EST Evidence Research Articles

Genome-wide identification and evolution of TC1/Mariner in the silkworm (Bombyx mori) genome.

TC1/Mariner transposons belong to class II transposable elements (TEs) that use DNA-mediated "cut and paste" mechanism to transpose, and they have been identified in almost all organisms. Although silkworm (Bombyx mori) has a large amount of TC1/Mariner elements, the genome wide information of this superfamily in the silkworm is unknown. In this study, we have identified 2670 TC1/Mariner (Bmmar) elements in the silkworm genome. All the TEs were classified into 22 families by means of fgclust, a tool of repetitive sequence classification, seven of which was first reported in this study. Phylogenetic and structure analyses based on the catalytic domain (DDxD/E) of transposase sequences indicated that all members of TC1/Mariner were grouped into five subgroups: Mariner, Tc1, maT, DD40D and DD41D/E. Of these five subgroups, maT rather than Mariner possessed most members of TC1/Mariner (51.23%) in the silkworm genome. In particular, phylogenetic analysis and structure analysis revealed that Bmmar15 (DD40D) formed a new basal subgroup of TC1/Mariner element in insects, which was referred to as bmori. Furthermore, we concluded that DD40D appeared to intermediate between mariner and Tc1. Finally, we estimated the insertion time for each copy of TC1/Mariner in the silkworm and found that most of members were dramatically amplified during a period from 0 to 1 mya. Moreover, the detailed functional data analysis showed that Bmmar1, Bmmar6 and Bmmar9 had EST evidence and intact transposases. These implied that TC1/Mariner might have potential transpositional activity. In conclusion, this study provides some new insights into the landscape, origin and evolution of TC1/Mariner in the insect genomes.

Genome-wide analysis of cytochrome P450 monooxygenase genes in the tobacco

Plant cytochrome P450 monooxygenases (CYP) constitute a large superfamily of heme-thiolate proteins, which are involved in a wide range of metabolic pathways. In this study, comparative genomic approaches were used to analyze tobacco CYP genes and their expression patterns. Based on analysis of the tobacco genomic DNA sequences that are currently available, 263 P450 genes that belong to 44 distinct clans were identified. EST evidence from 173 of the CYPs suggested that these genes are transcribed. Sequence features and secondary structures of the tobacco P450 genes were further analyzed through comparison with known P450 proteins. The expression profiles of 73 P450 genes were subsequently investigated by analyses of tobacco microarray data and RT-PCR. The results showed a variety of expression patterns of these genes in different tissues with a number of genes expressed in a tissue-specific manner. This study has set a foundation for further studies on functions of P450 genes in tobacco.

AmoebaDB and MicrosporidiaDB: functional genomic resources for Amoebozoa and Microsporidia species

AmoebaDB (http://AmoebaDB.org) and MicrosporidiaDB (http://MicrosporidiaDB.org) are new functional genomic databases serving the amoebozoa and microsporidia research communities, respectively. AmoebaDB contains the genomes of three Entamoeba species (E. dispar, E. invadens and E. histolityca) and microarray expression data for E. histolytica. MicrosporidiaDB contains the genomes of Encephalitozoon cuniculi, E. intestinalis and E. bieneusi. The databases belong to the National Institute of Allergy and Infectious Diseases (NIAID) funded EuPathDB (http://EuPathDB.org) Bioinformatics Resource Center family of integrated databases and assume the same architectural and graphical design as other EuPathDB resources such as PlasmoDB and TriTrypDB. Importantly they utilize the graphical strategy builder that affords a database user the ability to ask complex multi-data-type questions with relative ease and versatility. Genomic scale data can be queried based on BLAST searches, annotation keywords and gene ID searches, GO terms, sequence motifs, protein characteristics, phylogenetic relationships and functional data such as transcript (microarray and EST evidence) and protein expression data. Search strategies can be saved within a user’s profile for future retrieval and may also be shared with other researchers using a unique strategy web address.

