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[
J Biol Chem,
2014]
In a process known as quorum sensing, bacteria use chemicals called autoinducers for cell-cell communication. Population-wide detection of autoinducers enables bacteria to orchestrate collective behaviors. In the animal kingdom detection of chemicals is vital for success in locating food, finding hosts, and avoiding predators. This behavior, termed chemotaxis, is especially well studied in the nematode Caenorhabditis elegans. Here we demonstrate that the Vibrio cholerae autoinducer (S)-3-hydroxytridecan-4-one, termed CAI-1, influences chemotaxis in C. elegans. C. elegans prefers V. cholerae that produces CAI-1 over a V. cholerae mutant defective for CAI-1 production. The position of the CAI-1 ketone moiety is the key feature driving CAI-1-directed nematode behavior. CAI-1 is detected by the C. elegans amphid sensory neuron AWC(ON). Laser ablation of the AWC(ON) cell, but not other amphid sensory neurons, abolished chemoattraction to CAI-1. These analyses define the structural features of a bacterial-produced signal and the nematode chemosensory neuron that permit cross-kingdom interaction.
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[
International Worm Meeting,
2013]
Bacterial group behaviors are governed by a process called quorum sensing, in which bacteria produce, secrete, and detect extracellular signal molecules called autoinducers (AIs). Vibrios produce multiple AIs, some enable intra-species communication and others that promote inter-species communication. Vibrio cholerae produces an intra-species AI called CAI-1 that is a 13 carbon long fatty acyl molecule and the interspecies signal called AI-2 that is a boron-containing furanone. The information contained in the AIs is funneled into a shared phosphorelay signaling cascade that controls virulence, biofilm formation, and other traits. The bacteriovorous nematode, Caenorhabditis elegans, also uses small molecules to interpret its environment. A class of C. elegans-derived molecules called ascarosides influence nematode behaviors including attraction, repulsion, and mating. The presence of bacteria stimulates chemotaxis, egg-laying, and feeding in C. elegans, however, the bacteria-produced molecules that the nematode detects to control these phenotypes are largely unknown. We demonstrate that in addition to playing a vital role in quorum-sensing-regulated behaviors in V. cholerae, CAI-1 also influences behavior in C. elegans. C. elegans is more strongly attracted to V. cholerae than to its food source E. coli HB101 and C. elegans prefers V. cholerae that produces CAI-1 over a V. cholerae mutant for CAI-1 production. Consistent with this finding, robust chemoattraction occurs to synthetic CAI-1. CAI-1 is detected by the sensory neuron AWCON. Laser ablation of this cell, but not other amphid sensory neurons, abolished chemoattraction to CAI-1. To define which moieties of CAI-1 are crucial for recognition by C. elegans, we synthesized CAI-1 analogs and tested whether they promote chemoattraction. The fatty-acid chain length as and the precise position of the CAI-1 ketone group are the key features required for mediating CAI-1-directed nematode behavior. Together, these analyses define a bacteria-produced signal and the nematode detection apparatus that permit interkingdom communication.
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[
J Mol Histol,
2005]
NF-Y is a conserved trimer with histone-like subunits that binds and activates the common CCAAT promoter element.C.elegansNF-Y genes present two CeNF-YAs, a unique feature in kingdoms other than plants, one CeNF-YB and one CeNF-YC. The expression of both CeNF-YAs is restricted to the gonads and developing embryos, whereas the histone-like CeNF-YB- and CeNF-YC are also present in the pharyngeal bulb, in the neurons of ganglia surrounding the pharynx and in sensory organs of the head. Moreover, in infertile, 12-day-old worms, expression of the three subunits falls dramatically in the gonads. Our data indicate that NF-Y is not ubiquitously expressed.
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[
Biochim Biophys Acta,
2016]
BothDrosophila melanogaster and Caenorhabditis elegans (C. elegans) are useful model organisms to study in vivo roles of NF-Y during development. Drosophila NF-Y (dNF-Y) consists of three subunits dNF-YA, dNF-YB and dNF-YC. In some tissues, dNF-YC-related protein Mes4 may replace dNF-YC in dNF-Y complex. Studies with eye imaginal disc-specific dNF-Y-knockdown flies revealed that dNF-Y positively regulates the sevenless gene encoding a receptor tyrosine kinase, a component of the ERK pathway and negatively regulates the Sensless gene encoding a transcription factor to ensure proper development of R7 photoreceptor cells together with proper R7 axon targeting. dNF-Y also controls the Drosophila Bcl-2 (debcl) to regulate apoptosis. In thorax development, dNF-Y is necessary for both proper Drosophila JNK (basket) expression and JNK signaling activity that is responsible for thorax development. Drosophila
p53 gene was also identified as one of the dNF-Y target genes in this system. C. elegans contains two forms of NF-YA subunit, CeNF-YA1 and CeNF-YA2. C. elegans NF-Y (CeNF-Y) therefore consists of CeNF-YB, CeNF-YC and either CeNF-YA1 or CeNF-YA2. CeNF-Y negatively regulates expression of the Hox gene
egl-5 (ortholog of Drosophila Abdominal-B) that is involved in tail patterning. CeNF-Y also negatively regulates expression of the
tbx-2 gene that is essential for development of the pharyngeal muscles, specification of neural cell fate and adaptation in olfactory neurons. Negative regulation of the expression of
egl-5 and
tbx-2 by CeNF-Y provides new insight into the physiological meaning of negative regulation of gene expression by NF-Y during development. In addition, studies on NF-Y in platyhelminths are also summarized.
