Cloning, sequence analysis, and tissue expression of marmoset paraoxonase 1.

Paraoxonase (PON) plays roles in the metabolism of organophosphate xenobiotics and drugs. Despite the importance of marmosets for research into drug metabolism and pharmacokinetics, marmoset paraoxonase has not yet been fully characterized. Consequently, we identified the PON1 gene in the marmoset genome by sequence homology analysis. Marmoset PON1 cDNA containing an open reading frame (1065 bp) was successfully cloned from marmoset liver by reverse transcription-polymerase chain reaction. The deduced amino acid sequence (355 amino acids) has approximately 93% identity with the human ortholog and contains important amino acid residues for substrate binding and calcium ion coordination. According to a phylogenetic tree of PON1 amino acid sequences constructed using data from seven animal species, marmoset PON1 is closer to human PON1 than it is to the PON1 orthologs of experimental animals such as pigs, rabbits, rats, and mice. Marmoset PON1 mRNA was predominantly expressed in liver among the five tissues examined. Marmoset PON1 protein secreted into plasma was detected by immunoblotting. The paraoxon-hydrolyzing activity in plasma was higher in marmosets than in humans. Based on these data, we concluded that marmoset and human PON1 have similar characteristics with regard to genomic structure, amino acid sequences, and tissue distribution.

Medicinal Materials DNA Barcode Database (MMDBD) version 1.5-one-stop solution for storage, BLAST, alignment and primer design.

Authentication of medicinal materials by deoxyribonucleic acid (DNA) technology is gaining popularity. In 2010, our team has created Medicinal Materials DNA Barcode Database (MMDBD) version 1.0 to provide an interactive database for documenting DNA barcode sequences of medicinal materials. This database now contains DNA barcode sequences of medicinal materials listed in the Chinese Pharmacopoeia, Dietary Supplements Compendium and Herbal Medicine Compendium of the US Pharmacopoeia and selected adulterants. The data archive is regularly updated and currently it stores 62 011 DNA sequences of 2111 medicinal materials. Our team has recently completed the major improvement on the interfaces and incorporated essential bioinformatics tools to facilitate the authentication work. MMDBD version 1.5 contains detailed information of each medicinal material including their material names, medical part, pharmacopeia information, biological classification in rank of family and status on the Convention on International Trade in Endangered Species of Wild Fauna and Flora and the International Union for Conservation of Nature's Red List of Threatened Species, if any. DNA sequences can be retrieved by search in Latin scientific name, Chinese name, family name, material name, medical part and simplified Chinese character stroke. A `BLAST'-based engine for searching DNA sequences is included in the MMDBD version 1.5. Since primer design is a key step in DNA barcoding authentication, we have integrated the `Clustal Omega alignment tool' and `Primer3' in the form of web interface. These new tools facilitate multiple sequence comparison and the design of primers for amplification of a target DNA barcode region, allowing DNA barcoding authentication.

Reproductive Long Intergenic Noncoding RNAs Exhibit Male Gamete Specificity and Polycomb Repressive Complex 2-Mediated Repression.

Long noncoding RNAs (lncRNAs) have been characterized extensively in animals and are involved in several processes, including homeobox gene expression and X-chromosome inactivation. In comparison, there has been much less detailed characterization of plant lncRNAs, and the number of distinct lncRNAs encoded in plant genomes and their regulation by developmental and epigenetic mechanisms remain largely unknown. Here, we analyzed transcriptome data from Asian rice (Oryza sativa) and identified 6,309 long intergenic noncoding RNAs (lincRNAs), focusing on their expression in reproductive tissues and organs. Most O. sativa lincRNAs were expressed in a highly tissue-specific manner, with an unexpectedly high fraction specifically expressed in male gametes. Mutation of a component of the Polycomb Repressive Complex2 (PRC2) resulted in derepression of another large class of lincRNAs, whose expression is correlated with H3K27 trimethylation in developing panicles. Overlap with the sperm cell-specific lincRNAs suggests that epigenetic repression of lincRNAs in the panicles was partially relieved in the male germline. Expression of a subset of lincRNAs also showed modulation by drought in reproductive tissues. Comparison with other cereal genomes showed that the lincRNAs generally have low levels of conservation at both the sequence and structural levels. Use of a novelty detection support vector machine model enabled the detection of nucleotide sequence and structural homology in ∼10% and ∼4% of the lincRNAs in genomes of purple false brome (Brachypodium distachyon) and maize (Zea mays), respectively. This is the first study to report on a large number of lncRNAs that are targets of repression by PRC2 rather than mediating regulation via PRC2. That the vast majority of the lincRNAs reported here do not overlap with those of other rice studies indicates that these are a significant addition to the known lincRNAs in rice.

