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PubMed2005

Structure and architecture of the maize genome.

Maize (Zea mays or corn) plays many varied and important roles in society. It is not only an important experimental model plant, but also a major livestock feed crop and a significant source of industrial products such as sweeteners and ethanol. In this study we report the systematic analysis of contiguous sequences of the maize genome. We selected 100 random regions averaging 144 kb in size, representing about 0.6% of the genome, and generated a high-quality dataset for sequence analysis. This sampling contains 330 annotated genes, 91% of which are supported by expressed sequence tag data from maize and other cereal species. Genes averaged 4 kb in size with five exons, although the largest was over 59 kb with 31 exons. Gene density varied over a wide range from 0.5 to 10.7 genes per 100 kb and genes did not appear to cluster significantly. The total repetitive element content we observed (66%) was slightly higher than previous whole-genome estimates (58%-63%) and consisted almost exclusively of retroelements. The vast majority of genes can be aligned to at least one sequence read derived from gene-enrichment procedures, but only about 30% are fully covered. Our results indicate that much of the increase in genome size of maize relative to rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) is attributable to an increase in number of both repetitive elements and genes.

Base Composition↗
PubMed1998

Functional analysis of the Escherichia coli genome using the sequence-to-structure-to-function paradigm: identification of proteins exhibiting the glutaredoxin/thioredoxin disulfide oxidoreductase activity.

The application of an automated method for the screening of protein activity based on the sequence-to-structure-to-function paradigm is presented for the complete Escherichia coli genome. First, the structure of the protein is identified from its sequence using a threading algorithm, which aligns the sequences to the best matching structure in a structural database and extends sequence analysis well beyond the limits of local sequence identity. Then, the active site is identified in the resulting sequence-to-structure alignment using a "fuzzy functional form" (FFF), a three-dimensional descriptor of the active site of a protein. Here, this sequence-to-structure-to-function concept is applied to analysis of the complete E. coli genome, i.e. all E. coli open reading frames (ORFs) are screened for the thiol-disulfide oxidoreductase activity of the glutaredoxin/thioredoxin protein family. We show that the method can identify the active sites in ten sequences that are known to or proposed to exhibit this activity. Furthermore, oxidoreductase activity is predicted in two other sequences that have not been identified previously. This method distinguishes protein pairs with similar active sites from proteins pairs that are just topological cousins, i.e. those having similar global folds, but not necessarily similar active sites. Thus, this method provides a novel approach for extraction of active site and functional information based on three-dimensional structures, rather than simple sequence analysis. Prediction of protein activity is fully automated and easily extendible to new functions. Finally, it is demonstrated here that the method can be applied to complete genome database analysis.

Algorithms↗
PubMed2004

FusionDB: a database for in-depth analysis of prokaryotic gene fusion events.

FusionDB (http://igs-server.cnrs-mrs.fr/FusionDB/) constitutes a resource dedicated to in-depth analysis of bacterial and archaeal gene fusion events. Such events can provide the 'Rosetta stone' in the search for potential protein-protein interactions, as well as metabolic and regulatory networks. However, the false positive rate of this approach may be quite high, prompting a detailed scrutiny of putative gene fusion events. FusionDB readily provides much of the information required for that task. Moreover, FusionDB extends the notion of gene fusion from that of a single gene to that of a family of genes by assembling pairs of genes from different genomes that belong to the same Cluster of Orthogonal Groups (COG). Multiple sequence alignments and phylogenetic tree reconstruction for the N- and C-terminal parts of these 'COG fusion' events are provided to distinguish single and multiple fusion events from cases of gene fission, pseudogenes and other false positives. Finally, gene fusion events with matches to known structures of heterodimers in the Protein Data Bank (PDB) are identified and may be visualized. FusionDB is fully searchable with access to sequence and alignment data at all levels. A number of different scores are provided to easily differentiate 'real' from 'questionable' cases, especially when larger database searches are performed. FusionDB is cross-linked with the 'Phylogenomic Display of Bacterial Genes' (PhydBac) online web server. Together, these servers provide the complete set of information required for in-depth analysis of non-homology-based gene function attribution.

Artificial Gene Fusion↗
PubMed2004

AGenDA: gene prediction by cross-species sequence comparison.

