Detection of induced mutations in CaFAD2 genes by next-generation sequencing leading to the production of improved oil composition in Crambe abyssinica.

Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.

Functional analysis of the omega-6 fatty acid desaturase (CaFAD2) gene family of the oil seed crop Crambe abyssinica.

BACKGROUND: Crambe abyssinica produces high erucic acid (C22:1, 55-60%) in the seed oil, which can be further increased by reduction of polyunsaturated fatty acid (PUFA) levels. The omega-6 fatty acid desaturase enzyme (FAD2) is known to be involved in PUFA biosynthesis. In crambe, three CaFAD2 genes, CaFAD2-C1, CaFAD2-C2 and CaFAD2-C3 are expressed. RESULTS: The individual effect of each CaFAD2 gene on oil composition was investigated through studying transgenic lines (CaFAD2-RNAi) for differential expression levels in relation to the composition of seed-oil. Six first generation transgenic plants (T1) showed C18:1 increase (by 6% to 10.5%) and PUFA reduction (by 8.6% to 10.2%). The silencing effect in these T1-plants ranged from the moderate silencing (40% to 50% reduction) of all three CaFAD2 genes to strong silencing (95% reduction) of CaFAD2-C3 alone. The progeny of two T1-plants (WG4-4 and WG19-6) was further analysed. Four or five transgene insertions are characterized in the progeny (T2) of WG19-6 in contrast to a single insertion in the T2 progeny of WG4-4. For the individual T2-plants of both families (WG19-6 and WG4-4), seed-specific silencing of CaFAD2-C1 and CaFAD2-C2 was observed in several individual T2-plants but, on average in both families, the level of silencing of these genes was not significant. A significant reduction in expression level (P < 0.01) in both families was only observed for CaFAD2-C3 together with significantly different C18:1 and PUFA levels in oil. CONCLUSIONS: CaFAD2-C3 expression is highly correlated to levels of C18:1 (r = -0.78) and PUFA (r = 0.75), which suggests that CaFAD2-C3 is the most important one for changing the oil composition of crambe.

Structural insight into the recognition of the H3K4me3 mark by the TFIID subunit TAF3.

Trimethylation of lysine residue K4 of histone H3 (H3K4me3) strongly correlates with active promoters for RNA polymerase II-transcribed genes. Several reader proteins, including the basal transcription factor TFIID, for this nucleosomal mark have been identified. Its TAF3 subunit specifically binds the H3K4me3 mark via its conserved plant homeodomain (PHD) finger. Here, we report the solution structure of the TAF3-PHD finger and its complex with an H3K4me3 peptide. Using a combination of NMR, mutagenesis, and affinity measurements, we reveal the structural basis of binding affinity, methylation-state specificity, and crosstalk with asymmetric dimethylation of R2. A unique local structure rearrangement in the K4me3-binding pocket of TAF3 due to a conserved sequence insertion underscores the requirement for cation-pi interactions by two aromatic residues. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 "methyl-methyl" switch in the histone code dynamically regulates TFIID-promoter association.

Versatile conjugation of octreotide to dendrimers by cycloaddition ("click") chemistry to yield high-affinity multivalent cyclic Peptide dendrimers.

The somatostatin analogue Tyr(3)-octreotide, which has a high binding affinity for the SSTR2 receptor (somatostatin receptor subtype 2) expressed on tumor cells, is used clinically for the diagnosis and treatment of a variety of neuroendocrine tumors and gastrointestinal disorders. There is growing interest in the development of multivalent peptide systems, because they may have enhanced binding affinity compared to monovalent analogues. In this report, we describe the design and synthesis of a series of Tyr(3)-octreotide-containing monomeric, dimeric, and tetrameric dendrimeric conjugates. These multivalent dendrimeric cyclic peptides were obtained using Cu(I)-catalyzed 1,3-dipolar cycloaddition between peptidyl azides and dendrimeric alkynes. Their affinities for the SSTR2 receptor were determined by a competitive binding assay on rat brain sections.

Microwave-assisted click polymerization for the synthesis of Abeta(16-22) cyclic oligomers and their self-assembly into polymorphous aggregates.

