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Background: The Qinghai-Tibet Plateau (QTP) is the highest plateau in the world, and the microorganisms there play vital ecological roles in the global biogeochemical cycle; however, detailed information on the microbial communities in QTP is still lacking. Results: Here, we performed a landscape survey of the microorganisms in Bamucuo, Tibet, resulting in 160,212 (soil) and 135,994 (water) contigs by shotgun metagenomic methods, and generated 75 nearly complete metagenome-assembled genomes (MAGs). Proteobacteria, Actinobacteria and Firmicutes were found to be the three most dominant bacterial phyla, while Euryarchaeota was the most dominant archaeal phylum. Surprisingly, Pandoravirus salinus was found in the soil microbial community. The KEGG annotations showed that the genes related to metabolism accounted for 62.9% (soil) and 58.4% (water), respectively. Of the 75 MAGs, 63 were found to contain the genes related to all the six carbon fixation pathways, and the heavy metal resistance genes, the pressure response subsystem, and the secondary metabolite biosynthesis gene clusters (BGCs) were discovered to be abundant. Conclusions: We concluded that the microorganisms in Bamucuo fix carbon mainly through the 3-hydroxypropionic bi-cycle pathway; this study, for the first time, characterized the microbial community composition and metabolic capacity in QTP high-altitude areas (with an altitude of 4,555 meters), confirmed that QTP is a huge and valuable resource bank in which more new non-resistant antibiotics and many other bioactive substances could be developed, and provided the expanded information for further microbial community studies in QTP.

The histone variant H3.3 is deposited across active genes, regulatory regions, and telomeres. It remains unclear how H3.3 interacts with chromatin modifying enzymes and thereby modulates gene activity. In this study, we performed a co-immunoprecipitation-mass spectrometry analysis of proteins associated with H3.3-containing nucleosomes and identified the nucleosome remodeling and deacetylase complex (NuRD) as a major H3.3-interactor. We show that the H3.3-NuRD interaction is dependent on the H3.3 lysine 4 residue and that NuRD binding occurs when lysine 4 is in its unmodified state. The majority of NuRD binding colocalizes with H3.3 and directly correlates with gene activity. H3.3 depletion led to reduced levels of NuRD at sites previously occupied by H3.3, as well as a global decrease in histone marks associated with gene activation. Our results demonstrate the importance of H3.3 in the maintenance of the cellular epigenetic landscape and reveal a highly prevalent interaction between the histone variant H3.3 and the multiprotein complex NuRD.

The Antarctic icefish, a family (Channichthyidae) of teleosts within the perciform suborder Notothenioidei, are the only known vertebrates without oxygen-transporting haemoglobins and that are largely devoid of circulating erythrocytes. To elucidate the evo-devo mechanisms underpinning the suppressed erythropoiesis in the icefish, we conducted comparative studies on the transcriptomes and microRNAomes of the primary haematopoietic tissues between an icefish (Chionodraco hamatus) and two red-blooded notothenioids (Trematomus bernacchii and Gymnodraco acuticeps). We identified substantial remodelling of the haematopoietic programs in the icefish through which erythropoiesis is selectively suppressed. Experimental verification showed that erythropoietic suppression in the icefish may be attributable to the upregulation of TGF-β signalling, which coincides with reductions in multiple transcription factors essential for erythropoiesis and the upregulation of hundreds of microRNAs, the majority (> 80%) of which potentially target erythropoiesis regulating factors. Of the six microRNAs selected for verification, three miRNAs (miR-152, miR-1388 and miR-16b) demonstrated suppressive functions on GATA1 and ALAS2, which are two factors important for erythroid differentiation, resulting in reduced numbers of erythroids in microinjected zebra fish embryos. Codon substitution analyses of the genes of the TGF-β superfamily revealed signs of positive selection in TGF-β1 and endoglin in the lineages leading to Antarctic notothenioids. Both genes are previously known to function in erythropoietic suppression. These findings implied a general trend of erythropoietic suppression in the cold-adapted notothenioid lineages through evolutionary modulation of the multi-functional TGF-β signalling pathway. This trend is more pronounced in the haemoglobin-less icefish, which may pre-emptively hinder the otherwise defective erythroids from production.

