CO2 conversion to isocyanate via multiple N-Si bond cleavage at a bulky uranium(III) complex.
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2015 Oct 28;51(84):15454-7. doi: 10.707c.CO2 conversion to isocyanate via multiple N-Si bond cleavage at a bulky uranium(III) complex.1, , , , , .1Univ. Grenoble Alpes, INAC-SCIB, F-38000 Grenoble, France.AbstractThe reaction of the sterically saturated uranium(III) tetrasilylamido complex [K(18c6)][U(N(SiMe3)2)4] with CO2 leads to CO2 insertion into the U-N bond affording the stable U(IV) isocyanate complex [K(18c6)][U(N(SiMe3)2)3(NCO)2]n that was crystallographically characterized. DFT studies indicate that the reaction involves the [2+2] cyclo-addition of a double bond of O=CO to the U-N(SiMe3)2 bond and proceeds to the final product through multiple silyl migration steps.PMID:
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External link. Please review our .Copy number variation of individual cattle genomes using next-generation sequencing.
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):778-90. doi: 10.1101/gr.. Epub
2012 Feb 2.Copy number variation of individual cattle genomes using next-generation sequencing.1, , , , , , , , , , , , , , .1USDA-ARS, ANRI, Bovine Functional Genomics Laboratory, Beltsville, Maryland 20705, USA.AbstractCopy number variations (CNVs) affect a wide range
however, CNVs in or near segmental duplication regions are often intractable. Using a read depth approach based on next-generation sequencing, we examined genome-wide copy number differences among five taurine (three Angus, one Holstein, and one Hereford) and one indicine (Nelore) cattle. Within mapped chromosomal sequence, we identified 1265 CNV regions comprising ~55.6-Mbp sequence--476 of which (~38%) have not previously been reported. We validated this sequence-based CNV call set with array comparative genomic hybridization (aCGH), quantitative PCR (qPCR), and fluorescent in situ hybridization (FISH), achieving a validation rate of 82% and a false positive rate of 8%. We further estimated absolute copy numbers for genomic segments and annotated genes in each individual. Surveys of the top 25 most variable genes revealed that the Nelore individual had the lowest copy numbers in 13 cases (~52%, χ(2) P-value &0.05). In contrast, genes related to pathogen- and parasite-resistance, such as CATHL4 and ULBP17, were highly duplicated in the Nelore individual relative to the taurine cattle, while genes involved in lipid transport and metabolism, including APOL3 and FABP2, were highly duplicated in the beef breeds. These CNV regions also harbor genes like BPIFA2A (BSP30A) and WC1, suggesting that some CNVs may be associated with breed-specific differences in adaptation, health, and production traits. By providing the first individualized cattle CNV and segmental duplication maps and genome-wide gene copy number estimates, we enable future CNV studies into highly duplicated regions in the cattle genome.PMID:
[PubMed - indexed for MEDLINE] Individualized cattle CNV map. The Btau_4.0 assembly is represented as black bars with assembly gaps indicated by white boxes on the chromosomes. Larger bars intersecting the chromosomes represent the previously discovered WSSD (red), WGAC (blue), and WSSD/WGAC joint-prediction (purple) regions. Tracks underneath the chromosomes represent the CNV data sets (in order from top to bottom) for DTTRACE, merged CNVRs from all data sets, BINE, BTAN1, BTAN2, BTAN3, and BTHO. The colors for each bar in the animal data set tracks represent the average estimated CN for each CNV as shown in the legend. The merged CNVR track does not have CN information and is uniformly colored brown.Genome Res. ):778-790.Correlation between computational predictions and experimental validations. (A) A good agreement of lengths (r = 0.904) exists between previously discovered WSSD+, WGAC+, and predicted DTTRACE duplications. (B) Calculated digital aCGH probe values (BTAN2_ngs) were compared with probe log2 ratios from a whole-genome aCGH (BTAN2_whole). Digital aCGH values were estimated using a log2 ratio of the 1-kbp CN windows from BTAN2 divided by CN estimates from DTTRACE. A moderate correlation (r = 0.524) was found for aCGH probe values and digital aCGH values within CNV intervals &20 kbp that had fewer than 80% of their lengths occupied by common repeats.Genome Res. ):778-790.Computational predictions and aCGH validations of segmental duplication copy number differences for six