Brassinosteroids (BRs) are phytohormones which regulate various developmental processes in plants. They are exceptional phytohormones, as they do not undergo long-distance transport between plant organs. However, knowledge about the function of the enzymes that catalyse BR biosynthesis (particularly its early stages) in cereal crops remains limited. Therefore, this study identifies and analyses the function of the HvDWARF5 (HvDWF5) gene, involved in the early stage of BR biosynthesis in barley (Hordeum vulgare), an important cereal crop, using the TILLING (Targeting Induced Local Lesions IN Genomes) approach. The detailed functional analysis allowed for the identification of various mutations in different gene fragments. The influence of these mutations on plant architecture, reproduction, and yield was characterised. Moreover, effects of the missense and intron retention mutations on sequence and splicing of the HvDWF5 transcript, sequence and predicted structure of the encoded HvDWF5 enzyme, and accumulation of endogenous BR were determined. Some of the barley mutants identified in this study showed semi-dwarfism, a trait of particular importance for cereal breeding and yield. However, unlike other BR mutants in cereals, this did not negatively affect grain size or weight. It indicated that mutations in this gene allow for a balance between plant height reduction and maintenance of grain size. Thus, the results of this study provide a novel insight into the role of the HvDWF5 gene in the BR biosynthesis-dependent regulation of architecture and reproduction of the important cereal crop - barley.
- MeSH
- brassinosteroidy * metabolismus biosyntéza MeSH
- fenotyp MeSH
- ječmen (rod) * genetika metabolismus růst a vývoj MeSH
- jedlá semena genetika metabolismus růst a vývoj MeSH
- mutace * genetika MeSH
- regulace genové exprese u rostlin MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné geny MeSH
- rostlinné proteiny * genetika metabolismus MeSH
- semena rostlinná genetika růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- brassinosteroidy * MeSH
- regulátory růstu rostlin MeSH
- rostlinné proteiny * MeSH
Light-harvesting protein LHCB5 is one of the three minor antenna proteins (LHCB4-6) that connect the core (C) of photosystem II (PSII) with strongly (S) and moderately (M) bound peripheral trimeric antennae (LHCIIs), forming a dimeric PSII supercomplex known as C2S2M2. Plants lacking LHCB4 and LHCB6 do not form C2S2M2, indicating that these minor antenna proteins are crucial for C2S2M2 assembly. However, studies on antisense asLhcb5 plants suggest this may not apply to LHCB5. Using mild clear-native PAGE (CN-PAGE) and electron microscopy (EM), we separated and structurally characterized the C2S2M2 supercomplex from the Arabidopsis lhcb5 mutant. When compared with wild type (WT), the C2S2M2 supercomplexes in the lhcb5 mutant have slightly different positions of S and M trimers and are generally smaller and present in the thylakoid membrane at higher density. Using CN-PAGE, we did not observe any PSII megacomplexes in the lhcb5 mutant, although they are routinely detected by this method in WT. However, we identified the megacomplexes directly in thylakoid membranes via EM, indicating that the megacomplexes are formed but are too labile to be separated. While in WT, both parallel- and non-parallel-associated PSII supercomplexes can be detected in the thylakoid membrane (Nosek et al., 2017, Plant Journal 89, pp. 104-111), only the parallel-associated PSII supercomplexes were found in the lhcb5 mutant. This finding suggests that the formation of non-parallel-associated PSII supercomplexes depends on the presence of LHCB5. The presence of large PSII supercomplexes and megacomplexes, even though less stable, could explain the WT-like photosynthetic characteristics of the lhcb5 mutant.
