New features of the Blocks Database servers.

Blocks are ungapped multiple sequence alignments representing conserved protein regions, and the Blocks Database consists of blocks from documented protein families. World Wide Web (http://www. blocks.fhcrc.org) and Email (blocks@blocks.fhcrc.org) servers provide tools for homology searching and for analyzing protein family relationships. New enhancements include a multiple alignment processor that extends the use of these tools to imported multiple alignments of families not present in the database and a PCR primer designer that implements a new strategy for gene isolation.     

Superior performance in protein homology detection with the Blocks Database servers.

The Blocks Database World Wide Web (http://www.blocks.fhcrc.org ) and Email (blocks@blocks.fhcrc.org) servers provide tools for the detection and analysis of protein homology based on alignment blocks representing conserved regions of proteins. During the past year, searching has been augmented by supplementation of the Blocks Database with blocks from the Prints Database, for a total of 4754 blocks from 1163 families. Blocks from both the Blocks and Prints Databases and blocks that are constructed from sequences submitted to Block Maker can be used for blocks-versus-blocks searching of these databases with LAMA, and for viewing logos and bootstrap trees. Sensitive searches of up-to-date protein sequence databanks are carried out via direct links to the MAST server using position-specific scoring matrices and to the BLAST and PSI-BLAST servers using consensus-embedded sequence queries. Utilizing the trypsin family to evaluate performance, we illustrate the superiority of blocks-based tools over expert pairwise searching or Hidden Markov Models.     

Blocks+: a non-redundant database of protein alignment blocks derived from multiple compilations.

MOTIVATION: As databanks grow, sequence classification and prediction of function by searching protein family databases becomes increasingly valuable. The original Blocks Database, which contains ungapped multiple alignments for families documented in Prosite, can be searched to classify new sequences. However, Prosite is incomplete, and families from other databases are now available to expand coverage of the Blocks Database. RESULTS: To take advantage of protein family information present in several existing compilations, we have used five databases to construct Blocks+, a unified database that is built on the PROTOMAT/BLOSUM scoring model and that can be searched using a single algorithm for consistent sequence classification. The LAMA blocks-versus-blocks searching program identifies overlapping protein families, making possible a non-redundant hierarchical compilation. Blocks+ consists of all blocks derived from PROSITE, blocks from Prints not present in PROSITE, blocks from Pfam-A not present in PROSITE or Prints, and so on for ProDom and Domo, for a total of 1995 protein families represented by 8909 blocks, doubling the coverage of the original Blocks Database. A challenge for any procedure aimed at non-redundancy is to retain related but distinct families while discarding those that are duplicates. We illustrate how using multiple compilations can minimize this potential problem by examining the SNF2 family of ATPases, which is detectably similar to distinct families of helicases and ATPases. AVAILABILITY: http://blocks.fhcrc.org/     

CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) PCR primer design

We have developed a new primer design strategy for PCR amplification of distantly related gene sequences based on consensus-degenerate hybrid oligonucleotide primers (CODEHOPs). An interactive program has been written to design CODEHOP PCR primers from conserved blocks of amino acids within multiply-aligned protein sequences. Each CODEHOP consists of a pool of related primers containing all possible nucleotide sequences encoding 3-4 highly conserved amino acids within a 3' degenerate core. A longer 5' non-degenerate clamp region contains the most probable nucleotide predicted for each flanking codon. CODEHOPs are used in PCR amplification to isolate distantly related sequences encoding the conserved amino acid sequence. The primer design software and the CODEHOP PCR strategy have been utilized for the identification and characterization of new gene orthologs and paralogs in different plant, animal and bacterial species. In addition, this approach has been successful in identifying new pathogen species. The CODEHOP designer (http://blocks.fhcrc.org/codehop.html) is linked to BlockMaker and the Multiple Alignment Processor within the Blocks Database World Wide Web (http://blocks.fhcrc.org).     

Searching databases of conserved sequence regions by aligning protein multiple-alignments.

