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PubMed2006

Advances in the Exon-Intron Database (EID).

Investigation of exon-intron gene structures is a non-trivial task due to enormous expansions of the eukaryotic genomes, great variety of gene forms, and the imperfectness in sequence data. A number of available informational systems on various gene characteristics complement each other and are indispensable for many genomic studies. Among them, the Exon-Intron Database (EID) is a good choice for large-scale computational examination of exon/intron structure and splicing. It has many internal filters that control for sequence quality, consistency of gene descriptions, accordance to standards, and possible errors. New innovations in EID are described. The collection of exons and introns has been extended beyond coding regions and current versions of EID contain data on untranslated regions of gene sequences as well. Intron-less genes are included as a special part of EID. For species with entirely sequenced genomes, species-specific databases have been generated. A novel Mammalian Orthologous Intron Database (MOID) has been introduced which includes the full set of introns that come from orthologous genes that have the same positions relative to the reading frames. Examples of statistical analyses of gene sequences using EID are provided. We present the latest data on our comparison of intron positions in 11,025 orthologous genes of human, mouse and rat, and find no convincing cases of intron gain. We discuss relevant data-quality issues of genomic databases. In particular, 5% of genes in genomic databases contain internal stop codons. This fact is due to a combination of biological reasons and also to errors in sequence annotations. The EID is freely available at www.meduohio.edu/bioinfo/eid/.

Base Sequence↗
PubMed2005

Gene and alternative splicing annotation with AIR.

Designing effective and accurate tools for identifying the functional and structural elements in a genome remains at the frontier of genome annotation owing to incompleteness and inaccuracy of the data, limitations in the computational models, and shifting paradigms in genomics, such as alternative splicing. We present a methodology for the automated annotation of genes and their alternatively spliced mRNA transcripts based on existing cDNA and protein sequence evidence from the same species or projected from a related species using syntenic mapping information. At the core of the method is the splice graph, a compact representation of a gene, its exons, introns, and alternatively spliced isoforms. The putative transcripts are enumerated from the graph and assigned confidence scores based on the strength of sequence evidence, and a subset of the high-scoring candidates are selected and promoted into the annotation. The method is highly selective, eliminating the unlikely candidates while retaining 98% of the high-quality mRNA evidence in well-formed transcripts, and produces annotation that is measurably more accurate than some evidence-based gene sets. The process is fast, accurate, and fully automated, and combines the traditionally distinct gene annotation and alternative splicing detection processes in a comprehensive and systematic way, thus considerably aiding in the ensuing manual curation efforts.

Alternative Splicing↗
PubMed2000

Is "junk" DNA mostly intron DNA?

Among higher eukaryotes, very little of the genome codes for protein. What is in the rest of the genome, or the "junk" DNA, that, in Homo sapiens, is estimated to be almost 97% of the genome? Is it possible that much of this "junk" is intron DNA? This is not a question that can be answered just by looking at the published data, even from the finished genomes. One cannot assume that there are no genes in a sequenced region, just because no genes were annotated. We introduce another approach to this problem, based on an analysis of the cDNA-to-genomic alignments, in all of the complete or nearly-complete genomes from the multicellular organisms. Our conclusion is that, in animals but not in plants, most of the "junk" is intron DNA.

Animals↗
PubMed2001

Kiddo, a new transposable element family closely associated with rice genes.

The promoter region of the rice ubiquitin2 (rubq2) gene was found to be polymorphic between japonica (T309) and indica (IR24) lines as the result of a 270-bp deletion in T309. A TTATA footprint in the T309 rubq2 promoter suggested that an excision event had occurred, and inspection of the 270-bp region present in IR24 revealed that it had all the characteristics of a miniature inverted repeat transposable element (MITE). Database searches showed that this element is a member of a new MITE family, which we have named Kiddo. Thirty-five complete Kiddo sequences were identified in existing rice genomic sequence databases. They could be arranged into four groups, within-group sequence identity was over 90%, with 65-75% identity between groups. The high sequence similarity within a group indicates that some Kiddo members were recently mobile and may still be active. An additional 24 decayed Kiddo sequences were detected. Interestingly, approximately 80% of 18 Kiddo members from annotated accessions lie within 530 bp of a coding sequence. That approximately 40% of Kiddo members present in genic regions reside in introns suggests that Kiddo transposition entails the use of both DNA and RNA intermediates, and may provide some insight into the origins of individual groups. DNA blot analysis showed that Kiddo is a rice-specific element, although one sequence with limited (72%) similarity to Kiddo group A was detected as a wheat EST. Kiddo family members may represent new molecular and phylogenetic markers, as well as representing valuable materials for studying the molecular mechanisms of MITE transposition.

