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PubMed2006

Djinn Lite: a tool for customised gene transcript modelling, annotation-data enrichment and exploration.

BACKGROUND: There is an ever increasing rate of data made available on genetic variation, transcriptomes and proteomes. Similarly, a growing variety of bioinformatic programs are becoming available from many diverse sources, designed to identify a myriad of sequence patterns considered to have potential biological importance within inter-genic regions, genes, transcripts, and proteins. However, biologists require easy to use, uncomplicated tools to integrate this information, visualise and print gene annotations. Integrating this information usually requires considerable informatics skills, and comprehensive knowledge of the data format to make full use of this information. Tools are needed to explore gene model variants by allowing users the ability to create alternative transcript models using novel combinations of exons not necessarily represented in current database deposits of mRNA/cDNA sequences. RESULTS: Djinn Lite is designed to be an intuitive program for storing and visually exploring of custom annotations relating to a eukaryotic gene sequence and its modelled gene products. In particular, it is helpful in developing hypothesis regarding alternate splicing of transcripts by allowing the construction of model transcripts and inspection of their resulting translations. It facilitates the ability to view a gene and its gene products in one synchronised graphical view, allowing one to drill down into sequence related data. Colour highlighting of selected sequences and added annotations further supports exploration, visualisation of sequence regions and motifs known or predicted to be biologically significant. CONCLUSION: Gene annotating remains an ongoing and challenging task that will continue as gene structures, gene transcription repertoires, disease loci, protein products and their interactions become more precisely defined. Djinn Lite offers an accessible interface to help accumulate, enrich, and individualize sequence annotations relating to a gene, its transcripts and translations. The mechanism of transcript definition and creation, and subsequent navigation and exploration of features, are very intuitive and demand only a short learning curve. Ultimately, Djinn Lite can form the basis for providing valuable clues to plan new experiments, providing storage of sequences and annotations for dedication to customised projects. The application is appropriate for Windows 98-ME-2000-XP-2003 operating systems.

Alternative Splicing↗
PubMed2003

A new approach for gene annotation using unambiguous sequence joining.

The problem addressed by this paper is accurate and automatic gene annotation following precise identification/ annotation of exon and intron boundaries of biologically verified nucleotide sequences using the alignment of human genomic DNA to curated mRNA transcripts. We provide a detailed description of a new cDNA/DNA homology gene annotation algorithm that combines the results of BLASTN searches and spliced alignments. Compared to other programs currently in use, annotation quality is significantly increased through the unambiguous junction of genomic DNA sequences. We also address gene annotation with both non-canonic splice sites and short exons. The approach has been tested on the Genie learning subset as well as full-scale human RefSeq, and has demonstrated performance as high as 97%.

Algorithms↗
PubMed2002

Chromosomal DNA deletions explain phenotypic characteristics of two antigenic variants, phase II and RSA 514 (crazy), of the Coxiella burnetii nine mile strain.

After repeated passages through embyronated eggs, the Nine Mile strain of Coxiella burnetii exhibits antigenic variation, a loss of virulence characteristics, and transition to a truncated lipopolysaccharide (LPS) structure. In two independently derived strains, Nine Mile phase II and RSA 514, these phenotypic changes were accompanied by a large chromosomal deletion (M. H. Vodkin and J. C. Williams, J. Gen. Microbiol. 132:2587-2594, 1986). In the work reported here, additional screening of a cosmid bank prepared from the wild-type strain was used to map the deletion termini of both mutant strains and to accumulate all the segments of DNA that comprise the two deletions. The corresponding DNAs were then sequenced and annotated. The Nine Mile phase II deletion was completely nested within the deletion of the RSA 514 strain. Basic alignment and homology studies indicated that a large group of LPS biosynthetic genes, arranged in an apparent O-antigen cluster, was deleted in both variants. Database homologies identified, in particular, mannose pathway genes and genes encoding sugar methylases and nucleotide sugar epimerase-dehydratase proteins. Candidate genes for addition of sugar units to the core oligosaccharide for synthesis of the rare sugar 6-deoxy-3-C-methylgulose (virenose) were identified in the deleted region. Repeats, redundancies, paralogous genes, and two regions with reduced G+C contents were found within the deletions.

Antigenic Variation↗
PubMed2001

Two large Arabidopsis thaliana gene families are homologous to the Brassica gene superfamily that encodes pollen coat proteins and the male component of the self-incompatibility response.