A general searching method for comparing multiple sequence alignments was developed to detect sequence relationships between conserved protein regions. Multiple alignments are treated as sequences of amino acid distributions and aligned by comparing pairs of such distributions. Four different comparison measures were tested and the Pearson correlation coefficient chosen. The method is sensitive, detecting weak sequence relationships between protein families. Relationships are detected beyond the range of conventional sequence database searches, illustrating the potential usefulness of the method. The previously undetected relation between flavoprotein subunits of two oxidoreductase families points to the potential active site in one of the families. The similarity between the bacterial RecA, DnaA and Rad51 protein families reveals a region in DnaA and Rad51 proteins likely to bind and unstack single-stranded DNA. Helix--turn--helix DNA binding domains from diverse proteins are readily detected and shown to be similar to each other. Glycosylasparaginase and gamma-glutamyltransferase enzymes are found to be similar in their proteolytic cleavage sites. The method has been fully implemented on the World Wide Web at URL: http://blocks.fhcrc.org/blocks-bin/LAMAvsearch.     

SIFT: Predicting amino acid changes that affect protein function

Single nucleotide polymorphism (SNP) studies and random mutagenesis projects identify amino acid substitutions in protein-coding regions. Each substitution has the potential to affect protein function. SIFT (Sorting Intolerant From Tolerant) is a program that predicts whether an amino acid substitution affects protein function so that users can prioritize substitutions for further study. We have shown that SIFT can distinguish between functionally neutral and deleterious amino acid changes in mutagenesis studies and on human polymorphisms. SIFT is available at http://blocks.fhcrc.org/sift/SIFT.html.     

Periodic distributions of hydrophobic amino acids allows the definition of fundamental building blocks to align distantly related proteins

Several studies on large and small families of proteins proved in a general manner that hydrophobic amino acids are globally conserved even if they are subjected to high rate substitution. Statistical analysis of amino acids evolution within blocks of hydrophobic amino acids detected in sequences suggests their usage as a basic structural pattern to align pairs of proteins of less than 25% sequence identity, with no need of knowing their 3D structure. The authors present a new global alignment method and an automatic tool for Proteins with HYdrophobic Blocks ALignment (PHYBAL) based on the combinatorics of overlapping hydrophobic blocks. Two substitution matrices modeling a different selective pressure inside and outside hydrophobic blocks are constructed, the Inside Hydrophobic Blocks Matrix and the Outside Hydrophobic Blocks Matrix, and a 4D space of gap values is explored. PHYBAL performance is evaluated against Needleman and Wunsch algorithm run with Blosum 30, Blosum 45, Blosum 62, Gonnet, HSDM, PAM250, Johnson and Remote Homo matrices. PHYBAL behavior is analyzed on eight randomly selected pairs of proteins of >30% sequence identity that cover a large spectrum of structural properties. It is also validated on two large datasets, the 127 pairs of the Domingues dataset with >30% sequence identity, and 181 pairs issued from BAliBASE 2.0 and ranked by percentage of identity from 7 to 25%. Results confirm the importance of considering substitution matrices modeling hydrophobic contexts and a 4D space of gap values in aligning distantly related proteins. Two new notions of local and global stability are defined to assess the robustness of an alignment algorithm and the accuracy of PHYBAL. A new notion, the SAD-coefficient, to assess the difficulty of structural alignment is also introduced. PHYBAL has been compared with Hydrophobic Cluster Analysis and HMMSUM methods.     

A transition probability model for amino acid substitutions from blocks.

Substitution matrices have been useful for sequence alignment and protein sequence comparisons. The BLOSUM series of matrices, which had been derived from a database of alignments of protein blocks, improved the accuracy of alignments previously obtained from the PAM-type matrices estimated from only closely related sequences. Although BLOSUM matrices are scoring matrices now widely used for protein sequence alignments, they do not describe an evolutionary model. BLOSUM matrices do not permit the estimation of the actual number of amino acid substitutions between sequences by correcting for multiple hits. The method presented here uses the Blocks database of protein alignments, along with the additivity of evolutionary distances, to approximate the amino acid substitution probabilities as a function of actual evolutionary distance. The PMB (Probability Matrix from Blocks) defines a new evolutionary model for protein evolution that can be used for evolutionary analyses of protein sequences. Our model is directly derived from, and thus compatible with, the BLOSUM matrices. The model has the additional advantage of being easily implemented.     