Arabidopsis Proteins↗
PubMed2005

Closing in on the C. elegans ORFeome by cloning TWINSCAN predictions.

The genome of Caenorhabditis elegans was the first animal genome to be sequenced. Although considerable effort has been devoted to annotating it, the standard WormBase annotation contains thousands of predicted genes for which there is no cDNA or EST evidence. We hypothesized that a more complete experimental annotation could be obtained by creating a more accurate gene-prediction program and then amplifying and sequencing predicted genes. Our approach was to adapt the TWINSCAN gene prediction system to C. elegans and C. briggsae and to improve its splice site and intron-length models. The resulting system has 60% sensitivity and 58% specificity in exact prediction of open reading frames (ORFs), and hence, proteins-the best results we are aware of any multicellular organism. We then attempted to amplify, clone, and sequence 265 TWINSCAN-predicted ORFs that did not overlap WormBase gene annotations. The success rate was 55%, adding 146 genes that were completely absent from WormBase to the ORF clone collection (ORFeome). The same procedure had a 7% success rate on 90 Worm Base "predicted" genes that do not overlap TWINSCAN predictions. These results indicate that the accuracy of WormBase could be significantly increased by replacing its partially curated predicted genes with TWINSCAN predictions. The technology described in this study will continue to drive the C. elegans ORFeome toward completion and contribute to the annotation of the three Caenorhabditis species currently being sequenced. The results also suggest that this technology can significantly improve our knowledge of the "parts list" for even the best-studied model organisms.

Animals↗
PubMed2004

Application of comparative genomics to the analysis of vertebrate regulatory elements.

Gene regulatory regions (also known as 'cis-regulatory modules') in vertebrates are poorly understood and annotated by comparison with protein-coding sequences. The short and degenerate sequences of regulatory elements and their distribution over large intergenic and intronic regions pose a major challenge to genomics scientists. Comparative genomics can be used to identify putative regulatory regions, and to analyse regulatory regions into their constituent transcription factor binding sites. There is need for high throughput assay systems to analyse the function of predicted vertebrate gene regulatory regions.

Animals↗
PubMed2006

Prediction of genomic functional elements.

As the number of sequenced genomes increases, the ability to deduce genome function becomes increasingly salient. For many genome sequences, the only annotation that will be available for the foreseeable future will be based on computational predictions and comparisons with functional elements in related species. Here we discuss computational approaches for automated genome-wide annotation of functional elements in mammalian genomes. These include methods for ab initio and comparative gene-structure predictions. Gene features such as intron splice sites, 3' untranslated regions, promoters, and cis-regulatory elements are discussed, as is a novel method for predicting DNaseI hypersensitive sites. Recent methodologies for predicting noncoding RNA genes, including microRNA genes and their targets, are also reviewed.

Animals↗
PubMed2007

Comparative genomic analysis of prion genes.

