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[
Genetics,
2020]
Differential gene expression across cell types underlies the development and cell physiology in multicellular organisms. <i>C. elegans</i> is a powerful, extensively used model to address these biological questions. A remaining bottleneck relates, however, to the difficulty to obtain comprehensive tissue-specific gene transcription data, since available methods are still challenging to execute and/or require large worm populations. Here, we introduce the <u>R</u>N<u>A</u><u>P</u>ol Dam<u>ID</u> (RAPID) approach, in which the Dam methyltransferase is fused to a ubiquitous RNA polymerase subunit in order to create transcriptional footprints <i>via</i> methyl marks on the DNA of transcribed genes. To validate the method, we determined the polymerase footprints in whole animals, sorted embryonic blastomeres and in different tissues from intact young adults by driving Dam fusion expression tissue-specifically. We obtained meaningful transcriptional footprints in line with RNA-seq studies in whole animals or specific tissues. To challenge the sensitivity of RAPID and demonstrate its utility to determine novel tissue-specific transcriptional profiles, we determined the transcriptional footprints of the pair of XXX neuroendocrine cells, representing 0.2% of the somatic cell content of the animals. We identified 2362 candidate genes with putatively active transcription in XXX cells, among which the few known markers for these cells. Using transcriptional reporters for a subset of new hits, we confirmed that the majority of them were expressed in XXX and identified novel XXX-specific markers. Taken together, our work establishes RAPID as a valid method for the determination of polymerase footprints in specific tissues of <i>C. elegans</i> without the need for cell sorting or RNA tagging.
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[
Proc Natl Acad Sci U S A,
2022]
In Darwin's and Mendel's times, researchers investigated a wealth of organisms, chosen to solve particular problems for which they seemed especially well suited. Later, a focus on a few organisms, which are accessible to systematic genetic investigations, resulted in larger repertoires of methods and applications in these few species. Genetic animal model organisms with large research communities are the nematode <i>Caenorhabditis elegans</i>, the fly <i>Drosophila melanogaster</i>, the zebrafish <i>Danio rerio,</i> and the mouse <i>Mus musculus.</i> Due to their specific strengths, these model organisms have their strongest impacts in rather different areas of biology. <i>C. elegans</i> is unbeatable in the analysis of cell-to-cell contacts by saturation mutagenesis, as worms can be grown very fast in very high numbers. In <i>Drosophila</i>, a rich pattern is generated in the embryo as well as in adults that is used to unravel the underlying mechanisms of morphogenesis. The transparent larvae of zebrafish are uniquely suited to study organ development in a vertebrate, and the superb versatility of reverse genetics in the mouse made it the model organism to study human physiology and diseases. The combination of these models allows the in-depth genetic analysis of many fundamental biological processes using a plethora of different methods, finally providing many specific approaches to combat human diseases. The plant model <i>Arabidopsis thaliana</i> provides an understanding of many aspects of plant biology that might ultimately be useful for breeding crops.
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[
Genes Nutr,
2006]
Beyond our inherited genetic make-up environmental factors are central for health and disease and finally determine our life span. Amongst the environmental factors nutrition plays a prominent role in affecting a variety of degenerative processes that are linked to aging. The exponential increase of non-insulin-dependent diabetes mellitus in industrialized nations as a consequence of a long-lasting caloric supernutrition is an expression of this environmental challenge that also affects aging processes. The most consistent effects along the environmental factors that slow down aging - from simple organisms to rodents and primates - have been observed for caloric restriction. In the yeast Saccharomyces cerevisiae, the fruit fly Drosophila melanogaster and the nematode Caenorhabditis elegans, sirtuins (silencing information regulators) have been identified to mediate as "molecular sensors" the effects of caloric restriction on aging processes. Sirtuins are NAD(+)-dependent deacetylases that are activated when e.g. cell energy status is low and the NAD(+) over NADH ratio is high. As a consequence transcription rates of a variety of genes including that of the apoptosis inducing p(53) gene are reduced. Moreover, in C. elegans, sirtuins were shown to interact with proteins of the insulin/IGF-1 signaling cascade of which several members are known to extend life span of the nematodes when mutated. Downstream targets of this pathway include genes that encode antioxidative enzymes such as Superoxide dismutase (SOD) whose transcription is activated when receptor activation by insulin/IGF is low or when sirtuins are active and the ability of cells to resist oxidative damage appears to determine their life span. Amongst dietary factors that activate sirtuins are certain polyphenols such as quercetin and resveratrol. Whereas their ability to affect life span has been demonstrated in simple organisms, their efficacy in mammals awaits proof of principle.