Automatic gene prediction is one of the major challenges in computational sequence analysis. Traditional approaches to gene finding rely on statistical models derived from previously known genes. By contrast, a new class of comparative methods relies on comparing genomic sequences from evolutionary related organisms to each other. These methods are based on the concept of phylogenetic footprinting: they exploit the fact that functionally important regions in genomic sequences are usually more conserved than non-functional regions. We created a WWW-based software program for homology-based gene prediction at BiBiServ (Bielefeld Bioinformatics Server). Our tool takes pairs of evolutionary related genomic sequences as input data, e.g. from human and mouse. The server runs CHAOS and DIALIGN to create an alignment of the input sequences and subsequently searches for conserved splicing signals and start/stop codons near regions of local sequence conservation. Genes are predicted based on local homology information and splice signals. The server returns predicted genes together with a graphical representation of the underlying alignment. The program is available at http://bibiserv.TechFak.Uni-Bielefeld.DE/agenda/.

Animals↗
PubMedDate not supplied

A simple method for aligning many protein sequences.

A simple extension of the Needleman and Wunsch algorithm for aligning pairs of protein sequences allows it to be used for the efficient generation of very large multiple-sequence alignments whose members are similar. This technique could have applications in a broad range of high-volume genomics projects.

Algorithms↗
PubMed2002

TLFAM--a new set of protein family databases.

PFAM is a popular and effective database of Hidden Markov Models (HMMs), which represent a wide range of protein families. Here, we introduce TLFAM as a more specific set of HMM databases. Analyses of bacterial genomes using TLFAM-Pro show better scores, E-values, and alignment lengths than those using the more generalized PFAM. Since PFAM will still find hits that TLFAM-Pro will not, we recommend that they be used jointly, rather than exclusively. This method provides the best features of both databases. This method has been extended to a number of other organism types, such as archaea, and the databases are freely available to interested researchers.

Databases, Protein↗
PubMed2003

MicroRNA targets in Drosophila.

BACKGROUND: The recent discoveries of microRNA (miRNA) genes and characterization of the first few target genes regulated by miRNAs in Caenorhabditis elegans and Drosophila melanogaster have set the stage for elucidation of a novel network of regulatory control. We present a computational method for whole-genome prediction of miRNA target genes. The method is validated using known examples. For each miRNA, target genes are selected on the basis of three properties: sequence complementarity using a position-weighted local alignment algorithm, free energies of RNA-RNA duplexes, and conservation of target sites in related genomes. Application to the D. melanogaster, Drosophila pseudoobscura and Anopheles gambiae genomes identifies several hundred target genes potentially regulated by one or more known miRNAs. RESULTS: These potential targets are rich in genes that are expressed at specific developmental stages and that are involved in cell fate specification, morphogenesis and the coordination of developmental processes, as well as genes that are active in the mature nervous system. High-ranking target genes are enriched in transcription factors two-fold and include genes already known to be under translational regulation. Our results reaffirm the thesis that miRNAs have an important role in establishing the complex spatial and temporal patterns of gene activity necessary for the orderly progression of development and suggest additional roles in the function of the mature organism. In addition the results point the way to directed experiments to determine miRNA functions. CONCLUSIONS: The emerging combinatorics of miRNA target sites in the 3' untranslated regions of messenger RNAs are reminiscent of transcriptional regulation in promoter regions of DNA, with both one-to-many and many-to-one relationships between regulator and target. Typically, more than one miRNA regulates one message, indicative of cooperative translational control. Conversely, one miRNA may have several target genes, reflecting target multiplicity. As a guide to focused experiments, we provide detailed online information about likely target genes and binding sites in their untranslated regions, organized by miRNA or by gene and ranked by likelihood of match. The target prediction algorithm is freely available and can be applied to whole genome sequences using identified miRNA sequences.

3' Untranslated Regions↗
PubMed2004

Defining relationships between the known members of the cytochrome P450 3A subfamily, including five putative chimpanzee members.

An analysis of the cytochrome P450 3A subfamily (CYP3A) was undertaken in order to define relationships across species among subfamily members. Some members were excluded due to incomplete sequences, while others were held in abeyance because of their almost complete homology. This is the first publication of five chimpanzee CYP3A genes-CYP3A4, CYP3A5, CYP3A7, CYP3A43, and CYP3A67. This project utilized two approaches for characterizing possible relationships-phylogenetic analysis and genomic structure. For the phylogenetic analysis, both nucleotide and amino acid sequences were aligned in silico using the CLUSTAL algorithm, and then visually inspected for accuracy. Three different computer software packages were utilized: MEGA 2.1, TREECON 1.3b, and PHYLIP 3.5. Multiple methods were used: neighbor-joining (NJ), minimum evolution (ME), maximum parsimony (MP), and maximum likelihood (ML). The resulting topologies were compared against each other to define the consensus topology. In addition, the chimpanzee, human, mouse, and rat genome databases were searched for intron/exon information pertaining to the included genes. Both methods suggest the same conclusion, defining orthologs is plausible between similar species (i.e., mouse and rat), but is less useful between species of different orders (i.e., primate and rodent) or classes (i.e., mammal and avian).

