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[
Cell,
2019]
In this issue, Moore etal. and Posner etal., provide evidence for how the activity of the nervous system in C.elegans results in gene expression changes in the germline to pass on parental experiences and learned behavior to their progeny.
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Cortez, Angel, Hastie, Eric, Attix, Haley, Zarilla, Kathy, Cho, Martin, George, Alex, Panchal, Henali
[
MicroPubl Biol,
2021]
Research experiences in community college lead to increased retention in science, technology, engineering, and mathematics (STEM) (Nerio et al., 2019). This two week undergraduate research experience (URE) was designed to enhance laboratory skills in students with limited prior exposure, introduce developmental biology and genetics in a model organism system (C. elegans), and encourage participation in generation of data for a micropublication. The University of North Carolina at Chapel Hill and Durham Technical Community College partnered to host the URE for two weeks, for two hours, 4 days a week to limit lab time for students who work full time jobs. Here, we report our findings comparing early developmental cell division of wild type N2 embryos and a wild caught strain that was obtained from soil outside of Loeb Hall in Woods Hole, MA in 2017. The strain, originally called WH strain, was grown on OP50 and survived, suggesting it is a bacteriovore. The WH nematode lays embryos at the one cell stage, making early divisions observable without the dissection or bleaching required for the N2 strain. Students used primers to amplify the 18S ribosomal subunit geneused in phylogenetic analysis of taxafrom extracted genomic DNA and sent the product for sequencing (Floyd et al., 2005). The hairpin 17 region was selected to display a comparison because of high conservation (Nyaku et al., 2013). BLAST results for the N2 strain matched N2 and results for the wild caught WH strain matched with the nematode strain Acrobeloides sp. LKC 27 (a match of 99.7% and E value of 0), available from the Caenorhabditis Genetics Center. LKC 27 was isolated from a western corn rootworm from a Brookings, SD insectary in 2003 (personal communication with Dr. Lynn Carta, USDA-ARS). Students concluded that additional loci need to be examined to determine the relationship of the WH strain to LKC 27.
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[
J Nematol,
2006]
Chitinolytic microflora may contribute to biological control of plant-parasitic nematodes by causing decreased egg viability through degradation of egg shells. Here, the influence of Lysobacter enzymogenes strain C3 on Caenorhabditis elegans, Heterodera schachtii, Meloidogyne javanica, Pratylenchus penetrans, and Aphelenchoides fragariae is described. Exposure of C. elegans to L. enzymogenes strain C3 on agar resulted in almost complete elimination of egg production and death of 94% of hatched juveniles after 2 d. Hatch of H. schachtii eggs was about 50% on a lawn of L. enzymogenes strain C3 on agar as compared to 80% on a lawn of E. coli. Juveniles that hatched on a lawn of L. enzymogenes strain C3 on agar died due to disintegration of the cuticle and body contents. Meloidogyne javanica juveniles died after 4 d exposure to a 7-d-old chitin broth culture of L. enzymogenes strain C3. Immersion of A. fragariae, M. javanica, and P. penetrans juveniles and adults in a nutrient broth culture of L. enzymogenes strain C3 led to rapid death and disintegration of the nematodes. Upon exposure to L. enzymogenes strain C3 cultures in nutrient broth, H. schachtii juveniles were rapidly immobilized and then lysed after three days. The death and disintegration of the tested nematodes suggests that toxins and enzymes produced by this strain are active against a range of nematode species.
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[
STAR Protoc,
2021]
Animal experiences, including learned behaviors, can be passed down to several generations of progeny in a phenomenon known as transgenerational epigenetic inheritance. Yet, little is known regarding the molecular mechanisms regulating physiologically relevant transgenerational memories. Here, we present a method for <i>Caenorhabditis elegans</i> in which worms learn to avoid the pathogen <i>Pseudomonas aeruginosa</i> (PA14). Unlike previous protocols, this training paradigm, either using PA14 lawns or through exposure to a PA14 small RNA (P11), induces memory in four generations of progeny. For complete details on the use and execution of this protocol, please refer to Moore etal. (2019) and Kaletsky etal. (2020).