- MeSH
- Arabidopsis * genetika metabolismus MeSH
- fotosystém II (proteinový komplex) * metabolismus genetika MeSH
- mutace genetika MeSH
- proteiny huseníčku * metabolismus genetika MeSH
- proteiny vázající chlorofyl MeSH
- světlosběrné proteinové komplexy * metabolismus genetika MeSH
- tylakoidy * metabolismus ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fotosystém II (proteinový komplex) * MeSH
- LHCB5 protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku * MeSH
- proteiny vázající chlorofyl MeSH
- světlosběrné proteinové komplexy * MeSH
Indole-3-acetic acid (IAA), the most common form of auxin, is involved in a great range of plant physiological processes. IAA is synthesized from the amino acid tryptophan and can be transported and inactivated in a myriad of ways. Despite intense research efforts, there are still dark corners in our comprehension of IAA metabolism and its interplays with other pathways. Genetic screens are a powerful tool for unbiasedly looking for new players in a given biological process. However, pleiotropism of auxin-related phenotypes and indirect effects make it necessary to incorporate additional screening steps to specifically find mutants affected in IAA homeostasis. We previously developed and validated a high-throughput methodology to simultaneously quantify IAA, key precursors, and inactive forms from as little as 10 mg of fresh tissue. We have carried out a genetic screening to identify mutants involved in IAA metabolism. Auxin reporters DR5pro:VENUS and 35Spro:DII-VENUS were EMS-mutagenized and subjected to a parallel morphological and reporter-signal pre-screen. We then obtained the auxin metabolite profile of 325 M3 selected lines and used multivariate data analysis to identify potential IAA-metabolism mutants. To test the screening design, we identified the causal mutations in three of the candidate lines by mapping-by-sequencing: dii365.3, dii571.1 and dr693. These carry new alleles of CYP83A1, MIAO, and SUPERROOT2, respectively, all of which have been previously involved in auxin homeostasis. Our results support the suitability of this approach to find new genes involved in IAA metabolism.
BACKGROUND: Through the agnostic screening of patients with uncharacterised disease phenotypes for an upregulation of type I interferon (IFN) signalling, we identified a cohort of individuals heterozygous for mutations in PTPN1, encoding the protein-tyrosine phosphatase 1B (PTP1B). We aimed to describe the clinical phenotype and molecular and cellular pathology of this new disease. METHODS: In this case series, we identified patients and collected clinical and neuroradiological data through collaboration with paediatric neurology and clinical genetics colleagues across Europe (Czechia, France, Germany, Italy, Slovenia, and the UK) and Israel. Variants in PTPN1 were identified by exome and directed Sanger sequencing. The expression of IFN-stimulated genes was determined by quantitative (q) PCR or NanoString technology. Experiments to assess RNA and protein expression and to investigate type 1 IFN signalling were undertaken in patient fibroblasts, hTERT-immortalised BJ-5ta fibroblasts, and RPE-1 cells using CRISPR-Cas9 editing and standard cell biology techniques. FINDINGS: Between Dec 20, 2013, and Jan 11, 2023, we identified 12 patients from 11 families who were heterozygous for mutations in PTPN1. We found ten novel or very rare variants in PTPN1 (frequency on gnomAD version 4.1.0 of <1·25 × 10:sup>-6). Six variants were predicted as STOP mutations, two involved canonical splice-site nucleotides, and two were missense substitutions. In three patients, the variant occurred de novo, whereas in nine affected individuals, the variant was inherited from an asymptomatic parent. The clinical phenotype was characterised by the subacute onset (age range 1-8 years) of loss of motor and language skills in the absence of seizures after initially normal development, leading to spastic dystonia and bulbar involvement. Neuroimaging variably demonstrated cerebral atrophy (sometimes unilateral initially) or high T2 white matter signal. Neopterin in CSF was elevated in all ten patients who were tested, and all probands demonstrated an upregulation of IFN-stimulated genes in whole blood. Although clinical stabilisation and neuroradiological improvement was seen in both treated and untreated patients, in six of eight treated patients, high-dose corticosteroids were judged clinically to result in an improvement in neurological status. Of the four asymptomatic parents tested, IFN signalling in blood was normal (three patients) or minimally elevated (one patient). Analysis of patient blood and fibroblasts showed that tested PTPN1 variants led to reduced levels of PTPN1 mRNA and PTP1B protein, and in-vitro assays demonstrated that loss of PTP1B function was associated with impaired negative regulation of type 1 IFN signalling. INTERPRETATION: PTPN1 haploinsufficiency causes a type 1 IFN-driven autoinflammatory encephalopathy. Notably, some patients demonstrated stabilisation, and even recovery, of neurological function in the absence of treatment, whereas in others, the disease appeared to be responsive to immune suppression. Prospective studies are needed to investigate the safety and efficacy of specific immune suppression approaches in this disease population. FUNDING: The UK Medical Research Council, the European Research Council, and the Agence Nationale de la Recherche.
