Showing papers on "Mutant published in 2015"
TL;DR: It is shown that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose, and the activation of a compensatory network to buffer against deleterious mutations was not observed after translational or transcriptional knockdown.
Abstract: Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.
996 citations
TL;DR: It is suggested that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.
705 citations
TL;DR: A genome-wide length-dependent increase in gene expression is identified in MeCP2 mutant mouse models and human RTT brains, and it is found that long genes as a population are enriched for neuronal functions and selectively expressed in the brain.
Abstract: Rett syndrome is caused by mutation of the MECP2 gene that codes for a protein that binds methylated DNA; this study reveals that MeCP2 affects the expression of long genes, which often serve neuronal functions Autism-related Rett syndrome is caused by disruption of the MECP2 gene, which codes for a methyl-DNA binding protein, but how MECP2 may control transcription of other genes has remained unclear Now Michael Greenberg and colleagues show that disruption of the Mecp2 gene in a mouse model and in human Rett syndrome leads to preferential upregulation of longer genes, and that these often serve neuronal functions Further data indicate that decreasing the expression of long genes, via hypomethylation of the dinucleotide CA, attenuates Rett-related dysfunctions in cultured neurons lacking MECP2 Disruption of the MECP2 gene leads to Rett syndrome (RTT), a severe neurological disorder with features of autism1 MECP2 encodes a methyl-DNA-binding protein2 that has been proposed to function as a transcriptional repressor, but despite numerous mouse studies examining neuronal gene expression in Mecp2 mutants, no clear model has emerged for how MeCP2 protein regulates transcription3,4,5,6,7,8,9 Here we identify a genome-wide length-dependent increase in gene expression in MeCP2 mutant mouse models and human RTT brains We present evidence that MeCP2 represses gene expression by binding to methylated CA sites within long genes, and that in neurons lacking MeCP2, decreasing the expression of long genes attenuates RTT-associated cellular deficits In addition, we find that long genes as a population are enriched for neuronal functions and selectively expressed in the brain These findings suggest that mutations in MeCP2 may cause neurological dysfunction by specifically disrupting long gene expression in the brain
511 citations
TL;DR: ABF1 is a functional homolog of AREB1, AREB2, and ABF3 in ABA-dependent gene expression from a comparative analysis between the areb 1 areb2 abf3 abf1 and areb1 areb 2 abF3 mutants.
Abstract: Under osmotic stress conditions such as drought and high salinity, the plant hormone abscisic acid (ABA) plays important roles in stress-responsive gene expression mainly through three bZIP transcription factors, AREB1/ABF2, AREB2/ABF4 and ABF3, which are activated by SNF1-related kinase 2s (SnRK2s) such as SRK2D/SnRK2.2, SRK2E/SnRK2.6 and SRK2I/SnRK2.3 (SRK2D/E/I). However, since the three AREB/ABFs are crucial, but not exclusive, for the SnRK2-mediated gene expression, transcriptional pathways governed by SRK2D/E/I are not fully understood. Here, we show that a bZIP transcription factor, ABF1, is a functional homolog of AREB1, AREB2 and ABF3 in ABA-dependent gene expression in Arabidopsis. Despite lower expression levels of ABF1 than those of the three AREB/ABFs, the areb1 areb2 abf3 abf1 mutant plants displayed increased sensitivity to drought and decreased sensitivity to ABA in primary root growth compared with the areb1 areb2 abf3 mutant. Genome-wide transcriptome analyses revealed that expression of downstream genes of SRK2D/E/I, which include many genes functioning in osmotic stress responses and tolerance such as transcription factors and LEA proteins, was mostly impaired in the quadruple mutant. Thus, these results indicate that the four AREB/ABFs are the predominant transcription factors downstream of SRK2D/E/I in ABA signalling in response to osmotic stress during vegetative growth.
428 citations
TL;DR: This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa.
Abstract: Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ∼2 weeks.
334 citations
TL;DR: A simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish and constitutes a unique tool to spatially control gene knockout and greatly broadens the scope of loss-of-function studies inZebrafish.
