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PubMed2005

Applications of DNA tiling arrays to experimental genome annotation and regulatory pathway discovery.

Microarrays have become a popular and important technology for surveying global patterns in gene expression and regulation. A number of innovative experiments have extended microarray applications beyond the measurement of mRNA expression levels, in order to uncover aspects of large-scale chromosome function and dynamics. This has been made possible due to the recent development of tiling arrays, where all non-repetitive DNA comprising a chromosome or locus is represented at various sequence resolutions. Since tiling arrays are designed to contain the entire DNA sequence without prior consultation of existing gene annotation, they enable the discovery of novel transcribed sequences and regulatory elements through the unbiased interrogation of genomic loci. The implementation of such methods for the global analysis of large eukaryotic genomes presents significant technical challenges. Nonetheless, tiling arrays are expected to become instrumental for the genome-wide identification and characterization of functional elements. Combined with computational methods to relate these data and map the complex interactions of transcriptional regulators, tiling array experiments can provide insight toward a more comprehensive understanding of fundamental molecular and cellular processes.

Gene Expression Profiling↗
PubMed2006

A novel nuclear-encoded mitochondrial poly(A) polymerase PAPD1 is a potential candidate gene for the extreme obesity related phenotypes in mammals.

People with obesity, especially extreme obesity, are at risk for many health problems. However, the responsible genes remain unknown in >95% of severe obesity cases. Our previous genome-wide scan of Wagyu x Limousin F2 cattle crosses with extreme phenotypes revealed a molecular marker significantly associated with intramuscular fat deposition. Characterization of this marker showed that it is orthologous to the human gene KIAA1462 located on HSA10p11.23, where a major quantitative trait locus for morbid obesity has been reported. The newly identified mitochondrial poly(A) polymerase associated domain containing 1 (PAPD1) gene, which is located near this marker, is particularly interesting because the polymerase is required for the polyadenylation and stabilization of mammalian mitochondrial mRNAs. In the present study, both cDNA and genomic DNA sequences were annotated for the bovine PAPD1 gene and ten genetic markers were detected in the promoter and exon 1 region. Among seven markers assayed on approximately 250 Wagyu x Limousin F2 animals, two single nucleotide polymorphisms (SNPs) in the promoter region were significantly associated with intramuscular fat (P<0.05). However, there was a significant interaction (P<0.05) between a third SNP, which causes an amino acid change in coding exon 1, and each of these two promoter SNPs on intramuscular fat deposition. In particular, the differences between double heterozygous animals at two polymorphic sites and the slim genotype animals exceeded 2.3 standard deviations for the trait in both cases. Our study provides evidence for a new mechanism--the involvement of compound heterosis in extreme obesity, which warrants further examination.

Adipose Tissue↗
PubMed2005

Organization of the sex-ratio meiotic drive region in Drosophila simulans.

Sex-ratio meiotic drive is the preferential transmission of the X chromosome by XY males, which occurs in several Drosophila species and results in female-biased progeny. Although the trait has long been known to exist, its molecular basis remains completely unknown. Here we report a fine-mapping experiment designed to characterize the major drive locus on a sex-ratio X chromosome of Drosophila simulans originating from the Seychelles (XSR6). This primary locus was found to contain two interacting elements at least, both of which are required for drive expression. One of them was genetically tracked to a tandem duplication containing six annotated genes (Trf2, CG32712, CG12125, CG1440, CG12123, org-1), and the other to a candidate region located approximately 110 kb away and spanning seven annotated genes. RT-PCR showed that all but two of these genes were expressed in the testis of both sex-ratio and standard males. In situ hybridization to polytene chromosomes revealed a complete association of the duplication with the sex-ratio trait in random samples of X chromosomes from Madagascar and Reunion.

Animals↗
PubMed2005

A molecular-capsular-type prediction system for 90 Streptococcus pneumoniae serotypes using partial cpsA-cpsB sequencing and wzy- or wzx-specific PCR.

