Abstract：
All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).
摘要：
Naegleria滋养体的所有核糖体基因都保持在含有封闭的环状染色体外核糖体DNA（rDNA）的元件（CERE）中。虽然对CERE知之甚少，对三种Naegleria物种的完整基因组序列分析清楚地表明，核基因组中没有rDNA顺反子。此外，一个单一的DNA复制起点已经被定位在N.gruberiCERE中，支持CERE独立于核基因组复制的假设。这种CERE特征表明，可能可以使用工程CERE将外源蛋白引入Naegleria滋养体。作为探索在Naegleria中使用CERE作为载体的第一步，我们开发了一种用克隆到pGEM7zf（pGRUB）中的N.gruberiCERE的分子克隆转染N.gruberi的方案。转染后，pGRUB很容易在N.gruberi滋养体中检测到至少七个传代，以及通过包装和包装。作为一种控制，用骨架载体转染滋养体，pGEM7zf+,没有N.gruberi序列(pGEM)。在转染入N.gruberi后的第一次传代后未检测到pGEM，表明它无法在真核生物中复制。这些研究描述了Naegleria滋养体的转染方案，并证明pGRUB中的细菌质粒序列不会抑制转染的CERE克隆的成功转染和复制。此外，这种转染方案对于理解驱动其在滋养体中复制的CERE的最小序列至关重要，以及识别非核糖体序列（NRS）中的调节区。