An in silico platform for predicting, screening and designing of antihypertensive peptides.

High blood pressure or hypertension is an affliction that threatens millions of lives worldwide. Peptides from natural origin have been shown recently to be highly effective in lowering blood pressure. In the present study, we have framed a platform for predicting and designing novel antihypertensive peptides. Due to a large variation found in the length of antihypertensive peptides, we divided these peptides into four categories (i) Tiny peptides, (ii) small peptides, (iii) medium peptides and (iv) large peptides. First, we developed SVM based regression models for tiny peptides using chemical descriptors and achieved maximum correlation of 0.701 and 0.543 for dipeptides and tripeptides, respectively. Second, classification models were developed for small peptides and achieved maximum accuracy of 76.67%, 72.04% and 77.39% for tetrapeptide, pentapeptide and hexapeptides, respectively. Third, we have developed a model for medium peptides using amino acid composition and achieved maximum accuracy of 82.61%. Finally, we have developed a model for large peptides using amino acid composition and achieved maximum accuracy of 84.21%. Based on the above study, a web-based platform has been developed for locating antihypertensive peptides in a protein, screening of peptides and designing of antihypertensive peptides.

H2rs: deducing evolutionary and functionally important residue positions by means of an entropy and similarity based analysis of multiple sequence alignments.

BACKGROUND: The identification of functionally important residue positions is an important task of computational biology. Methods of correlation analysis allow for the identification of pairs of residue positions, whose occupancy is mutually dependent due to constraints imposed by protein structure or function. A common measure assessing these dependencies is the mutual information, which is based on Shannon's information theory that utilizes probabilities only. Consequently, such approaches do not consider the similarity of residue pairs, which may degrade the algorithm's performance. One typical algorithm is H2r, which characterizes each individual residue position k by the conn(k)-value, which is the number of significantly correlated pairs it belongs to. RESULTS: To improve specificity of H2r, we developed a revised algorithm, named H2rs, which is based on the von Neumann entropy (vNE). To compute the corresponding mutual information, a matrix A is required, which assesses the similarity of residue pairs. We determined A by deducing substitution frequencies from contacting residue pairs observed in the homologs of 35 809 proteins, whose structure is known. In analogy to H2r, the enhanced algorithm computes a normalized conn(k)-value. Within the framework of H2rs, only statistically significant vNE values were considered. To decide on significance, the algorithm calculates a p-value by performing a randomization test for each individual pair of residue positions. The analysis of a large in silico testbed demonstrated that specificity and precision were higher for H2rs than for H2r and two other methods of correlation analysis. The gain in prediction quality is further confirmed by a detailed assessment of five well-studied enzymes. The outcome of H2rs and of a method that predicts contacting residue positions (PSICOV) overlapped only marginally. H2rs can be downloaded from http://www-bioinf.uni-regensburg.de. CONCLUSIONS: Considering substitution frequencies for residue pairs by means of the von Neumann entropy and a p-value improved the success rate in identifying important residue positions. The integration of proven statistical concepts and normalization allows for an easier comparison of results obtained with different proteins. Comparing the outcome of the local method H2rs and of the global method PSICOV indicates that such methods supplement each other and have different scopes of application.

Molecular cloning of a plasma membrane Ca²âº ATPase (PMCA) from Y-organs of the blue crab (Callinectes sapidus), and determination of spatial and temporal patterns of PMCA gene expression.