In a previous study, a molecular capsular type (MCT) prediction system for 51 Streptococcus pneumoniae serotypes was developed based on a combination of partial cpsA-cpsB sequencing and serotype(s)/group(s)-specific PCR. In this study, another 169 S. pneumoniae isolates were added to the existing database of 427 isolates, including representatives of all 39 serotypes not previously studied. In addition to the authors' own limited sequence data for all 90 serotypes, cpsA-cpsB sequence data published by the S. pneumoniae capsular loci-sequencing group (http://www.sanger.ac.uk/Projects/S_pneumoniae/CPS/) at the Sanger Institute or available from GenBank were incorporated into the database. All serotypes, except 25A, were represented by at least two isolates. The number of sequence types identified was 138, of which 110 corresponded to single conventional serotypes (CSs); of these, 57 were represented by two or more isolates. Twenty-six sequence types were shared by between two and four CSs. To resolve these shared cpsA-cpsB sequence types and increase the discriminatory power of our system, the genes encoding the capsular polysaccharide flippase (wzx) and polymerase (wzy) were annotated and 24 new serotype(s)/group(s)-specific PCRs targeting wzy and two targeting wzx were designed. Using both cpsA-cpsB sequencing and wzx/wzy PCR, MCT correctly predicted the CSs of 516 (73 %) and the serogroup of an additional 155 (22 %) of the 708 isolates evaluated. For 5 % of isolates, MCT could not distinguish between members of five serotype pairs (37 isolates) containing members of different serogroups. Although further study of the relationship between MCT and CS is needed, this system now allows serotype or serogroup identification of 95 % of S. pneumoniae isolates.

Bacterial Capsules↗
PubMed2005

Tiling microarray analysis of rice chromosome 10 to identify the transcriptome and relate its expression to chromosomal architecture.

BACKGROUND: Sequencing and annotation of the genome of rice (Oryza sativa) have generated gene models in numbers that top all other fully sequenced species, with many lacking recognizable sequence homology to known genes. Experimental evaluation of these gene models and identification of new models will facilitate rice genome annotation and the application of this knowledge to other more complex cereal genomes. RESULTS: We report here an analysis of the chromosome 10 transcriptome of the two major rice subspecies, japonica and indica, using oligonucleotide tiling microarrays. This analysis detected expression of approximately three-quarters of the gene models without previous experimental evidence in both subspecies. Cloning and sequence analysis of the previously unsupported models suggests that the predicted gene structure of nearly half of those models needs improvement. Coupled with comparative gene model mapping, the tiling microarray analysis identified 549 new models for the japonica chromosome, representing an 18% increase in the annotated protein-coding capacity. Furthermore, an asymmetric distribution of genome elements along the chromosome was found that coincides with the cytological definition of the heterochromatin and euchromatin domains. The heterochromatin domain appears to associate with distinct chromosome level transcriptional activities under normal and stress conditions. CONCLUSION: These results demonstrated the utility of genome tiling microarray in evaluating annotated rice gene models and in identifying novel transcriptional units. The tiling microarray sanalysis further revealed a chromosome-wide transcription pattern that suggests a role for transposable element-enriched heterochromatin in shaping global transcription in response to environmental changes in rice.

Chromosomes, Plant↗
PubMed1998

Molecular biology and pathogenicity of mycoplasmas.

The recent sequencing of the entire genomes of Mycoplasma genitalium and M. pneumoniae has attracted considerable attention to the molecular biology of mycoplasmas, the smallest self-replicating organisms. It appears that we are now much closer to the goal of defining, in molecular terms, the entire machinery of a self-replicating cell. Comparative genomics based on comparison of the genomic makeup of mycoplasmal genomes with those of other bacteria, has opened new ways of looking at the evolutionary history of the mycoplasmas. There is now solid genetic support for the hypothesis that mycoplasmas have evolved as a branch of gram-positive bacteria by a process of reductive evolution. During this process, the mycoplasmas lost considerable portions of their ancestors' chromosomes but retained the genes essential for life. Thus, the mycoplasmal genomes carry a high percentage of conserved genes, greatly facilitating gene annotation. The significant genome compaction that occurred in mycoplasmas was made possible by adopting a parasitic mode of life. The supply of nutrients from their hosts apparently enabled mycoplasmas to lose, during evolution, the genes for many assimilative processes. During their evolution and adaptation to a parasitic mode of life, the mycoplasmas have developed various genetic systems providing a highly plastic set of variable surface proteins to evade the host immune system. The uniqueness of the mycoplasmal systems is manifested by the presence of highly mutable modules combined with an ability to expand the antigenic repertoire by generating structural alternatives, all compressed into limited genomic sequences. In the absence of a cell wall and a periplasmic space, the majority of surface variable antigens in mycoplasmas are lipoproteins. Apart from providing specific antimycoplasmal defense, the host immune system is also involved in the development of pathogenic lesions and exacerbation of mycoplasma induced diseases. Mycoplasmas are able to stimulate as well as suppress lymphocytes in a nonspecific, polyclonal manner, both in vitro and in vivo. As well as to affecting various subsets of lymphocytes, mycoplasmas and mycoplasma-derived cell components modulate the activities of monocytes/macrophages and NK cells and trigger the production of a wide variety of up-regulating and down-regulating cytokines and chemokines. Mycoplasma-mediated secretion of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-1 (IL-1), and IL-6, by macrophages and of up-regulating cytokines by mitogenically stimulated lymphocytes plays a major role in mycoplasma-induced immune system modulation and inflammatory responses.

