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PubMed1998

Recognition of human genes by stochastic parsing.

A gene finding system, GeneDecoder, based on a parsing technique using a stochastic grammar and dictionary of genetic words is introduced. The structure of human genes are expressed by a stochastic grammar and a dictionary, whose components are the genetic words consisting of genetic phonemes, built as hidden Markov models (HMMs). The HMMs represent the nucleotide acid bases, the codons, and the amino acids. The genetic words in the dictionary are described by the sequence of these HMMs and represent exons, introns, intergenic regions, tRNA regions and signals in DNA sequences. The statistics between these regions are expressed by the grammar, which is a stochastic network of the genetic words. Using the same kind of technique of speech recognition by HMMs with a word dictionary and a grammar, the stochastic network of genetic words enables the motif dictionary to be used during the parsing of the DNA sequences. At the same time, stochastic features of donor/acceptor sites, information of the di-codon statistics, and other important features are integrated into stochastic scores during the parsing. As a result, while the system parses DNA sequences and finds the exon/intron structures, the protein motifs are automatically annotated in the regions. It helps to identify the functions of the genes and reduces the cost of homology search for each hypothetical coding regions. This method is different from simply using the information of homology search. This method uses the information of the motif patterns during the parsing process, but searching the motif patterns after/before finding the coding regions cannot directly affect the parsing process itself. Experimental results have shown that this method reasonably finds and annotates the motifs in the exons in the DNA sequence of human.

Amino Acid Sequence↗
PubMed2000

The molecular evolution of signal peptides.

Signal peptides direct mature peptides to their appropriate cellular location, after which they are cleaved off. Very many random alternatives can serve the same function. Of all coding sequences, therefore, signal peptides might come closest to being neutrally evolving. Here we consider this issue by examining the molecular evolution of 76 mouse-rat orthologues, each with defined signal peptides. Although they do evolve rapidly, they evolve about half as fast as neutral sequences. This indicates that a substantial proportion of mutations must be under stabilizing selection. A few putative signal sequences lack a hydrophobic core and these tend to be more slowly evolving than others, indicating even stronger stabilizing selection. However, closer scrutiny suggests that some of these represent mis-annotations in GenBank. It is also likely that some of the substitutions are not neutral. We find, for example, that the rate of protein evolution correlates with that of the mature peptide. This may be a result of compensatory evolution. We also find that signal peptides of immune genes tend to be faster evolving than the average, which suggests an association with antagonistic co-evolution. Previous reports also indicated that the signal peptide of the imprinted gene, Igf2r, is also unusually fast evolving. This, it was hypothesized, might also be indicative of antagonistic co-evolution. Comparison of Igf2r's signal peptide evolution shows that, although it is not an outlier, its rate of evolution is comparable to that of many of the faster evolving immune system signal sequences and 5/6 of the amino acid changes do not conserve hydrophobicity. This is at least suggestive that there is something unusual about Igf2r's signal sequence.

Amino Acid Substitution↗
PubMed2002

Modeling the percolation of annotation errors in a database of protein sequences.

Public sequence databases contain information on the sequence, structure and function of proteins. Genome sequencing projects have led to a rapid increase in protein sequence information, but reliable, experimentally verified, information on protein function lags a long way behind. To address this deficit, functional annotation in protein databases is often inferred by sequence similarity to homologous, annotated proteins, with the attendant possibility of error. Now, the functional annotation in these homologous proteins may itself have been acquired through sequence similarity to yet other proteins, and it is generally not possible to determine how the functional annotation of any given protein has been acquired. Thus the possibility of chains of misannotation arises, a process we term 'error percolation'. With some simple assumptions, we develop a dynamical probabilistic model for these misannotation chains. By exploring the consequences of the model for annotation quality it is evident that this iterative approach leads to a systematic deterioration of database quality.

Animals↗
PubMed2005

Bioinformatic analyses implicate the collaborating meiotic crossover/chiasma proteins Zip2, Zip3, and Spo22/Zip4 in ubiquitin labeling.

