Polyploidization has provided much genetic variation for plant adaptive evolution, but the mechanisms by which the molecular evolution of polyploid genomes establishes genetic architecture underlying species differentiation are unclear. Brassica is an ideal model to increase knowledge of polyploid evolution. Here we describe a draft genome sequence of Brassica oleracea, comparing it with that of its sister species B. rapa to reveal numerous chromosome rearrangements and asymmetrical gene loss in duplicated genomic blocks, asymmetrical amplification of transposable elements, differential gene co-retention for specific pathways and variation in gene expression, including alternative splicing, among a large number of paralogous and orthologous genes. Genes related to the production of anticancer phytochemicals and morphological variations illustrate consequences of genome duplication and gene divergence, imparting biochemical and morphological variation to B. oleracea. This study provides insights into Brassica genome evolution and will underpin research into the many important crops in this genus.
We report an aptamer discovery technology that reproducibly yields higher affinity aptamers in fewer rounds compared to conventional selection. Our method (termed particle display) transforms libraries of solution-phase aptamers into "aptamer particles", each displaying many copies of a single sequence on its surface. We then use fluorescence-activated cell sorting (FACS) to individually measure the relative affinities of >10(8) aptamer particles and sort them in a high-throughput manner. Through mathematical analysis, we identified experimental parameters that enable optimal screening, and demonstrate enrichment performance that exceeds the theoretical maximum achievable with conventional selection by many orders of magnitude. We used particle display to obtain high-affinity DNA aptamers for four different protein targets in three rounds, including proteins for which previous DNA aptamer selection efforts have been unsuccessful. We believe particle display offers an extraordinarily efficient mechanism for generating high-quality aptamers in a rapid and economic manner, towards accelerated exploration of the human proteome.
Amylomaize (AMM) was utilized as the substrate for cycloamylose (CA) production in this study. After debranching, AMM was incubated with 10 U/g of Thermus aquaticus 4-?-glucanotransferase (TA 4?GTase) for various reaction times in water or in DMSO reaction system. The maximum conversion yield of CA was greatly improved from 24.55% to 45.58% and from 27.40% to 47.25% after debranching in the water and DMSO reaction systems, respectively. Compared with the method that produced CA from commercial potato amylose, this method produced CA from a natural amylopectin containing starch with enhanced conversion yield after debranching. Meanwhile, we found that the minimum degree of polymerization (DP) of CA from TA 4?GTase treatment was 5, regardless of the reaction conditions. These results were different from those reported in the literature that stated the minimum DP of CA produced with a TA 4?GTase treatment was 22 regardless of the reaction conditions.
Alternative splicing is a tissue and developmental stage specific process and greatly increases the biodiversity of proteins. Besides the trans- and cis-factors on the genome level, the process of RNA splicing is also regulated by epigenetic factors. In the present work, we proposed a new method to predict exon skipping events by using the histone methylation and acetylation information. The maximum relevance minimum redundancy method followed by incremental feature selection was performed to select the optimal feature set. Based on the optimized features, our method obtained an overall accuracy of 68.5% in a 10-fold cross validation test for exon skipping event prediction. It is anticipated that our method may become a useful tool for alternative splicing events prediction and the selected optimal features will provide insights into the regulatory mechanisms of epigenetic factors in alternative splicing.
Soybean (Glycine max) is one of the most important crop plants for providing protein and oil. It is important to investigate soybean genome for its economic and scientific value. Polyploidy is a widespread and recursive phenomenon during plant evolution, and it could generate massive duplicated genes which is an important resource for genetic innovation. Improved sequence alignment criteria and statistical analysis are used to identify and characterize duplicated genes produced by polyploidization in soybean. Based on the collinearity method, duplicated genes by whole genome duplication account for 70.3% in soybean. From the statistical analysis of the molecular distances between duplicated genes, our study indicates that the whole genome duplication event occurred more than once in the genome evolution of soybean, which is often distributed near the ends of chromosomes.
