Random Amplified Polymorphic DNA Analysis of Heterodera cruciferae and H. schachtii populations

Heterodera schachtii and H. cruciferae are sympatric in California and frequently occur in the same field upon the same host. We have investigated the use of polymerase chain reaction (PCR) amplification of nematode DNA sequences to differentiate H. schachtii and H. cruciferae and to assess genetic variability within each species. Single, random oligodeoxyribonucleotide primers were used to generate PCR-amplified fragments, termed RAPD (random amplified polymorphic DNA) markers, from genomic DNA of each species. Each of 19 different random primers yielded from 2 to 12 fragments whose size ranged from 200 to 1,500 bp. Reproducible differences in fragment patterns allowed differentiation of the two species with each primer. Similarities and differences among six different geographic populations of H. schachtii were detected. The potential application of RAPD analysis to relationships among nematode populations was assessed through cluster analysis of these six different populations, with 78 scorable markers from 10 different random primers. DNA from single cysts was successfully amplified, and genetic variability was revealed within geographic populations. The use of RAPD markers to assess genetic variability is a simple, reproducible technique that does not require radioisotopes. This powerful new technique can be used as a diagnostic tool and should have broad application in nematology.     

Walking through the protein sequence space: towards new generation of the homology modeling

A new method is proposed to reveal apparent evolutionary relationships between protein fragments with similar 3D structures by finding "intermediate" sequences in the proteomic database. Instead of looking for homologies and intermediates for a whole protein domain, we build a chain of intermediate short sequences, which allows one to link similar structural modules of proteins belonging to the same or different families. Several such chains of intermediates can be combined into an evolutionary tree of structural protein modules. All calculations were made for protein fragments of 20 aa residues. Three evolutionary trees for different module structures are described. The aim of the paper is to introduce the new method and to demonstrate its potential for protein structural predictions. The approach also opens new perspectives for protein evolution studies.     

The isolation of structural genes from libraries of eucaryotic DNA

We present a procedure for eucaryotic structural gene isolation which involves the construction and screening of cloned libraries of genomic DNA. Large random DNA fragments are joined to phage lambda vectors by using synthetic DNA linkers. The recombinant molecules are packaged into viable phage particles in vitro and amplified to establish a permanent library. We isolated structural genes together with their associated sequences from three libraries constructed from Drosophila, silkmoth and rabbit genomic DNA. In particular, we obtained a large number of phage recombinants bearing the chorion gene sequence from the silkmoth library and several independent clones of β-globin genes from the rabbit library. Restriction mapping and hybridization studies reveal the presence of closely linked β-globin genes.     

Nucleic acids packaging processes: effects of adenine tracts and sequence-dependent curvature.

The effects of short runs of adenines (A-tracts) upon nucleic acids packaging processes and the properties of the resulting condensates were investigated by using random DNA sequences isolated from natural sources, as well as synthetic segments obtained by an extensive ligation of specific oligomers. Reiteration of short A-tracts (A(N) where N less than 3) within the DNA molecules is found to be compatible with a long-range chiral organization of the strands in the nucleic acid condensed phases. This chiral order, whose occurrence necessitates a high degree of flexibility, is shown, however, to differ from that exhibited by packed species originating from random AT-rich fragments; the altered patterns are interpreted in terms of a reduced overall flexibility of the DNA strands. Repetition of longer A-tracts (where N greater than 3), in which the distinct structural features that characterize this motif are fully expressed, results in a complete suppression of any chiral order in the packed particles, assigned to a significantly enhanced rigidity. DNA fragments where A-tracts are reiterated in phase, leading to a stable macroscopic curvature, are found to undergo condensation through altered pathways and to form toroidal shapes of unusually small dimensions. The results point towards the intriguing possibility that A-tracts and, in particular, the global, intrinsic curvature associated with such motifs, might be involved in the determination of nucleic acids packaging pathways, and underline the usefulness of defined sequences in the study of DNA condensation processes.     

Protein backbone structure determination using only residual dipolar couplings from one ordering medium

Residual dipolar couplings provide significant structural information for proteins in the solution state, which makes them attractive for the rapid determination of protein folds. Unfortunately, dipolar couplings contain inherent structural ambiguities which make them difficult to use in the absence of additional information. In this paper, we describe an approach to the construction of protein backbone folds using experimental dipolar couplings based on a bounded tree search through a structural database. We filter out false positives via an overlap similarity measure that insists that protein fragments assigned to overlapping regions of the sequence must have self-consistent structures. This allows us to determine a backbone fold (including the correct C-C bond orientations) using only residual dipolar coupling data obtained from one ordering medium. We demonstrate the applicability of the method using experimental data for ubiquitin.     

