Large-scale RNAi screens identify novel genes that interact with the C. elegans retinoblastoma pathway as well as splicing-related components with synMuv B activity

Uropathogenic Escherichia coli (UPEC) is a major bacterial pathogen that causes urinary tract infections (UTIs). The mouse is an available UTI model for studying the pathogenicity; however, Caenorhabditis elegans represents as an alternative surrogate host with the capacity for high-throughput analysis. Then, we established a simple assay for a UPEC infection model with C. elegans for large-scale screening. A total of 133 clinically isolated E. coli strains, which included UTI-associated and fecal isolates, were applied to demonstrate the simple pathogenicity assay. From the screening, several virulence factors (VFs) involved with iron acquisition (chuA, fyuA, and irp2) were significantly associated with high pathogenicity. We then evaluated whether the VFs in UPEC were involved in the pathogenicity. Mutants of E. coli UTI89 with defective iron acquisition systems were applied to a solid killing assay with C. elegans. As a result, the survival rate of C. elegans fed with the mutants significantly increased compared to when fed with the parent strain. The results demonstrated, the simple assay with C. elegans was useful as a UPEC infectious model. To our knowledge, this is the first report of the involvement of iron acquisition in the pathogenicity of UPEC in a C. elegans model.

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Frequent Germline Mutations and Somatic Repeat Instability in DNA Mismatch-Repair-Deficient Caenorhabditis elegans

Genetics ◽

10.1093/genetics/161.2.651 ◽

2002 ◽

Vol 161 (2) ◽

pp. 651-660

Author(s):

Marcel Tijsterman ◽

Joris Pothof ◽

Ronald H A Plasterk

Keyword(s):

Caenorhabditis Elegans ◽

Mismatch Repair ◽

Large Scale ◽

Somatic Tissue ◽

Repeat Instability ◽

Dna Mismatch ◽

C Elegans ◽

Colon Cancers ◽

Dna Repeat ◽

Female Germline

Abstract Mismatch-repair-deficient mutants were initially recognized as mutation-prone derivatives of bacteria, and later mismatch repair deficiency was found to predispose humans to colon cancers (HNPCC). We generated mismatch-repair-deficient Caenorhabditis elegans by deleting the msh-6 gene and analyzed the fidelity of transmission of genetic information to subsequent generations. msh-6-defective animals show an elevated level of spontaneous mutants in both the male and female germline; also repeated DNA tracts are unstable. To monitor DNA repeat instability in somatic tissue, we developed a sensitive system, making use of heat-shock promoter-driven lacZ transgenes, but with a repeat that puts this reporter gene out of frame. In genetic msh-6-deficient animals lacZ+ patches are observed as a result of somatic repeat instability. RNA interference by feeding wild-type animals dsRNA homologous to msh-2 or msh-6 also resulted in somatic DNA instability, as well as in germline mutagenesis, indicating that one can use C. elegans as a model system to discover genes involved in maintaining DNA stability by large-scale RNAi screens.

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Synaptonemal Complex dimerization regulates chromosome alignment and crossover patterning in meiosis

10.1101/2020.09.24.310540 ◽

2020 ◽

Author(s):

Spencer G. Gordon ◽

Lisa E. Kursel ◽

Kewei Xu ◽

Ofer Rog

Keyword(s):

Synaptonemal Complex ◽

Sequence Homology ◽

Genetic Information ◽

Large Scale ◽

Molecular Mechanisms ◽

Homologous Chromosomes ◽

C Elegans ◽

Local Sequence ◽

Chromosome Alignment ◽

Dimerization Interface

AbstractDuring sexual reproduction the parental homologous chromosomes find each other (pair) and align along their lengths by integrating local sequence homology with large-scale contiguity, thereby allowing for precise exchange of genetic information. The Synaptonemal Complex (SC) is a conserved zipper-like structure that assembles between the homologous chromosomes. This phase-separated interface brings chromosomes together and regulates exchanges between them. However, the molecular mechanisms by which the SC carries out these functions remain poorly understood. Here we isolated and characterized two mutations in the dimerization interface in the middle of the SC zipper in C. elegans. The mutations perturb both chromosome alignment and the regulation of genetic exchanges. Underlying the chromosome-scale phenotypes are distinct alterations to the way SC subunits interact with one another. We propose that the SC brings homologous chromosomes together through two biophysical activities: obligate dimerization that prevents assembly on unpaired chromosomes; and a tendency to phase-separate that extends pairing interactions along the entire length of the chromosomes.