cattle genomes. Depth-of-coverage tracks for DTTRACE, BINE, BTAN2, and BTHO are below a UCSC track for each investigated gene region. Regions colored in red on the plot indicate excessive read depth (& mean + 4 × STDEV), whereas gray regions indicate intermediate read depth (& mean + 3 × STDEV). Normal read depth values are colored green (mean ± 2 × STDEV). Digital aCGH tracks show the log2 ratio of the copy number of each listed animal compared to DTTRACE, with high values listed in green (&0.5); low values: red (&-0.5); and nominal values: gray (0.5 & x & -0.5). Whole-genome CGH array experiments, using Dominette as a reference sample in all cases, are listed below the digital aCGH experiments. Color schemes for the aCGH plots are the same as for the digital aCGH. Previously detected segmental duplications (SDs) are shown below the UCSC plot, if present in the region. (A) CNVs intersecting the BPIFA2A (BSP30A) locus (chr13:87495). A duplication of this region was predicted for all animals and was confirmed by whole-genome aCGH. (B) In the ULBP17 locus (chr9:99803), BINE was predicted to have a higher copy number than DTTRACE across the region from both read depth and aCGH experiments. (C) The promoter region of FABP2 (chr6:8288) was a predicted duplication in Dominette (H beef breed), BTAN2 (A beef), and BINE (N dual-purpose) but not in BTHO (H milk).Genome Res. ):778-790.Cluster analysis of copy number variable genes in individual cattle. (A) Copy number values for each animal were plotted within the AOX1 locus (chr2:84307) using the color scheme depicted in the legend. Heatmap boxes represent 1-kbp sliding, nonoverlapping windows in the region. The dendrogram indicates the hierarchical ordering of animals based on a Pearson's hierarchical clustering of the CN values within the region. Within AOX1, the last exons are predicted to have a higher CN in BINE than in any other animal. This observation was confirmed using aCGH and qPCR. (B) A heatmap of APOL3 reveals significantly higher CN in the three Angus animals (BTAN3, BTAN2, and BTAN1) for the first APOL3 transcript (NM_) than in the other breeds (chr5:17344).Genome Res. ):778-790.Publication TypesMeSH TermsSubstancesSecondary Source IDGrant SupportFull Text SourcesMiscellaneous
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External link. Please review our .An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi.
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):2725-37. doi: 10.1128/IAI.00376-15. Epub
2015 Apr 20.An Invertron-Like Linear Plasmid Mediates Intracellular Survival and Virulence in Bovine Isolates of Rhodococcus equi.1, 1, 2, 2, 3, 4, 2, 5.1Microbial Pathogenesis Unit, School of Biomedical Sciences and Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, United Kingdom.2Division of Infection and Immunity, Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom.3Microbial Pathogenesis Unit, School of Biomedical Sciences and Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, United Kingdom Division of Infection and Immunity, Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom.4School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland.5Microbial Pathogenesis Unit, School of Biomedical Sciences and Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, United Kingdom Division of Infection and Immunity, Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom Grupo de Patogenómica Bacteriana, Facultad de Veterinaria, Universidad de León, León, Spain v.boland@ed.ac.uk.AbstractWe report a novel host-associated virulence plasmid in Rhodococcus equi, pVAPN, carried by bovine isolates of this facultative intracellular pathogenic actinomycete. Surprisingly, pVAPN is a 120-kb invertron-like linear replicon unrelated to the circular virulence plasmids associated with equine (pVAPA) and porcine (pVAPB variant) R. equi isolates. pVAPN is similar to the linear plasmid pNSL1 from Rhodococcus sp. NS1 and harbors six new vap multigene family members (vapN to vapS) in a vap pathogenicity locus presumably acquired via en bloc mobilization from a direct predecessor of equine pVAPA. Loss of pVAPN rendered R. equi avirulent in macrophages and mice. Mating experiments using an in vivo transconjugant selection strategy demonstrated that pVAPN transfer is sufficient to confer virulence to a plasmid-cured R. equi recipient. Phylogenetic analyses assigned the vap multigene family complement from pVAPN, pVAPA, and pVAPB to seven monophyletic clades, each containing plasmid