BACKGROUND: The homologues of human disease genes are expected to contribute to better understanding of physiological and pathogenic processes. We made use of the present availability of vertebrate genomic sequences, and we have conducted the most comprehensive comparative genomic analysis of the prion protein gene PRNP and its homologues, shadow of prion protein gene SPRN and doppel gene PRND, and prion testis-specific gene PRNT so far. RESULTS: While the SPRN and PRNP homologues are present in all vertebrates, PRND is known in tetrapods, and PRNT is present in primates. PRNT could be viewed as a TE-associated gene. Using human as the base sequence for genomic sequence comparisons (VISTA), we annotated numerous potential cis-elements. The conserved regions in SPRNs harbour the potential Sp1 sites in promoters (mammals, birds), C-rich intron splicing enhancers and PTB intron splicing silencers in introns (mammals, birds), and hsa-miR-34a sites in 3'-UTRs (eutherians). We showed the conserved PRNP upstream regions, which may be potential enhancers or silencers (primates, dog). In the PRNP 3'-UTRs, there are conserved cytoplasmic polyadenylation element sites (mammals, birds). The PRND core promoters include highly conserved CCAAT, CArG and TATA boxes (mammals). We deduced 42 new protein primary structures, and performed the first phylogenetic analysis of all vertebrate prion genes. Using the protein alignment which included 122 sequences, we constructed the neighbour-joining tree which showed four major clusters, including shadoos, shadoo2s and prion protein-likes (cluster 1), fish prion proteins (cluster 2), tetrapode prion proteins (cluster 3) and doppels (cluster 4). We showed that the entire prion protein conformationally plastic region is well conserved between eutherian prion proteins and shadoos (18-25% identity and 28-34% similarity), and there could be a potential structural compatibility between shadoos and the left-handed parallel beta-helical fold. CONCLUSION: It is likely that the conserved genomic elements identified in this analysis represent bona fide cis-elements. However, this idea needs to be confirmed by functional assays in transgenic systems.

3' Untranslated Regions↗
PubMed2004

'Frankenstein genes', or the Mad Magazine version of the human pseudogenome.

Annotation of the human genome is inching forward. Seven human chromosomes have now been fully annotated, covering 17 per cent of the genome, and at least one chromosome has been re-annotated. The enormity of the task forces a dependence on automated tools for detecting and assembling the genes, followed by hand curation to correct errors and polish the gene models. The accuracy of gene prediction algorithms is very good for internal exons from intact genes, but these programs do peculiar and exasperating things to pseudogenes. These programs can actually resurrect pseudogenes from the dead, making them into viable gene models for intact proteins, albeit science-fictional proteins. This process is demonstrated for four human pseudogenes from the cytochrome P450 family and one putatively functional P450 gene, CYP2U1, having a non-consensus intron boundary. These examples are offered as a call-to-arms to improve pseudogene prediction as an art in itself, and not as a by-product of gene annotation. Failure to do so will flood the databases with thousands of false-positive predictions. Indeed, they are already there.

Amino Acid Sequence↗
PubMed2004

Predicting polymerase II core promoters by cooperating transcription factor binding sites in eukaryotic genes.

Several discriminate functions for predicting core promoters that based on the potential cooperation between transcription factor binding sites (TFBSs) are discussed. It is demonstrated that the promoter predicting accuracy is improved when the cooperation among TFBSs is taken into consideration. The core promoter region of a newly discovered gene CKLFSF1 is predicted to locate more than 1.5 kb far away from the 5' end of the transcript and in the last intron of its upstream gene, which is experimentally confirmed later. The core promoters of 3402 human RefSeq sequences, obtained by extending the mRNAs in human genome sequences, are predicted by our algorithm, and there are about 60% of the predicted core promoters locating within the +/- 500 bp region relative to the annotated transcription start site.

Algorithms↗
PubMed2003

ASAP: the Alternative Splicing Annotation Project.

Recently, genomics analyses have demonstrated that alternative splicing is widespread in mammalian genomes (30-60% of genes reported to have multiple isoforms), and may be one of their most important mechanisms of functional regulation. However, by comparison with other genomics data such as genome annotation, SNPs, or gene expression, there exists relatively little database infrastructure for the study of alternative splicing. We have constructed an online database ASAP (the Alternative Splicing Annotation Project) for biologists to access and mine the enormous wealth of alternative splicing information coming from genomics and proteomics. ASAP is based on genome-wide analyses of alternative splicing in human (30 793 alternative splice relationships found) from detailed alignment of expressed sequences onto the genomic sequence. ASAP provides precise gene exon-intron structure, alternative splicing, tissue specificity of alternative splice forms, and protein isoform sequences resulting from alternative splicing. Moreover, it can help biologists design probe sequences for distinguishing specific mRNA isoforms. ASAP is intended to be a community resource for collaborative annotation of alternative splice forms, their regulation, and biological functions. The URL for ASAP is http://www.bioinformatics.ucla.edu/ASAP.