326 citations
TL;DR: A drug-inducible small guide RNA (sgRNA) vector system allowing for ubiquitous and efficient gene deletion in murine and human cells mediates the efficient, temporally controlled deletion of MCL-1 in human Burkitt lymphoma cell lines that require this anti-apoptotic BCL-2 protein for sustained survival and growth.
317 citations
TL;DR: It is found that 2-HG depletion did not inhibit growth of several IDH1 mutant solid cancer types, and NAD+ depletion is identified as a metabolic susceptibility of IDH 1 mutant cancers.
311 citations
TL;DR: It is concluded that ABP1 is not a key component in auxin signaling or Arabidopsis development, and the induction of known auxin-regulated genes is similar in both wild-type and abp1 plants in response to auxin treatments.
Abstract: Auxin binding protein 1 (ABP1) has been studied for decades. It has been suggested that ABP1 functions as an auxin receptor and has an essential role in many developmental processes. Here we present our unexpected findings that ABP1 is neither required for auxin signaling nor necessary for plant development under normal growth conditions. We used our ribozyme-based CRISPR technology to generate an Arabidopsis abp1 mutant that contains a 5-bp deletion in the first exon of ABP1, which resulted in a frameshift and introduction of early stop codons. We also identified a T-DNA insertion abp1 allele that harbors a T-DNA insertion located 27 bp downstream of the ATG start codon in the first exon. We show that the two new abp1 mutants are null alleles. Surprisingly, our new abp1 mutant plants do not display any obvious developmental defects. In fact, the mutant plants are indistinguishable from wild-type plants at every developmental stage analyzed. Furthermore, the abp1 plants are not resistant to exogenous auxin. At the molecular level, we find that the induction of known auxin-regulated genes is similar in both wild-type and abp1 plants in response to auxin treatments. We conclude that ABP1 is not a key component in auxin signaling or Arabidopsis development.
309 citations
City of Hope National Medical Center1, Sichuan University2, Tuen Mun Hospital3, National Center for Toxicological Research4, Koç University5, Bilkent University6, Beckman Research Institute7, Boğaziçi University8, University of Paris9, University of Nebraska Medical Center10, Beijing Institute of Genomics11, Aarhus University Hospital12, Helsinki University Central Hospital13
TL;DR: The findings suggest that JAK-STAT pathway inhibition may represent a therapeutic strategy in NK/T-cell lymphomas and their cell lines through next generation and/or Sanger sequencing.
Abstract: Lymphomas arising from NK or gd-T cells are very aggressive diseases and little is known regarding their pathogenesis. Here we report frequent activating mutations of STAT3 and STAT5B in NK/T-cell lymphomas (n ¼ 51), gd-T-cell lymphomas (n ¼ 43) and their cell lines (n ¼ 9) through next generation and/or Sanger sequencing. STAT5B N642H is particularly frequent in all forms of gd-T-cell lymphomas. STAT3 and STAT5B mutations are associated with increased phosphorylated protein and a growth advantage to transduced cell lines or normal NK cells. Growth-promoting activity of the mutants can be partially inhibited by a JAK1/2 inhibitor. Molecular modelling and surface plasmon resonance measurements of the N642H mutant indicate a marked increase in binding affinity of the phosphotyrosine-Y699 with the mutant histidine. This is associated with the prolonged persistence of the mutant phosphoSTAT5B and marked increase of binding to target sites. Our findings suggest that JAK-STAT pathway inhibition may represent a therapeutic strategy.
308 citations
TL;DR: It is suggested that extracellular miRNAs can function in target cells and uncover a potential new mode of action for mutant KRAS in CRC.
Abstract: Mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. To test whether exosomal RNAs also contribute to changes in gene expression in recipient cells, and whether mutant KRAS might regulate the composition of secreted microRNAs (miRNAs), we compared small RNAs of cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status. We show that exosomal profiles are distinct from cellular profiles, and mutant exosomes cluster separately from wild-type KRAS exosomes. miR-10b was selectively increased in wild-type exosomes, while miR-100 was increased in mutant exosomes. Neutral sphingomyelinase inhibition caused accumulation of miR-100 only in mutant cells, suggesting KRAS-dependent miRNA export. In Transwell co-culture experiments, mutant donor cells conferred miR-100-mediated target repression in wild-type-recipient cells. These findings suggest that extracellular miRNAs can function in target cells and uncover a potential new mode of action for mutant KRAS in CRC.
