Metabolic versatility of the Riftia pachyptila endosymbiont revealed through metagenomics.

The facultative symbiont of Riftia pachyptila, named here Candidatus Endoriftia persephone, has evaded culture to date, but much has been learned regarding this symbiosis over the past three decades since its discovery. The symbiont population metagenome was sequenced in order to gain insight into its physiology. The population genome indicates that the symbionts use a partial Calvin-Benson Cycle for carbon fixation and the reverse TCA cycle (an alternative pathway for carbon fixation) that contains an unusual ATP citrate lyase. The presence of all genes necessary for heterotrophic metabolism, a phosphotransferase system, and dicarboxylate and ABC transporters indicate that the symbiont can live mixotrophically. The metagenome has a large suite of signal transduction, defence (both biological and environmental) and chemotaxis mechanisms. The physiology of Candidatus Endoriftia persephone is explored with respect to functionality while associated with a eukaryotic host, versus free-living in the hydrothermal environment.

Alternative splicing of mouse transcription factors affects their DNA-binding domain architecture and is tissue specific.

BACKGROUND: Analyzing proteins in the context of all available genome and transcript sequence data has the potential to reveal functional properties not accessible through protein sequence analysis alone. To analyze the impact of alternative splicing on transcription factor (TF) protein structure, we constructed a comprehensive database of splice variants in the mouse transcriptome, called MouSDB3 containing 461 TF loci. RESULTS: Our analysis revealed that 62% of these loci in MouSDB3 have variant exons, compared to 29% of all loci. These variant TF loci contain a total of 324 alternative exons, of which 23% are in-frame. When excluded, 80% of in-frame alternative exons alter the domain architecture of the protein as computed by SMART (simple modular architecture research tool). Sixty-eight % of these exons directly affect the coding regions of domains important for TF function. Seventy-five % of the domains affected are DNA-binding domains. Tissue distribution analyses of variant mouse TFs reveal that they have more alternatively spliced forms in 14 of the 18 tissues analyzed when compared to all the loci in MouSDB3. Further, TF isoforms are homogenous within a given single tissue and are heterogeneous across different tissues, indicating their tissue specificity. CONCLUSIONS: Our study provides quantitative evidence that alternative splicing preferentially adds or deletes domains important to the DNA-binding function of the TFs. Analyses described here reveal the presence of tissue-specific alternative splicing throughout the mouse transcriptome. Our findings provide significant biological insights into control of transcription and regulation of tissue-specific gene expression by alternative splicing via creation of tissue-specific TF isoforms.

Universal Reference RNA as a standard for microarray experiments.

BACKGROUND: Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR), developed with the goal of providing hybridization signal at each microarray probe location (spot). Measuring signal at each spot as the ratio of experimental RNA to reference RNA targets, rather than relying on absolute signal intensity, decreases variability by normalizing signal output in any two-color hybridization experiment. RESULTS: Human, mouse and rat URR (UHRR, UMRR and URRR, respectively) were prepared from pools of RNA derived from individual cell lines representing different tissues. A variety of microarrays were used to determine percentage of spots hybridizing with URR and producing signal above a user defined threshold (microarray coverage). Microarray coverage was consistently greater than 80% for all arrays tested. We confirmed that individual cell lines contribute their own unique set of genes to URR, arguing for a pool of RNA from several cell lines as a better configuration for URR as opposed to a single cell line source for URR. Microarray coverage comparing two separately prepared batches each of UHRR, UMRR and URRR were highly correlated (Pearson's correlation coefficients of 0.97). CONCLUSION: Results of this study demonstrate that large quantities of pooled RNA from individual cell lines are reproducibly prepared and possess diverse gene representation. This type of reference provides a standard for reducing variation in microarray experiments and allows more reliable comparison of gene expression data within and between experiments and laboratories.