Existing data indicate that a stage-specific increase in intracellular free Ca(2+) stimulates ecdysteroid production by crustacean molting glands (Y-organs). The concentration of Ca(2+) in cytosol is controlled mainly by proteins intrinsic to the plasma membrane and to the membranes of organelles. Several families of proteins are involved, including Ca(2+) channels, Ca(2+) pumps (ATPases), and Ca(2+) exchangers. The family of Ca(2+) pumps includes plasma membrane calcium ATPases (PMCAs). As a step toward understanding the involvement of calcium signaling in regulation of ecdysteroidogenesis, we used a PCR-based cloning strategy (RT-PCR followed by 3'- and 5'-RACE) to clone from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative PMCA. The 4292 base pair (bp) cDNA includes a 3510 bp open reading frame encoding a 1170-residue protein (Cas-PMCA). The conceptually translated protein has a relative molecular mass of 128.8×10(3) and contains all signature domains of an authentic PMCA, including ten transmembrane domains and a calmodulin binding site. The predicted membrane topography of Cas-PMCA is as expected for an authentic PMCA protein. A phylogenetic analysis of nonredundant amino acid sequences of PMCA proteins from different species showed Cas-PMCA clusters with other arthropod PMCA proteins. An assessment of tissue distribution showed the Cas-PMCA transcript to be broadly distributed in both neural and non-neural tissues. Studies using quantitative real-time PCR revealed stage-specific changes in Cas-PMCA abundance during the molting cycle, with peak expression occurring during premolt stage D2, a pattern consistent with the hypothesis that Cas-PMCA functions to maintain cellular Ca(2+) homeostasis in Y-organs.

PASS-bis: a bisulfite aligner suitable for whole methylome analysis of Illumina and SOLiD reads.

SUMMARY: The sequencing of bisulfite-treated DNA (Bi-Seq) is becoming a gold standard for methylation studies. The mapping of Bi-Seq reads is complex and requires special alignment algorithms. This problem is particularly relevant for SOLiD color space, where the bisulfite conversion C/T changes two adjacent colors into 16 possible combinations. Here, we present an algorithm that efficiently aligns Bi-Seq reads obtained either from SOLiD or Illumina. An accompanying methylation-caller program creates a genomic view of methylated and unmethylated Cs on both DNA strands. AVAILABILITY AND IMPLEMENTATION: The algorithm has been implemented as an option of the program PASS, freely available at http://pass.cribi.unipd.it. CONTACT: pass@cribi.unipd.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular cloning and characterization of estrogen receptor gene in the scallop Chlamys farreri: expression profiles in response to endocrine disrupting chemicals.

In order to gain insights into the mechanism of sex steroid signaling in molluscs, the full-length cDNA of estrogen receptor (ER) was isolated and characterized from Chlamys farreri for the first time. The positions of cysteine residues and other residues around them that constitute the two zinc finger motifs and the P-box are conserved. Phylogenetic analysis revealed that the CfER is an ortholog of the other mollusk ERs. Tissue distribution analysis of the CfER mRNA revealed that the expression of ER mRNA was observed in various tissues, and highest in the gonad of males and females. C. farreri were exposed for 10 days to endocrine disrupting chemicals including Benzo(a)pyrene (B(a)p) and polybrominated diphenyl ethers (BDE-47). B(a)p exposure at 0.4 and 2 µg/L caused significant increase in mRNA expression of ER and VTG, but B(a)p at 10 µg/L down-regulated CfER and VTG mRNA expression compared to control. Varying increase of ER and VTG mRNA transcripts was resulted in by BDE-47 at 0.1, 1 and 10 µg/L. These results elucidate potential roles of CfER induced by xenobiotics in C. farreri and can be helpful for investigating the mechanism of sex steroid signaling in bivalve mollusks.

Molecular cloning, characterization and expression of heat shock protein 70 gene from the oyster Crassostrea hongkongensis responding to thermal stress and exposure of Cu(2+) and malachite green.

Heat shock protein 70 (HSP70) acts mostly as a molecular chaperone and plays a key role in the process of protecting cells by facilitating the folding of nascent peptides and the cellular stress response. The cDNA of the oyster Crassostrea hongkongensis hsp70 (designated chhsp70) was cloned with the techniques of homological cloning and rapid amplification of cDNA ends (RACE). The full-length chhsp70 cDNA was 2251bp, consisting of a 130bp 5'-UTR, 216bp 3'-UTR with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1905bp, which encoded a polypeptide of 634 amino acids. Three classical HSP signature motifs were detected in ChHSP70, i.e., DLGTT-S-V, IFDLGGGTFDVSIL and VVLVGGSTRIPKIQK. BLAST analysis revealed that the ChHSP70 shared high identity with other bivalve HSP70. The phylogenetic analysis indicated that the ChHSP70 was a member of the HSP70 family. The chhsp70 mRNA transcripts were quantified by fluorescent real time RT-PCR under both unstressed and stressed conditions, i. e., heat shock and exposure to Cu(2+) and malachite green. Basal expression level was similar in mantle, gill, digestive gland, and heart, but higher in muscle than that in the others. A similar trend showed that the chhsp70 mRNA expression significantly increased at 3-6h, then dropped and returned to control level at 24h in the five tissues and organs mentioned above after heat shock. A clearly time-dependent expression pattern of chhsp70 mRNA in digestive gland and gill of the oyster was observed after exposure of Cu(2+) and malachite green. In the two tissues, the chhsp70 mRNA level reached the maximum at 6h after malachite green exposure and on day 4 after Cu(2+) exposure, and then decreased progressively to the control level. The results indicated that ChHSP70 of the oyster is an inducible protein, and plays an important role in response to the Cu(2+) and malachite green polluted stress, so chhsp70 might be used as a potential molecular biomarker of above pollutants.

