26 results on '"Harold E. Smith"'
Search Results
2. Identification of Suppressors oftop-2Embryonic Lethality inCaenorhabditis elegans
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David Schulman, Harold E. Smith, Christine K. Rourke, Aimee Jaramillo-Lambert, Thomas Wilmoth, Alekya Bheemreddy, Andy Golden, Dina Collins, and Nirajan Bhandari
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Genetics ,topoisomerase ii ,0303 health sciences ,biology ,Cas9 ,c. elegans ,Topoisomerase ,QH426-470 ,biology.organism_classification ,03 medical and health sciences ,0302 clinical medicine ,Meiosis ,RNA interference ,top-2 ,Homologous chromosome ,biology.protein ,meiosis ,DNA supercoil ,Molecular Biology ,Gene ,030217 neurology & neurosurgery ,Genetics (clinical) ,Caenorhabditis elegans ,030304 developmental biology - Abstract
Topoisomerase II is an enzyme with important roles in chromosome biology. This enzyme relieves supercoiling and DNA and RNA entanglements generated during mitosis. Recent studies have demonstrated that Topoisomerase II is also involved in the segregation of homologous chromosomes during the first meiotic division. However, the function and regulation of Topoisomerase II in meiosis has not been fully elucidated. Here, we conducted a genetic suppressor screen in Caenorhabditis elegans to identify putative genes that interact with topoisomerase II during meiosis. Using a temperature-sensitive allele of topoisomerase II, top-2(it7ts), we identified eleven suppressors of top-2-induced embryonic lethality. We used whole-genome sequencing and a combination of RNAi and CRISPR/Cas9 genome editing to identify and validate the responsible suppressor mutations. We found both recessive and dominant suppressing mutations that include one intragenic and 10 extragenic loci. The extragenic suppressors consist of a known Topoisomerase II-interacting protein and two novel interactors. We anticipate that further analysis of these suppressing mutations will provide new insights into the function of Topoisomerase II during meiosis.
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- 2020
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3. Identification of Suppressors of
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Nirajan, Bhandari, Christine, Rourke, Thomas, Wilmoth, Alekya, Bheemreddy, David, Schulman, Dina, Collins, Harold E, Smith, Andy, Golden, and Aimee, Jaramillo-Lambert
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Meiosis ,topoisomerase II ,top-2 ,Mutant Screen Report ,C. elegans ,Animals ,Mitosis ,meiosis ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Alleles - Abstract
Topoisomerase II is an enzyme with important roles in chromosome biology. This enzyme relieves supercoiling and DNA and RNA entanglements generated during mitosis. Recent studies have demonstrated that Topoisomerase II is also involved in the segregation of homologous chromosomes during the first meiotic division. However, the function and regulation of Topoisomerase II in meiosis has not been fully elucidated. Here, we conducted a genetic suppressor screen in Caenorhabditis elegans to identify putative genes that interact with topoisomerase II during meiosis. Using a temperature-sensitive allele of topoisomerase II, top-2(it7ts), we identified eleven suppressors of top-2-induced embryonic lethality. We used whole-genome sequencing and a combination of RNAi and CRISPR/Cas9 genome editing to identify and validate the responsible suppressor mutations. We found both recessive and dominant suppressing mutations that include one intragenic and 10 extragenic loci. The extragenic suppressors consist of a known Topoisomerase II-interacting protein and two novel interactors. We anticipate that further analysis of these suppressing mutations will provide new insights into the function of Topoisomerase II during meiosis.
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- 2020
4. A Genetic Analysis of the Caenorhabditis elegans Detoxification Response
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Johji Miwa, Tetsunari Fukushige, Harold E. Smith, John A. Hanover, and Michael W. Krause
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0301 basic medicine ,Genetics ,Mutation ,biology ,Mutant ,Regulator ,medicine.disease_cause ,biology.organism_classification ,03 medical and health sciences ,030104 developmental biology ,Phase II Detoxification ,medicine ,Allele ,Gene ,Transcription factor ,Caenorhabditis elegans - Abstract
Oxidative damage contributes to human diseases of aging including diabetes, cancer, and cardiovascular disorders. Reactive oxygen species resulting from xenobiotic and endogenous metabolites are sensed by a poorly understood process, triggering a cascade of regulatory factors and leading to the activation of the transcription factor Nrf2 (Nuclear factor-erythroid-related factor 2, SKN-1 in Caenorhabditis elegans). Nrf2/SKN-1 activation promotes the induction of the phase II detoxification system that serves to limit oxidative stress. We have extended a previous C. elegans genetic approach to explore the mechanisms by which a phase II enzyme is induced by endogenous and exogenous oxidants. The xrep (xenobiotics response pathway) mutants were isolated as defective in their ability to properly regulate the induction of a glutathione S-transferase (GST) reporter. The xrep-1 gene was previously identified as wdr-23, which encodes a C. elegans homolog of the mammalian β-propeller repeat-containing protein WDR-23. Here, we identify and confirm the mutations in xrep-2, xrep-3, and xrep-4. The xrep-2 gene is alh-6, an ortholog of a human gene mutated in familial hyperprolinemia. The xrep-3 mutation is a gain-of-function allele of skn-1. The xrep-4 gene is F46F11.6, which encodes a F-box-containing protein. We demonstrate that xrep-4 alters the stability of WDR-23 (xrep-1), a key regulator of SKN-1 (xrep-3). Epistatic relationships among the xrep mutants and their interacting partners allow us to propose an ordered genetic pathway by which endogenous and exogenous stressors induce the phase II detoxification response.