DNA methylation is an essential epigenetic mechanism involved in multiple biological processes. However, the relationship between DNA methylation and cold acclimation remains poorly understood. In this study, Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) was performed to reveal a genome-wide methylation profile of zebrafish (Danio rerio) embryonic fibroblast cells (ZF4) and its variation under cold pressure. MeDIP-seq assay was conducted with ZF4 cells cultured at appropriate temperature of 28°C and at low temperature of 18°C for 5 (short-term) and 30 (long-term) days, respectively. Our data showed that DNA methylation level of whole genome increased after a short-term cold exposure and decreased after a long-term cold exposure. It is interesting that metabolism of folate pathway is significantly hypomethylated after short-term cold exposure, which is consistent with the increased DNA methylation level. 21% of methylation peaks were significantly altered after cold treatment. About 8% of altered DNA methylation peaks are located in promoter regions, while the majority of them are located in non-coding regions. Methylation of genes involved in multiple cold responsive biological processes were significantly affected, such as anti-oxidant system, apoptosis, development, chromatin modifying and immune system suggesting that those processes are responsive to cold stress through regulation of DNA methylation. Our data indicate the involvement of DNA methylation in cellular response to cold pressure, and put a new insight into the genome-wide epigenetic regulation under cold pressure.

a b s t r a c t We evaluated the effect of transgenic cottons (Bacillus thuringiensis (Bt) and Bt with cowpea trypsin inhibitor (Bt-CpTI)) on soil ecosystems after three and four years of growth. The persistence and the rela- tionships among insecticidal crystal proteins 1Ac (Cry1Ac) and cowpea trypsin inhibitor (CpTI) proteins derived from transgenic cottons, microbial properties, and enzymatic activities in the rhizosphere soil were investigated in the repeated cultivation experiment. The Cry1Ac and CpTI proteins persisted in the rhizosphere soil of transgenic cotton, whereas no proteins were detected in the rhizosphere soil of non- transgenic cotton. The persistence of CpTI inhibited the degradation of Cry1Ac protein in the rhizosphere soil of Bt-CpTI cotton. Repeated measures analysis of variance indicated that the year of cultivation, the variety of cotton, the transgenic trait, and their interactions significantly influenced the microbial proper- ties and the enzymatic activities in rhizosphere soil. Overall, the repeated cultivation of transgenic cotton had significantly negative effects on the microbial properties and the enzymatic activities in rhizosphere soil compared to those in the rhizosphere soil of non-transgenic cotton.

We describe a new method for sequencing-based cross-species transcriptome comparisons and define a new metric for evaluating gene expression across species using protein-coding families as units of comparison. Using this measure transcriptomes from different species were evaluated by mapping them to gene families and integrating the mapping results with expression data. Statistical tests were applied to the transcriptome evaluation results to identify differentially expressed families. A Perl program named Pro-Diff was compiled to implement this method. To evaluate the method and provide an example of its use, two liver EST transcriptomes from two closely related fish that live in different temperature zones were compared. One EST library was from a recent sequencing project of Dissosticus mawsoni, a fish that lives in cold Antarctic sea waters, while the other was newly sequenced data (available at: http://www.fishgenome.org/ polarbank/) from Notothenia angustata, a species that lives in temperate near-shore water of southern New Zealand. Results from the comparison were consistent with results inferred from phenotype differences and also with our previously published Gene Ontology-based method. The Pro-Diff program and operation manual can be downloaded from: http://www.fishgenome.org/download/Prodiff.rar.

Hepcidin is a small bioactive peptide with dual roles as an antimicrobial peptide and as the principal hormonal regulator of iron homeostasis in human and mouse. Hepcidin homologs of very similar structures are found in lower vertebrates, all comprise approximately 20-25 amino acids with 8 highly conserved cysteines forming 4 intramolecular disulfide bonds, giving hepcidin a hairpin structure. Hepcidins are particularly diverse in teleost fishes, which may be related to the diversity of aquatic environments with varying degree of pathogen challenge, oxygenation, and iron concentration, factors known to alter hepcidin expression in mammals. We characterized the diversity of hepcidin genes of the Antarctic notothenioid fishes that are endemic to the world's coldest and most oxygen-rich marine water. Notothenioid fishes have at least 4 hepcidin variants, in 2 distinctive structural types. Type I hepcidins comprise 3 distinct variants that are homologs of the widespread 8-cysteine hepcidins. Type II is a novel 4-cysteine variant and therefore only 2 possible disulfide bonds, highly expressed in hematopoietic tissues. Analyses of d(N)/d(S) substitution rate ratios and likelihood ratio test under site-specific models detected significant signal of positive Darwinian selection on the mature hepcidin-coding sequence, suggesting adaptive evolution of notothenioid hepcidins. Genomic polymerase chain reaction and Southern hybridization showed that the novel type II hepcidin occurs exclusively in lineages of the Antarctic notothenioid radiation but not in the basal non-Antarctic taxa, and lineage-specific positive selection was detected on the branch leading to the type II hepcidin clade under branch-site models, suggesting adaptive evolution of the reduced cysteine variant in response to the polar environment. We also isolated a structurally distinct 4-cysteine (4cys) hepcidin from an Antarctic eelpout that is unrelated to the notothenioids but inhabits the same freezing water. Neighbor-Joining (NJ) analyses of teleost hepcidins showed that the eelpout 4cys variant arose independently from the notothenioid version, which lends support to adaptive evolution of reduced cysteine hepcidin variants on cold selection. The NJ tree also showed taxonomic-specific expansions of hepcidin variants, indicating that duplication and diversification of hepcidin genes play important roles in evolutionary response to diverse ecological conditions.