Mutations in SLC30A10 cause parkinsonism and dystonia with hypermanganesemia, polycythemia, and chronic liver disease.

Manganese is essential for several metabolic pathways but becomes toxic in excessive amounts. Manganese levels in the body are therefore tightly regulated, but the responsible protein(s) remain incompletely known. We studied two consanguineous families with neurologic disorders including juvenile-onset dystonia, adult-onset parkinsonism, severe hypermanganesemia, polycythemia, and chronic hepatic disease, including steatosis and cirrhosis. We localized the genetic defect by homozygosity mapping and then identified two different homozygous frameshift SLC30A10 mutations, segregating with disease. SLC30A10 is highly expressed in the liver and brain, including in the basal ganglia. Its encoded protein belongs to a large family of membrane transporters, mediating the efflux of divalent cations from the cytosol. We show the localization of SLC30A10 in normal human liver and nervous system, and its depletion in liver from one affected individual. Our in silico analyses suggest that SLC30A10 possesses substrate specificity different from its closest (zinc-transporting) homologs. We also show that the expression of SLC30A10 and the levels of the encoded protein are markedly induced by manganese in vitro. The phenotype associated with SLC30A10 mutations is broad, including neurologic, hepatic, and hematologic disturbances. Intrafamilial phenotypic variability is also present. Chelation therapy can normalize the manganesemia, leading to marked clinical improvements. In conclusion, we show that SLC30A10 mutations cause a treatable recessive disease with pleomorphic phenotype, and provide compelling evidence that SLC30A10 plays a pivotal role in manganese transport. This work has broad implications for understanding of the manganese biology and pathophysiology in multiple human organs.

RRBSMAP: a fast, accurate and user-friendly alignment tool for reduced representation bisulfite sequencing.

SUMMARY: Reduced representation bisulfite sequencing (RRBS) is a powerful yet cost-efficient method for studying DNA methylation on a genomic scale. RRBS involves restriction-enzyme digestion, bisulfite conversion and size selection, resulting in DNA sequencing data that require special bioinformatic handling. Here, we describe RRBSMAP, a short-read alignment tool that is designed for handling RRBS data in a user-friendly and scalable way. RRBSMAP uses wildcard alignment, and avoids the need for any preprocessing or post-processing steps. We benchmarked RRBSMAP against a well-validated MAQ-based pipeline for RRBS read alignment and observed similar accuracy but much improved runtime performance, easier handling and better scaling to large sample sets. In summary, RRBSMAP removes bioinformatic hurdles and reduces the computational burden of large-scale epigenome association studies performed with RRBS. AVAILABILITY: http://rrbsmap.computational-epigenetics.org/ http://code.google.com/p/bsmap/ CONTACT: wl1@bcm.tmc.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Molecular authentication of the medicinal herb Ruta graveolens (Rutaceae) and an adulterant using nuclear and chloroplast DNA markers.

Dried parts of different plant species often look alike, especially in powdered form, making them very difficult to identify. Ruta graveolens, sold as a dried medicinal herb, can be adulterated with Euphorbia dracunculoides. The genomic DNA was isolated from the leaf powder (100 mg each) using the modified CTAB method. Internal transcribed spacer sequences of nuclear ribosomal DNA (nrDNA-ITS), and chloroplast spacer sequences (rpoB and rpoC1) are regarded as potential genes for plant DNA barcoding. We amplified and sequenced these spacer sequences and confirmed the sequences with a BLAST search. Sequence alignment was performed using ClustalX to look for differences in the sequences. A DNA marker was developed based on rpoB and rpoC1 of the nrDNA-ITS for the identification of the adulterant E. dracunculoides in samples of R. graveolens that are sold in local herbal markets. Sequence-characterized amplified region markers of 289 and 264 bp for R. graveolens and 424 bp for E. dracunculoides were developed from dissimilar sequences of this nrDNA-ITS to speed up the authentication process. This marker successfully distinguished these species in extracted samples with as little as 5 ng DNA/µL extract.