- MeSH
- dítě MeSH
- haploinsuficience * genetika MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mutace genetika MeSH
- nemoci mozku genetika MeSH
- neurozánětlivé nemoci genetika MeSH
- předškolní dítě MeSH
- tyrosinfosfatasa nereceptorového typu 1 * genetika MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- PTPN1 protein, human MeSH Prohlížeč
- tyrosinfosfatasa nereceptorového typu 1 * MeSH
Rasopathies are genetic disorders often associated with developmental delay and intellectual disability. Noonan syndrome (NS) is one of the most common Rasopathies, caused by mutations in PTPN11 in more than 50% of cases. In mammalian neurons, PTPN11 controls the trafficking of postsynaptic glutamate receptors. This process is disrupted in neurons expressing PTPN11 variants associated with Rasopathies and is thought to contribute to the cognitive impairments in Noonan syndrome. Recent work revealed presynaptic impairments upon expression of RASopathy-linked PTPN11 variants in Drosophila. However, the presynaptic role of PTPN11 has not yet been addressed in mammals. Here, we investigated membrane trafficking of synaptic vesicles in cultured mouse cortical neurons expressing Rasopathy-associated PTPN11D61Y variant. We observed a significantly smaller readily releasable and total recycling pool of synaptic vesicles. The drop in synaptic vesicle release competence was accompanied by a decreased rate of SV retrieval. Interestingly, the presynaptic phenotype was evident in mature (DIV21) but not in immature (DIV12) neurons. Thus, our data reveal importance of balanced PTPN11 activity for normal trafficking of neurotransmitter-filled synaptic vesicles in the presynaptic ending of mature neurons.
- MeSH
- kultivované buňky MeSH
- mutace genetika MeSH
- myši MeSH
- neurony metabolismus MeSH
- stárnutí genetika metabolismus MeSH
- synaptické vezikuly * metabolismus MeSH
- tyrosinfosfatasa nereceptorového typu 11 * metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- Ptpn11 protein, mouse MeSH Prohlížeč
- tyrosinfosfatasa nereceptorového typu 11 * MeSH
Formation of the apical hook in etiolated dicot seedlings results from differential growth in the hypocotyl apex and is tightly controlled by environmental cues and hormones, among which auxin and gibberellins (GAs) play an important role. Cell expansion is tightly regulated by the cell wall, but whether and how feedback from this structure contributes to hook development are still unclear. Here, we show that etiolated seedlings of the Arabidopsis (Arabidopsis thaliana) quasimodo2-1 (qua2) mutant, defective in pectin biosynthesis, display severe defects in apical hook formation and maintenance, accompanied by loss of asymmetric auxin maxima and differential cell expansion. Moreover, qua2 seedlings show reduced expression of HOOKLESS1 (HLS1) and PHYTOCHROME INTERACTING FACTOR4 (PIF4), which are positive regulators of hook formation. Treatment of wild-type seedlings with the cellulose inhibitor isoxaben (isx) also prevents hook development and represses HLS1 and PIF4 expression. Exogenous GAs, loss of DELLA proteins, or HLS1 overexpression partially restore hook development in qua2 and isx-treated seedlings. Interestingly, increased agar concentration in the medium restores, both in qua2 and isx-treated seedlings, hook formation, asymmetric auxin maxima, and PIF4 and HLS1 expression. Analyses of plants expressing a Förster resonance energy transfer-based GA sensor indicate that isx reduces accumulation of GAs in the apical hook region in a turgor-dependent manner. Lack of the cell wall integrity sensor THESEUS 1, which modulates turgor loss point, restores hook formation in qua2 and isx-treated seedlings. We propose that turgor-dependent signals link changes in cell wall integrity to the PIF4-HLS1 signaling module to control differential cell elongation during hook formation.
