The R2R3-MYB genes regulate pigmentation and morphogenesis of flowers, including flower and cell shape, and therefore have importance in the development of new varieties of orchids. However, new variety development is limited by the long breeding time required in orchids. In this study, we identified a cDNA, DhMYB1, that is expressed during flower development in a hybrid orchid, Dendrobium hybrida (Dendrobium bobby messina X Dendrobium chao phraya) then used the direct application of dsRNA to observe the effect of gene silencing on flower phenotype and floral epidermal cell shape.
Twenty percent of genes that encode for hypothetical proteins from Klebsiella pneumoniae MGH78578 were identified, leading to KPN00728 and KPN00729 after bioinformatics analysis. Both open reading frames showed high sequence homology to Succinate dehydrogenase Chain C (SdhC) and D (SdhD) from Escherichia coli. Recently, KPN00729 was assigned as SdhD. KPN00728 thus remains of particular interest as no annotated genes from the complete genome sequence encode for SdhC. We discovered KPN00728 has a missing region with conserved residues important for ubiquinone (UQ) and heme group binding. Structure and function prediction of KPN00728 coupled with secondary structure analysis and transmembrane topology showed KPN00728 adopts SDH-(subunit C)-like structure. To further probe its functionality, UQ was docked on the built model (consisting KPN00728 and KPN00729) and formation of hydrogen bonds between UQ and Ser27, Arg31 (KPN00728) and Tyr84 (KPN00729) further reinforces and supports that KPN00728 is indeed SDH. This is the first report on the structural and function prediction of KPN00728 of K. pneumoniae MGH78578 as SdhC.
Here we report the draft genomes and annotation of four N-acyl homoserine lactone (AHL)-producing members from the family Sphingomonadaceae. Comparative genomic analyses of 62 Sphingomonadaceae genomes were performed to gain insights into the distribution of the canonical luxI/R-type quorum sensing (QS) network within this family. Forty genomes contained at least one luxR homolog while the genome of Sphingobium yanoikuyae B1 contained seven Open Reading Frames (ORFs) that have significant homology to that of luxR. Thirty-three genomes contained at least one luxI homolog while the genomes of Sphingobium sp. SYK6, Sphingobium japonicum, and Sphingobium lactosutens contained four luxI. Using phylogenetic analysis, the sphingomonad LuxR homologs formed five distinct clades with two minor clades located near the plant associated bacteria (PAB) LuxR solo clade. This work for the first time shows that 13 Sphingobium and one Sphingomonas genome(s) contain three convergently oriented genes composed of two tandem luxR genes proximal to one luxI (luxR-luxR-luxI). Interestingly, luxI solos were identified in two Sphingobium species and may represent species that contribute to AHL-based QS system by contributing AHL molecules but are unable to perceive AHLs as signals. This work provides the most comprehensive description of the luxI/R circuitry and genome-based taxonomical description of the available sphingomonad genomes to date indicating that the presence of luxR solos and luxI solos are not an uncommon feature in members of the Sphingomonadaceae family.
A Cucumber green mottle mosaic virus (CGMMV) was used to present a truncated dengue virus type 2 envelope (E) protein binding region from amino acids 379 to 423 (EB4). The EB4 gene was inserted at the terminal end of the CGMMV coat protein (CP) open reading frame (ORF). Read-through sequences of TMV or CGMMV, CAA-UAG-CAA-UUA, or AAA-UAG-CAA-UUA were, respectively, inserted in between the CP and the EB4 genes. The chimeric clones, pRT, pRG, and pCG+FSRTRE, were transcribed into full-length capped recombinant CGMMV transcripts. Only constructs with the wild-type CGMMV read-through sequence yielded infectious viruses following infection of host plant, muskmelon (Cucumis melo) leaves. The ratio of modified to unmodified CP for the read-through expression clone developed was also found to be approximately 1:1, higher than what has been previously reported. It was also observed that infectivity was not affected by differences in pI between the chimera and its wild counterpart. Analysis of recombinant viruses after 21-days-postinculation (dpi) revealed that deletions occurred resulting in partial reversions of the viral population to near wild type and suggesting that this would be the limiting harvest period for obtaining true to type recombinants with this construct.
The king cobra (Ophiophagus hannah) is widely distributed throughout many parts of Asia. This study aims to investigate the complexity of Malaysian Ophiophagus hannah (MOh) venom for a better understanding of king cobra venom variation and its envenoming pathophysiology. The venom gland transcriptome was investigated using the Illumina HiSeq™ platform, while the venom proteome was profiled by 1D-SDS-PAGE-nano-ESI-LCMS/MS.
