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PubMed2005

Morphological irregularities and features of resistance to apoptosis in the dcp-1/pita double mutated egg chambers during Drosophila oogenesis.

In the present study, we demonstrate the most novel characteristic morphological features of Drosophila egg chambers lacking both dcp-1 and pita functions in the germline cells. Dcp-1 is an effector caspase and it has been previously shown to play an important role during Drosophila oogenesis [McCall and Steller, 1998 : Science 279 : 230-234; Laundrie et al., 2003 : Genetics 165 : 1881-1888; Peterson et al., 2003 : Dev Biol 260 : 113-123]. The completion of sequencing and annotation of the Drosophila genome has revealed that the dcp-1 gene is nested within an intron of another distinct gene, called pita, a member of the C2H2 zinc finger protein family that regulates transcriptional initiation. The dcp-1(-/-)/pita(-/-) nurse cells exhibit euchromatic nuclei (delay of apoptosis) during the late stages of oogenesis, as revealed by conventional light and electron microscopy. The phalloidin-FITC staining discloses significant defects in actin cytoskeleton arrangement. The actin bundles fail to organize properly and the distribution of actin filaments in the ring canals is changed compared to the wild type. The oocyte and the chorion structures have been also modified. The oocyte nucleus is out of position and the chorion appears to contain irregular foldings, while the respiratory filaments obtain an altered morphology. The dcp-1(-/-)/pita(-/-) egg chambers do not exhibit the rare events of spontaneously induced apoptosis, observed for the wild type flies, during mid-oogenesis. Interestingly, the mutated egg chambers are protected by staurosporine-induced apoptosis in a percentage of 40%, strongly suggesting the essential role of dcp-1 and/or pita during mid-oogenesis.

Actins↗
PubMed2006

Reverse transcriptase template switching and false alternative transcripts.

Reverse transcriptase (RT) can switch from one template to another in a homology-dependent manner. In the study of eukaryotic transcripts, this propensity of RT can produce an artificially deleted cDNA, which can be wrongly interpreted as an alternative transcript. Here, we have investigated the presence of such template-switching artifacts in cDNA databases, by scanning a collection of human splice sites (Information for the Coordinates of Exons, ICE database). We have confirmed several cases at the experimental level. Artifacts represent a significant portion of apparently spliced sequences using noncanonical splice signals but are rare in the context of the whole database. However, care should be taken in the annotation of alternative transcripts, especially when the RT used is poorly thermostable and when the putative intron is flanked by direct repeats, which are the substrate for template switching.

Alternative Splicing↗
PubMed2006

Large-scale trends in the evolution of gene structures within 11 animal genomes.

We have used the annotations of six animal genomes (Homo sapiens, Mus musculus, Ciona intestinalis, Drosophila melanogaster, Anopheles gambiae, and Caenorhabditis elegans) together with the sequences of five unannotated Drosophila genomes to survey changes in protein sequence and gene structure over a variety of timescales--from the less than 5 million years since the divergence of D. simulans and D. melanogaster to the more than 500 million years that have elapsed since the Cambrian explosion. To do so, we have developed a new open-source software library called CGL (for "Comparative Genomics Library"). Our results demonstrate that change in intron-exon structure is gradual, clock-like, and largely independent of coding-sequence evolution. This means that genome annotations can be used in new ways to inform, corroborate, and test conclusions drawn from comparative genomics analyses that are based upon protein and nucleotide sequence similarities.

Animals↗
PubMed2026

The Role of Small Segmental Duplications in Generating Identical Isoforms Through Alternative Splicing Sites.