DOI: http://dx.doi.org/10.7554/eLife.07197.001
TL;DR: It is shown that SL-dependent regulation of shoot branching in Arabidopsis requires three D53-like proteins, SUPPRESSOR of MORE AXILLARY GROWTH2-LIKE6 (SMXL6), SMXL7, and SMXL8; this requires D14 and MAX2 and TPR2-dependent transcriptional repression activity and interaction with D14 in a GR24-responsive manner.
Abstract: Strigolactones (SLs) are carotenoid-derived phytohormones that control many aspects of plant development, including shoot branching, leaf shape, stem secondary thickening, and lateral root growth. In rice (Oryza sativa), SL signaling requires the degradation of DWARF53 (D53), mediated by a complex including D14 and D3, but in Arabidopsis thaliana, the components and mechanism of SL signaling involving the D3 ortholog MORE AXILLARY GROWTH2 (MAX2) are unknown. Here, we show that SL-dependent regulation of shoot branching in Arabidopsis requires three D53-like proteins, SUPPRESSOR OF MORE AXILLARY GROWTH2-LIKE6 (SMXL6), SMXL7, and SMXL8. The smxl6 smxl7 smxl8 triple mutant suppresses the highly branched phenotypes of max2 and the SL-deficient mutant max3. Overexpression of a mutant form of SMXL6 that is resistant to SL-induced ubiquitination and degradation enhances shoot branching. Exogenous application of the SL analog rac-GR24 causes ubiquitination and degradation of SMXL6, 7, and 8; this requires D14 and MAX2. D53-like SMXLs form complexes with MAX2 and TOPLESS-RELATED PROTEIN2 (TPR2) and interact with D14 in a GR24-responsive manner. Furthermore, D53-like SMXLs exhibit TPR2-dependent transcriptional repression activity and repress the expression of BRANCHED1. Our findings reveal that in Arabidopsis, D53-like SMXLs act with TPR2 to repress transcription and so allow lateral bud outgrowth but that SL-induced degradation of D53-like proteins activates transcription to inhibit outgrowth.
TL;DR: It is shown that long-term HSP90 inhibition significantly extends the survival of mutp53 Q/− (R248Q allele) and H/H (R172H allele) mice by 59% and 48%, respectively, but not their corresponding p53−/− littermates, and drug activity correlates with induction ofmutp53 degradation, tumour apoptosis and prevention of T-cell lymphomagenesis.
Abstract: Novel hotspot mutant p53 gain-of-function mouse model shows that tumours depend on its sustained expression, and genetic and pharmacological approaches reveal mutant p53 as an actionable cancer drug target The tumour suppressor TP53 is one of the most frequently mutated genes in human cancer Although some genetic alterations cause its complete loss, many are gain-of-function mutations in which mutant p53 protein gains new properties that promote tumorigenesis Ute Moll and colleagues now demonstrate in a mouse model that expression of such a p53 mutant is needed not only for tumour initiation but also for tumour maintenance They further show that they can target mutant p53 indirectly by inhibiting a chaperone called Hsp90 which is needed to keep mutant p53 protein stable In a mouse model of lymphoma, this approach led to degradation of mutant p53 and reduced tumour growth Missense mutations in p53 generate aberrant proteins with abrogated tumour suppressor functions that can also acquire oncogenic gain-of-function activities that promote malignant progression, invasion, metastasis and chemoresistance1,2,3,4,5 Mutant p53 (mutp53) proteins undergo massive constitutive stabilization specifically in tumours, which is the key requisite for the acquisition of gain-of-functions activities6,7,8 Although currently 11 million patients worldwide live with tumours expressing highly stabilized mutp53, it is unknown whether mutp53 is a therapeutic target in vivo Here we use a novel mutp53 mouse model expressing an inactivatable R248Q hotspot mutation (floxQ) to show that tumours depend on sustained mutp53 expression Upon tamoxifen-induced mutp53 ablation, allotransplanted and autochthonous tumours curb their growth, thus extending animal survival by 37%, and advanced tumours undergo apoptosis and tumour regression or stagnation The HSP90/HDAC6 chaperone machinery, which is significantly upregulated in cancer compared with normal tissues, is a major determinant of mutp53 stabilization9,10,11,12 We show that long-term HSP90 inhibition significantly extends the survival of mutp53 Q/− (R248Q allele2) and H/H (R172H allele3) mice by 59% and 48%, respectively, but not their corresponding p53−/− littermates This mutp53-dependent drug effect occurs in H/H mice treated with 17DMAG+SAHA and in H/H and Q/− mice treated with the potent Hsp90 inhibitor ganetespib Notably, drug activity correlates with induction of mutp53 degradation, tumour apoptosis and prevention of T-cell lymphomagenesis These proof-of-principle data identify mutp53 as an actionable cancer-specific drug target
TL;DR: Targeted mutagenesis using TALENs is a useful approach to creating important agronomic traits inFragrant rice by disrupting the OsBADH2 gene, which results in the synthesis of 2-acetyl-1-pyrroline, which is a major fragrance compound.
