A novel in vitro Caenorhabditis elegans transcription system.
A novel in vitro Caenorhabditis elegans transcription system.
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DOI：
10.1186/s12860-020-00332-8
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发表时间：
2020-11-30
影响因子：
2.8
通讯作者：
Sun J
中科院分区：
文献类型：
--
作者：
Wibisono P;Liu Y;Sun J
秀丽隐杆线虫是生物学研究的优秀模式生物，但其对真核转录机制生化阐明的贡献有限。线虫生化研究的最大障碍之一是由于其周围角质层较厚而很难获得具有功能活性的核提取物。 Lichtsteiner和Tjian曾在20世纪90年代开发出秀丽隐杆线虫体外转录系统，但尚未得到广泛应用，很可能是因为转录反应是用胚胎核提取物而不是幼虫或成虫的核提取物重建的，而用于制备核提取物的杜恩斯均质化方法可能会导致蛋白质不稳定。除了杜恩斯均质化之外，还开发了几种其他技术来破坏蠕虫，但在使用这些方法破坏蠕虫后没有重建转录反应。尚未建立能够有效制备来自幼虫或成虫的功能活性核提取物的线虫转录系统。此外，作为传统放射性检测替代方案的用于检测转录的非放射性方法也需要适应这样的体外系统。通过采用 Balch 均质化，我们实现了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性的核提取物。使用这种核提取物成功地重建了体外转录反应。此外，我们的系统采用了基于 PCR 的非放射性检测方法来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线虫转录，结果表明铜绿假单胞菌感染以启动子或基因特异性方式改变转录活性。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，该系统用幼虫或成虫的核提取物重建转录反应，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化研究秀丽隐杆线虫转录机制和基因表达调控。在我们的系统中有效制备功能活性核提取物填补了秀丽隐杆线虫生化研究的技术空白，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用途。在线版本包含可在 10.1186/s12860-020-00332-8 获取的补充材料。
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. The online version contains supplementary material available at 10.1186/s12860-020-00332-8.
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DOI：
10.1895/wormbook.1.45.2
发表时间：
2013-06-04
期刊：
WormBook : the online review of C. elegans biology
影响因子：
--
作者：
Reinke V;Krause M;Okkema P
通讯作者：
Okkema P
DOI：
10.1073/pnas.96.2.715
发表时间：
1999-01-19
影响因子：
11.1
作者：
Tan, MW;Mahajan-Miklos, S;Ausubel, FM
通讯作者：
Ausubel, FM
影响因子：
2.9
作者：
Bhaskaran, Shylesh;Butler, Jeffrey A.;Rea, Shane L.
通讯作者：
Rea, Shane L.
DOI：
10.1016/0005-2787(74)90182-8
发表时间：
1974-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
COCHETME.M;CHAMBON, P
通讯作者：
CHAMBON, P
影响因子：
2.7
作者：
PowellCoffman, JA;Knight, J;Wood, WB
通讯作者：
Wood, WB