DOMINANT SUPPRESSORS OF A MUSCLE MUTANT DEFINE AN ESSENTIAL GENE OF CAENORHABDITIS ELEGANS

Genetics ◽  
1982 ◽  
Vol 101 (2) ◽  
pp. 211-225
Author(s):  
Iva S Greenwald ◽  
H Robert Horvitz
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Family ◽  
Protein Complex ◽  
Essential Gene ◽  
Null Alleles ◽  
Missense Mutations ◽  
Suppressor Activity ◽  
C Elegans ◽  
Suppressor Mutations ◽  
Dominant Suppressor

ABSTRACT The sup-11 I locus of C. elegans was defined by rare dominant suppressors of unc-93(e1500) III, a mutation that affects muscle structure. All ten of these dominant suppressors have a recessive "scrawny" phenotype. Two additional classes of sup-11 alleles were identified. One class, null alleles, was obtained by reversion of the dominant suppressor activity. These null alleles are recessive embryonic lethals, indicating that sup-11 is an essential gene. Members of the second class, rare semidominant revertants of the "scrawny" phenotype, are partial suppressors of unc-93(e1500). The genetic properties of the dominant suppressor mutations suggest that they are rare missense mutations that confer a novel activity to the sup-11 protein. We consider some of the ways that sup-11 alleles might suppress unc-93(e1500), including the possibilities that the altered sup-11 proteins restore function to a protein complex or are modified products of a gene that is a member of an unc-93 gene family.

scholarly journals Insights into the Involvement of Spliceosomal Mutations in Myelodysplastic Disorders from Analysis of SACY-1/DDX41 in Caenorhabditis elegans

Genetics ◽  
2020 ◽  
Vol 214 (4) ◽  
pp. 869-893 ◽  
Author(s):  
Tatsuya Tsukamoto ◽  
Micah D. Gearhart ◽  
Seongseop Kim ◽  
Gemechu Mekonnen ◽  
Caroline A. Spike ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Myelodysplastic Syndromes ◽  
Missense Mutations ◽  
Synthetic Lethal ◽  
C Elegans ◽  
Link Type ◽  
Dead Box ◽  
Synthetic Lethal Interactions ◽  
Biochemical Analyses ◽  
Cellular Phenotypes

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia (AML). DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase that is recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of AML. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here, we conduct a comprehensive analysis of the Caenorhabditis elegans DDX41 ortholog, SACY-1. Biochemical analyses defined SACY-1 as a component of the C. elegans spliceosome, and genetic analyses revealed synthetic lethal interactions with spliceosomal components. We used the auxin-inducible degradation system to analyze the consequence of SACY-1 depletion on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-dependent and splicing-independent mechanisms. Altered 3′ splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 in the second step of splicing. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. The transcriptome changes observed after SACY-1 depletion suggest that disruption of the spliceosome induces a stress response, which could contribute to the cellular phenotypes conferred by sacy-1 mutant alleles. Multiple sacy-1/ddx41 missense mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that their incorporation into the spliceosome is detrimental. Antagonistic variants that perturb the function of the spliceosome may be relevant to the disease-causing mutations, including DDX41, affecting highly conserved components of the spliceosome in humans.

scholarly journals The Caenorhabditis elegans rol-6 gene, which interacts with the sqt-1 collagen gene to determine organismal morphology, encodes a collagen.

1990 ◽  
Vol 10 (5) ◽  
pp. 2081-2089 ◽  
Author(s):  
J M Kramer ◽  
R P French ◽  
E C Park ◽  
J J Johnson
Keyword(s):  
Caenorhabditis Elegans ◽  
Restriction Site ◽  
Genetic Interaction ◽  
The Other ◽  
C Elegans ◽  
Allele Specific ◽  
Specific Restriction ◽  
The Right ◽  
Sequence Similarities ◽  
Dominant Suppressor

