MOLECULAR GENETICS OF LEISHMANIA USING DNA TRANSFECTION

我们的实验室已经开发了一种DNA载体pR-NEO，以及允许 将这种DNA可重复地稳定导入人类寄生虫 用氨基糖苷类G418 a选择后的大型利什曼原虫 这个过程称为转染。 初步数据显示，这种DNA存在于 在染色体外复制，表达正确加工的mRNA 带有连接到杂合基因上的小外显子序列， 正常DHFR-TS mRNA的编码区被编码区取代 新霉素磷酸转移酶基因，并表达这种酶的水平， 足以赋予对药物G418（一种氨基糖苷类相关的 新霉素）。 这些特征允许分离转染的细胞， 平板液体培养。 此外，该质粒构建体可以增加 在拷贝数中响应于增加的药物压力，并且可以 从转染的利什曼原虫中重新分离并重新导入E.杆菌 以供进一步分析。 这些发现使我们能够利用这些DNA 启动利什曼原虫的功能性基因研究 提出了四个基本的实验领域： 1)转染方法的优化和进一步发展， 包括瞬态分析的发展。 2)分析转染DNA的命运和结构，以及 其中编码的杂合RNA的结构和加工。 3)pR-NEO载体的解剖，以描绘最小的 遗传元件，如起源、启动子、加工位点和元件 给予忠实的隔离。 将注意测试 目前的分子模型，如多顺反子前mRNA的出现， 从远端5'启动子表达。 4)pR-NEO载体的操作方式将允许 乘客DNA分子如cDNA或基因组DNA的表达 片段 这种方法可能最终允许隔离和功能 其他利什曼原虫基因的测试。

Our lab has developed a DNA vector, pR-NEO, and methods which allow the reproducible stable introduction of this DNA into the human parasite Leishmania major following selection with the aminoglycoside G418, a process termed transfection. Preliminary data show that this DNA exists extra-chromosomally, replicates, expresses a correctly processed mRNA bearing the mini-exon sequence attached to a hybrid gene in which the coding region of the normal DHFR-TS mRNA is replaced by the coding region of neomycin phosphotransferase gene, and expresses this enzyme at a level sufficient to confer resistance to the drug G418 (an aminoglycoside related to neomycin). These features permit the isolation of transfected cells, in liquid culture of on plates. Moreover, this plasmid construct can increase in copy number in response to increased drug pressure, and can be reisolated from the transfected Leishmania and reintroduced into E. coli for further analysis. These findings allow us to propose to use this DNA to initiate functional genetic studies of Leishmania. Four basic areas of experimentation are proposed: 1) optimization and further development of the methods of transfection, including the development of transient assays. 2) analysis of the fate and structure of the transfected DNAs, and the structure and processing of hybrid RNAs encoded therein. 3) dissection of the pR-NEO vector in an effort to delineate minimal genetic elements such as origins, promoters, processing sites and elements conferring faithful segregation. Attention will be given to testing current molecular models such as the occurrence of poly-cistronic pre-mRNAs expressed from a distant 5' promoter. 4) manipulation of the pR-NEO vector in a way that will allow the expression of passenger DNA molecules, such as cDNAs or genomic DNA segments. This approach may ultimately permit the isolation and functional testing of other Leishmania genes.