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- 2017
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5. Mapping Challenging Mutations by Whole-Genome Sequencing
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Andy Golden, Aimee Jaramillo-Lambert, Amy S. Fabritius, and Harold E. Smith
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0301 basic medicine ,Genotype ,ved/biology.organism_classification_rank.species ,Genomics ,Genome-wide association study ,Computational biology ,Investigations ,QH426-470 ,Biology ,Polymorphism, Single Nucleotide ,03 medical and health sciences ,0302 clinical medicine ,complex alleles ,Genetics ,Animals ,Allele ,Caenorhabditis elegans ,Model organism ,Molecular Biology ,Alleles ,variant detection ,Genetics (clinical) ,Exome sequencing ,Genes, Dominant ,030304 developmental biology ,Whole genome sequencing ,0303 health sciences ,Genome ,ved/biology ,Chromosome Mapping ,High-Throughput Nucleotide Sequencing ,Limiting ,SNP mapping ,biology.organism_classification ,Forward genetics ,forward genetics ,030104 developmental biology ,Mutation ,Mutation (genetic algorithm) ,Identification (biology) ,030217 neurology & neurosurgery ,Genome-Wide Association Study ,Genetic screen - Abstract
Whole-genome sequencing provides a rapid and powerful method for identifying mutations on a global scale, and has spurred a renewed enthusiasm for classical genetic screens in model organisms. The most commonly characterized category of mutation consists of monogenic, recessive traits, due to their genetic tractability. Therefore, most of the mapping methods for mutation identification by whole-genome sequencing are directed toward alleles that fulfill those criteria (i.e., single-gene, homozygous variants). However, such approaches are not entirely suitable for the characterization of a variety of more challenging mutations, such as dominant and semi-dominant alleles or multigenic traits. Therefore, we have developed strategies for the identification of those classes of mutations, using polymorphism mapping inCaenorhabditis elegansas our model for validation. We also report an alternative approach for mutation identification from traditional recombinant crosses, and a solution to the technical challenge of sequencing sterile or terminally arrested strains where population size is limiting. The methods described herein extend the applicability of whole-genome sequencing to a broader spectrum of mutations, including classes that are difficult to map by traditional means.
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- 2016
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6. Rapid and Efficient Identification ofCaenorhabditis elegansLegacy Mutations Using Hawaiian SNP-Based Mapping and Whole-Genome Sequencing
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Andy Golden, Harold E. Smith, Aimee Jaramillo-Lambert, Abigail S. Fuchsman, and Amy S. Fabritius
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Male ,Mutant ,Eggshell formation ,Biology ,Polymorphism, Single Nucleotide ,03 medical and health sciences ,0302 clinical medicine ,RNA interference ,Genetics ,essential genes ,Animals ,CRISPR ,Selection, Genetic ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,CRISPR/Cas9 ,Molecular Biology ,Gene ,Alleles ,Genetics (clinical) ,030304 developmental biology ,Genome, Helminth ,0303 health sciences ,cdc-25 ,Genes, Essential ,Mutant Screen Reports ,Cas9 ,Genetic Complementation Test ,Hawaiian SNP mapping ,Chromosome Mapping ,High-Throughput Nucleotide Sequencing ,Genomics ,biology.organism_classification ,whole-genome sequencing ,Mutation ,Female ,CRISPR-Cas Systems ,030217 neurology & neurosurgery ,Genetic screen - Abstract
The production of viable embryos requires the coordination of many cellular processes, including protein synthesis, cytoskeletal reorganization, establishment of polarity, cell migration, cell division, and in Caenorhabditis elegans, eggshell formation. Defects in any of these processes can lead to embryonic lethality. We examined six temperature-sensitive mutants as well as one nonconditional mutant that were previously identified in genetic screens as either embryonic lethal (maternal-effect or zygotic lethal) or eggshell defective. The responsible molecular lesion for each had never been determined. After confirmation of temperature sensitivity and lethality, we performed whole-genome sequencing using a single-nucleotide polymorphism mapping strategy to pinpoint the molecular lesions. Gene candidates were confirmed by RNA interference phenocopy and/or complementation tests and one mutant was further validated by CRISPR (Clustered Regularly Interspaced Short Palidromic Repeats)/Cas9 gene editing. This approach identified new alleles of several genes that had only been previously studied by RNA interference depletion. Our identification of temperature-sensitive alleles for all of these essential genes provides an extremely useful tool for further investigation for the C. elegans community, such as the ability to address mutant phenotypes at various developmental stages and the ability to carry out suppressor/enhancer screens to identify other genes that function in a specific cellular process.
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- 2015
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7. A Genetic Analysis of the
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Tetsunari, Fukushige, Harold E, Smith, Johji, Miwa, Michael W, Krause, and John A, Hanover
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Nuclear Proteins ,Aldehyde Dehydrogenase ,Investigations ,Xenobiotics ,DNA-Binding Proteins ,Repressor Proteins ,Oxidative Stress ,Inactivation, Metabolic ,Mutation ,Animals ,Humans ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Metabolic Networks and Pathways ,Glutathione Transferase ,Transcription Factors - Abstract
Oxidative damage contributes to human diseases of aging including diabetes, cancer, and cardiovascular disorders. Reactive oxygen species resulting from xenobiotic and endogenous metabolites are sensed by a poorly understood process, triggering a cascade of regulatory factors and leading to the activation of the transcription factor Nrf2 (Nuclear factor-erythroid-related factor 2, SKN-1 in Caenorhabditis elegans). Nrf2/SKN-1 activation promotes the induction of the phase II detoxification system that serves to limit oxidative stress. We have extended a previous C. elegans genetic approach to explore the mechanisms by which a phase II enzyme is induced by endogenous and exogenous oxidants. The xrep (xenobiotics response pathway) mutants were isolated as defective in their ability to properly regulate the induction of a glutathione S-transferase (GST) reporter. The xrep-1 gene was previously identified as wdr-23, which encodes a C. elegans homolog of the mammalian β-propeller repeat-containing protein WDR-23. Here, we identify and confirm the mutations in xrep-2, xrep-3, and xrep-4. The xrep-2 gene is alh-6, an ortholog of a human gene mutated in familial hyperprolinemia. The xrep-3 mutation is a gain-of-function allele of skn-1. The xrep-4 gene is F46F11.6, which encodes a F-box-containing protein. We demonstrate that xrep-4 alters the stability of WDR-23 (xrep-1), a key regulator of SKN-1 (xrep-3). Epistatic relationships among the xrep mutants and their interacting partners allow us to propose an ordered genetic pathway by which endogenous and exogenous stressors induce the phase II detoxification response.