The rol-6 gene is one of the more than 40 loci in Caenorhabditis elegans that primarily affect organismal morphology. Certain mutations in the rol-6 gene produce animals that have the right roller phenotype, i.e., they are twisted into a right-handed helix. The rol-6 gene interacts with another gene that affects morphology, sqt-1; a left roller allele of sqt-1 acts as a dominant suppressor of a right roller allele of rol-6. The sqt-1 gene has previously been shown to encode a collagen. We isolated and sequenced the rol-6 gene and found that it also encodes a collagen. The rol-6 gene was identified by physical mapping of overlapping chromosomal deficiencies that cover the gene and by identification of an allele-specific restriction site alteration. The amino acid sequence of the collagen encoded by rol-6 is more similar to that of the sqt-1 collagen than to any of the other ten C. elegans cuticle collagen sequences compared. The locations of cysteine residues flanking the Gly-X-Y repeat regions of rol-6 and sqt-1 are identical, but differ from those in the other collagens. The sequence similarities between rol-6 and sqt-1 indicate that they represent a new collagen subfamily in C. elegans. These findings suggest that these two collagens physically interact, possibly explaining the genetic interaction seen between the rol-6 and sqt-1 genes.

scholarly journals An efficient genome editing strategy to generate putative null mutants in Caenorhabditis elegans using CRISPR/Cas9

2018 ◽  
Author(s):  
Han Wang ◽  
Heenam Park ◽  
Jonathan Liu ◽  
Paul W. Sternberg
Keyword(s):  
Dna Repair ◽  
Caenorhabditis Elegans ◽  
Genome Editing ◽  
Gene Function ◽  
Unique Sequence ◽  
Target Site ◽  
Null Alleles ◽  
Wild Type ◽  
C Elegans ◽  
Null Mutants

AbstractNull mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long stop knock-in (STOP-IN) cassette into the early exons of target genes. This cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette right at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two 35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2. We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for additional 20 genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.SummaryWe report a simple and efficient CRISPR/Cas9 genome editing strategy to generate putative null C. elegans mutants by inserting a small universal stop knock-in (STOP-IN) cassette with stop codons in three frames and frameshifts. The strategy is cloning-free, with the mixture consisting of preassembled Cas9 ribonucleoprotein and single stranded repair DNA oligos directly injected into gonads of adult C. elegans. The universal STOP-IN cassette also contains a unique sequence that simplifies detection of successful knock-in events via PCR and an exogenous Cas9 target sequence that allows further genome editing.

scholarly journals A SECOND INFORMATIONAL SUPPRESSOR, SUP-7 X, IN CAENORHABDITIS ELEGANS

Genetics ◽  
1981 ◽  
Vol 97 (2) ◽  
pp. 307-325
Author(s):  
Robert H Waterston
Keyword(s):  
Suppressor Gene ◽  
Null Alleles ◽  
Chain Gene ◽  
Wild Type ◽  
Heavy Chain Gene ◽  
Myosin Heavy Chain Gene ◽  
C Elegans ◽  
X Linkage ◽  
Suppressor Mutations ◽  
Dose Dependent

ABSTRACT More than 30 independent suppressor mutations have been obtained in the nematode C. elegans through reversion analysis of two unc-13 mutants. Many of the new isolates map to the region of the previously identified informational suppressor, sup-5 III (Waterston and Brenner 1978). Several of the other suppressor mutations map to the left half of the X-linkage group and define a second suppressor gene, sup-7 X. In tests against 40 mutations in six genes, the sup-7(st5) allele was found to suppress to a greater extent the same alleles acted on by sup-5(e1464). Like sup-5(e1464), sup-7(st5) acts on null alleles of the myosin heavy-chain gene unc-54 I (MacLeod et al. 1977; MacLeod, Waterston and Brenner 1977) and the putative paramyosin gene unc-15 I (Waterston et al. 1977). Chemical analysis of unc-15(e1214); sup-7(st5) animals show that paramyosin is restored to more than 30% of the wild-type level. —As was observed for sup-5(e1464), suppression by sup-7(st5) is dose dependent and is greater in animals grown at 15° than at 25°. However, associated with this increased suppression is a decreased viability of sup-7(st5) homozygotes. Reversion of the lethality has resulted in the isolation of deficiency mutations that complement st5 lethality, but lack suppressor function. These properties of sup-7(st5) suggest that it, like sup-5(e1464), is an informational suppressor of null alleles, and its reversion via deficiencies further narrows the possible explanations of its action.