Animals↗
PubMed2006

SNPSplicer: systematic analysis of SNP-dependent splicing in genotyped cDNAs.

Functional annotation of SNPs (as generated by HapMap (http://www.hapmap.org) for instance) is a major challenge. SNPs that lead to single amino acid substitutions, stop codons, or frameshift mutations can be readily interpreted, but these represent only a fraction of known SNPs. Many SNPs are located in sequences of splicing relevance-the canonical splice site consensus sequences, exonic and intronic splice enhancers or silencers (exonic splice enhancer [ESE], intronic splice enhancer [ISE], exonic splicing silencer [ESS], and intronic splicing silencer [ISS]), and others. We propose using sets of matching DNA and complementary DNA (cDNA) as a screening method to investigate the potential splice effects of SNPs in RT-PCR experiments with tissue material from genotyped sources. We have developed a software solution (SNPSplicer; http://www.ikmb.uni-kiel.de/snpsplicer) that aids in the rapid interpretation of such screening experiments. The utility of the approach is illustrated for SNPs affecting the donor splice sites (rs2076530:A>G, rs3816989:G>A) leading to the use of a cryptic splice site and exon skipping, respectively, and an exonic splice enhancer SNP (rs2274987:C/T), leading to inclusion of a new exon. We anticipate that this methodology may help in the functional annotation of SNPs in a more high-throughput fashion.

Alternative Splicing↗
PubMed2004

Discovery of a thermostable Baeyer-Villiger monooxygenase by genome mining.

Baeyer-Villiger monooxygenases represent useful biocatalytic tools, as they can catalyze reactions which are difficult to achieve using chemical means. However, only a limited number of these atypical monooxygenases are available in recombinant form. Using a recently described protein sequence motif, a putative Baeyer-Villiger monooxygenase (BVMO) was identified in the genome of the thermophilic actinomycete Thermobifida fusca. Heterologous expression of the respective protein in Escherichia coli and subsequent enzyme characterization showed that it indeed represents a BVMO. The NADPH-dependent and FAD-containing monooxygenase is active with a wide range of aromatic ketones, while aliphatic substrates are also converted. The best substrate discovered so far is phenylacetone (k(cat) = 1.9 s(-1), K(M) = 59 microM). The enzyme exhibits moderate enantioselectivity with alpha-methylphenylacetone (enantiomeric ratio of 7). In addition to Baeyer-Villiger reactions, the enzyme is able to perform sulfur oxidations. Different from all known BVMOs, this newly identified biocatalyst is relatively thermostable, displaying an activity half-life of 1 day at 52 degrees C. This study demonstrates that, using effective annotation tools, genomes can efficiently be exploited as a source of novel BVMOs.

Actinomycetales↗
PubMed2004

Comparative organization of wheat homoeologous group 3S and 7L using wheat-rice synteny and identification of potential markers for genes controlling xanthophyll content in wheat.

EST and genomic DNA sequencing efforts for rice and wheat have provided the basis for interpreting genome organization and evolution. In this study we have used EST and genomic sequencing information and a bioinformatic approach in a two-step strategy to align portions of the wheat and rice genomes. In the first step, wheat ESTs were used to identify rice orthologs and it was shown that wheat 3S and rice 1 contain syntenic units with intrachromosomal rearrangements. Further analysis using anchored rice contiguous sequences and TBLASTX alignments in a second alignment step showed interruptions by orthologous genes that map elsewhere in the wheat genome. This indicates that gene content and order is not as conserved as large chromosomal blocks as previously predicted. Similarly, chromosome 7L contains syntenic units with rice 6 and 8 but is interrupted by combinations of intrachromosomal and interchromosomal rearrangements involving syntenic units and single gene orthologs from other rice chromosome groups. We have used the rice sequence annotations to identify genes that can be used to develop markers linked to biosynthetic pathways on 3BS controlling xanthophyll production in wheat and thus involved in determining flour colour.

