Isolation of C. elegans Deletion Mutants Following ENU Mutagenesis and Thermostable Restriction Enzyme PCR Screening

2006 ◽  
Vol 32 (1) ◽  
pp. 083-086 ◽  
Author(s):  
Chunyi George Huang ◽  
Peter Agre ◽  
Kevin Strange ◽  
Todd Lamitina
Keyword(s):  
Restriction Enzyme ◽  
Deletion Mutants ◽  
Enu Mutagenesis ◽  
Pcr Screening ◽  
C Elegans

G3PP/PGP: A Novel Calorie Restriction Enzyme in C. Elegans

2021 ◽  
Vol 45 (7) ◽  
pp. S37
Author(s):  
Elite Possik ◽  
Clémence Schmitt ◽  
Anfal Al-Mass ◽  
Johanne Morin ◽  
Heidi Erb ◽  
...  
Keyword(s):  
Calorie Restriction ◽  
Restriction Enzyme ◽  
C Elegans

scholarly journals DNA Methylation on N6-Adenine Regulates the Hyphal Development during Dimorphism in the Early-Diverging Fungus Mucor lusitanicus

2021 ◽  
Vol 7 (9) ◽  
pp. 738
Author(s):  
Macario Osorio-Concepción ◽  
Carlos Lax ◽  
Eusebio Navarro ◽  
Francisco E. Nicolás ◽  
Victoriano Garre
Keyword(s):  
Wild Type Strain ◽  
Pathogenic Fungi ◽  
Infection Process ◽  
Deletion Mutants ◽  
Polar Growth ◽  
Wild Type ◽  
C Elegans ◽  
Double Deletion ◽  
Multiple Processes ◽  
Causative Agents

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales, causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxy adenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. The Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins similar to C. elegans NMAD-1 and ALKBH1, respectively. The function of these three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase in 6mA levels during the dimorphic transition, suggesting that 6mA is associated with dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction in polar growth, suggesting that 6mA also has a role during the spore–macrophage interaction that could be important in the infection process.

scholarly journals DNA Methylation on N6-Adenine Regulates the Hyphal Development During Dimorphism in the Early-Diverging Fungus Mucor lusitanicus

2021 ◽  
Author(s):  
Macario Osorio-Concepción ◽  
Carlos Lax ◽  
Eusebio Navarro ◽  
Francisco E. Nicolás ◽  
Victoriano Garre
Keyword(s):  
Wild Type Strain ◽  
Pathogenic Fungi ◽  
Infection Process ◽  
Deletion Mutants ◽  
Polar Growth ◽  
Wild Type ◽  
C Elegans ◽  
Double Deletion ◽  
Multiple Processes ◽  
Causative Agents

The epigenetic modifications control the pathogenicity of human pathogenic fungi, which have been poorly studied in Mucorales; causative agents of mucormycosis. This order belongs to a group referred to as early-diverging fungi that are characterized by high levels of N6-methyldeoxyadenine (6mA) in their genome with dense 6mA clusters associated with actively expressed genes. AlkB enzymes can act as demethylases of 6mA in DNA, with the most remarkable eukaryotic examples being mammalian ALKBH1 and Caenorhabditis elegans NMAD-1. Mucor lusitanicus (formerly M. circinelloides f. lusitanicus) genome contains one gene, dmt1, and two genes, dmt2 and dmt3, encoding proteins homologs to C. elegans NMAD-1 and ALKBH1, respectively. The function of the three genes was analyzed by the generation of single and double deletion mutants for each gene. Multiple processes were studied in the mutants, but defects were only found in single and double deletion mutants for dmt1. In contrast to the wild-type strain, dmt1 mutants showed an increase of 6mA levels during the dimorphic transition, suggesting that 6mA regulates dimorphism in M. lusitanicus. Furthermore, the spores of dmt1 mutants challenged with macrophages underwent a reduction of polar growth, suggesting that 6mA also has a role during the spore-macrophage interaction that could be important in the infection process.

