MOLECULAR GENETICS OF LEISHMANIA USING DNA TRANSFECTION

我们的实验室已经开发出一种DNA载体pr-neo，以及允许 将该DNA可重复稳定地导入人体寄生虫 重大利什曼原虫在氨基糖苷G418，a 这一过程称为转染法。初步数据显示，这种DNA存在 在染色体外，复制，表达正确处理的mRNA 带有与杂交基因相连的迷你外显子序列，在该基因中 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的表达，并在一定水平上表达该酶。 足以对药物G418(一种氨基糖苷类)产生抗药性 对新霉素)。这些特征允许分离转基因细胞。 平皿液体培养。此外，该质粒构建物可增加 在拷贝数量上应对药物压力的增加，并可 从转基因利什曼原虫中重新分离并重新导入大肠杆菌 以作进一步分析。这些发现使我们能够提议使用这种DNA 启动利什曼原虫的功能遗传学研究。 提出了实验的四个基本领域： 1)优化和进一步发展转染法， 包括发展瞬变测试。 2)分析转基因DNA的命运和结构，以及 其中编码的杂交RNA的结构和处理。 3)解剖Pr-neo向量，努力描绘最小 遗传要素，如起源、启动子、加工地点和要素 实行忠实的种族隔离。将注意测试 当前的分子模型，如多顺反子前mRNAs的发生 来自遥远的5‘端启动子的表达。 4)对Pr-neo向量的操作将允许 乘客DNA分子的表达，如cDNA或基因组DNA 细分市场。这种方法最终可能允许隔离和功能 检测利什曼原虫的其他基因。

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.