Nucleic Acid Databases Research Articles

The Leishmania major RNA Polymerase II Largest Subunit Lacks a Carboxy-Terminus Heptad Repeat Structure and its Encoding Gene is Linked with the Calreticulin Gene

The gene encoding the RNA polymerase II largest subunit (RPOIILS) has been isolated and sequenced from the kinetoplastid protozoan, Leishmania (Leishmania) major. The RPOIILS gene was shown to be present as a single copy and is composed of an uninterrupted open reading frame of 4.99 kb, specifying a protein 1663 aa in length with a predicted molecular mass of approximately 185 kDa. The carboxy terminus domain (CTD) of the RPOIILS from L. (L.) major, typical of the more evolutionary primitive protozoa, lacked a heptad repeat structure which is present in higher eukaryotes and some other protozoan phyla. Comparison of the predicted aa composition of the CTD from a diverse range of eukaryotic species revealed the abundance of Ser and Pro residues as the only discernible evolutionary conservative feature. A putative ATG start codon for an additional expressed sequence was located 1.1 kb downstream of the L. (L.) major RPOIILS gene stop codon. Nucleic acid database searches revealed the identity of this gene as that encoding the calcium binding protein calreticulin (CLT). The close proximity of the RPOIILS and CLT genes in L. (L.) major raises the possibility that these genes are transcribed as part of the same polycistronic unit.

Preliminary profile of the Cryptosporidium parvum genome: an expressed sequence tag and genome survey sequence analysis

Cryptosporidium parvum is a protozoan enteropathogen that infects humans and animals and causes a pronounced diarrheal disease that can be life-threatening in immunocompromised hosts. No specific chemo- or immunotherapies exist to treat cryptosporidiosis and little molecular information is available to guide development of such therapies. To accelerate gene discovery and identify genes encoding potential drug and vaccine targets we constructed sporozoite cDNA and genomic DNA sequencing libraries from the Iowa isolate of C. parvum and determined ∼2000 sequence tags by single-pass sequencing of random clones. Together, the 567 expressed sequence tags (ESTs) and 1507 genome survey sequences (GSSs) totaled one megabase (1 mb) of unique genomic sequence indicating that ∼10% of the 10.4 mb C. parvum genome has been sequence tagged in this gene discovery expedition. The tags were used to search the public nucleic acid and protein databases via BLAST analyses, and 180 ESTs (32%) and 277 GSSs (18%) exhibited similarity with database sequences at smallest sum probabilities P( N)≤10 −8. Some tags encoded proteins with clear therapeutic potential including S-adenosylhomocysteine hydrolase, histone deacetylase, polyketide/fatty-acid synthases, various cyclophilins, thrombospondin-related cysteine-rich protein and ATP-binding-cassette transporters. Several anonymous ESTs encoded proteins predicted to contain signal peptides or multiple transmembrane spanning segments suggesting they were destined for membrane-bound compartments, the cell surface or extracellular secretion. One-hundred four simple sequence repeats were identified within the nonredundant sequence tag collection with (TAA) ≥6/(TTA) ≥6 and (TA) ≥10/(AT) ≥10 being the most prevalent, occurring 40 and 15 times, respectively. Various cellular RNAs and their genes were also identified including the small and large ribosomal RNAs, five tRNAs, the U2 small nuclear RNA, and the small and large virus-like, double-stranded RNAs. This investigation has demonstrated that survey sequencing is an efficient procedure for gene discovery and genome characterization and has identified and sequence tagged many C. parvum genes encoding potential therapeutic targets.

SPAC: identification of polypeptides using their amino-acid composition

We describe here new software able to retrieve from protein or nucleic acid databases, the sequence corresponding to a protein or peptide whose only amino acid composition and molecular weight are known. This algorithm is particularly devoted to the retrieval of partial sequences, a task that other available software performs poorly. Its accuracy for the attribution of a protein fragment to a sequence could represent an easy and economical first tool upstream the use of more sophisticated and expensive methods in proteomic research. SPAC (Sequence Protein Alignment with Composition) is a shareware available software on web site http://www.univ-tln.fr/∼grillas/SPAC/. This web site also presents help and a detailed description of the algorithm and interface.