- MeSH
- Arabidopsis * genetika růst a vývoj metabolismus MeSH
- benzamidy MeSH
- buněčná stěna * metabolismus MeSH
- gibereliny metabolismus MeSH
- hypokotyl růst a vývoj genetika metabolismus MeSH
- kyseliny indoloctové * metabolismus MeSH
- mutace genetika MeSH
- pektiny metabolismus MeSH
- proteiny huseníčku * metabolismus genetika MeSH
- regulace genové exprese u rostlin * MeSH
- semenáček * genetika růst a vývoj metabolismus MeSH
- transkripční faktory bHLH * metabolismus genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- benzamidy MeSH
- gibereliny MeSH
- isoxaben MeSH Prohlížeč
- kyseliny indoloctové * MeSH
- pektiny MeSH
- PIF4 protein, Arabidopsis MeSH Prohlížeč
- proteiny huseníčku * MeSH
- transkripční faktory bHLH * MeSH
BACKGROUND: Colorectal cancer is still the second leading cause of cancer-related deaths and thus biomarkers allowing prediction of the resistance of patients to therapy and estimating their prognosis are needed. We designed a panel of 558 genes with pharmacogenomics records related to 5-fluorouracil resistance, genes important for sensitivity to other frequently used drugs, major oncodrivers, and actionable genes. We performed a target enrichment sequencing of DNA from tumors and matched blood samples of patients, and compared the results with patient prognosis stratified by systemic adjuvant chemotherapy. RESULTS: The median number of detected variants per tumor sample was 18.5 with 4 classified as having a high predicted functional effect and 14.5 moderate effect. APC, TP53, and KRAS were the most frequent mutated genes (64%, 59%, and 42% of mutated samples, respectively) followed by FAT4 (23%), FBXW7, and PIK3CA (16% for both). Patients with advanced stage III had more frequently APC, TP53, or KRAS mutations than those in stages I or II. KRAS mutation counts followed an increasing trend with grade (G1 < G2 < G3). The response to adjuvant therapy was worse in carriers of frameshift mutations in APC or 12D variant in KRAS, but none of these oncodrivers had prognostic value. Carriage of somatic mutations in any of the genes ABCA13, ANK2, COL7A1, NAV3, or UNC80 had prognostic relevance for worse overall survival (OS) of all patients. In contrast, mutations in FLG, GLI3, or UNC80 were prognostic in the same direction for patients untreated, and mutations in COL6A3, LRP1B, NAV3, RYR1, RYR3, TCHH, or TENM4 for patients treated with adjuvant therapy. The first association was externally validated. From all germline variants with high or moderate predicted functional effects (median 326 per patient), > 5% frequency and positive Manhattan plot based on 3-year RFS, rs72753407 in NFACS, rs34621071 in ERBB4, and rs2444274 in RIF1 were significantly associated with RFS, OS or both. CONCLUSIONS: The present study identified several putative somatic and germline genetic events with prognostic potential for colorectal cancer that should undergo functional characterization.