Structural and transcriptional analysis of plant genes encoding the bifunctional lysine ketoglutarate reductase saccharopine dehydrogenase enzyme

BackgroundAmong the dietary essential amino acids, the most severely limiting in the cereals is lysine. Since cereals make up half of the human diet, lysine limitation has quality/nutritional consequences. The breakdown of lysine is controlled mainly by the catabolic bifunctional enzyme lysine ketoglutarate reductase - saccharopine dehydrogenase (LKR/SDH). The LKR/SDH gene has been reported to produce transcripts for the bifunctional enzyme and separate monofunctional transcripts. In addition to lysine metabolism, this gene has been implicated in a number of metabolic and developmental pathways, which along with its production of multiple transcript types and complex exon/intron structure suggest an important node in plant metabolism. Understanding more about the LKR/SDH gene is thus interesting both from applied standpoint and for basic plant metabolism.ResultsThe current report describes a wheat genomic fragment containing an LKR/SDH gene and adjacent genes. The wheat LKR/SDH genomic segment was found to originate from the A-genome of wheat, and EST analysis indicates all three LKR/SDH genes in hexaploid wheat are transcriptionally active. A comparison of a set of plant LKR/SDH genes suggests regions of greater sequence conservation likely related to critical enzymatic functions and metabolic controls. Although most plants contain only a single LKR/SDH gene per genome, poplar contains at least two functional bifunctional genes in addition to a monofunctional LKR gene. Analysis of ESTs finds evidence for monofunctional LKR transcripts in switchgrass, and monofunctional SDH transcripts in wheat, Brachypodium, and poplar.ConclusionsThe analysis of a wheat LKR/SDH gene and comparative structural and functional analyses among available plant genes provides new information on this important gene. Both the structure of the LKR/SDH gene and the immediately adjacent genes show lineage-specific differences between monocots and dicots, and findings suggest variation in activity of LKR/SDH genes among plants. Although most plant genomes seem to contain a single conserved LKR/SDH gene per genome, poplar possesses multiple contiguous genes. A preponderance of SDH transcripts suggests the LKR region may be more rate-limiting. Only switchgrass has EST evidence for LKR monofunctional transcripts. Evidence for monofunctional SDH transcripts shows a novel intron in wheat, Brachypodium, and poplar.

Deep proteogenomics; high throughput gene validation by multidimensional liquid chromatography and mass spectrometry of proteins from the fungal wheat pathogen Stagonospora nodorum

BackgroundStagonospora nodorum, a fungal ascomycete in the class dothideomycetes, is a damaging pathogen of wheat. It is a model for necrotrophic fungi that cause necrotic symptoms via the interaction of multiple effector proteins with cultivar-specific receptors. A draft genome sequence and annotation was published in 2007. A second-pass gene prediction using a training set of 795 fully EST-supported genes predicted a total of 10762 version 2 nuclear-encoded genes, with an additional 5354 less reliable version 1 genes also retained.ResultsIn this study, we subjected soluble mycelial proteins to proteolysis followed by 2D LC MALDI-MS/MS. Comparison of the detected peptides with the gene models validated 2134 genes. 62% of these genes (1324) were not supported by prior EST evidence. Of the 2134 validated genes, all but 188 were version 2 annotations. Statistical analysis of the validated gene models revealed a preponderance of cytoplasmic and nuclear localised proteins, and proteins with intracellular-associated GO terms. These statistical associations are consistent with the source of the peptides used in the study. Comparison with a 6-frame translation of the S. nodorum genome assembly confirmed 905 existing gene annotations (including 119 not previously confirmed) and provided evidence supporting 144 genes with coding exon frameshift modifications, 604 genes with extensions of coding exons into annotated introns or untranslated regions (UTRs), 3 new gene annotations which were supported by tblastn to NR, and 44 potential new genes residing within un-assembled regions of the genome.ConclusionWe conclude that 2D LC MALDI-MS/MS is a powerful, rapid and economical tool to aid in the annotation of fungal genomic assemblies.

