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[
International Worm Meeting,
2007]
The investigation of parasitic nematode biology has been hampered by the lack of tools for genetic analysis and manipulation. Nematodes of the genus Parastrongyloides have an unusual life history composed of a conventional parasitic life cycle as gastrointestinal parasites and a completely free-living life cycle. The switch between life cycles is determined by environmental conditions (see Grant & Stasiuk, this meeting), so that under appropriate conditions these worms can be maintained indefinitely as free-living nematodes. We have taken advantage of this free-living life cycle to produce transgenic Parastrongyloides trichosuri, a parasite of the Australian brush tail possum. The free living female adult of P. trichosuri is similar to adult C. elegans hermaphrodites: the reproductive anatomy is a central vulva with bilaterally symmetrical gonad arms extending posterior and anterior. The gonad arms a reflexed, with a gonadal syncytium at the distal end of each arm. Transgenic worms were generated by microinjection of DNA into the gonadal syncytium and recovery of transgenic progeny in the F1. Inheritance of the transgene is consistent with the formation of extrachromosomal arrays, as in C. elegans , so that a proportion of progeny from a transgenic mother carry the transgene. Transmission of the transgene occurs in the parasitic as well as the free living life cycle. The transgene expression is low, perhaps a result of silencing, but using a constitutive promoter derived from the P. trichosuri orthologue of the
hsp-1 gene we have also shown expression in both the parasitic and the free-living life cycles. This technology may permit manipulation of the host-parasite relationship and/or the delivery of bioactive proteins to the host by a transgenic worm: we present evidence that a protein encoded by a transgene and expressed during the parasitic life cycle can elicit a biological response from the host infected with the transgenic worms.
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[
International Worm Meeting,
2015]
Alzheimer's disease (AD) is an age-related neurodegenerative disorder that is the most common cause of dementia in humans. A characteristic feature of AD is the formation of neuronal extracellular senile plaques composed of aggregates of fibrillar amyloid beta (Abeta) peptides. The senile plaques are found in a range amino-truncated and post translationally forms, with Abeta1-42 peptide being the most abundant species. These Abeta peptides have been proposed to contribute to the pathophysiology of the disease; however, there are few tools available to test this hypothesis directly. This project aims to use the model organism, Caenorhabditis elegans, as an in vivo system to study the toxicity associated with these Abeta species. Although C. elegans has been used previously, this is the first report of pan-neuronal transgenic expression of human Abeta1-42. C. elegans strains generated carrying transgenic Abeta1-42 were initially characterized to determine the number of copies of Abeta, from which a number of strains with different level of variation in copy number were identified. A selected strain was subsequently analyzed at the transcript level by quantitative real time PCR (qRT-PCR), which confirmed that that Abeta was being expressed. The effect of neuronal Abeta expression was further characterized using a chemotaxis behavioral assay, which demonstrated minor chemotactic defects in the transgenic C. elegans strains, for all the chemo-attractant compounds tested. Current work is focused on investigating different pan-neuronal gene promoter systems to influence the timing and level of Abeta expression. This work demonstrates the feasibility of expressing human Abeta1-42 in a tractable in vivo system, and in turn, provides a new tool to investigate the in vivo toxicity of Abeta to further enable the study of the molecular and cellular mechanisms underlying AD progression. These strains may also be used in subsequent screens to develop novel therapeutics to treat Alzheimer's disease.
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[
International C. elegans Meeting,
2001]
Helix-loop-helix (HLH) proteins are transcription factors that are involved in cellular specification, differentiation, and differential gene expression in development. Normal pancreatic functioning, brain and eye morphogenesis, and skeletal muscle development are just some aspects of human development that are regulated by HLH proteins. The nematode, Caenorhabditis elegans, is an ideal model to study myogenic related HLH proteins since it has a simple genetic system and is known to produce numerous transcriptional regulators. After identifying six DNA sequences that are likely to encode myogenic HLH genes in C. elegans, we used RT-PCR, RNA interference, and promoter fusions to determine which of the genes are expressed, and the timing and localization of their expression. (Research supported by NSF grant # MCB9986640 and by MBRS/RISE grant # R25GM58094)
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[
European Worm Meeting,
2006]
Petr Liby1, Marketa Kostrouchova1, Michal Pohludka1, Jaroslav Vohanka2, Eva Brozova2, Marta Kostrouchova2 and Zdenek Kostrouch1. BIR-1 is a member of the Inhibitors of Apoptosis protein family that is involved in the regulation of microtubule organization, condensation of chromosomes and cell division in C. elegans as well as in vertebrates. In C. elegans,
bir-1 is organized in an operon together with SKIP, a transcription and splicing cofactor.