type-specific allelic variants of a precursor vap gene carried by the nearest vap island ancestor. Deletion of vapN, the predicted "bovine-type" allelic counterpart of vapA, essential for virulence in pVAPA, abrogated pVAPN-mediated intramacrophage proliferation and virulence in mice. Our findings support a model in which R. equi virulence is conferred by host-adapted plasmids. Their central role is mediating intracellular proliferation in macrophages, promoted by a key vap determinant present in the common ancestor of the plasmid-specific vap islands, with host tropism as a secondary trait selected during coevolution with specific animal species. Copyright (C) 2015, American Society for Microbiology. All Rights Reserved.PMID:
[PubMed - indexed for MEDLINE] Detection of pVAPN by PFGE. (A) Genomic DNA of bovine isolate 1571 and equine isolate 103S; three and two independent lysates per strain are shown. Relevant positions of the lambda PFGE marker (New England BioLabs) are indicated. pVAPN is observable as a distinct band of ≈100 kb in the bovine isolate. (B) Southern blot analysis of bovine isolates PAM1571, PAM1533, and PAM1554 (strain 103S was used as a negative control). (Left) Relevant sections of the PFGE (right) membrane hybridized with a pVAPN-specific DNA probe (600-bp fragment encompassing the 3′ region of vapN and the 5′ region of vapQ). The arrow indicates the pVAPN band.Infect Immun. ):.Genome alignments of the linear virulence plasmid pVAPN, circular virulence plasmids pVAPA and pVAPB, and the respective closest homologs from nonpathogenic rhodococcal species (pNSL1 from Rhodococcus sp. NS1 [] and pREC1 from R. erythropolis []). The alignments were built with EasyFig (http://easyfig.sourceforge.net/). The circular plasmids (pVAPA, pVAPB, and pREC1) were linearized starting from the first conserved gene of the housekeeping backbone. Regions with significant similarity between plasmids are connected by gray stripes (tblastx E value threshold of 0.1); grayscale indicates percent similarity. ORFs are color coded as follows: hypothetical proteins (gray), conjugation or DNA replication/recombination/metabolism (red), DNA mobility genes (magenta), transcriptional regulators (blue), secreted proteins (dark green), membrane proteins (pale green), metabolic functions (yellow), vap family genes (black), and pseudogenes (brown). Green and pale red bars below the genes indicate conjugation and replication/partitioning functional modules, dashed underline indicates HGT regions identified by Alien_hunter (); and the triangle indicates a putative origin of replication. Abbreviations: 3oxoACPr, 3-oxoacyl-ACP 3oxoACPs, 3-oxoacyl-ACP Endo, Exo, Lig, LT, ly MT, Mtase, PK, PR, pentape ssRec, site- TPR, TPR FA met, fatty acid metabolism.Infect Immun. ):.ML trees of Rhodococcus plasmid backbones (A) and the R. equi vap multigene family (B). The Hasegawa-Kishino-Yano with gamma distribution (HKY+G) evolutionary model was used. (A) ML tree based on a concatenated alignment of orthologs from a selection of rhodococcal extrachromosomal replicons (total of 7,802 nucleotides). The genes used are indicated by dots in . Values &50 for 100 bootstrap replicates are indicated. Symbols: triangles, circles, circular plasmids. (B) vap family members derived from each of the predicted seven precursor vap genes in the MRCA of the extant pVAPA, pVAPB, and pVAPN PAIs are in gray balloons.Infect Immun. ):.pVAPN telomeric sequences. (A) ClustalΩ alignment of the left- and right-end 200 terminal nucleotides. Identical nucleotides are shaded (dark and light blue, purines and pyrimidines, respectively). Inverted repeats are indicated above the sequence. In red are four conserved palindromic sequences with the central motif GCTNCGC identified in the binding site of telomere-associated proteins involved in Streptomyces linear plasmid replication (). Several “GCTNCGC” palindromic sequences are normally present in the telomeres of rhodococcal linear plasmids () (see Fig. S2 in the supplemental material). (B) Secondary structures potentially formed by the palindromic sequences in pVAPN telomeres, as numbered in panel A. Structures were determined with mFold. Free energy (ΔG) values: -33.84 kcal/mol (left), -37.95 kcal/mol (right).Infect Immun. ):.Genetic structure of the vap PAIs from pVAPN (15.1 kb), pVAPA (21.5 kb), and pVAPB (15.9 kb). Color codes of genes: vap family (black), DNA conjugation/partitioning (red), DNA mobility/recombination (magenta), transcriptional