Manual annotation of the mouse genome: HAVANA and EUCOMM

AbstractThe European Conditional Mouse Mutagenesis (EUCOMM) project aims to establish a mutant resource containing up to 13,000 conditional mouse mutations in C57BL/6N embryonic stem cells. As part of this resource, up to 8,000 targeted conditional mutations will be generated for genes that can not be readily trapped by random gene trapping methods. Non-symmetrical critical exons are identified as part of the targeting approach, which when deleted produce a knockout (KO) transcript that is susceptible to nonsensemediated decay (NMD). The success of this approach is dependent on detailed and accurate genome annotation, which is provided by the Human and Vertebrate Analysis and Annotation (HAVANA) group at the Wellcome Trust Sanger Institute. Based on a combination of EST, cDNA and protein evidence, HAVANA produces high quality manual annotation of gene structures, that includes alternative splice variants, polyadenylation features, pseudogenes and gene family clusters. Using this annotation we are able to identify suitable exons for targeting, whilst minimising potential disruption to adjacent genomic features. Annotation of both gene and KO transcripts can be viewed through the Vertebrate Genome Annotation (VEGA) browser ("http://vega.sanger.ac.uk":http://vega.sanger.ac.uk).More information on the EUCOMM resource and the targeted genes is available at "http://www.eucomm.org":http://www.eucomm.org. Targeting vector design information can also be viewed through the Ensembl genome browser ("http://www.ensembl.org":http://www.ensembl.org) as DAS resources ("http://das.sanger.ac.uk/das/KO_designs":http://das.sanger.ac.uk/das/KO_designs ; "http://das.sanger.ac.uk/das/KO_vectors":http://das.sanger.ac.uk/das/KO_vectors). In addition to the EUCOMM project, HAVANA also contributes towards the mouse Consensus Coding DNA Sequence (CCDS) project and is involved in collaborations with RefSeq and MGI.

Manual annotation of the mouse genome: HAVANA and EUCOMM

The European Conditional Mouse Mutagenesis (EUCOMM) project aims to establish a mutant resource containing up to 13,000 conditional mouse mutations in C57BL/6N embryonic stem cells. As part of this resource, up to 8,000 targeted conditional mutations will be generated for genes that can not be readily trapped by random gene trapping methods. Non-symmetrical critical exons are identified as part of the targeting approach, which when deleted produce a knockout (KO) transcript that is susceptible to nonsensemediated decay (NMD). The success of this approach is dependent on detailed and accurate genome annotation, which is provided by the Human and Vertebrate Analysis and Annotation (HAVANA) group at the Wellcome Trust Sanger Institute. Based on a combination of EST, cDNA and protein evidence, HAVANA produces high quality manual annotation of gene structures, that includes alternative splice variants, polyadenylation features, pseudogenes and gene family clusters. Using this annotation we are able to identify suitable exons for targeting, whilst minimising potential disruption to adjacent genomic features. Annotation of both gene and KO transcripts can be viewed through the Vertebrate Genome Annotation (VEGA) browser ("http://vega.sanger.ac.uk":http://vega.sanger.ac.uk).More information on the EUCOMM resource and the targeted genes is available at "http://www.eucomm.org":http://www.eucomm.org. Targeting vector design information can also be viewed through the Ensembl genome browser ("http://www.ensembl.org":http://www.ensembl.org) as DAS resources ("http://das.sanger.ac.uk/das/KO_designs":http://das.sanger.ac.uk/das/KO_designs ; "http://das.sanger.ac.uk/das/KO_vectors":http://das.sanger.ac.uk/das/KO_vectors). In addition to the EUCOMM project, HAVANA also contributes towards the mouse Consensus Coding DNA Sequence (CCDS) project and is involved in collaborations with RefSeq and MGI.