Behavioural phenotyping of C. elegans on UV-killed E. coli mutants v1

2021 ◽  
Author(s):  
Saul Moore
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Deletion ◽  
Uv Light ◽  
Single Gene ◽  
Deletion Mutants ◽  
E Coli ◽  
C Elegans ◽  
Behavioural Phenotyping ◽  
Keio Collection ◽  
Single Gene Deletion

Protocol for screening candidate behaviour-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their effects on Caenorhabditis elegans behaviour when killed by ultraviolet (UV) light

Semaphorin 1a and semaphorin 1b are required for correct epidermal cell positioning and adhesion during morphogenesis in C. elegans

Development ◽  
2002 ◽  
Vol 129 (9) ◽  
pp. 2065-2078
Author(s):  
Val E. Ginzburg ◽  
Peter J. Roy ◽  
Joseph G. Culotti
Keyword(s):  
Axon Guidance ◽  
Transmembrane Proteins ◽  
Precursor Cell ◽  
Deletion Mutants ◽  
Normal Position ◽  
C Elegans ◽  
Seam Cell ◽  
Ray Cells ◽  
Cell Positioning ◽  
Anterior Posterior

The semaphorin family comprises secreted and transmembrane proteins involved in axon guidance and cell migration. We have isolated and characterized deletion mutants of C. elegans semaphorin 1a (Ce-sema-1a or smp-1) and semaphorin 1b (Ce-sema-1b or smp-2) genes. Both mutants exhibit defects in epidermal functions. For example, the R1.a-derived ray precursor cells frequently fail to change anterior/posterior positions completely relative to their sister tail lateral epidermal precursor cell R1.p, causing ray 1 to be formed anterior to its normal position next to ray 2. The ray cells, which normally separate from the lateral tail seam cell (SET) at the end of L4 stage, remains connected to the SET cell even in adult mutant males. The ray 1 defects are partially penetrant in each single Ce-sema-1 mutant at 20°C, but are greatly enhanced in Ce-sema-1 double mutants, suggesting that Ce-Sema-1a and Ce-Sema-1b function in parallel to regulate ray 1 position. Both mutants also have defects in other aspects of epidermal functions, including head and tail epidermal morphogenesis and touch cell axon migration, whereas, smp-1 mutants alone have defects in defecation and brood size. A feature of smp-1 mutants that is shared with mutants of mab-20 (which encodes Sema-2a) is the abnormal perdurance of contacts between epidermal cells.

scholarly journals Genome organization of thePseudomonas aeruginosanarrow host range plasmid R91–5 determined by deletion and cloning analysis

1985 ◽  
Vol 45 (2) ◽  
pp. 195-198 ◽  
Author(s):  
R. J. Moore ◽  
V. Krishnapillai
Keyword(s):  
Genetic Analysis ◽  
Host Range ◽  
Restriction Enzyme ◽  
Genome Organization ◽  
Conjugative Plasmid ◽  
Deletion Mutants ◽  
Broad Host Range ◽  
Dna Fragments ◽  
Narrow Host Range ◽  
Fertility Inhibition

SummaryBy physical and genetic analysis of deletion mutants of the narrow host range IncP-10P. aeruginosaconjugative plasmid R91–5 it has been shown that the phenotypes related to its transfer, namely the inhibition of the replication of the phage G101, entry exclusion and the fertility inhibition of the wide host range plasmid R18 map at kilobase coordinates 19·7–20·7, 18·5–19·7, 28·8–30·15 and/or 34·9–36·15, respectively. These locations have been confirmed by cloning restriction enzyme generated DNA fragments of R91–5 into a small broad host range, multicopy non-conjugative plasmid.

scholarly journals A simple and reliable method for creating PCR-detectable mutants in Arabidopsis with the polycistronic tRNA–gRNA CRISPR/Cas9 system

2019 ◽  
Vol 41 (10) ◽  
Author(s):  
Liangliang Hui ◽  
Min Zhao ◽  
Junqi He ◽  
Yingying Hu ◽  
Yanshuang Huo ◽  
...  
Keyword(s):  
Gene Editing ◽  
Reliable Method ◽  
Screening Methods ◽  
Deletion Mutants ◽  
Pcr Screening ◽  
Large Deletions ◽  
System A ◽  
Homozygous Mutant ◽  
Laborious Work ◽  
Rapid Tool