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- 2017
8. Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans
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Chih Yung S. Lee, Alexandre Paix, Harold E. Smith, Jarrett Smith, Deepika Calidas, Yuemeng Wang, Michael W. Krause, Helen Schmidt, Tu Lu, and Geraldine Seydoux
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CRISPR-Associated Proteins ,Oligonucleotides ,Gene Expression ,Investigations ,Biology ,Genome ,Homology directed repair ,03 medical and health sciences ,0302 clinical medicine ,Genome editing ,Genes, Reporter ,Genetics ,genome editing ,Animals ,CRISPR ,DNA Breaks, Double-Stranded ,Cas9 ,Caenorhabditis elegans ,Homologous Recombination ,Gene ,030304 developmental biology ,0303 health sciences ,Methods, Technology, and Resources ,Recombinational DNA Repair ,biology.organism_classification ,Non-homologous end joining ,homology-directed repair ,Mutagenesis, Insertional ,Gene Targeting ,Codon, Terminator ,short homology arms ,Gene Deletion ,030217 neurology & neurosurgery - Abstract
Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (∼30–60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline.
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- 2014
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9. Identification of Suppressors ofmbk-2/DYRKby Whole-Genome Sequencing
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Kevin F. O’ Connell, Harold E. Smith, Jennifer T. Wang, Michael L. Stitzel, Geraldine Seydoux, Yuemeng Wang, and Dominique Rasoloson
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Molecular Sequence Data ,Single-nucleotide polymorphism ,Investigations ,Biology ,Polymorphism, Single Nucleotide ,law.invention ,suppressors ,law ,Catalytic Domain ,Genetics ,Animals ,MBK-2 ,SNP ,Amino Acid Sequence ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,Gene ,Genetics (clinical) ,Whole genome sequencing ,Genome, Helminth ,Transition (genetics) ,Gene Expression Regulation, Developmental ,Epistasis, Genetic ,Sequence Analysis, DNA ,Protein-Tyrosine Kinases ,single nucleotide polymorphism mapping ,biology.organism_classification ,Phenotype ,whole-genome sequencing ,DYRK kinase ,C. elegans ,Suppressor ,RNA Interference - Abstract
Screening for suppressor mutations is a powerful method to isolate genes that function in a common pathway or process. Because suppressor mutations often do not have phenotypes on their own, cloning of suppressor loci can be challenging. A method combining whole-genome sequencing (WGS) and single nucleotide polymorphism (SNP) mapping (WGS/SNP mapping) was developed to identify mutations with visible phenotypes in C. elegans. We show here that WGS/SNP mapping is an efficient method to map suppressor mutations without the need for previous phenotypic characterization. Using RNA-mediated interference to test candidate loci identified by WGS/SNP mapping, we identified 10 extragenic and six intragenic suppressors of mbk-2, a DYRK family kinase required for the transition from oocyte to zygote. Remarkably, seven suppressors are mutations in cell-cycle regulators that extend the timing of the oocyte-to-zygote transition.
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- 2014
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10. The Paired-box protein PAX-3 regulates the choice between lateral and ventral epidermal cell fates in C. elegans
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Kenneth W. Thompson, Jessica S. Dymond, Harold E. Smith, Pradeep M. Joshi, Lakshmi Gorrepati, Michael W. Krause, and David M. Eisenmann
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0301 basic medicine ,Cell type ,animal structures ,Embryo, Nonmammalian ,Cell ,Cell fate determination ,Biology ,Article ,Vulva ,Animals, Genetically Modified ,03 medical and health sciences ,Precursor cell ,medicine ,Animals ,Paired Box Transcription Factors ,Cell Lineage ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,Transcription factor ,Genetics ,Epidermis (botany) ,Cell Biology ,biology.organism_classification ,Embryonic stem cell ,Cell biology ,Luminescent Proteins ,030104 developmental biology ,medicine.anatomical_structure ,Epidermal Cells ,Microscopy, Fluorescence ,Larva ,embryonic structures ,Mutation ,Female ,RNA Interference ,Epidermis ,Developmental Biology - Abstract
The development of the single cell layer skin or hypodermis of Caenorhabditis elegans is an excellent model for understanding cell fate specification and differentiation. Early in C. elegans embryogenesis, six rows of hypodermal cells adopt dorsal, lateral or ventral fates that go on to display distinct behaviors during larval life. Several transcription factors are known that function in specifying these major hypodermal cell fates, but our knowledge of the specification of these cell types is sparse, particularly in the case of the ventral hypodermal cells, which become Vulval Precursor Cells and form the vulval opening in response to extracellular signals. Previously, the gene pvl-4 was identified in a screen for mutants with defects in vulval development. We found by whole genome sequencing that pvl-4 is the Paired-box gene pax-3, which encodes the sole PAX-3 transcription factor homolog in C. elegans. pax-3 mutants show embryonic and larval lethality, and body morphology abnormalities indicative of hypodermal cell defects. We report that pax-3 is expressed in ventral P cells and their descendants during embryogenesis and early larval stages, and that in pax-3 reduction-of-function animals the ventral P cells undergo a cell fate transformation and express several markers of the lateral seam cell fate. Furthermore, forced expression of pax-3 in the lateral hypodermal cells causes them to lose expression of seam cell markers. We propose that pax-3 functions in the ventral hypodermal cells to prevent these cells from adopting the lateral seam cell fate. pax-3 represents the first gene required for specification solely of the ventral hypodermal fate in C. elegans providing insights into cell type diversification.
- Published
- 2016
11. Library Construction for Mutation Identification by Whole-Genome Sequencing
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Harold E. Smith
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Cancer genome sequencing ,Whole genome sequencing ,Genetics ,Genome ,Shotgun sequencing ,DNA Mutational Analysis ,High-Throughput Nucleotide Sequencing ,Genomics ,Biology ,Disease gene identification ,Article ,Mutation (genetic algorithm) ,Mutation ,Animals ,Caenorhabditis elegans ,Illumina dye sequencing ,Exome sequencing ,Gene Library - Abstract
Next-generation sequencing provides a rapid and powerful method for mutation identification. Herein is described a workflow for sample preparation to allow the simultaneous mapping and identification of candidate mutations by whole-genome sequencing in Caenorhabditis elegans. The protocol is designed for small numbers of worms to accommodate classes of mutations, such as lethal and sterile alleles, that are difficult to identify by traditional means.