Zip2 and Zip3 are meiosis-specific proteins that, in collaboration with several partners, act at the sites of crossover-designated, axis-associated recombinational interactions to mediate crossover/chiasma formation. Here, Spo22 (also called Zip4) is identified as a probable functional collaborator of Zip2/3. The molecular roles of Zip2, Zip3, and Spo22/Zip4 are unknown. All three proteins are part of a small evolutionary cohort comprising similar homologs in four related yeasts. Zip3 is shown to contain a RING finger whose structural features most closely match those of known ubiquitin E3s. Further, Zip3 exhibits major domainal homologies to Rad18, a known DNA-binding ubiquitin E3. Also described is an approach to the identification and mapping of repeated protein sequence motifs, Alignment Based Repeat Annotation (ABRA), that we have developed. When ABRA is applied to Zip2 and Spo22/Zip4, they emerge as a 14-blade WD40-like repeat protein and a 22-unit tetratricopeptide repeat protein, respectively. WD40 repeats of Cdc20, Cdh1, and Cdc16 and tetratricopeptide repeats of Cdc16, Cdc23, and Cdc27, all components of the anaphase-promoting complex, are also analyzed. These and other findings suggest that Zip2, Zip3, and Zip4 act together to mediate a process that involves Zip3-mediated ubiquitin labeling, potentially as a unique type of ubiquitin-conjugating complex.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and characterization of Tap, a putative multidrug efflux pump present in Mycobacterium fortuitum and Mycobacterium tuberculosis.

A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N'-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced into M. smegmatis. Further characterization of this plasmid allowed the identification of the M. fortuitum tap gene. A homologous gene in the M. tuberculosis H37Rv genome has been identified. The M. tuberculosis tap gene (Rv1258 in the annotated sequence of the M. tuberculosis genome) was cloned and conferred low-level resistance to tetracycline when introduced into M. smegmatis. The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns. Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected. In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing the tap genes were decreased. When tetracycline accumulation experiments were carried out with the M. fortuitum tap gene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP. We conclude that the Tap proteins of the opportunistic organism M. fortuitum and the important pathogen M. tuberculosis are probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.

Amino Acid Sequence↗
PubMed2005

Dissimilation of cysteate via 3-sulfolactate sulfo-lyase and a sulfate exporter in Paracoccus pantotrophus NKNCYSA.

Paracoccus pantotrophus NKNCYSA utilizes (R)-cysteate (2-amino-3-sulfopropionate) as a sole source of carbon and energy for growth, with either nitrate or molecular oxygen as terminal electron acceptor, and the specific utilization rate of cysteate is about 2 mkat (kg protein)(-1). The initial degradative reaction is catalysed by an (R)-cysteate : 2-oxoglutarate aminotransferase, which yields 3-sulfopyruvate. The latter was reduced to 3-sulfolactate by an NAD-linked sulfolactate dehydrogenase [3.3 mkat (kg protein)(-1)]. The inducible desulfonation reaction was not detected initially in cell extracts. However, a strongly induced protein with subunits of 8 kDa (alpha) and 42 kDa (beta) was found and purified. The corresponding genes had similarities to those encoding altronate dehydratases, which often require iron for activity. The purified enzyme could then be shown to convert 3-sulfolactate to sulfite and pyruvate and it was termed sulfolactate sulfo-lyase (Suy). A high level of sulfite dehydrogenase was also induced during growth with cysteate, and the organism excreted sulfate. A putative regulator, OrfR, was encoded upstream of suyAB on the reverse strand. Downstream of suyAB was suyZ, which was cotranscribed with suyB. The gene, an allele of tauZ, encoded a putative membrane protein with transmembrane helices (COG2855), and is a candidate to encode the sulfate exporter needed to maintain homeostasis during desulfonation. suyAB-like genes are widespread in sequenced genomes and environmental samples where, in contrast to the current annotation, several presumably encode the desulfonation of 3-sulfolactate, a component of bacterial spores.

Amino Acid Sequence↗
PubMed2003

New technologies to assess genotype-phenotype relationships.