Detailed characterization of the mouse embryonic stem cell transcriptome reveals novel genes and intergenic splicing associated with pluripotency

BackgroundTranscriptional control of embryonic stem (ES) cell pluripotency has been a subject of intense study. Transcriptional regulators including Oct4 (Oct3/4 index), Sox2 and Nanog are fundamental for maintaining the undifferentiated state. However, the ES cell transcriptome is not limited to their targets, and exhibits considerable complexity when assayed with microarray, MPSS, cDNA/EST sequencing, and SAGE technologies. To identify novel genes associated with pluripotency, we globally searched for ES transcripts not corresponding to known genes, validated their sequences, determined their expression profiles, and employed RNAi to test their function.ResultsGene Identification Signature (GIS) analysis, a SAGE derivative distinguished by paired 5' and 3' transcript end tags, identified 153 candidate novel transcriptional units (TUs) distinct from known genes in a mouse E14 ES mRNA library. We focused on 16 TUs free of artefacts and mapping discrepancies, five of which were validated by RTPCR product sequencing. Two of the TUs were revealed by annotation to represent novel protein-coding genes: a PRY-domain cluster member and a KRAB-domain zinc finger. The other three TUs represented intergenic splicing events involving adjacent, functionally unrelated protein-coding genes transcribed in the same orientation, with one event potentially encoding a fusion protein containing domains from both component genes (Clk2 and Scamp3). Expression profiling using embryonic samples and adult tissue panels confirmed that three of the TUs were unique to or most highly expressed in ES cells. Expression levels of all five TUs dropped dramatically during three distinct chemically induced differentiation treatments of ES cells in culture. However, siRNA knockdowns of the TUs did not alter mRNA levels of pluripotency or differentiation markers, and did not affect cell morphology.ConclusionTranscriptome libraries retain considerable potential for novel gene discovery despite massive recent cDNA and EST sequencing efforts; cDNA and EST evidence for these ES cell TUs had been limited or absent. RTPCR and full-length sequencing remain essential in resolving the bottleneck between numerous candidate novel transcripts inferred from high-throughput sequencing and the small fraction that can be validated. RNAi results indicate that, despite their strong association with pluripotency, these five transcriptomic novelties may not be required for maintaining it.

Refined annotation and assembly of the Tetrahymena thermophila genome sequence through EST analysis, comparative genomic hybridization, and targeted gap closure

BackgroundTetrahymena thermophila, a widely studied model for cellular and molecular biology, is a binucleated single-celled organism with a germline micronucleus (MIC) and somatic macronucleus (MAC). The recent draft MAC genome assembly revealed low sequence repetitiveness, a result of the epigenetic removal of invasive DNA elements found only in the MIC genome. Such low repetitiveness makes complete closure of the MAC genome a feasible goal, which to achieve would require standard closure methods as well as removal of minor MIC contamination of the MAC genome assembly. Highly accurate preliminary annotation of Tetrahymena's coding potential was hindered by the lack of both comparative genomic sequence information from close relatives and significant amounts of cDNA evidence, thus limiting the value of the genomic information and also leaving unanswered certain questions, such as the frequency of alternative splicing.ResultsWe addressed the problem of MIC contamination using comparative genomic hybridization with purified MIC and MAC DNA probes against a whole genome oligonucleotide microarray, allowing the identification of 763 genome scaffolds likely to contain MIC-limited DNA sequences. We also employed standard genome closure methods to essentially finish over 60% of the MAC genome. For the improvement of annotation, we have sequenced and analyzed over 60,000 verified EST reads from a variety of cellular growth and development conditions. Using this EST evidence, a combination of automated and manual reannotation efforts led to updates that affect 16% of the current protein-coding gene models. By comparing EST abundance, many genes showing apparent differential expression between these conditions were identified. Rare instances of alternative splicing and uses of the non-standard amino acid selenocysteine were also identified.ConclusionWe report here significant progress in genome closure and reannotation of Tetrahymena thermophila. Our experience to date suggests that complete closure of the MAC genome is attainable. Using the new EST evidence, automated and manual curation has resulted in substantial improvements to the over 24,000 gene models, which will be valuable to researchers studying this model organism as well as for comparative genomics purposes.

