4D bioinformatics: a new look at the ribosome as an example

We have adapted the Java Molecular Viewer (JMV) to virtual reality display environments, through a number of extensions to the Java 3D code. Phylogenetic information derived from multiple alignments (temporal information) can be overlaid onto molecule structures (spatial information). The number of sequences included in the underlying multiple alignment can be changed instantaneously, resulting in dynamical updates of the displayed information. JMV was also extended to handle an infinite number of objects (molecules) in the same display. The objects can be manipulated in six degrees of freedom simultaneously or independently. We have used the small subunit ribosomal RNA to demonstrate the system (http:// cave.ucalgary.ca), which can be used for any molecule with a resolved structure.     

High spontaneous mutation rate in the hyperthermophilic archaeon Sulfolobus solfataricus is mediated by transposable elements

We have isolated uracil-auxotrophic mutants of the hyperthermophilic archaeon Sulfolobus solfataricus in order to explore the genomic stability and mutational frequencies of this organism and to identify complementable recipients for a selectable genetic transformation system. Positive selection of spontaneous mutants resistant to 5-fluoroorotate yielded uracil auxotrophs with frequencies of between 10(-4) and 10(-5) per sensitive, viable cell. Four different, nonhomologous insertion sequences (ISs) were identified at different positions within the chromosomal pyrEF locus of these mutants. They ranged in size from 1,058 to 1,439 bp and possessed properties typical of known transposable elements, i.e., terminal inverted repeats, flanking duplicated target sequences, and putative transposase genes encoding motifs that are indicative of the IS4-IS5 IS element families. Between 12 and 25 copies of each IS element were found in chromosomal DNAs by Southern analyses. While characteristic fingerprint patterns created by IS element-specific probes were observed with genomic DNA of different S. solfataricus strains, no homologous sequences were identified in DNA of other well-characterized strains of the order Sulfolobales.     

Comparative analysis of metagenomes from three methanogenic hydrocarbon-degrading enrichment cultures with 41 environmental samples

Methanogenic hydrocarbon metabolism is a key process in subsurface oil reservoirs and hydrocarbon-contaminated environments and thus warrants greater understanding to improve current technologies for fossil fuel extraction and bioremediation. In this study, three hydrocarbon-degrading methanogenic cultures established from two geographically distinct environments and incubated with different hydrocarbon substrates (added as single hydrocarbons or as mixtures) were subjected to metagenomic and 16S rRNA gene pyrosequencing to test whether these differences affect the genetic potential and composition of the communities. Enrichment of different putative hydrocarbon-degrading bacteria in each culture appeared to be substrate dependent, though all cultures contained both acetate- and H₂-utilizing methanogens. Despite differing hydrocarbon substrates and inoculum sources, all three cultures harbored genes for hydrocarbon activation by fumarate addition (bssA, assA, nmsA) and carboxylation (abcA, ancA), along with those for associated downstream pathways (bbs, bcr, bam), though the cultures incubated with hydrocarbon mixtures contained a broader diversity of fumarate addition genes. A comparative metagenomic analysis of the three cultures showed that they were functionally redundant despite their enrichment backgrounds, sharing multiple features associated with syntrophic hydrocarbon conversion to methane. In addition, a comparative analysis of the culture metagenomes with those of 41 environmental samples (containing varying proportions of methanogens) showed that the three cultures were functionally most similar to each other but distinct from other environments, including hydrocarbon-impacted environments (for example, oil sands tailings ponds and oil-affected marine sediments). This study provides a basis for understanding key functions and environmental selection in methanogenic hydrocarbon-associated communities.     

Semantic Web Service provision: a realistic framework for Bioinformatics programmers

Several semantic Web Services clients for Bioinformatics have been released, but to date no support systems for service providers have been described. We have created a framework ('MobyServlet') that very simply allows an existing Java application to conform to the MOBY-S semantic Web Services protocol. Using an existing Java program for codon-pair bias determination as an example, we enumerate the steps required for MOBY-S compliance. With minimal programming effort, such a deployment has the advantages of: (1) wider exposure to the user community by automatic inclusion in all MOBY-S client programs and (2) automatic interoperability with other MOBY-S services for input and output. Complex on-line analysis will become easier for biologists as more developers adopt MOBY-S. AVAILABILITY: The framework and documentation are freely available from the Java developer's section of http://www.biomoby.org/.     

Bioinformatics visualization and integration with open standards: the Bluejay genomic browser

We have created a new Java-based integrated computational environment for the exploration of genomic data, called Bluejay. The system is capable of using almost any XML file related to genomic data. Non-XML data sources can be accessed via a proxy server. Bluejay has several features, which are new to Bioinformatics, including an unlimited semantic zoom capability, coupled with Scalable Vector Graphics (SVG) outputs; an implementation of the XLink standard, which features access to MAGPIE Genecards as well as any BioMOBY service accessible over the Internet; and the integration of gene chip analysis tools with the functional assignments. The system can be used as a signed web applet, Web Start, and a local stand-alone application, with or without connection to the Internet. It is available free of charge and as open source via http://bluejay.ucalgary.ca.     