The accelerating pace of the discovery of genes has far surpassed our capabilities to understand their biological function--in other words, the phenotypes they engender. We need efficient and comprehensive large-scale phenotyping technologies. This presents a difficult challenge because phenotypes are numerous and diverse, and they can be observed and annotated at the molecular, cellular and organismal level. New technologies and approaches will therefore be required. Here, I describe recent efforts to develop new and efficient technologies for assessing cellular phenotypes.

Escherichia coli↗
PubMed2005

Evolutionary profiles from the QR factorization of multiple sequence alignments.

We present an algorithm to generate complete evolutionary profiles that represent the topology of the molecular phylogenetic tree of the homologous group. The method, based on the multidimensional QR factorization of numerically encoded multiple sequence alignments, removes redundancy from the alignments and orders the protein sequences by increasing linear dependence, resulting in the identification of a minimal basis set of sequences that spans the evolutionary space of the homologous group of proteins. We observe a general trend that these smaller, more evolutionarily balanced profiles have comparable and, in many cases, better performance in database searches than conventional profiles containing hundreds of sequences, constructed in an iterative and computationally intensive procedure. For more diverse families or superfamilies, with sequence identity <30%, structural alignments, based purely on the geometry of the protein structures, provide better alignments than pure sequence-based methods. Merging the structure and sequence information allows the construction of accurate profiles for distantly related groups. These structure-based profiles outperformed other sequence-based methods for finding distant homologs and were used to identify a putative class II cysteinyl-tRNA synthetase (CysRS) in several archaea that eluded previous annotation studies. Phylogenetic analysis showed the putative class II CysRSs to be a monophyletic group and homology modeling revealed a constellation of active site residues similar to that in the known class I CysRS.

Algorithms↗
PubMed2005

EST sequencing and time course microarray hybridizations identify more than 700 Medicago truncatula genes with developmental expression regulation in flowers and pods.

To evaluate the molecular mechanisms during pod and seed formation in legumes, starting with the development of reproductive organs, we constructed two cDNA libraries from developing flowers (MtFLOW) and pods including seeds (MtPOSE) of the model plant Medicago truncatula Gaertner. A total of 2,516 expressed sequence tags (ESTs) clustered into 1,776 nonredundant sequences (2k-set), which were annotated and assigned to functional classes. While about 30% of the ESTs encoded proteins of yet unknown function, typical annotations pointed to seed storage proteins, LTPs and lipoxygenases. The 2k-set was used to upgrade Mt6k-RIT microarrays (Küster et al. in J Biotechnol 108: 95, 2004) to Mt8k versions representing approximately 6,300 nonredundant M. truncatula genes. These were used to perform time course expression profiling studies based on hybridizations of samples that covered eight different developmental stages from flower buds to almost mature pods versus leaves as a common reference. About 180 up- and 70 downregulated genes were typically found for each stage and in total, 782 genes were either twofold up- or downregulated in at least one of the eight stages investigated. Based on this set, a combination of self-organizing map and hierarchical clustering revealed genes displaying expression regulation during characteristic stages of M. truncatula flower and pod development. Amongst those, several genes encoded proteins related to seed metabolism and development including novel regulators and proteins involved in signaling.

Expressed Sequence Tags↗
PubMed2002

Altered expression of novel genes in the cerebral cortex following experimental brain injury.

Damage to the cerebral cortex results in neurological impairments such as motor, attention, memory and executive dysfunctions. To examine the molecular mechanisms contributing to these deficits, mRNA expression was profiled using high-density cDNA microarray hybridization after experimental cortical impact injury in mice. The mRNA levels at 2 h, 6 h, 24 h, 3 days and 14 days after injury were compared with those of control animals. This revealed 86 annotated genes and 24 expression sequence tags (ESTs) as being differentially expressed with a 1.5-fold or greater change. Quantitative real-time PCR analysis was used to independently verify these results for selected genes. Seven functional classes of genes were found to be altered following injury, including transcription factors, signal transduction genes and inflammatory proteins. While a few of these genes have been previously reported to be differentially regulated following injury, the most of the genes have not been previously implicated in traumatic brain injury (TBI) pathophysiology. For example, consistent with previous reports, the transcription factor c-jun and the neurotrophic factor bdnf mRNA levels were altered as a result of TBI. Among the novel genes, the mRNA levels for the high mobility group protein 1 (hmg-1), the regulator of G-protein signaling 2 (rgs-2), the transforming growth factor beta inducible early growth response (tieg), the inhibitor of DNA binding 3 (id3), and the heterogeneous nuclear ribonucleoprotein H (hnrnp h) were changed following injury. The functional significance of these genes in neurite outgrowth, neuronal regeneration, and plasticity following injury are discussed.