- Klíčová slova
- Carcinoma, Colorectal, Drug, Oncodriver, Pharmacogene, Prognosis, Resistance, Sequencing,
- MeSH
- chemorezistence genetika MeSH
- dospělí MeSH
- F-Box a WD repetice obsahující protein 7 genetika MeSH
- farmakogenetika metody MeSH
- fluoruracil terapeutické užití MeSH
- fosfatidylinositol-3-kinasy třídy I MeSH
- kolorektální nádory * genetika farmakoterapie patologie MeSH
- lidé středního věku MeSH
- lidé MeSH
- mutace genetika MeSH
- nádorové biomarkery genetika MeSH
- nádorový supresorový protein p53 genetika MeSH
- prognóza MeSH
- protein familiární adenomatózní polypózy genetika MeSH
- protoonkogenní proteiny p21(ras) genetika MeSH
- senioři MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- APC protein, human MeSH Prohlížeč
- F-Box a WD repetice obsahující protein 7 MeSH
- fluoruracil MeSH
- fosfatidylinositol-3-kinasy třídy I MeSH
- KRAS protein, human MeSH Prohlížeč
- nádorové biomarkery MeSH
- nádorový supresorový protein p53 MeSH
- PIK3CA protein, human MeSH Prohlížeč
- protein familiární adenomatózní polypózy MeSH
- protoonkogenní proteiny p21(ras) MeSH
- TP53 protein, human MeSH Prohlížeč
- MeSH
- astrocytom * genetika patologie MeSH
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- MAP kinasový signální systém * fyziologie genetika MeSH
- mutace genetika MeSH
- nádory mozku * genetika patologie MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- dopisy MeSH
Mitosis is a crucial stage in the cell cycle, controlled by a vast network of regulators responding to multiple internal and external factors. The fission yeast Schizosaccharomyces pombe demonstrates catastrophic mitotic phenotypes due to mutations or drug treatments. One of the factors provoking catastrophic mitosis is a disturbed lipid metabolism, resulting from, for example, mutations in the acetyl-CoA/biotin carboxylase (cut6), fatty acid synthase (fas2, also known as lsd1) or transcriptional regulator of lipid metabolism (cbf11) genes, as well as treatment with inhibitors of fatty acid synthesis. It has been previously shown that mitotic fidelity in lipid metabolism mutants can be partially rescued by ammonium chloride supplementation. In this study, we demonstrate that mitotic fidelity can be improved by multiple nitrogen sources. Moreover, this improvement is not limited to lipid metabolism disturbances but also applies to a number of unrelated mitotic mutants. Interestingly, the partial rescue is not achieved by restoring the lipid metabolism state, but rather indirectly. Our results highlight a novel role for nitrogen availability in mitotic fidelity.
- Klíčová slova
- Closed mitosis, Cut, Lipid metabolism, Mitotic catastrophe, Nitrogen availability, TOR,
- MeSH
- dusík * metabolismus MeSH
- metabolismus lipidů * MeSH
- mitóza * MeSH
- mutace genetika MeSH
- Schizosaccharomyces pombe - proteiny * metabolismus genetika MeSH
- Schizosaccharomyces * metabolismus genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- dusík * MeSH
- Schizosaccharomyces pombe - proteiny * MeSH
CDK13-related disorder, also known as congenital heart defects, dysmorphic facial features and intellectual developmental disorder (CHDFIDD) is associated with mutations in the CDK13 gene encoding transcription-regulating cyclin-dependent kinase 13 (CDK13). Here, we focused on the development of craniofacial structures and analyzed early embryonic stages in CHDFIDD mouse models, with one model comprising a hypomorphic mutation in Cdk13 and exhibiting cleft lip/palate, and another model comprising knockout of Cdk13, featuring a stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically expressed at high levels in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13 deficiency leads to development of hypoplastic branches of the trigeminal nerve including the maxillary branch. Additionally, we detected significant changes in the expression levels of genes involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes in the expression pattern of other key face-specific genes (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in the regulation of craniofacial morphogenesis.
- Klíčová slova
- Axons, CDK13, Craniofacial development, Neurite outgrowth, Orofacial clefts, Trigeminal ganglion,
- MeSH
- cyklin-dependentní kinasy metabolismus genetika MeSH
- embryo savčí metabolismus MeSH
- embryonální vývoj * genetika MeSH
- fenotyp MeSH
- lebka embryologie patologie MeSH
- mentální retardace genetika MeSH
- modely nemocí na zvířatech * MeSH
- mutace genetika MeSH
- myši MeSH
- nervus trigeminus embryologie MeSH
- neurogeneze * genetika MeSH
- obličej embryologie abnormality MeSH
- protein doublecortin MeSH
- rozštěp patra genetika patologie embryologie MeSH
- rozštěp rtu genetika patologie embryologie MeSH
- vývojová regulace genové exprese * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- cyklin-dependentní kinasy MeSH
- Dcx protein, mouse MeSH Prohlížeč
- protein doublecortin MeSH