The male component of the self-incompatibility response in Brassica has recently been shown to be encoded by the S locus cysteine-rich gene (SCR). SCR is related, at the sequence level, to the pollen coat protein (PCP) gene family whose members encode small, cysteine-rich proteins located in the proteo-lipidic surface layer (tryphine) of Brassica pollen grains. Here we show that the Arabidopsis genome includes two large gene families with homology to SCR and to the PCP gene family, respectively. These genes are poorly predicted by gene-identification algorithms and, with few exceptions, have been missed in previous annotations. Based on sequence comparison and an analysis of the expression patterns of several members of each family, we discuss the possible functions of these genes. In particular, we consider the possibility that SCR-related genes in Arabidopsis may encode ligands for the S gene family of receptor-like kinases in this species.

Alleles↗
PubMed2005

Small-world network approach to identify key residues in protein-protein interaction.

We show that protein complexes can be represented as small-world networks, exhibiting a relatively small number of highly central amino-acid residues occurring frequently at protein-protein interfaces. We further base our analysis on a set of different biological examples of protein-protein interactions with experimentally validated hot spots, and show that 83% of these predicted highly central residues, which are conserved in sequence alignments and nonexposed to the solvent in the protein complex, correspond to or are in direct contact with an experimentally annotated hot spot. The remaining 17% show a general tendency to be close to an annotated hot spot. On the other hand, although there is no available experimental information on their contribution to the binding free energy, detailed analysis of their properties shows that they are good candidates for being hot spots. Thus, highly central residues have a clear tendency to be located in regions that include hot spots. We also show that some of the central residues in the protein complex interfaces are central in the monomeric structures before dimerization and that possible information relating to hot spots of binding free energy could be obtained from the unbound structures.

Amino Acids↗
PubMed2006

Target selection and deselection at the Berkeley Structural Genomics Center.

At the Berkeley Structural Genomics Center (BSGC), our goal is to obtain a near-complete structural complement of proteins in the minimal organisms Mycoplasma genitalium and M. pneumoniae, two closely related pathogens. Current targets for structure determination have been selected in six major stages, starting with those predicted to be most tractable to high throughput study and likely to yield new structural information. We report on the process used to select these proteins, as well as our target deselection procedure. Target deselection reduces experimental effort by eliminating targets similar to those recently solved by the structural biology community or other centers. We measure the impact of the 69 structures solved at the BSGC as of July 2004 on structure prediction coverage of the M. pneumoniae and M. genitalium proteomes. The number of Mycoplasma proteins for which the fold could first be reliably assigned based on structures solved at the BSGC (24 M. pneumoniae and 21 M. genitalium) is approximately 25% of the total resulting from work at all structural genomics centers and the worldwide structural biology community (94 M. pneumoniae and 86 M. genitalium) during the same period. As the number of structures contributed by the BSGC during that period is less than 1% of the total worldwide output, the benefits of a focused target selection strategy are apparent. If the structures of all current targets were solved, the percentage of M. pneumoniae proteins for which folds could be reliably assigned would increase from approximately 57% (391 of 687) at present to around 80% (550 of 687), and the percentage of the proteome that could be accurately modeled would increase from around 37% (254 of 687) to about 64% (438 of 687). In M. genitalium, the percentage of the proteome that could be structurally annotated based on structures of our remaining targets would rise from 72% (348 of 486) to around 76% (371 of 486), with the percentage of accurately modeled proteins would rise from 50% (243 of 486) to 58% (283 of 486). Sequences and data on experimental progress on our targets are available in the public databases TargetDB and PEPCdb.

Amino Acids↗
PubMed2006

Evolutionary trace report_maker: a new type of service for comparative analysis of proteins.

: Evolutionary trace report_maker offers a new type of service for researchers investigating the function of novel proteins. It pools, from different sources, information about protein sequence, structure and elementary annotation, and to that background superimposes inference about the evolutionary behavior of individual residues, using real-valued evolutionary trace method. As its only input it takes a Protein Data Bank identifier or UniProt accession number, and returns a human-readable document in PDF format, supplemented by the original data needed to reproduce the results quoted in the report.

Algorithms↗
PubMed2004

The Centre for Modeling Human Disease Gene Trap resource.