Abstract: Summary Fragrant rice is favoured worldwide because of its agreeable scent. The presence of a defective badh2 allele encoding betaine aldehyde dehydrogenase (BADH2) results in the synthesis of 2-acetyl-1-pyrroline (2AP), which is a major fragrance compound. Here, transcription activatorlike effector nucleases (TALENs) were engineered to target and disrupt the OsBADH2 gene. Six heterozygous mutants (30%) were recovered from 20 transgenic hygromycin-resistant lines. Sanger sequencing confirmed that these lines had various indel mutations at the TALEN target site. All six transmitted the BADH2 mutations to the T1 generation; and four T1 mutant lines tested also efficiently transmitted the mutations to the T2 generation. Mutant plants carrying only the desired DNA sequence change but not the TALEN transgene were obtained by segregation in the T1 and T2 generations. The 2AP content of rice grains of the T1 lines with homozygous mutations increased from 0 to 0.35–0.75 mg/kg, which was similar to the content of a positive control variety harbouring the badh2-E7 mutation. We also simultaneously introduced three different pairs of TALENs targeting three separate rice genes into rice cells by bombardment and obtained lines with mutations in one, two and all three genes. These results indicate that targeted mutagenesis using TALENs is a useful approach to creating important agronomic traits.
TL;DR: This review article comprehensively discusses the current strategies targeting oncogenic mutant p53 in cancers, with special focus on compounds that restore wild-type p53 transcriptional activity of Mutant p53 and those reducing mutants p53 levels.
Abstract: Among genetic alterations in human cancers, mutations in the tumor suppressor p53 gene are the most common, occurring in over 50% of human cancers. The majority of p53 mutations are missense mutations and result in the accumulation of dysfunctional p53 protein in tumors. These mutants frequently have oncogenic gain-of-function activities and exacerbate malignant properties of cancer cells, such as metastasis and drug resistance. Increasing evidence reveals that stabilization of mutant p53 in tumors is crucial for its oncogenic activities, while depletion of mutant p53 attenuates malignant properties of cancer cells. Thus, mutant p53 is an attractive druggable target for cancer therapy. Different approaches have been taken to develop small-molecule compounds that specifically target mutant p53. These include compounds that restore wild-type conformation and transcriptional activity of mutant p53, induce depletion of mutant p53, inhibit downstream pathways of oncogenic mutant p53, and induce synthetic lethality to mutant p53. In this review article, we comprehensively discuss the current strategies targeting oncogenic mutant p53 in cancers, with special focus on compounds that restore wild-type p53 transcriptional activity of mutant p53 and those reducing mutant p53 levels.
TL;DR: In this paper, the authors examined in vivo hematopoietic consequences of the most common U2AF1 mutation using a doxycycline-inducible transgenic mouse model.
TL;DR: This study targeted the tomato RIN gene, which encodes a MADS-box transcription factor regulating fruit ripening, and targeted three regions within the gene, confirming the applicability of the CRISPR/Cas9 mutagenesis system in tomato.