Amino Acid Sequence↗
PubMed2004

Efficient filtering methods for clustering cDNAs with spliced sequence alignment.

MOTIVATION: Clustering sequences of a full-length cDNA library into alternative splice form candidates is a very important problem. RESULTS: We developed a new efficient algorithm to cluster sequences of a full-length cDNA library into alternative splice form candidates. Current clustering algorithms for cDNAs tend to produce too many clusters containing incorrect splice form candidates. Our algorithm is based on a spliced sequence alignment algorithm that considers splice sites. The spliced sequence alignment algorithm is a variant of an ordinary dynamic programming algorithm, which requires O(nm) time for checking a pair of sequences where n and m are the lengths of the two sequences. Since the time bound is too large to perform all-pair comparison for a large set of sequences, we developed new techniques to reduce the computation time without affecting the accuracy of the output clusters. Our algorithm was applied to 21 076 mouse cDNA sequences of the FANTOM 1.10 database to examine its performance and accuracy. In these experiments, we achieved about 2-12-fold speedup against a method using only a traditional hash-based technique. Moreover, without using any information of the mouse genome sequence data or any gene data in public databases, we succeeded in listing 87-89% of all the clusters that biologists have annotated manually. AVAILABILITY: We provide a web service for cDNA clustering located at https://access.obigrid.org/ibm/cluspa/, for which registration for the OBIGrid (http://www.obigrid.org) is required.

Algorithms↗
PubMed2006

ARFA: a program for annotating bacterial release factor genes, including prediction of programmed ribosomal frameshifting.

UNLABELLED: Correct annotation of genes encoding release factors in bacterial genomes is often complicated by utilization of +1 programmed ribosomal frameshifting during synthesis of release factor 2, RF2. In the absence of robust computational approaches for predicting ribosomal frameshifting, the success of proper annotation depends on annotators' familiarity with this phenomenon. Here we describe a novel computer tool that allows automatic discrimination of genes encoding class-I bacterial release factors, RF1, RF2 and RFH. Most usefully, this program identifies and automatically annotates +1 frameshifting in RF2 encoding genes. Comparison of ARFA performance with existing annotations of bacterial genomes revealed that only 20% of RF2 genes utilizing ribosomal frameshifting during their expression are annotated correctly. AVAILABILITY: The PHP based web interface of ARFA and the source code are located at http://recode.genetics.utah.edu/arfa

Base Sequence↗
PubMed1994

The Ribonuclease P database.

The Ribonuclease P Sequence database is a compilation of RNase P sequences, sequence alignments, secondary structures, three-dimensional models, and accessory information. In its initial form, the database contains information on RNase P RNA in bacteria and archaea, and RNase P protein in bacteria. The sequences themselves are presented phylogenetically ordered and aligned. The database also contains secondary structures of bacterial and archaeal RNAs, including specially annotated 'reference' secondary structures of Escherichia coli and Bacillus subtilis RNase P RNAs, a minimum phylogenetic consensus structure, and coordinates for models of three-dimensional structure.

Bacillus subtilis↗
PubMed2003

Refined annotation of the Arabidopsis genome by complete expressed sequence tag mapping.

Expressed sequence tags (ESTs) currently encompass more entries in the public databases than any other form of sequence data. Thus, EST data sets provide a vast resource for gene identification and expression profiling. We have mapped the complete set of 176,915 publicly available Arabidopsis EST sequences onto the Arabidopsis genome using GeneSeqer, a spliced alignment program incorporating sequence similarity and splice site scoring. About 96% of the available ESTs could be properly aligned with a genomic locus, with the remaining ESTs deriving from organelle genomes and non-Arabidopsis sources or displaying insufficient sequence quality for alignment. The mapping provides verified sets of EST clusters for evaluation of EST clustering programs. Analysis of the spliced alignments suggests corrections to current gene structure annotation and provides examples of alternative and non-canonical pre-mRNA splicing. All results of this study were parsed into a database and are accessible via a flexible Web interface at http://www.plantgdb.org/AtGDB/.

Alternative Splicing↗
PubMed2003

GeneFizz: A web tool to compare genetic (coding/non-coding) and physical (helix/coil) segmentations of DNA sequences. Gene discovery and evolutionary perspectives.