Gene trap mutagenesis of mouse embryonic stem cells generates random loss-of-function mutations, which can be identified by a sequence tag and can often report the endogenous expression of the mutated gene. The Centre for Modeling Human Disease is performing expression- and sequence-based screens of gene trap insertions to generate new mouse mutations as a resource for the scientific community. The gene trap insertions are screened using multiplexed in vitro differentiation and induction assays, and sequence tags are generated to complement expression profiles. Researchers may search for insertions in genes expressed in target cell lineages, under specific in vitro conditions, or based upon sequence identity via an online searchable database (http://www.cmhd.ca/sub/genetrap.asp). The clones are available as a resource to researchers worldwide to help to functionally annotate the mammalian genome and will serve as a source to test candidate loci identified by phenotype-driven mutagenesis screens.

Animals↗
PubMed2000

Bioinformatics, target discovery and the pharmaceutical/biotechnology industry.

With the first draft of the human genome now available a directed genome-wide mining strategy is being implemented by many pharmaceutical and biotechnology companies in order to identify novel members of the most therapeutically relevant target families. At the same time there is an increasing amount of annotation relevant to the human genome sequence entering into the public domain. The ability to identify protein families on a genome-wide scale can only be done at speed by using high-throughput computational approaches. This review describes many of the latest algorithmic developments in this field and shows how they can be best put to use for target identification and prioritization.

Algorithms↗
PubMed2002

Expression of human smooth muscle calponin in transgenic mice revealed with a bacterial artificial chromosome.

Defining regulatory elements governing cell-restricted gene expression can be difficult because cis-elements may reside tens of kilobases away from start site(s) of transcription. Artificial chromosomes, which harbor hundreds of kilobases of genomic DNA, preserve a large sequence landscape containing most, if not all, regulatory elements controlling the expression of a particular gene. Here, we report on the use of a bacterial artificial chromosome (BAC) to begin understanding the in vivo regulation of smooth muscle calponin (SM-Calp). Long and accurate polymerase chain reaction, sequencing, and in silico analyses facilitated the complete sequence annotation of a BAC harboring human SM-Calp (hSM-Calp). RNase protection, in situ hybridization, Western blotting, and immunohistochemistry assays showed the BAC clone faithfully expressed hSM-Calp in both cultured cells and transgenic mice. Moreover, expression of hSM-Calp mirrored that of endogenous mouse SM-Calp suggesting that all cis-regulatory elements governing hSM-Calp expression in vivo were contained within the BAC. These BAC mice represent a new model system in which to systematically assess regulatory elements governing SM-Calp transcription in vivo.

Animals↗
PubMed2002

Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2.

The Drosophila Genome Project website contains an annotated gene (CG14575) for a G protein-coupled receptor. We cloned this receptor and found that the cloned cDNA did not correspond to the annotated gene; it partly contained different exons and additional exons located at the 5(')-end of the annotated gene. We expressed the coding part of the cloned cDNA in Chinese hamster ovary cells and found that the receptor was activated by two neuropeptides, capa-1 and -2, encoded by the Drosophila capability gene. Database searches led to the identification of a similar receptor in the genome from the malaria mosquito Anopheles gambiae (58% amino acid residue identities; 76% conserved residues; and 5 introns at identical positions within the two insect genes). Because capa-1 and -2 and related insect neuropeptides stimulate fluid secretion in insect Malpighian (renal) tubules, the identification of this first insect capa receptor will advance our knowledge on insect renal function.

Amino Acid Sequence↗
PubMed2004

Highly specific and accurate selection of siRNAs for high-throughput functional assays.

MOTIVATION: Small interfering RNA (siRNA) is widely used in functional genomics to silence genes by decreasing their expression to study the resulting phenotypes. The possibility of performing large-scale functional assays by gene silencing accentuates the necessity of a software capable of the high-throughput design of highly specific siRNA. The main objective sought was the design of a large number of siRNAs with appropriate thermodynamic properties and, especially, high specificity. Since all the available procedures require, to some extent, manual processing of the results to guarantee specific results, specificity constitutes to date, the major obstacle to the complete automation of all the steps necessary for the selection of optimal candidate siRNAs. RESULT: Here, we present a program that for the first time completely automates the search for siRNAs. In SiDE, the most complete set of rules for the selection of siRNA candidates (including G+C content, nucleotides at determined positions, thermodynamic properties, propensity to form internal hairpins, etc.) is implemented and moreover, specificity is achieved by a conceptually new method. After selecting possible siRNA candidates with the optimal functional properties, putative unspecific matches, which can cause cross-hybridization, are checked in databases containing a unique entry for each gene. These truly non-redundant databases are constructed from the genome annotations (Ensembl). Also intron/exon boundaries, presence of polymorphisms (single nucleotide polymorphisms) specificity for either gene or transcript, and other features can be selected to be considered in the design of siRNAs. AVAILABILITY: The program is available as a web server at http://side.bioinfo.cnio.es. The program was written under the GPL license. CONTACT: jdopazo@cnio.es.