A field test of porous carbonated blocks used as artificial reef in seaweed beds of <Emphasis Type="Italic">Ecklonia cava</Emphasis>

Marine forests are the main primary producer in coastal waters, supplying food to fish and shellfish as well as providing their spawning and growing sites. It is important to conserve marine forest in order to protect coastal marine environments. A “Marine Block” comprised of steelmaking slag particles combined by CaCO₃ has several merits: (1) solidification of CO₂, (2) stability and safety, (3) porous materials covered with CaCO₃, (4) harmony with the seabed environment, (5) recycling of iron by-products, and (6) the ability to be mass produced in coastal ironworks. The objectives of this study were to observe the algal succession, the growth of Ecklonia cava and other attached organisms on Marine Blocks and concrete blocks compared to natural seaweed beds. In November 2001, five 1 m³ Marine Blocks and five 1 m³ concrete blocks were installed close to E. cava beds around the coastal frontage of Jogashima at the mouth of Tokyo Bay. Scuba-diving observations were continued until April 2006. In the field, a large number of seaweed and animal species were observed on the Marine Blocks, and it was found that the speed of succession, the plant length and the wet weight of E. cava were greater on Marine Blocks than on concrete blocks. The main advantages of Marine Blocks are considered to be the recycling of steelmaking slag, the absorption and solidification of atmospheric CO₂, and the conservation of coastal environments.     

Genetic analysis of gliadin components in winter wheat using two-dimensional polyacrylamide gel electrophoresis

Summary Blocks of gliadin components found both in a number of varieties and in single F₂ grains of winter wheat intervarietal hybrids have been studied by two-dimensional electrophoresis combining electrophoresis in acidic aluminium-lactate buffer (pH3.1) and SDS-electrophoresis. Gliadin components (spots) have been shown to be inherited as linked groups (blocks), codominantly and in accordance with a gene dosage in triploid endosperm. Blocks include components differing in their electrophoretic mobility and molecular weight. Some allelic variants of blocks differ only in presence of few additional components or in the electrophoretic mobility of components with similar molecular weights; other variants may contain no similar components. Apparently, in the course of evolution, mutations in individual genes of gliadin-coding loci and processes changing the number of expressing genes and the sizes of their structural part occurred.     

Occurrence of chloroplast ribosome recognition sites within conserved elements of the RNA genomes of carlaviruses

The nucleotide sequences upstream from the carlavirus open reading frames were examined for direct sequence homology. Blocks of homology were evident upstream from the 25 K ORFs of potato virus S (PVS), potato virus M (PVM) and lily symptomless virus (LSV), and upstream from the coat protein initiation codons of PVS, PVM, LSV, carnation latent virus and Helenium virus S. These blocks, which correspond to the 5′-terminal regions of the subgenomic RNAs, were shown to contain potential ribosome recognition sequences. The distances between the binding sites and initiation codons ranged from 20 to 40 nucleotides on the viral RNAs. Whilst the majority of chloroplasts mRNAs have a distance of 8 nucleotides between binding site and initiation codon, the remaining have a distance of 23 nucleotides which is similar to that reported here for the carlaviruses.     

Collection of Gravitropic Effectors from Mucilage of Electrotropically-stimulated Roots of Zea mays L.

We placed agar blocks adjacent to tips of electrotropicallystimulated primary roots of Zea mays. Blocks placed adjacentto the anode-side of the roots for 3 h induced significant curvaturewhen subsequently placed asymmetrically on tips of vertically-orientedroots. Curvature was always toward the side of the root ontowhich the agar block was placed. Agar blocks not contactingroots and blocks placed adjacent to the cathode-side of electrotropicallystimulated roots did not induce significant curvature when placedasymmetrically on tips of vertically-oriented roots. Atomicabsorption spectrophotometry indicated that blocks adjacentto the anode-side of electrotropically-stimulated roots containedsignificantly more calcium than (1) blocks not contacting roots,and (2) blocks contacting the cathode-side of roots. These resultsdemonstrate the presence of a gradient of endogenous Ca in mucilageof electrotropically-stimulated roots (i.e. roots undergoinggravitropic-like curvature). Zea mays, corn, mucilage, root gravitropism, electrotropism     

Proteomics analysis of two heat shock proteins in insects

Heat shock proteins (HSPs) are found in all living organisms, from bacteria to humans, are expressed under stress. In this study, characterization of two families of HSP including HSP60 and HSP70 protein was compared in different insect species from different orders. According to the conserved motifs analysis, none of the motifs were shared by all insects of two protein families but each family had their own common motifs. Functional and structural analyses were carried out on seven different insect species from each protein family as the representative samples. These analyses were performed via ExPASy database tools. The tertiary structure of Drosophila melanogater as the sample of each protein family were predicted by the Phyre2 and TM-score servers then their qualities were verified by SuperPose and PROCHECK. The tertiary structures were predicted through the “c4pj1E” model (PDB Accession Code: 4pj1) in HSP60 family and “c3d2fC” model (PDB Accession Code: 3d2f) in HSP70 family. The protein phylogenetic tree was constructed using the Neighbor-joining (NJ) method by Molecular Evolutionary Genetic Analysis (MEGA) 6.06. According to the results, there was a high identity of HSP60 and HSP70 families so that they should be derived from a common ancestor however they belonged to separate groups. In protein–protein interaction analysis by STRING 10.0, 10 common enriched pathways of biological process, molecular function and Kyoto Encyclopedia of Genes and Genomes (KEGG) were identified in D. melanogaster in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of insects and other organisms.

