Stable expression of the bacterial neo r gene in Leishmania enriettii
Stable expression of the bacterial neo r gene in Leishmania enriettii
复制标题
细菌neo r基因在恩里氏利什曼原虫中的稳定表达
作者：
A. Laban;J. Tobin;M. Lafaille;D. Wirth
由于缺乏在寄生原生动物中引入和表达修饰基因或外源基因的方法，寄生原生动物的分子遗传学研究一直受到阻碍。最近的两篇报道描述了细菌氯霉素乙酰转移酶(CAT)基因在寄生虫特异性序列控制下的瞬时表达1，2。现在我们描述了一个可选择的标记，新霉素抗性基因(Neo R)在恩氏利什曼原虫中的稳定表达。将含有neo r基因的嵌合基因插入到细胞中，获得了稳定表达neo r基因的耐药恩氏杆菌。这项工作的一个目的是分析信使RNA的反式剪接所需的序列，因为尝试泛体有一种新的RNA反式剪接过程，最初在布鲁氏锥虫3-5中描述，随后在其他几种锥虫中描述，包括6号锥虫。许多尝试-泛体基因以串联阵列排列，基因间序列既包含用于添加剪接前导序列的剪接受体位点，也包含推测的多聚腺苷化位点6。从几个不同的新城疫霉菌株系中分离到的信使核糖核酸在α-微管蛋白基因间隔区的剪接受体位置上含有与neo r基因连接的剪接前导序列。Theneo-r mRNA也被多腺化。质粒DNA存在于耐药生物体内，似乎是染色体外的。这些方法的发展使反式剪接所需序列的功能分析成为可能。
MOLECULAR genetic studies in parasitic protozoa have been hindered by the lack of methods for the introduction and expression of modified or foreign genes in these organisms. Two recent reports described the transient expression of the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of parasite-specific sequences1,2. We now describe the stable expression of a selectable marker, the gene for neomycin resistance (neo r) in Leishmania enriettii. A chimaeric gene containing the neo r gene inserted between two α-tubulin intergenic sequences was introduced into the cells and drug-resistant L. enriettii were observed which stably expressed theneo r gene. One goal of this work was to analyse the sequences necessary fortrans-splicing of messenger RNA, as try-panosomatids have a novel process of RNA trans-splicing, described initially in Trypanosome brucei 3–5 and subsequently in several other trypanosomatids, including L. enriettii 6. Many try-panosomatid genes are arranged in tandem arrays and the intergenic sequences contain both the splice acceptor site for the addition of the spliced leader sequence and a putative polyadenylation site6. Messenger RNA isolated from several differentneo r L. enrietii lines contained the spliced leader sequence joined to the neo r gene at the position of the splice acceptor site in the α-tubulin intergenic sequence. Theneo r mRNA was also polyadenylated. Plasmid DNA is present within the drug-resistant organisms and appears to be extrachromosomal. The development of these methods allows the functional analysis of sequences necessary for trans-splicing.
编码 RNA 剪接前导序列的利什曼原虫基因的克隆和表征。
影响因子：
14.9
作者：
Miller,SI;Landfear,SM;Wirth,DF
通讯作者：
Wirth,DF