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- 2015
12. Promotion of Bone Morphogenetic Protein Signaling by Tetraspanins and Glycosphingolipids
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Katharine Constas, Devin E. McMahon, Dennis Liu, Michael W. Krause, Harold E. Smith, Nirav M. Amin, Emad Alam, Sinthu Ranjan, Chenxi Tian, Erich M. Schwarz, Neta Shwartz, Zhiyu Liu, Lindsey C. Szymczak, Sijung Yun, Arielle Schaeffer, Amanda S. Lindy, Herong Shi, Sunny Sheth, Jingpeng He, Saad Kubba, Jun Liu, Nimra Amir Dad, Stephanie Zimmerman, Taner Aydin, and Yevgeniy Plavskin
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Genetic Markers ,Cancer Research ,endocrine system ,lcsh:QH426-470 ,Tetraspanins ,Molecular Sequence Data ,Notch signaling pathway ,Biology ,Bone morphogenetic protein ,Sensitivity and Specificity ,Glycosphingolipids ,03 medical and health sciences ,0302 clinical medicine ,Tetraspanin ,immune system diseases ,Genes, Reporter ,Transforming Growth Factor beta ,Genetics ,Animals ,Amino Acid Sequence ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Molecular Biology ,Transcription factor ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,0303 health sciences ,virus diseases ,Transforming growth factor beta ,Sequence Analysis, DNA ,3. Good health ,BMPR2 ,Cell biology ,lcsh:Genetics ,Phenotype ,Gene Expression Regulation ,030220 oncology & carcinogenesis ,embryonic structures ,Bone Morphogenetic Proteins ,Mutation ,biology.protein ,Signal transduction ,Transforming growth factor ,Research Article ,Signal Transduction ,Transcription Factors - Abstract
Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGFβ) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like “Sma/Mab” signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development., Author Summary The bone morphogenetic protein (BMP) signaling pathway is required for multiple developmental processes during metazoan development. Various diseases, including cancer, can result from mis-regulation of the BMP pathway. Thus, it is critical to identify factors that ensure proper regulation of BMP signaling. Using the nematode C. elegans, we have devised a highly specific and sensitive genetic screen to identify new modulators in the BMP pathway. Through this screen, we identified three conserved tetraspanin molecules as novel factors that function to promote BMP signaling in a living organism. We further showed that these three tetraspanins likely form a complex and function together with glycosphingolipids to promote BMP signaling. Recent studies have implicated several tetraspanins in cancer initiation, progression and metastasis in mammals. Our findings suggest that the involvement of tetraspanins in cancer may partially be due to their function in modulating the activity of BMP signaling.
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- 2015
13. Evaluating alignment and variant-calling software for mutation identification in C. elegans by whole-genome sequencing
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Sijung Yun and Harold E. Smith
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0301 basic medicine ,Nematoda ,Computer science ,DNA Mutational Analysis ,Gene Identification and Analysis ,lcsh:Medicine ,medicine.disease_cause ,Computer Architecture ,Database and Informatics Methods ,Invertebrate Genomics ,lcsh:Science ,Genetics ,Mutation ,Multidisciplinary ,High-Throughput Nucleotide Sequencing ,Software Engineering ,Animal Models ,Genomics ,Experimental Organism Systems ,Mutation (genetic algorithm) ,Engineering and Technology ,Identification (biology) ,Sequence Analysis ,Research Article ,Computer and Information Sciences ,Multiple Alignment Calculation ,Bioinformatics ,Single-nucleotide polymorphism ,Sequence alignment ,Research and Analysis Methods ,Instruction Pipelines ,03 medical and health sciences ,Model Organisms ,Computational Techniques ,Genetic variation ,medicine ,Animals ,Allele ,Caenorhabditis elegans ,Mutation Detection ,Pipelines (Computing) ,Whole genome sequencing ,Software Tools ,lcsh:R ,Organisms ,Biology and Life Sciences ,Computational Biology ,Genome Analysis ,Invertebrates ,Split-Decomposition Method ,030104 developmental biology ,Animal Genomics ,Caenorhabditis ,lcsh:Q ,Sequence Alignment ,Software ,Reference genome - Abstract
Whole-genome sequencing is a powerful tool for analyzing genetic variation on a global scale. One particularly useful application is the identification of mutations obtained by classical phenotypic screens in model species. Sequence data from the mutant strain is aligned to the reference genome, and then variants are called to generate a list of candidate alleles. A number of software pipelines for mutation identification have been targeted to C. elegans, with particular emphasis on ease of use, incorporation of mapping strain data, subtraction of background variants, and similar criteria. Although success is predicated upon the sensitive and accurate detection of candidate alleles, relatively little effort has been invested in evaluating the underlying software components that are required for mutation identification. Therefore, we have benchmarked a number of commonly used tools for sequence alignment and variant calling, in all pair-wise combinations, against both simulated and actual datasets. We compared the accuracy of those pipelines for mutation identification in C. elegans, and found that the combination of BBMap for alignment plus FreeBayes for variant calling offers the most robust performance.
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- 2017
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14. Identifying insertion mutations by whole-genome sequencing
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Harold E. Smith
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Transposable element ,Genetics ,Whole genome sequencing ,DNA, Bacterial ,Genome ,Mutagenesis (molecular biology technique) ,DNA ,Sequence Analysis, DNA ,Biology ,General Biochemistry, Genetics and Molecular Biology ,DNA sequencing ,Article ,Mutagenesis, Insertional ,DNA Transposable Elements ,Escherichia coli ,Animals ,Insertion ,Mobile genetic elements ,Caenorhabditis elegans ,Gene ,Sequence Alignment ,Genome, Bacterial ,Biotechnology - Abstract
Insertion mutagenesis via mobile genetic element is a common technique for the analysis of gene function in model organisms. Next-generation sequencing offers an attractive approach for localizing the site of insertion, but alignment-based mapping of mobile genetic elements is challenging. A computational method for identifying insertion sites is reported herein. The technique was validated by mapping transposons in both bacterial and nematode species. The approach should be extensible to other systems that employ mobile genetic elements to generate mutations.