Human utilization of the mulberry-silkworm interaction started at least 5,000 years ago and greatly influenced world history through the Silk Road. Complementing the silkworm genome sequence, here we describe the genome of a mulberry species Morus notabilis. In the 330-Mb genome assembly, we identify 128 Mb of repetitive sequences and 29,338 genes, 60.8% of which are supported by transcriptome sequencing. Mulberry gene sequences appear to evolve ~3 times faster than other Rosales, perhaps facilitating the species spread worldwide. The mulberry tree is among a few eudicots but several Rosales that have not preserved genome duplications in more than 100 million years; however, a neopolyploid series found in the mulberry tree and several others suggest that new duplications may confer benefits. Five predicted mulberry miRNAs are found in the haemolymph and silk glands of the silkworm, suggesting interactions at molecular levels in the plant-herbivore relationship. The identification and analyses of mulberry genes involved in diversifying selection, resistance and protease inhibitor expressed in the laticifers will accelerate the improvement of mulberry plants.
In this research work, dextran was extracted from deteriorated sugarcane by alcohol precipitation and purified by gel filtration chromatography. Total acid hydrolysis and enzymatic degradation were utilized to confirm the purity of separated polysaccharide. Using the more recently available techniques such as ((1)H,(13)C) and two-dimensional (COSY and HMQC) NMR spectral analysis, methylation GC-MS and MALDI-TOF mass spectrometry, the structure of sugarcane dextran (SC-Dex) was investigated. On the basis of all spectra, SC-Dex showed a branched polysaccharide that contained only d-glucose residues in consecutive ?-(1-6) linkages in the main chain with ?-(1-3) branches. Methylation analysis showed that, the degree of ?-(1-3) branching levels was 4.37%. Several structural fragments were identified from MALDI-TOF spectrum with peak-to-peak mass difference of 162gmol(-1), which confirmed that the repeat unit in SC-Dex was d-glucose. The surface morphology of SC-Dex, revealed the spherically shaped and porous structure. Using HPSEC-MALLS-RI system, the average molecular weight of SC-Dex was estimated to be 1.753×10(6)gmol(-1) with an index of polydispersity value of 1.069.
Affinity reagents that bind to specific molecular targets are an essential tool for both diagnostics and targeted therapeutics. There is a particular need for advanced technologies for the generation of reagents that specifically target cell-surface markers, because transmembrane proteins are notoriously difficult to express in recombinant form. We have previously shown that microfluidics offers many advantages for generating affinity reagents against purified protein targets, and we have now significantly extended this approach to achieve successful in vitro selection of T7 phage-displayed peptides that recognize markers expressed on live, adherent cells within a microfluidic channel. As a model, we have targeted neuropilin-1 (NRP-1), a membrane-bound receptor expressed at the surface of human prostate carcinoma cells that plays central roles in angiogenesis, cell migration, and invasion. We show that, compared to conventional biopanning methods, microfluidic selection enables more efficient discovery of peptides with higher affinity and specificity by providing controllable and reproducible means for applying stringent selection conditions against minimal amounts of target cells without loss. Using our microfluidic system, we isolate peptide sequences with superior binding affinity and specificity relative to the well known NRP-1-binding RPARPAR peptide. As such microfluidic systems can be used with a wide range of biocombinatorial libraries and tissue types, we believe that our method represents an effective approach toward efficient biomarker discovery from patient samples.
We report the annotation and analysis of the draft genome sequence of Brassica rapa accession Chiifu-401-42, a Chinese cabbage. We modeled 41,174 protein coding genes in the B. rapa genome, which has undergone genome triplication. We used Arabidopsis thaliana as an outgroup for investigating the consequences of genome triplication, such as structural and functional evolution. The extent of gene loss (fractionation) among triplicated genome segments varies, with one of the three copies consistently retaining a disproportionately large fraction of the genes expected to have been present in its ancestor. Variation in the number of members of gene families present in the genome may contribute to the remarkable morphological plasticity of Brassica species. The B. rapa genome sequence provides an important resource for studying the evolution of polyploid genomes and underpins the genetic improvement of Brassica oil and vegetable crops.
The effects of cyclodextrins and derivatives on the activity and structure of pullulanase were investigated in this study. Our results showed that cyclodextrins and derivatives decreased the activity of pullulanase. This decrease was attributed to the interaction between the hydrophobic cavities of cyclodextrins and pullulanase. The hydrophobic cavity was confirmed to encapsulate the groups of pullulanase molecules by the addition of competitive guests. The results obtained from fluorescence spectroscopy analysis showed that ?-CD showed more efficient interactions with pullulanase molecules and the side chain groups of cyclodextrin significantly prevented the interaction between the hydrophobic cavities of ?-CD and pullulanase molecules. These findings suggest that the geometric dimension of hydrophobic cavities was crucial for matching between cyclodextrins and pullulanase and steric hindrance caused by side chains led to the decrease of the interaction.
