Identification of small RNAs abundant in Burkholderia cenocepacia biofilms reveal putative regulators with a potential role in carbon and iron metabolism

Small RNAs play a regulatory role in many central metabolic processes of bacteria, as well as in developmental processes such as biofilm formation. Small RNAs of Burkholderia cenocepacia, an opportunistic pathogenic beta-proteobacterium, are to date not well characterised. To address that, we performed genome-wide transcriptome structure analysis of biofilm grown B. cenocepacia J2315. 41 unannotated short transcripts were identified in intergenic regions of the B. cenocepacia genome. 15 of these short transcripts, highly abundant in biofilms, widely conserved in Burkholderia sp. and without known function, were selected for in-depth analysis. Expression profiling showed that most of these sRNAs are more abundant in biofilms than in planktonic cultures. Many are also highly abundant in cells grown in minimal media, suggesting they are involved in adaptation to nutrient limitation and growth arrest. Their computationally predicted targets include a high proportion of genes involved in carbon metabolism. Expression and target genes of one sRNA suggest a potential role in regulating iron homoeostasis. The strategy used for this study to detect sRNAs expressed in B. cenocepacia biofilms has successfully identified sRNAs with a regulatory function

Peptides encoded by short ORFs control development and define a new eukaryotic gene family

Despite recent advances in developmental biology and in genomics, key questions remain regarding the organisation of cells into embryos. One possibility is that novel types of genes might await discovery and could provide some of the answers. Genome annotation depends strongly on comparison with previously known gene sequences, and so genes having previously uncharacterised structure and function can be missed. Here we present the characterisation of tarsal-less, a new such type of gene. Tarsal-less has two unusual features: first, it contains more than one coding unit, a structure more similar to some bacterial genes. Second, it codes for small peptides rather than proteins, and in fact these peptides represent the smallest gene products known to date. Functional analysis of this gene in the fruitfly Drosophila shows that it has important functions throughout development, including tissue morphogenesis and pattern formation. We identify genes similar to tarsal-less in other species, and thus define a tarsal-less-related gene family. We expect that a combination of bioinformatic and functional methods, such as the ones we use in this study, will identify and characterize more genes of this type. Potentially, thousands of such new genes may exist

Prioritisation of candidate genes for psychiatric disorders

The application of genome-wide association studies and next-generation sequencing has had limited success in identifying causal genes for complex diseases. Bipolar disorder is one such disease whose aetiology has not been elucidated despite the application of these technologies. Candidate gene prioritisation offers a solution to limit the vast amount of possible candidate genes produced from the combination of data sources. Current prioritisation tools rely heavily on previous data and thus do not perform well for poorly characterised diseases such as bipolar disorder. Here we have developed Data Integrated Genetics, DIG, a new candidate gene prioritisation tool designed specifically for complex genetic diseases. Given a user-specified disease query, DIG initially data-mines literature, linkage, homolog and sequence data to create a pool of possible candidates. The tool filters out likely false positives by removing pseudogenes. A unique data integration method is used to rank the remaining list of genes. Additionally, ranking is validated by tissue expression and single nucleotide polymorphism annotation. DIG exhibited comparable performance to existing tools when evaluated with four complex diseases. Eight novel genes were identified when DIG was applied to bipolar disorder, of which the Huntingtin gene poses as an exciting avenue for new aetiology research. The ease of use and realistic number of possible candidates given in the DIG results make this tool highly useful for research application in the study of complex genetic diseases. DIG is freely available from http://www.cbio.uct.ac.za/DIG

NET-GE: a novel NETwork-based Gene Enrichment for detecting biological processes associated to Mendelian diseases

Enrichment analysis is a widely applied procedure for shedding light on the molecular mechanisms and functions at the basis of phenotypes, for enlarging the dataset of possibly related genes/proteins and for helping interpretation and prioritization of newly determined variations. Several standard and Network-based enrichment methods are available. Both approaches rely on the annotations that characterize the genes/proteins included in the input set; network based ones also include in different ways physical and functional relationships among different genes or proteins that can be extracted from the available biological networks of interactions

Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence

Bis-(3 ',5 ') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K-d similar to 2 mu M). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence

Gene Expression in the Choanoderm

The body plan of sponges (phylum Porifera) is an outlier among modern animals and is thought to have special evolutionary significance. Sponges lack muscles, nerves and a gut. Instead, they are composed of few cell types and simple tissues that function to pump water through an internal canal network where bacterial prey are filtered by a specialized tissue called the choanoderm. The choanoderm is composed of cells with striking similarity to choanoflagellates, the unicellular relatives of animals. Thus, the traditional view is that the sponge choanoderm is a useful model of the first animal epithelial tissues. Using the freshwater sponge Ephydatia muelleri, we have performed gene expression analysis of the choanoderm tissue and have begun to develop an experimental method to validate and characterize the function of candidate choanoderm genes. The data suggest that the choanoderm may be the only metazoan tissue not reliant on the classical cadherin/catenin complex for cell adhesion. Yet we find evidence for conserved developmental mechanisms and other structural features such as epithelial polarity and microvillar organization. Finally, we will explore the possibility that genes unique to choanoflagellates and sponges, have conserved functions in the choanoderm tissue. This prediction derives from the hypothesized homology of these putatively ancient cell types

Genomic Analysis of Drosophila Neuronal Remodeling: A Role for the RNA-Binding Protein Boule as a Negative Regulator of Axon Pruning

Drosophila mushroom body (MB) {gamma} neurons undergo axon pruning during metamorphosis through a process of localized degeneration of specific axon branches. Developmental axon degeneration is initiated by the steroid hormone ecdysone, acting through a nuclear receptor complex composed of USP (ultraspiracle) and EcRB1 (ecdysone receptor B1) to regulate gene expression in MB {gamma} neurons. To identify ecdysone-dependent gene expression changes in MB {gamma} neurons at the onset of axon pruning, we use laser capture microdissection to isolate wild-type and mutant MB neurons in which EcR (ecdysone receptor) activity is genetically blocked, and analyze expression changes by microarray. We identify several molecular pathways that are regulated in MB neurons by ecdysone. The most striking observation is the upregulation of genes involved in the UPS (ubiquitin–proteasome system), which is cell autonomously required for {gamma} neuron pruning. In addition, we characterize the function of Boule, an evolutionarily conserved RNA-binding protein previously implicated in spermatogenesis in flies and vertebrates. boule expression is downregulated by ecdysone in MB neurons at the onset of pruning, and forced expression of Boule in MB {gamma} neurons is sufficient to inhibit axon pruning. This activity is dependent on the RNA-binding domain of Boule and a conserved DAZ (deleted in azoospermia) domain implicated in interactions with other RNA-binding proteins. However, loss of Boule does not result in obvious defects in axon pruning or morphogenesis of MB neurons, suggesting that it acts redundantly with other ecdyonse-regulated genes. We propose a novel function for Boule in the CNS as a negative regulator of developmental axon pruning

Gene expression profiles in rat brain disclose CNS signature genes and regional patterns of functional specialisation