Alternative splicing plays a crucial role in expanding proteomic diversity but can also generate identical isoforms under certain conditions. While mutually exclusive splicing of tandem exons has occasionally been reported to produce identical isoforms, the extent to which other splicing events contribute to this phenomenon remains unclear. In this study, we demonstrate that alternative 5' and 3' splice site selection can also lead to the formation of identical isoforms, providing an additional type of splicing event for functional redundancy in transcriptomes. To address this, we analyzed reference genome annotations from 15 plant species, including Arabidopsis thaliana and wheat (Triticum aestivum), obtained from the RefSeq database. Identical isoforms were computationally defined as transcripts with distinct exon-intron structures but identical coding sequences. Our analysis reveals that the majority of alternative 5' and 3' fragments originate from small segmental duplications, suggesting that sequence repetition within gene regions facilitates the emergence of such splicing patterns. We also observed differences in the annotated 5' UTRs of some identical isoforms. However, since the alternative splicing sites themselves were not located within UTRs, these differences may reflect annotation uncertainty rather than genuine AS-derived variation. Given that UTR predictions in reference databases are not always precise, such observations should be interpreted cautiously. Expression analysis using an isoform-specific k-mer approach confirmed that identical isoforms can be differentially regulated. These findings suggest that, beyond expanding protein diversity, alternative splicing can also generate redundant isoforms that are differentially expressed at the RNA level, indicating potential regulatory roles. By elucidating the structural and regulatory factors contributing to the formation and retention of identical isoforms, our study provides new insights into the evolutionary and functional significance of alternative splicing in plants.

Alternative Splicing↗
PubMed2004

Gene structure prediction using an orthologous gene of known exon-intron structure.

Given the availability of complete genome sequences from related organisms, sequence conservation can provide important clues for predicting gene structure. In particular, one should be able to leverage information about known genes in one species to help determine the structures of related genes in another. Such an approach is appealing in that high-quality gene prediction can be achieved for newly sequenced species, such as mouse and puffer fish, using the extensive knowledge that has been accumulated about human genes. This article reports a novel approach to predicting the exon-intron structures of mouse genes by incorporating constraints from orthologous human genes using techniques that have previously been exploited in speech and natural language processing applications. The approach uses a context-free grammar to parse a training corpus of annotated human genes. A statistical training procedure produces a weighted recursive transition network (RTN) intended to capture the general features of a mammalian gene. This RTN is expanded into a finite state transducer (FST) and composed with an FST capturing the specific features of the human orthologue. This model includes a trigram language model on the amino acid sequence as well as exon length constraints. A final stage uses the free software package ClustalW to align the top n candidates in the search space. For a set of 98 orthologous human-mouse pairs, we achieved 96% sensitivity and 97% specificity at the exon level on the mouse genes, given only knowledge gleaned from the annotated human genome.

Algorithms↗
PubMed2000

Genie--gene finding in Drosophila melanogaster.

A hidden Markov model-based gene-finding system called Genie was applied to the genomic Adh region in Drosophila melanogaster as a part of the Genome Annotation Assessment Project (GASP). Predictions from three versions of the Genie gene-finding system were submitted, one based on statistical properties of coding genes, a second included EST alignment information, and a third that integrated protein sequence homology information. All three programs were trained on the provided Drosophila training data. In addition, promoter assignments from an integrated neural network were submitted. The gene assignments overlapped >90% of the 222 annotated genes and 26 possibly novel genes were predicted, of which some might be overpredictions. The system correctly identified the exon boundaries of 70% of the exons in cDNA-confirmed genes and 77% of the exons with the addition of EST sequence alignments. The best of the three Genie submissions predicted 19 of the annotated 43 gene structures entirely correct (44%). In the promoter category, only 30% of the transcription start sites could be detected, but by integrating this program as a sensor into Genie the false-positive rate could be dropped to 1/16,786 (0.006%). The results of the experiment on the long contiguous genomic sequence revealed some problems concerning gene assembly in Genie. The results were used to improve the system. We show that Genie is a robust hidden Markov model system that allows for a generalized integration of information from different sources such as signal sensors (splice sites, start codon, etc.), content sensors (exons, introns, intergenic) and alignments of mRNA, EST, and peptide sequences. The assessment showed that Genie could effectively be used for the annotation of complete genomes from higher organisms.

Animals↗
PubMed2003

TRACTS: A program to map oligopurine.oligopyrimidine and other binary DNA tracts.

