Molecular Biology

Background: The organic solute transporter (OSTalpha-OSTbeta) is a heteromeric transporter that is expressed on the basolateral membrane of epithelium in intestine, kidney, liver, testis and adrenal gland and facilitates efflux of bile acids and other steroid solutes. Both subunits are required for plasma membrane localization of the functional transporter but it is unclear how and where the subunits interact and whether glycosylation is required for functional activity. We sought to examine these questions for the human OSTalpha-OSTbeta transporter using the human hepatoma cell line, HepG2, and COS7 cells transfected with constructs of human OSTalpha-FLAG and OSTbeta-Myc. Results: Tunicamycin treatment demonstrated that human OSTalpha is glycosylated. In COS7 cells Western blotting identified the unglycosylated form (~31 kD), the core precursor form (~35 kD), and the mature, complex glycoprotein (~40 kD). Immunofluorescence of both cells indicated that, in the presence of OSTbeta, the alpha subunit could still be expressed on the plasma membrane after tunicamycin treatment. Furthermore, the functional uptake of 3H-estrone sulfate was unchanged in the absence of N-glycosylation. Co-immunoprecipitation indicates that the immature form of OSTalpha interact with OSTbeta. However, immunoprecipitation of OSTbeta using an anti-Myc antibody did not co-precipitate the mature, complex glycosylated form of OSTalpha, suggesting that the primary interaction occurs early in the biosynthetic pathway and may be transient. Conclusions: In conclusion, human OSTalpha is a glycoprotein that requires interaction with OSTbeta to reach the plasma membrane. However, glycosylation of OSTalpha is not necessary for interaction with the beta subunit or for membrane localization or function of the heteromeric transporter.

Background: Because of its polygenic nature, Alzheimer's disease (AD) is believed to be caused not by defects in single genes, but rather by variations in a large number of genes and their complex interactions. A systems biology approach, such as the generation of a network of co-expressed genes and the identification of functional modules and cis-regulatory elements, to extract insights and knowledge from microarray data will lead to a better understanding of complex diseases such as AD. In this study, we perform a series of analyses using co-expression networks, cis-regulatory elements, and functions of co-expressed gene modules, to analyse single-cell gene expression data from normal and AD-affected subjects. Results: We identified six co-expressed gene modules, each of which represented a biological process perturbed in AD. AD-related genes, such as APOE, A2M, PON2 and MAP4, and cardiovascular diseases (CVD)-associated genes, including COMT, CBS and WNK1, all congregated in a single module. Some of the disease-related genes were hub genes while many of them were directly connected to one or more hub genes. Further investigation of this disease-associated module revealed cis-regulatory elements that match to the binding sites of transcription factors involved in AD and CVD. Conclusions: Our results show the extensive links between AD and CVD at the co-expression and co-regulation levels, providing further evidence for the hypothesis that CVD and AD are linked. Our results support the notion that diseases in which the same set of biochemical pathways are affected may tend to co-occur with each other.

Controlled simulations of genome evolution are useful for benchmarking tools. However, many simulators lack extensibility and cannot measure parameters directly from data. These issues are addressed by three new open-source programs: gsimulator (for neutrally-evolving DNA), simgram (for generic structured features) and simgenome (for syntenic genome blocks). Each offers algorithms for parameter measurement and reconstruction of ancestral sequence. All three tools outperform the leading neutral DNA simulator (DAWG) in benchmarks. The programs are available at http://biowiki.org/SimulationTools.