Tetrahymena Metallothioneins Fall into Two Discrete Subfamilies

BackgroundMetallothioneins are ubiquitous small, cysteine-rich, multifunctional proteins which can bind heavy metals.Methodology/Principal FindingsWe report the results of phylogenetic and gene expression analyses that include two new Tetrahymena thermophila metallothionein genes (MTT3 and MTT5). Sequence alignments of all known Tetrahymena metallothioneins have allowed us to rationalize the structure of these proteins. We now formally subdivide the known metallothioneins from the ciliate genus Tetrahymena into two well defined subfamilies, 7a and 7b, based on phylogenetic analysis, on the pattern of clustering of Cys residues, and on the pattern of inducibility by the heavy metals Cd and Cu. Sequence alignment also reveals a remarkably regular, conserved and hierarchical modular structure of all five subfamily 7a MTs, which include MTT3 and MTT5. The former has three modules, while the latter has only two. Induction levels of the three T. thermophila genes were determined using quantitative real time RT-PCR. Various stressors (including heavy metals) brought about dramatically different fold-inductions for each gene; MTT5 showed the highest fold-induction. Conserved DNA motifs with potential regulatory significance were identified, in an unbiased way, upstream of the start codons of subfamily 7a MTs. EST evidence for alternative splicing in the 3′ UTR of the MTT5 mRNA with potential regulatory activity is reported.Conclusion/SignificanceThe small number and remarkably regular structure of Tetrahymena MTs, coupled with the experimental tractability of this model organism for studies of in vivo function, make it an attractive system for the experimental dissection of the roles, structure/function relationships, regulation of gene expression, and adaptive evolution of these proteins, as well as for the development of biotechnological applications for the environmental monitoring of toxic substances.

Molecular cloning and characterization of IL-15Rα gene in rainbow trout ( Oncorhynchus mykiss)

The α-chain of IL-15 receptor complex serves as a high-affinity, specific subunit for IL-15 binding. It shares functional and structural features with IL-2 receptor α-chain. Here we report for the first time that the molecular cloning, characterization and sequence analysis of the teleostean IL-15Rα from Oncorhynchus mykiss. The full-length cDNA comprises of a 5′ untranslated region (UTR) of 167 bp, an open reading frame of 732 bp and a large 3′UTR of 630 bp, constitutively expressed in all the tissues examined. Another two variants are found derived from alternative splicing. Two copies of rainbow trout IL-15Rα (rtIL-15Rα) were detected in the genome by Southern blot hybridization. Moreover, EST evidence is also traced from other fishes. The deduced rtIL-15Rα of 243 amino acids contains a 17aa signal peptide at N-terminus and a transmembrane region at C-terminus, as well as a typical sushi domain included in the extracellular region. Phylogenetic tree analysis groups rtIL-15Rα with other IL-15Rαs but separates it from IL-2Rαs. In addition, a putative sequence was found in the sushi domain which is conserved and versatile among all species and this might relate to cytokine recognition.

Sweetness and light: illuminating the honey bee genome

come this, Martin Beye supplied AT-rich DNA isolated fromis the first hymenopteran andthe fifth insect genome to be sequenced (Honey BeeGenome Sequencing Consortium, 2006) in what promisesto be a swarm of insect genome sequences expected toappear over the next few years (Table 1). The Honey BeeGenome Sequencing Project (HBGSP) was conceptualizedover a period from 1998 to 2001 by the community atcourses, conferences and workshops (Robinson, 1999;Maleszka, 2000; Pennisi, 2001). In addition, initial effortswere directed at physical and genetic maps of the genome(Estoup

Identification and evolutionary analysis of novel exons and alternative splicing events using cross-species EST-to-genome comparisons in human, mouse and rat