In contrast to ?-, ?- and ?-cyclodextrins, little information is available on the isolation and separation of cycloamylose (CA) with degree of polymerization (DP) larger than 22. The objective of the current study was to develop a new iodine affinity capillary electrophoresis (CE) for separation of CA with DP of 22-42, which was based on the formation of CA-iodine inclusion complexes, CA with twisted conformations made complicated mobility behaviors on CE instead of merely size dependent. The influences of iodide/iodine ratio, iodine concentration, pH, ion strength of running phosphate buffer, voltage, and temperature on the peak resolution and electrophoretic mobility were further investigated. Our results suggest that iodine affinity capillary electrophoresis provides a versatile and selective tool for the isolation and analysis of CA with DP from 22 to 42.
Hardness, springiness and water retention of konjac glucomannan gel (g-KGM) as a novel carrier material for time-temperature integrator (TTI) in aseptic processing were determined and compared with those of sodium alginate gel (g-SA). Hardness of both g-KGM and g-SA increased with temperature: values of g-SA were significantly higher (p < 0.05) than those of g-KGM at all temperatures. No significant difference in springiness between g-KGM and g-SA from 40 °C to 90 °C and significant differences (p < 0.05) between 100 °C and 140 °C were found. Water retention property of g-KGM was lower than that of g-SA between 60 °C and 100 °C, but much higher between 100 °C and 140 °C. Heat transfer tests performed on g-KGM alone as well as on g-KGM as a carrier, embedded with TTI, ?-amylase as an integrator, indicated that g-KGM was suitable for industrial ultrahigh temperature sterilization test.
The effects of production parameters on the biotransformation of sucrose were investigated to enhance the yield of neo-FOS by Xanthophyllomyce dendrorhous cells. Cells showed optimal beta-fructofuranosidase activity at neutral pH condition and the yield of neo-FOS showed no significant differences between buffer and buffer-free systems. Cell concentration negatively affected the maximum neo-FOS yield. Sucrose concentration positively increased the maximum yield of neo-FOS. Elevating the reaction temperature to 30 degrees C, the neo-FOS productivity increased 1.85-fold compared with that at 20 degrees C. Meanwhile, cell age of 32 h enabled the biotransformation of sucrose more efficiently. In addition, free cells exhibited a higher productivity over immobilized cells. The maximum neo-FOS concentration finally reached 227.72 g/l from 400 g/l sucrose under the optimal conditions.
Polyploidy often confers emergent properties, such as the higher fibre productivity and quality of tetraploid cottons than diploid cottons bred for the same environments. Here we show that an abrupt five- to sixfold ploidy increase approximately 60?million years (Myr) ago, and allopolyploidy reuniting divergent Gossypium genomes approximately 1-2 Myr ago, conferred about 30-36-fold duplication of ancestral angiosperm (flowering plant) genes in elite cottons (Gossypium hirsutum and Gossypium barbadense), genetic complexity equalled only by Brassica among sequenced angiosperms. Nascent fibre evolution, before allopolyploidy, is elucidated by comparison of spinnable-fibred Gossypium herbaceum A and non-spinnable Gossypium longicalyx F genomes to one another and the outgroup D genome of non-spinnable Gossypium raimondii. The sequence of a G. hirsutum A(t)D(t) (in which t indicates tetraploid) cultivar reveals many non-reciprocal DNA exchanges between subgenomes that may have contributed to phenotypic innovation and/or other emergent properties such as ecological adaptation by polyploids. Most DNA-level novelty in G. hirsutum recombines alleles from the D-genome progenitor native to its New World habitat and the Old World A-genome progenitor in which spinnable fibre evolved. Coordinated expression changes in proximal groups of functionally distinct genes, including a nuclear mitochondrial DNA block, may account for clusters of cotton-fibre quantitative trait loci affecting diverse traits. Opportunities abound for dissecting emergent properties of other polyploids, particularly angiosperms, by comparison to diploid progenitors and outgroups.
