Identification of the causative mutation (using Whole Genome Sequencing) in Arabidopsis EMS mutants showing subcellular trafficking defects
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ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE146989 | GEO | 2020/03/16
REPOSITORIES: GEO
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Project description:Identification of the causative mutation (using Whole Genome Sequencing) in Arabidopsis EMS mutants showing subcellular trafficking defects
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Project description:Parkinson's disease (PD) is the most common neurodegenerative movement disorder, characterized by progressive loss of dopaminergic neurons in the substantia nigra, intraneuronal deposition of misfolded proteins known as Lewy bodies, and chronic neuroinflammation. PD can arise from monogenic mutations, but in most cases, the etiology is unclear. Viral infection is gaining increasing attentions as a trigger of PD. In this study, we investigated whether the PD-causative 620 aspartate (D) to asparagine (N) mutation in the vacuolar protein sorting 35 ortholog (Vps35) precipitated herpes simplex virus (HSV) infection. We observed that ectopic expression of Vps35 significantly reduced the proliferation and release of HSV-1 virions; the D620N mutation rendered Vps35 a partial loss of such inhibitory effects. Tetherin is a host cell protein capable of restricting the spread of encapsulated viruses including HSV-1 and SARS-Cov-2, both of which are implicated in the development of parkinsonism. Compared with cells overexpressing wildtype Vps35, cells expressing mutant Vps35 with D620N had less Tetherin on cell surfaces. Real-time and static cell imaging revealed that Tetherin recycled through Vps35-positive endosomes. Expression of Vps35 with D620N reduced endosomal dynamics and frequency of motile Tetherin-containing vesicles, a sign of defective production of recycling carriers. Our study suggests that the D620N mutation in Vps35 hinders Tetherin trafficking to cell surfaces and facilitates virus spread.
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Project description:Induced mutation is an important approach for creating novel plant germplasms. The introduction of dwarf or semi-dwarf genes into wheat has led to great advancements in yield improvement. In this study, four elite dwarf wheat mutants, named dm1-dm4, induced from γ-ray irradiation or ethyl methanesulfonate (EMS) mutagenesis, were used to identify transcriptome variations and dwarfing mechanisms. The results showed that the hotspot regions of mutations distributed on the chromosomes were consistent among the four mutant lines and these regions were mainly located around the 50, 360 and 400 Mb positions of chromosome 1A and the distal regions of chromosomes 2A and 2BL. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that 'protein processing in endoplasmic reticulum' was the most common significantly enriched pathway based on the differentially expressed genes (DEGs) between wildtype (WT) and the mutants. Notably, 18 out of 20 genes involved in this process encode heat shock proteins (HSPs). The results implied that HSPs might participate in wheat dwarfism response and function in the dwarfism process through protein folding and/or degradation. Moreover, seven genes in dm4 involved in modulating auxin levels were down-regulated and dm4 was more sensitive to auxin treatment compared with WT, indicating the important roles of auxin in regulation of dwarf phenotype in dm4. This study not only identified transcriptome sequence variation induced by physical and chemical mutagenesis but also revealed potential dwarfing mechanisms in the wheat mutant lines.
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Project description:C. trachomatis HtrA mutants generated using EMS mutagenesis
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Project description:Reducing lignin concentration in lignocellulosic biomass can increase forage digestibility for ruminant livestock and saccharification yields of biomass for bioenergy. In sorghum (Sorghum bicolor (L.) Moench) and several other C4 grasses, brown midrib (bmr) mutants have been shown to reduce lignin concentration. Putative bmr mutants isolated from an EMS-mutagenized population were characterized and classified based on their leaf midrib phenotype and allelism tests with the previously described sorghum bmr mutants bmr2, bmr6, and bmr12. These tests resulted in the identification of additional alleles of bmr2, bmr6, and bmr12, and, in addition, six bmr mutants were identified that were not allelic to these previously described loci. Further allelism testing among these six bmr mutants showed that they represented four novel bmr loci. Based on this study, the number of bmr loci uncovered in sorghum has doubled. The impact of these lines on agronomic traits and lignocellulosic composition was assessed in a 2-yr field study. Overall, most of the identified bmr lines showed reduced lignin concentration of their biomass relative to wild-type (WT). Effects of the six new bmr mutants on enzymatic saccharification of lignocellulosic materials were determined, but the amount of glucose released from the stover was similar to WT in all cases. Like bmr2, bmr6, and bmr12, these mutants may affect monolignol biosynthesis and may be useful for bioenergy and forage improvement when stacked together or in combination with the three previously described bmr alleles.