To characterize the dengue epidemic that recently occurred in Malaysia, we sequenced cDNAs from nine 1993-1994 dengue virus type-3 (DEN-3) isolates in Malaysia (DEN-3 was the most common type in Malaysia during this period). Nucleic acid sequences (720 nucleotides in length) from the nine isolates, encompassing the precursor of membrane protein (preM) and membrane (M) protein genes and part of the envelope (E) protein gene were aligned with various reference DEN-3 sequences to generate a neighbor-joining phylogenetic tree. According to the constructed tree, the nine Malaysian isolates were grouped into subtype II, which comprises Thai isolates from 1962 to 1987. Five earlier DEN-3 virus Malaysian isolates from 1974 to 1981 belonged to subtype I. The present data indicate that the recent dengue epidemic in Malaysia was due to the introduction of DEN-3 viruses previously endemic to Thailand.
The Rab GTPase family plays a vital role in several plant physiological processes including fruit ripening. Fruit softening during ripening involves trafficking of cell wall polymers and enzymes between cellular compartments. Mango, an economically important fruit crop, is known for its delicious taste, exotic flavour and nutritional value. So far, there is a paucity of information on the mango Rab GTPase family. In this study, 23 genes encoding Rab proteins were identified in mango by a comprehensive in silico approach. Sequence alignment and similarity tree analysis with the model plant Arabidopsis as a reference enabled the bona fide assignment of the deduced mango proteins to classify into eight subfamilies. Expression analysis by RNA-Sequencing (RNA-Seq) showed that the Rab genes were differentially expressed in ripe and unripe mangoes suggesting the involvement of vesicle trafficking during ripening. Interaction analysis showed that the proteins involved in vesicle trafficking and cell wall softening were interconnected providing further evidence of the involvement of the Rab GTPases in fruit softening. Correlation analyses showed a significant relationship between the expression level of the RabA3 and RabA4 genes and fruit firmness at the unripe stage of the mango varieties suggesting that the differences in gene expression level might be associated with the contrasting firmness of these varieties. This study will not only provide new insights into the complexity of the ripening-regulated molecular mechanism but also facilitate the identification of potential Rab GTPases to address excessive fruit softening.
Transporters mediate the movement of compounds across the membranes that separate the cell from its environment and across the inner membranes surrounding cellular compartments. It is estimated that one third of a proteome consists of membrane proteins, and many of these are transport proteins. Given the increase in the number of genomes being sequenced, there is a need for computational tools that predict the substrates that are transported by the transmembrane transport proteins. In this paper, we present TranCEP, a predictor of the type of substrate transported by a transmembrane transport protein. TranCEP combines the traditional use of the amino acid composition of the protein, with evolutionary information captured in a multiple sequence alignment (MSA), and restriction to important positions of the alignment that play a role in determining the specificity of the protein. Our experimental results show that TranCEP significantly outperforms the state-of-the-art predictors. The results quantify the contribution made by each type of information used.
High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.
Several anuran groups of Laurasian origin are each co-distributed in four isolated regions of the Northern Hemisphere: central/southern Europe and adjacent areas, Korean Peninsula and adjacent areas, Indo-Malaya, and southern North America. Similar distribution patterns have been observed in diverse animal and plant groups. Savage [Savage, J.M., 1973. The geographic distribution of frogs: patterns and predictions. In: Vial, J.L. (Ed.), Evolutionary Biology of the Anurans. University of Missouri Press, Columbia, pp. 351-445] hypothesized that the Miocene global cooling and increasing aridities in interiors of Eurasia and North America caused a southward displacement and range contraction of Laurasian frogs (and other groups). We use the frog genus Bombina to test Savage's biogeographical hypothesis. A phylogeny of Bombina is reconstructed based on three mitochondrial and two nuclear gene fragments. The genus is divided into three major clades: an Indo-Malaya clade includes B. fortinuptialis, B. lichuanensis, B. maxima, and B. microdeladigitora; a European clade includes B. bombina, B. pachypus, and B. variegata; and a Korean clade contains B. orientalis. The European and Korean clades form sister-group relationship. Molecular dating of the phylogenetic tree using the penalized likelihood and Bayesian analyses suggests that the divergence between the Indo-Malaya clade and other Bombina species occurred 5.9-28.6 million years ago. The split time between the European clade and the Korean clade is estimated at 5.1-20.9 million years ago. The divergence times of these clades are not significantly later than the timing of Miocene cooling and drying, and therefore can not reject Savage's hypothesis. Some other aspects of biogeography of Bombina also are discussed. The Korean Peninsula and the Shandong Peninsula might have supplied distinct southern refugia for B. orientalis during the Pleistocene glacial maxima. In the Indo-Malaya clade, the uplift of the Tibetan Plateau might have promoted the split between B. maxima and the other species.
Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.
Brugia malayi is a filarial nematode, which causes lymphatic filariasis in humans. In 1995, the disease has been identified by the World Health Organization (WHO) as one of the second leading causes of permanent and long-term disability and thus it is targeted for elimination by year 2020. Therefore, accurate filariasis diagnosis is important for management and elimination programs. A recombinant antigen (BmR1) from the Bm17DIII gene product was used for antibody-based filariasis diagnosis in "Brugia Rapid". However, the structure and dynamics of BmR1 protein is yet to be elucidated. Here we study the three dimensional structure and dynamics of BmR1 protein using comparative modeling, threading and ab initio protein structure prediction. The best predicted structure obtained via an ab initio method (Rosetta) was further refined and minimized. A total of 5 ns molecular dynamics simulation were performed to investigate the packing of the protein. Here we also identified three epitopes as potential antibody binding sites from the molecular dynamics average structure. The structure and epitopes obtained from this study can be used to design a binder specific against BmR1, thus aiding future development of antigen-based filariasis diagnostics to complement the current diagnostics.
A key event in the domestication and breeding of the oil palm Elaeis guineensis was loss of the thick coconut-like shell surrounding the kernel. Modern E. guineensis has three fruit forms, dura (thick-shelled), pisifera (shell-less) and tenera (thin-shelled), a hybrid between dura and pisifera. The pisifera palm is usually female-sterile. The tenera palm yields far more oil than dura, and is the basis for commercial palm oil production in all of southeast Asia. Here we describe the mapping and identification of the SHELL gene responsible for the different fruit forms. Using homozygosity mapping by sequencing, we found two independent mutations in the DNA-binding domain of a homologue of the MADS-box gene SEEDSTICK (STK, also known as AGAMOUS-LIKE 11), which controls ovule identity and seed development in Arabidopsis. The SHELL gene is responsible for the tenera phenotype in both cultivated and wild palms from sub-Saharan Africa, and our findings provide a genetic explanation for the single gene hybrid vigour (or heterosis) attributed to SHELL, via heterodimerization. This gene mutation explains the single most important economic trait in oil palm, and has implications for the competing interests of global edible oil production, biofuels and rainforest conservation.
Hortaea werneckii is an environmental dematiaceous fungus found in the halophilic environment. It causes tinea nigra. We report the isolation of H. werneckii from blood and splenic abscess of two patients with acute myelomonocytic leukaemia. H. werneckii grew at room temperature but not at 37 degrees C, it was identified by biochemical tests, growth characteristics and the presence of conspicuous collarette intercalary on dividing yeast cells. The use of specific oligonucleotide primer Hor-F (5'-TGGACACCTTCA TAACTCTTG-3') and Hor-R (5'-TCACAACGCTTAGAGACGG-3') confirmed the two isolates were H. werneckii. The sequence for 281 nucleotide of HW299 and HW403 were 99% identical but differed only in one nucleotide. In vitro anti-fungal susceptibility testing showed that the isolates were resistant to amphotericin B and flucytosine.