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Proc Natl Acad Sci U S A,
1978]
Embryogenesis of the free-living soil nematode Caenorhabditis elegans produces a juvenile having about 550 cells at hatching. We have determined the lineages of 182 cells by tracing the divisions of individual cells in living embryos. An invariant pattern of cleavage divisions of the egg generates a set of stem cells. These stem cells are the founders of six stem cell lineages. Each lineage has its own clock-i.e., an autonomous rhythm of synchronous cell divisions. The rhythms are maintained in spite of extensive cellular rearrangement. The rate and the orientation of the cell divisions of the cell lineages are essentially invariant among individuals. Thus, the destiny of cells seems to depend primarily on their lineage history. The anterior position of the site of origin of the stem cells in the egg relates to the rate of the cell cycle clock, suggesting intracellular preprogramming of the uncleaved egg. We used a technique that allows normal embryogenesis, from the fertilized egg to hatching, outside the parent under a cover glass. Embryogenesis was followed microscopically with Nomarski interference optics and high-resolution vidoe recording.
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Proc Natl Acad Sci U S A,
2021]
Electrical synapses are specialized structures that mediate the flow of electrical currents between neurons and have well known roles in synchronizing the activities of neuronal populations, both by mediating the current transfer from more active to less active neurons and by shunting currents from active neurons to their less active neighbors. However, how these positive and negative functions of electrical synapses are coordinated to shape rhythmic synaptic outputs and behavior is not well understood. Here, using a combination of genetics, behavioral analysis, and live calcium imaging in <i>Caenorhabditis elegans</i>, we show that electrical synapses formed by the gap junction protein INX-1/innexin couple the presynaptic terminals of a pair of motor neurons (AVL and DVB) to synchronize their activation in response to a pacemaker signal. Live calcium imaging reveals that <i>
inx-1</i>/innexin mutations lead to asynchronous activation of AVL and DVB, due, in part, to loss of AVL-mediated activation of DVB by the pacemaker. In addition, loss of <i>
inx-1</i> leads to the ectopic activation of DVB at inappropriate times during the cycle through the activation of the L-type voltage-gated calcium channel EGL-19. We propose that electrical synapses between AVL and DVB presynaptic terminals function to ensure the precise and robust execution of a specific step in a rhythmic behavior by both synchronizing the activities of presynaptic terminals in response to pacemaker signaling and by inhibiting their activation in between cycles when pacemaker signaling is low.
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RNA,
2007]
The GLD-2 family of poly(A) polymerases add successive AMP monomers to the 3'' end of specific RNAs, forming a poly(A) tail. Here, we identify a new group of GLD-2-related nucleotidyl transferases from Arabidopsis, Schizosaccharomyces pombe, Caenorhabditis elegans, and humans. Like GLD-2, these enzymes are template independent and add nucleotides to the 3'' end of an RNA substrate. However, these new enzymes, which we refer to as poly(U) polymerases, add poly(U) rather than poly(A) to their RNA substrates.
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[
Development,
2007]
Oocytes in the C. elegans gonad enlarge rapidly. During the stage of enlargement, they are transcriptionally quiescent, and it is not understood how they acquire large quantities of materials such as mRNA and protein. Enlarging oocytes are connected via cytoplasmic bridges to a large, younger population of transcriptionally active germ cells at various stages of mitosis and meiosis. We show here that there is a general streaming of gonad cytoplasm towards and into the enlarging oocytes, originating primarily from pachytene-stage germ cells. Because previous studies suggested that most or all of the pachytene germ cells have the potential to differentiate into oocytes, the pachytene cells appear to function transiently as nurse cells. Somatic gonadal cells that surround the germ cells do not appear essential for streaming. Instead, materials appear to be pulled into oocytes by forces generated either in, or adjacent to, the enlarging oocytes themselves. Streaming appears to be driven by the actomyosin cytoskeleton, although we show that populations of both microfilaments and microtubules are oriented in the direction of flow. Our study shows that oocyte enlargement in C. elegans differs significantly from that in Drosophila, where a small number of specialized nurse cells expel their contents into the enlarging oocyte.