Chemical compound navigator: a web-based chem-BLAST, chemical taxonomy-based search engine for browsing compounds

A novel technique to annotate, query, and analyze chemical compounds has been developed and is illustrated by using the inhibitor data on HIV protease-inhibitor complexes. In this method, all chemical compounds are annotated in terms of standard chemical structural fragments. These standard fragments are defined by using criteria, such as chemical classification; structural, chemical, or functional groups; and commercial, scientific or common names or synonyms. These fragments are then organized into a data tree based on their chemical substructures. Search engines have been developed to use this data tree to enable query on inhibitors of HIV protease (http://xpdb.nist.gov/hivsdb/hivsdb.html). These search engines use a new novel technique, Chemical Block Layered Alignment of Substructure Technique (Chem-BLAST) to search on the fragments of an inhibitor to look for its chemical structural neighbors. This novel technique to annotate and query compounds lays the foundation for the use of the Semantic Web concept on chemical compounds to allow end users to group, sort, and search structural neighbors accurately and efficiently. During annotation, it enables the attachment of "meaning" (i.e., semantics) to data in a manner that far exceeds the current practice of associating "metadata" with data by creating a knowledge base (or ontology) associated with compounds. Intended users of the technique are the research community and pharmaceutical industry, for which it will provide a new tool to better identify novel chemical structural neighbors to aid drug discovery.     

Novel structural tree of β-proteins containing abcd units

A database of 528 β-proteins and β-domains containing abcd units, including 244 nonhomologous ones, was compiled from the Protein Data Bank (total 1511 PDB entries). A novel structural tree of this class of proteins was constructed to include 153 possible polypeptide chain folds. A structural classification of β-proteins containing abcd units was proposed on the basis of the tree. The database and the structural tree are available at http://strees.protres.ru/.     

Combining evolutionary and structural information for local protein structure prediction

We study the effects of various factors in representing and combining evolutionary and structural information for local protein structural prediction based on fragment selection. We prepare databases of fragments from a set of non-redundant protein domains. For each fragment, evolutionary information is derived from homologous sequences and represented as estimated effective counts and frequencies of amino acids (evolutionary frequencies) at each position. Position-specific amino acid preferences called structural frequencies are derived from statistical analysis of discrete local structural environments in database structures. Our method for local structure prediction is based on ranking and selecting database fragments that are most similar to a target fragment. Using secondary structure type as a local structural property, we test our method in a number of settings. The major findings are: (1) the COMPASS-type scoring function for fragment similarity comparison gives better prediction accuracy than three other tested scoring functions for profile-profile comparison. We show that the COMPASS-type scoring function can be derived both in the probabilistic framework and in the framework of statistical potentials. (2) Using the evolutionary frequencies of database fragments gives better prediction accuracy than using structural frequencies. (3) Finer definition of local environments, such as including more side-chain solvent accessibility classes and considering the backbone conformations of neighboring residues, gives increasingly better prediction accuracy using structural frequencies. (4) Combining evolutionary and structural frequencies of database fragments, either in a linear fashion or using a pseudocount mixture formula, results in improvement of prediction accuracy. Combination at the log-odds score level is not as effective as combination at the frequency level. This suggests that there might be better ways of combining sequence and structural information than the commonly used linear combination of log-odds scores. Our method of fragment selection and frequency combination gives reasonable results of secondary structure prediction tested on 56 CASP5 targets (average SOV score 0.77), suggesting that it is a valid method for local protein structure prediction. Mixture of predicted structural frequencies and evolutionary frequencies improve the quality of local profile-to-profile alignment by COMPASS.     

Novel structural tree for beta-proteins containing abcd-units

A database of 528 beta-proteins and beta-domains containing abcd-units (among them 244 are nonhomologous) has been compiled from the Protein Data Bank (total 1511 PDB entries). A novel structural tree for this structural class of proteins that is composed of 153 possible polypeptide chain folds has been constructed. The structural classification of beta-proteins containing abcd-unit based on the structural tree has been developed. Both the database and the structural tree are accessible at the web-site (http://strees.protres.ru/).     