- Published
- 2011
15. Requirement for the ERI/DICER complex in endogenous RNA interference and sperm development in Caenorhabditis elegans
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Harold E. Smith, Derek M. Pavelec, Jennifer Lachowiec, Thomas F. Duchaine, and Scott Kennedy
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Male ,Ribonuclease III ,Small interfering RNA ,Molecular Sequence Data ,Investigations ,Mice ,RNA interference ,Gene expression ,Genetics ,Gene silencing ,Animals ,Humans ,Amino Acid Sequence ,RNA, Messenger ,RNA, Small Interfering ,Caenorhabditis elegans Proteins ,Spermatogenesis ,Caenorhabditis elegans ,biology ,fungi ,RNA ,biology.organism_classification ,Spermatozoa ,Caenorhabditis ,Organ Specificity ,Exoribonucleases ,biology.protein ,RNA Interference ,Dicer - Abstract
Small regulatory RNAs are key regulators of gene expression. One class of small regulatory RNAs, termed the endogenous small interfering RNAs (endo siRNAs), is thought to negatively regulate cellular transcripts via an RNA interference (RNAi)-like mechanism termed endogenous RNAi (endo RNAi). A complex of proteins composed of ERI-1/3/5, RRF-3, and DICER (the ERI/DICER complex) mediates endo RNAi processes in Caenorhabditis elegans. We conducted a genetic screen to identify additional components of the endo RNAi machinery. Our screen recovered alleles of eri-9, which encodes a novel DICER-interacting protein, and a missense mutation within the helicase domain of DICER [DCR-1(G492R)]. ERI-9(−) and DCR-1(G492) animals exhibit defects in endo siRNA expression and a concomitant failure to regulate mRNAs that exhibit sequence homology to these endo siRNAs, indicating that ERI-9 and the DCR-1 helicase domain function in the C. elegans endo RNAi pathway. We define a subset of Eri mutant animals (including eri-1, rrf-3, eri-3, and dcr-1, but not eri-9 or ergo-1) that exhibit temperature-sensitive, sperm-specific sterility and defects in X chromosome segregation. Among these mutants we find multiple aberrations in sperm development beginning with cytokinesis and extending through terminal differentiation. These results identify novel components of the endo RNAi machinery, demonstrate differential requirements for the Eri factors in the sperm-producing germline, and begin to delineate the functional requirement for the ERI/DICER complex in sperm development.
- Published
- 2009
16. E1 Ubiquitin-Activating Enzyme UBA-1 Plays Multiple Roles throughout C. elegans Development
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Madhura Kulkarni and Harold E. Smith
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Male ,Cancer Research ,Embryo, Nonmammalian ,Indoles ,Developmental Biology/Germ Cells ,Ubiquitin-activating enzyme ,Mutant ,Ubiquitin-Activating Enzymes ,Ubiquitin-conjugating enzyme ,medicine.disease_cause ,0302 clinical medicine ,Ubiquitin ,Genetics (clinical) ,Caenorhabditis elegans ,Genes, Helminth ,0303 health sciences ,Mutation ,Hydrolysis ,Homozygote ,Ubiquitin-Protein Ligase Complexes ,Spermatozoa ,Ubiquitin ligase ,Genetics and Genomics/Gene Function ,Meiosis ,Biochemistry ,Research Article ,Heterozygote ,lcsh:QH426-470 ,Ubiquitin-Protein Ligases ,Biology ,Anaphase-Promoting Complex-Cyclosome ,03 medical and health sciences ,Genetics ,medicine ,Animals ,Caenorhabditis elegans Proteins ,Molecular Biology ,Ubiquitins ,Ecology, Evolution, Behavior and Systematics ,Alleles ,030304 developmental biology ,Fluorescent Dyes ,Genetic Complementation Test ,Ubiquitination ,biology.organism_classification ,lcsh:Genetics ,Proteasome ,biology.protein ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Poly-ubiquitination of target proteins typically marks them for destruction via the proteasome and provides an essential mechanism for the dynamic control of protein levels. The E1 ubiquitin-activating enzyme lies at the apex of the ubiquitination cascade, and its activity is necessary for all subsequent steps in the reaction. We have isolated a temperature-sensitive mutation in the Caenorhabditis elegans uba-1 gene, which encodes the sole E1 enzyme in this organism. Manipulation of UBA-1 activity at different developmental stages reveals a variety of functions for ubiquitination, including novel roles in sperm fertility, control of body size, and sex-specific development. Levels of ubiquitin conjugates are substantially reduced in the mutant, consistent with reduced E1 activity. The uba-1 mutation causes delays in meiotic progression in the early embryo, a process that is known to be regulated by ubiquitin-mediated proteolysis. The uba-1 mutation also demonstrates synthetic lethal interactions with alleles of the anaphase-promoting complex, an E3 ubiquitin ligase. The uba-1 mutation provides a sensitized genetic background for identifying new in vivo functions for downstream components of the ubiquitin enzyme cascade, and it is one of the first conditional mutations reported for the essential E1 enzyme in a metazoan animal model., Author Summary Proteins that control an organism's development must first be turned on at the proper time and place, and then turned off when they are no longer needed. One of the “off” signals occurs through the attachment of a small protein, known as ubiquitin, to the target protein, which typically leads to the destruction of the target. Attachment of ubiquitin is controlled by a series of enzymes, the first of which is known as E1. Most organisms have a single gene for the E1 enzyme, and its activity is crucial for the degradation of a wide range of target proteins throughout development. We have identified a temperature-sensitive mutation in the E1 enzyme of the nematode Caenorhabditis elegans. By manipulating the growth temperature, we have determined the various functions of E1 at different stages of development. We find that this enzyme controls embryonic and larval development, sperm fertility, and body size. We also characterized sex-specific roles for E1; males exhibit progressive paralysis and defects in the tail, which is used for mating. In addition to the knowledge gained, this mutation provides a means of identifying both the functions of other ubiquitin enzymes during development as well as the target proteins that are marked for destruction.