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RNA interference in Caenorhabditis elegans: Uptake, mechanism, and regulation

Parasitology ◽

10.1017/s0031182011001788 ◽

2011 ◽

Vol 139 (5) ◽

pp. 560-573 ◽

Cited By ~ 35

Author(s):

JIMMY J. ZHUANG ◽

CRAIG P. HUNTER

Keyword(s):

Rna Interference ◽

Genetic Analysis ◽

Large Scale ◽

Parasitic Nematodes ◽

Loss Of Function ◽

Uptake Mechanism ◽

Delivery Methods ◽

C Elegans ◽

Systemic Rnai ◽

Powerful Research Tool

SUMMARYRNA interference (RNAi) is a powerful research tool that has enabled molecular insights into gene activity, pathway analysis, partial loss-of-function phenotypes, and large-scale genomic discovery of gene function. While RNAi works extremely well in the non-parasitic nematode C. elegans, it is also especially useful in organisms that lack facile genetic analysis. Extensive genetic analysis of the mechanisms, delivery and regulation of RNAi in C. elegans has provided mechanistic and phenomenological insights into why RNAi is so effective in this species. These insights are useful for the testing and development of RNAi in other nematodes, including parasitic nematodes where more effective RNAi would be extremely useful. Here, we review the current advances in C. elegans for RNA delivery methods, regulation of cell autonomous and systemic RNAi phenomena, and implications of enhanced RNAi mutants. These discussions, with a focus on mechanism and cross-species application, provide new perspectives for optimizing RNAi in other species.

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The receptor tyrosine kinase HIR-1 coordinates HIF-independent responses to hypoxia and extracellular matrix injury

Science Signaling ◽

10.1126/scisignal.aat0138 ◽

2018 ◽

Vol 11 (550) ◽

pp. eaat0138

Author(s):

Roman Vozdek ◽

Yong Long ◽

Dengke K. Ma

Keyword(s):

Extracellular Matrix ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Large Scale ◽

Hypoxia Inducible Factor ◽

Metabolic Adaptation ◽

Loss Of Function ◽

Ecm Remodeling ◽

C Elegans ◽

Heme Peroxidases

Inadequate tissue oxygen, or hypoxia, is a central concept in the pathophysiology of ischemic disorders and cancer. Hypoxia promotes extracellular matrix (ECM) remodeling, cellular metabolic adaptation, and cancer cell metastasis. To discover new pathways through which cells respond to hypoxia, we performed a large-scale forward genetic screen inCaenorhabditis elegansand identified a previously uncharacterized receptor tyrosine kinase named HIR-1. Loss of function inhir-1phenocopied the impaired ECM integrity associated with hypoxia or deficiency in the oxygen-dependent dual oxidase, heme peroxidases, or cuticular collagens involved in ECM homeostasis. Genetic suppressor screens identified NHR-49 and MDT-15 as transcriptional regulators downstream of HIR-1. Furthermore,hir-1mutants showed defects in adapting to and recovering from prolonged severe hypoxia. We propose thatC. elegansHIR-1 coordinates hypoxia-inducible factor–independent responses to hypoxia and hypoxia-associated ECM remodeling through mechanisms that are likely conserved in other organisms.

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Expression Trapping: Identification of Novel Genes Expressed in Hematopoietic and Endothelial Lineages by Gene Trapping in ES Cells

Blood ◽

10.1182/blood.v92.12.4622 ◽

1998 ◽

Vol 92 (12) ◽

pp. 4622-4631 ◽

Cited By ~ 45

Author(s):

William L. Stanford ◽

Georgina Caruana ◽

Katherine A. Vallis ◽

Maneesha Inamdar ◽

Michihiro Hidaka ◽

...

Keyword(s):

Large Scale ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Gene Trap ◽

Novel Genes ◽

Specific Expression ◽

Blood Island

Abstract We have developed a large-scale, expression-based gene trap strategy to perform genome-wide functional analysis of the murine hematopoietic and vascular systems. Using two different gene trap vectors, we have isolated embryonic stem (ES) cell clones containing lacZreporter gene insertions in genes expressed in blood island and vascular cells, muscle, stromal cells, and unknown cell types. Of 79 clones demonstrating specific expression patterns, 49% and 16% were preferentially expressed in blood islands and/or the vasculature, respectively. The majority of ES clones that expressedlacZ in blood islands also expressed lacZ upon differentiation into hematopoietic cells on OP9 stromal layers. Importantly, the in vivo expression of the lacZ fusion products accurately recapitulated the observed in vitro expression patterns. Expression and sequence analysis of representative clones suggest that this approach will be useful for identifying and mutating novel genes expressed in the developing hematopoietic and vascular systems.