A program to map the locations and frequencies of DNA tracts composed of only two bases ('Binary DNA') is described. The program, TRACTS (URL http://bioportal.weizmann.ac.il/tracts/tracts.html and/or http://bip.weizmann.ac.il/miwbin/servers/tracts) is of interest because long tracts composed of only two bases are highly over-represented in most genomes. In eukaryotes, oligopurine.oligopyrimidine tracts ('R.Y tracts') are found in the highest excess. In prokaryotes, W tracts predominate (A,T 'rich'). A pre-program, ANEX, parses database annotation files of GenBank and EMBL, to produce a convenient one-line list of every gene (exon, intron) in a genome. The main unit lists and analyzes tracts of the three possible binary pairs (R.Y, K.M and S;W). As an example, the results of R.Y tract mapping of mammalian gene p53 is described.

Base Composition↗
PubMed2005

HOPPSIGEN: a database of human and mouse processed pseudogenes.

Processed pseudogenes result from reverse transcribed mRNAs. In general, because processed pseudogenes lack promoters, they are no longer functional from the moment they are inserted into the genome. Subsequently, they freely accumulate substitutions, insertions and deletions. Moreover, the ancestral structure of processed pseudogenes could be easily inferred using the sequence of their functional homologous genes. Owing to these characteristics, processed pseudogenes represent good neutral markers for studying genome evolution. Recently, there is an increasing interest for these markers, particularly to help gene prediction in the field of genome annotation, functional genomics and genome evolution analysis (patterns of substitution). For these reasons, we have developed a method to annotate processed pseudogenes in complete genomes. To make them useful to different fields of research, we stored them in a nucleic acid database after having annotated them. In this work, we screened both mouse and human complete genomes from ENSEMBL to find processed pseudogenes generated from functional genes with introns. We used a conservative method to detect processed pseudogenes in order to minimize the rate of false positive sequences. Within processed pseudogenes, some are still having a conserved open reading frame and some have overlapping gene locations. We designated as retroelements all reverse transcribed sequences and more strictly, we designated as processed pseudogenes, all retroelements not falling in the two former categories (having a conserved open reading or overlapping gene locations). We annotated 5823 retroelements (5206 processed pseudogenes) in the human genome and 3934 (3428 processed pseudogenes) in the mouse genome. Compared to previous estimations, the total number of processed pseudogenes was underestimated but the aim of this procedure was to generate a high-quality dataset. To facilitate the use of processed pseudogenes in studying genome structure and evolution, DNA sequences from processed pseudogenes, and their functional reverse transcribed homologs, are now stored in a nucleic acid database, HOPPSIGEN. HOPPSIGEN can be browsed on the PBIL (Pole Bioinformatique Lyonnais) World Wide Web server (http://pbil.univ-lyon1.fr/) or fully downloaded for local installation.

Animals↗
PubMed2004

Genome SEGE: a database for 'intronless' genes in eukaryotic genomes.

BACKGROUND: A number of completely sequenced eukaryotic genome data are available in the public domain. Eukaryotic genes are either 'intron containing' or 'intronless'. Eukaryotic 'intronless' genes are interesting datasets for comparative genomics and evolutionary studies. The SEGE database containing a collection of eukaryotic single exon genes is available. However, SEGE is derived using GenBank. The redundant, incomplete and heterogeneous qualities of GenBank data are a bottleneck for biological investigation in comparative genomics and evolutionary studies. Such studies often require representative gene sets from each genome and this is possible only by deriving specific datasets from completely sequenced genome data. Thus Genome SEGE, a database for 'intronless' genes in completely sequenced eukaryotic genomes, has been constructed. AVAILABILITY: http://sege.ntu.edu.sg/wester/intronless DESCRIPTION: Eukaryotic 'intronless' genes are extracted from nine completely sequenced genomes (four of which are unicellular and five of which are multi-cellular). The complete dataset is available for download. Data subsets are also available for 'intronless' pseudo-genes. The database provides information on the distribution of 'intronless' genes in different genomes together with their length distributions in each genome. Additionally, the search tool provides pre-computed PROSITE motifs for each sequence in the database with appropriate hyperlinks to InterPro. A search facility is also available through the web server. CONCLUSIONS: The unique features that distinguish Genome SEGE from SEGE is the service providing representative 'intronless' datasets for completely sequenced genomes. 'Intronless' gene sets available in this database will be of use for subsequent bio-computational analysis in comparative genomics and evolutionary studies. Such analysis may help to revisit the original genome data for re-examination and re-annotation.