Animals↗
PubMed2003

Detecting putative orthologs.

We developed an algorithm that improves upon the common procedure of taking reciprocal best blast hits(rbh) in the identification of orthologs. The method-reciprocal smallest distance algorithm (rsd)-relies on global sequence alignment and maximum likelihood estimation of evolutionary distances to detect orthologs between two genomes. rsd finds many putative orthologs missed by rbh because it is less likely than rbh to be misled by the presence of a close paralog.

Algorithms↗
PubMed1994

Species-specific amplification of tRNA-derived short interspersed repetitive elements (SINEs) by retroposition: a process of parasitization of entire genomes during the evolution of salmonids.

Fourteen members of the Hpa I subfamilies of tRNA-derived SINEs in particular salmonid species were isolated from genomic libraries of chum salmon, kokanee, coho salmon, masu salmon, and steelhead. Alignment of the sequences of these 14 members, together with those of 4 members already published, 3 of which were previously demonstrated to have been amplified specifically in certain lineages, revealed the presence of five subfamilies with particular diagnostic nucleotides. The amplification of members of the same subfamily in different salmonid lineages and the amplification of members of different subfamilies in the same salmonid lineage suggest that multiple dispersed loci were responsible for amplification or, alternatively, that SINEs were transmitted horizontally between species. These two possibilities are not mutually exclusive. Our results also indicate that the Hpa I SINEs in salmonids behave like parasites. The amplification of these SINEs is ongoing and continues to shape the evolution of salmonid genomes.

Animals↗
PubMed2002

Computational analysis of core promoters in the Drosophila genome.

BACKGROUND: The core promoter, a region of about 100 base-pairs flanking the transcription start site (TSS), serves as the recognition site for the basal transcription apparatus. Drosophila TSSs have generally been mapped by individual experiments; the low number of accurately mapped TSSs has limited analysis of promoter sequence motifs and the training of computational prediction tools. RESULTS: We identified TSS candidates for about 2,000 Drosophila genes by aligning 5' expressed sequence tags (ESTs) from cap-trapped cDNA libraries to the genome, while applying stringent criteria concerning coverage and 5'-end distribution. Examination of the sequences flanking these TSSs revealed the presence of well-known core promoter motifs such as the TATA box, the initiator and the downstream promoter element (DPE). We also define, and assess the distribution of, several new motifs prevalent in core promoters, including what appears to be a variant DPE motif. Among the prevalent motifs is the DNA-replication-related element DRE, recently shown to be part of the recognition site for the TBP-related factor TRF2. Our TSS set was then used to retrain the computational promoter predictor McPromoter, allowing us to improve the recognition performance to over 50% sensitivity and 40% specificity. We compare these computational results to promoter prediction in vertebrates. CONCLUSIONS: There are relatively few recognizable binding sites for previously known general transcription factors in Drosophila core promoters. However, we identified several new motifs enriched in promoter regions. We were also able to significantly improve the performance of computational TSS prediction in Drosophila.

Animals↗
PubMed1995

Comparison of the 5'-flanking sequences of the human and bovine von Willebrand factor-encoding genes reveals alternation of highly homologous domains with species-specific Alu-type repeats.

