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[
Arch Ophthalmol,
1985]
A 14-year-old native of Ethiopia with previously treated onchocerciasis moved to California where he was examined for evidence of persisting nematode infestation. Skin and conjunctival biopsy specimens initially disclosed no abnormalities. Subsequently, conjunctival nodules developed, and a biopsy specimen of one of these revealed microfilariae of Onchocerca volvulus lying adjacent to a necrotic eosinophilic granulomatous inflammatory nodule. To our knowledge, nodules of this type have not heretofore been reported to be a notable feature of ocular onchocerciasis. This type of inflammation has a relationship to degenerating microfilaria in onchocerciasis and in other nematode infestation.
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George, Alex, Attix, Haley, Cortez, Angel, Cho, Martin, Zarilla, Kathy, Panchal, Henali, Hastie, Eric
[
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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[
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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[
C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany,
2010]
The two centrosomes that are present during mitosis in most animal cells differ in age. Indeed, each centrosome contains one procentriole that formed in the very same cell cycle and one centriole that formed at an earlier stage: in the preceding cell cycle for one centrosome (the younger one) and at an earlier cell cycle for the other one (the older one). Results in flies and mice established that in some asymmetric stem cell divisions, the older centrosome is invariably inherited by the cell that keeps the stem cell fate. The relevance of this pattern of centriole inheritance and its importance for cell fate determination remains incompletely understood. The sperm donates the only two centrioles present in newly fertilized C. elegans embryos. These centrioles recruit pericentriolar material from maternal stores and enter the canonical centrosome duplication cycle, wh ich results in the formation of two centrosomes, each with a procentriole and a centriole, which ensure bipolar spindle assembly during mitosis. It is not known whether the two paternally contributed centrioles are inherited in a random fashion in later stages of development. To address this question, we performed marked mating experiments, whereby males expressing the centriolar marker GFP-SAS-4 were mated with wild-type hermaphrodites. As a result, the paternally contributed centrioles can be tracked in the embryo. Preliminary findings indicate that the inheritance of the paternally contributed centrioles is random during the first two embryonic divisions. We are extending this analysis to blastomeres at later stages of embryonic development.
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Slack, Frank J, Hillier, LaDeana, Kato, Masaomi, Ratsch, Gunnar, Leng, J, Gerstein, Mark, Ewing, Brent, High, Amber, Wang, Guilin, Robilotto, Rebecca, MacCoss, Michael, Zeller, Georg, Reinke, Valerie, Spencer, W Clay, Thompson, Owen, Strasbourger, Pnina, Henz, S, Green, Phil, Waterston, Robert H, Miller, III, David M, Merrihew, Gennifer
[
International Worm Meeting,
2011]
As part of the modENCODE consortium, we are characterizing the C. elegans transcriptome using tiling arrays, RNA-seq, RT-PCR and mass spectrometry. Our earlier studies on whole animals of various stages and conditions and on specific cells and tissues led to a much improved set of protein coding genes covering greater than 95% of all genes including more than 12,413 trans-spliced leaders, 20,515 different trans-spliced transcript start sites, 28,199 polyA sites, 111,786 confirmed splice junctions, >7,000 inferred non-coding (nc) RNAs, and over 50 new miRNAs (1-5). More recently, we have (1) analyzed biological replicates with RNA-seq for different stages and conditions, validating the observed expression levels; (2) closed gaps in RNA-seq coverage of weakly expressed genes with RT-PCR; (3) characterized the RNA content of more finely staged embryos with RNA-seq; (4) tested methods that deplete rRNA to allow direct analysis by RNA-seq of ncRNAs and smaller samples, such as specific embryonic cells and tissues; (5) analyzed polyA+ RNA from selected stages of C. briggsae, C. remanei, C. brenneri and C. japonica; (6) analyzed miRNAs under additional stresses and conditions; and (7) characterized the proteins present in 12 size fractions from 16 different stages and conditions. All of the data are available through the modENCODE Data Coordinating Center and increasingly through WormBase. Our goal is to provide the community with a comprehensive description of the transcripts of the C. elegans genome, providing information about their specific utilization where possible. References 1. Hillier et al. Genome Research PMID: 19181841 2. Gerstein et al Science PMID: 21177976 3. Lu et al. Genome Reseaarch PMID: 21177971 4. Allen et al. Genome Research PMID: 21177958 5. Spencer et al. Genome Research PMID: 21177967.