Databases, Genetic↗
PubMed2003

Reinvestigation of the Saccharomyces cerevisiae genome annotation by comparison to the genome of a related fungus: Ashbya gossypii.

BACKGROUND: The recently sequenced genome of the filamentous fungus Ashbya gossypii revealed remarkable similarities to that of the budding yeast Saccharomyces cerevisiae both at the level of homology and synteny (conservation of gene order). Thus, it became possible to reinvestigate the S. cerevisiae genome in the syntenic regions leading to an improved annotation. RESULTS: We have identified 23 novel S. cerevisiae open reading frames (ORFs) as syntenic homologs of A. gossypii genes; for all but one, homologs are present in other eukaryotes including humans. Other comparisons identified 13 overlooked introns and suggested 69 potential sequence corrections resulting in ORF extensions or ORF fusions with improved homology to the syntenic A. gossypii homologs. Of the proposed corrections, 25 were tested and confirmed by resequencing. In addition, homologs of nearly 1,000 S. cerevisiae ORFs, presently annotated as hypothetical, were found in A. gossypii at syntenic positions and can therefore be considered as authentic genes. Finally, we suggest that over 400 S. cerevisiae ORFs that overlap other ORFs in S. cerevisiae and for which no homolog can be detected in A. gossypii should be regarded as spurious. CONCLUSIONS: Although, the S. cerevisiae genome is rightly considered as one of the most accurately sequenced and annotated eukaryotic genomes, we have shown that it still benefits substantially from comparison to the completed sequence and syntenic gene map of A. gossypii, an evolutionarily related fungus. This type of approach will strongly support the annotation of more complex genomes such as the human and murine genomes.

Amino Acid Sequence↗
PubMed2005

Protein encoding genes in an ancient plant: analysis of codon usage, retained genes and splice sites in a moss, Physcomitrella patens.

BACKGROUND: The moss Physcomitrella patens is an emerging plant model system due to its high rate of homologous recombination, haploidy, simple body plan, physiological properties as well as phylogenetic position. Available EST data was clustered and assembled, and provided the basis for a genome-wide analysis of protein encoding genes. RESULTS: We have clustered and assembled Physcomitrella patens EST and CDS data in order to represent the transcriptome of this non-seed plant. Clustering of the publicly available data and subsequent prediction resulted in a total of 19,081 non-redundant ORF. Of these putative transcripts, approximately 30% have a homolog in both rice and Arabidopsis transcriptome. More than 130 transcripts are not present in seed plants but can be found in other kingdoms. These potential "retained genes" might have been lost during seed plant evolution. Functional annotation of these genes reveals unequal distribution among taxonomic groups and intriguing putative functions such as cytotoxicity and nucleic acid repair. Whereas introns in the moss are larger on average than in the seed plant Arabidopsis thaliana, position and amount of introns are approximately the same. Contrary to Arabidopsis, where CDS contain on average 44% G/C, in Physcomitrella the average G/C content is 50%. Interestingly, moss orthologs of Arabidopsis genes show a significant drift of codon fraction usage, towards the seed plant. While averaged codon bias is the same in Physcomitrella and Arabidopsis, the distribution pattern is different, with 15% of moss genes being unbiased. Species-specific, sensitive and selective splice site prediction for Physcomitrella has been developed using a dataset of 368 donor and acceptor sites, utilizing a support vector machine. The prediction accuracy is better than those achieved with tools trained on Arabidopsis data. CONCLUSION: Analysis of the moss transcriptome displays differences in gene structure, codon and splice site usage in comparison with the seed plant Arabidopsis. Putative retained genes exhibit possible functions that might explain the peculiar physiological properties of mosses. Both the transcriptome representation (including a BLAST and retrieval service) and splice site prediction have been made available on http://www.cosmoss.org, setting the basis for assembly and annotation of the Physcomitrella genome, of which draft shotgun sequences will become available in 2005.