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Project description:The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.
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Project description:BackgroundCystic fibrosis (CF) is a rare condition in Asians. Since 1985, only about 30 Chinese patients have been reported with molecular confirmation.MethodUsing our in-house next-generation sequencing (NGS) pipeline for childhood bronchiectasis, we identified disease-causing CFTR mutations in CF patients in Hong Kong. After identifying p.I1023R in multiple patients, haplotype analysis was performed with genome-wide microarray to ascertain the likelihood of this being a founder mutation. We also assessed the processing and gating activity of the mutant protein by Western hybridization and patch-clamp test.ResultsMolecular diagnoses were confirmed in four patients, three of whom shared a missense mutation: CFTR:c.3068T>G:p.I1023R. The results suggested that p.I1023R is a founder mutation in southern Han Chinese. In addition, the processing and gating activity of the mutant protein was assessed by gel electrophoresis and a patch-clamp test. The mutant protein exhibited trafficking defects, suggesting that the dysfunction is caused by reduced cell surface expression of the fully glycosylated proteins.ConclusionTogether with other previously reported mutations, the specific founder mutation presented herein suggests a unique CFTR mutation spectrum in the southern Chinese populations, and this finding has vital implications for improving molecular testing and mutation-specific treatments for Chinese patients with CF.
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Project description:We have previously identified embryo-specific (emb) mutations that resulted in maize kernels containing abnormal embryos with normal-appearing endosperm among the progeny of active Robertson's Mutator stocks. Our rationale for the mutant screen described here is that it should be possible to produce ethyl methane sulfonate (EMS)-induced emb mutations at a frequency higher than that obtained by transposon mutagenesis and with greater ease. This proved to be the case when we screened for mutations that are embryo-specific among progeny of materials generated with EMS-treated pollen. The EMS-induced emb mutation frequency reported here is nearly three times the 4.5% we obtained with the transposable element stocks. The 45 mutants reported here were all tested for germination capacity and nearly all were lethal. The embryo phenotypes of 34 mutations were examined by dissection of the mature embryos. All were found to be retarded in development and morphologically abnormal. Half of the mutants in this group were blocked in the proembryo and transition stages. They likely include mutations in nuclear genes coding for plastid proteins. The other 17 are mainly blocked in the coleoptilar stage, or in later stages with a low frequency. This group likely includes mutations in genes regulating the completion of shoot apical meristem (SAM) development and accompanying morphogenetic events. Most of the complementation tests using 19 of the mutations in 35 unique combinations complimented each other, except for two pairs of mutations with similar phenotypes. Our results provide additional evidence for the presence of many emb loci in the maize genome.
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Project description:Bilateral alternation of muscle contractions requires reciprocal inhibition between the two sides of the hindbrain and spinal cord, and disruption of this inhibition should lead to simultaneous activation of bilateral muscles. At 1 day after fertilization, wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. Similar simultaneous contractions are observed in wild-type embryos treated with strychnine, a blocker of the inhibitory glycine receptor (GlyR). This result suggests that glycinergic synaptic transmission is defective in beo mutants. Muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the CNS. Recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. Furthermore, immunostaining with an antibody against GlyR showed clusters in wild-type neurons but not in beo neurons. These data suggest that the failure of GlyRs to aggregate at synaptic sites causes impairment of glycinergic transmission and abnormal behavior in beo mutants. Indeed, mutations in the GlyR beta-subunit, which are thought to be required for proper localization of GlyRs, were identified as the basis for the beo mutation. These data demonstrate that GlyRbeta is essential for physiologically relevant clustering of GlyRs in vivo. Because GlyR mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, the zebrafish beo mutant should be a useful animal model for this condition.
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