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[
East Asia Worm Meeting,
2010]
The study of protein interactions in the physiological context is a valuable tool for the investigation of biological regulatory mechanisms. Here, we are interested in the interactions between molecular motors and small adaptor proteins that might have a regulatory function. For example, our previous study shows a regulatory function of an active zone protein liprin-alpha (SYD-2) on UNC-104 (KIF1A). However, factors that control UNC-104/SYD-2 interactions are not known. Similarly, it is known that UNC-16 (JIP3) acts as a scaffold for kinesin-1 (KLC-2) regulating the transport of synaptic vesicles; while it is unknown which factors regulate the UNC-16/KLC-2 interaction. To investigate factors that activate or suppress the interactions between these kinesins and their adaptor proteins, we use a novel method BiFC (Bimolecular fluorescence complementation) in combination with forward and reverse mutagenesis. Here, we fuse fluorescent protein complementary fragments (hybrids of fluorophores) to each protein in the complex. In detail, we express the N-terminal half of a YFP fused to one protein and at the same time the C-terminal half fused to another protein in the complex. As it is known that YFP-N can complement with YFP-C to make a functional YFP, this method enables us to investigate physical interactions between two proteins in the living worm. Specifically, we use a native, pan-neuronal promoter pUnc104 to drive the expression of the following proteins in the nervous system of C. elegans: YN::SYD-2/UNC-104::YC, UNC-16::YN/KLC-2::YC as well as UNC-104::YN/UNC-104::YC. As a positive control we use bJUN::YN and bFOS::YC that are known to express and strongly interact in the nucleus. The investigation of UNC-104/UNC-104 interaction is of special interest as in the literature it is highly discussed whether this kinesin-3 exists as a monomer or dimer when activated or deactivated. The next important step in this research will be to use forward (EMS) and reverse (genome-wide RNAi screen) mutagenesis to identify genes that might disrupt the interaction between the BiFC pairs. Therefore we would be able to investigate novel regulators in the kinesin/adaptor protein complexes.
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[
EMBO J,
2013]
A key finding of modern ageing research is that our limitation in lifespan is more than the result of accumulated organismal decay. Lifespan is regulated by genetically defined chemosensory and endocrine pathways, which integrate signals that reflect the internal and external status of the animal. New findings by Liu and Cai unravel a role for the environmental gases oxygen and carbon dioxide in the regulation of lifespan homeostasis and thus a novel function of oxygen-chemosensory neurons in C. elegans.
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[
Crit Rev Biochem Mol Biol,
2012]
The CCAAT box promoter element and NF-Y, the transcription factor (TF) that binds to it, were among the first cis-elements and trans-acting factors identified; their interplay is required for transcriptional activation of a sizeable number of eukaryotic genes. NF-Y consists of three evolutionarily conserved subunits: a dimer of NF-YB and NF-YC which closely resembles a histone, and the "innovative" NF-YA. In this review, we will provide an update on the functional and biological features that make NF-Y a fundamental link between chromatin and transcription. The last 25 years have witnessed a spectacular increase in our knowledge of how genes are regulated: from the identification of cis-acting sequences in promoters and enhancers, and the biochemical characterization of the corresponding TFs, to the merging of chromatin studies with the investigation of enzymatic machines that regulate epigenetic states. Originally identified and studied in yeast and mammals, NF-Y - also termed CBF and CP1 - is composed of three subunits, NF-YA, NF-YB and NF-YC. The complex recognizes the CCAAT pentanucleotide and specific flanking nucleotides with high specificity (Dorn et al., 1997; Hatamochi et al., 1988; Hooft van Huijsduijnen et al, 1987; Kim & Sheffery, 1990). A compelling set of bioinformatics studies clarified that the NF-Y preferred binding site is one of the most frequent promoter elements (Suzuki et al., 2001, 2004; Elkon et al., 2003; Marino-Ramirez et al., 2004; FitzGerald et al., 2004; Linhart et al., 2005; Zhu et al., 2005; Lee et al., 2007; Abnizova et al., 2007; Grskovic et al., 2007; Halperin et al., 2009; Hakkinen et al., 2011). The same consensus, as determined by mutagenesis and SELEX studies (Bi et al., 1997), was also retrieved in ChIP-on-chip analysis (Testa et al., 2005; Ceribelli et al., 2006; Ceribelli et al., 2008; Reed et al., 2008). Additional structural features of the CCAAT box - position, orientation, presence of multiple Transcriptional Start Sites - were previously reviewed (Dolfini et al., 2009) and will not be considered in detail here.
