Sequence Analysis of KpnI Repeat Sequences to Revisit the Phylogeny of the Genus Carthamus L.

Repetitive DNA sequences constitute a significant proportion of eukaryotic genomes. Knowledge about the distribution of repetitive DNA sequences is necessary in order to gain insights into the organization, evolution and behavior of eukaryotic genomes. Therefore, we used two repetitive DNA sequences pCtKpnI-I and pCtKpnI-II, earlier reported in Carthamus tinctorius L. to study the phylogeny and to revise the taxonomic status of the taxa belonging to the genus. The study unraveled two major lines within the genus Carthamus; one line included all the diploid taxa (2n?=?24) and the other line comprised the taxa with 2n?=?20 and the polyploid taxa (2n?=?44 and 64). The results of the present study will prove useful in molecular breeding for improving some targeted agronomic traits in genus Carthamus.     

Creating hierarchical models of protein families based on Expressed Sequence Tags: the "Sprockets" analysis pipeline

We have created an analysis pipeline called Sprockets, which can be used to classify proteins into various hierarchical “families”, and build searchable models of these families. The construction of these families is based on data from Expressed Sequence Tags (ESTs) and Coding DNA Sequences (CDSs), making Sprockets clusters especially suitable for studying gene families in organisms for which the completely sequenced genome does not (yet) exist. The pipeline consists of two main parts: pair-wise analysis and grouping of sequences with Z-score statistics, followed by hierarchical splitting of clusters into alignable protein families. Various computational and statistical techniques applied in Sprockets allow it to act like a massive and selective multiple sequence alignment engine for combining individual sequence collections and related public sequences. The end result is a database of gene Hidden Markov Models, each related to the other by three levels of similarity: secondary structure, function and evolutionary origin. For a sample 20,000 EST set from Lactuca spp., Sprockets provided a 9% improvement in mapping of function to unknown sequences over traditional pair-wise search methods and InterPro mapping.     

Rapid method for detection of restriction endonuclease-producing strains in enteropathogenic bacteria

An improved rapid method is described for the detection of restriction endonucleases in enteropathogenic bacteria including Salmonella and Escherichia coli. With the improved method, at least six restriction endonucleases with different specificity were found in 415 strains tested. Five of them were shown to be isoschizomers of known restriction endonucleases, while one seemed to be novel. Among the isoschizomers, SthI endonuclease (isoschizomer of KpnI) in Salmonella thompson appears to be useful; unlike KpnI, SthI generates DNA fragments with a 5′-protruding end.     

Molecular evolution and structural variations in nuclear encoded chloroplast localized heat shock protein 26 (sHSP26) from genetically diverse wheat species

Heat shock proteins are an important class of molecular chaperones known to impart tolerance under high temperature stress. sHSP26, a member of small heat shock protein subfamily is specifically involved in protecting plant’s photosynthetic machinery. The present study aimed at identifying and characterizing sequence and structural variations in sHSP26 from genetically diverse progenitor and non-progenitor species of wheat. In silico analysis identified three paralogous copies of TaHSP26 to reside on short arm of chromosome 4A while one homeologue each was localized on long arm of chromosome 4B and 4D of cultivated bread wheat. Wild DD-genome donor Aegilops tauschii carried an additional sHSP26 gene (AET4Gv20569400) which was absent in the cultivated DD genome of bread wheat. In vitro amplification of this novel gene in wild accessions of Ae. tauschii and synthetic hexaploid wheat but not in cultivated bread wheat validated this finding. Further, significant length polymorphism could be identified in exon1 from diverse sHSP26 sequences. Multiple sequence alignment of procured sequences revealed numerous sSNPs and nsSNPs. D3A, P125 L, Q242 K were designated as homeolog specific- while A49 G as non-progenitor specific amino acid replacements. A 9-bp indel in TmHSP26-1(GA) translated into a deletion of SPM amino acid segment in chloroplast specific conserved consensus region III. High degree of divergence in nucleotide sequence between cultivated and wild species appeared in the form of higher ω values (Ka/Ks >1) indicating positive selection during the course of evolution. Phylogenetic analysis elucidated ancestral relationships between wheat sHSP26 proteins and orthologous proteins across plant kingdom. Overall, data mining approach may be employed as an effective pre-breeding strategy to identify and mobilize novel stress responsive genes and distinct allelic variants from wider germplasm collections of wheat to enhance climate resilience of present day elite wheat cultivars.     