von Willebrand factor (vWF), a multimeric glycoprotein important for hemostasis, is specifically synthesized in endothelial cells and in platelet precursors (megakaryocytes). Recent studies from two laboratories, including ours, were published regarding the cell-specific transcription of reporter genes controlled by the human (hu) vWF promoter in transfected bovine (bo) endothelial cells and cells of non-endothelial origins. In order to verify that the regulatory domains previously characterized in the 5' region of hu vWF are also present in bo vWF, we have sequenced 1.9 kb upstream from the cap site, plus five exons. The comparison of human and bovine exons two to five shows homology of 83% at the nucleotide (nt) level and 78% at the deduced amino-acid sequence level. The bovine and human exons one, which are non-coding and span 233 and 250 bp, respectively, are only 64% homologous. In the first exon, potentially involved in endothelial-cell-specific transcription, the binding site for factor Sp1 is present in bo vWF, whereas the GATA sequence is replaced by a GACA sequence. The sequence corresponding to the human basal promoter, located between nt -89 and +19, is well conserved with 82% homology. However, the human TAATTA sequence (at nt -32) considered to be a TATA box, is replaced by TCATTA, and the CCAAT element at nt -18 is replaced by CCTGT. Among domains involved in transcription, the negative regulatory domain located 5' from the core promoter is highly conserved. The bovine sequence upstream from the first intron can be aligned with the human sequence up to nt -656 which is located in a polymorphic poly(GT)18-26 sequence. At this site, the bovine DNA contains an insertion of 523 bp which corresponds to a bovine Alu-type art2 repeat of 331 bp flanked by bovine microsatellites. The art2 sequence is an Alu-type repeat in artiodactyls with at least 100,000 copies in the bovine genome. Upstream from this insertion, 368 bp of the bovine sequence can be aligned with the human counterpart up to a 9-bp element which flanks an human Alu repeat which is absent from the bovine DNA. Upstream of the human Alu insertion and a duplicate of the 9-bp element, the two sequences are again homologous.

Amino Acid Sequence↗
PubMed2006

Efficient q-gram filters for finding all epsilon-matches over a given length.

Fast and exact comparison of large genomic sequences remains a challenging task in biosequence analysis. We consider the problem of finding all epsilon-matches between two sequences, i.e., all local alignments over a given length with an error rate of at most epsilon. We study this problem theoretically, giving an efficient q-gram filter for solving it. Two applications of the filter are also discussed, in particular genomic sequence assembly and BLAST-like sequence comparison. Our results show that the method is 25 times faster than BLAST, while not being heuristic.

Algorithms↗
PubMed2004

Optimal spaced seeds for homologous coding regions.

Optimal spaced seeds were developed as a method to increase sensitivity of local alignment programs similar to BLASTN. Such seeds have been used before in the program PatternHunter, and have given improved sensitivity and running time relative to BLASTN in genome-genome comparison. We study the problem of computing optimal spaced seeds for detecting homologous coding regions in unannotated genomic sequences. By using well-chosen seeds, we are able to improve the sensitivity of coding sequence alignment over that of TBLASTX, while keeping runtime comparable to BLASTN. We identify good seeds by first giving effective hidden Markov models of conservation in alignments of homologous coding regions. We give an efficient algorithm to compute the optimal spaced seed when conservation patterns are generated by these models. Our results offer the hope of improved gene finding due to fewer missed exons in DNA/DNA comparison, and more effective homology search in general, and may have applications outside of bioinformatics.

Algorithms↗
PubMed2001

Biased distribution of inverted and direct Alus in the human genome: implications for insertion, exclusion, and genome stability.

Alu sequences, the most abundant class of large dispersed DNA repeats in human chromosomes, contribute to human genome dynamics. Recently we reported that long inverted repeats, including human Alus, can be strong initiators of genetic change in yeast. We proposed that the potential for interactions between adjacent, closely related Alus would influence their stability and this would be reflected in their distribution. We have undertaken an extensive computational analysis of all Alus (the database is at http://dir.niehs.nih.gov/ALU) to better understand their distribution and circumstances under which Alu sequences might affect genome stability. Alus separated by <650 bp were categorized according to orientation, length of regions sharing high sequence identity, distance between highly identical regions, and extent of sequence identity. Nearly 50% of all Alu pairs have long alignable regions (>275 bp), corresponding to nearly full-length Alus, regardless of orientation. There are dramatic differences in the distributions and character of Alu pairs with closely spaced, nearly identical regions. For Alu pairs that are directly repetitive, approximately 30% have highly identical regions separated by <20 bp, but only when the alignments correspond to near full-size or half-size Alus. The opposite is found for the distribution of inverted repeats: Alu pairs with aligned regions separated by <20 bp are rare. Furthermore, closely spaced direct and inverted Alus differ in their truncation patterns, suggesting differences in the mechanisms of insertion. At larger distances, the direct and inverted Alu pairs have similar distributions. We propose that sequence identity, orientation, and distance are important factors determining insertion of adjacent Alus, the frequency and spectrum of Alu-associated changes in the genome, and the contribution of Alu pairs to genome instability. Based on results in model systems and the present analysis, closely spaced inverted Alu pairs with long regions of alignment are likely at-risk motifs (ARMs) for genome instability.