TL;DR: AtABCC3 detoxifies cadmium by transporting phytochelatin–cadmium complexes into the vacuoles, and it can functionally complement abcc1 abcc2 mutants.
Abstract: The heavy metal cadmium (Cd) is a widespread environmental contaminant with harmful effects on living cells. In plants, phytochelatin (PC)-dependent Cd detoxification requires that PC-Cd complexes are transported into vacuoles. Here, it is shown that Arabidopsis thaliana seedlings defective in the ABCC transporter AtABCC3 (abcc3) have an increased sensitivity to different Cd concentrations, and that seedlings overexpressing AtABCC3 (AtABCC3ox) have an increased Cd tolerance. The cellular distribution of Cd was analysed in protoplasts from abcc3 mutants and AtABCC3 overexpressors grown in the presence of Cd, by means of the Cd-specific fluorochromes 5-nitrobenzothiazole coumarin (BTC-5N) and Leadmium™ Green AM dye. This analysis revealed that Cd is mostly localized in the cytosol of abcc3 mutant protoplasts whereas there is an increase in vacuolar Cd in protoplasts from AtABCC3ox plants. Overexpression of AtABCC3 in cad1-3 mutant seedlings defective in PC production and in plants treated with l-buthionine sulphoximine (BSO), an inhibitor of PC biosynthesis, had no effect on Cd tolerance, suggesting that AtABCC3 acts via PCs. In addition, overexpression of AtABCC3 in atabcc1 atabcc2 mutant seedlings defective in the Cd transporters AtABCC1 and AtABCC2 complements the Cd sensitivity of double mutants, but not in the presence of BSO. Accordingly, the level of AtABCC3 transcript in wild type seedlings was lower than that of AtABCC1 and AtABCC2 in the absence of Cd but higher after Cd exposure, and even higher in atabcc1 atabcc2 mutants. The results point to AtABCC3 as a transporter of PC-Cd complexes, and suggest that its activity is regulated by Cd and is co-ordinated with the activity of AtABCC1/AtABCC2.
TL;DR: It is reported that ABI1 (ABA-INSENSITIVE 1) can interact with the U-box E3 ligases PUB12 and PUB13, but is ubiquitinated only when it interacts with ABA receptors in an in vitro assay.
Abstract: Clade A protein phosphatase 2Cs (PP2Cs) are abscisic acid (ABA) co-receptors that block ABA signalling by inhibiting the downstream protein kinases. ABA signalling is activated after PP2Cs are inhibited by ABA-bound PYR/PYL/RCAR ABA receptors (PYLs) in Arabidopsis. However, whether these PP2Cs are regulated by other factors remains unknown. Here, we report that ABI1 (ABA-INSENSITIVE 1) can interact with the U-box E3 ligases PUB12 and PUB13, but is ubiquitinated only when it interacts with ABA receptors in an in vitro assay. A mutant form of ABI1-1 that is unable to interact with PYLs is more stable than the wild-type protein. Both ABI1 degradation and all tested ABA responses are reduced in pub12 pub13 mutants compared with the wild type. Introducing the abi1-3 loss-of-function mutation into pub12 pub13 mutant recovers the ABA-insensitive phenotypes of the pub12 pub13 mutant. We thus uncover an important regulatory mechanism for regulating ABI1 levels by PUB12 and PUB13.
TL;DR: The functions and mechanisms of wild-type and mutant p53 in metabolic regulation are reviewed, and their potential roles in tumorigenesis are discussed.
TL;DR: It is demonstrated that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen and has important roles in redox homeostasis in all forms of life.
Abstract: Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathione-dependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen.
TL;DR: This study uncovered a regulatory mechanism, consisting of two calcineurin B-like (CBL) Ca sensors partnering with four CBL-interacting protein kinases (CIPKs) forming a CBL–CipK network that allows plant cells to sequester the extra Mg2+ into vacuoles, thereby protecting plants from high-Mg toxicity.