Identification and functional analysis of a novel von Willebrand factor mutation in a family with type 2A von Willebrand disease

von Willebrand factor (VWF) is essential for normal hemostasis. VWF gene mutations cause the hemorrhagic von Willebrand disease (VWD). In this study, a 9-year-old boy was diagnosed as type 2A VWD, based on a history of abnormal bleeding, low plasma VWF antigen and activity, low plasma factor VIII activity, and lack of plasma high-molecular-weight (HMW) VWF multimers. Sequencing analysis detected a 6-bp deletion in exon 28 of his VWF gene, which created a mutant lacking D1529V1530 residues in VWF A2 domain. This mutation also existed in his family members with abnormal bleedings but not in >60 normal controls. In transfected HEK293 cells, recombinant VWF ΔD1529V1530 protein had markedly reduced levels in the conditioned medium (42±4% of wild-type (WT) VWF, p<0.01). The mutant VWF in the medium had less HMW multimers. In contrast, the intracellular levels of the mutant VWF in the transfected cells were significantly higher than that of WT (174±29%, p<0.05), indicating intracellular retention of the mutant VWF. In co-transfection experiments, the mutant reduced WT VWF secretion from the cells. By immunofluorescence staining, the retention of the mutant VWF was identified within the endoplasmic reticulum (ER). Together, we identified a unique VWF mutation responsible for the bleeding phenotype in a patient family with type 2A VWD. The mutation impaired VWF trafficking through the ER, thereby preventing VWF secretion from the cells. Our results illustrate the diversity of VWF gene mutations, which contributes to the wide spectrum of VWD.     

A comparative, BAC end sequence enabled map of the genome of the American mink (Neovison vison)

In this report we present the results of the analysis of approximately 2.7 Mb of genomic information for the American mink (Neovison vison) derived through BAC end sequencing. Our study, which encompasses approximately 1/1000^th of the mink genome, suggests that simple sequence repeats (SSRs) are less common in the mink than in the human genome, whereas the average GC content of the mink genome is slightly higher than that of its human counterpart. The 2.7 Mb mink genomic dataset also contained 2,416 repeat elements (retroids and DNA transposons) occupying almost 31% of the sequence space. Among repeat elements, LINEs were over-represented and endogenous viruses (aka LTRs) under-represented in comparison to the human genome. Finally, we present a virtual map of the mink genome constructed with reference to the human and canine genome assemblies using a comparative genomics approach and incorporating over 200 mink BESs with unique hits to the human genome.     

NUCLEOTIDE SEQUENCES OF SMALL-SUBUNIT AND INTERNAL TRANSCRIBED SPACER REGIONS OF NUCLEAR rRNA GENES SUPPORT THE AUTONOMY OF SOME GENERA OF THE GELIDIALES (RHODOPHYTA)

The lack of homogeneity in all previously proposed, distinguishing characteristics has left the relationships of taxa within the Gelidiales as one of the most enduring taxonomic uncertainties in the Rhodophyta. Although a precise knowledge of the taxonomy of commercially harvested members of the Gelidiales would assist resource management, agronomic practices, and marketing, even the distinction between two major groups, Gelidium and Pterocladia, has long remained controversial. In this study, the 18S ribosomal RNA (rRNA), internal transcribed spacer 1 (ITS1), 5.8S rRNA, and ITS2 regions of Gelidium latifolium (Greville) Bornet et Thuret, G. sesquipedale (Clemente) Thuret in Bornet et Thuret, G. vagum Okamura, Pterocladia lucida (Brown ex Turner) J. Agardh, and a recent segregate from Pterocladia, Pterocladiella capillacea (Gmelin) Santelices et Hommersand, were sequenced and analyzed. The ITS1, 5.8S rRNA, and ITS2 regions of G. arbuscula (Montagne) B=orgesen, G. canariensis (Grunow) Seoane-Camba, G. capense (Gmelin) Silva, and G. pristoides (Turner) Kützing were also sequenced. Phylogenetic analyses based on the 18S rRNA genes from four Gelidium species, Pterocladia lucida, and Pterocladiella capillacea , compared with 18S rRNA genes from several other red algae confirmed the division between Gelidium and the Pterocladia/Pterocladiella isolates and were consistent with the recently proposed segregation of Pterocladiella from Pterocladia. Analyses based on the ITS regions of seven Gelidium species, Pterocladia lucida, and Pterocladiella capillacea were completely consistent with the conclusions drawn from the 18S rRNA data. There were extensive length and sequence differences between Gelidium and Pterocladia/Pterocladiella . In addition, there were larger sequence differences between Pterocladiella and Pterocladia than exist among Gelidium isolates, in keeping with the recently proposed separation of the former two taxa.