Identification and functional characterization of a novel α-conotoxin (EIIA) from Conus ermineus

Nicotinic acetylcholine receptors (nAChRs) are one of the most important families in the ligand-gated ion channel superfamily due to their involvement in primordial brain functions and in several neurodegenerative pathologies. The discovery of new ligands which can bind with high affinity and selectivity to nAChR subtypes is of prime interest in order to study these receptors and to potentially discover new drugs for treating various pathologies. Predatory cone snails of the genus Conus hunt their prey using venoms containing a large number of small, highly structured peptides called conotoxins. Conotoxins are classified in different structural families and target a large panel of receptors and ion channels. Interestingly, nAChRs represent the only subgroup for which Conus has developed seven distinct families of conotoxins. Conus venoms have thus received much attention as they could represent a potential source of selective ligands of nAChR subtypes. We describe the mass spectrometric-based approaches which led to the discovery of a novel α-conotoxin targeting muscular nAChR from the venom of Conus ermineus. The presence of several posttranslational modifications complicated the N-terminal sequencing. To discriminate between the different possible sequences, analogs with variable N-terminus were synthesized and fragmented by MS/MS. Understanding the fragmentation pathways in the low m/z range appeared crucial to determine the right sequence. The biological activity of this novel α-conotoxin (α-EIIA) that belongs to the unusual α4/4 subfamily was determined by binding experiments. The results revealed not only its selectivity for the muscular nAChR, but also a clear discrimination between the two binding sites described for this receptor.     

Single molecule force spectroscopy reveals the context dependent folding pathway of the C-terminal fragment of Top7

Top7 is a de novo designed protein with atomic level accuracy and shows a folded structure not found in nature. Previous studies showed that the folding of Top7 is not cooperative and involves various folding intermediate states. In addition, various fragments of Top7 were found to fold on their own in isolation. These features displayed by Top7 are distinct from those of naturally occurring proteins of a similar size and suggest a rough folding energy landscape. However, it remains unknown if and how the intra-polypeptide chain interactions among the neighboring sequences of Top7 affect the folding of these Top7 fragments. Here we used single-molecule optical tweezers to investigate the folding–unfolding pathways of full length Top7 as well as its C-terminal fragment (CFr) in different sequence environments. Our results showed that the mechanical folding of Top7 involves an intermediate state that likely involves non-native interactions/structure. More importantly, we found that the folding of CFr is entirely dependent upon its sequence context in which it is located. When in isolation, CFr indeed folds into a cooperative structure showing near-equilibrium unfolding–folding transitions at ∼6.5 pN in OT experiments. However, CFr loses its autonomous cooperative folding ability and displays a folding pathway that is dependent on its interactions with its neighboring sequence/structure. This context-dependent folding dynamics and pathway of CFr are distinct from those of naturally occurring proteins and highlight the critical importance of intra-chain interactions in shaping the overall energy landscape and the folding pathway of Top7. These new insights may have important implications on the de novo design of proteins.

Optical tweezers experiments reveal that the folding of the C-terminal fragment of Top7 (cFr) is context-dependent. Depending on its neighboring sequence, cFr shows very different folding pathways and folding kinetics.      

Electrochemical DNA biosensor for the detection of Listeria Monocytogenes using toluidine blue as a hybridization indicator

An electrochemical DNA biosensor was established for the determination of actin-assembly inducing protein (actA) gene sequences from Listeria monocytogenes and its polymerase chain reaction (PCR) product. The actA gene probe sequences were covalently immobilized on the surface of the mercaptoacetic acid self-assembled gold electrode with the help of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), which was further used to hybridize with the target sequence. Toluidine blue (TB) was used as an effective electrochemical indicator for the discrimination of the hybridization reaction on the electrode surface, which had stronger interaction with double-stranded DNA (dsDNA) than single-stranded DNA (ssDNA). The electrochemical parameters of TB on DNA modified electrodes were carefully calculated. Based on the different electrochemical responses of TB on DNA modified electrodes, the actA gene sequences can be detected in the concentration range from 1.0 × 10^-7 to 8.0 × 10^-5 M. The PCR product of Listeria monocytogenes was successfully detected by the proposed electrochemical biosensor.     