Abstract: Although Mg(2+) is essential for a myriad of cellular processes, high levels of Mg(2+) in the environment, such as those found in serpentine soils, become toxic to plants. In this study, we identified two calcineurin B-like (CBL) proteins, CBL2 and CBL3, as key regulators for plant growth under high-Mg conditions. The Arabidopsis mutant lacking both CBL2 and CBL3 displayed severe growth retardation in the presence of excess Mg(2+), implying elevated Mg(2+) toxicity in these plants. Unexpectedly, the cbl2 cbl3 mutant plants retained lower Mg content than wild-type plants under either normal or high-Mg conditions, suggesting that CBL2 and CBL3 may be required for vacuolar Mg(2+) sequestration. Indeed, patch-clamp analysis showed that the cbl2 cbl3 mutant exhibited reduced Mg(2+) influx into the vacuole. We further identified four CBL-interacting protein kinases (CIPKs), CIPK3, -9, -23, and -26, as functionally overlapping components downstream of CBL2/3 in the signaling pathway that facilitates Mg(2+) homeostasis. The cipk3 cipk9 cipk23 cipk26 quadruple mutant, like the cbl2 cbl3 double mutant, was hypersensitive to high-Mg conditions; furthermore, CIPK3/9/23/26 physically interacted with CBL2/3 at the vacuolar membrane. Our results thus provide evidence that CBL2/3 and CIPK3/9/23/26 constitute a multivalent interacting network that regulates the vacuolar sequestration of Mg(2+), thereby protecting plants from Mg(2+) toxicity.
TL;DR: Results suggest that OsHKT1;1 has a role in controlling Na+ concentration and preventing sodium toxicity in leaf blades and is regulated by the OsMYBc transcription factor.
Abstract: Sodium transporters play key roles in plant tolerance to salt stress. Here, we report that a member of the High-Affinity K(+) Transporter (HKT) family, OsHKT1;1, in rice (Oryza sativa 'Nipponbare') plays an important role in reducing Na(+) accumulation in shoots to cope with salt stress. The oshkt1;1 mutant plants displayed hypersensitivity to salt stress. They contained less Na(+) in the phloem sap and accumulated more Na(+) in the shoots compared with the wild type. OsHKT1;1 was expressed mainly in the phloem of leaf blades and up-regulated in response to salt stress. Using a yeast one-hybrid approach, a novel MYB coiled-coil type transcription factor, OsMYBc, was found to bind to the OsHKT1;1 promoter. In vivo chromatin immunoprecipitation and in vitro electrophoresis mobility shift assays demonstrated that OsMYBc binds to AAANATNC(C/T) fragments within the OsHKT1;1 promoter. Mutation of the OsMYBc-binding nucleotides resulted in a decrease in promoter activity of OsHKT1;1. Knockout of OsMYBc resulted in a reduction in NaCl-induced expression of OsHKT1;1 and salt sensitivity. Taken together, these results suggest that OsHKT1;1 has a role in controlling Na(+) concentration and preventing sodium toxicity in leaf blades and is regulated by the OsMYBc transcription factor.
TL;DR: Melatonin caused translocation of NPR1 (nonexpressor of PR1) protein from the cytoplasm into the nucleus indicating that melatonin‐elicited pathogen resistance in response to avirulent pathogen attack is SA‐dependent in Arabidopsis.
Abstract: Serotonin N-acetyltransferase (SNAT) is the penultimate enzyme in the melatonin biosynthesis pathway in plants. We examined the effects of SNAT gene inactivation in two Arabidopsis T-DNA insertion mutant lines. After inoculation with the avirulent pathogen Pseudomonas syringe pv. tomato DC3000 harboring the elicitor avrRpt2 (Pst-avrRpt2), melatonin levels in the snat knockout mutant lines were 50% less than in wild-type Arabidopsis Col-0 plants. The snat knockout mutant lines exhibited susceptibility to pathogen infection that coincided with decreased induction of defense genes including PR1, ICS1, and PDF1.2. Because melatonin acts upstream of salicylic acid (SA) synthesis, the reduced melatonin levels in the snat mutant lines led to decreased SA levels compared to wild-type, suggesting that the increased pathogen susceptibility of the snat mutant lines could be attributed to decreased SA levels and subsequent attenuation of defense gene induction. Exogenous melatonin treatment failed to induce defense gene expression in nahG Arabidopsis plants, but restored the induction of defense gene expression in the snat mutant lines. In addition, melatonin caused translocation of NPR1 (nonexpressor of PR1) protein from the cytoplasm into the nucleus indicating that melatonin-elicited pathogen resistance in response to avirulent pathogen attack is SA-dependent in Arabidopsis.