We report on the design, synthesis, and structural analysis of cyclic oligomers with an amyloidogenic peptide sequence as the repeating unit to obtain novel self-assembling bionanomaterials. The peptide was derived from the Alzheimer Abeta(16-22) sequence since its strong tendency to form antiparallel beta-sheets ensured the formation of intermolecular hydrogen bridges on which the supramolecular assembly of the individual cyclic oligomers was based. The synthesis of the cyclic oligomers was performed via a microwave-assisted Cu(I)-catalyzed 1,3-dipolar cycloaddition reaction of azido-Lys-Leu-Val-Phe-Phe-Ala-Glu-propargyl amide as the monomer. The formation of cyclic oligomers, up to pentamers (35 amino acid residues), was verified by MALDI-TOF analysis and the individual cyclic monomer and dimer could be isolated by HPLC. Gelation behavior and the self-assembly of the linear monomer and the cyclic monomer and dimer were studied by TEM, FTIR and CD. Significant differences were observed in the morphology of the supramolecular aggregates of these three peptides that could be explained by alterations of the hydrogen bond network.

Synthesis and characterization of biodegradable peptide-based polymers prepared by microwave-assisted click chemistry.

In this study, the microwave-assisted copper(I)-catalyzed 1,3-dipolar cycloaddition reaction was used to synthesize peptide triazole-based polymers from two novel peptide-based monomers: azido-phenylalanyl-alanyl-lysyl-propargyl amide (1) and azido-phenylalanyl-alanyl-glycolyl-lysyl-propargyl amide (2). The selected monomers have sites for enzymatic degradation as well as for chemical hydrolysis to render the resulting polymer biodegradable. Depending on the monomer concentration in DMF, the molecular mass of the polymers could be tailored between 4.5 and 13.9 kDa (corresponding with 33-100 amino acid residues per polymer chain). As anticipated, both polymers can be enzymatically degraded by trypsin and chymotrypsin, whereas the ester bond in the polymer of 2 undergoes chemical hydrolysis under physiological conditions, as was shown by a ninhydrin-based colorimetric assay and MALDI-TOF analysis. In conclusion, the microwave-assisted copper(I)-catalyzed 1,3-dipolar cycloaddition reaction is an effective tool for synthesizing biodegradable peptide polymers, and it opens up new approaches toward the synthesis of (novel) designed biomedical materials.

Synthesis of peptide-based polymers by microwave-assisted cycloaddition backbone polymerization.

The optimized reaction conditions for the Cu(I)-catalyzed N-->C polymerization of azido-phenylalanyl-alanyl-propargyl amide to yield either high molecular weight linear polymers or medium-sized cyclic polymers is described. These reaction conditions will be applied to tailor the synthesis, properties, and structure of biologically relevant peptide-based biopolymers.

Synthesis of DOTA-conjugated multivalent cyclic-RGD peptide dendrimers via 1,3-dipolar cycloaddition and their biological evaluation: implications for tumor targeting and tumor imaging purposes.

This report describes the design and synthesis of a series of alpha(V)beta(3) integrin-directed monomeric, dimeric and tetrameric cyclo[Arg-Gly-Asp-d-Phe-Lys] dendrimers using "click chemistry". It was found that the unprotected N-epsilon-azido derivative of cyclo[Arg-Gly-Asp-d-Phe-Lys] underwent a highly chemoselective conjugation to amino acid-based dendrimers bearing terminal alkynes using a microwave-assisted Cu(I)-catalyzed 1,3-dipolar cycloaddition. The alpha(V)beta(3) binding characteristics of the dendrimers were determined in vitro and their in vivoalpha(V)beta(3) targeting properties were assessed in nude mice with subcutaneously growing human SK-RC-52 tumors. The multivalent RGD-dendrimers were found to have enhanced affinity toward the alpha(V)beta(3) integrin receptor as compared to the monomeric derivative as determined in an in vitro binding assay. In case of the DOTA-conjugated (111)In-labeled RGD-dendrimers, it was found that the radiolabeled multimeric dendrimers showed specifically enhanced uptake in alpha(V)beta(3) integrin expressing tumors in vivo. These studies showed that the tetrameric RGD-dendrimer had better tumor targeting properties than its dimeric and monomeric congeners.