Animals↗
PubMed2005

Adding some SPICE to DAS.

UNLABELLED: The distributed annotation system (DAS) defines a communication protocol used to exchange biological annotations. It is motivated by the idea that annotations should not be provided by single centralized databases but instead be spread over multiple sites. Data distribution, performed by DAS servers, is separated from visualization, which is carried out by DAS clients. The original DAS protocol was designed to serve annotation of genomic sequences. We have extended the protocol to be applicable to macromolecular structures. Here we present SPICE, a new DAS client that can be used to visualize protein sequence and structure annotations. AVAILABILITY: http://www.efamily.org.uk/software/dasclients/spice/

Algorithms↗
PubMed2005

ESTviewer: a web interface for visualizing mouse, rat, cattle, pig and chicken conserved ESTs in human genes and human alternatively spliced variants.

ESTviewer is a web application for interactively visualizing human gene structures, with emphasis on mammalian and avian expressed sequence tags (ESTs) that are conserved in the human genome and alternatively spliced (AS) variants. AS variants from the UCSC, Vega and PSEP annotations are presented in this application for comparison. EST data from six species, human, mouse, rat, cattle, pig and chicken, are mapped to the human genome to show cross-species EST conservation in annotated exonic and intronic regions. Cross-species EST conservation is evolutionarily and functionally important because it represents the effects of selection pressure on genic regions and transcriptome over evolutionary time. Emphatically, ESTviewer provides a convenient tool to compare highly conserved non-human ESTs and human AS variants. The application takes human gene accession Ids or coordinates of genomic sequences as inputs and presents annotated gene structures and their AS variants. In addition, the lengths and percentages of human genic regions covered by ESTs are displayed to show the level of EST coverage of different species. The percentages of the UCSC, Vega and PSEP annotated exons covered by ESTs of the six studied species are also displayed in the interface.

Animals↗
PubMed2002

Gene expression profiling of prolonged cold ischemia and reperfusion in murine heart transplants.

BACKGROUND: Heart transplantation causes complex changes in the biological homeostasis of the graft. Current knowledge is restricted to a few genes and regulation of certain factors involved in ischemia-reperfusion (I/R) injury. Efficient strategies to prevent I/R injury, however, require a better understanding of its mechanisms. Using cDNA microarrays, we investigated gene expression profiles of murine cardiac isografts. METHODS: For microarray hybridization experiments, chips with 8,734 individual target sequences were used. Messenger RNA was extracted from hearts subjected to warm ischemia and different time periods of reperfusion or to prolonged cold ischemia or warm ischemia and transplantation. Native hearts served as controls. RESULTS: A set of 68 sequences was regulated in all hearts. In addition, grafts without cold ischemia showed differential expression of 65 sequences, which were not found in hearts transplanted after cold storage, and which in turn had 38 sequences regulated and not detected in grafts without cold ischemia. Overall, approximately 50% of regulated transcripts are expressed sequence tags (ESTs) with unknown function. Annotated genes encoded immune modulators (20% of sequences), receptor proteins, structural proteins, and proteins involved in metabolism. CONCLUSION: Our data demonstrate expression profiles of hearts subjected to prolonged cold ischemia or transplantation in an isogeneic setting. We have defined functional complexes and detected a substantial amount of ESTs encoding novel proteins. These studies may provide a molecular basis for further functional experiments and may help identify potential targets for modulation of postischemic inflammation.