regulators (blue), other regulators (cyan), membrane proteins (green), metabolic reactions (yellow). Orthologs are in the same color shade and linked by gray bands. ORFs encoding hypothetical proteins are represented in light blue-gray and in white if they are outside the PAI. White arrowheads point to the first and last genes of the consensus PAI. The traA pseudogene/phage excisionase-rep-copG HGT cluster presumably acquired by pVAPN from the pVAPA backbone is boxed. The figure also schematizes the probable evolutionary relationships of the vap multigene family as inferred from phylogenetic analyses (; see also Fig. S4 and S5 in the supplemental material) and PAI the model minimizes the number of vap gene loss events. Solid lines/arrows connect vap genes belonging to the same monophyletic group (thus likely representing allelic variants of a common vap gene ancestor). Curved lines/arrows indicate vap gene duplications within a PAI. Crosses denote vap genes that were lost, and asterisks indicate pseudogenes. Two alternative evolutionary paths are shown for vapA-vapB-vapK1-vapK2-vapN (see the legend of Fig. S5 in the supplemental material for additional details). The black dots indicate the non-vap genes used for the MLSA shown in Fig. S4B in the supplemental material.Infect Immun. ):.Hypothetical reconstruction of vap PAI evolution. (A) Model of vap multigene family evolution. Lines indicate the evolutionary path of the vap genes between ancestral PAIs, the most recent common ancestor (MRCA), and extant PAIs. Pre-pVAPA designates the hypothetical direct precursor of the current pVAPA PAI. Gene duplication events are indicated by red squares, gene loss events by crosses, and pseudogenes by asterisks and white borders. (B) Fate of the vap PAI and R. equi virulence plasmid evolution. (a) Acquisition by rhodococcal circular replicon of vap PAI ancestor conferring the ability to
(b) mobilization of vap PAI from pre-pVAPA plasmid to rhodoco (c) evolution of species specificity.Infect Immun. ):.Intracellular proliferation in murine (J774A.1) and human (THP-1) macrophages. Data are expressed as normalized IGC values (see Materials and Methods). Means of data from three duplicate experiments ± standard errors are shown. Statistical significance was analyzed by 2-way ANOVA; P values determined by ?idák post hoc multiple-comparison tests at each time point are shown if ≤0.05. (A) Plasmidless derivative and in-frame ΔvapN mutant of bovine isolate 1571. Two-way ANOVA P values = 0.0007 for J774A.1 and 0.0160 for THP-1. (B) Plasmidless derivative and in-frame ΔvapA mutant of equine isolate 103S. Two-way ANOVA P values = 0.0112 for J774A.1 cells and &0.0001 for THP-1 cells.Infect Immun. ):.Competitive virulence assay in mouse lung. BALB/c mice (n = 4 per time point) were infected intranasally with a ≈1:1 mixture of the test bacteria, and the competing populations were monitored 60 min after infection (t = 0) and then on four consecutive days. Bar height denotes total lung CFU, and the light and dark gray areas within bars indicate the proportions of the competing bacteria. Corresponding CI values are shown in . (A) Competition between wild-type bovine isolate 1571 and the isogenic plasmidless derivative 1571- at an infection dose of 3.7 × 107 CFU/mouse (2.3 × 107 and 1.4 × 107 CFU, respectively). (B) Competition between the avirulent 1571- strain and an in-frame 1571ΔvapN deletion mutant at an infection dose of 7.8 × 107 CFU/mouse (3.2 × 107 and 4.6 × 107 CFU, respectively).Infect Immun. ):.Transfer of pVAPN by mating confers virulence to a plasmid-negative R. equi recipient strain. (A) In vivo selection of pVAPN transconjugants in mice. Note the progressive enrichment of the recipient 103S-RmpR strain upon acquisition of the pVAPN plasmid. Time zero is 60 min after infection. (B) Intracellular proliferation in J774A.1 macrophages. Acquisition of pVAPN (and control pVAPA) promotes intracellular proliferation in the recipient 103S-RmpR strain. Data are expressed as normalized IGC values (see Materials and Methods). Means of data from three duplicate experiments ± P values (determined by 2-way ANOVA and ?idák post hoc multiple-comparison tests) are indicated. ns, not significant.Infect Immun. ):.Publication TypesMeSH TermsSubstancesSecondary Source IDGrant SupportFull Text Sources
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External link. Please review our .RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and Flowering.