Multiple Metal-Binding Cores Are Required for Metalloregulation by M-box Riboswitch RNAs

Riboswitches are regulatory RNAs that control downstream gene expression in response to direct association with intracellular metabolites or metals. Typically, riboswitch aptamer domains bind to a single small-molecule metabolite. In contrast, an X-ray crystallographic structural model for the M-box riboswitch aptamer revealed the absence of an organic metabolite ligand but the presence of at least six tightly associated magnesiums. This observation agrees well with the proposed role of the M-box riboswitch in functioning as a sensor of intracellular magnesium, although additional nonspecific metal interactions are also undoubtedly required for these purposes. To gain greater functional insight into the metalloregulatory capabilities of M-box RNAs, we sought to determine whether all or a subset of the RNA-chelated magnesium ions were required for riboswitch function. To accomplish this task, each magnesium-binding site was simultaneously yet individually perturbed through random incorporation of phosphorothioate nucleotide analogues, and RNA molecules were investigated for their ability to fold in varying levels of magnesium. These data revealed that all of the magnesium ions observed in the structural model are important for magnesium-dependent tertiary structure formation. Additionally, these functional data revealed a new core of potential metal-binding sites that are likely to assist formation of key tertiary interactions and were previously unobserved in the structural model. It is clear from these data that M-box RNAs require specific binding of a network of metal ions for partial fulfillment of their metalloregulatory functions.     

A partial genetic linkage map of slash pine (Pinus elliottii Engelm. var. elliottii) based on random amplified polymorphic DNAs

A set of 420 random, 10-base, oligonucleotide primers was screened for random amplified polymorphic DNA (RAPD) fragments within a sample of eight megagametophyte DNAs of a single slash pine (Pinus elliottii Engelm. var. elliottii) tree. The apparently repeatable RAPD fragments were further characterized within a sample of 68 megagametophytes from the same tree. Fragments segregating in a 11, present-to-absent, ratio were classified and mapped using multi-point linkage analysis. The analysis revealed 13 linkage groups of at least three loci, ranging in size from 28 to 68 cM, and nine linked pairs of loci. The 22 groups and pairs included 73 RAPD markers and covered a genetic map distance of approximately 782 cM. Genome size estimates, based on linkage data, ranged from 2880 to 3360 cM. Using a 30-cM map scale and including the 24 unlinked markers and the ends of the 13 linkage groups and nine linked pairs, the set of RAPD markers accounts for approximately 2160 cM or 64–75% of the genome. This extent of genomic coverage should allow for the efficient mapping of genes responsible for a reaction to the causal agent of fusiform rust disease, Cronartium quercuum (Berk.) Miyabe ex Shirai f. sp. fusiforme.     

Self-assembly of thin plates from micrococcal nuclease-digested chromatin of metaphase chromosomes

The three-dimensional organization of the enormously long DNA molecules packaged within metaphase chromosomes has been one of the most elusive problems in structural biology. Chromosomal DNA is associated with histones and different structural models consider that the resulting long chromatin fibers are folded forming loops or more irregular three-dimensional networks. Here, we report that fragments of chromatin fibers obtained from human metaphase chromosomes digested with micrococcal nuclease associate spontaneously forming multilaminar platelike structures. These self-assembled structures are identical to the thin plates found previously in partially denatured chromosomes. Under metaphase ionic conditions, the fragments that are initially folded forming the typical 30-nm chromatin fibers are untwisted and incorporated into growing plates. Large plates can be self-assembled from very short chromatin fragments, indicating that metaphase chromatin has a high tendency to generate plates even when there are many discontinuities in the DNA chain. Self-assembly at 37°C favors the formation of thick plates having many layers. All these results demonstrate conclusively that metaphase chromatin has the intrinsic capacity to self-organize as a multilayered planar structure. A chromosome structure consistent of many stacked layers of planar chromatin avoids random entanglement of DNA, and gives compactness and a high physical consistency to chromatids.     

SST: an algorithm for finding near-exact sequence matches in time proportional to the logarithm of the database size

MOTIVATION: Searches for near exact sequence matches are performed frequently in large-scale sequencing projects and in comparative genomics. The time and cost of performing these large-scale sequence-similarity searches is prohibitive using even the fastest of the extant algorithms. Faster algorithms are desired. RESULTS: We have developed an algorithm, called SST (Sequence Search Tree), that searches a database of DNA sequences for near-exact matches, in time proportional to the logarithm of the database size n. In SST, we partition each sequence into fragments of fixed length called 'windows' using multiple offsets. Each window is mapped into a vector of dimension 4(k) which contains the frequency of occurrence of its component k-tuples, with k a parameter typically in the range 4-6. Then we create a tree-structured index of the windows in vector space, with tree-structured vector quantization (TSVQ). We identify the nearest neighbors of a query sequence by partitioning the query into windows and searching the tree-structured index for nearest-neighbor windows in the database. When the tree is balanced this yields an O(logn) complexity for the search. This complexity was observed in our computations. SST is most effective for applications in which the target sequences show a high degree of similarity to the query sequence, such as assembling shotgun sequences or matching ESTs to genomic sequence. The algorithm is also an effective filtration method. Specifically, it can be used as a preprocessing step for other search methods to reduce the complexity of searching one large database against another. For the problem of identifying overlapping fragments in the assembly of 120 000 fragments from a 1.5 megabase genomic sequence, SST is 15 times faster than BLAST when we consider both building and searching the tree. For searching alone (i.e. after building the tree index), SST 27 times faster than BLAST. AVAILABILITY: Request from the authors.     

