A newly-developed community microarray resource for transcriptome profiling in Brassica species enables the confirmation of Brassica-specific expressed sequences

<p>Abstract</p> <p>Background</p> <p>The <it>Brassica </it>species include an important group of crops and provide opportunities for studying the evolutionary consequences of polyploidy. They are related to <it>Arabidopsis thaliana</it>, for which the first complete plant genome sequence was obtained and their genomes show extensive, although imperfect, conserved synteny with that of <it>A. thaliana</it>. A large number of EST sequences, derived from a range of different <it>Brassica </it>species, are available in the public database, but no public microarray resource has so far been developed for these species.</p> <p>Results</p> <p>We assembled unigenes using ~800,000 EST sequences, mainly from three species: <it>B. napus</it>, <it>B. rapa </it>and <it>B. oleracea</it>. The assembly was conducted with the aim of co-assembling ESTs of orthologous genes (including homoeologous pairs of genes in <it>B. napus </it>from each of the A and C genomes), but resolving assemblies of paralogous, or paleo-homoeologous, genes (<it>i.e</it>. the genes related by the ancestral genome triplication observed in diploid <it>Brassica </it>species). 90,864 unique sequence assemblies were developed. These were incorporated into the BAC sequence annotation for the <it>Brassica rapa </it>Genome Sequencing Project, enabling the identification of cognate genomic sequences for a proportion of them. A 60-mer oligo microarray comprising 94,558 probes was developed using the unigene sequences. Gene expression was analysed in reciprocal resynthesised <it>B. napus </it>lines and the <it>B. oleracea </it>and <it>B. rapa </it>lines used to produce them. The analysis showed that significant expression could consistently be detected in leaf tissue for 35,386 unigenes. Expression was detected across all four genotypes for 27,355 unigenes, genome-specific expression patterns were observed for 7,851 unigenes and 180 unigenes displayed other classes of expression pattern. Principal component analysis (PCA) clearly resolved the individual microarray datasets for <it>B. rapa</it>, <it>B. oleracea </it>and resynthesised <it>B. napus</it>. Quantitative differences in expression were observed between the resynthesised <it>B. napus </it>lines for 98 unigenes, most of which could be classified into non-additive expression patterns, including 17 that showed cytoplasm-specific patterns. We further characterized the unigenes for which A genome-specific expression was observed and cognate genomic sequences could be identified. Ten of these unigenes were found to be <it>Brassica</it>-specific sequences, including two that originate from complex loci comprising gene clusters.</p> <p>Conclusion</p> <p>We succeeded in developing a <it>Brassica </it>community microarray resource. Although expression can be measured for the majority of unigenes across species, there were numerous probes that reported in a genome-specific manner. We anticipate that some proportion of these will represent species-specific transcripts and the remainder will be the consequence of variation of sequences within the regions represented by the array probes. Our studies demonstrated that the datasets obtained from the arrays can be used for typical analyses, including PCA and the analysis of differential expression. We have also demonstrated that <it>Brassica</it>-specific transcripts identified <it>in silico </it>in the sequence assembly of public EST database accessions are indeed reported by the array. These would not be detectable using arrays designed using <it>A. thaliana </it>sequences.</p

A Viral Discovery Methodology for Clinical Biopsy Samples Utilising Massively Parallel Next Generation Sequencing

Here we describe a virus discovery protocol for a range of different virus genera, that can be applied to biopsy-sized tissue samples. Our viral enrichment procedure, validated using canine and human liver samples, significantly improves viral read copy number and increases the length of viral contigs that can be generated by de novo assembly. This in turn enables the Illumina next generation sequencing (NGS) platform to be used as an effective tool for viral discovery from tissue samples

Analyses of pig genomes provide insight into porcine demography and evolution

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model

In Mitochondria β-Actin Regulates mtDNA Transcription and Is Required for Mitochondrial Quality Control

Summary: In eukaryotic cells, actin regulates both cytoplasmic and nuclear functions. However, whether actin-based structures are present in the mitochondria and are involved in mitochondrial functions has not been investigated. Here, using wild-type β-actin +/+ and knockout (KO) β-actin −/− mouse embryonic fibroblasts we show evidence for the defect in maintaining mitochondrial membrane potential (MMP) in β-actin-null cells. MMP defects were associated with impaired mitochondrial DNA (mtDNA) transcription and nuclear oxidative phosphorylation (OXPHOS) gene expression. Using super-resolution microscopy we provided direct evidence on the presence of β-actin-containing structures inside mitochondria. Large aggregates of TFAM-stained nucleoids were observed in bulb-shaped mitochondria in KO cells, suggesting defects in mitochondrial nucleoid segregation without β-actin. The observation that mitochondria-targeted β-actin rescued mtDNA transcription and MMP suggests an indispensable functional role of a mitochondrial β-actin pool necessary for mitochondrial quality control. : Molecular Biology; Cell Biology; Functional Aspects of Cell Biology Subject Areas: Molecular Biology, Cell Biology, Functional Aspects of Cell Biolog