Background: Chimeric proteins obtained by the fusion of a G protein-coupled receptor (GPCR) sequence to the N-terminus of the G protein alpha-subunit have been extensively used to investigate several aspects of GPCR signalling. Although both the receptor and the G protein generally maintain a fully functional state in such polypeptides, original observations made using a chimera between the beta2-adrenergic receptor (beta2AR) and Galphas indicated that the fusion to the alpha-subunit resulted in a marked reduction of receptor desensitization and down-regulation. To further investigate this phenomenon, we have compared the rates of internalization and recycling between wild-type and Galphas-fused beta2AR. Results: The rate of agonist-induced internalization, measured as the disappearance of cell surface immunofluorescence in HEK293 cells permanently expressing N-terminus tagged receptors, was reduced three-fold by receptor-G protein fusion. However, both fused and non-fused receptors translocated to the same endocytic compartment, as determined by dual-label confocal analysis of cells co-expressing both proteins and transferrin co-localization. Receptor recycling, determined as the reversion of surface immunofluorescence following the addition of antagonist to cells that were previously exposed to agonist, markedly differed between wild-type and fused receptors. While most of the internalized beta2AR returned rapidly to the plasma membrane, beta2AR-Galphas did not recycle, and the observed slow recovery for the fusion protein immunofluorescence was entirely accounted for by protein synthesis. Conclusion: The covalent linkage between beta2AR and Galphas does not appear to alter the initial endocytic translocation of the two proteins, although there is reduced efficiency. It does, however, completely disrupt the process of receptor and G protein recycling. We conclude that the physical separation between receptor and Galpha is not necessary for the transit to early endosomes, but is an essential requirement for the correct post-endocytic sorting and recycling of the two proteins.

Condensin and cohesin are loaded onto yeast chromosomes by a common mechanism at RNA polymerase III transcribed genes. Whereas cohesin translocates from these loading sites to mediate cohesion at secondary locations, condensin remains, bringing distant sites together into clusters.

Background: Telomeres prevent the ends of eukaryotic chromosomes from being recognised as damaged DNA and protect against cancer and ageing. When telomere structure is perturbed, a co-ordinated series of events promote arrest of the cell cycle so that cells carrying damaged telomeres do not divide. In order to better understand the eukaryotic response to telomere damage, budding yeast strains harbouring a temperature sensitive allele of an essential telomere capping gene (cdc13-1) were subjected to a transcriptomic study. Results: The genome-wide response to uncapped telomeres in yeast cdc13-1 strains, which have telomere capping defects at temperatures above ~27oC, was determined. Telomere uncapping in cdc13-1 strains is associated with the differential expression of over 600 transcripts. Transcripts affecting responses to DNA damage and diverse environmental stresses were statistically over-represented. BNA2, required for the biosynthesis of NAD+, is highly and significantly up-regulated upon telomere uncapping in cdc13-1 strains. We find that deletion of BNA2 and NPT1, which is also involved in NAD+ synthesis, suppresses the temperature sensitivity of cdc13-1 strains, indicating that NAD+ metabolism may be linked to telomere end protection. Conclusion: Our data support the hypothesis that the response to telomere uncapping is related to but distinct from the response to non-telomeric double strand breaks. The induction of environmental stress responses may be a conserved feature of the eukaryotic response to telomere damage. BNA2 which is involved in NAD+ synthesis plays previously unidentified roles in the cellular response to telomere uncapping.

Background: The pax2/5/8 genes belonging to the PAX family of transcription factors are key developmental regulators that are involved in the patterning of various embryonic tissues. More particularly, their function in inner ear specification has been widely described. However, little is known about the direct downstream targets and so far, no global approaches have been performed to identify these target genes in this particular tissue. Results: Here we present an original bioinformatics pipeline composed of comparative genomics, database querying and text mining tools, which is designed to rapidly and specifically discover PAX2/5/8 direct downstream targets involved in inner ear development. We provide evidence supported by experimental validation in medaka fish that brain 2 (POU domain, class 3, transcription factor 2), claudin-7, secretory pathway component sec31-like and meteorin-like precursor are novel direct downstream targets of PAX2/5/8. Conclusion: This study illustrates the power of extensive mining of public data repositories using bioinformatics methods to provide answers for a specific biological question. It furthermore demonstrates how the usage of such a combinatorial approach is advantageous for the biologist in terms of experimentation time and costs.

Background: In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane. Results: Here, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11) from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28. Conclusions: Nutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources.