Determining the roles of different chain fragments in recognition of immunoglobulin fold

We examine sequence-to-structure specificity of beta-structural fragments of immunoglobulin domains. The structure specificity of separate chain fragments is estimated by computing the Z-score values in recognition of the native structure in gapless threading tests. To improve the accuracy of our calculations we use energy averaging over diverse homologs of immunoglobulin domains. We show that the interactions between residues of beta-structure are more determinant in recognition of the native structure than the interactions within the whole chain molecule. This result distinguishes immunoglobulins from more typical proteins where the interactions between residues of the whole chain normally recognize the native fold more accurately than interactions between the residues of the secondary structure residues alone [Reva,B. and Topiol,S. (2000) BIOCOMPUTING: Proceedings of the Pacific Symposium. World Scientific Publishing Co., pp. 168-178]. We also find that the predominant contributions of the secondary structure are produced by the four central beta-strands that form the core of the molecule. The results of this study allow us through quantitative means to understand the architecture of immunoglobulin molecules. Comparing the fold recognition data for different chain fragments one can say that beta-strands form a rigid frame for immunoglobulin molecules, whereas loops, with no structural role, can develop a broad variety of binding specificities. It is well known that protein function is determined by specific portions of a protein chain. This study suggests that the whole protein structure can be predominantly determined by a few fragments of chain which form the structural framework of the molecule. This idea may help in better understanding the mechanisms of protein evolution: strengthening a protein structure in the key framework-forming regions allows mutations and flexibility in other chain regions.     

Effect of forest fragment size on tree diversity and population structure of humid subtropical forest of Meghalaya,India

The present study was conducted in subtropical humid forests of Meghalaya to study the distributional pattern of species, floristic composition, community structure and tree population structure.Forest fragments of varying sizes(0.5 ha, 1 ha, 2 ha and 5 ha) were used in the study.All of the forest fragments are distributed within the same altitudinal range, and had similar rainfall and temperature regimes.Four forest fragments were sampled using random quadrats to analyze the impact of fragment size on tree...     

iBitter-SCM: Identification and characterization of bitter peptides using a scoring card method with propensity scores of dipeptides

In general, hydrolyzed proteins, plant-derived alkaloids and toxins displays unpleasant bitter taste. Thus, the perception of bitter taste plays a crucial role in protecting animals from poisonous plants and environmental toxins. Therapeutic peptides have attracted great attention as a new drug class. The successful identification and characterization of bitter peptides are essential for drug development and nutritional research. Owing to the large volume of peptides generated in the post-genomic era, there is an urgent need to develop computational methods for rapidly and effectively discriminating bitter peptides from non-bitter peptides. To the best of our knowledge, there is yet no computational model for predicting and analyzing bitter peptides using sequence information. In this study, we present for the first time a computational model called the iBitter-SCM that can predict the bitterness of peptides directly from their amino acid sequence without any dependence on their functional domain or structural information. iBitter-SCM is a simple and effective method that was built using the scoring card method (SCM) with estimated propensity scores of amino acids and dipeptides. Our benchmarking results demonstrated that iBitter-SCM achieved an accuracy and Matthews coefficient correlation of 84.38% and 0.688, respectively, on the independent dataset. Rigorous independent test indicated that iBitter-SCM was superior to those of other widely used machine-learning classifiers (e.g. k-nearest neighbor, naive Bayes, decision tree and random forest) owing to its simplicity, interpretability and implementation. Furthermore, the analysis of estimated propensity scores of amino acids and dipeptides were performed to provide a better understanding of the biophysical and biochemical properties of bitter peptides. For the convenience of experimental scientists, a web server is provided publicly at http://camt.pythonanywhere.com/iBitter-SCM. It is anticipated that iBitter-SCM can serve as an important tool to facilitate the high-throughput prediction and de novo design of bitter peptides.     

Protein structural change upon ligand binding correlates with enzymatic reaction mechanism

Overall structural changes of enzymes in response to ligand binding were investigated by database analysis of 62 non-redundant enzymes whose ligand-unbound and ligand-bound forms were available in the Protein Data Bank. The results of analysis indicate that transferases often undergo large rigid-body domain motions upon ligand binding, while other enzymes, most typically, hydrolases, change their structures to a small extent. It was also found that the solvent accessibility of the substrate molecule was low in transferases but high in hydrolases. These differences are explained by the enzymatic reaction mechanisms. The transferase reaction requires the catalytic groups to be insulated from the water environment, and thus transferases bury the ligand molecule inside the protein by closing the cleft. On the other hand, the hydrolase reaction involves the surrounding water molecules and occurs at the protein surface, requiring only a small structural change.     

