Transformation of Mitochondria in Kinetoplastid Parasites

描述(申请人提供)：该项目建议开发动质体寄生虫线粒体的转化方法。动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(动泡体DNA或kDNA)由数千个称为小环和20-30个最大环的小DNA组成，编码与氧化代谢有关的蛋白质和线粒体核糖体RNA的基因。KDNA的复制和上环基因的表达包括一些基本序列，如启动小环和上环复制的复制起点，以及蛋白质表达、核糖体RNA合成和编辑上环转录本所需的引导RNA的合成所需的启动子序列。要分析这些DNA元件的功能，就必须能够在体外操纵这些序列，并能够将这些序列重新引入线粒体。通过DNA电穿孔将动质体寄生虫的细胞核转化到细胞核中，在分析染色体基因的调控和表达方面取得了巨大的成功。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粒体内线粒体基因的类似研究。最近的两项进展为转化动质体线粒体提供了一种很有前途的方法。首先，将DNA导入酵母和衣藻这种单细胞藻类线粒体的粒子枪方法的发展，应该适用于动质体线粒体的转化。其次，塔伦托利什曼原虫上颌环的一个基因编码一种突变形式的细胞色素c，已被证明对药物抗霉素A具有抗药性。该突变是通过在药物存在下塔伦托利什曼原虫的长期生长而选择的。我们建议用包裹了DNA的钨球进行粒子轰击，以获得含有该突变基因的质粒和/或线性DNA片段，用于转化野生型狼毒。DNA构建体可利用微环或上环复制起始点作为线粒体外切体进行复制，或通过重组插入上环作为完整的DNA片段。加入其他启动子序列，包括12S核糖体RNA的强启动子，可以提高抗霉素抗性基因的表达水平。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 DNA序列的方法。线粒体DNA携带着寄生虫新陈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.