A novel in vitro Caenorhabditis elegans transcription system

Abstract

AbstractBackgroundCaenorhabditis elegansis 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 forC. elegansbiochemical studies is the high difficulty of obtaining functionally active nuclear extract due to its thick surrounding cuticle. AC. elegansin 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. AC. eleganstranscription 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.ResultsBy 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 affectsC. eleganstranscription revealed thatPseudomonas aeruginosainfection changes transcription activity in a promoter- or gene-specific manner.ConclusionsIn this study, we developed an in vitroC. eleganstranscription 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 studyingC. eleganstranscription mechanisms and gene expression regulation. The effective preparation of functionally active nuclear extract in our system fills a technical gap in biochemical studies ofC. elegansand will expand the usefulness of this model organism in addressing many biological questions beyond transcription.