bir-1 inhibition induces developmental defects including dpy and egl phenotypes. Here we studied the effect of
bir-1 inhibition by RNAi in L1 larval stage using whole genome microarrays (Affymetrix). Microarrays detected changes in expression of several developmentally active collagen genes in
bir-1 inhibited larvae. We further characterized the effect of
bir-1 inhibition on selected collagen gene expression and induction of dpy phenotype. Acknowledgement: We thank Drs. A. Fire for GFP construct, vectors and host used in RNAi and M.W. Krause for support and advice. We thank the NIDDK Microarrays facility for performing the microarrays analysis. The work was supported by the grant 303/03/0333 from the Czech Science Foundation, by the grant NC-7554 from the Ministry of Health of the Czech Republic and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.
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[
International Worm Meeting,
2019]
Dopamine is an important neurotransmitter that regulates a myriad of brain functions ranging from motor control to memory and learning. Dysregulation of dopamine is associated with multiple disorders including Parkinson's disease (PD), attention deficit hyperactivity disorder (ADHD), addiction, and schizophrenia, among others. In order to better understand dopamine function at the molecular level, we are investigating the roles and regulation of dopaminergic genes using Caenorhabditis elegans (C. elegans) as a model system. We are conducting swimming induced paralysis (SWIP) assays using a computer-aided video analysis system, allowing us to collect more nuanced data to uncover new phenotypes and discover roles for genes involved in dopaminergic signaling. We have selected eight genes to focus on in this study. Each of these eight genes is highly enriched in dopamine neurons, based on RNASeq data from our laboratory, and mutant strains for these genes show accelerated swimming-induced paralysis when compared to the wild-type strain, N2, a phenotype characteristic of hyperdopaminergic signaling. Based on our computer-aided video analysis, we propose to gain more insight into specific rates and patterns of paralysis of these mutant strains in an effort to identify novel genes critical for dopaminergic functioning. Funding support from NSF CREST Grant #HRD1547757, NIH BD2K R25 Grant #1R25MD010396-01, and DOE Title VII Grant MD-HBCU #P382G090004.
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[
International C. elegans Meeting,
2001]
To build upon knowledge gained from the genome of C. elegans , we have begun generating Expressed Sequence Tags (ESTs) from parasitic (and free-living) nematodes. This project will generate >225,000 5' ESTs from 14 species by 2003. Additionally, the Sanger Centre and Edinburgh Univ. will complete 80,000 ESTs from 7 species. Through these combined efforts, we anticipate the identification of >80,000 new nematode genes. At the GSC, approximately 35,000 ESTs have been generated to date including sequences from Ancylostoma caninum, Heterodera glycines, Meloidogyne incognita and javanica, Parastrongyloides trichosuri, Pristionchus pacificus, Strongyloides stercoralis and ratti, Trichinella spiralis, and Zeldia punctata . We will report on our progress in sequence analysis, including the creation of the NemaGene gene index for each species by EST clustering and consensus sequence generation, identification of common and rare transcripts, and identification of genes with orthologues in C. elegans and other nematodes. All sequences are publicly available at www.ncbi.nlm.nih.gov/dbEST. NemaGene sequences and project details are available at WWW.NEMATODE.NET. We would like to thank collaborators who have provided materials and ideas for this project including Prema Arasu, David Bird, Rick Davis, Warwick Grant, John Hawdon, Doug Jasmer, Andrew Kloek, Thomas Nutman, Charlie Opperman, Alan Scott, Ralf Sommer, and Mark Viney. This work is funded by NIH-AI-46593, NSF-0077503, and a Merck / Helen Hay Whitney Foundation fellowship.
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[
International C. elegans Meeting,
1995]
The
lin-12 gene plays a central role in several different cell fate decisions requiring cell interactions and may encode a receptor for intercellular signals.
lin-12(
n676n930) is a hypomorphic allele of
lin-12 at 25C. Five sel genes, including
sel-9 and
sel-10, were isolated in a screen for suppressors of
lin-12(
n676n930), and therefore may contribute to cell fate decisions mediated by
lin-12 (see also abstract by Grant and Greenwald).