Background: Reversible phosphorylation of proteins is involved in a wide range of processes ranging from signaling cascades to the regulation of protein complex assembly. Little is known about the structure and evolution of phosphorylation networks. Recent high-throughput phosphoproteomics studies have resulted in the rapid accumulation of phosphopeptide datasets for many model organisms. We here exploit these novel data for the comparative analysis of phosphorylation events between difference species of eukaryotes. Results: Comparison of phosphoproteomics datasets of six eukaryotes yields an overlap ranging from approximately 700 sites for human and mouse (two large datasets of closely related species) to a single site for fish and yeast (distantly related as well as two of the smallest datasets). Some conserved events seem surprisingly old: those shared by plant and animals suggests conservation over a billion years time-scale. In spite of the hypothesized incomprehensive nature of phosphoproteomics datasets and differences in experimental procedures, we show that the overlap between phosphoproteomes is larger than expected by chance and indicates increased functional relevance. Despite the dynamic nature of the evolution of phosphorylation, the relative overlap between the different datasets is identical to the phylogeny of the species studied. Conclusions: This analysis provides a framework for the generation of biological insights by the comparative analysis of high-throughput phosphoproteomics datasets. We expect the rapidly growing amount of data from high-throughput mass spectrometry analysis to make comparative phosphoproteomics a powerful tool for understanding the evolutionary and functional dynamics of reversible phosphorylation.

A report of the Biochemical Society meeting 'The Molecular Biology of Archaea', St Andrews, UK, 19-21 August 2008.

In the current financial crisis, the house-price 'bubble' has burst with spectacular effects. Big science projects in biology may be on their way to becoming the next scientific bubble.

Background: Defensins, small endogenous peptides with antimicrobial activity, are a pivotal components of innate immune response. A large cluster of defensin genes is located on human chromosome 8p and among them, the beta defensin 1 (DEFB1) promoter has been deeply studied since specific polymorphisms and haplotypes associate with asthma and atopy, susceptibility to severe sepsis, as well as HIV and Candida infection predisposition. Results: Here, we characterize the sequence variation and haplotype structure of the DEFB1 promoter region in 6 human populations. In all of them, we observed high levels of nucleotide variation, an excess of intermediate-frequency alleles, reduced population differentiation and a genealogy with common haplotypes separated by deep branches. Indeed a significant departure from the expectation of evolutionary neutrality was observed in all populations and the possibility that this is due to demographic history alone was ruled out. Also, we verified that the selection signature is restricted to the promoter region and not due to a linked balanced polymorphism. A phylogeny-based estimation indicated that the two major haplotype clades separated around 4.5 million years ago, approximately the time when the human and chimpanzee lineages split. Conclusions: Altogether these features represent strong molecular signatures of long-term balancing selection, a process that is thought to be extremely rare outside MHC genes. Our data indicate that the DEFB1 promoter region carries functional variants and support previous hypotheses whereby alleles predisposing to atopic disorders are widespread in modern societies because they conferred resistance to pathogens in ancient settings.

Background: Carnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/sphecules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36), are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined. Results: Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs) and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC) that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM), but not with the sorting and assembly machinery (SAM). Conclusions: Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer membrane also sheds light on the mechanisms involved in sorting of host-cell membrane proteins to mitochondria, a process that has been largely unexplored in plants.

Background: The evolutionary rate of a protein is a basic measure of evolution at the molecular level. Previous studies have shown that genes expressed in the brain have significantly lower evolutionary rates than those expressed in somatic tissues. Results: We study the evolutionary rates of genes expressed in 21 different human brain regions. We find that genes highly expressed in the more recent cortical regions of the brain have lower evolutionary rates than genes highly expressed in sub-cortical regions. This may partially result from the observation that genes which are highly expressed in cortical regions tend to be highly expressed in sub-cortical regions, and thus their evolution faces a richer set of functional constraints. The frequency of mammalian-specific and primate- specific genes is higher in the highly expressed gene sets of sub-cortical brain regions than in those of cortical brain regions. The basic inverse correlation between evolutionary rate and gene expression is significantly stronger in brain vs non-brain tissues, and in cortical vs sub-cortical regions. Extending upon this cortical/sub-cortical trend, this inverse correlation is generally more marked for tissues that are located higher along the cranial vertical axis during development, giving rise to the possibility that these tissues are also more evolutionary recent. Conclusions: We find that (a) cortically-expressed genes are more conserved than sub-cortical ones, and that (b) gene expression levels exert stronger constraints on sequence evolution in cortical vs sub-cortical regions. Taken together, these findings suggest that cortically-expressed genes are under stronger selective pressure than sub-cortically expressed genes.

