Caenorhabditis elegans is an excellent model organism for biological research, but its contributions to biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest obstacles for C. elegans biochemical studies is the high difficulty of obtaining functionally active nuclear extract due to its thick surrounding cuticle. A C. elegans in vitro transcription system was once developed by Lichtsteiner and Tjian in the 1990s, but it has not become widely used, most likely because the transcription reactions were re-constituted with nuclear extract from embryos, not from larval or adult worms, and the method of Dounce homogenization used to prepare the nuclear extract could lead to protein instability. Besides Dounce homogenization, several other techniques were developed to break worms, but no transcription reactions were re-constituted following worm disruption using these approaches. A C. elegans transcription system with effective preparation of functionally active nuclear extract from larval or adult worms has yet to be established. Additionally, non-radioactive methods for detecting transcription as alternatives to the conventional radioactive detection also need to be adapted into such an in vitro system. By employing Balch homogenization, we achieved effective disruption of larval and adult worms and obtained functionally active nuclear extract through subcellular fractionation. In vitro transcription reactions were successfully re-constituted using such nuclear extract. Furthermore, a PCR-based non-radioactive detection method was adapted into our system to either qualitatively or quantitatively detect transcription. Using this system to assess how pathogen infection affects C. elegans transcription revealed that Pseudomonas aeruginosa infection changes transcription activity in a promoter- or gene-specific manner. In this study, we developed an in vitro C. elegans transcription system that re-constitutes transcription reactions with nuclear extract of larval or adult worms and can both qualitatively and quantitatively detect transcription activity using non-radioactive approaches. This in vitro system is useful for biochemically studying C. elegans transcription mechanisms and gene expression regulation. The effective preparation of functionally active nuclear extract in our system fills a technical gap in biochemical studies of C. elegans and will expand the usefulness of this model organism in addressing many biological questions beyond transcription.

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新型体外秀丽隐杆线虫转录系统

秀丽隐杆线虫是用于生物学研究的极好的模型生物，但是其对真核转录机制的生物化学阐明的贡献是有限的。秀丽隐杆线虫生化研究的最大障碍之一是由于其周围表皮较厚，很难获得具有功能活性的核提取物。线虫体外转录系统由Lichtsteiner和Tjian于1990年代开发，但尚未广泛使用，这很可能是因为转录反应是用胚胎的核提取物而不是幼虫或成年蠕虫重建的，用Dounce匀浆法制备核提取物可能导致蛋白质不稳定。除了Dounce均质化外，还开发了其他几种技术来破坏蠕虫，但是使用这些方法在蠕虫破坏后没有重新构建转录反应。有效建立来自幼虫或成虫的功能活性核提取物的秀丽隐杆线虫转录系统尚未建立。另外，还需要将用于检测转录的非放射性方法作为常规放射性检测的替代方法，以适应这种体外系统。通过使用Balch匀浆，我们实现了对幼虫和成虫的有效破坏，并通过亚细胞分级分离获得了功能活跃的核提取物。使用这种核提取物成功地重构了体外转录反应。此外，将基于PCR的非放射性检测方法应用于我们的系统，以定性或定量检测转录。使用该系统评估病原体感染如何影响秀丽隐杆线虫的转录，表明铜绿假单胞菌感染以启动子或基因特异性方式改变了转录活性。在这项研究中，我们开发了一种体外秀丽隐杆线虫转录系统，该系统利用幼虫或成年蠕虫的核提取物重新构成转录反应，并且可以使用非放射性方法定性和定量检测转录活性。该体外系统可用于生物化学研究秀丽隐杆线虫的转录机制和基因表达调控。在我们系统中有效制备功能活跃的核提取物，填补了秀丽隐杆线虫生化研究中的技术空白，并将扩大该模型生物在解决转录以外的许多生物学问题方面的实用性。