Molecular modeling of 2-nitropropane dioxygenase domain of Mycobacterium tuberculosis H37Rv and docking of herbal ligands.

The 3D structure of enoyl reductase (ER) domain generated by the SWISS MODEL server contains the 2-nitropropane dioxygenase (2NPD) structure displaying the TIM barrel fold. Though TIM barrel fold is made up of both main and inserted domains, in our study, we could only predict the structure of the main domain, which had central barrel of eight beta-strands surrounded by eight alpha-helices. Superimposition of the 2NPD region of ER domain of Mycobacterium tuberculosis H37Rv on to the corresponding region of 2UVA_G revealed a good structural alignment between the two, suggesting this template to be a good structural homologue. Among various herbal ligands that were screened as inhibitors, daucosterol was found to bind in closest proximity to the flavin mono nucleotide (FMN) binding site with the lowest docking energy.

Alternative splicing determines mRNA translation initiation and function of human K(2P)10.1 K+ channels.

Potassium-selective ion channels regulate cardiac and neuronal excitability by stabilizing the resting membrane potential and by modulating shape and frequency of action potentials. The delicate control of membrane voltage requires structural and functional diversity of K+ channel subunits expressed in a given cell. Here we reveal a previously unrecognized biological mechanism. Tissue-specific mRNA splicing regulates alternative translation initiation (ATI) of human K(2P)10.1 K+ background channels via recombination of 5 nucleotide motifs. ATI-dependent expression of full-length protein or truncated subunits initiated from two downstream start codons determines macroscopic current amplitudes and biophysical properties of hK(2P)10.1 channels. The interaction between hK(2P)10.1 mRNA splicing, translation and function increases K+ channel complexity and is expected to contribute to electrophysiological plasticity of excitable cells.

Detection of rotavirus in food associated with a gastroenteritis outbreak in a mother and child sanatorium.

In the course of a rotavirus outbreak in a mother and child sanatorium 74 food samples from the sanatorium kitchen were taken and tested for rotavirus. Rotavirus particles were isolated from 25 g food samples by a simple method including ultrafiltration, originally designed for the detection of norovirus in various food matrices. Rotavirus was successfully detected in a sample of potato stew by conventional RT-seminested-PCR. Sequence comparison of the amplification products obtained from the potato stew and a stool sample from an infected child verified that the two viruses were identical.

Reduced amino acid alphabets exhibit an improved sensitivity and selectivity in fold assignment.

MOTIVATION: Many proteins with vastly dissimilar sequences are found to share a common fold, as evidenced in the wealth of structures now available in the Protein Data Bank. One idea that has found success in various applications is the concept of a reduced amino acid alphabet, wherein similar amino acids are clustered together. Given the structural similarity exhibited by many apparently dissimilar sequences, we undertook this study looking for improvements in fold recognition by comparing protein sequences written in a reduced alphabet. RESULTS: We tested over 150 of the amino acid clustering schemes proposed in the literature with all-versus-all pairwise sequence alignments of sequences in the Distance mAtrix aLIgnment database. We combined several metrics from information retrieval popular in the literature: mean precision, area under the Receiver Operating Characteristic curve and recall at a fixed error rate and found that, in contrast to previous work, reduced alphabets in many cases outperform full alphabets. We find that reduced alphabets can perform at a level comparable to full alphabets in correct pairwise alignment of sequences and can show increased sensitivity to pairs of sequences with structural similarity but low-sequence identity. Based on these results, we hypothesize that reduced alphabets may also show performance gains with more sophisticated methods such as profile and pattern searches. AVAILABILITY: A table of results as well as the substitution matrices and residue groupings from this study can be downloaded from (http://www.rpgroup.caltech.edu/publications/supplements/alphabets).

Efficient algorithms to explore conformation spaces of flexible protein loops.