Alternative Splicing↗
PubMed2005

Functional characterization of XendoU, the endoribonuclease involved in small nucleolar RNA biosynthesis.

XendoU is the endoribonuclease involved in the biosynthesis of a specific subclass of Xenopus laevis intron-encoded small nucleolar RNAs. XendoU has no homology to any known cellular RNase, although it has sequence similarity with proteins tentatively annotated as serine proteases. It has been recently shown that XendoU represents the cellular counterpart of a nidovirus replicative endoribonuclease (NendoU), which plays a critical role in viral replication and transcription. In this paper, we combined prediction and experimental data to define the amino acid residues directly involved in XendoU catalysis. Specifically, we find that XendoU residues Glu-161, Glu-167, His-162, His-178, and Lys-224 are essential for RNA cleavage, which occurs in the presence of manganese ions. Furthermore, we identified the RNA sequence required for XendoU binding and showed that the formation of XendoU-RNA complex is Mn2+-independent.

Amino Acid Sequence↗
PubMed2006

GAPP: a fully automated software for the confident identification of human peptides from tandem mass spectra.

This paper introduces the genome annotating proteomic pipeline (GAPP), a totally automated publicly available software pipeline for the identification of peptides and proteins from human proteomic tandem mass spectrometry data. The pipeline takes as its input a series of MS/MS peak lists from a given experimental sample and produces a series of database entries corresponding to the peptides observed within the sample, along with related confidence scores. The pipeline is capable of finding any peptides expected, including those that cross intron-exon boundaries, and those due to single nucleotide polymorphisms (SNPs), alternate splicing, and post-translational modifications (PTMs). GAPP can therefore be used to re-annotate genomes, and this is supported through the inclusion of a Distributed Annotation System (DAS) server, which allows the peptides identified by the pipeline to be displayed in their genomic context within the Ensembl genome browser. GAPP is freely available via the web, at www. gapp.info.

Alternative Splicing↗
PubMed2006

Accurate and unambiguous tag-to-gene mapping in serial analysis of gene expression.

BACKGROUND: In this study, we present a robust and reliable computational method for tag-to-gene assignment in serial analysis of gene expression (SAGE). The method relies on current genome information and annotation, incorporation of several new features, and key improvements over alternative methods, all of which are important to determine gene expression levels more accurately. The method provides a complete annotation of potential virtual SAGE tags within a genome, along with an estimation of their confidence for experimental observation that ranks tags that present multiple matches in the genome. RESULTS: We applied this method to the Saccharomyces cerevisiae genome, producing the most thorough and accurate annotation of potential virtual SAGE tags that is available today for this organism. The usefulness of this method is exemplified by the significant reduction of ambiguous cases in existing experimental SAGE data. In addition, we report new insights from the analysis of existing SAGE data. First, we found that experimental SAGE tags mapping onto introns, intron-exon boundaries, and non-coding RNA elements are observed in all available SAGE data. Second, a significant fraction of experimental SAGE tags was found to map onto genomic regions currently annotated as intergenic. Third, a significant number of existing experimental SAGE tags for yeast has been derived from truncated cDNAs, which are synthesized through oligo-d(T) priming to internal poly-(A) regions during reverse transcription. CONCLUSION: We conclude that an accurate and unambiguous tag mapping process is essential to increase the quality and the amount of information that can be extracted from SAGE experiments. This is supported by the results obtained here and also by the large impact that the erroneous interpretation of these data could have on downstream applications.

Chromosome Mapping↗
PubMed2003

cDNA2Genome: a tool for mapping and annotating cDNAs.