Embarking on rice functional genomics via cDNA microarray: use of 3' UTR probes for specific gene expression analysis.

Because rice is the major source of food for half of the world population, a comprehensive understanding of its genome will have a tremendous impact on the status of agriculture in the Twenty-first Century. The Rice Genome Research Program (RGP) has undertaken an extensive rice genome analysis since its initial launching in 1991 that has resulted in the establishment of a catalog of rice genes, a high-density linkage map, and a YAC-based physical map. An enormous collection of expressed sequence tags (ESTs) was generated from large-scale cDNA sequencing using cDNA libraries derived from rice callus cultured in different media and tissues such as root, shoot, leaf and panicle. This included more than 9000 partial cDNA sequences corresponding to unique genes have been identified. Based on the results of large-scale cDNA analysis, microarray can be used to monitor gene expression profiles and to initiate functional analysis of the rice genome. We initiated a rice cDNA microarray project beginning in April 1999 that aims to elucidate the function of all genes in rice using a gene expression monitoring system. The National Institute of Agrobiological Resources (NIAR) and the Institute of the Society for Techno-innovation of Agriculture, Forestry and Fisheries (STAFF) are jointly conducting this project in collaboration with 64 research institutions all over Japan. The research areas and investigator/institutions involved in this collaboration are listed in http://microarray.rice.dna.affrc.go.jp/. Homology search of all ESTs was performed by BLASTN and about over 9000 clones with high similarity were clustered. Clustered clones were analyzed for sequence similarity against public protein and nucleic acid databases. Then BLASTN and BLASTX were performed and clones with high similarity were identified putatively. One

The IMB Jena Image Library of biological macromolecules.

The IMB Jena Image Library of Biological Macro-molecules (http://www. imb-jena.de/IMAGE.html ) is aimed at a better dissemination of information on three-dimensional biopolymer structures with an emphasis on visualization and analysis. It provides access to all structure entries deposited at the Protein Data Bank (PDB) and Nucleic Acid Database (NDB). By combining automatic and manual processing it is possible to keep pace with the rapidly growing number of known biopolymer structures and to provide, for selected entries, information not available from automatic procedures. Each entry page contains basic information on the structure, various visualization and analysis tools as well as links to other databases. The visualization techniques adopted include static mono/stereo raster or vector graphics representations, virtual reality modeling (VRML), RasMol/Chime scripts and Java applets. A helix and bending analysis tool provides consistent information on about 750 DNA and RNA duplex structures. Access to metal-containing PDB entries is possible via the Periodic Table of Elements. Finally, general information on amino acids, cis -peptide bonds, structural elements in proteins, base pairs, nucleic acid model conformations and experimental methods for biopolymer structure determination is provided.

Protein-directed DNA structure II. Raman spectroscopy of a leucine zipper bZIP complex.

Mechanisms of transcription may involve protein-directed changes in DNA structure and DNA-directed changes in protein structure. We have employed Raman spectroscopy to characterize vibrational signatures associated with such induced molecular fitting for two classes of transcription factors-the basic leucine-zipper (bZIP) motif and the high-mobility-group (HMG) box-each with a DNA target site. Results for bZIP are described here; findings for the HMG-box are reported in the preceding paper in this issue [Benevides, J. M., Chan, G., Lu, X.-J., Olson, W. K., Weiss, M. A., and Thomas, G. J., Jr. (2000) Biochemistry 39, 537-547]. The yeast activator GCN4 provides a well-studied example of bZIP recognition, wherein B-DNA serves essentially as a template for protein folding. Analysis of Raman spectra of the 57-residue GCN4 bZIP domain, its AP-1 binding site, and their specific complex confirms a DNA-induced increase in alpha-helicity, attributable to folding of GCN4 basic arms with virtually no change in B-DNA structure, consistent with previous X-ray and NMR structure determinations. The absence of DNA perturbations in the bZIP model contrasts sharply with the HMG box, where DNA structure perturbations predominate. The bZIP and HMG-box models represent two opposing extremes in a range of induced fits identifiable by Raman spectroscopy. Previously characterized lambda repressor/operator complexes [Benevides, J. M., Weiss, M. A., and Thomas, G. J. (1994) J. Biol. Chem. 269, 10869-10878] occupy an intermediate position within this range. A comprehensive tabulation of Raman markers proposed as diagnostic of different protein/DNA recognition motifs is presented. The results are analyzed in terms of available DNA crystal structures (Nucleic Acid Database) to identify details of DNA conformation that correlate with specific Raman recognition markers.