BackgroundAlternative splicing (AS) is important for evolution and major biological functions in complex organisms. However, the extent of AS in mammals other than human and mouse is largely unknown, making it difficult to study AS evolution in mammals and its biomedical implications.ResultsHere we describe a cross-species EST-to-genome comparison algorithm (ENACE) that can identify novel exons for EST-scanty species and distinguish conserved and lineage-specific exons. The identified exons represent not only novel exons but also evolutionarily meaningful AS events that are not previously annotated. A genome-wide AS analysis in human, mouse and rat using ENACE reveals a total of 758 novel cassette-on exons and 167 novel retained introns that have no EST evidence from the same species. RT-PCR-sequencing experiments validated ~50 ~80% of the tested exons, indicating high presence of exons predicted by ENACE. ENACE is particularly powerful when applied to closely related species. In addition, our analysis shows that the ENACE-identified AS exons tend not to pass the nonsynonymous-to-synonymous substitution ratio test and not to contain protein domain, implying that such exons may be under positive selection or relaxed negative selection. These AS exons may contribute to considerable inter-species functional divergence. Our analysis further indicates that a large number of exons may have been gained or lost during mammalian evolution. Moreover, a functional analysis shows that inter-species divergence of AS events may be substantial in protein carriers and receptor proteins in mammals. These exons may be of interest to studies of AS evolution. The ENACE programs and sequences of the ENACE-identified AS events are available for download.ConclusionENACE can identify potential novel cassette exons and retained introns between closely related species using a comparative approach. It can also provide information regarding lineage- or species-specificity in transcript isoforms, which are important for evolutionary and functional studies.

Primate-Specific Endogenous Cis-Antisense Transcription in the Human 5q31 Protocadherin Gene Cluster

Protocadherins (PCDH), localized to synaptic junctions, contribute to the formation of neuronal networks during brain development; thus, it is speculated that protocadherins may play a role in evolution of neuronal complexity. While protocadherin genes are highly conserved in vertebrates, EST evidence from the locus suggests apparently species-specific cis-antisense transcripts. Novel cis-antisense transcripts, which partially overlap the PCDHalpha12 variable exon, PCDHbeta3 single-exon gene, and PCDHpsi5 unprocessed pseudogene in the human 5q31 PCDHalpha/beta/gamma gene cluster and which are coexpressed with sense-strand transcripts in fetal and adult brain, were identified computationally and validated by gene-specific strand-specific reverse transcriptase PCR (SSRTPCR) and sequencing. Absence of antisense transcripts arising from equivalent genomic locations in mouse indicates that the antisense transcripts originated in the primates after the primate-rodent divergence. Furthermore, not all expected orthologues of human sense and antisense PCDH transcripts were detected in rhesus macaque brain, implying that protocadherin expression patterns differ between primate species. RT followed by quantitative real-time PCR (QPCR) analysis of the three genes in the brain of all three species, and of the PCDHbeta15 gene paralogous to PCDHpsi5 in human and rhesus, revealed that the presence of antisense transcripts was significantly associated with lower sense expression levels across all orthologues. This inverse relationship, along with the pattern of sense and antisense coexpression in the brain, is consistent with a regulatory role for the primate-specific PCDH cis-antisense transcripts, which may represent recent evolutionary inventions modulating the activity of this conserved gene cluster.

Recognition of Unknown Conserved Alternatively Spliced Exons

The split structure of most mammalian protein-coding genes allows for the potential to produce multiple different mRNA and protein isoforms from a single gene locus through the process of alternative splicing (AS). We propose a computational approach called UNCOVER based on a pair hidden Markov model to discover conserved coding exonic sequences subject to AS that have so far gone undetected. Applying UNCOVER to orthologous introns of known human and mouse genes predicts skipped exons or retained introns present in both species, while discriminating them from conserved noncoding sequences. The accuracy of the model is evaluated on a curated set of genes with known conserved AS events. The prediction of skipped exons in the ~1% of the human genome represented by the ENCODE regions leads to more than 50 new exon candidates. Five novel predicted AS exons were validated by RT-PCR and sequencing analysis of 15 introns with strong UNCOVER predictions and lacking EST evidence. These results imply that a considerable number of conserved exonic sequences and associated isoforms are still completely missing from the current annotation of known genes. UNCOVER also identifies a small number of candidates for conserved intron retention.