Algorithms↗
PubMed2005

Functional insights from the distribution and role of homopeptide repeat-containing proteins.

Expansion of "low complex" repeats of amino acids such as glutamine (Poly-Q) is associated with protein misfolding and the development of degenerative diseases such as Huntington's disease. The mechanism by which such regions promote misfolding remains controversial, the function of many repeat-containing proteins (RCPs) remains obscure, and the role (if any) of repeat regions remains to be determined. Here, a Web-accessible database of RCPs is presented. The distribution and evolution of RCPs that contain homopeptide repeats tracts are considered, and the existence of functional patterns investigated. Generally, it is found that while polyamino acid repeats are extremely rare in prokaryotes, several eukaryote putative homologs of prokaryote RCP-involved in important housekeeping processes-retain the repetitive region, suggesting an ancient origin for certain repeats. Within eukarya, the most common uninterrupted amino acid repeats are glutamine, asparagines, and alanine. Interestingly, while poly-Q repeats are found in vertebrates and nonvertebrates, poly-N repeats are only common in more primitive nonvertebrate organisms, such as insects and nematodes. We have assigned function to eukaryote RCPs using Online Mendelian Inheritance in Man (OMIM), the Human Reference Protein Database (HRPD), FlyBase, and Wormpep. Prokaryote RCPs were annotated using BLASTp searches and Gene Ontology. These data reveal that the majority of RCPs are involved in processes that require the assembly of large, multiprotein complexes, such as transcription and signaling.

Amino Acid Sequence↗
PubMed2005

cpbAg1 encodes an active carboxypeptidase B expressed in the midgut of Anopheles gambiae.

We previously used differential display to identify several Anopheles gambiae genes, whose expression in the mosquito midgut was regulated upon ingestion of Plasmodium falciparum. Here, we report the characterization of one of these genes, cpbAg1, which codes for the first zinc-carboxypeptidase B identified in An. gambiae and in any insect. Expression of cpbAg1 in baculovirus gave rise to an active enzyme, and determination of the N-terminal amino acids confirmed that CPBAg1 contains a signal peptide and a pro-peptide, typical features of digestive zinc carboxypeptidases. cpbAg1 mRNA was mainly produced in the mosquito midgut, where it accumulated in unfed females and was rapidly down-regulated upon blood feeding. Annotation of the An. gambiae genome predicts twenty-three sequences coding for zinc-carboxypeptidases of which only two (cpbAg1 and cpbAg2) are expressed at a significant level in the mosquito midgut.

Amino Acid Sequence↗
PubMed2006

Analysis of expressed sequence tags from a naked foraminiferan Reticulomyxa filosa.

Foraminifers are a major component of modern marine ecosystems and one of the most important oceanic producers of calcium carbonate. They are a key phylogenetic group among amoeboid protists, but our knowledge of their genome is still mostly limited to a few conserved genes. Here, we report the first study of expressed genes by means of expressed sequence tag (EST) from the freshwater naked foraminiferan Reticulomyxa filosa. Cluster analysis of 1630 valid ESTs enabled the identification of 178 groups of related sequences and 871 singlets. Approximately 50% of the putative unique 1059 ESTs could be annotated using Blast searches against the protein database SwissProt + TrEMBL. The EST database described here is the first step towards gene discovery in Foraminifera and should provide the basis for new insights into the genomic and transcriptomic characteristics of these interesting but poorly understood protists.

Animals↗
PubMed2004

Microarray analyses identify molecular biomarkers of Atlantic salmon macrophage and hematopoietic kidney response to Piscirickettsia salmonis infection.