01 Jan 2015
TL;DR: It is proposed that binding of metal ions, most probably zinc, to conserved cysteinyl residues stabilizes the tertiary structure of wild-type p53.
Abstract: In human tumors, many different point mutations of the /;5.) gene knock out suppressor function and induce the p53 polypeptide to adopt an immunologically distinct, "mutant" conformation. Here we show that ex posure to the metal ( delator 1,10-phenanthroline induces wild-type p53 to adopt the mutant conformation and that this process is reversible. Con version to mutant phenotype also occurs after exposure to (a) an organic mercurial reagent targeting cysteinyl residues and (/>I low concentrations of mercury or cadmium. We propose that binding of metal ions, most probably zinc, to conserved cysteinyl residues stabilizes the tertiary struc ture of wild-type p53.
TL;DR: Results suggest how a single-base-pair mutation can cause a dramatic epigenetic switch and monoallelic expression in cancer cell lines.
Abstract: Somatic mutations in the promoter of the gene for telomerase reverse transcriptase (TERT) are the most common noncoding mutations in cancer. They are thought to activate telomerase, contributing to proliferative immortality, but the molecular events driving TERT activation are largely unknown. We observed in multiple cancer cell lines that mutant TERT promoters exhibit the H3K4me2/3 mark of active chromatin and recruit the GABPA/B1 transcription factor, while the wild-type allele retains the H3K27me3 mark of epigenetic silencing; only the mutant promoters are transcriptionally active. These results suggest how a single-base-pair mutation can cause a dramatic epigenetic switch and monoallelic expression.
TL;DR: The pleiotropic phenotype of the mutant demonstrated the importance of this glycosyltranferase in the physiology and virulence of the bacterium, and the fpgA mutant strain could be considered a good candidate for the design of an attenuated vaccine.
Abstract: Flavobacterium psychrophilum is an important fish pathogen, responsible for Cold Water Disease, with a significant economic impact on salmonid farms worldwide. In spite of this, little is known about the bacterial physiology and pathogenesis mechanisms, maybe because it is difficult to manipulate, being considered a fastidious microorganism. Mutants obtained using a Tn4351 transposon were screened in order to identify those with alteration in colony morphology, colony spreading and extracellular proteolytic activity, amongst other phenotypes. A F. psychrophilum mutant lacking gliding motility showed interruption of the FP1638 locus that encodes a putative type-2 glycosyltransferase (from here on referred to as fpgA gene, Flavobacterium psychrophilum glycosyltransferase). Additionally, the mutant also showed a decrease in the extracellular proteolytic activity as a consequence of down regulation in the fpgA mutant background of the fpp2-fpp1 operon promoter, responsible for the major extracellular proteolytic activity of the bacterium. The protein glycosylation profile of the parental strain showed the presence of a 22 kDa glycosylated protein which is lost in the mutant. Complementation with the fpgA gene led to the recovery of the wild-type phenotype. LD50 experiments in the rainbow trout infection model show that the mutant was highly attenuated. The pleiotropic phenotype of the mutant demonstrated the importance of this glycosyltranferase in the physiology and virulence of the bacterium. Moreover, the fpgA mutant strain could be considered a good candidate for the design of an attenuated vaccine.
TL;DR: In this paper, the authors identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene, named HaABCA2. All mutations truncate the ABCA2 protein.