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):294-309. doi: 10.1104/pp.15.01280. Epub
2015 Nov 17.RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and Flowering.1, 1, 2, 1.1Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853 (X.S., A.G., M.R.H., S.B.).2Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853 (X.S., A.G., M.R.H., S.B.) mrh5@cornell.edu.AbstractPlant RNA editosomes modify cytidines (C) to uridines (U) at specific sites in plastid and mitochondrial transcripts. Members of the RNA-editing factor interacting protein (RIP) family and Organelle RNA Recognition Motif-containing (ORRM) family are essential components of the Arabidopsis (Arabidopsis thaliana) editosome. ORRM2 and ORRM3 have been recently identified as minor mitochondrial editing factors whose silencing reduces editing efficiency at ~6% of the mitochondrial C targets. Here we report the identification of ORRM4 (for organelle RRM protein 4) as a novel, major mitochondrial editing factor that controls ~44% of the mitochondrial editing sites. C-to-U conversion is reduced, but not eliminated completely, at the affected sites. The orrm4 mutant exhibits slower growth and delayed flowering time. ORRM4 affects editing in a site-specific way, though orrm4 mutation affects editing of the entire transcript of certain genes. ORRM4 contains an RRM domain at the N terminus and a Gly-rich domain at the C terminus. The RRM domain provides the editing activity of ORRM4, whereas the Gly-rich domain is required for its interaction with ORRM3 and with itself. The presence of ORRM4 in the editosome is further supported by its interaction with RIP1 in a bimolecular fluorescence complementation assay. The identification of ORRM4 as a major mitochondrial editing factor further expands our knowledge of the composition of the RNA editosome and reveals that adequate mitochondrial editing is necessary for normal plant development. (C) 2016 American Society of Plant Biologists. All Rights Reserved.PMID:
[PubMed - in process] Transient silencing of ORRM4 causes mitochondrial editing defects. A, Relative RNA expression level of ORRM4 measured by . B, Twenty-two editing sites that experienced a significant decrease of editing extent ≥ 10% upon the silencing of ORRM4. Editing extents were measured by . Two biological replicates were used in each silencing experiment. Not inoculated, Plants not inoculated with Agrobacterium; GFPsil, plants inoculated with Agrobacterium carrying a GFP- ORRM2sil, plants inoculated with Agrobacteria harboring an ORRM2/GFP c ORRM3sil, plants inoculated with Agrobacteria harboring an ORRM3/GFP c ORRM4sil, plants inoculated with Agrobacterium carrying an ORRM4/GFP cosilencing construct.Plant Physiol. ):294-309.ORRM4 gene model, RNA expression level, and editing extents in the orrm4 mutant. A, Gene structure of ORRM4. Triangle indicates the locus of the T-DNA insertion. Dashed box shows the gene-specific region selected for the
experiment. B, Relative ORRM4 expression level measured by . ORRM4 expression is reduced to an undetectable level in orrm4 homozygous mutants relative to wild-type plants (n = 3). ***, Significance of P & 0.001. C, ORRM4 mutant plants recapitulate the mitochondrial editing defects in the ORRM4-silenced plants assayed by . Not inoculated, Plants not inoculated with Agrobacterium (n = 2); GFP_sil, plants inoculated with Agrobacterium carrying a GFP-silencing construct (n = 2); wt, wild-type Arabidopsis (n = 2); ORRM4sil_1, one plant inoculated with Agrobacterium carrying an ORRM4/GFP c orrm4, orrm4 homozygous mutant plants (n = 2).Plant Physiol. ):294-309.orrm4 mutation or silencing affects editing extent in a site-specific way. A, Proportion of sites affected by orrm4 mutation on each