Communicated by Ramaswamy H. Sarma     

Blocks of gliadin components in winter wheat detected by one-dimensional polyacrylamide gel electrophoresis

Summary Inheritance of gliadin components in winter wheat has been studied by one-dimensional polyacrylamide gel electrophoresis. Single F₂ grains from 36 intervarietal hybrid combinations have been analysed. The genetic analysis has revealed blocks, including 1–6 gliadin components, which are inherited as individual mendelian traits. About 80 variants of blocks have been detected. On the basis of the allelism test they are grouped into 6 series in accordance with the number of known gliadin-coding loci located on chromosomes of the homoeologous groups 1 and 6. Each series includes 8–18 blocks controlled by different alleles of one gliadin-coding locus. Blocks of components have been confirmed to be inherited codominantly in accordance to the gene dose in the triploid endosperm. The highest similarity between members of one series is observed in groups of blocks controlled by chromosomes ID and 6D. On the contrary, many blocks controlled by chromosomes 1A and 1B have no bands in common. The presented catalogue of blocks of components may be used to make up gliadin genetic formulae and to compare electrophoregrams obtained by different authors. Blocks of gliadin components are suitable genetic markers for use in revealing and studying heterogeneity of wheat varieties, in tracing their origin, in identifying recombinations, translocations and substitutions of the genetic material and in solving many other problems of the origin, evolution and selection of hexaploid wheat.     

Binding MOAD (Mother Of All Databases)

Binding MOAD (Mother of All Databases) is the largest collection of high-quality, protein-ligand complexes available from the Protein Data Bank. At this time, Binding MOAD contains 5331 protein-ligand complexes comprised of 1780 unique protein families and 2630 unique ligands. We have searched the crystallography papers for all 5000+ structures and compiled binding data for 1375 (26%) of the protein-ligand complexes. The binding-affinity data ranges 13 orders of magnitude. This is the largest collection of binding data reported to date in the literature. We have also addressed the issue of redundancy in the data. To create a nonredundant dataset, one protein from each of the 1780 protein families was chosen as a representative. Representatives were chosen by tightest binding, best resolution, etc. For the 1780 "best" complexes that comprise the nonredundant version of Binding MOAD, 475 (27%) have binding data. This significant collection of protein-ligand complexes will be very useful in elucidating the biophysical patterns of molecular recognition and enzymatic regulation. The complexes with binding-affinity data will help in the development of improved scoring functions and structure-based drug discovery techniques. The dataset can be accessed at http://www.BindingMOAD.org.     

General framework for developing and evaluating database scoring algorithms using the TANDEM search engine

MOTIVATION: Tandem mass spectrometry (MS/MS) identifies protein sequences using database search engines, at the core of which is a score that measures the similarity between peptide MS/MS spectra and a protein sequence database. The TANDEM application was developed as a freely available database search engine for the proteomics research community. To extend TANDEM as a platform for further research on developing improved database scoring methods, we modified the software to allow users to redefine the scoring function and replace the native TANDEM scoring function while leaving the remaining core application intact. Redefinition is performed at run time so multiple scoring functions are available to be selected and applied from a single search engine binary. We introduce the implementation of the pluggable scoring algorithm and also provide implementations of two TANDEM compatible scoring functions, one previously described scoring function compatible with PeptideProphet and one very simple scoring function that quantitative researchers may use to begin their development. This extension builds on the open-source TANDEM project and will facilitate research into and dissemination of novel algorithms for matching MS/MS spectra to peptide sequences. The pluggable scoring schema is also compatible with related search applications P3 and Hunter, which are part of the X! suite of database matching algorithms. The pluggable scores and the X! suite of applications are all written in C++. AVAILABILITY: Source code for the scoring functions is available from http://proteomics.fhcrc.org     

Cryopreservation of Cortical Tissue Blocks for the Generation of Highly Enriched Neuronal Cultures

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank''s Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1°C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.