Piscirickettsia salmonis is the intracellular bacterium that causes salmonid rickettsial septicemia, an infectious disease that kills millions of farmed fish each year. The mechanisms used by P. salmonis to survive and replicate within host cells are not known. Piscirickettsiosis causes severe necrosis of hematopoietic kidney. Microarray-based experiments with QPCR validation were used to identify Atlantic salmon macrophage and hematopoietic kidney genes differentially transcribed in response to P. salmonis infection. Infections were confirmed by microscopy and RT-PCR with pathogen-specific primers. In infected salmon macrophages, 71 different transcripts were upregulated and 31 different transcripts were downregulated. In infected hematopoietic kidney, 30 different transcripts were upregulated and 39 different transcripts were downregulated. Ten antioxidant genes, including glutathione S-transferase, glutathione reductase, glutathione peroxidase, and cytochrome b558 alpha- and beta-subunits, were upregulated in infected macrophages but not in infected hematopoietic kidney. Changes in redox status of infected macrophages may allow these cells to tolerate P. salmonis infection, raising the possibility that treatment with antioxidants may reduce hematopoietic tissue damage caused by this rickettsial infection. The downregulation of transcripts involved in adaptive immune responses (e.g., T cell receptor alpha-chain and C-C chemokine receptor 7) in infected hematopoietic kidney but not in infected macrophages may contribute to infection-induced kidney tissue damage. Molecular biomarkers of P. salmonis infection, characterized by immune-relevant functional annotations and high fold differences in expression between infected and noninfected samples, may aid in the development of anti-piscirickettsial vaccines and therapeutics.

Animals↗
PubMed2003

Reevaluation of human cytomegalovirus coding potential.

The Bio-Dictionary-based Gene Finder was used to reassess the coding potential of the AD169 laboratory strain of human cytomegalovirus and sequences in the Toledo strain that are missing in the laboratory strain of the virus. The gene-finder algorithm assesses the potential of an ORF to encode a protein based on matches to a database of amino acid patterns derived from a large collection of proteins. The algorithm was used to score all human cytomegalovirus ORFs with the potential to encode polypeptides >/=50 aa in length. As a further test for functionality, the genomes of the chimpanzee, rhesus, and murine cytomegaloviruses were searched for orthologues of the predicted human cytomegalovirus ORFs. The analysis indicates that 37 previously annotated ORFs ought to be discarded, and at least nine previously unrecognized ORFs with relatively strong coding potential should be added. Thus, the human cytomegalovirus genome appears to contain approximately 192 unique ORFs with the potential to encode a protein. Support for several of the predictions of our in silico analysis was obtained by sequencing several domains within a clinical isolate of human cytomegalovirus.

Algorithms↗
PubMed2025

Identification and classification of ion-channels across the tree of life provide functional insights into understudied CALHM channels.

The ion channel (IC) genes encoded in the human genome play fundamental roles in cellular functions and disease and are one of the largest classes of druggable proteins. However, limited knowledge of the diverse molecular and cellular functions carried out by ICs presents a major bottleneck in developing selective chemical probes for modulating their functions in disease states. The wealth of sequence data available on ICs from diverse organisms provides a valuable source of untapped information for illuminating the unique modes of channel regulation and functional specialization. However, the extensive diversification of IC sequences and the lack of a unified resource present a challenge in effectively using existing data for IC research. Here, we perform integrative mining of available sequence, structure, and functional data on 419 human ICs across disparate sources, including extensive literature mining by leveraging advances in large language models to annotate and curate the full complement of the "channelome". We employ a well-established orthology inference approach to identify and extend the IC orthologs across diverse organisms to above 48,000. We show that the depth of conservation and taxonomic representation of IC sequences can further be translated to functional similarities by clustering them into functionally relevant groups, which can be used for downstream functional prediction on understudied members. We demonstrate this by delineating co-conserved patterns characteristic of the understudied family of the Calcium Homeostasis Modulator (CALHM) family of ICs. Through mutational analysis of co-conserved residues altered in human diseases and electrophysiological studies, we show that these evolutionarily-constrained residues play an important role in channel gating functions. Thus, by providing new tools and resources for performing large comparative analyses on ICs, this study addresses the unique needs of the IC community and provides the groundwork for accelerating the functional characterization of dark channels for therapeutic intervention.

CALHM1↗