Mining of EST-SSR Markers of Musa and Their Transferability Studies Among the Members of Order the Zingiberales

Expressed sequence tags (ESTs) databases of 11 Musa complementary DNA libraries were retrieved from National Center of Biotechnology Information and used for mining simple sequence repeats (SSRs). Out of 21,056 unique ESTs, SSR regions were found only in 5,158 ESTs. Among these SSR containing ESTs, the occurrence of trinucleotide repeats are the most abundant followed by mono-, di-, tetra-, hexa-, and pentanucleotides. Moreover, this study showed that the rate of class II SSRs (<20 nucleotides) was higher than the class I SSRs (<20 nucleotides), and proportion of class I and II SSRs as abundant for tri-repeats. As a representative sample, primers were synthesized for 24 ESTs, carrying >12 nucleotides of SSR region, and tested among the various genomic group of Musa accessions. The result showed that 88 % of primers were functional primers, and 43 % are showing polymorphism among the Musa accessions. Transferability studies of Musa EST-SSRs among the genera of the order Zingiberales exhibited 100 and 58 % transferability in Musaceae and Zingiberaceae, respectively. The sequence comparison of SSR regions among the different Musa accessions confirmed that polymorphism is mainly due to the variation in repeat length. High percentage of cross-species, cross-genera, and cross-family transferability also suggested that these Musa EST-SSR markers will be a valuable resource for the comparative mapping by developing COS markers, in evolutionary studies and in improvement of the members of Zingiberaceae and Musaceae.     

Sequence-Tunable Phase Behavior and Intrinsic Fluorescence in Dynamically Interacting Peptides

A conceptual framework towards understanding biological condensed phases is emerging, derived from biological, biomimetic, and synthetic sequences. However, de novo peptide condensate design remains a challenge due to an incomplete understanding of the structural and interactive complexity. We designed peptide modules based on a simple repeat motif composed of tripeptide spacers (GSG, SGS, GLG) interspersed with adhesive amino acids (R/H and Y). We show, using sequence editing and a combination of computation and experiment, that n→π* interactions in GLG backbones are a dominant factor in providing sufficient backbone structure, which in turn regulates the water interface, collectively promoting liquid droplet formation. Moreover, these R(GLG)Y and H(GLG)Y condensates unexpectedly display sequence-dependent emission that is a consequence of their non-covalent network interactions, and readily observable by confocal microscopy.     

Development and Characterization of Microsatellite Markers (SSR) in Sesamum (Sesamum indicum L.) Species

Microsatellites, also known as simple sequence repeats (SSRs), are the class of repetitive DNA sequences present throughout the genome of many plant and animal species. Recent advances in molecular genetics had been the introduction of microsatellite markers to investigate the genetic structuring of natural plant populations. We have employed an enrichment strategy for microsatellite isolation by using multi-enzymes digestion, microsatellite oligoprobes, and streptavidin magnetic beads in Sesamum (Sesamum indicum L.). More than 200 SSR motifs were detected (SSR motifs ??2 repeat units or 6?bp); 80?% of the clones contained SSR motifs. When regarding SSRs with four or more repeat units and a minimum length of 10?bp, 132 of them showed repeats. Eighteen SSR markers were initially characterized for optimum annealing temperature using a gradient PCR technique. Among the 18 SSR markers characterized, five were found to be polymorphic and used to analyze 60 Sesamum germplasm accessions. The maximum number of alleles detected was four with a single primer and the least number of two alleles with three primers with an average PIC value of 0.77. SSRs are a valuable tool for estimating genetic diversity and analyzing the evolutionary and historical development of cultivars at the genomic level in sesame breeding programs.     

