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[
International Worm Meeting,
2003]
Respiratory Complex II, or succinate dehydrogenase/fumarate reductase (SDH/FR), of the mitochondrial electron transport chain catalyses either oxidation of succinate under aerobic conditions or reduction of fumarate under microaerobic conditions. The Complex is composed of four polypeptides, Fp, Ip, cybL and cybS. Fp and Ip form the catalytic site while the cyb subunits catalyse electron transport. We chose the Fp subunit for RNA interference (RNAi) studies as it is expected to be a critical component in determining the catalytic function of Complex II. While C. elegans normally lives aerobically, with SDH activity predominating, worms can also survive microaerobic conditions, excreting metabolic end-products indicative of FR activity. We aim to assess which Fp subunit is responsible for FR activity. Three genes encode putative Fp subunits in C. elegans. We silenced the expression of each gene, individually and in combination, using the RNAi feeding method. No obvious phenotype was evident for any gene silenced individually, but simultaneous knockdown of the two closely-related Fp genes (C03G5.1 and C34B2.7) that have high homology to SDH/FR Fp subunits from nematode parasites, induced an 80% decrease in fecundity. The two genes are thus functionally redundant and important under aerobiosis. We also tested worm viability in the presence of sodium azide, an inhibitor of respiratory Complex IV. To date we have observed that silencing expression of the third putative SDH/FR Fp gene (F48E8.3) induced an increased trend in mortality. Further work is planned to identify which gene encodes the Fp subunit catalysing FR activity in the nematode.
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Nagai T, Teramoto T, Araki S, Zhao Y, Chang YF, Nakano M, Campbell RE, Ishihara T, Abdelfattah AS, Fujiwara M, Wu J
[
Science,
2011]
Engineered fluorescent protein (FP) chimeras that modulate their fluorescence in response to changes in calcium ion (Ca(2+)) concentration are powerful tools for visualizing intracellular signaling activity. However, despite a decade of availability, the palette of single FP-based Ca(2+) indicators has remained limited to a single green hue. We have expanded this palette by developing blue, improved green, and red intensiometric indicators, as well as an emission ratiometric indicator with an 11,000% ratio change. This series enables improved single-color Ca(2+) imaging in neurons and transgenic Caenorhabditis elegans. In HeLa cells, Ca(2+) was imaged in three subcellular compartments, and, in conjunction with a cyan FP-yellow FP-based indicator, Ca(2+) and adenosine 5'-triphosphate were simultaneously imaged. This palette of indicators paints the way to a colorful new era of Ca(2+) imaging.
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[
Genetics,
2019]
CRISPR-based genome editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry, or wrmScarlet in <i>Caenorhabditis elegans</i> This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, ssDNA donors (200 bp) are used to insert the 5' and 3' fragments of the FP in the locus of interest. In the second step, these sequences act as homology regions for homology-directed repair using a dsDNA donor (PCR product) containing the middle fragment, thus completing the FP sequence. In Nested CRISPR, the first step involving ssDNA donors is a well-established method that yields high editing efficiencies, and the second step is reliable because it uses universal crRNAs and PCR products. We have also used Nested CRISPR in a non-essential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step. In the search for modifications to optimize the method, we tested synthetic sgRNAs, but did not observe a significant increase in efficiency. To streamline the approach, we combined all Step 1 and Step 2 reagents in a single injection and were successful in 3 of 5 loci tested with editing efficiencies of up to 20%. Finally, we discuss the prospects of this method in the future.
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[
Mol Biochem Parasitol,
1994]
Complex II in adult mitochondria of the parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron-transport observed in these organelles. In the present study, cDNAs for the flavoprotein (Fp) subunits of complex II have been isolated, cloned and sequenced from both A. suum and the aerobic, free-living nematode, Caenorhabditis elegans. Additional sequence at the 3' end of the mRNAs was determined by the Rapid Amplification of cDNA Ends (RACE). Nucleotide sequence analysis of the A. suum cDNAs revealed a 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1935 nucleotides and a 3' untranslated region of 616 nucleotides including a poly(A) tail from a polyadenylation signal (AATAAA). The open reading frame encoded a 645 amino acid sequence, including a 30 amino acid mitochondrial presequence. The amino acid sequences for the Fp subunits from both organisms were very similar, even though the ascarid enzyme functions physiologically as a fumarate reductase and the C. elegans enzyme a succinate dehydrogenase. The ascarid sequence was much less similar to the Escherichia coli fumarate reductase. The sensitivity of other Fp subunits to sulfhydryl reagents appears to reside in a cysteine immediately preceding a conserved arginine in the putative active site. In both nematode sequences, this cysteine is replaced by serine even though the succinate dehydrogenase activity of both enzymes is still sensitive to sulfhydryl inhibition. A cysteine six residues upstream of the serine may be involved in the sulfhydryl sensitivity of the nematode enzymes. Surprisingly, in contrast to succinate dehydrogenase activity, the fumarate reductase activity of the ascarid enzyme was not sensitive to sulfhydryl inhibition, suggesting that the mechanism of the two reactions involves separate catalytic processes.
