Characterization of Mutations Induced by Ethyl Methanesulfonate, UV, and Trimethylpsoralen in the Nematode Caenorhabditis elegans

Gengyo-Ando, Keiko; Mitani, Shohei

doi:10.1006/bbrc.2000.2260

Cited by 197 publications

(147 citation statements)

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“…The mutated alleles were obtained from a trimethylpsoralen/ultraviolet mutagenized library, as described previously 36 and identified by PCR amplification and all mutants were backcrossed with N2 at least twice before analysis. Detailed information of primer sets used is available in Supplementary Materials and Methods.…”

Section: Discussionmentioning

confidence: 99%

Reduced expression of BTBD10, an Akt activator, leads to motor neuron death

et al. 2012

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BTBD10, an Akt interactor, activates Akt by decreasing the protein phosphatase 2A-mediated dephosphorylation and inactivation of Akt. Overexpression of BTBD10 suppresses motor neuron death that is induced by a familial amyotrophic lateral sclerosis (ALS)-linked superoxide dismutase 1 (SOD1) mutant, G93A-SOD1 in vitro. In this study, we further investigated the BTBD10-mediated suppression of motor neuron death. We found that the small interfering RNA-mediated inhibition of BTBD10 expression led to the death of cultured motor neurons. In Caenorhabditis elegans (C. elegans), disruption of the btbd-10 gene caused not only loss of neurons, including both motor and touch-receptor neurons, but also a locomotion defect. In addition, we found that the expression of BTBD10 was generally decreased in the motor neurons from patients of sporadic ALS and transgenic mice overexpressing G93A-SOD1 (G93A-SOD1-transgenic mice). Collectively, these results suggest that the reduced expression of BTBD10 leads to motor neuron death both in vitro and in vivo. A gain-of-toxic-function of an intracellular molecule or a loss of normal function of an essential intracellular molecule, including an intracellular prosurvival signal, contributes to cell death. The Akt family of proteins (Akt1, 2 and 3 in mammalian cells) provides a major intracellular prosurvival signal by phosphorylating many target proteins. 1 The major upstream activator of Akt is the phosphatidylinositol 3-kinase (PI3K)-mediated signal that activates Akt by phosphorylation. Akt's activity is also regulated by protein phosphatase-mediated dephosphorylation. 2 In our previous study, we showed that BTBD10 activates Akt by inhibiting protein phosphatase 2A (PP2A)-mediated Akt dephosphorylation. 3 Recent advances in genetics and neuroscience have suggested that both gains of toxic and/or losses of normal function of intracellular molecules contribute to the pathogenesis of neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is a representative motor neuron-specific neurodegenerative disease. 4 Approximately 10% of ALS cases are genetically inherited. 4,5 Dominant mutations in the superoxide dismutase 1 (SOD1) gene (the first identified familial ALS (FALS)-linked gene) account for approximately 20% of FALS cases. The gain-of-toxic-function of SOD1, caused by a FALS-linked mutation, is thought to be closely linked to the ALS pathogenesis. The second identified FALS-linked gene, a FALS-linked mutation of which results in an autosomal-recessive phenotype, encodes a protein named alsin. 6,7 Loss of alsin function, caused by a FALSlinked mutation, leads to the abnormal endolysosomal trafficking and contributes to motor neuron death. 8 We have independently shown that wild-type alsin suppresses mutant SOD1-induced neuronal cell death by activating a prosurvival pathway involving Rac1, PI3K and Akt3, and that FALS-linked mutations in the gene abolish the alsin function. 9,10 To further characterize the Akt-mediated prosurvival pathways that are linked to the ALS pathogenesi...

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Section: Discussionmentioning

confidence: 99%

Reduced expression of BTBD10, an Akt activator, leads to motor neuron death

et al. 2012

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“…Endogenous G4 DNA loci were assayed using a PCR-based approach 39,40 . Genomic DNA was isolated either from single worms or pools of worms and subjected to nested rounds of PCRs with primers that flank a G4 motif; amplicons are typically 1 kb in size.…”

Section: Methodsmentioning

confidence: 99%

A Polymerase Theta-dependent repair pathway suppresses extensive genomic instability at endogenous G4 DNA sites

et al. 2014

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Genomes contain many sequences that are intrinsically difficult to replicate. Tracts of tandem guanines, for instance, have the potential to adopt stable G-quadruplex structures, which are prone to cause genome alterations. Here we describe G4 DNA-induced mutagenesis in Caenorhabditis elegans and identify a non-canonical DNA break repair mechanism that generates deletions characterized by an extremely narrow size distribution, minimal homology of exactly one nucleotide at the junctions, and by the occasional presence of templated insertions. This typical mutation profile is fully dependent on the A-family polymerase Theta, the absence of which leads to profound loss of sequences surrounding G4 motifs. Theta-mediated end-joining prevails over non-homologous end joining and homologous recombination and prevents genomic havoc at replication fork barriers at the expense of small deletions. G4 DNA-induced deletions also manifest in the genomes of wild isolates of C. elegans, indicating a protective role for this pathway during evolution.

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“…Several deletion alleles used in this work were isolated as previously described (Gengyo-Ando & Mitani 2000). The strains include: N2 Bristol wild -type, ift-81(tm2355), ift-81(tm2356), ift-81(tm2389), ift-74(tm2394), ift-74(tm2397), tm2394;tm2355, unc-86(eDf25), osm-1(p808), osm-1(p816), osm-5(p803), osm-5(ok451), osm-6(p811), che-2(e1033), che-13(e1805), che-13(n1520), tm2355;tmEx1455[pFX_venusT-ift-81(FL) (ift-81::venus), pFX_YFPT-unc-122], tm2355;tmEx1457[pFX_venusT, pFX_YFPT-unc-122], tm2394;tmEx1458[pFX_venusT-ift-74(FL) (ift-74::venus), pFX_YFPT-unc-122] andtm2394;tmEx1460[pFX_venusT, pFX_YFPT-unc-122], tmEx1446[pFX_venusT-ift-81(FL) (ift-81::venus), pRF4], tmEx1452 , (osm-6::venus), pRF4], tmEx1415 , pRF4], tmEx1444 (osm-9::venus), pRF4], tm2355;tmEx1444, tm2394;tmEx1444, tmEx1407[pFX_venusT-ift-81(aa1) (ift-81p::venus), pRF4], tm2355; tmEx1407 and tm2394; tmEx1407.…”

Section: Strainsmentioning

confidence: 99%