The Pacific oyster Crassostrea gigas belongs to one of the most species-rich but genomically poorly explored phyla, the Mollusca. Here we report the sequencing and assembly of the oyster genome using short reads and a fosmid-pooling strategy, along with transcriptomes of development and stress response and the proteome of the shell. The oyster genome is highly polymorphic and rich in repetitive sequences, with some transposable elements still actively shaping variation. Transcriptome studies reveal an extensive set of genes responding to environmental stress. The expansion of genes coding for heat shock protein 70 and inhibitors of apoptosis is probably central to the oysters adaptation to sessile life in the highly stressful intertidal zone. Our analyses also show that shell formation in molluscs is more complex than currently understood and involves extensive participation of cells and their exosomes. The oyster genome sequence fills a void in our understanding of the Lophotrochozoa.
Nucleic acid-based aptamers possess many useful features that make them a promising alternative to antibodies and other affinity reagents, including well-established chemical synthesis, reversible folding, thermal stability and low cost. However, the selection process typically used to generate aptamers (SELEX) often requires significant resources and can fail to yield aptamers with sufficient affinity and specificity. A number of seminal theoretical models and numerical simulations have been reported in the literature offering insights into experimental factors that govern the effectiveness of the selection process. Though useful, these previous models have not considered the full spectrum of experimental factors or the potential impact of tuning these parameters at each round over the course of a multi-round selection process. We have developed an improved mathematical model to address this important question, and report that both target concentration and the degree of non-specific background binding are critical determinants of SELEX efficiency. Although smaller target concentrations should theoretically offer superior selection outcome, we show that the level of background binding dramatically affect the target concentration that will yield maximum enrichment at each round of selection. Thus, our model enables experimentalists to determine appropriate target concentrations as a means for protocol optimization. Finally, we perform a comparative analysis of two different selection methods over multiple rounds of selection, and show that methods with inherently lower background binding offer dramatic advantages in selection efficiency.
In our current research work, the effect of ultrasound irradiation on the enzymatic activity and enzymatic hydrolysis kinetic parameters of dextran catalysis by dextranase were investigated. Furthermore, the effects of ultrasound irradiation on the structure of dextranase were investigated with the aid of fluorescence spectroscopy and circular dichroism (CD) spectroscopy. The maximum activity of dextranase was observed when the sample was treated with ultrasound at 25 kHz, 40 W for 15 min, under which the enzyme activity increased by 13.43% compared the routine thermal incubation at 50 °C. Experimental Kinetics results, demonstrated that, both the V(max) and K(M) values of dextranase increased with ultrasound-treated compared with the incubation at 50 °C. Likewise, both the catalytic and specificity constants were higher under the effects of an ultrasonic field, indicating that, the substrate is converted into the product at an increased rate when compared with the incubation at 50 °C. On the other hand, fluorescence and CD spectra reflected that the ultrasound irradiation had increased the number of tryptophan on dextranase surface with increased ?-helix by 15.74% and reduced random coil by 5.41% upon ultrasound-treated dextranase protein compared to the control, which were helpful for the improvement of its activity.
Many analytical techniques benefit greatly from the use of affinity reagent pairs, wherein each reagent recognizes a discrete binding site on a target. For example, antibody pairs have been widely used to dramatically increase the specificity of enzyme linked immunosorbent assays (ELISA). Nucleic acid-based aptamers offer many advantageous features relative to protein-based affinity reagents, including well-established chemical synthesis, thermostability, and low production cost. However, the generation of suitable aptamer pairs has posed a significant challenge, and few such pairs have been reported to date. To address this important challenge, we present multivalent aptamer isolation systematic evolution of ligands by exponential enrichment (MAI-SELEX), a technique designed for the efficient selection of aptamer pairs. In contrast to conventional selection methods, our method utilizes two selection modules to generate separate aptamer pools that recognize distinct binding sites on a single target. Using MAI-SELEX, we have isolated two groups of 2-fluoro-modified RNA aptamers that specifically recognize the ?V or ?3 subunits of integrin ?V?3. These aptamers exhibit low nanomolar affinities for their targets, with minimal cross-reactivity to other closely related integrin homologues. Moreover, we show that these aptamer pairs do not interfere with each others binding and effectively detect the target even in complex mixtures such as undiluted serum.
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