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[
International Worm Meeting,
2011]
Operons are common in prokaryotic genomes but, except for Nematoda, are uncommon in eukaryotes. Nematoda "operons" comprise gene clusters with short, approx. 100bp, intergenic regions. A discrete nascent transcript, transcribed from a major upstream promoter, undergoes trans-splicing and 5' addition of one of two possible spliced-leader sequences, followed by cis-splicing and finally translation of mature mRNA. Operons are common in C. elegans and approx. 15% of genes are clustered into operons. The significance of Nematoda operons is unclear although evidence suggests they may have arisen as a consequence of genome evolution and compaction - the "easy come, slow go" scenario. The classical view of operon transcription is of an upstream promoter generating a single "operon" transcript, however evidence suggests operon gene transcription maybe more complex. For example, microarray data indicates that correlated operon gene expression is weaker with increasing intergenic distance suggesting expression is influenced by intergenic sequence-located elements. Further evidence comes from comparison of expression data between fluorescent protein (FP) transcriptional reporters driven either by the upstream "promoter" or intergenic "internal promoter" sequences. These two reporter types generated different expression patterns indicating the presence of regulatory elements within the operon. As an alternative to these transcriptional reporters dissection of operon-clustered gene expression could be investigated using translational reporters generated directly from genomic clones. As they contain 5' and 3' flanking and intronic sequences such genomic clone-based reporters are more likely to contain all, or many, of the regulatory elements associated with the gene of interest (GOI). Furthermore, these reporters are ideal for analyzing operon transcriptional complexity as the size of the fosmid insert (approx. 40kb) easily accommodates an intact operon. Tagging each operon gene with a different FP would permit operon gene expression analyses within the genomic context of that operon. We have used counter-selection recombineering to insert seamlessly commercially-synthesized, C. elegans-codon-optimized FP genes into the 3' ends of genes within a number of different 3-gene operons each contained within a different fosmid clone. The suitability of these FP genes, as reporters for multiple tagging has been validated by both in vivo and in vitro experiments. Operon targets, with confirmed gene structures and preliminary expression pattern data available from published literature, have been identified and the corresponding, multi-gene-tagged fosmid-based reporters created in readiness for expression pattern analyses.
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[
Genetics,
2015]
A central goal in the development of genome engineering technology is to reduce the time and labor required to produce custom genome modifications. Here we describe a new selection strategy for producing fluorescent protein (FP) knock-ins using CRISPR/Cas9-triggered homologous recombination. We have tested our approach in Caenorhabditis elegans. This approach has been designed to minimize hands-on labor at each step of the procedure. Central to our strategy is a newly developed self-excising cassette (SEC) for drug selection. SEC consists of three parts: a drug-resistance gene, a visible phenotypic marker, and an inducible Cre recombinase. SEC is flanked by LoxP sites and placed within a synthetic intron of a fluorescent protein tag, resulting in an FP-SEC module that can be inserted into any C. elegans gene. Upon heat shock, SEC excises itself from the genome, leaving no exogenous sequences outside the fluorescent protein tag. With our approach, one can generate knock-in alleles in any genetic background, with no PCR screening required and without the need for a second injection step to remove the selectable marker. Moreover, this strategy makes it possible to produce a fluorescent protein fusion, a transcriptional reporter and a strong loss-of-function allele for any gene of interest in a single injection step.