Alternative Splicing↗
PubMed2000

Analysis of canonical and non-canonical splice sites in mammalian genomes.

A set of 43 337 splice junction pairs was extracted from mammalian GenBank annotated genes. Expressed sequence tag (EST) sequences support 22 489 of them. Of these, 98.71% contain canonical dinucleotides GT and AG for donor and acceptor sites, respectively; 0.56% hold non-canonical GC-AG splice site pairs; and the remaining 0.73% occurs in a lot of small groups (with a maximum size of 0.05%). Studying these groups we observe that many of them contain splicing dinucleotides shifted from the annotated splice junction by one position. After close examination of such cases we present a new classification consisting of only eight observed types of splice site pairs (out of 256 a priori possible combinations). EST alignments allow us to verify the exonic part of the splice sites, but many non-canonical cases may be due to intron sequencing errors. This idea is given substantial support when we compare the sequences of human genes having non-canonical splice sites deposited in GenBank by high throughput genome sequencing projects (HTG). A high proportion (156 out of 171) of the human non-canonical and EST-supported splice site sequences had a clear match in the human HTG. They can be classified after corrections as: 79 GC-AG pairs (of which one was an error that corrected to GC-AG), 61 errors that were corrected to GT-AG canonical pairs, six AT-AC pairs (of which two were errors that corrected to AT-AC), one case was produced from non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two cases left of supported non-canonical splice sites. If we assume that approximately the same situation is true for the whole set of annotated mammalian non-canonical splice sites, then the 99.24% of splice site pairs should be GT-AG, 0.69% GC-AG, 0.05% AT-AC and finally only 0.02% could consist of other types of non-canonical splice sites. We analyze several characteristics of EST-verified splice sites and build weight matrices for the major groups, which can be incorporated into gene prediction programs. We also present a set of EST-verified canonical splice sites larger by two orders of magnitude than the current one (22 199 entries versus approximately 600) and finally, a set of 290 EST-supported non-canonical splice sites. Both sets should be significant for future investigations of the splicing mechanism.

Animals↗
PubMed2005

FeatureExtract--extraction of sequence annotation made easy.

Work on a large number of biological problems benefits tremendously from having an easy way to access the annotation of DNA sequence features, such as intron/exon structure, the contents of promoter regions and the location of other genes in upsteam and downstream regions. For example, taking the placement of introns within a gene into account can help in a phylogenetic analysis of homologous genes. Designing experiments for investigating UTR regions using PCR or DNA microarrays require knowledge of known elements in UTR regions and the positions and strandness of other genes nearby on the chromosome. A wealth of such information is already known and documented in databases such as GenBank and the NCBI Human Genome builds. However, it usually requires significant bioinformatics skills and intimate knowledge of the data format to access this information. Presented here is a highly flexible and easy-to-use tool for extracting feature annotation from GenBank entries. The tool is also useful for extracting datasets corresponding to a particular feature (e.g. promoters). Most importantly, the output data format is highly consistent, easy to handle for the user and easy to parse computationally. The FeatureExtract web server is freely available for both academic and commercial use at http://www.cbs.dtu.dk/services/FeatureExtract/.

Chromosomes↗
PubMed2000

Gene structure prediction by spliced alignment of genomic DNA with protein sequences: increased accuracy by differential splice site scoring.