scholarly journals Ce-emerin and LEM-2: essential roles in Caenorhabditis elegans development, muscle function, and mitosis

2012 ◽  
Vol 23 (4) ◽  
pp. 543-552 ◽  
Author(s):  
Rachel Barkan ◽  
Adam J. Zahand ◽  
Kfir Sharabi ◽  
Ayelet T. Lamm ◽  
Naomi Feinstein ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Caenorhabditis Elegans ◽  
Life Span ◽  
Muscle Activity ◽  
Striated Muscle ◽  
Null Alleles ◽  
Cell Nuclei ◽  
C Elegans ◽  
Dividing Cells ◽  
Mesodermal Lineage

Emerin and LEM2 are ubiquitous inner nuclear membrane proteins conserved from humans to Caenorhabditis elegans. Loss of human emerin causes Emery-Dreifuss muscular dystrophy (EDMD). To test the roles of emerin and LEM2 in somatic cells, we used null alleles of both genes to generate C. elegans animals that were either hypomorphic (LEM-2–null and heterozygous for Ce-emerin) or null for both proteins. Single-null and hypomorphic animals were viable and fertile. Double-null animals used the maternal pool of Ce-emerin to develop to the larval L2 stage, then arrested. Nondividing somatic cell nuclei appeared normal, whereas dividing cells had abnormal nuclear envelope and chromatin organization and severe defects in postembryonic cell divisions, including the mesodermal lineage. Life span was unaffected by loss of Ce-emerin alone but was significantly reduced in LEM-2–null animals, and double-null animals had an even shorter life span. In addition to striated muscle defects, double-null animals and LEM-2–null animals showed unexpected defects in smooth muscle activity. These findings implicate human LEM2 mutations as a potential cause of EDMD and further suggest human LEM2 mutations might cause distinct disorders of greater severity, since C. elegans lacking only LEM-2 had significantly reduced life span and smooth muscle activity.

A single histone H1 isoform (H1.1) is essential for chromatin silencing and germline development in Caenorhabditis elegans

Development ◽  
2001 ◽  
Vol 128 (7) ◽  
pp. 1069-1080 ◽  
Author(s):  
M.A. Jedrusik ◽  
E. Schulze
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Family ◽  
Molecular Mechanisms ◽  
Histone H1 ◽  
Specific Aspect ◽  
Polycomb Group ◽  
Germline Development ◽  
C Elegans ◽  
Proliferation And Differentiation ◽  
Germline Proliferation

In remarkable contrast to somatic cells, the germline of the nematode Caenorhabditis elegans efficiently silences transgenic DNA. The molecular mechanisms responsible for this have been shown to implicate chromatin proteins encoded by the mes genes (Kelly, W. G. and Fire, A. (1998) Development 125, 2451–2456), of which two are the C. elegans homologs of Polycomb Group gene transcriptional repressors. We have analyzed the contribution of the histone H1 gene family to this specific aspect of germ cells in C. elegans. We show with isotype-specific double stranded RNA-mediated interference (RNAi) that a single member of this gene family (H1.1) is essential for the repression of a silenced reporter-transgene in the germline of hermaphrodites and males, whereas no change is found in the somatic expression of this reporter. Additionally, RNA-mediated interference with H1.1 gene expression can cause a phenotype with severe affection of germline proliferation and differentiation in the hermaphrodite, and even sterility (5%-11% penetrance). These and further features observed in histone H1.1 RNAi experiments are also characteristic of the mes phenotype (Garvin, C., Holdeman, R. and Strome, S. (1998) Genetics 148, 167–185), which is believed to result from the desilencing of genes required for somatic differentiation in the germline. Our observations therefore support this interpretation of the mes phenotype and they identify a single histone H1 isoform (H1.1) as a new component specifically involved in chromatin silencing in the germline of C. elegans.