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[
Genetics,
2023]
I outline a streamlined method to insert large, single-copy transgenes into the C. elegans genome using Recombination-Mediated Cassette Exchange (RMCE) that relies solely on drug selection yielding a homozygous fluorescent protein (FP) marked transgene in 3 generations (8 days) at high efficiency (>1 insertion per 2 injected P0 animals). Landing sites for this approach are available on four chromosomes in several configurations which yield lines marked in distinct cell types. An array of vectors permit creating transgenes using a variety of selection methods (HygR, NeoR, PuroR, and
unc-119) that yield lines expressing different colored FP tagged lines (BFP, GFP, mNG, and Scarlet). Although these transgenes retain a plasmid backbone and a selection marker, the inclusion of these sequences typically does not alter the expression of several cell specific promoters tested. However, in certain orientations promoters exhibits crosstalk with adjacent transcription units. In cases where crosstalk is problematic, the loxP-flanked fluorescent marker, plasmid backbone and hygR gene can be excised by crossing through germline Cre expressing lines also created using this technique. Finally, genetic and molecular reagents designed to facilitate customization of both targeting vectors and landing sites are also described. Together, the rRMCE toolbox provides a platform for developing further innovative uses of RMCE to create complex genetically engineered tools.
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Smogorzewska A, Schlabach M, Elledge SJ, Kunkel TA, Lach FP, Colaiacovo MP, Harper JW, Saito TT, Desetty R, Sowa ME, Clark AB
[
Mol Cell,
2010]
The Fanconi anemia (FA) pathway is responsible for interstrand crosslink repair. At the heart of this pathway is the FANCI-FAND2 (ID) complex, which, upon ubiquitination by the FA core complex, travels to sites of damage to coordinate repair that includes nucleolytic modification of the DNA surrounding the lesion and translesion synthesis. How the ID complex regulates these events is unknown. Here we describe a shRNA screen that led to the identification of two nucleases necessary for crosslink repair, FAN1 (KIAA1018) and EXDL2. FAN1 colocalizes at sites of DNA damage with the ID complex in a manner dependent on FAN1's ubiquitin-binding domain (UBZ), the ID complex, and monoubiquitination of FANCD2. FAN1 possesses intrinsic 5'-3' exonuclease activity and endonuclease activity that cleaves nicked and branched structures. We propose that FAN1 is a repair nuclease that is recruited to sites of crosslink damage in part through binding the ubiquitinated ID complex through its UBZ domain.
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[
Phytomedicine,
2016]
BACKGROUND: Propolis is a bioactive natural product collected by honeybees (Apis mellifera) from plant sources. PURPOSE: This study was undertaken to determine the effect of propolis extracts from arid region of Argentina, on the activity/expression of pro-inflammatory enzymes, and as potential free radical scavenger, antifungal and anthelmintic agent as well as to get a first insight into the polyphenolic profile of the active fractions. STUDY DESIGN/METHODS: Two propolis samples were collected in different time from hives located in Tucuman, Argentina. They are representative of the collection time of the raw material for phytotherapeutical purposes. Ethanolic extracts from both propolis were obtained. The PEEs were analyzed for total polyphenol (TP), non-flavonoid phenols (NFP) and flavonoid (FP) content followed by HPLC-DAD analysis and identification of components by HPLC-MS/MS(n). The potentiality as anti-inflammatory (LOX, COX, iNOS enzymes), antioxidant, antifungal and nematicidal was determined. RESULTS: PEEs contain high levels of TP, NFP and FP, including cinnamic acid, caffeic acid prenyl ester, caffeoyl dihydrocaffeate and caffeic acid 3,4-dihydroxyphenethyl ester, liquiritigenin, 2',4'-dihydroxychalcone and 2',4'-dihydroxy-3'-methoxychalcone. The PEEs in vitro reduced the activity of LOX and COX-2. Pretreatment of RAW 264.7 cells with PEEs before the induction of inflammatory state, inhibited NO overproduction and the iNOS protein expression was significantly decreased. The PEEs exhibited antioxidant, antifungal (Candida sp.) and nematicidal effect (C. elegans). CONCLUSION: These findings show the potential use of characterized PEEs from arid regions of Argentina as phytomedicine.
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[
Gene,
2010]
To meet the increasing need of simultaneously co-expressing two different genes in the same cell of transgenic Caenorhabditis elegans, here, we report the establishment of dicistronic vectors that contain an intercistronic region (ICR) of the C. elegans operon, CEOP5428. In these vectors, a green fluorescence protein (GFP) and a red FP (RFP) genes were placed in the first and second cistrons, respectively, which were separated by the ICR. Driven by the fibrillarin (
fib-1) or
myo-2 promoter, the GFP- and RFP-fusion proteins were consistently co-expressed in the entire worm cells or in the pharynx muscle cells of the transgenic worms, respectively. Our work demonstrates that ICR-containing dicistronic vectors could be developed into versatile co-expression systems in C. elegans for functional analysis of genes of interest.