transcript. Each bar represents a transcript color-coded according to the complex to which it belongs. B, The decreased editing and the alteration of editing extents of sites on the rpl5 transcript measured by . C, The decreases in editing caused by ORRM4 silencing are highly correlated with the decreases caused by the orrm4 mutation on the rpl5 transcript. D, Editing at site nad4 C1101 is significantly reduced while editing at two sites next to it is not. E, Reduction of editing is observed at site ccmFc C160, but not at ccmFc C155. Not inoc, Plants not inoculated with Agrobacterium (n = 2); GFP_sil, plants inoculated with Agrobacterium carrying a GFP-silencing construct (n = 2); wt, wild-type Arabidopsis (n = 2); ORRM4sil_1, one plant inoculated with Agrobacterium carrying an ORRM4/GFP c orrm4 mutants (n = 2), Δorrm4 = (% editing of wt - % editing of orrm4)/% ΔORRM4sil_1 = (% editing of Not inoc - % editing of ORRM4sil_1)/% editing of Not inoc.Plant Physiol. ):294-309.orrm4 mutation can affect the transcript abundance. A, RNA blots hybridized with nad4, nad7, rps4, or rpl5 wt, three wild- orrm4-/-, three homozygous mutant plants. The negative of the ethidium bromide staining gel in the bottom panel serves as a loading control. B, Quantification of the transcript abundance visualized in the RNA blots and normalized to the amount of RNA loaded demonstrates a significant reduction for nad4 and rps4 (**P & 0.01), but not for nad7 or rpl5.Plant Physiol. ):294-309.orrm4 mutant plants show a slow-growth and late-flowering phenotype. A, Plant growth phenotype of orrm4 homozygous mutants (bottom) and wild-type plants (top) grown at 14 h of light per day for 29 d. B, Fresh weight of orrm4 homozygous mutants and wild-type plants grown at 14 h of light per day for 24 d, 31 d, and 37 d, respectively (n = 6). C, orrm4 homozygous mutants flower late compared to the wild-type plants. First bud, Days until visible flower buds in the c 1 cm, days until inflorescence stem reached 1 first flower, days until first open flower (n = 30). D and E, The fresh weight and the number of total leaves of the orrm4 mutants versus the wild-type plants at the opening of the first flower (n = 30). ** P & 0.01, ***P & 0.001.Plant Physiol. ):294-309.Stable expression of ORRM4 driven by a 35S promoter in orrm4 homozygous mutants restores the editing defects caused by the orrm4 mutation. A, Editing of rpl5 C59 assayed by . B, Editing of rps4 C235 assayed by . C, Editing of cox2 C138 assayed by . orrm4-/-, Three plants from the T1 segregating population that do not contain the ORRM4 orrm4-/- w/ 35S::ORRM4, plants from the T1 segregating population that carry one or two copies of ORRM4 transgene driven by a 35S promoter in the orrm4 wt, wild-type plants. Editing extents are shown as dark orange bars in complemented lines with excess editing of rpl5 C59 and rps4 C235 and decreased editing of cox2 C138. D, ORRM4 expression measured by . The level of ORRM4 expression was normalized to the average detected in wild-type plants. The average expression in wild-type plants was arbitrarily chosen to be 1 (n = 3, log10 scale).Plant Physiol. ):294-309.The genetic interaction between ORRM4 and other mitochondrial editing factors. A, The correlation between percent of reduced sites per gene affected in the orrm4 mutant versus that in the rip1 mutant. B, Number of mitochondrial sites affected in rip1, rip3, or orrm4 mutants. C, Number of mitochondrial sites affected in ORRM2-silenced, ORRM3-silenced, or orrm4 mutant plants. Numbers in overlap region indicates the sites under the control of both/all. Number outside the circles shows the sites that are not affected by any of the three mutations.Plant Physiol. ):294-309.ORRM4 interacts with RIP1, ORRM3, and ORRM4 when transiently coexpressed in N. benthamiana leaves by