Internal correlations in minor groove profiles of experimental and computed B-DNA conformations

It has been noticed that converged conformations of B-DNA oligomers obtained in MD calculations often have very small atom position rmsd values from the canonical B-DNA and all helical parameters close to the standard values, but their minor grooves tend to be somewhat narrower. This apparent bias disappears, however, when C5′ rather than phosphorus atoms are used for measuring the groove width. At the origin of this effect is the specific orientation of phosphate groups in the canonical B-DNA model which maximizes their separation across the minor groove. When measured by C5′ traces, minor groove profiles of experimental structures available in the Nucleic Acids Database show much less tendency to narrow below the canonical width. Correlation analysis reveals a high degree of correspondence in shapes of minor grooves of calculated and experimental single-crystal structures of B-DNA oligomers.

Protein-DNA interactions: a structural analysis

A detailed analysis of the DNA-binding sites of 26 proteins is presented using data from the Nucleic Acid Database (NDB) and the Protein Data Bank (PDB). Chemical and physical properties of the protein-DNA interface, such as polarity, size, shape, and packing, were analysed. The DNA-binding sites shared common features, comprising many discontinuous sequence segments forming hydrophilic surfaces capable of direct and water-mediated hydrogen bonds. These interface sites were compared to those of protein-protein binding sites, revealing them to be more polar, with many more intermolecular hydrogen bonds and buried water molecules than the protein-protein interface sites. By looking at the number and positioning of protein residue-DNA base interactions in a series of interaction footprints, three modes of DNA binding were identified (single-headed, double-headed and enveloping). Six of the eight enzymes in the data set bound in the enveloping mode, with the protein presenting a large interface area effectively wrapped around the DNA. A comparison of structural parameters of the DNA revealed that some values for the bound DNA (including twist, slide and roll) were intermediate of those observed for the unbound B-DNA and A-DNA. The distortion of bound DNA was evaluated by calculating a root-mean-square deviation on fitting to a canonical B-DNA structure. Major distortions were commonly caused by specific kinks in the DNA sequence, some resulting in the overall bending of the helix. The helix bending affected the dimensions of the grooves in the DNA, allowing the binding of protein elements that would otherwise be unable to make contact. From this structural analysis a preliminary set of rules that govern the bending of the DNA in protein-DNA complexes, are proposed.

The PIR-International Protein Sequence Database.

The Protein Information Resource (PIR; http://www-nbrf.georgetown. edu/pir/) supports research on molecular evolution, functional genomics, and computational biology by maintaining a comprehensive, non-redundant, well-organized and freely available protein sequence database. Since 1988 the database has been maintained collaboratively by PIR-International, an international association of data collection centers cooperating to develop this resource during a period of explosive growth in new sequence data and new computer technologies. The PIR Protein Sequence Database entries are classified into superfamilies, families and homology domains, for which sequence alignments are available. Full-scale family classification supports comparative genomics research, aids sequence annotation, assists database organization and improves database integrity. The PIR WWW server supports direct on-line sequence similarity searches, information retrieval, and knowledge discovery by providing the Protein Sequence Database and other supplementary databases. Sequence entries are extensively cross-referenced and hypertext-linked to major nucleic acid, literature, genome, structure, sequence alignment and family databases. The weekly release of the Protein Sequence Database can be accessed through the PIR Web site. The quarterly release of the database is freely available from our anonymous FTP server and is also available on CD-ROM with the accompanying ATLAS database search program.

