods for altering the DNA sequence of the mitochondrial DNA in parasites commonly found in tropical and sub-tropical areas of the world. The mitochondrial DNA carries the genetic information for the parasite's machinery for metabolism and energy production. An understanding of the molecular and biochemical basis for energy production in these parasites will aid in the development of novel drugs for the treatment of parasitic diseases.