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[
International C. elegans Meeting,
1999]
One component of the basal transcription machinery is a 700kD complex called TFIID. TFIID is composed of TATA-binding protein (TBP; CeTBP in C. elegans 1 ) and several TBP-associated factors. The prevailing view is that this complex is expressed in all cells, at all stages, and that it plays an important role in promoter recognition and recruitment of additional components of the basal machinery to target genes. We have identified a variant of TBP (called
tlp-1 for T BP- l ike p rotein) in a yeast two-hybrid screen designed to identify proteins that interact with conserved C-terminal sequences of the pharynx identity factor PHA-4 2 . TBP variants have also been found in flies, humans and rodents 3 . Given the ubiquitous and fundamental role of TBP during transcription, why do animals need a second TBP-like protein? Our data suggest that
tlp-1 has an essential function during embryogenesis. First, we examined
tlp-1 expression. We defined the
tlp-1 gene structure and constructed a
tlp-1::GFP chimera. This gene is expressed widely from early embryogenesis until adulthood. By contrast, CeTBP::GFP expression does not initiate until the 3-fold stage. Second, we used RNA interference to determine the loss-of-function phenotypes of
tlp-1 and CeTBP . The phenotype of most
tlp-1(RNAi) embryos mimics the effects of blocking RNA polymerase II 4 : expression of an early zygotic GFP reporter construct is extinguished, E and MS blastomeres fail to gastrulate normally, and embryos arrest at about the 100-cell stage with little overt differentiation. CeTBP(RNAi) embryos, on the other hand, progress through embryogenesis and arrest as fully differentiated L1 larvae. In summary, we propose that i) PHA-4 activates transcription by targeting the basal transcription machinery via the PHA-4 C-terminus and ii) variant TBP-like proteins play an essential role during embryogenesis to mediate RNA polymerase II-dependent transcription. 1. Lichsteiner and Tjian, 1993. 2. Thanks to Bob Barstead and Phillip James for yeast two-hybrid reagents. 3. Crowley et al., 1993; Hansen et al., 1997; Ohbayashi et al., 1999. 4. Edgar et al, 1994; Powell-Coffman et al., 1996. Thanks to Spencer Wright for technical assistance.
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[
West Coast Worm Meeting,
2004]
As reported previously in worm meetings, to identify additional genes that act in the late stage of vulval differentiation and that act in P cell generation, we have performed two screens for suppressors of the Muv phenotype of a lf allele of
lin-31 , which acts downstream of the MAPK in the vulval induction pathway. After screening more than 100,000 haploid genomes, we isolated 10 suppressor mutations that have now been determined to be alleles of six genes. All alleles suppress the Muv phenotype ranging from 100% to less than 10%. We have used traditional and SNP mapping methods to locate these genes in the genome. Three of the genes have been cloned and studied extensively, and two others have been located to specific cosmid regions. Phenotype characterization and genetic interaction analysis suggest that none of these genes act directly downstream of the RTK/Ras-mediated cell signalling pathway for vulval induction. Instead, some of these may act in specifying the competence of vulval precursor cells for induction or morphogenesis. Positional cloning of a gene defined by two of the suppressors indicated that these are alleles of the
let-21 gene (2) which encodes an exchange factor for Rho GTPases that is homologous to mouse ECT2 and Drosophila Pebble. One of the alleles,
ku427 , appears to cause more P specific defects: Pn.p cells are frequently missing due to defects in migration as well as cytokinesis of P cells. This result is consistent with our previously published result that Rho family GTPases are involved in P cell migration and cytokinesis (3). While LET-21 appears to function both in cytokinesis and migration of P cells, UNC-73, an exchange factor that may act on both RHO and RAC small GTPases, seems to act only on migration. (1) Miller et al. (1993) Gene Dev. 7, 933-947. (2) Ohmachi et al 2000 MWM, Canevascini et al 2003 IWM (3) Spencer et al. (2001). Proc. Natl. Acad. Sci. USA 98, 13132-13137.
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[
International Worm Meeting,
2013]
Embryonic development is a tightly controlled and regulated process in many species, with a complex network of transcription factors regulating gene activity. In C. elegans, the process is so tightly controlled that the lineage of cell divisions is invariant and has been fully mapped (Sulston et al, 1983). The set of genetic messages at an embryo's earliest stage is completely maternal, transitioning over time to being generated completely from its own DNA. Determining which genes become active at specific time points provides insight into when key regulatory events occur, and determining the total network of gene expression in each cell type over time will help map out developmental processes unique to each tissue. Previous studies have utilized a combination of FACS, SAGE, and microarrays (Meissner et al, 2009; Spencer et al, 2011) to gain insight into what genes are expressed in late and mixed stage embryonic tissues. More recently, RNA-Seq has been used for expression studies, producing gene expression data with advantages over SAGE and microarrays such as a broader range of expression levels, differentiation between gene isoforms, and information on every transcript expressed (reviewed in Wang et al, 2009). We have previously shown the ability to isolate time synchronized whole embryos at specific time points throughout embryonic development (see abstract from Max Boeck), and we aim to do the same with isolated tissue types by utilizing a collection of GFP and mCherry labeled C. elegans strains (Murray et al, 2012; Sarov et al, 2012) that label individual cells and tissues. Using FACS to isolate specific cell subsets at discrete time points and RNA-Seq for transcript identification and quantification, we will be able to create a map of embryonic gene expression not only specific to individual cells and tissues, but specific to those cell and tissue types over time. This will build upon and complement previous data by adding temporal information and added sensitivity. Clustering of genes with similar expression patterns to genes with known roles in development, combined with emerging ChIP-Seq data, will help identify novel genes involved in specific developmental processes.