Pyric tree spatial patterning interactions in historical and contemporary mixed conifer forests,California, USA

Tree spatial patterns in dry coniferous forests of the western United States, and analogous ecosystems globally, were historically aggregated, comprising a mixture of single trees and groups of trees. Modern forests, in contrast, are generally more homogeneous and overstocked than their historical counterparts. As these modern forests lack regular fire, pattern formation and maintenance is generally attributed to fire. Accordingly, fires in modern forests may not yield historically analogous patterns. However, direct observations on how selective tree mortality among pre‐existing forest structure shapes tree spatial patterns is limited. In this study, we (a) simulated fires in historical and contemporary counterpart plots in a Sierra Nevadan mixed‐conifer forest, (b) estimated tree mortality, and (c) examined tree spatial patterns of live trees before and after fire, and of fire‐killed trees. Tree mortality in the historical period was clustered and density‐dependent, because trees were aggregated and segregated by tree size before fire. Thus, fires maintained an aggregated distribution of tree groups. Tree mortality in the contemporary period was widespread, except for dispersed large trees, because most trees were a part of large, interconnected tree groups. Thus, postfire tree patterns were more uniform and devoid of moderately sized tree groups. Postfire tree patterns in the historical period, unlike the contemporary period, were within the historical range of variability identified for the western United States. This divergence suggests that decades of forest dynamics without significant disturbances have altered the historical means of pyric pattern formation. Our results suggest that ecological silvicultural treatments, such as forest restoration thinnings, which emulate qualities of historical forests may facilitate the reintroduction of fire as a means to reinforce forest structural heterogeneity.     

Understanding 3D structural complexity of individual Scots pine trees with different management history

Tree functional traits together with processes such as forest regeneration, growth, and mortality affect forest and tree structure. Forest management inherently impacts these processes. Moreover, forest structure, biodiversity, resilience, and carbon uptake can be sustained and enhanced with forest management activities. To assess structural complexity of individual trees, comprehensive and quantitative measures are needed, and they are often lacking for current forest management practices. Here, we utilized 3D information from individual Scots pine (Pinus sylvestris L.) trees obtained with terrestrial laser scanning to, first, assess effects of forest management on structural complexity of individual trees and, second, understand relationship between several tree attributes and structural complexity. We studied structural complexity of individual trees represented by a single scale‐independent metric called “box dimension.” This study aimed at identifying drivers affecting structural complexity of individual Scots pine trees in boreal forest conditions. The results showed that thinning increased structural complexity of individual Scots pine trees. Furthermore, we found a relationship between structural complexity and stem and crown size and shape as well as tree growth. Thus, it can be concluded that forest management affected structural complexity of individual Scots pine trees in managed boreal forests, and stem, crown, and growth attributes were identified as drivers of it.     

Excitation energy transfer and chlorophyll orientation in the green alga Chlamydomonas reinhardi

We have investigated the process of intermolecular excitation energy transfer and the relative orientation of the chlorophyll molecules in the unicellular green alga Chlamydomonas reinhardi. The principal experiments involved in vivo measurements of the fluorescence polarization as a function of the exciting-light wavelength in the presence and in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. We found that as the fluorescence lifetime increases upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea that the degree of fluorescence polarization decreases over the excitation region from 600 to 660 nm. This result, we argue, implies that a Förster mechanism of excitation energy transfer is involved for Photosystem II chlorophyll molecules absorbing primarily below 660 nm. We must add that our results do not exclude the possibility of a delocalized transfer process from being involved as well. Fluorescence polarization measurements using chloroplast fragments are also discussed in terms of a Förster transfer mechanism. As the excitation wavelength approaches 670 nm the fluorescence polarization is nearly constant upon the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea.Experiments performed using either vertically or horizontally polarized exciting light show that the fluorescence polarization increases as the exciting light wavelength increases from 650 to 673 nm. This suggests the possibility that chlorophyll molecules absorbing at longer wavelengths have a higher degree of relative order. Furthermore, these studies imply that chlorophyll molecules exist in discrete groups that are characterized by different absorption maxima and by different degrees of the fluorescence polarization. In view of these results we discuss different models for the Photosystem II antenna system and energy transfer between different groups of optically distinguishable chlorophyll molecules.