Background: A large proportion of species-specific exons are alternatively spliced. In primates, Alu elements play a crucial role in the process of exon creation but many new exons have appeared through other mechanisms. Despite many recent studies, it is still unclear which are the splicing regulatory requirements for de novo exonization and how splicing regulation changes throughout an exon's lifespan. Results: Using comparative genomics, we have defined sets of exons with different evolutionary ages. Younger exons have weaker splice-sites and lower absolute values for the relative abundance of putative splicing regulators between exonic and adjacent intronic regions, indicating a less consolidated splicing regulation. This relative abundance is shown to increase with exon age leading to higher exon inclusion. We show that this local difference in the density of regulators might be of biological significance, as it outperforms other measures in real versus pseudo exon classification. We apply this new measure to the specific case of the exonization of anti-sense Alu elements and show that they are characterized by a general lack of exonic splicing silencers. Conclusion: Our results suggest that specific sequence environments are required for exonization and that these can change with time. We propose a model of exon creation and establishment in human genes, in which splicing decisions depend on the relative local abundance of regulatory motifs. Using this model, we provide further explanation why Alu elements serve as a major substrate for exon creation in primates. Finally, we discuss the benefits of integrating such information in gene prediction.

Background: Understanding the response of complex biochemical networks to genetic perturbations and environmental variability is a fundamental challenge in biology. Integration of high-throughput experimental assays and genome-scale computational methods is likely to produce insight otherwise unreachable, but specific examples of such integration have only just begun to be explored. Results: In this study, we measured growth phenotypes of 465 Saccharomyces cerevisiae gene deletion mutants under 16 metabolically relevant conditions and integrated them with the corresponding flux balance model predictions. We first used discordance between experimental results and model predictions to guide a stage of experimental refinement, which resulted in a significant improvement in the quality of the experimental data. Next, we used discordance still present in the refined experimental data to assess the reliability of yeast metabolism models under different conditions. In addition to estimating predictive capacity based on growth phenotypes, we sought to explain these discordances by examining predicted flux distributions visualized through a new, freely available platform. This analysis led to insight into the glycerol utilization pathway and the potential effects of metabolic shortcuts on model results. Finally, we used model predictions and experimental data to discriminate between alternative raffinose catabolism routes. Conclusions: Our study demonstrates how a new level of integration between high throughput measurements and flux balance model predictions can improve understanding of both experimental and computational results. The added value of a joint analysis is a more reliable platform for specific testing of biological hypotheses, such as the catabolic routes of different carbon sources.

Our knowledge on tissue- and disease-specific functions of human genes is rather limited and highly context-specific. Here, we developed a method for the comparison of mRNA expression levels of most human genes across 9,783 Affymetrix gene expression array experiments representing 43 normal human tissue types, 68 cancer types, and 64 other diseases. This database of gene expression patterns in normal human tissues and pathological conditions covers 113 million datapoints and is available at the GeneSapiens website.

A report of the Cold Spring Harbor Laboratory meeting 'Mechanisms and Models of Cancer', Cold Spring Harbor, USA, 13-17 August 2008.

Massively parallel short-tag sequencing of cDNA libraries - RNAseq - is being used to study the dynamics and complexity of eukaryotic transcriptomes, giving new biological insights into the 'active genome'.

NetGrep (http://genomics.princeton.edu/singhlab/netgrep/) is a system for searching protein interaction networks for matches to user-supplied "network schemas." Each schema consists of descriptions of proteins (e.g., their molecular functions or putative domains) along with the desired topology and types of interactions among them. Schemas can thus describe domain-domain interactions, signaling and regulatory pathways, or more complex network patterns. NetGrep provides an advanced graphical interface for specifying schemas and fast algorithms for extracting their matches.