Sequestration within biomolecular condensates inhibits Aβ-42 amyloid formation

Biomolecular condensates are emerging as an efficient strategy developed by cells to control biochemical reactions in space and time by locally modifying composition and environment. Yet, local increase in protein concentration within these compartments could promote aberrant aggregation events, including the nucleation and growth of amyloid fibrils. Understanding protein stability within the crowded and heterogeneous environment of biological condensates is therefore crucial, not only when the aggregation-prone protein is the scaffold element of the condensates but also when proteins are recruited as client molecules within the compartments. Here, we investigate the partitioning and aggregation kinetics of the amyloidogenic peptide Abeta42 (Aβ-42), the peptide strongly associated with Alzheimer''s disease, recruited into condensates based on low complexity domains (LCDs) derived from the DEAD-box proteins Laf1, Dbp1 and Ddx4, which are associated with biological membraneless organelles. We show that interactions between Aβ-42 and the scaffold proteins promote sequestration and local increase of the peptide concentration within the condensates. Yet, heterotypic interactions within the condensates inhibit the formation of amyloid fibrils. These results demonstrate that biomolecular condensates could sequester aggregation-prone proteins and prevent aberrant aggregation events, despite the local increase in their concentration. Biomolecular condensates could therefore work not only as hot-spots of protein aggregation but also as protective reservoirs, since the heterogenous composition of the condensates could prevent the formation of ordered fibrillar aggregates.

Biomolecular condensates sequester an aggregation-prone peptide and prevent its aggregation, showing that heterotypic interactions within the condensates can prevent the formation of amyloid fibrils, despite the local increase in concentration.     

Laminated morphology of nontwisting beta-sheet fibrils constructed via peptide self-assembly

A synthetic peptide has been de novo designed that self-assembles into beta-sheet fibrils exhibiting a nontwisted, stacked morphology. The stacked morphology is constituted by 2.5 nm wide filaments that laterally associate to form flat fibril laminates exceeding 50 nm in width and micrometers in length. The height of each fibril is limited to the length of exactly one peptide monomer in an extended beta-strand conformation, approximately 7 nm. Once assembled, these highly ordered, 2-D structures are stable over a wide range of pH and temperature and exhibit characteristics similar to those of amyloid fibrils. Furthermore, the rate of assembly and degree of fibril lamination can be controlled with kinetic parameters of pH and temperature. Finally, the presence of a diproline peptide between two beta-sheet-forming strands in the peptide sequence is demonstrated to be an important factor in promoting the nontwisting, laminated fibril morphology.     

Antibody-free reading of the histone code using a simple chemical sensor array

The histone code refers to the complex network of histone post-translational modifications that control gene expression and are of high interest as drivers of a large number of human diseases. We report here on a mix-and-match toolkit of readily available dyes and calixarene host molecules that can be combined to form dye-displacement sensors that respond to a wide variety of cationic peptides. Using the data from only two or three such simple supramolecular sensors as a chemical sensor array produces fingerprints of data that discriminate robustly among many kinds of histone code elements. "Reads" that are accomplished include the discrimination of unmethylated, mono-, di-, and trimethylated lysines on a single histone tail sequence, identification of different modifications and combinations of modifications on a single histone tail sequence, identification of a single modification type in several different sequence contexts, and identification of isomeric dimethylarginine modifications. Reads that are sometimes troublesome for antibodies are achieved. We also report on the ability of the sensor array to report simultaneously on the concentrations and identities of histone modifications. This sensor array discriminates between post-translationally modified analytes without being limited to partners that contain a single, programmed binding interaction.     

Highly Diastereoselective Synthesis of Syn‐1,3‐Dihydroxyketone Motifs from Propargylic Alcohols via Spiroepoxide Intermediates

Syn dihydroxyketone motifs are embedded in a wide range of biologically active natural products, however the development of stereoselective synthetic methods to assemble these structures has proven a challenging task. We report a highly diastereoselective method for the synthesis of syn dihydroxyketones from propargylic alcohols, with wide scope for application in natural product synthesis. The reaction sequence involves regioselective cyclisation of propargylic alcohols with incorporation of a triketone to give enol dioxolanes that are then diastereoselectively epoxidised to form unusual spiroepoxide intermediates. Hydrolysis affords syn dihydroxyketones as essentially single diastereisomers. The reaction sequence is operationally simple, of wide substrate scope, and remarkably can be efficiently carried out as a one‐pot process with no loss of overall yield or diastereoselectivity.     

