Thermal stress responses in the bacterial biosphere of the Great Barrier Reef sponge, Rhopaloeides odorabile.

Marine sponges are diverse, abundant and provide a crucial coupling point between benthic and pelagic habitats due to their high filtration rates. They also harbour extensive microbial communities, with many microbial phylotypes found exclusively in sponge hosts and not in the seawater or surrounding environment, i.e. so-called sponge-specific clusters (SCs) or sponge- and coral-specific clusters (SCCs). We employed DNA (16S rRNA gene) and RNA (16S rRNA)-based amplicon pyrosequencing to investigate the effects of sublethal thermal stress on the bacterial biosphere of the Great Barrier Reef sponge Rhopaloeides odorabile. A total of 8381 operational taxonomic units (OTUs) (97% sequence similarity) were identified, affiliated with 32 bacterial phyla from seawater samples, 23 bacterial phyla from sponge DNA extracts and 18 bacterial phyla from sponge RNA extracts. Sublethal thermal stress (31°C) had no effect on the present and/or active portions of the R. odorabile bacterial community but a shift in the bacterial assemblage was observed in necrotic sponges. Over two-thirds of DNA and RNA sequences could be assigned to previously defined SCs/SCCs in healthy sponges whereas only 12% of reads from necrotic sponges could be assigned to SCs/SCCs. A rapid decline in host health over a 1°C temperature increment suggests that sponges such as R. odorabile may be highly vulnerable to the effects of global climate change.

NRED: a database of long noncoding RNA expression.

In mammals, thousands of long non-protein-coding RNAs (ncRNAs) (>200 nt) have recently been described. However, the biological significance and function of the vast majority of these transcripts remain unclear. We have constructed a public repository, the Noncoding RNA Expression Database (NRED), which provides gene expression information for thousands of long ncRNAs in human and mouse. The database contains both microarray and in situ hybridization data, much of which is described here for the first time. NRED also supplies a rich tapestry of ancillary information for featured ncRNAs, including evolutionary conservation, secondary structure evidence, genomic context links and antisense relationships. The database is available at http://jsm-research.imb.uq.edu.au/NRED, and the web interface enables both advanced searches and data downloads. Taken together, NRED should significantly advance the study and understanding of long ncRNAs, and provides a timely and valuable resource to the scientific community.

Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation.

The transcriptional networks that regulate embryonic stem (ES) cell pluripotency and lineage specification are the subject of considerable attention. To date such studies have focused almost exclusively on protein-coding transcripts. However, recent transcriptome analyses show that the mammalian genome contains thousands of long noncoding RNAs (ncRNAs), many of which appear to be expressed in a developmentally regulated manner. The functions of these remain untested. To identify ncRNAs involved in ES cell biology, we used a custom-designed microarray to examine the expression profiles of mouse ES cells differentiating as embryoid bodies (EBs) over a 16-d time course. We identified 945 ncRNAs expressed during EB differentiation, of which 174 were differentially expressed, many correlating with pluripotency or specific differentiation events. Candidate ncRNAs were identified for further characterization by an integrated examination of expression profiles, genomic context, chromatin state, and promoter analysis. Many ncRNAs showed coordinated expression with genomically associated developmental genes, such as Dlx1, Dlx4, Gata6, and Ecsit. We examined two novel developmentally regulated ncRNAs, Evx1as and Hoxb5/6as, which are derived from homeotic loci and share similar expression patterns and localization in mouse embryos with their associated protein-coding genes. Using chromatin immunoprecipitation, we provide evidence that both ncRNAs are associated with trimethylated H3K4 histones and histone methyltransferase MLL1, suggesting a role in epigenetic regulation of homeotic loci during ES cell differentiation. Taken together, our data indicate that long ncRNAs are likely to be important in processes directing pluripotency and alternative differentiation programs, in some cases through engagement of the epigenetic machinery.

Rapid identification of single nucleotide substitutions using SeqDoC.

Identification and characterization of nucleotide substitutions in DNA sequences for single nucleotide polymorphism or point mutation detection can be a time consuming and sometimes inaccurate process, particularly in relatively low-throughput situations where fully automated solutions may not be appropriate. SeqDoC provides a simple web-based application to simplify this identification process, by using direct subtractive comparison of the raw sequence traces to highlight differences characteristic of nucleotide substitutions. Sequencing artefacts, such as variable peak separation and signal strength, are compensated for with moving window normalisation functions, whereas the signal to noise ratio of the comparison trace is greatly enhanced by applying an algorithm to emphasise features associated with nucleotide substitutions. Analysis of the output is simple and intuitive, permitting rapid identification of points of difference between the reference and test sequence traces.

Genome-wide review of transcriptional complexity in mouse protein kinases and phosphatases.

BACKGROUND: Alternative transcripts of protein kinases and protein phosphatases are known to encode peptides with altered substrate affinities, subcellular localizations, and activities. We undertook a systematic study to catalog the variant transcripts of every protein kinase-like and phosphatase-like locus of mouse http://variant.imb.uq.edu.au. RESULTS: By reviewing all available transcript evidence, we found that at least 75% of kinase and phosphatase loci in mouse generate alternative splice forms, and that 44% of these loci have well supported alternative 5' exons. In a further analysis of full-length cDNAs, we identified 69% of loci as generating more than one peptide isoform. The 1,469 peptide isoforms generated from these loci correspond to 1,080 unique Interpro domain combinations, many of which lack catalytic or interaction domains. We also report on the existence of likely dominant negative forms for many of the receptor kinases and phosphatases, including some 26 secreted decoys (seven known and 19 novel: Alk, Csf1r, Egfr, Epha1, 3, 5,7 and 10, Ephb1, Flt1, Flt3, Insr, Insrr, Kdr, Met, Ptk7, Ptprc, Ptprd, Ptprg, Ptprl, Ptprn, Ptprn2, Ptpro, Ptprr, Ptprs, and Ptprz1) and 13 transmembrane forms (four known and nine novel: Axl, Bmpr1a, Csf1r, Epha4, 5, 6 and 7, Ntrk2, Ntrk3, Pdgfra, Ptprk, Ptprm, Ptpru). Finally, by mining public gene expression data (MPSS and microarrays), we confirmed tissue-specific expression of ten of the novel isoforms. CONCLUSION: These findings suggest that alternative transcripts of protein kinases and phosphatases are produced that encode different domain structures, and that these variants are likely to play important roles in phosphorylation-dependent signaling pathways.