Several applications in biology - e.g., incorporation of protein flexibility in ligand docking algorithms, interpretation of fuzzy X-ray crystallographic data, and homology modeling - require computing the internal parameters of a flexible fragment (usually, a loop) of a protein in order to connect its termini to the rest of the protein without causing any steric clash. One must often sample many such conformations in order to explore and adequately represent the conformational range of the studied loop. While sampling must be fast, it is made difficult by the fact that two conflicting constraints - kinematic closure and clash avoidance - must be satisfied concurrently. This paper describes two efficient and complementary sampling algorithms to explore the space of closed clash-free conformations of a flexible protein loop. The "seed sampling" algorithm samples broadly from this space, while the "deformation sampling" algorithm uses seed conformations as starting points to explore the conformation space around them at a finer grain. Computational results are presented for various loops ranging from 5 to 25 residues. More specific results also show that the combination of the sampling algorithms with a functional site prediction software (FEATURE) makes it possible to compute and recognize calcium-binding loop conformations. The sampling algorithms are implemented in a toolkit (LoopTK), which is available at https://simtk.org/home/looptk.

Antibody CDR H3 modeling rules: extension for the case of absence of Arg H94 and Asp H101.

The third complementary determining region of the immunoglobulin heavy chain (CDR H3) is one of the more difficult structures to model due to genetic reasons. However, the conformation of proximal to beta-framework ("torso") part of the CDR H3 is very predictable. Current "CDR's canonical classes" theory is based on identifying the key positions, H94 and H101. We can determine the CDR H3 "torso" structure if arginine or lysine is present in the H94 position and/or aspartic acid in the H101 position. We target the case characterized by the absence of key residues in both the H94 and H101 positions. There has not been discussion on this case in the literature. 51 CDR H3 structures of this nature are analyzed and we established new sequence-structure rules. These rules contribute to more accurate modeling of the antibody's structure.

Integrating protein structures and precomputed genealogies in the Magnum database: examples with cellular retinoid binding proteins.

BACKGROUND: When accurate models for the divergent evolution of protein sequences are integrated with complementary biological information, such as folded protein structures, analyses of the combined data often lead to new hypotheses about molecular physiology. This represents an excellent example of how bioinformatics can be used to guide experimental research. However, progress in this direction has been slowed by the lack of a publicly available resource suitable for general use. RESULTS: The precomputed Magnum database offers a solution to this problem for ca. 1,800 full-length protein families with at least one crystal structure. The Magnum deliverables include 1) multiple sequence alignments, 2) mapping of alignment sites to crystal structure sites, 3) phylogenetic trees, 4) inferred ancestral sequences at internal tree nodes, and 5) amino acid replacements along tree branches. Comprehensive evaluations revealed that the automated procedures used to construct Magnum produced accurate models of how proteins divergently evolve, or genealogies, and correctly integrated these with the structural data. To demonstrate Magnum's capabilities, we asked for amino acid replacements requiring three nucleotide substitutions, located at internal protein structure sites, and occurring on short phylogenetic tree branches. In the cellular retinoid binding protein family a site that potentially modulates ligand binding affinity was discovered. Recruitment of cellular retinol binding protein to function as a lens crystallin in the diurnal gecko afforded another opportunity to showcase the predictive value of a browsable database containing branch replacement patterns integrated with protein structures. CONCLUSION: We integrated two areas of protein science, evolution and structure, on a large scale and created a precomputed database, known as Magnum, which is the first freely available resource of its kind. Magnum provides evolutionary and structural bioinformatics resources that are useful for identifying experimentally testable hypotheses about the molecular basis of protein behaviors and functions, as illustrated with the examples from the cellular retinoid binding proteins.

Prediction of DNA-binding residues from sequence features.

Protein-DNA interaction plays a pivotal role in transcriptional regulation, DNA metabolism and chromatin formation. Although structural data are available for a few hundreds of protein-DNA complexes, the molecular recognition pattern is still poorly understood. With the rapid accumulation of sequence data from many genomes, it is important to develop predictive methods for identification of potential DNA-binding residues in proteins. In this study, neural networks have been trained using five sequence-derived features for prediction of DNA-binding residues. These features include the molecular mass, hydrophobicity index, side chain pKa value, solvent accessible surface area and conservation score of an amino acid. Interestingly, the side chain pKa value appears to be the best feature for prediction, suggesting that the ionization state of amino acid side chains is important for DNA-binding. The predictive performance is enhanced by using multiple features for classifier construction. The classifier that has been constructed using all the five features predicts at 72.71% sensitivity and 67.73% specificity. This is by far the most accurate classifier reported for prediction of DNA-binding residues from sequence data. The classifier has also been evaluated by using the Receiver Operating Characteristic curve and by examining the predictions made for different classes of DNA-binding proteins. Supplementary materials including the datasets are available at http://bioinformatics.ksu.edu/pdi/feature.html.