Dissociation energies of X-H bonds in amino acids

In biochemistry, free radicals are versatile species which can perform diverse functions including: signaling, synthesis, and destructive modification. It is of interest to understand how radicals behave within all biomolecules and specifically within peptides and proteins. The 20 standard amino acids contain a wide range of chemical structures, which give proteins their complexity and ultimately their functionality. Many factors influence how radicals interact with these complex molecules, including the bond dissociation energies (BDEs) for homolytically cleaving any X-H bonds. The BDEs provide a simple measure for comparing the thermodynamic favorability of abstracting hydrogen atoms from various sites within a protein. BDEs for abstractable hydrogen atoms have been calculated for each amino acid, the peptide backbone, and peptide termini in order to compile a roadmap of the relative thermodynamics which influence protein radical chemistry. With this information it is possible to gain insight into what contributions both kinetics and thermodynamics will make to various radical mediated reaction pathways.     

Analysis of sequence repeats of proteins in the PDB

Internal repeats in protein sequences play a significant role in the evolution of protein structure and function. Applications of different bioinformatics tools help in the identification and characterization of these repeats. In the present study, we analyzed sequence repeats in a non-redundant set of proteins available in the Protein Data Bank (PDB). We used RADAR for detecting internal repeats in a protein, PDBeFOLD for assessing structural similarity, PDBsum for finding functional involvement and Pfam for domain assignment of the repeats in a protein. Through the analysis of sequence repeats, we found that identity of the sequence repeats falls in the range of 20–40% and, the superimposed structures of the most of the sequence repeats maintain similar overall folding. Analysis sequence repeats at the functional level reveals that most of the sequence repeats are involved in the function of the protein through functionally involved residues in the repeat regions. We also found that sequence repeats in single and two domain proteins often contained conserved sequence motifs for the function of the domain.     

Detection of single and multiple polymorphic loci by synthetic tandem repeats of short oligonucleotides

Loci containing tandem repeats of short sequences are sometimes associated with a high level of polymorphism due to variations in the number of repeats. The different variants can be easily characterized by Southern blotting when the repeats span a range from a few hundred bases to a few kilobases, and probes derived from such tandem repeats constitute convenient genetic markers. These structures, usually called minisatellites, are best documented in the human genome, where their number has been estimated to be at least 1500. However, their role and mode of evolution are poorly understood. We are developing tools to evaluate the number of such redundant sequences in a genome and to gain access to new polymorphic loci. Our strategy is based on the use of polymers of oligonucleotides as DNA probes for hybridization on Southern blots. In a previous report, we made polymers with random units of 14 bp and showed that they detect multiple polymorphic loci on human genomic DNA. At present, we are testing the effect of an increase in the complexity of the polymer, as obtained by the use of a longer random unit, and the effect of slight sequence modifications to a particular tandem repeat sequence. In addition, some of these synthetic probes can detect a single polymorphic locus and directly provide new genetic markers.     

Sequential allylic C-H amination/vinylic C-H arylation: a strategy for unnatural amino acid synthesis from α-olefins

Tandem reaction sequences that selectively convert multiple C-H bonds of abundant hydrocarbon feedstocks to functionalized materials enable rapid buildup of molecular complexity in an economical way. A tandem C-H amination/vinylic C-H arylation reaction sequence is described under Pd(II)/sulfoxide-catalysis that furnishes a wide range of α- and β-homophenylalanine precursors from commodity α-olefins and readily available aryl boronic acids. General routes to enantiopure amino acid esters and densely functionalized homophenylalanine derivatives are demonstrated.     

Diversity-oriented synthesis of 3-iodochromones and heteroatom analogues via ICl-induced cyclization

The ICl-induced cyclization of heteroatom-substituted alkynones provides a simple, highly efficient approach to various 3-iodochromones and analogues. This process is run under mild conditions, tolerates various functional groups, and generally provides chromones in good to excellent yields. Subsequent palladium-catalyzed transformations afford a rapid increase in molecular complexity and a convenient preparation of a wide range of functionally substituted chromones, furans, and polycyclic compounds. Iodothiochromenones and iodoquinolinones are also prepared by similar ICl-induced cyclizations.     