Gene identification in genomic DNA from eukaryotes is complicated by the vast combinatorial possibilities of potential exon assemblies. If the gene encodes a protein that is closely related to known proteins, gene identification is aided by matching similarity of potential translation products to those target proteins. The genomic DNA and protein sequences can be aligned directly by scoring the implied residues of in-frame nucleotide triplets against the protein residues in conventional ways, while allowing for long gaps in the alignment corresponding to introns in the genomic DNA. We describe a novel method for such spliced alignment. The method derives an optimal alignment based on scoring for both sequence similarity of the predicted gene product to the protein sequence and intrinsic splice site strength of the predicted introns. Application of the method to a representative set of 50 known genes from Arabidopsis thaliana showed significant improvement in prediction accuracy compared to previous spliced alignment methods. The method is also more accurate than ab initio gene prediction methods, provided sufficiently close target proteins are available. In view of the fast growth of public sequence repositories, we argue that close targets will be available for the majority of novel genes, making spliced alignment an excellent practical tool for high-throughput automated genome annotation.

Algorithms↗
PubMed2006

Conservation patterns in different functional sequence categories of divergent Drosophila species.

We have explored the distributions of fully conserved ungapped blocks in genome-wide pair-wise alignments of recently completed species of Drosophila: D. melanogaster, D. yakuba, D. ananassae, D. pseudoobscura, D. virilis, and D. mojavensis. Based on these distributions we have found that nearly every functional sequence category possesses its own distinctive conservation pattern, sometimes independent of the overall sequence conservation level. In the coding and regulatory regions, the ungapped blocks were longer than in introns, UTRs, and nonfunctional sequences. At the same time, the blocks in the coding regions carried a 3N + 2 signature characteristic of synonymous substitutions in the third-codon position. Larger block sizes in transcription regulatory regions can be explained by the presence of conserved arrays of binding sites for transcription factors. We also have shown that the longest ungapped blocks, or "ultraconserved" sequences, are associated with specific gene groups, including those encoding ion channels and components of the cytoskeleton. We discuss how restraining conservation patterns may help in mapping functional sequence categories and improve genome annotation.

Animals↗
PubMed2004

Thermodynamic modeling of donor splice site recognition in pre-mRNA.

When eukaryotic genes are edited by the spliceosome, the first step in intron recognition is the binding of a U1 small nuclear RNA with the donor ( 5(') ) splice site. We model this interaction thermodynamically to identify splice sites. Applied to a set of 65 annotated genes, our "finding with binding" method achieves a significant separation between real and false sites. Analyzing binding patterns allows us to discard a large number of decoy sites. Our results improve statistics-based methods for donor site recognition, demonstrating the promise of physical modeling to find functional elements in the genome.

Base Pairing↗
PubMed1996

LALNVIEW: a graphical viewer for pairwise sequence alignments.

LALNVIEW is a graphical program for visualising local alignments between two sequences (protein or nucleic acids). Sequences are represented by coloured rectangles to give an overall picture of their similarities. LALNVIEW can display sequence features (exon, intron, active site, domain, propeptide, etc.) along with the alignment. When using LALNVIEW through our Web servers, sequence features are automatically extracted from database annotations (SWISS-PROT, GenBank, EMBL or HOVERGEN) and displayed with the alignment. LALNVIEW is a useful tool for analysing pairwise sequence alignments and for making the link between sequence homology and what is known about the structure or function of sequences. LALNVIEW executables for UNIX, Macintosh and PC computers are freely available from our server (http:// expasy.hcuge.ch/sprot/lalnview.html).

Acyltransferases↗
PubMed2004

Recent advances in gene structure prediction.

De novo gene predictors are programs that predict the exon-intron structures of genes using the sequences of one or more genomes as their only input. In the past two years, dual-genome de novo predictors, which exploit local rates and patterns of mutation inferred from alignments between two genomes, have led to significant improvements in accuracy. Systems that exploit more than two genomes simultaneously have only recently begun to appear and are not yet competitive on practical tasks, but offer the greatest hope for near-term improvements. Dual-genome de novo prediction for compact eukaryotic genomes such as those of Arabidopsis thaliana and Caenorhabditis elegans is already quite accurate. Although mammalian gene prediction lags behind in accuracy, it is yielding ever more useful results. Coupled with significant improvements in pseudogene detection methods, which have eliminated many false positives, we have reached the point where de novo gene predictions are being used as hypotheses to drive experimental annotation via systematic RT-PCR and sequencing.

Animals↗