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[
International Worm Meeting,
2003]
In order to better understand cell fate determination in Caenorhabditis elegans, we are conducting a functional analysis of LIN-31, a winged-helix transcription factor (WH TF) that acts as a tissue-specific effector of the conserved Ras/MAP kinase signaling pathway to promote or suppress vulval cell fates in the development of the hermaphrodite vulva (Miller et al., Genes and Dev., 7:933, 1993). In addition to a DNA-binding domain (DBD), the LIN-31 protein contains several regions of interest: a small acidic-rich region, four MAP kinase consensus phosphorylation sites, and a small region at the C-terminus that displays homology with a subset of WH proteins. These regions could play a number of roles, from transcriptional activation to an interaction domain for LIN-1, which is known to heterodimerize with LIN-31 (Tan et al., Cell, 93:569,1998). Using site-directed mutagenesis techniques, specific mutations were introduced into the gene at these regions of interest. Stable transgenic lines were created through germline microinjection of mutant plasmids into animals with no functional LIN-31. Through phenotypic analysis of multiple transgenic lines, we are beginning to better understand the functional significance and contribution of each of these different sites to LIN-31 function. Our results thus far support the current model (Miller et al., 1993; Tan et al., 1998; Miller et al., Genetics, 156:1595, 2000), that LIN-31 has two functions: 1) to activate vulval cell fates in P5.p, P6.p and P7.p; and 2) to repress vulval cell fates in P3.p, P4.p, and P8.p.In addition, we are initiating an in vitro functional analysis of LIN-31 protein. We used a bacterial expression system to produce GST::LIN-31 fusion protein. Using electrophoretic mobility shift assays, we have determined that wild-type LIN-31 protein is able to specifically bind the promoter of another WH TF target. LIN-31's ability to interact with this promoter was disrupted when 1) LIN-31 carried a previously characterized point mutation in the DBD believed to disrupt its interaction with the target DNA (Miller et al., 2000) and 2) when the promoter sequence contained base substitutions. We are now in the process of creating, expressing, and purifying mutant GST::LIN-31 fusion proteins in order to investigate LIN-31 sequences required for heterodimerization with LIN-1.
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[
West Coast Worm Meeting,
2002]
In order to better understand cell fate determination in Caenorhabditis elegans, we are conducting a functional analysis of LIN-31, a winged-helix transcription factor (WH TF) that acts as a tissue-specific effector of the conserved Ras/MAP kinase signaling pathway to promote or suppress vulval cell fates in the development of the hermaphrodite vulva (Miller et al., Genes and Dev., 7:933, 1993). In addition to a DNA-binding domain (DBD), the LIN-31 protein contains several regions of interest: a small acidic-rich region, four MAP kinase consensus phosphorylation sites, and a small region at the C-terminus that displays homology with a subset of WH proteins. These regions could play a number of roles, from transcriptional activation to an interaction domain for LIN-1, which is known to heterodimerize with LIN-31 (Tan et al., Cell, 93:569,1998). Using site-directed mutagenesis techniques, specific mutations were introduced into the gene at these regions of interest. Stable transgenic lines were created through germline microinjection of mutant plasmids into animals with no functional LIN-31 protein. Through phenotypic analysis of multiple transgenic lines, we are beginning to better understand the functional significance and contribution of each of these different sites to LIN-31 function. Our results thus far support the current model (Miller et al., 1993; Tan et al., 1998; Miller et al., Genetics, 156:1595, 2000), that LIN-31 has two functions: 1) to activate vulval cell fates in P5.p, P6.p and P7.p; and 2) to repress vulval cell fates in P3.p, P4.p, and P8.p. In addition, we are initiating a functional analysis of LIN-31 protein using two assays: ability to bind a putative DNA target sequence and ability to heterodimerize with LIN-1. We used a bacterial expression system to produce GST::LIN-31 fusion protein. Using gel-shift assays, we confirmed function of wild-type protein by demonstrating its ability to bind the transthyretin (TTR) promoter, a consensus sequence recognized by HNF-3, another WH TF sharing DBD sequence homology (Costa et al., Mol. Cell. Biol., 9:1415, 1989). We are now in the process of creating, expressing, and purifying GST::LIN-31 fusion proteins carrying specific mutations, including two point mutations in the DBD believed to disrupt interaction of the LIN-31 with its target DNA (Miller et al., 2000). These mutant proteins will allow us to test in vitro their ability to bind the TTR promoter and to heterodimerize with LIN-1.
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[
PLoS One,
2012]
Programmed cell death (apoptosis) is essential for the development and homeostasis of metazoans. The central step in the execution of programmed cell death is the activation of caspases. In C. elegans, the core cell death regulators EGL-1(a BH3 domain-containing protein), CED-9 (Bcl-2), and CED-4 (Apaf-1) act in an inhibitory cascade to activate the CED-3 caspase. Here we have identified an additional component
eif-3.K (eukaryotic translation initiation factor 3 subunit k) that acts upstream of
ced-3 to promote programmed cell death. The loss of
eif-3.K reduced cell deaths in both somatic and germ cells, whereas the overexpression of
eif-3.K resulted in a slight but significant increase in cell death. Using a cell-specific promoter, we show that
eif-3.K promotes cell death in a cell-autonomous manner. In addition, the loss of
eif-3.K significantly suppressed cell death-induced through the overexpression of
ced-4, but not
ced-3, indicating a distinct requirement for
eif-3.K in apoptosis. Reciprocally, a loss of
ced-3 suppressed cell death induced by the overexpression of
eif-3.K. These results indicate that
eif-3.K requires
ced-3 to promote programmed cell death and that
eif-3.K acts upstream of
ced-3 to promote this process. The EIF-3.K protein is ubiquitously expressed in embryos and larvae and localizes to the cytoplasm. A structure-function analysis revealed that the 61 amino acid long WH domain of EIF-3.K, potentially involved in protein-DNA/RNA interactions, is both necessary and sufficient for the cell death-promoting activity of EIF-3.K. Because human eIF3k was able to partially substitute for C. elegans
eif-3.K in the promotion of cell death, this WH domain-dependent EIF-3.K-mediated cell death process has potentially been conserved throughout evolution.