assays. Each confocal image shows the merge of GFP signal (green) and chlorophyll autofluorescence (red). Scale bar (white line) represents 20 μm. White arrowheads point to the GFP signals indicating the interaction. A and B, ORRM4 does not interact with MEF9 serving as a negative control. C, ORRM4 interacts with RIP1. D, RIP1 forms homo-dimers. E, ORRM4 forms hetero-dimers with ORRM3. F, ORRM4 forms homo-dimers.Plant Physiol. ):294-309.In the
assay, ORRM4 interacts with ORRM3 and itself, and its C-terminal
domain is required for the interaction. A, ORRM4 forms hetero-dimers with ORRM3. B, ORRM4 forms homo-dimers. C, ORRM4 does not interact with ORRM2 in yeast. D, ORRM4 does not interact with MEF1 or MEF20 in yeast. E, The C-terminal
domain of ORRM4 is required for its interaction with itself, whereas its N-terminal RRM domain is not. F, The N-terminal RRM domain of ORRM3 interacts with the C-terminal
domain of ORRM4. EM, Yeasts transformed with a vector carrying an empty GW cassette as a negative control.Plant Physiol. ):294-309.The N-terminal RRM domain of ORRM4 is able to rescue RNA editing activity in the orrm4 mutant, whereas the C-terminal
domain does not. A, The motif diagram of ORRM1, ORRM2, ORRM3, and ORRM4. B, Constructs used for transformation. nORRM4, Amino acids 1 to 112; cORRM4, amino acids 113 to 290; mtp, amino acids 1 to 29 of the nuclear-encoded coxIV from yeast. C, Editing of rps4 C77 and rpl5 C59 assayed by . D, Editing of rps4 C77, rps4 C235, and rpl5 C59 assayed by . wt, Wild-type plants (n = 2); orrm4 homozygous mutants (n = 2); orrm4 w/ 35S::nORRM4, orrm4 homozygous mutants transformed with the N-terminal RRM domain of ORRM4 under a 35S orrm4 w/ 35S::mtp-cORRM4, orrm4 homozygous mutants transformed with the C-terminal
domain of ORRM4 under the control of a 35S promoter and an mtp. E, ORRM4 expression measured by . The level of ORRM4 expression was normalized to the average detected in wild-type plants. The average expression in wild-type plants was arbitrarily chosen to be one (two plants assayed for wild type and orrm4; no. of technical replicates, three). The expression of the two constructs was measured with two different set of primers.Plant Physiol. ):294-309.Stable expression of ORRM3 or the N-terminal RRM domain of ORRM3 driven by a 35S promoter is able to rescue RNA editing activity in the orrm4 mutant. A, Constructs used for transformation. nORRM3, Amino acids 1 to 120; ORRM3, the coding region of ORRM3. B, Editing of rps4 C77, rpl5 C59, and cox2 C138 assayed by . Left, Editing in mutant plants transformed with the full-length ORRM3; right, editing in mutant plants transformed with the N-terminal RRM domain of ORRM3; wt, wild-type plants (n = 2); orrm4 homozygous mutants (n = 2); orrm4 w/ 35S::ORRM3, orrm4 homozygous mutants transformed with the coding region of ORRM3 under a 35S orrm4 w/ 35S::nORRM3, orrm4 homozygous mutants transformed with the N-terminal RRM domain of ORRM3 under a 35S promoter. C, ORRM3 expression measured by . The level of ORRM3 expression was normalized to the average detected in wild-type plants. The average expression in wild-type plants was arbitrarily chosen to be one (n = 3).Plant Physiol. ):294-309.Model of the mitochondrial editosome that operates on ccmC C618 based on protein-protein interaction data. Data are from
assays ( ; ). The stoichiometry of the components is not indicated, as it is unknown. The editable C at position 618 on the ccmC transcript is represented by a star. The cis-element upstream of the target C is shown as a red bar.Plant Physiol. ):294-309.Publication TypesFull Text SourcesMiscellaneous
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