Permeable trypanosome cells as a model system for transcription and trans-splicing.
Permeable trypanosome cells as a model system for transcription and trans-splicing.
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可渗透的锥虫细胞作为转录和转拼的模型系统。
DOI：
10.1093/nar/18.11.3319
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发表时间：
1990
影响因子：
14.9
通讯作者：
Tschudi,C
中科院分区：
文献类型：
--
作者：
Ullu,E;Tschudi,C
我们已经建立了布氏锥虫可渗透细胞的条件来研究转录和转剪接。我们发现，单价离子的浓度，以及在较小程度上，二价离子的浓度对许多不同基因的表达起着关键作用。最值得注意的是，剪接的前导（SL）RNA的合成在20 mM KCl时是最佳的，而较高的钾浓度是抑制性的。此外，MgCl 2浓度高于3 mM时，会导致3′端缩短的SL RNA物质的积累，之前曾报道其不参与反式剪接（20）。使用SL RNA合成的最佳条件，我们观察到新合成的α-微管蛋白RNA的精确反式剪接。此外，我们检测到SL内含子既以分支Y结构的形式连接到高分子量RNA，又作为自由的线性分子，其迅速翻转。此外，抑制SL RNA合成的离子浓度仅产生未剪接的α-微管蛋白RNA，从而证明转录和反式剪接可以解偶联。
We have established conditions forTrypanosoma bruceipermeable cells to study transcription and transsplicing. We found that the concentration of monovalent and, to a lesser extent, divalent ions plays a critical role for the expression of a number of different genes. Most remarkably, the synthesis of the spliced leader (SL) RNA was optimal at 20 mM KCI, whereas higher potassium concentrations were inhibitory. In addition, MgCL2concentrations above 3 mM led to the accumulation of a 3′ end shortened SL RNA species, which has been previously reported not to participate in trans-splicing (20). Using conditions optimal for the synthesis of the SL RNA, we observed accurate transsplicing of newly-synthesized α-tubulin RNA. Moreover, we detected the SL intron both joined to high molecular weight RNAs in the form of branched Y-structures and as a free linear molecule, which rapidly turned over. Furthermore, ionic concentrations that inhibit the synthesis of the SL RNA produced exclusively unspliced α-tubulin RNA, thus demonstrating that transcription and trans-splicing can be uncoupled.