Identification and analysis of alternative splicing events conserved in human and mouse.

Alternative pre-mRNA splicing affects a majority of human genes and plays important roles in development and disease. Alternative splicing (AS) events conserved since the divergence of human and mouse are likely of primary biological importance, but relatively few of such events are known. Here we describe sequence features that distinguish exons subject to evolutionarily conserved AS, which we call alternative conserved exons (ACEs), from other orthologous human/mouse exons and integrate these features into an exon classification algorithm, acescan. Genome-wide analysis of annotated orthologous human-mouse exon pairs identified approximately 2,000 predicted ACEs. Alternative splicing was verified in both human and mouse tissues by using an RT-PCR-sequencing protocol for 21 of 30 (70%) predicted ACEs tested, supporting the validity of a majority of acescan predictions. By contrast, AS was observed in mouse tissues for only 2 of 15 (13%) tested exons that had EST or cDNA evidence of AS in human but were not predicted ACEs, and AS was never observed for 11 negative control exons in human or mouse tissues. Predicted ACEs were much more likely to preserve the reading frame and less likely to disrupt protein domains than other AS events and were enriched in genes expressed in the brain and in genes involved in transcriptional regulation, RNA processing, and development. Our results also imply that the vast majority of AS events represented in the human EST database are not conserved in mouse.

PlantGDB, plant genome database and analysis tools.

PlantGDB (http://www.plantgdb.org/) is a database of molecular sequence data for all plant species with significant sequencing efforts. The database organizes EST sequences into contigs that represent tentative unique genes. Contigs are annotated and, whenever possible, linked to their respective genomic DNA. Genome sequence fragments are assembled similarly. The goal of the PlantGDB web site is to establish the basis for identifying sets of genes common to all plants or specific to particular species by integrating a number of bioinformatics tools that facilitate gene prediction and cross- species comparisons. For species with large-scale genome sequencing efforts, PlantGDB provides genome browsing capabilities that integrate all available EST and cDNA evidence for current gene models (for Arabidopsis thaliana, see the AtGDB site at http://www.plantgdb.org/AtGDB/).

Book Review: Est Evidence 5: Linking Medical Research to Practice

Book Review: Est Evidence 5: Linking Medical Research to Practice

Optimal spliced alignment of homologous cDNA to a genomic DNA template.

Supplementary cDNA or EST evidence is often decisive for discriminating between alternative gene predictions derived from computational sequence inspection by any of a number of requisite programs. Without additional experimental effort, this approach must rely on the occurrence of cognate ESTs for the gene under consideration in available, generally incomplete, EST collections for the given species. In some cases, particular exon assignments can be supported by sequence matching even if the cDNA or EST is produced from non-cognate genomic DNA, including different loci of a gene family or homologous loci from different species. However, marginally significant sequence matching alone can also be misleading. We sought to develop an algorithm that would simultaneously score for predicted intrinsic splice site strength and sequence matching between the genomic DNA template and a related cDNA or EST. In this case, weakly predicted splice sites may be chosen for the optimal scoring spliced alignment on the basis of surrounding sequence matching. Strongly predicted splice sites will enter the optimal spliced alignment even without strong sequence matching. We designed a novel algorithm that produces the optimal spliced alignment of a genomic DNA with a cDNA or EST based on scoring for both sequence matching and intrinsic splice site strength. By example, we demonstrate that this combined approach appears to improve gene prediction accuracy compared with current methods that rely only on either search by content and signal or on sequence similarity. The algorithm is available as a C subroutine and is implemented in the SplicePredictor and GeneSeqer programs. The source code is available via anonymous ftp from ftp. zmdb.iastate.edu. Both programs are also implemented as a Web service at http://gremlin1.zool.iastate.edu/cgi-bin/s p.cgiand http://gremlin1.zool.iastate.edu/cgi-bin/g s.cgi, respectively. vbrendel@iastate.edu