Mutational analysis of whole mitochondrial DNA in patients with MELAS and MERRF diseases

Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make the exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mtDNA mutations in 61 Korean unrelated families (or isolated patients) with MELAS or MERRF. In particular, the mtDNA sequences were completely determined for 49 patients. From the mutational analysis of mtDNA obtained from blood, 5 confirmed pathogenic mutations were identified in 17 families, and 4 unreported pathogenically suspected mutations were identified in 4 families. The m.3243A>G in the tRNA^Leu(UUR) was predominantly observed in 10 MELAS families, and followed by m.8344A>G in the tRNA^Lys of 4 MERRF families. Most pathogenic mutations showed heteroplasmy, and the rates were considerably different within the familial members. Patients with a higher rate of mutations showed a tendency of having more severe clinical phenotypes, but not in all cases. This study will be helpful for the molecular diagnosis of mitochondrial diseases, as well as establishment of mtDNA database in Koreans.     

In silico characterization of alkaline proteases from different species of Aspergillus

A total of 49 protein sequences of alkaline proteases retrieved from GenBank representing different species of Aspergillus have been characterized for various physiochemical properties, homology search, multiple sequence alignment, motif, and super family search and phylogenetic tree construction. The sequence level homology was obtained among different groups of alkaline protease enzymes, viz alkaline serine protease, oryzin, calpain-like protease, serine protease, subtilisin-like alkaline proteases. Multiple sequence alignment of alkaline protease protein sequence of different Aspergillus species revealed a stretch of conserved region for amino acid residues from 69 to 110 and 130–204. The phylogenetic tree constructed indicated several Aspergillus species-specific clusters for alkaline proteases namely Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Aspergillus clavatus. The distributions of ten commonly observed motifs were analyzed among these proteases. Motif 1 with a signature amino acid sequence of 50 amino acids, i.e., ASFSNYGKVVDIFAPGQDILSCWIGSTTATNTISGTSMATPHIVGLSCYL, was uniformly observed in proteases protein sequences indicating its involvement with the structure and enzymatic function. Motif analysis of acidic proteases of Aspergillus and bacterial alkaline proteases has revealed different signature amino acid sequences. The superfamily search for these proteases revealed the presence of subtilases, serine-carboxyl proteinase, calpain large subunit, and thermolysin-like superfamilies with 45 representing the subtilases superfamily.     

The influence of the purine 2-amino group on DNA conformation and stability-II: Synthesis and physical characterization of d[CGT(2-NH2)ACG], d[CGU(2-NH2)ACG], and d[CGT(2-NH2)AT(2-NH2)ACG]

Synthesis of an N-protected derivative of 2-amino-2'-deoxyadenosine (dA') from 2'-deoxyguanosine is reported. The syntheses of several oligodeoxynucleotides containing this modified nucleoside are described, together with physical characterization via melting studies and CD conformational analysis. As expected, the 2-amino group is seen to add to the duplex stability. Although the sequence d(TA')₃ was found to undergo a salt-induced conformational transition, mixed sequences such as d(CGTA'CG) did not display this behavior. All guanine residues present in these sequences were O⁶ protected, either with the cyanoethyl group or the 4-nitrophenylethyl group, to eliminate guanine degradation during phosphorylation and condensation reactions. Procedures for the introduction and removal of these O⁶ protecting groups are described.     