Abstract: The use of conventional chemical insecticides and bacterial toxins to control lepidopteran pests of global agriculture has imposed significant selection pressure leading to the rapid evolution of insecticide resistance. Transgenic crops (e.g., cotton) expressing the Bt Cry toxins are now used world wide to control these pests, including the highly polyphagous and invasive cotton bollworm Helicoverpa armigera. Since 2004, the Cry2Ab toxin has become widely used for controlling H. armigera, often used in combination with Cry1Ac to delay resistance evolution. Isolation of H. armigera and H. punctigera individuals heterozygous for Cry2Ab resistance in 2002 and 2004, respectively, allowed aspects of Cry2Ab resistance (level, fitness costs, genetic dominance, complementation tests) to be characterised in both species. However, the gene identity and genetic changes conferring this resistance were unknown, as was the detailed Cry2Ab mode of action. No cross-resistance to Cry1Ac was observed in mutant lines. Biphasic linkage analysis of a Cry2Ab-resistant H. armigera family followed by exon-primed intron-crossing (EPIC) marker mapping and candidate gene sequencing identified three independent resistance-associated INDEL mutations in an ATP-Binding Cassette (ABC) transporter gene we named HaABCA2. A deletion mutation was also identified in the H. punctigera homolog from the resistant line. All mutations truncate the ABCA2 protein. Isolation of further Cry2Ab resistance alleles in the same gene from field H. armigera populations indicates unequal resistance allele frequencies and the potential for Bt resistance evolution. Identification of the gene involved in resistance as an ABC transporter of the A subfamily adds to the body of evidence on the crucial role this gene family plays in the mode of action of the Bt Cry toxins. The structural differences between the ABCA2, and that of the C subfamily required for Cry1Ac toxicity, indicate differences in the detailed mode-of-action of the two Bt Cry toxins.
TL;DR: It is shown that the TLE6 mutation is gender-specific and leads to the earliest known human embryonic lethality phenotype, in this first report of a human defect in a member of the subcortical maternal subcritical maternal complex.
Abstract: Embryonic lethality is a recognized phenotypic expression of individual gene mutations in model organisms. However, identifying embryonic lethal genes in humans is challenging, especially when the phenotype is manifested at the preimplantation stage. In an ongoing effort to exploit the highly consanguineous nature of the Saudi population to catalog recessively acting embryonic lethal genes in humans, we have identified two families with a female-limited infertility phenotype. Using autozygosity mapping and whole exome sequencing, we map this phenotype to a single mutation in TLE6, a maternal effect gene that encodes a member of the subcortical maternal complex in mammalian oocytes. Consistent with the published phenotype of mouse Tle6 mutants, embryos from female patients who are homozygous for the TLE6 mutation fail to undergo early cleavage, with resulting sterility. The human mutation abrogates TLE6 phosphorylation, a step that is reported to be critical for the PKA-mediated progression of oocyte meiosis II. Furthermore, the TLE6 mutation impairs its binding to components of the subcortical maternal complex. In this first report of a human defect in a member of the subcortical maternal subcritical maternal complex, we show that the TLE6 mutation is gender-specific and leads to the earliest known human embryonic lethality phenotype.
TL;DR: Experimental analysis of the complete set of genomic regions necessary for survival in a cyanobacterium achieved by screening for the fitness of hundreds of thousands of mutants provides a snapshot of the essential genes and intergenic regions necessary to live the photosynthetic lifestyle.
Abstract: Synechococcus elongatus PCC 7942 is a model organism used for studying photosynthesis and the circadian clock, and it is being developed for the production of fuel, industrial chemicals, and pharmaceuticals. To identify a comprehensive set of genes and intergenic regions that impacts fitness in S. elongatus, we created a pooled library of ∼ 250,000 transposon mutants and used sequencing to identify the insertion locations. By analyzing the distribution and survival of these mutants, we identified 718 of the organism's 2,723 genes as essential for survival under laboratory conditions. The validity of the essential gene set is supported by its tight overlap with well-conserved genes and its enrichment for core biological processes. The differences noted between our dataset and these predictors of essentiality, however, have led to surprising biological insights. One such finding is that genes in a large portion of the TCA cycle are dispensable, suggesting that S. elongatus does not require a cyclic TCA process. Furthermore, the density of the transposon mutant library enabled individual and global statements about the essentiality of noncoding RNAs, regulatory elements, and other intergenic regions. In this way, a group I intron located in tRNA(Leu), which has been used extensively for phylogenetic studies, was shown here to be essential for the survival of S. elongatus. Our survey of essentiality for every locus in the S. elongatus genome serves as a powerful resource for understanding the organism's physiology and defines the essential gene set required for the growth of a photosynthetic organism.