- Published
- 2008
17. Critical contact residues that mediate polymerization of nematode major sperm protein
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Harold E. Smith and Antonio del Castillo-Olivares
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Male ,macromolecular substances ,Biology ,Biochemistry ,law.invention ,Protein filament ,Affinity chromatography ,law ,parasitic diseases ,Animals ,Protein Interaction Domains and Motifs ,Amino Acids ,Cytoskeleton ,Caenorhabditis elegans ,Molecular Biology ,Amoeboid movement ,Cell Biology ,Helminth Proteins ,In vitro ,Recombinant Proteins ,Cell biology ,Major sperm protein ,Polymerization ,Amino Acid Substitution ,Recombinant DNA - Abstract
The polymerization of protein filaments provides the motive force in a variety of cellular processes involving cell motility and intracellular transport. Regulated assembly and disassembly of the major sperm protein (MSP) underlies amoeboid movement in nematode sperm, and offers an attractive model system for characterizing the biomechanical properties of filament formation and force generation. To that end, structure-function studies of MSP from the nematode Caenorhabditis elegans have been performed. Recombinant MSP was purified from Escherichia coli using a novel affinity chromatography technique, and filament assembly was assessed by in vitro polymerization in the presence of polyethylene glycol. Prior molecular studies and structure from X-ray crystallography have implicated specific residues in protein-protein interactions necessary for filament assembly. Purified MSP containing substitutions in these residues fails to form filaments in vitro. Short peptides based on predicted sites of interaction also effectively disrupt MSP polymerization. These results confirm the structural determination of intermolecular contacts and demonstrate the importance of these residues in MSP assembly.
- Published
- 2007
18. Expression of a unique drug-resistant Hsp90 ortholog by the nematode Caenorhabditis elegans
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Cynthia L, David, Harold E, Smith, Deborah A, Raynes, Elizabeth J, Pulcini, and Luke, Whitesell
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Reticulocytes ,Lactams, Macrocyclic ,Blotting, Western ,Molecular Sequence Data ,Quinones ,Sequence Homology ,Original Articles ,Flow Cytometry ,Adenosine Triphosphate ,Drug Resistance, Neoplasm ,Benzoquinones ,Animals ,Humans ,Electrophoresis, Polyacrylamide Gel ,Amino Acid Sequence ,HSP90 Heat-Shock Proteins ,Caenorhabditis elegans ,Cells, Cultured ,Protein Binding - Abstract
In all species studied to date, the function of heat shock protein 90 (Hsp90), a ubiquitous and evolutionarily conserved molecular chaperone, is inhibited selectively by the natural product drugs geldanamycin (GA) and radicicol. Crystal structures of the N-terminal region of yeast and human Hsp90 have revealed that these compounds interact with the chaperone in a Bergerat-type adenine nucleotide–binding fold shared throughout the gyrase, Hsp90, histidine kinase mutL (GHKL) superfamily of adenosine triphosphatases. To better understand the consequences of disrupting Hsp90 function in a genetically tractable multicellular organism, we exposed the soil-dwelling nematode Caenorhabditis elegans to GA under a variety of conditions designed to optimize drug uptake. Mutations in the gene encoding C elegans Hsp90 affect larval viability, dauer development, fertility, and life span. However, exposure of worms to GA produced no discernable phenotypes, although the amino acid sequence of worm Hsp90 is 85% homologous to that of human Hsp90. Consistent with this observation, we found that solid phase–immobilized GA failed to bind worm Hsp90 from worm protein extracts or when translated in a rabbit reticulocyte lysate system. Further, affinity precipitation studies using chimeric worm-vertebrate fusion proteins or worm C-terminal truncations expressed in reticulocyte lysate revealed that the conserved nucleotide-binding fold of worm Hsp90 exhibits the novel ability to bind adenosine triphosphate but not GA. Despite its unusual GA resistance, worm Hsp90 appeared fully functional when expressed in a vertebrate background. It heterodimerized with its vertebrate counterpart and showed no evidence of compromising its essential cellular functions. Heterologous expression of worm Hsp90 in tumor cells, however, did not render them GA resistant. These findings provide new insights into the nature of unusual N-terminal nucleotide-binding fold of Hsp90 and suggest that target-related drug resistance is unlikely to emerge in patients receiving GA-like chemotherapeutic agents.
- Published
- 2003
19. Identification of protein-protein interactions of the major sperm protein (MSP) of Caenorhabditis elegans
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Samuel Ward and Harold E. Smith
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Male ,Models, Molecular ,Protein Folding ,Molecular Sequence Data ,Immunoglobulin domain ,Biology ,complex mixtures ,Models, Biological ,Protein Structure, Secondary ,Protein–protein interaction ,Suppression, Genetic ,Bacterial Proteins ,Structural Biology ,Cell Movement ,Genes, Reporter ,parasitic diseases ,Animals ,Amino Acid Sequence ,Caenorhabditis elegans ,neoplasms ,Molecular Biology ,Actin ,Serine Endopeptidases ,DNA-binding domain ,Helminth Proteins ,biology.organism_classification ,Molecular biology ,Spermatozoa ,Recombinant Proteins ,Cell biology ,Major sperm protein ,Mutagenesis ,Protein folding ,Repressor lexA ,Dimerization ,Sequence Alignment - Abstract
In nematodes, sperm are amoeboid cells that crawl via an extended pseudopod. Unlike those in other crawling cells, this pseudopod contains little or no actin; instead, it utilizes the major sperm protein (MSP). In vivo and in vitro studies of Ascaris suum MSP have demonstrated that motility occurs via the regulated assembly and disassembly of MSP filaments. Filaments composed of MSP dimers are thought to provide the motive force. We have employed the yeast two-hybrid system to investigate MSP-MSP interactions and provide insights into the process of MSP filament formation. Fusions of the Caenorhabditis elegans msp-142 gene to both the lexA DNA binding domain (LEXA-MSP) and a transcriptional activation domain (AD-MSP) interact to drive expression of a lacZ reporter construct. A library of AD-MSP mutants was generated via mutagenic PCR and screened for clones that fail to interact with LEXA-MSP. Single missense mutations were identified and mapped to the crystal structure of A. suum MSP. Two classes of mutations predicted from the structure were recovered: changes in residues critical for the overall fold of the protein, and changes in residues in the dimerization interface. Multiple additional mutations were obtained in the two carboxy-terminal β strands, a region not predicted to be involved in protein folding or dimer formation. Size fractionation of bacterially expressed MSPs indicates that mutations in this region do not abolish dimer formation. A number of compensating mutations that restore the interaction also map to this region. The data suggest that the carboxy-terminal β strands are directly involved in interactions required for MSP filament assembly.
