A novelin vitro Caenorhabditis eleganstranscription system

Abstract

11Caenorhabditis elegans is an excellent model organism for biological research, but its contributions to 12 biochemical elucidation of eukaryotic transcription mechanisms have been limited. One of the biggest 13 obstacles for biochemical studies of C. elegans is the high difficulty of preparing functionally active nuclear 14 extract due to its thick surrounding cuticle. By employing Balch homogenization, we have achieved effective 15 disruption of larval and adult worms and have obtained functionally active nuclear extract through subcellular 16 fractionation. In vitro transcription reactions were successfully re-constituted using such nuclear extract. 17Furthermore, two non-radioactive detection methods, PCR and qRT-PCR, have been adapted into our system to 18 qualitatively and quantitatively detect transcription, respectively. Using this system to assess how pathogen 19 infection affects C. elegans transcription revealed that Pseudomonas aeruginosa infection increased 20 transcription activity. Our in vitro system is useful for biochemically studying C. elegans transcription 21 mechanisms and gene expression regulations. The effective preparation of functionally active nuclear extract in 22 our system fills a technical gap in biochemical studies of C. elegans and will expand the usefulness of this 23 model organism in addressing many biological questions beyond transcription. 24 25 2 Keywords 26 in vitro transcription, Caenorhabditis elegans, Balch homogenizer, subcellular fractionation, non-radioactive 27 detection 28 29 Background 30Caenorhabditis elegans is a free-living, 1-millimeter-long, nematode worm found in soil and decaying organic 31 matter. In 1963, Sydney Brenner proposed research into C. elegans, stating that "I would like to tame a small 32 metazoan organism to study development directly" (1). Since then, C. elegans has been used as a model 33 organism to address a wide range of biological questions such as those relating to development, metabolism, 34 neurobiology, and aging. The nematode has many characteristics that make it an excellent model system, 35 including, but not limited to, its rapid (3-day) life cycle, small size, ease of laboratory cultivation, genetic 36 tractability, invariant lineage, effectiveness of RNA interference, and transparent body that allows for 37 monitoring development or gene expression with single-cell resolution. Research involving the use of C. 38 elegans, including Brenner's work on organ development, was awarded with the Nobel Prize in 2002, 2006, and 39 2008.40 41Because of its genomic simplicity and physical characteristics, C. elegans offers a unique system to study 42 transcription mechanisms and regulation. For example, mutations in pre-initiation complex genes have been 43 recovered in genetic screens of C. elegans and have linked regulation that involves these factors to specific 44 biological processes (2); the nematode is transparent throughout its entire life cycle making it an ideal system to 45 use fluorescent protein reporter genes to monitor g...

Isolation of Active <em>Caenorhabditis elegans</em> Nuclear Extract and Reconstitution for <em>In Vitro</em> Transcription

“…Here a protocol for consistent isolation of functionally active nuclear extract from L4 C. elegans is described. Although this protocol focused on visualizing transcription activity, the cDNA produced post-transcriptionally can be quantified using RT-qPCR to obtain a more precise measurement of the transcription activity 8 . This method of isolating nuclear proteins from C. elegans can help expand the study of the eukaryotic transcription machinery.…”

Neuronal GPCR regulates the specificity of innate immunity against pathogen infection

“…Because of the high importance of transcription in the fundamental functions of life and the potential regulatory role of NMUR-1 in this process, we next examined how NMUR-1, or the lack thereof, impacts transcription. To this end, we developed a novel in vitro transcription system to measure C. elegans transcriptional activity (Wibisono et al, 2020). One of the biggest obstacles for biochemical studies of C. elegans is the high difficulty of preparing functionally active nuclear extract due to its thick surrounding cuticle.…”

“…In our transcription system, Balch homogenization was employed to effectively disrupt worms, and functionally active nuclear extracts were obtained through subcellular fractionation. Subsequently, the nuclear extracts were used to re-constitute in vitro transcription reactions with a linear DNA template containing the worm Δpes-10 promoter, followed by PCR-based, non-radioactive detection of transcripts (Wibisono et al, 2020). Using this system, we were able to compare the general transcriptional activity of nmur-1(ok1387) animals with that of wild-type animals at the whole-organism level.…”

“…To quantitatively compare the transcriptional activity of wild-type and mutant nuclear extracts, we next titrated the DNA template with varying amounts of each extract and used qRT-PCR to quantify transcripts at each titration point. The resulting data were then fitted with the Michaelis-Menten model and the non-linear least-squares method, as we described previously (Wibisono et al, 2020). The titration curves and two titration parameters, namely, the maximum yield and the amount of nuclear extract needed to reach 50% of the maximum yield, are shown in Fig.…”

“…In vitro transcription assays were performed following our published protocol (Wibisono et al, 2020). Briefly, nuclear extracts were prepared from synchronized young adult animals using Balch homogenization.…”