The Nucleic Acid Database: A Resource for Nucleic Acid Science

The Nucleic Acid Database (NDB) distributes information about nucleic acid-containing structures. Here the information content of the database as well as the query capabilities are described. A summary of how the technology developed by this project has been used to develop other macromolecular databases is given.

Water molecules in DNA recognition II: a molecular dynamics view of the structure and hydration of the trp operator

The structure and hydration of the DNA duplex d-(AGCGTACTAGTACGCT)2corresponding to the trp operator fragment used in the crystal structure of the half site complex (PDB entry 1TRR) was studied by a 1.4 ns molecular dynamics simulation in water. The simulation, starting from a B-DNA conformation, used a non-bonded cutoff of 1.4 nm with a reaction field correction and resulted in a stable trajectory. The average DNA conformation obtained was closer to the ones found in the crystal structures of the complexes (PDB entries 1TRO and 1TRR) than to the crystal structure of unbound trp operator (Nucleic Acid Database entry BDJ061). The DNA hydration was characterized in terms of hydrogen bond percentages and corresponding residence times. The residence times of water molecules within 0.35 nm of the DNA non-exchangeable protons were calculated for comparison with NMR measurements of intermolecular water-DNA NOEs and nuclear magnetic relaxation dispersion measurements. No significant difference was found between major and minor groove hydration. The DNA donors and acceptors were hydrogen bonded to water molecules for 77(±19)% of the time on average. The average residence time of the hydrogen bonded water molecules was 11(±11) ps with a maximum of 223 ps. When all water molecules within NOE distance (0.35 nm) of non-exchangeable protons were considered, the average residence times increased to an average of 100(±4) ps and a maximum of 608 ps. These results agree with the experimental NMR results of Sunnerhagen et al. which did not show any evidence for water molecules bound with more than 1 ns residence time on the DNA surface. The exchange of hydration water from the DNA occurred in the major groove primarily through direct exchange with the bulk solvent, while access to and from the minor groove frequently proceeded via pathways involving ribose O3′ and O4′ and phosphate O2P oxygen atoms. The most common water diffusion pathways in the minor groove were perpendicular to the groove direction. In general, water molecules visited only a limited number of sites in the DNA grooves before exiting. The hydrogen bonding sites, where hydrogen bonds could be formed with donor and acceptor groups of the DNA, were filled with water molecules with an average B-factor value of 0.58 mn2. No special values were observed at any of the sites, where water molecules were observed both in the trp repressor/operator co-crystals and in the crystal structure of unbound DNA.

PQS: a protein quaternary structure file server

Part of the procedure outlined here uses a modification to the WHATIF program specially written for this purpose by R. Hooft at EMBL Heidelberg. J. Westbrook of the Nucleic Acid Data Base group at the University of Rutgers (http:// ndbserver.rutgers.edu) made available a machine-readable list of the quaternary states for all nucleic acid and protein–nucleic acid complexes held in the PDB that was used in the annotation and checking stage described here.

Striking Evolutionary Conservation of a cis-Element Related to Nuclear Receptor Target Sites and Present in TR2 Orphan Receptor Genes

A systematic scanning of nucleic acid databases for DNA elements made of combinations of RGGTCA nuclear receptor half sites, has revealed that identical 19 nucleotide-long motifs composed of two inverted RGGTCA sites with a spacing of 7 nucleotides (IR7), are present upstream of the regions coding for the human TR2 and of the sea urchin SpSHR2 orphan receptors. We have developed an experimental strategy based on PCR, to check if this IR7 could correspond to an unusually long cis-element, conserved along evolution and regulating the TR2 genes. We found that indeed IR7 is present in the 5′ untranslated region of TR2 genes from all species tested, includingXenopus,rainbow trout, zebrafish and mouse. The exact conservation throughout the animal kingdom of such a long, non repetitive and non coding genomic region, highly suggests that it should ensure important biological functions. In addition, this work has allowed the identification of a new, non coding, upstream exon in the mouse TR2 gene present in testicular TR2 mRNAs.