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[
European Worm Meeting,
2006]
Marketa Kostrouchova1, Petr Liby1, Marta Kostrouchova2 and Zdenek Kostrouch1. Valproic acid (VPA, 2-propylpentanoic acid), a drug widely used in the treatment of epilepsy and bipolar disorder, has been shown to posses anti-cancer activity. VPA suppresses tumor growth, induces tumor cell differentiation and reduces the formation of metastases. Multiple mechanisms were shown to be part of VPAs mode of action, including inhibition of histone deacetylase activity, inhibition of glycogen synthase 3 and interaction with the regulation of nuclear hormone receptors. We studied the effect of VPA on the development and gene expression in C. elegans. VPA delivered to worms by microinjections to the ovarial syncytium or by incubating worms in various concentrations of VPA (1 to 10 mM) caused a dose dependent delay of larval development and lethality in two developmental stages: in late embryogenesis and in L1 larval stage. Surprisingly, most animals were able to overcome the effect of VPA, suggesting a compensatory or balancing mechanism. Microarray experiments show that genes affected by VPA can be divided to at least three different categories suggesting multiple mechanisms to be involved in VPA mode of action. Acknowledgement: We thank Dr. M.W. Krause for support and advice. We thank the NIDDK Microarrays facility for performing the microarrays analysis. The work was supported by the grant 303/03/0333 from the Czech Science Foundation, by the grant NC-7554 from the Ministry of Health of the Czech Republic and by the grant 0021620806 from the Ministry of Education, Youth and Sports of the Czech Republic.
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[
International C. elegans Meeting,
2001]
The helix-loop-helix (HLH) family of transcription factors is responsible for the expression of genes that regulate many aspects of development in eukaryotes including cellular differentiation and specification, morphogenesis, and growth. The nematode, Caenorhabditis elegans, possesses many different transcription factors, including HLH proteins, many of which have been identified and characterized. Through Blast searches, we have identified putative HLH-encoding genes in the C. elegans genome, some of which are homologous, at varying degrees of identity and similarity, to members of the Achaete-Scute (As), Hairy-related (Hes), and Atonal-related (Ato) subfamilies regulating neurogenesis. While As and Ato-related proteins form heterodimers with ubiquitous HLH proteins in order to facilitate transcription, Hes proteins dimerize with universal HLH proteins to repress transcription of a target gene. We have sought to determine which of the hypothetical genes are expressed in wild-type C. elegans by RT-PCR and by Northern blotting, and to determine the spatial and temporal expression patterns using in situ hybridization. We have also performed RNA interference assays to assign a preliminary function to the genes that are expressed. (Supported by NSF grant MCB9986640 and by MBRS RISE grant R25GM58904)
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[
International C. elegans Meeting,
1995]
We are using free-living nematodes to identify genes likely to be responsible for ivermectin resistance in parasitic nematodes. Previous studies revealed i) that low-level (5-15X) resistance in C. elegans is very common, conferred by recessive mutations in >30 genes, ii) that dominant resistance is less frequent, we find 6,000 recessives for each dominant mutation and iii) that high level resistance (>100X) is very rare, requiring recessive mutations in more than one gene. We observed a similar pattern of resistance in other free-living nematodes. Peter Hunt and Warwick Grant discovered that most low level resistance mutations also confer defects in dye-filling of the amphid neurons (Johnson, et al. WBG 13(3), 70). Many ivermectin resistance mutations are now known to be alleles of che, osm and dyf genes that control the dye-filling phenotype (Starich et al (1995) Genetics 139:171). Complementary results with other species show: i) that resistance in species that dye-fill is accompanied by dye-filling defects and iii) that species that do not dye-fill are "naturally" resistant to ivermectin. We are currently studying two new forms of resistance: 1) Dyf(+) low level resistance -- some mutations are unusual alleles of known genes -- and 2) mid-level (20-40X) resistance - - some mutations are in genes that contribute to high level resistance, other mutations are in new genes. Finally, we have been measuring the ivermectin resistance allele frequency in wild nematode populations. In one Dfy(+) diecious nematode species (not yet identified) we isolated 4 resistant strains (all Dyf) from the inbred progeny of 93 fertilized females -- apparent allele frequency in the wild: ~0.01 (4/93x4).