The GeneFizz (http://pbga.pasteur.fr/GeneFizz) web tool permits the direct comparison between two types of segmentations for DNA sequences (possibly annotated): the coding/non-coding segmentation associated with genomic annotations (simple genes or exons in split genes) and the physics-based structural segmentation between helix and coil domains (as provided by the classical helix-coil model). There appears to be a varying degree of coincidence for different genomes between the two types of segmentations, from almost perfect to non-relevant. Following these two extremes, GeneFizz can be used for two purposes: ab initio physics-based identification of new genes (as recently shown for Plasmodium falciparum) or the exploration of possible evolutionary signals revealed by the discrepancies observed between the two types of information.

Algorithms↗
PubMed2003

Multi-species sequence comparison: the next frontier in genome annotation.

Multi-species comparisons of DNA sequences are more powerful for discovering functional sequences than pairwise DNA sequence comparisons. Most current computational tools have been designed for pairwise comparisons, and efficient extension of these tools to multiple species will require knowledge of the ideal evolutionary distance to choose and the development of new algorithms for alignment, analysis of conservation, and visualization of results.

Animals↗
PubMed2005

Ribosome builder: a software project to simulate the ribosome.

The Ribosome Builder is a software project that provides tools and techniques to create dynamic models of macromolecular systems from the rapidly growing numbers of atomic structural models. It includes a computer program that allows the user to assemble the multiple molecular components within a 3D space and to define the hypothetical interactions of these components with the initial goal of understanding protein translation at an atomic level of detail. The program employs a simplified molecular dynamics forcefield that can simulate the long time-scale events, such as docking of translation factors and mRNA translocation. An embedded scripting language and Application Programming Interface (API) enable the creation of Steered Molecular Dynamics (SMD) simulations through the programmable application of external forces and torques on atoms and bonds. A graphical interface is provided for displaying and interacting with models, recording movies of molecular dynamics movements, and creating annotated 3D simulations of complex macromolecular events. Initial applications of the project include simulation of tetraloop folding, docking of an mRNA on the 30S subunit and a schematic simulation of the translation elongation cycle. The program is an open source project released under the GNU public license.

Computer Simulation↗
PubMed2005

Rv0216, a conserved hypothetical protein from Mycobacterium tuberculosis that is essential for bacterial survival during infection, has a double hotdog fold.

The Mycobacterium tuberculosis genome contains about 4000 genes, of which approximately a third code for proteins of unknown function or are classified as conserved hypothetical proteins. We have determined the three-dimensional structure of one of these, the rv0216 gene product, which has been shown to be essential for M. tuberculosis growth in vivo. The structure exhibits the greatest similarity to bacterial and eukaryotic hydratases that catalyse the R-specific hydration of 2-enoyl coenzyme A. However, only part of the catalytic machinery is conserved in Rv0216 and it showed no activity for the substrate crotonyl-CoA. The structure of Rv0216 allows us to assign new functional annotations to a family of seven other M. tuberculosis proteins, a number if which are essential for bacterial survival during infection and growth.

Acyl Coenzyme A↗
PubMed2006

EGenBio: a data management system for evolutionary genomics and biodiversity.

BACKGROUND: Evolutionary genomics requires management and filtering of large numbers of diverse genomic sequences for accurate analysis and inference on evolutionary processes of genomic and functional change. We developed Evolutionary Genomics and Biodiversity (EGenBio; http://egenbio.lsu.edu) to begin to address this. DESCRIPTION: EGenBio is a system for manipulation and filtering of large numbers of sequences, integrating curated sequence alignments and phylogenetic trees, managing evolutionary analyses, and visualizing their output. EGenBio is organized into three conceptual divisions, Evolution, Genomics, and Biodiversity. The Genomics division includes tools for selecting pre-aligned sequences from different genes and species, and for modifying and filtering these alignments for further analysis. Species searches are handled through queries that can be modified based on a tree-based navigation system and saved. The Biodiversity division contains tools for analyzing individual sequences or sequence alignments, whereas the Evolution division contains tools involving phylogenetic trees. Alignments are annotated with analytical results and modification history using our PRAED format. A miscellaneous Tools section and Help framework are also available. EGenBio was developed around our comparative genomic research and a prototype database of mtDNA genomes. It utilizes MySQL-relational databases and dynamic page generation, and calls numerous custom programs. CONCLUSION: EGenBio was designed to serve as a platform for tools and resources to ease combined analysis in evolution, genomics, and biodiversity.

Animals↗