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Doucette-Stamm L, Lamesch PE, Reboul J, Temple GF, Hartley JL, Brasch MA, Hill DE, Vaglio P, Thierry-Mieg N, Shin-i T, Lee H, Moore T, Vandenhaute J, Kohara Y, Vidal M, Jackson C, Thierry-Mieg J, Tzellas N, Thierry-Mieg D, Hitti J
[
Nat Genet,
2001]
The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or "ORFeomes," need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.AD - Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.FAU - Reboul, JAU - Reboul JFAU - Vaglio, PAU - Vaglio PFAU - Tzellas, NAU - Tzellas NFAU - Thierry-Mieg, NAU - Thierry-Mieg NFAU - Moore, TAU - Moore TFAU - Jackson, CAU - Jackson CFAU - Shin-i, TAU - Shin-i TFAU - Kohara, YAU - Kohara YFAU - Thierry-Mieg, DAU - Thierry-Mieg DFAU - Thierry-Mieg, JAU - Thierry-Mieg JFAU - Lee, HAU - Lee HFAU - Hitti, JAU - Hitti JFAU - Doucette-Stamm, LAU - Doucette-Stamm LFAU - Hartley, J LAU - Hartley JLFAU - Temple, G FAU - Temple GFFAU - Brasch, M AAU - Brasch MAFAU - Vandenhaute, JAU - Vandenhaute JFAU - Lamesch, P EAU - Lamesch PEFAU - Hill, D EAU - Hill DEFAU - Vidal, MAU - Vidal MLA - engID - R21 CA81658 A 01/CA/NCIID - RO1 HG01715-01/HG/NHGRIPT - Journal ArticleCY - United StatesTA - Nat GenetJID - 9216904SB - IM
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[
J Biol Chem,
1998]
Tyrosine O-sulfation, a common post-translational modification in eukaryotes, is mediated by Golgi enzymes that catalyze the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues in polypeptides. We recently isolated cDNAs encoding human and mouse tyrosylprotein sulfotransferase-1 (Ouyang, Y. B., Lane, W. S., and Moore, K. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2896-2901). Here we report the isolation of cDNAs encoding a second tyrosylprotein sulfotransferase (TPST), designated TPST-2. The human and mouse TPST-2 cDNAs predict type II transmembrane proteins of 377 and 376 amino acid residues, respectively. The cDNAs encode functional N-glycosylated enzymes when expressed in mammalian cells. In addition, preliminary analysis indicates that TPST-1 and TPST-2 have distinct specificities toward peptide substrates. The human TPST-2 gene is on chromosome 22q12.1, and the mouse gene is in the central region of chromosome 5. We have also identified a cDNA that encodes a TPST in the nematode Caenorhabditis elegans that maps to the right arm of chromosome III. Thus, we have identified two new members of a class of membrane-bound sulfotransferases that catalyze tyrosine O-sulfation. These enzymes may catalyze tyrosine O-sulfation of a variety of protein substrates involved in diverse physiologic functions.
-
[
International C. elegans Meeting,
2001]
We are investigating how genes predicted to be involved protein degradation effect embryogenesis in Caenorhabditis elegans . Within the cell, protein degradation is primarily accomplished through the ubiquitin-proteasome pathway. Studies in other systems show that E2 and E3 enzymes work in tandem to attach ubiquitin to a specific protein substrate, thereby condemning the substrate to degradation by the proteasome. We have identified 26 potential E2 genes within the completed genome of C. elegans . We are assessing the function of these genes through the use of RNAi-mediated interference (RNAi). E3 ligases are less conserved and more numerous than E2s. One class of E3 enzymes contains proteins with RING finger domains. We have previously identified 112 genes containing a RING finger in the C. elegans database. Four of the RING finger proteins were found to be required for embryogenesis (Moore, ECWM 2000, 154). By comparing E2 RNAi phenotypes with the RING finger mutant phenotypes, we hope to determine which E2 ubiquitin-conjugating enzymes partner with specific RING finger proteins. One of the four essential RING finger containing genes is
par-2 , a gene involved in establishing anterior-posterior polarity in the embryo. PAR-2 protein is localized asymmetrically to the posterior cortex in embryos. In order to understand if protein degradation is involved in PAR-2 localization, we are using a transgenic strain expressing PAR-2:GFP to observe PAR-2 localization in E2 RNAi embryos.