scholarly journals Distinct effects of tubulin isotype mutations on neurite growth in Caenorhabditis elegans

2017 ◽  
Vol 28 (21) ◽  
pp. 2786-2801 ◽  
Author(s):  
Chaogu Zheng ◽  
Margarete Diaz-Cuadros ◽  
Ken C. Q. Nguyen ◽  
David H. Hall ◽  
Martin Chalfie
Keyword(s):  
Caenorhabditis Elegans ◽  
Neuronal Development ◽  
Function Analysis ◽  
Critical Role ◽  
Neurite Growth ◽  
Cellular Level ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Exterior Surface ◽  
C Elegans

Tubulins, the building block of microtubules (MTs), play a critical role in both supporting and regulating neurite growth. Eukaryotic genomes contain multiple tubulin isotypes, and their missense mutations cause a range of neurodevelopmental defects. Using the Caenorhabditis elegans touch receptor neurons, we analyzed the effects of 67 tubulin missense mutations on neurite growth. Three types of mutations emerged: 1) loss-of-function mutations, which cause mild defects in neurite growth; 2) antimorphic mutations, which map to the GTP binding site and intradimer and interdimer interfaces, significantly reduce MT stability, and cause severe neurite growth defects; and 3) neomorphic mutations, which map to the exterior surface, increase MT stability, and cause ectopic neurite growth. Structure-function analysis reveals a causal relationship between tubulin structure and MT stability. This stability affects neuronal morphogenesis. As part of this analysis, we engineered several disease-associated human tubulin mutations into C. elegans genes and examined their impact on neuronal development at the cellular level. We also discovered an α-tubulin (TBA-7) that appears to destabilize MTs. Loss of TBA-7 led to the formation of hyperstable MTs and the generation of ectopic neurites; the lack of potential sites for polyamination and polyglutamination on TBA-7 may be responsible for this destabilization.

scholarly journals Transcriptome changes during the initiation and progression of Duchenne muscular dystrophy in Caenorhabditis elegans

2020 ◽  
Vol 29 (10) ◽  
pp. 1607-1623 ◽  
Author(s):  
Heather C Hrach ◽  
Shannon O’Brien ◽  
Hannah S Steber ◽  
Jason Newbern ◽  
Alan Rawls ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Disease Progression ◽  
Screening Method ◽  
Dystrophin Gene ◽  
Specific Gene ◽  
Missense Mutations ◽  
Compensatory Mechanisms ◽  
C Elegans

Abstract Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease characterized by progressive muscle degeneration. The condition is driven by nonsense and missense mutations in the dystrophin gene, leading to instability of the sarcolemma and skeletal muscle necrosis and atrophy. Resulting changes in muscle-specific gene expression that take place in dystrophin’s absence remain largely uncharacterized, as they are potentially obscured by the chronic inflammation elicited by muscle damage in humans. Caenorhabditis elegans possess a mild inflammatory response that is not active in the muscle, and lack a satellite cell equivalent. This allows for the characterization of the transcriptome rearrangements affecting disease progression independently of inflammation and regeneration. In effort to better understand these dynamics, we have isolated and sequenced body muscle-specific transcriptomes from C. elegans lacking functional dystrophin at distinct stages of disease progression. We have identified an upregulation of genes involved in mitochondrial function early in disease progression, and an upregulation of genes related to muscle repair in later stages. Our results suggest that in C. elegans, dystrophin may have a signaling role early in development, and its absence may activate compensatory mechanisms that counteract muscle degradation caused by loss of dystrophin. We have also developed a temperature-based screening method for synthetic paralysis that can be used to rapidly identify genetic partners of dystrophin. Our results allow for the comprehensive identification of transcriptome changes that potentially serve as independent drivers of disease progression and may in turn allow for the identification of new therapeutic targets for the treatment of DMD.

scholarly journals The unc-8 and sup-40 genes regulate ion channel function in Caenorhabditis elegans motorneurons.