Alu Elements↗
PubMed2006

Evaluation of affymetrix severe acute respiratory syndrome resequencing GeneChips in characterization of the genomes of two strains of coronavirus infecting humans.

Severe acute respiratory syndrome (SARS) was discovered during a recent global outbreak of atypical pneumonia. A number of immunologic and molecular studies of the clinical samples led to the conclusion that a novel coronavirus (SARS-CoV) was associated with the outbreak. Later, a SARS resequencing GeneChip was developed by Affymetrix to characterize the complete genome of SARS-CoV on a single GeneChip. The present study was carried out to evaluate the performance of SARS resequencing GeneChips. Two human SARS-CoV strains (CDC#200301157 and Urbani) were resequenced by the SARS GeneChips. Five overlapping PCR amplicons were generated for each strain and hybridized with these GeneChips. The successfully hybridized GeneChips generated nucleotide sequences of nearly complete genomes for the two SARS-CoV strains with an average call rate of 94.6%. Multiple alignments of nucleotide sequences obtained from SARS GeneChips and conventional sequencing revealed full concordance. Furthermore, the GeneChip-based analysis revealed no additional polymorphic sites. The results of this study suggest that GeneChip-based genome characterization is fast and reproducible. Thus, SARS resequencing GeneChips may be employed as an alternate tool to obtain genome sequences of SARS-CoV strains pathogenic for humans in order to further understand the transmission dynamics of these viruses.

Genome, Viral↗
PubMed2001

Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene.

A notable difficulty in annotating genomic sequence is identifying the correct start codon in a gene. An important such case has been found with KRIT1, the cerebral cavernous malformation type 1 (CCM1) gene. Analysis of human and mouse genomic sequence encompassing the region containing KRIT1/Krit1 using exon/gene-prediction and comparative alignment programs revealed putative exons upstream of the previously described first exon. These additional candidate exons show significant matches to mouse and human ESTs that are contiguous with and extend upstream from the previously designated 5' end of the KRIT1 cDNA sequence. RT-PCR and 5'RACE experiments confirm the presence of four additional upstream coding exons that encode an additional 207 amino acids. Importantly, a novel frameshift mutation in one of these newly identified KRIT1 exons has been found in a CCM1 family. These data establish the authentic KRIT1 amino acid sequence and suggest that the additional KRIT1 exons may harbor mutations in other CCM1 families. In addition, these results provide another example of the utility of rigorous computational and comparative sequence analysis for refining gene structure.

Alternative Splicing↗
PubMed2005

The phylogenetic distribution and evolutionary origins of endocannabinoid signalling.

The endocannabinoid signalling system in mammals comprises several molecular components, including cannabinoid receptors (e.g. CB1, CB2), putative endogenous ligands for these receptors [e.g. anandamide, 2-arachidonoylglycerol (2-AG)] and enzymes involved in the biosynthesis and inactivation of anandamide (e.g. NAPE-PLD, FAAH) and 2-AG (e.g. DAG lipase, MGL). In this review we examine the occurrence of these molecules in non-mammalian organisms (in particular, animals and plants) by surveying published data and by basic local alignment search tool (BLAST) analysis of the GenBank database and of genomic sequence data from several vertebrate and invertebrate species. We conclude that the ability of cells to synthesise molecules that are categorised as "endocannabinoids" in mammals is an evolutionarily ancient phenomenon that may date back to the unicellular common ancestor of animals and plants. However, exploitation of these molecules for intercellular signalling may have occurred independently in different lineages during the evolution of the eukaryotes. The CB1- and CB2-type receptors that mediate effects of endocannabinoids in mammals occur throughout the vertebrates, and an orthologue of vertebrate cannabinoid receptors was recently identified in the deuterostomian invertebrate Ciona intestinalis (CiCBR). However, orthologues of the vertebrate cannabinoid receptors are not found in protostomian invertebrates (e.g. Drosophila, Caenorhabditis elegans). Therefore, it is likely that a CB1/CB2-type cannabinoid receptor originated in a deuterostomian invertebrate. This phylogenetic information provides a basis for exploitation of selected non-mammalian organisms as model systems for research on endocannabinoid signalling.

Amidohydrolases↗