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Expression Trapping: Identification of Novel Genes Expressed in Hematopoietic and Endothelial Lineages by Gene Trapping in ES Cells

Blood ◽

10.1182/blood.v92.12.4622.424k23_4622_4631 ◽

1998 ◽

Vol 92 (12) ◽

pp. 4622-4631 ◽

Cited By ~ 4

Author(s):

William L. Stanford ◽

Georgina Caruana ◽

Katherine A. Vallis ◽

Maneesha Inamdar ◽

Michihiro Hidaka ◽

...

Keyword(s):

Large Scale ◽

Es Cells ◽

Expression Patterns ◽

Embryonic Stem ◽

Gene Trap ◽

Novel Genes ◽

Specific Expression ◽

Blood Island

We have developed a large-scale, expression-based gene trap strategy to perform genome-wide functional analysis of the murine hematopoietic and vascular systems. Using two different gene trap vectors, we have isolated embryonic stem (ES) cell clones containing lacZreporter gene insertions in genes expressed in blood island and vascular cells, muscle, stromal cells, and unknown cell types. Of 79 clones demonstrating specific expression patterns, 49% and 16% were preferentially expressed in blood islands and/or the vasculature, respectively. The majority of ES clones that expressedlacZ in blood islands also expressed lacZ upon differentiation into hematopoietic cells on OP9 stromal layers. Importantly, the in vivo expression of the lacZ fusion products accurately recapitulated the observed in vitro expression patterns. Expression and sequence analysis of representative clones suggest that this approach will be useful for identifying and mutating novel genes expressed in the developing hematopoietic and vascular systems.

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Large-scale analyses of human microbiomes reveal thousands of small, novel genes and their predicted functions

10.1242/prelights.7731 ◽

2019 ◽

Author(s):

Ganesh Kadamur

Keyword(s):

Large Scale ◽

Novel Genes

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Large-scale Gene Knockdown in C. elegans Using dsRNA Feeding Libraries to Generate Robust Loss-of-function Phenotypes

Journal of Visualized Experiments ◽

10.3791/50693 ◽

2013 ◽

Author(s):

Kathryn N. Maher ◽

Mary Catanese ◽

Daniel L. Chase

Keyword(s):

Large Scale ◽

Gene Knockdown ◽

Loss Of Function ◽

C Elegans

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NemaLife: A structured microfluidic culture device optimized for aging studies in crawling C. elegans

10.1101/675827 ◽

2019 ◽

Cited By ~ 4

Author(s):

Mizanur Rahman ◽

Hunter Edwards ◽

Nikolajs Birze ◽

Rebecca Gabrilska ◽

Kendra P. Rumbaugh ◽

...

Keyword(s):

Large Scale ◽

The Body ◽

Physiological Data ◽

Aging Research ◽

C Elegans ◽

Age Related ◽

Technology Platforms ◽

Aging Studies ◽

Pharyngeal Pumping ◽

Culture Maintenance

AbstractCaenorhabditis elegans is a powerful animal model in aging research. Standard longevity assays on agar plates involve the tedious task of picking and transferring animals to prevent younger progeny from contaminating age-synchronized adult populations. Large-scale studies employ progeny-blocking drugs or sterile mutants to avoid progeny contamination, but such manipulations change adult physiology and alter the influence of reproduction on normal aging. Moreover, for some agar growth-based technology platforms, such as automated lifespan machines, reagents such as food or drugs cannot be readily added/removed after initiation of the study. Current microfluidic approaches are well-suited to address these limitations, but in their liquid-based environments animals swim rather than crawl, introducing swim-induced stress in the lifespan analysis. Here we report a simple microfluidic device that we call NemaLife that features: 1) an optimized micropillar arena in which animals can crawl, 2) sieve channels that separate progeny and prevent the loss of adults from the arena during culture maintenance, and 3) ports which allow rapid accessibility to feed the adult-only population and introduce reagents as needed. Culture maintenance and liquid manipulation are performed with simple hand-held syringes to facilitate integration of our technology into general laboratory protocols. Additionally, device geometry and feeding protocols were designed to emulate the body gait, locomotion, and lifespan of animals reared on agar. We validated our approach with longevity analyses of classical aging mutants (daf-2, age-1, eat-2, and daf-16) and animals subjected to RNAi knockdown of age-related genes (age-1 and daf-16). We also showed that healthspan measures such as pharyngeal pumping and tap-induced stimulated reversals can be scored across the lifespan. Overall, the capacity to generate reliable lifespan and physiological data from the NemaLife chip underscores the potential of this device to accelerate healthspan and lifespan investigations in C. elegans.

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