Genetics ◽  
1995 ◽  
Vol 139 (3) ◽  
pp. 1261-1272 ◽  
Author(s):  
W Shreffler ◽  
T Magardino ◽  
K Shekdar ◽  
E Wolinsky
Keyword(s):  
Caenorhabditis Elegans ◽  
Ion Channel ◽  
Gene Family ◽  
Gene Products ◽  
Dominant Mutation ◽  
Common Component ◽  
Channel Function ◽  
Suppressor Mutations ◽  
Neuronal Types ◽  
Dominant Mutants

Abstract Two Caenorhabditis elegans genes, unc-8 and sup-40, have been newly identified, by genetic criteria, as regulating ion channel function in motorneurons. Two dominant unc-8 alleles cause motorneuron swelling similar to that of other neuronal types in dominant mutants of the deg-1 gene family, which is homologous to a mammalian gene family encoding amiloride-sensitive sodium channel subunits. As for previously identified deg-1 family members, unc-8 dominant mutations are recessively suppressed by mutations in the mec-6 gene, which probably encodes a second type of channel component. An unusual dominant mutation, sup-41 (lb125), also co-suppresses unc-8 and deg-1, suggesting the existence of yet another common component of ion channels containing unc-8 or deg-1 subunits. Dominant, transacting, intragenic suppressor mutations have been isolated for both unc-8 and deg-1, consistent with the idea that, like their mammalian homologues, the two gene products function as multimers. The sup-40 (lb130) mutation dominantly suppresses unc-8 motorneuron swelling and produces a novel swelling phenotype in hypodermal nuclei. sup-40 may encode an ion channel component or regulator that can correct the osmotic defect caused by abnormal unc-8 channels.

scholarly journals eat-5 and unc-7 represent a multigene family in Caenorhabditis elegans involved in cell-cell coupling.

1996 ◽  
Vol 134 (2) ◽  
pp. 537-548 ◽  
Author(s):  
T A Starich ◽  
R Y Lee ◽  
C Panzarella ◽  
L Avery ◽  
J E Shaw
Keyword(s):  
Green Fluorescent Protein ◽  
Caenorhabditis Elegans ◽  
Gene Family ◽  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Amino Acid Sequences ◽  
C Elegans ◽  
Pharyngeal Muscles ◽  
Green Fluorescent ◽  
Posterior Pharynx

The Drosophila melanogaster genes Passover and l(1)ogre and the Caenorhabditis elegans gene unc-7 define a gene family whose function is not known. We have isolated and characterized the C. elegans gene eat-5, which is required for synchronized pharyngeal muscle contractions, and find that it is a new member of this family. Simultaneous electrical and video recordings reveal that in eat-5 mutants, action potentials of muscles in the anterior and posterior pharynx are unsynchronized. Injection of carboxyfluorescein into muscles of the posterior pharynx demonstrates that all pharyngeal muscles are dye-coupled in wild-type animals; in eat-5 mutants, however, muscles of the anterior pharynx are no longer dye-coupled to posterior pharyngeal muscles. We show that a gene fusion of eat-5 to the green fluorescent protein is expressed in pharyngeal muscles. unc-7 and eat-5 are two of at least sixteen members of this family in C. elegans as determined by database searches and PCR-based screens. The amino acid sequences of five of these members in C. elegans have been deduced from cDNA sequences. Polypeptides of the family are predicted to have four transmembrane domains with cytoplasmic amino and carboxyl termini. We have constructed fusions of one of these polypeptides with beta-galactosidase and with green fluorescent protein. The fusion proteins appear to be localized in a punctate pattern at or near plasma membranes. We speculate that this gene family is required for the formation of gap junctions.

Sign in / Sign up

Export Citation Format

Share Document