Evidence for conservation and selection of upstream open reading frames suggests probable encoding of bioactive peptides.

BACKGROUND: Approximately 40% of mammalian mRNA sequences contain AUG trinucleotides upstream of the main coding sequence, with a quarter of these AUGs demarcating open reading frames of 20 or more codons. In order to investigate whether these open reading frames may encode functional peptides, we have carried out a comparative genomic analysis of human and mouse mRNA 'untranslated regions' using sequences from the RefSeq mRNA sequence database. RESULTS: We have identified over 200 upstream open reading frames which are strongly conserved between the human and mouse genomes. Consensus sequences associated with efficient initiation of translation are overrepresented at the AUG trinucleotides of these upstream open reading frames, while comparative analysis of their DNA and putative peptide sequences shows evidence of purifying selection. CONCLUSION: The occurrence of a large number of conserved upstream open reading frames, in association with features consistent with protein translation, strongly suggests evolutionary maintenance of the coding sequence and indicates probable functional expression of the peptides encoded within these upstream open reading frames.

PineappleDB: an online pineapple bioinformatics resource.

BACKGROUND: A world first pineapple EST sequencing program has been undertaken to investigate genes expressed during non-climacteric fruit ripening and the nematode-plant interaction during root infection. Very little is known of how non-climacteric fruit ripening is controlled or of the molecular basis of the nematode-plant interaction. PineappleDB was developed to provide the research community with access to a curated bioinformatics resource housing the fruit, root and nematode infected gall expressed sequences. DESCRIPTION: PineappleDB is an online, curated database providing integrated access to annotated expressed sequence tag (EST) data for cDNA clones isolated from pineapple fruit, root, and nematode infected root gall vascular cylinder tissues. The database currently houses over 5600 EST sequences, 3383 contig consensus sequences, and associated bioinformatic data including splice variants, Arabidopsis homologues, both MIPS based and Gene Ontology functional classifications, and clone distributions. The online resource can be searched by text or by BLAST sequence homology. The data outputs provide comprehensive sequence, bioinformatic and functional classification information. CONCLUSION: The online pineapple bioinformatic resource provides the research community with access to pineapple fruit and root/gall sequence and bioinformatic data in a user-friendly format. The search tools enable efficient data mining and present a wide spectrum of bioinformatic and functional classification information. PineappleDB will be of broad appeal to researchers investigating pineapple genetics, non-climacteric fruit ripening, root-knot nematode infection, crassulacean acid metabolism and alternative RNA splicing in plants.

SeqDoC: rapid SNP and mutation detection by direct comparison of DNA sequence chromatograms.

BACKGROUND: This paper describes SeqDoC, a simple, web-based tool to carry out direct comparison of ABI sequence chromatograms. This allows the rapid identification of single nucleotide polymorphisms (SNPs) and point mutations without the need to install or learn more complicated analysis software. RESULTS: SeqDoC produces a subtracted trace showing differences between a reference and test chromatogram, and is optimised to emphasise those characteristic of single base changes. It automatically aligns sequences, and produces straightforward graphical output. The use of direct comparison of the sequence chromatograms means that artefacts introduced by automatic base-calling software are avoided. Homozygous and heterozygous substitutions and insertion/deletion events are all readily identified. SeqDoC successfully highlights nucleotide changes missed by the Staden package 'tracediff' program. CONCLUSION: SeqDoC is ideal for small-scale SNP identification, for identification of changes in random mutagenesis screens, and for verification of PCR amplification fidelity. Differences are highlighted, not interpreted, allowing the investigator to make the ultimate decision on the nature of the change.

CATMA: a complete Arabidopsis GST database.

The Complete Arabidopsis Transcriptome Micro Array (CATMA) database contains gene sequence tag (GST) and gene model sequences for over 70% of the predicted genes in the Arabidopsis thaliana genome as well as primer sequences for GST amplification and a wide range of supplementary information. All CATMA GST sequences are specific to the gene for which they were designed, and all gene models were predicted from a complete reannotation of the genome using uniform parameters. The database is searchable by sequence name, sequence homology or direct SQL query, and is available through the CATMA website at http://www.catma.org/.

BACFinder: genomic localisation of large insert genomic clones based on restriction fingerprinting.

We have developed software that allows the prediction of the genomic location of a bacterial artificial chromosome (BAC) clone, or other large genomic clone, based on a simple restriction digest of the BAC. The mapping is performed by comparing the experimentally derived restriction digest of the BAC DNA with a virtual restriction digest of the whole genome sequence. Our trials indicate that this program identified the genomic regions represented by BAC clones with a degree of accuracy comparable to that of end-sequencing, but at considerably less cost. Although the program has been developed principally for use with Arabidopsis BACs, it should align large insert genomic clones to any fully sequenced genome.