- Published
- 1998
20. SPE-44 Implements Sperm Cell Fate
- Author
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Harold E. Smith, Diane C. Shakes, Katie Guevel, and Madhura Kulkarni
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Male ,Cancer Research ,Transcription, Genetic ,Cellular differentiation ,Gene Identification and Analysis ,Gene Expression ,Cell Fate Determination ,Germline ,Molecular cell biology ,0302 clinical medicine ,Stem cell fate specification ,Genetics (clinical) ,Caenorhabditis elegans ,Genetics ,Regulation of gene expression ,0303 health sciences ,biology ,Stem Cells ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Animal Models ,Spermatozoa ,Chromatin ,Cell biology ,Caenorhabditis ,Meiosis ,Cellular Types ,Cell Division ,Signal Transduction ,Research Article ,Organelle assembly ,lcsh:QH426-470 ,DNA transcription ,Molecular Genetics ,03 medical and health sciences ,Model Organisms ,Animals ,Cell Lineage ,Gene Regulation ,Caenorhabditis elegans Proteins ,Spermatogenesis ,Biology ,Molecular Biology ,Ecology, Evolution, Behavior and Systematics ,Cytokinesis ,030304 developmental biology ,Cell Cycle Checkpoints ,Sex Determination Processes ,biology.organism_classification ,Sperm ,lcsh:Genetics ,Germ Cells ,Gene Function ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
The sperm/oocyte decision in the hermaphrodite germline of Caenorhabditis elegans provides a powerful model for the characterization of stem cell fate specification and differentiation. The germline sex determination program that governs gamete fate has been well studied, but direct mediators of cell-type-specific transcription are largely unknown. We report the identification of spe-44 as a critical regulator of sperm gene expression. Deletion of spe-44 causes sperm-specific defects in cytokinesis, cell cycle progression, and organelle assembly resulting in sterility. Expression of spe-44 correlates precisely with spermatogenesis and is regulated by the germline sex determination pathway. spe-44 is required for the appropriate expression of several hundred sperm-enriched genes. The SPE-44 protein is restricted to the sperm-producing germline, where it localizes to the autosomes (which contain sperm genes) but is excluded from the transcriptionally silent X chromosome (which does not). The orthologous gene in other Caenorhabditis species is similarly expressed in a sex-biased manner, and the protein likewise exhibits autosome-specific localization in developing sperm, strongly suggestive of an evolutionarily conserved role in sperm gene expression. Our analysis represents the first identification of a transcriptional regulator whose primary function is the control of gamete-type-specific transcription in this system., Author Summary Stem cells give rise to the variety of specialized cell types within an organism. The decision to adopt a particular cell fate, a process known as specification or determination, requires the coordinated expression of all of the genes needed for that specialized cell to develop and function properly. Understanding the mechanisms that govern these patterns of gene expression is critical to our understanding of stem cell fate specification. We study this process in a nematode species that makes both sperm and eggs from the same stem cell population. We have identified a gene, named spe-44, that is required for the proper expression of sperm genes (but not egg genes). Mutation in spe-44 produces sterile sperm with developmental defects. spe-44 is controlled by factors that govern the sperm/egg decision, and its function in controlling sperm gene expression appears to be conserved in other nematode species.
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- 2012
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21. Regulation of sperm gene expression by the GATA factor ELT-1
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Antonio del Castillo-Olivares, Harold E. Smith, and Madhura Kulkarni
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Male ,endocrine system ,Cell fate determination ,Biology ,GATA Transcription Factors ,Article ,03 medical and health sciences ,0302 clinical medicine ,Germ line ,Two-Hybrid System Techniques ,Animals ,Transgenes ,GATA factor ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Promoter Regions, Genetic ,Spermatogenesis ,Molecular Biology ,Transcription factor ,Gene ,reproductive and urinary physiology ,Oligonucleotide Array Sequence Analysis ,030304 developmental biology ,Genetics ,Regulation of gene expression ,0303 health sciences ,urogenital system ,Computational Biology ,Promoter ,Cell Biology ,Spermatozoa ,Sperm ,3. Good health ,Gene Expression Regulation ,Germ-line sex determination ,GATA transcription factor ,RNA Interference ,Gene expression ,Gene Deletion ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Cell fate specification is mediated primarily through the expression of cell-type-specific genes. The regulatory pathway that governs the sperm/egg decision in the hermaphrodite germ line of Caenorhabditis elegans has been well characterized, but the transcription factors that drive these developmental programs remain unknown. We report the identification of ELT-1, a GATA transcription factor that specifies hypodermal fate in the embryo, as a regulator of sperm-specific transcription in the germ line. Computational analysis identified a conserved bipartite sequence element that is found almost exclusively in the promoters of a number of sperm genes. ELT-1 was recovered in a yeast one-hybrid screen for factors that bind to that sperm consensus site. In vitro assays defined the sperm consensus sequence as an optimal binding site for ELT-1. We determined that expression of elt-1 is elevated in the sperm-producing germ line, and that ELT-1 is required for sperm function. Deletion of the ELT-1 binding site from a sperm promoter abrogates sperm-specific expression of a reporter transgene. This work demonstrates a role for the ELT-1 transcription factor in sperm, and provides a critical link between the germ line sex determination program and gamete-specific gene expression.
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22. A global profile of germline gene expression in C-elegans
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Carrie Van Doren, Rebecca Begley, Valerie Reinke, John Wang, Stuart K. Kim, Elizabeth B. Davis, Steven J.M. Jones, Stewart Scherer, Harold E. Smith, Jeremy Nance, and Samuel Ward
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Male ,medicine.medical_specialty ,X Chromosome ,Cell Cycle Proteins ,Germline ,Molecular genetics ,Gene expression ,medicine ,Animals ,Drosophila Proteins ,RNA-Induced Silencing Complex ,RNA, Messenger ,Caenorhabditis elegans ,Caenorhabditis elegans Proteins ,Spermatogenesis ,Gene ,Molecular Biology ,X chromosome ,Genetics ,biology ,Receptors, Notch ,Gene Expression Profiling ,Gene Expression Regulation, Developmental ,Membrane Proteins ,Proteins ,Helminth Proteins ,Cell Biology ,biology.organism_classification ,Spermatozoa ,Gene expression profiling ,Cytoskeletal Proteins ,Argonaute Proteins ,Oocytes ,DNA microarray - Abstract
We used DNA microarrays to profile gene expression patterns in the C. elegans germline and identified 1416 germline-enriched transcripts that define three groups. The sperm-enriched group contains an unusually large number of protein kinases and phosphatases. The oocyte-enriched group includes potentially new components of embryonic signaling pathways. The germline-intrinsic group, defined as genes expressed similarly in germlines making only sperm or only oocytes, contains a family of piwi-related genes that may be important for stem cell proliferation. Finally, examination of the chromosomal location of germline transcripts revealed that sperm-enriched and germline-intrinsic genes are nearly absent from the X chromosome, but oocyte-enriched genes are not.