Protease catalysis mediated by a substrate mimetic: a novel enzymatic approach to the synthesis of carboxylic acid amides

We present a protease-based method for the coupling of non-coded and non-amino-acid-derived amines with carboxy components. The key feature of this approach is the combination of the substrate-mimetic strategy with the ability of the cysteine protease clostripain to accept a wide spectrum of amines. Firstly, we tested the use of the 4-guanidinophenyl ester leaving group to mediate acceptance of non-coded and non-amino-acid-derived acyl residues. This employed beta-amino acid and simple carboxylic acid moieties as acyl donors, and several amino acid and peptide units as acyl acceptors. The study was completed by the use of non-amino-acid-derived acyl acceptors comprising simple amines, amino alcohols, and diamines. The results indicate that the approach presented is a useful strategy for the synthesis of peptide isosteres, peptide analogues, and organic amides. These last open a new range of synthetic applications of proteases completely beyond peptide synthesis, achieving efficient and selective acylations of non-amino-acid-derived amines under extraordinarily mild reaction conditions.     

Scaling of folding times with protein size

Current experimental data show a 9-orders-of-magnitude span in the folding times of proteins. Such a wide range is typically considered a direct consequence of the complexity in structural and sequence patterns of natural proteins. By using a database of 69 proteins and peptides analyzed experimentally, we observe that the folding time scales with the number of residues in the protein. The correlation coefficient is 0.74 or higher, and indicates that it is possible to predict the folding time of a protein with a precision of approximately 1.1 times decades from just its size. A simple thermodynamic analysis of this correlation suggests that the smallest proteins are expected to have very marginal free energy barriers to folding.     

Novel numerical and graphical representation of DNA sequences and proteins

We have introduced novel numerical and graphical representations of DNA, which offer a simple and unique characterization of DNA sequences. The numerical representation of a DNA sequence is given as a sequence of real numbers derived from a unique graphical representation of the standard genetic code. There is no loss of information on the primary structure of a DNA sequence associated with this numerical representation. The novel representations are illustrated with the coding sequences of the first exon of beta-globin gene of half a dozen species in addition to human. The method can be extended to proteins as is exemplified by humanin, a 24-aa peptide that has recently been identified as a specific inhibitor of neuronal cell death induced by familial Alzheimer's disease mutant genes.     

SSR-Based DNA Fingerprinting and Diversity Assessment Among Indian Germplasm of <Emphasis Type="Italic">Euryale ferox</Emphasis>: an Aquatic Underutilized and Neglected Food Crop

Euryale ferox is native to Southeast Asia and China, and it is one of the important aquatic food crops propagated mostly in eastern part of India. The aim of the present study was to characterize and evaluate the genetic diversity of ex situ collections of E. ferox germplasm from different geographical states of India using microsatellite (simple sequence repeats (SSRs)) markers. Ten SSR markers were analyzed to assess DNA fingerprinting and genetic diversity of 16 cultivated germplasm of E. ferox. Total 37 polymorphic alleles were recorded with an average of 3.7 allele frequency per primer. The polymorphic information content value varied from 0.204 to 0.735 with mean of 0.448. A high range of heterozygosity (Ho 0.228; He 0.512) was detected in the present study. The neighbor-joining (N-J) tree and the principle coordinate analysis showed that the germplasm divided in to three main clusters. The results of the present investigation comply that SSR markers are effective for computing genetic assessment of genetic diversity and similarity with classifying cultivated varieties of E. ferox. Evaluation of genetic diversity among Indian E. ferox germplasm could provide useful information for genetic improvement.     

Novel numerical and graphical representation of DNA sequences and proteins

We have introduced novel numerical and graphical representations of DNA, which offer a simple and unique characterization of DNA sequences. The numerical representation of a DNA sequence is given as a sequence of real numbers derived from a unique graphical representation of the standard genetic code. There is no loss of information on the primary structure of a DNA sequence associated with this numerical representation. The novel representations are illustrated with the coding sequences of the first exon of β-globin gene of half a dozen species in addition to human. The method can be extended to proteins as is exemplified by humanin, a 24-aa peptide that has recently been identified as a specific inhibitor of neuronal cell death induced by familial Alzheimer's disease mutant genes.     

人类Y染色体回文序列中的重复序列与碱基关联

利用信息论和统计学的方法并结合生物学的特征研究人类Y染色体回文序列的互信息、“n字”熵、条件熵,定量分析了回文序列的长程关联和短程关联,发现其中既存在长程关联也存在短程关联,并且它们主要是由序列中的重复序列引起的．研究表明重复序列含量越高碱基之间的关联越强．