Abstract To develop an easy and robust method for creating genetically stable and easily detectable Arabidopsis mutants, we adopted the polycistronic tRNA–gRNA CRISPR/Cas9 (PTG/Cas9) system, a multiplex gene-editing tool in rice, with PTOX as the reporter gene. The PTG/Cas9 system has a great potential in generating large deletions detectable by PCR, which greatly simplifies the laborious work of mutant screening. We constructed a PTOX–PTG/Cas9 system with five gRNAs and introduced it into Arabidopsis. At T1 generation, 24.4% of transgenic plants were chimeric with PCR-detectable deletions in PTOX locus, but no homozygous mutant was found, indicating that gene editing occurred predominantly in somatic cells. After a self-cross propagation, 60% of T1 chimeric plants were able to produce homozygous, heterozygous, or bi-allelic ptox offsprings. Inheritable homozygous ptox mutants without Cas9 gene can be obtained earliest at T2 generation. We further targeted five other genes using the same procedure and achieved homozygous Cas9-free mutants with large deletions for all genes within three generations. We established a standard and reliable protocol to generate stable inherited deletion mutants in 2–3 generations along with simple PCR screening methods. We conclude that the rice PTG/Cas9 system is an efficient, easy, and rapid tool to edit genes in Arabidopsis. We propose that it could be applied to other genes in Arabidopsis, and it might have the potential to edit genes in other plant species as well.

scholarly journals Dietary supplementation with PUFAs rescues the eggshell defects caused by seipin mutations in C. elegans

2020 ◽  
Author(s):  
Xiaofei Bai ◽  
Leng-Jie Huang ◽  
Sheng-Wen Chen ◽  
Ben Nebenfuehr ◽  
Brian Wysolmerski ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fatty Acid Biosynthesis ◽  
Gene Expression Pattern ◽  
Dietary Supplementation ◽  
Embryonic Lethality ◽  
Permeability Barrier ◽  
Deletion Mutants ◽  
Functional Roles ◽  
C Elegans ◽  
Barrier Formation

AbstractSEIPIN, an ER membrane protein, plays critical roles in lipid droplet (LD) formation and lipid storage. Dysfunction of SEIPIN causes a variety of human diseases, including lipodystrophy, neuropathies, and male and female infertility. However, the cellular and molecular mechanisms of SEIPIN in causing these diseases are poorly understood. To address such mechanisms, we investigated the functional roles of R01B10.6 (seip-1), the sole SEIPIN1 ortholog in C. elegans, using CRISPR/Cas9 gene editing, and transcriptional assays. SEIP-1::mScarlet is widely expressed throughout development in C. elegans. Three full gene deletion mutants, generated by CRISPR/Cas9, displayed penetrant embryonic lethality. EM imaging and the visualization of reporter genes revealed that the lipid-rich permeability barrier, the innermost layer of the C. elegans embryonic eggshell, was defective or missing. Intriguingly, depletion of SEIP-1 revealed a perturbed gene expression pattern for fatty acid biosynthesis enzymes, in agreement with the disrupted permeability barrier formation phenotype of the embryos. Lastly, dietary supplementation of PUFAs rescued the embryonic lethality and defective permeability barrier in the deletion mutants. In sum, our study suggests that SEIP-1 may maternally regulate LD biogenesis and maintain lipid homeostasis to orchestrate the formation of the lipid-rich permeability barrier, which is crucial for eggshell formation and embryogenesis.

Restriction enzyme mapping of the DNA of Streptomyces bacteriophage B alpha and its deletion derivatives

1982 ◽  
Vol 152 (3) ◽  
pp. 1288-1291
Author(s):  
H Ishihara ◽  
M M Nakano ◽  
H Ogawara
Keyword(s):  
Restriction Enzyme ◽  
Cleavage Site ◽  
Restriction Enzymes ◽  
Deletion Mutants ◽  
Restriction Map ◽  
Restriction Enzyme Mapping ◽  
Enzyme Mapping

Cleavage analysis of actinophage B alpha DNA was done with several restriction enzymes, and a restriction map of the DNA was determined. The DNA appeared to carry cohesive ends. Deletion mutants of actinophage B alpha were isolated by five cycles of treatment with 15 mM PPi. Both mutants had deletions of 2.5 of 1.8 megadaltons near one end of the genome, and one of them lost the single EcoRI cleavage site.

Antioxidant rescue of C. elegans behaviour on Keio E. coli mutants v1

2021 ◽  
Author(s):  
Saul Moore
Keyword(s):  
Caenorhabditis Elegans ◽  
Gene Deletion ◽  
Single Gene ◽  
Deletion Mutants ◽  
E Coli ◽  
C Elegans ◽  
Keio Collection ◽  
Single Gene Deletion

Protocol for screening candidate behaviour-modifying E. coli BW25113 single-gene deletion mutants from the 'Keio Collection', to investigate their effects on Caenorhabditis elegans behaviour in the presence of antioxidants.

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