Background: The mammalian brain is divided into distinct regions with structural and neurophysiological differences. As a result, gene expression is likely to vary between regions in relation to their cellular composition and neuronal function. In order to improve our knowledge and understanding of regional patterns of gene expression in the CNS, we have generated a global map of gene expression in selected regions of the adult rat brain (frontomedial-, temporal- and occipital cortex, hippocampus, striatum and cerebellum; both right and left sides) as well as in three major non-neural tissues (spleen, liver and kidney) using the Applied Biosystems Rat Genome Survey Microarray. Results: By unsupervised hierarchical clustering, we found that the transcriptome within a region was highly conserved among individual rats and that there were no systematic differences between the two hemispheres (right versus left side). Further, we identified distinct sets of genes showing significant regional enrichment. Functional annotation of each of these gene sets clearly reflected several important physiological features of the region in question, including synaptic transmission within the cortex, neurogenesis in hippocampus and G-protein-mediated signalling in striatum. In addition, we were able to reveal potentially new regional features, such as mRNA transcription- and neurogenesis-annotated activities in cerebellum and differential use of glutamate signalling between regions. Finally, we determined a set of 'CNSsignature' genes that uncover characteristics of several common neuronal processes in the CNS, with marked overrepresentation of specific features of synaptic transmission, ion transport and cell communication, as well as numerous novel unclassified genes. Conclusion: We have generated a global map of gene expression in the rat brain and used this to determine functional processes and pathways that have a regional preference or ubiquitous distribution within the CNS, respectively. The existence of shared specialised neuronal activities in CNS is interesting in a context of potential functional redundancy, and future studies should further explore the overall characteristics of CNS-specific versus region-specific gene profiles in the brain

Identification of candidate effector genes of <i>Pratylenchus penetrans</i>

Pratylenchus penetrans is one of the most important species of root lesion nematodes (RLNs) because of its detrimental and economic impact in a wide range of crops. Similar to other plant‐parasitic nematodes (PPNs), P. penetrans harbours a significant number of secreted proteins that play key roles during parasitism. Here, we combined spatially and temporally resolved next‐generation sequencing datasets of P. penetrans to select a list of candidate genes aimed at the identification of a panel of effector genes for this species. We determined the spatial expression of transcripts of 22 candidate effectors within the oesophageal glands of P. penetrans by in situ hybridization. These comprised homologues of known effectors of other PPNs with diverse putative functions, as well as novel pioneer effectors specific to RLNs. It is noteworthy that five of the pioneer effectors encode extremely proline‐rich proteins. We then combined in situ localization of effectors with available genomic data to identify a non‐coding motif enriched in promoter regions of a subset of P. penetrans effectors, and thus a putative hallmark of spatial expression. Expression profiling analyses of a subset of candidate effectors confirmed their expression during plant infection. Our current results provide the most comprehensive panel of effectors found for RLNs. Considering the damage caused by P. penetrans, this information provides valuable data to elucidate the mode of parasitism of this nematode and offers useful suggestions regarding the potential use of P. penetrans‐specific target effector genes to control this important pathogen

A genomic approach to the study of Tribolium castaneum genetics, development & evolution

During the last decade, Tribolium castaneum has become the insect of choice for comparative genetics and developmental studies outside of drosophilids. Until recently, most molecular studies have focused on the comparative analysis of early development with a focus on segmentation and homeotic genes. In order to acquire independent knowledge on the genetic basis of insect development, a genomic approach consisting of EST and BAC-ends sequencing projects has been initiated in Tribolium. The EST project resulted in the production of 2,246 random sequences representing 488 non-redundant EST contigs. Of those, 280 sequences were selected, along with 86 independently cloned putative transcription factors, and further characterized by in situ hybridization. Expression analysis led to the identification of at least 25 novel genes putatively involved in diverse aspects of Tribolium embryonic development such as segmentation, appendage development, neurogenesis, myogenesis and terminal patterning. Comparative evolutionary analysis of the EST sequences verified that Tribolium is a slow evolving species when compared to dipterans. As predicted by the neutral theory, the data also revealed that evolutionary rates are a composite measure of both gene and species specific rates. To date, the BAC-ends sequencing project resulted in the production of 8,640 sequences covering 2.9% of the Tribolium genome. A functional analysis of a subset of these BAC-end sequences (BES) allowed the identification of 486 putative ORFs. It is estimated that of the 53,000 BES to be produced, 6,900 ORFs will be found, comprising 18% of the genome. Random sequencing of ESTs and production of BES are shown to be powerful ways to identify new genes, to help mapping the Tribolium genome and to identify coding regions in genomic sequences