Biological entities as chemical reactors for synthesis of nanomaterials: Progress,challenges and future perspective

Synthesis of nanomaterials is being gained extensive attention in the fields of chemistry, applied physics, catalysis, drug delivery and the most important in diagnosis and therapeutic applications. Recently, many reports have been published on physical and chemical synthesis of magnetic as well as metallic nanoparticles (NPs) with viable surface functionalization, but still there is a dire need of such strategies that can combine synthetic methodology with stable surface modification found in nature. Synthesis of NPs via biological methods is the possible way to solve these barriers. However, systematized summary and outlooks of NPs synthesis via biological entities with various influencing factors e.g. temperature, pH, concentration of reactants and reaction time has rarely been reported. This review will present the distinct advantages of biological synthesis of NPs over physical and chemical methods. It will also highlight the recent progress on synthesis of NPs via various biological systems i.e. plant, fungus, bacteria, and yeast. Furthermore, it will explain various factors that control the size, shape, and morphology of these NPs. Finally, it would present the future perspectives of green chemistry for the development of nano-science and -biotechnology.     

Highly fluorescence quenching graphene oxide-based oligodeoxynucleotide hairpin systems for probing CNG DNA repeat sequences

In this study we synthesized a novel graphene-oxide (GO) based CNG repeat hairpin probing system capable of detecting target CAG and CTG DNA repeat sequences. The fluorescence of the 30-mer CNG repeat hairpin structure was quenched dramatically by GO in the absence of the target sequence, with a high quenching constant [K = 0.030 (mg/mL)^?1]. We optimized the quenching behavior of this probing system by using graphene oxide (GO) to induce a high degree of discrimination factor (44.6 times) between the fluorescence of the target sequence and that of other non-target sequences. All detection process is explained by displacement mechanism using adsorption, desorption, and hybridization of probe with target DNA sequence on the GO. Graphene-oxide (GO) based CNG repeat hairpin probing system exhibited high sensitivity and selectivity to the target CNG repeat sequence and its detection process is so simple and quick.     

Antibacterial study of Eucalyptus grandis fabricated zinc oxide and magnesium doped zinc oxide nanoparticles and its characterization

Mg-doped zinc oxide and zinc oxide nanoparticles were prepared by using methanolic seed extract from the Eucalyptus grandis plant via a green approach. Phytoconstituents present in seed extract act as capping and stabilizing agents for the biosynthesis of nanoparticles. Doping of Mg to zinc oxide nanoparticles increases the bandgap energy, thus enhancing its chemical, physical and optical properties. Further, it was characterized by various techniques such as scanning electron microscopy giving morphological information about the wurtzite hexagonal structure of bio-synthesized nanoparticles. X-ray diffraction technique tells about the crystalline nature of particles and the average crystallite size for zinc oxide and doped zinc oxide nanoparticles. Mg as a dopant enhances the properties of nanoparticles, thus making it more efficiently applicable as an antibacterial agent against Escherichia coli, gram-negative bacteria.     

硫代反义寡核苷酸药物FT19有关物质的分析

为了进行硫代反义寡核苷酸药物FT19的质量控制研究,建立了用阴离子交换高效液相色谱(AX-HPLC)和毛细管凝胶电泳(CGE)分析自行合成的FT19有关物质的方法。设计合成了FT19的硫代不完全序列(P=O)1和短序列(n-x)并将它们作为已知杂质。在AX-HPLC上,使用的分析柱为DNA Pac PA-100 (4 mm×250 mm);流动相A为10 mmol/L NaOH-0.1 mol/L NaCl,流动相B为10 mmol/L NaOH-3 mol/L NaCl;梯度洗脱条件为流动相B液在8 min内从60%升至100%;流速1 mL/min;柱温40 ℃;检测波长为260 nm。CGE所用毛细管规格为内径100 μm,总长度为31 cm,有效长度为20 cm;电泳缓冲溶液为三羟甲基氨基甲烷（Tris）-硼酸-7 mol/L尿素,pH 8.5;采用电动进样,进样电压-10 kV;分离胶为250 g/L的聚丙烯酰胺;检测波长为254 nm。结果表明,FT19与硫代不完全的(P=O)1序列在AX-HPLC上能够达到基线分离,与短序列(n-1)在CGE上能达到基线分离。说明AX-HPLC和CGE联合应用能够很好地分析FT19中的有关物质。