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Cytoskeleton (Hoboken),
2013]
The gelsolin homology (GH) domain has been found to date exclusively in actin-binding proteins. In humans, three copies of the domain are present in CapG, five copies in supervillin, and six copies each in adseverin, gelsolin, flightless I and the villins: villin, advillin and villin-like protein. Caenorhabditis elegans contains a four-GH-domain protein, GSNL-1. These architectures are predicted to have arisen from gene triplication followed by gene duplication to result in the six-domain protein. The subsequent loss of one, two or three domains produced the five-, four-, and three-domain proteins, respectively. Here we conducted BLAST and hidden Markov based searches of UniProt and NCBI databases to identify novel gelsolin domain containing proteins. The variety in architectures suggests that the GH domain has been tested in many molecular constructions during evolution. Of particular note is flightless-like I protein (FLIIL1) from Entamoeba histolytica, which combines a leucine rich repeats (LRR) domain, seven GH domains, and a headpiece domain, thus combining many of the features of flightless I with those of villin or supervillin. As such, the GH domain superfamily appears to have developed along complex routes. The distribution of these proteins was analyzed in the 343 completely sequenced genomes, mapped onto the tree of life, and phylogenetic trees of the proteins were constructed to gain insight into their evolution. 2013 Wiley Periodicals, Inc.
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[
Bioinformatics,
2007]
MOTIVATION: Despite many years of research on how to properly align sequences in the presence of sequencing errors, alternative splicing and micro-exons, the correct alignment of mRNA sequences to genomic DNA is still a challenging task. RESULTS: We present a novel approach based on large margin learning that combines accurate plice site predictions with common sequence alignment techniques. By solving a convex optimization problem, our algorithm - called PALMA - tunes the parameters of the model such that true alignments score higher than other alignments. We study the accuracy of alignments of mRNAs containing artificially generated micro-exons to genomic DNA. In a carefully designed experiment, we show that our algorithm accurately identifies the intron boundaries as well as boundaries of the optimal local alignment. It outperforms all other methods: for 5702 artificially shortened EST sequences from C. elegans and human it correctly identifies the intron boundaries in all except two cases. The best other method is a recently proposed method called exalin which misaligns 37 of the sequences. Our method also demonstrates robustness to mutations, insertions and deletions, retaining accuracy even at high noise levels. AVAILABILITY: Datasets for training, evaluation and testing, additional results and a stand-alone alignment tool implemented in C++ and python are available at
http://www.fml.mpg.de/raetsch/projects/palma. -
[
J Neurogenet,
2020]
Mechanosensation such as touch, hearing and proprioception, is functionally regulated by mechano-gated ion channels through the process of transduction. Mechano-gated channels are a subtype of gated ion channels engaged in converting mechanical stimuli to chemical or electrical signals thereby modulating sensation. To date, a few families of mechano-gated channels (DEG/ENaC, TRPN, K<sub>2</sub>P, TMC and Piezo) have been identified in eukaryotes. Using a tractable genetic model organism <i>Caenorhabditis elegans</i>, the molecular mechanism of mechanosensation have been the focus of much research to comprehend the process of mechanotransduction. Comprising of almost all metazoans classes of ion channels, transporters and receptors, <i>C. elegans</i> is a powerful genetic model to explore mechanosensitive behaviors such as touch sensation and proprioception. The nematode relies primarily on its sensory abilities to survive in its natural environment. Genetic screening, calcium imaging and electrophysiological analysis have established that ENaC proteins and TRPN channel (TRP-4 protein) can characterize mechano-gated channels in <i>C. elegans</i>. A recent study reported that TMCs are likely the pore-forming subunit of a mechano-gated channel in <i>C. elegans</i>. Nevertheless, it still remains unclear whether Piezo as well as other candidate proteins can form mechano-gated channels in <i>C. elegans</i>.