Animals↗
PubMed2005

Accurate prediction of orthologous gene groups in microbes.

We present a new computational method for the prediction of orthologous gene groups for microbial genomes based on the prediction of co-occurrences of homologous genes. The method is inspired by the observation that homologous genes are highly likely to be orthologous if their neighboring genes are also homologous. Based on co-occurrences of homologous genes, we have grouped the (predicted) operons of 77 selected sequenced microbial genomes so that operons of the same group are highly likely to be functionally similar or related. We then cluster the homologous genes in the same operon group so that genes of the same cluster are highly likely to be similar in terms of their sequences and functions, i.e., they are predicted to be orthologous genes. By comparing our predicted orthologous gene groups with the COG assignments and NCBI annotations, we conclude that our method is promising to provide more accurate and specific predictions than the existing methods.

Algorithms↗
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↗
PubMed2005

The latest buzz in comparative genomics.

A second species of fruit fly has just been added to the growing list of organisms with complete and annotated genome sequences. The publication of the Drosophila pseudoobscura sequence provides a snapshot of how genomes have changed over tens of millions of years and sets the stage for the analysis of more fly genomes.

Animals↗
PubMed2000

Genome annotation assessment in Drosophila melanogaster.

Computational methods for automated genome annotation are critical to our community's ability to make full use of the large volume of genomic sequence being generated and released. To explore the accuracy of these automated feature prediction tools in the genomes of higher organisms, we evaluated their performance on a large, well-characterized sequence contig from the Adh region of Drosophila melanogaster. This experiment, known as the Genome Annotation Assessment Project (GASP), was launched in May 1999. Twelve groups, applying state-of-the-art tools, contributed predictions for features including gene structure, protein homologies, promoter sites, and repeat elements. We evaluated these predictions using two standards, one based on previously unreleased high-quality full-length cDNA sequences and a second based on the set of annotations generated as part of an in-depth study of the region by a group of Drosophila experts. Although these standard sets only approximate the unknown distribution of features in this region, we believe that when taken in context the results of an evaluation based on them are meaningful. The results were presented as a tutorial at the conference on Intelligent Systems in Molecular Biology (ISMB-99) in August 1999. Over 95% of the coding nucleotides in the region were correctly identified by the majority of the gene finders, and the correct intron/exon structures were predicted for >40% of the genes. Homology-based annotation techniques recognized and associated functions with almost half of the genes in the region; the remainder were only identified by the ab initio techniques. This experiment also presents the first assessment of promoter prediction techniques for a significant number of genes in a large contiguous region. We discovered that the promoter predictors' high false-positive rates make their predictions difficult to use. Integrating gene finding and cDNA/EST alignments with promoter predictions decreases the number of false-positive classifications but discovers less than one-third of the promoters in the region. We believe that by establishing standards for evaluating genomic annotations and by assessing the performance of existing automated genome annotation tools, this experiment establishes a baseline that contributes to the value of ongoing large-scale annotation projects and should guide further research in genome informatics.

Alcohol Dehydrogenase↗
PubMed2006

Discovery of a substrate selectivity switch in tyrosine ammonia-lyase, a member of the aromatic amino acid lyase family.

Tyrosine ammonia-lyase (TAL) is a recently described member of the aromatic amino acid lyase family, which also includes phenylalanine (PAL) and histidine ammonia-lyases (HAL). TAL is highly selective for L-tyrosine, and synthesizes 4-coumaric acid as a protein cofactor or antibiotic precursor in microorganisms. In this report, we identify a single active site residue important for substrate selection in this enzyme family. Replacing the active site residue His89 with Phe in TAL completely switched its substrate selectivity from tyrosine to phenylalanine, thereby converting it into a highly active PAL. When a corresponding mutation was made in PAL, the enzyme lost PAL activity and gained TAL activity. The discovered substrate selectivity switch is a rare example of a complete alteration of substrate specificity by a single point mutation. We also show that the identity of the amino acid at the switch position can serve as a guide to predict substrate specificities of annotated aromatic amino acid lyases in genome sequences.

Amino Acid Sequence↗