Der p 2 is a major dust mite allergen and >80% of mite allergic individuals have specific IgE to this allergen. Although it is well characterized in terms of allergenicity, there is still some ambiguity in terms of its biological function. Three-dimensional structural analysis of Der p 2 and its close homologues indicate the presence of a hydrophobic cavity which can potentially bind to lipid molecules. In this study, we aimed to identify the potential ligand of Der p 2. Using a liposome pulldown assay, we show that recombinant Der p 2 binds to liposomes prepared with exogenous cholesterol in a dose dependent fashion. Next, an ELISA based assay using immobilized lipids was used to study binding specificities of other lipid molecules. Cholesterol was the preferred ligand of Der p 2 among 11 different lipids tested. Two homologues of Der p 2, Der f 2 and Der f 22 also bound to cholesterol. Further, using liquid chromatography-mass spectrometry (LC-MS), we confirmed that cholesterol is the natural ligand of Der p 2. Three amino acid residues of Der p 2, V104, V106 and V110 are possible cholesterol binding sites, as alanine mutations of these residues showed a significant decrease in binding (p
Like other nematodes, both L(3) and adult Teladosagia circumcincta secrete or excrete NH(3)/NH(4)(+), but the reactions involved in the production are unclear. Glutamate dehydrogenase is a significant source NH(3)/NH(4)(+) in some species, but previous reports indicate that the enzyme is absent from L(3)Haemonchus contortus. We show that glutamate dehydrogenase was active in both L(3) and adult T. circumcincta. The apparent K(m)s of the L(3) enzyme differed from those of the adult enzyme, the most significant of these being the increase in the K(m) for NH(4)(+) from 18mM in L(3) to 49mM in adults. The apparent V(max) of the oxidative deamination reaction was greater than that of the reductive reaction in L(3), but this was reversed in adults. The activity of the oxidative reaction of the L(3) enzyme was not affected by adenine nucleotides, but that of the reductive reaction was stimulated significantly by either ADP or ATP. The L(3) enzyme was more active with NAD(+) than it was with NADP(+), although the activities supported by NADH and NADPH were similar at saturating concentrations. While the activity of the oxidative reaction was sufficient to account for the NH(3)/NH(4)(+) efflux we have previously reported, the reductive amination reaction was likely to be more active.
The American mink (Neovison vison) is a semiaquatic species of mustelid native to North America. It's an important animal for the fur industry. Many efforts have been made to locate genes influencing fur quality and color, but this search has been impeded by the lack of a reference genome. Here we present the first draft genome of mink. In our study, two mink individuals were sequenced by Illumina sequencing with 797 Gb sequence generated. Assembly yielded 7,175 scaffolds with an N50 of 6.3 Mb and length of 2.4 Gb including gaps. Repeat sequences constitute around 31% of the genome, which is lower than for dog and cat genomes. The alignments of mink, ferret and dog genomes help to illustrate the chromosomes rearrangement. Gene annotation identified 21,053 protein-coding sequences present in mink genome. The reference genome's structure is consistent with the microsatellite-based genetic map. Mapping of well-studied genes known to be involved in coat quality and coat color, and previously located fur quality QTL provide new knowledge about putative candidate genes for fur traits. The draft genome shows great potential to facilitate genomic research towards improved breeding for high fur quality animals and strengthen our understanding on evolution of Carnivora.
DARPP-32 (dopamine and adenosine 3', 5'-monophosphate-regulated phosphoprotein of 32 kDa), which belongs to PPP1R1 gene family, is known to act as an important integrator in dopamine-mediated neurotransmission via the inhibition of protein phosphatase-1 (PP1). Besides its neuronal roles, this protein also behaves as a key player in pathological and pharmacological aspects. Use of bioinformatics and phylogenetics approaches to further characterize the molecular features of DARPP-32 can guide future works. Predicted phosphorylation sites on DARPP-32 show conservation across vertebrates. Phylogenetics analysis indicates evolutionary strata of phosphorylation site acquisition at the C-terminus, suggesting functional expansion of DARPP-32, where more diverse signalling cues may involve in regulating DARPP-32 in inhibiting PP1 activity. Moreover, both phylogenetics and synteny analyses suggest de novo origination of PPP1R1 gene family via chromosomal rearrangement and exonization.
A maltogenic amylase (MAG1) from alkaliphilic Bacillus lehensis G1 was cloned, expressed in Escherichia coli, purified and characterised for its hydrolysis and transglycosylation properties. The enzyme exhibited high stability at pH values from 7.0 to 10.0. The hydrolysis of β-cyclodextrin (β-CD) produced malto-oligosaccharides of various lengths. In addition to hydrolysis, MAG1 also demonstrated transglycosylation activity for the synthesis of longer malto-oligosaccharides. The thermodynamic equilibrium of the multiple reactions was shifted towards synthesis when the reaction conditions were optimised and the water activity was suppressed, which resulted in a yield of 38% transglycosylation products consisting of malto-oligosaccharides of various lengths. Thin layer chromatography and high-performance liquid chromatography analyses revealed the presence of malto-oligosaccharides with a higher degree of polymerisation than maltoheptaose, which has never been reported for other maltogenic amylases. The addition of organic solvents into the reaction further suppressed the water activity. The increase in the transglycosylation-to-hydrolysis ratio from 1.29 to 2.15 and the increased specificity toward maltopentaose production demonstrated the enhanced synthetic property of the enzyme. The high transglycosylation activity of maltogenic amylase offers a great advantage for synthesising malto-oligosaccharides and rare carbohydrates.
The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.