Analysis of expressed genes in ethylene-treated potato leaves using expressed sequence tags

To isolate useful and interesting plant genes in large quantities, random sequencing of cDNA clones from potato leaf library treated with ethylene was performed. Partial sequences of randomly selected 210 clones with the insert of longer than 500 base pair (bp) as well as poly (A) tail have been compared with sequences in GeneBank, EMBL and DDBJ nucleic acid databases and fostered 193 expressed sequence tags (ESTs). The 210 cDNA clones identified are related to various aspect of metabolic pathways such as glycolysis, amino acid synthesis, translation mechanism, ribosome synthesis, hormone response, stress response, regulation of gene expression, and signal transduction. Among the 193 ESTs, 12 ESTs (29 cDNA clones) appeared more than once and 181 ESTs appeared once regarded as a solitary group. Out of 210 clones, 29 clones (13.8%) have no similarity to the known nucleotide sequences and could serve as a potentially useful resource for plant molecular biology referring to particular genes. Nucleotide sequencing to generate more ESTs from ethylene-induced as well as non-induced potato leaf is in progress as well.

Increment of efficiency in the identification of noble genes by colony hybridization assay. (Screening of low redundant clones from human fetal cDNA library).

For the rapid identification of noble genes in a specific tissue by computer analysis from the cDNA sequences determined by single-pass cDNA sequencing, clone redundancy was one of the major obstacles. To facilitate the efficiency in identification of noble genes, it was necessary to reduce the number of clones to be sequenced by eliminating the redundant clones for a rapid analysis. In order to increase the probability of isolating noble sequences from the cDNA clones of human fetal liver tissue origin, colony hybridization assay was adopted and redundant clones were efficiently removed. Four cDNA clones highly redundant in the human fetal liver cDNA libraries including alpha-globin, gamma-globin, serum albumin and H19 RNA sequences were selected as the probes. Two hundreds and sixty two cDNA clones were randomly selected and tested with the probes for hybridization properties. The identity of each cDNA clone giving positive or negative signals in the hybridization assay was determined by DNA homology search with the nucleic acid databases. Among the 76 clones giving positive signals, 57 clones (75%) were found to be identical to the probe sequences and could be eliminated by colony hybridization assay before neucleotide sequencing.

ENHANCEMENT OF MOUSE CDC46 GENE EXPRESSION IN THE OSTEOBLAST BY LASER IRRADIATION

To identify any gene whose expression is enhanced by irradiation with low incident levels of laser energy, we previously constructed the cDNA library of the osteoblastic cell line MC3T3-E1, using a stepwise subtraction procedure between cells with and without laser irradiation. In the present study, we focused on one cDNA clone, designated as MCL-60. Our preliminary sequencing experiment indicated that this clone exhibited high homology with the gene coding for mouse CDC46 (cell division cycle 46 gene which is involved in the initiation of DNA replication of eukaryotic cells. To confirm the gene product, the DNA sequence of the cDNA clone MCL-60 was determined and assessed in the GenBank and EMBL nucleic acid databases. The transcription level of the cDNA clone MCL-60 wag examined by Northern blot analysis. The DNA sequence of clone MCL-60 exhibited 99.6% homology with that of CDC46 /MCM5 (minichromosome maintenance 5), and the deduced amino acid sequence exhibited 100% homology. Higher CDC46 mRNA levels were observed in laser irradiated cells compared to the levels in non-irradiated cells. Furthermore, DNA synthesis was increased by laser irradiation. These findings suggest that low incident levels of laser irradiation may play a principal role in stimulating proliferation of osteoblasts with enhancement of the gene expression of CDC46.