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[
MicroPubl Biol,
2018]
Disrupting the function of sensory neurons of C. elegans can increase their lifespan (Apeld and Kenyon 1999). This effect is not limited to large-scale disruption, as ablation of single pairs of neurons have been shown to modify lifespan (Alcedo and Kenyon 2004; Lee and Kenyon 2009; Liu and Cai 2013). We tested whether silencing the neuron pair ASI with the tetanus toxin light chain (Tetx), as opposed to ablating it, could increase lifespan. Tetanus toxin disrupts neurotransmission by blocking the release of both small clear-core vesicles and large dense-core vesicles, but should not affect communication via gap junctions (Schiavo et al. 1992; McMahon et al. 1992). We expressed GFP::Tetx using the ASI-specific promoter
pgpa-4 (Figure Panel A) and conducted lifespan assays comparing animals with high fluorescence and undetectable fluorescence. Tetx in ASI extended lifespan in otherwise wild-type animals (Figure Panel B, Table 1, 14.9% average median lifespan increase across 5 replicates).
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Redemann, Stefanie, Ernst, Susanne, Ayloo, Swathi, Bringmann, Henrik, Schloissnig, Siegfried, Pozniakowski, Andrej, Hyman, Anthony A
[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
The variation of the expression level of a protein could provide a powerful tool to study protein function. However, there is no method that allows the precise control of protein levels under a native promotor in eukaryotes. We developed a method, which enables us to fine tune the protein expression levels in C. elegans by using synthetic genes with adapted codons. By modifying the codon usage of a gene, the Codon adaptation index (CAI) can be changed and the level of protein expression can be controlled. We used this method to regulate the expression of the G-protein regulator GPR-1/2, which is involved in force generation during spindle positioning in the first asymmetric cell division in C. elegans. By gradually increasing the amount of GPR-1/2, we found that the amount of force acting on the spindle in C. elegans embryos is directly related to the amount of the G protein regulator GPR1/2 in the cell. In C. elegans GPR-1/2 is found in a complex, the force-generating complex,which is thought to consist of at least three proteins: GPR-1/2, LIN-5 and a G-alpha protein. Since increasing the amount of GPR1/2 is sufficient to increase the force, this suggests that the other proteins are there in excess and that GPR-1/2 is the limiting component. The modification of the CAI is a good example of how the ability to over-express proteins is essential for identifying components that are limiting as opposed to permissive for force generation. This method provides the first way to control the level of protein expression levels in C. elegans, and the first method for overexpression of proteins in the C. elegans germline. With this method the protein levels of a protein of interest can be varied, while maintaining all the wild type genetic regulation and the wild type protein sequence.
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Xu MJ, Chen MX, Zhang YN, Zhang LL, Ai L, Chen SH, Tian LG, Chen JX, Guo J, Cai YC, Zhu XQ
[
Parasitol Res,
2011]
Trichinella spiralis is an important zoonotic nematode causing trichinellosis which is associated with human diseases such as malaise, anorexia, nausea, vomiting, abdominal pain, fever, diarrhea, and constipation. microRNAs (miRNAs) are endogenous small non-coding RNAs that play important roles in the regulation of gene expression. The objective of the present study was to examine the miRNA expression profile of the larvae of T. spiralis by Solexa deep sequencing combined with stem-loop real-time polymerase chain reaction (PCR) analysis. T. spiralis larvae were collected from the skeletal muscle of naturally infected pigs in Henan province, China, by artificial digestion using pepsin. The specific identity of the T. spiralis larvae was confirmed by PCR amplification and subsequent sequence analysis of the internal transcribed spacer of ribosomal DNA. A total of 17,851,693 reads with 2,773,254 unique reads were obtained. Eleven conserved miRNAs from 115 unique xsmall RNAs (sRNAs) and 12 conserved miRNAs from 130 unique sRNAs were found by BLAST analysis against the known miRNAs of Caenorhabditis elegans ( ftp://ftp.ncbi.nih.gov/genomes/Caenorhabditis_elegans ) and Brugia malayi dataset (
http://www.ncbi.nlm.nih.gov/genomeprj?Db=genomeprj&cmd=ShowDetailView&TermToSearch=9549 ) in miRBase, respectively. One novel miRNA with 12 precursors were identified and certified using the reference genome of B. malayi, while no novel miRNA was found when using the reference genome of C. elegans. Nucleotide bias analysis showed that the uracil was the prominent nucleotide, particularly at the 1st, 6th, 18th, and 23th positions, which were almost at the beginning, middle, and the end of the conserved miRNAs. The identification and characterization of T. spiralis miRNAs provides a new resource to study regulation of genes and their networks in T. spiralis.