A mixed community of actinomycetes produce multiple antibiotics for the fungus farming ant Acromyrmex octospinosus

This work was supported by a UEA-funded PhD studentship (JB) and an MRC Milstein award, G0801721 (MIH, RJMG and DY). MIH is a Research Councils UK Fellow. DY also received support from the Yunnan provincial government (20080A001) and the Chinese Academy of Sciences (0902281081).Background: Attine ants live in an intensely studied tripartite mutualism with the fungus Leucoagaricus gongylophorus, which provides food to the ants, and with antibiotic-producing actinomycete bacteria. One hypothesis suggests that bacteria from the genus Pseudonocardia are the sole, co-evolved mutualists of attine ants and are transmitted vertically by the queens. A recent study identified a Pseudonocardia-produced antifungal, named dentigerumycin, associated with the lower attine Apterostigma dentigerum consistent with the idea that co-evolved Pseudonocardia make novel antibiotics. An alternative possibility is that attine ants sample actinomycete bacteria from the soil, selecting and maintaining those species that make useful antibiotics. Consistent with this idea, a Streptomyces species associated with the higher attine Acromyrmex octospinosus was recently shown to produce the well-known antifungal candicidin. Candicidin production is widespread in environmental isolates of Streptomyces, so this could either be an environmental contaminant or evidence of recruitment of useful actinomycetes from the environment. It should be noted that the two possibilities for actinomycete acquisition are not necessarily mutually exclusive. Results: In order to test these possibilities we isolated bacteria from a geographically distinct population of A. octospinosus and identified a candicidin-producing Streptomyces species, which suggests that they are common mutualists of attine ants, most probably recruited from the environment. We also identified a Pseudonocardia species in the same ant colony that produces an unusual polyene antifungal, providing evidence for co-evolution of Pseudonocardia with A. octospinosus. Conclusions: Our results show that a combination of co-evolution and environmental sampling results in the diversity of actinomycete symbionts and antibiotics associated with attine ants.Publisher PDFPeer reviewe

β-actin regulates a heterochromatin landscape essential for optimal induction of neuronal programs during direct reprograming.

During neuronal development, β-actin serves an important role in growth cone mediated axon guidance. Consistent with this notion, in vivo ablation of the β-actin gene leads to abnormalities in the nervous system. However, whether β-actin is involved in the regulation of neuronal gene programs is not known. In this study, we directly reprogramed β-actin+/+ WT, β-actin+/- HET and β-actin-/- KO mouse embryonic fibroblast (MEFs) into chemically induced neurons (CiNeurons). Using RNA-seq analysis, we profiled the transcriptome changes among the CiNeurons. We discovered that induction of neuronal gene programs was impaired in KO CiNeurons in comparison to WT ones, whereas HET CiNeurons showed an intermediate levels of induction. ChIP-seq analysis of heterochromatin markers demonstrated that the impaired expression of neuronal gene programs correlated with the elevated H3K9 and H3K27 methylation levels at gene loci in β-actin deficient MEFs, which is linked to the loss of chromatin association of the BAF complex ATPase subunit Brg1. Together, our study shows that heterochromatin alteration in β-actin null MEFs impedes the induction of neuronal gene programs during direct reprograming. These findings are in line with the notion that H3K9Me3-based heterochromatin forms a major epigenetic barrier during cell fate change

Fibroblast Differentiation and Matrix Remodeling Impaired under Simulated Microgravity in 3D Cell Culture Model

Exposure to microgravity affects astronauts’ health in adverse ways. However, less is known about the extent to which fibroblast differentiation during the wound healing process is affected by the lack of gravity. One of the key steps of this process is the differentiation of fibroblasts into myofibroblasts, which contribute functionally through extracellular matrix production and remodeling. In this work, we utilized collagen-based three-dimensional (3D) matrices to mimic interstitial tissue and studied fibroblast differentiation under simulated microgravity (sµG). Our results demonstrated that alpha-smooth muscle actin (αSMA) expression and translocation of Smad2/3 into the cell nucleus were reduced upon exposure to sµG compared to the 1g control, which suggests the impairment of fibroblast differentiation under sµG. Moreover, matrix remodeling and production were decreased under sµG, which is in line with the impaired fibroblast differentiation. We further investigated changes on a transcriptomic level using RNA sequencing. The results demonstrated that sµG has less effect on fibroblast transcriptomes, while sµG triggers changes in the transcriptome of myofibroblasts. Several genes and biological pathways found through transcriptome analysis have previously been reported to impair fibroblast differentiation. Overall, our data indicated that fibroblast differentiation, as well as matrix production and remodeling, are impaired in 3D culture under sµG conditions