BACKGROUND: In the last years several high-throughput cDNA sequencing projects have been funded worldwide with the aim of identifying and characterizing the structure of complete novel human transcripts. However some of these cDNAs are error prone due to frameshifts and stop codon errors caused by low sequence quality, or to cloning of truncated inserts, among other reasons. Therefore, accurate CDS prediction from these sequences first require the identification of potentially problematic cDNAs in order to speed up the posterior annotation process. RESULTS: cDNA2Genome is an application for the automatic high-throughput mapping and characterization of cDNAs. It utilizes current annotation data and the most up to date databases, especially in the case of ESTs and mRNAs in conjunction with a vast number of approaches to gene prediction in order to perform a comprehensive assessment of the cDNA exon-intron structure. The final result of cDNA2Genome is an XML file containing all relevant information obtained in the process. This XML output can easily be used for further analysis such us program pipelines, or the integration of results into databases. The web interface to cDNA2Genome also presents this data in HTML, where the annotation is additionally shown in a graphical form. cDNA2Genome has been implemented under the W3H task framework which allows the combination of bioinformatics tools in tailor-made analysis task flows as well as the sequential or parallel computation of many sequences for large-scale analysis. CONCLUSIONS: cDNA2Genome represents a new versatile and easily extensible approach to the automated mapping and annotation of human cDNAs. The underlying approach allows sequential or parallel computation of sequences for high-throughput analysis of cDNAs.

Chromosome Mapping↗
PubMed2001

Genomic sequence and expression analyses of human chromatin assembly factor 1 p150 gene.

Chromatin assembly factor-1 (CAF-1) plays essential roles in eukaryotic chromatin assembly during DNA replication (Smith and Stillman, 1989. Cell 58, 15-25), (Krude, 1999. Eur. J. Biochem. 263, 1-5). Its p150 subunit, involved in interaction with histone H3 and H4, is critical to the CAF-1 nucleosome assembly activity. In this study, we sequenced a 96-kb genomic DNA region that includes a 42.8-kb CAF-1 p150 subunit gene (CHAF1A), and a 41.1-kb EEN gene. A scripted bioinformatics analysis pipeline (research agent) has been set up to annotate the BAC sequence with a set of integrated algorithms. The CAF-1 p150 subunit gene contains 15 exons and 14 introns. The promoter region is characterized by deletional analyses, revealing a potential repressor. Tissue-correlated alternative splicing forms of the transcript was initially identified by EST clustering analysis, then confirmed by RT-PCR which resulted more splicing forms than computational prediction.

3T3 Cells↗
PubMed2006

A web-based search engine for triplex-forming oligonucleotide target sequences.

Triplex technology offers a useful approach for site-specific modification of gene structure and function both in vitro and in vivo. Triplex-forming oligonucleotides (TFOs) bind to their target sites in duplex DNA, thereby forming triple-helical DNA structures via Hoogsteen hydrogen bonding. TFO binding has been demonstrated to site-specifically inhibit gene expression, enhance homologous recombination, induce mutation, inhibit protein binding, and direct DNA damage, thus providing a tool for gene-specific manipulation of DNA. We have developed a flexible web-based search engine to find and annotate TFO target sequences within the human and mouse genomes. Descriptive information about each site, including sequence context and gene region (intron, exon, or promoter), is provided. The engine assists the user in finding highly specific TFO target sequences by eliminating or flagging known repeat sequences and flagging overlapping genes. A convenient way to check for the uniqueness of a potential TFO binding site is provided via NCBI BLAST. The search engine may be accessed at spi.mdanderson.org/tfo.

Animals↗
PubMed2006

A computational approach for identifying pseudogenes in the ENCODE regions.

BACKGROUND: Pseudogenes are inheritable genetic elements showing sequence similarity to functional genes but with deleterious mutations. We describe a computational pipeline for identifying them, which in contrast to previous work explicitly uses intron-exon structure in parent genes to classify pseudogenes. We require alignments between duplicated pseudogenes and their parents to span intron-exon junctions, and this can be used to distinguish between true duplicated and processed pseudogenes (with insertions). RESULTS: Applying our approach to the ENCODE regions, we identify about 160 pseudogenes, 10% of which have clear 'intron-exon' structure and are thus likely generated from recent duplications. CONCLUSION: Detailed examination of our results and comparison of our annotation with the GENCODE reference annotation demonstrate that our computation pipeline provides a good balance between identifying all pseudogenes and delineating the precise structure of duplicated genes.

Computational Biology↗