Use of a 3D structure data base for understanding sequence-dependent conformational aspects of DNA

The roll-twist-slide correlation in the DNA crystal structures that are collected in the Nucleic Acid Data Base is analyzed in order to obtain a general understanding of the effects of the nucleotide sequence on the 3D structure of a dinucleotide step. It is concluded that the differences between the pyrimidine bases and the purine bases in terms of their physical shapes are the major factors that determine the stereochemical characteristics of the steps through base to backbone and base to base interactions. The characteristics are further modulated by the differences between the A:T and G:C base-pairs, which can be explained by enhancement of the purine-pyrimidine asymmetry in the A:T base-pair.

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor.

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.

Helix bending as a factor in protein/DNA recognition

Normal vectors perpendicular to individual base pairs are a powerful tool for studying the bending behavior of B-DNA, both in the form of normal vector plots and in matrices that list angles between vectors for all possible base pair combinations. A new analysis program, FREEHELIX, has been written for this purpose, and applied to 86 examples of sequence-specific protein/DNA complexes whose coordinates are on deposit in the Nucleic Acid Data Base. Bends in this sample of 86 structures almost invariably follow from roll angles between adjacent base pairs; tilt makes no net contribution. Roll in a direction compressing the broad major groove is much more common than that which compresses the minor groove. Three distinct types of B-DNA bending are observed, each with a different molecular origin: (1) Localized kinking is produced by large roll at single steps or at two steps separated by one turn of helix. (2) Smooth, planar curvature is produced by positive and negative roll angles spaced a half-turn apart, with random side-to-side zigzag roll at intermediate points, rather than a tilt contribution that might have been expected theoretically. (3) Three-dimensional writhe results from significant roll angles at a continuous series of steps. Writhe need not change the overall direction of helix axis, if it is continued indefinitely or for an integral number of helical turns. A-DNA itself can be formally considered as possessing uniform, continuous writhe that yields no net helix bending. Smooth curvature is the most intricate deformation of the three, and is least common. Writhe is the simplest deformation and is most common; indeed, a low level of continuous writhe is the normal condition of an otherwise unbent B-DNA helix of general sequence. With one exception, every example of major kinking in this sample of 86 structures involves a pyrimidine–purine step: C–A/T–G, T–A, or C–G. Purine–purine steps, especially A–A, show the least tendency toward roll deformations. © 1998 John Wiley & Sons, Inc. Biopoly 44: 361–403, 1997

Helix bending as a factor in protein/DNA recognition.

Normal vectors perpendicular to individual base pairs are a powerful tool for studying the bending behavior of B-DNA, both in the form of normal vector plots and in matrices that list angles between vectors for all possible base pair combinations. A new analysis program, FREEHELIX, has been written for this purpose, and applied to 86 examples of sequence-specific protein/DNA complexes whose coordinates are on deposit in the Nucleic Acid Data Base. Bends in this sample of 86 structures almost invariably follow from roll angles between adjacent base pairs; tilt makes no net contribution. Roll in a direction compressing the broad major groove is much more common than that which compresses the minor groove. Three distinct types of B-DNA bending are observed, each with a different molecular origin: (1) Localized kinking is produced by large roll at single steps or at two steps separated by one turn of helix. (2) Smooth, planar curvature is produced by positive and negative roll angles spaced a half-turn apart, with random side-to-side zigzag roll at intermediate points, rather than a tilt contribution that might have been expected theoretically. (3) Three-dimensional writhe results from significant roll angles at a continuous series of steps. Writhe need not change the overall direction of helix axis, if it is continued indefinitely or for an integral number of helical turns. A-DNA itself can be formally considered as possessing uniform, continuous writhe that yields no net helix bending. Smooth curvature is the most intricate deformation of the three, and is least common. Writhe is the simplest deformation and is most common; indeed, a low level of continuous writhe is the normal condition of an otherwise unbent B-DNA helix of general sequence. With one exception, every example of major kinking in this sample of 86 structures involves a pyrimidine-purine step: C-A/T-G, T-A, or C-G. Purine-purine steps, especially A-A, show the least tendency toward roll deformations.