23. Data on Lipodystrophy Reported by Researchers at University of Florida (Identification of Genetic Suppressors for a Bscl2 Lipodystrophy Pathogenic Variant In Caenorhabditis Elegans).
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LIPID metabolism disorders ,LIPODYSTROPHY ,DIGESTIVE system diseases ,LIFE sciences ,MEDICAL sciences - Abstract
Researchers at the University of Florida have identified genetic suppressors for a lipodystrophy pathogenic variant in Caenorhabditis elegans, a model organism for studying human diseases. Lipodystrophy is a group of metabolic disorders characterized by abnormal fat distribution in the body. The researchers conducted a chemical mutagenesis screen and identified five suppressor lines that restored embryonic viability and improved phenotypes associated with the lipodystrophy variant. This study provides valuable insights into potential genetic interactors and pathways involved in lipodystrophy. [Extracted from the article]
- Published
- 2024
24. Mutation of NEKL-4/NEK10 and TTLL genes suppress neuronal ciliary degeneration caused by loss of CCPP-1 deglutamylase function.
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Power, Kade M., Akella, Jyothi S., Gu, Amanda, Walsh, Jonathon D., Bellotti, Sebastian, Morash, Margaret, Zhang, Winnie, Ramadan, Yasmin H., Ross, Nicole, Golden, Andy, Smith, Harold E., Barr, Maureen M., and O'Hagan, Robert
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MUCOCILIARY system ,POST-translational modification ,POLYCYSTIC kidney disease ,SOCIAL degeneration ,CAENORHABDITIS elegans ,PROTEIN stability ,CARRIER proteins - Abstract
Ciliary microtubules are subject to post-translational modifications that act as a "Tubulin Code" to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in a tubulin tyrosine ligase-like glutamylase gene. We show that mutation in ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of ciliary dye filling and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 localizes to the ciliary base but does not localize to cilia, suggesting an indirect role in ciliary processes. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into the roles of the tubulin code in human ciliopathies and neurodegenerative diseases. Author summary: Cilia are microtubule-based organelles that play essential roles in human development and health. Ciliopathies are caused by abnormalities in the structure or function of primary cilia, with polycystic kidney disease (PKD) being a common clinical phenotype. As cilia are found on most non-dividing cells in the human body, ciliopathies often display extrarenal manifestations including neurological disorders and retinal degeneration. The Tubulin Code–combinatorial use of tubulin isotypes and post-translational modifications–dictates ciliary structure, motor-based transport, and function. Mutation in the tubulin deglutamylase ccpp-1 (cytosolic carboxypeptidase) results in ciliary hyperglutamylation and degeneration. C. elegans ccpp-1 ciliary degeneration is suppressed by a mutation in any of three TTLL (tubulin tyrosine ligase-like) glutamylase genes, indicating that regulated glutamylation is critically important for ciliary homeostasis. Pathological hyperglutamylation caused by CCP deglutamylase mutations are associated with human retinal degeneration and murine progressive neurodegeneration and sperm immotility. ccpp-1 ciliary degeneration is also suppressed by a mutation in the kinase NEKL-4/NEK10. NEK kinases are implicated in polycystic kidney disease and other ciliopathies and NEKL-4/NEK10 is important for ciliary stability in C. elegans. By identifying C. elegans genes important for neuronal and ciliary stability, "the worm" may inform research into human ciliopathies and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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25. Study Findings from University of Florida Broaden Understanding of Arthrogryposis (A Mutation In F-actin Polymerization Factor Suppresses the Distal Arthrogryposis Type 5 Piezo2 Pathogenic Variant In Caenorhabditis Elegans).
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CAENORHABDITIS elegans ,ARTHROGRYPOSIS ,F-actin ,LIFE sciences ,MEDICAL sciences ,POLYMERIZATION ,OVULATION ,GAIN-of-function mutations - Abstract
A study conducted by researchers at the University of Florida has shed light on the underlying mechanisms of distal arthrogryposis type 5 (DA5) and other PIEZO-associated diseases. The researchers used CRISPR/Cas9 gene editing to create four disease models and performed a genetic suppressor screen to identify potential suppressors of the pathogenic variant pezo-1(R2405P). They found that the C. elegans gene gex-3, which is an ortholog of human NCKAP1, acts as a suppressor of the pathogenic variant. This discovery suggests that the PIEZO channel coordinates with the cytoskeleton regulator to maintain the F-actin network, providing insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases. [Extracted from the article]
- Published
- 2024
26. Evaluating alignment and variant-calling software for mutation identification in C. elegans by whole-genome sequencing.
- Author
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Smith, Harold E. and Yun, Sijung
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CAENORHABDITIS elegans genetics ,GENETIC mutation ,NUCLEOTIDE sequence ,PHENOTYPES ,GENE mapping ,GENETIC software - Abstract
Whole-genome sequencing is a powerful tool for analyzing genetic variation on a global scale. One particularly useful application is the identification of mutations obtained by classical phenotypic screens in model species. Sequence data from the mutant strain is aligned to the reference genome, and then variants are called to generate a list of candidate alleles. A number of software pipelines for mutation identification have been targeted to C. elegans, with particular emphasis on ease of use, incorporation of mapping strain data, subtraction of background variants, and similar criteria. Although success is predicated upon the sensitive and accurate detection of candidate alleles, relatively little effort has been invested in evaluating the underlying software components that are required for mutation identification. Therefore, we have benchmarked a number of commonly used tools for sequence alignment and variant calling, in all pair-wise combinations, against both simulated and actual datasets. We compared the accuracy of those pipelines for mutation identification in C. elegans, and found that the combination of BBMap for alignment plus FreeBayes for variant calling offers the most robust performance. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
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