Background Massively parallel sequencing readouts of epigenomic assays are enabling integrative genome-wide analyses of genomic and epigenomic variation. Pash 3.0 performs sequence comparison and read mapping and can be employed as a module within diverse configurable analysis pipelines, including ChIP-Seq and methylome mapping by whole-genome bisulfite sequencing. Results Pash 3.0 generally matches the accuracy and speed of niche programs for fast mapping of short reads, and exceeds their performance on longer reads generated by a new generation of massively parallel sequencing technologies. By exploiting longer read lengths, Pash 3.0 maps reads onto the large fraction of genomic DNA that contains repetitive elements and polymorphic sites, including indel polymorphisms. Conclusions We demonstrate the versatility of Pash 3.0 by analyzing the interaction between CpG methylation, CpG SNPs, and imprinting based on publicly available whole-genome shotgun bisulfite sequencing data. Pash 3.0 makes use of gapped k-mer alignment, a non-seed based comparison method, which is implemented using multi-positional hash tables. This allows Pash 3.0 to run on diverse hardware platforms, including individual computers with standard RAM capacity, multi-core hardware architectures and large clusters.

Cephalochordates, urochordates, and vertebrates comprise the three extant groups of chordates. Although higher morphological and developmental similarity exists between cephalochordates and vertebrates, molecular phylogeny studies have instead suggested that the morphologically simplified urochordates are the closest relatives to vertebrates. MicroRNAs (miRNAs) are regarded as the major factors driving the increase of morphological complexity in early vertebrate evolution, and are extensively characterized in vertebrates and in a few species of urochordates. However, the comprehensive set of miRNAs in the basal chordates, namely the cephalochordates, remains undetermined. Through extensive sequencing of a small RNA library and genomic homology searches, we characterized 100 miRNAs from the cephalochordate amphioxus, Branchiostoma japonicum, and B. floridae. Analysis of the evolutionary history of the cephalochordate miRNAs showed that cephalochordates possess 54 miRNA families homologous to those of vertebrates, which is threefold higher than those shared between urochordates and vertebrates. The miRNA contents demonstrated a clear correlation between the extent of miRNA overlapping and morphological similarity among the three chordate groups, providing a strong evidence of miRNAs being the major genetic factors driving morphological complexity in early chordate evolution.

The antifreeze glycoprotein-fortified Antarctic notothenioid fishes comprise the predominant fish suborder in the isolated frigid Southern Ocean. Their ecological success undoubtedly entailed evolutionary acquisition of a full suite of cold-stable functions besides antifreeze protection. Prior studies of adaptive changes in these teleost fishes generally examined a single genotype or phenotype. We report here the genome-wide investigations of transcriptional and genomic changes associated with Antarctic notothenioid cold adaptation. We sequenced and characterized 33,560 ESTs from four tissues of the Antarctic notothenioid Dissostichus mawsoni and derived 3,114 nonredundant protein gene families and their expression profiles. Through comparative analyses of same-tissue transcriptome profiles of D. mawsoni and temperate/tropical teleost fishes, we identified 177 notothenioid protein families that were expressed many fold over the latter, indicating cold-related up-regulation. These up-regulated gene families operate in protein biosynthesis, protein folding and degradation, lipid metabolism, antioxidation, antiapoptosis, innate immunity, choriongenesis, and others, all of recognizable functional importance in mitigating stresses in freezing temperatures during notothenioid life histories. We further examined the genomic and evolutionary bases for this expressional up-regulation by comparative genomic hybridization of DNA from four pairs of Antarctic and basal non-Antarctic notothenioids to 10,700 D. mawsoni cDNA probes and discovered significant to astounding (3- to >300-fold, P < 0.05) Antarctic-specific duplications of 118 protein-coding genes, many of which correspond to the up-regulated gene families. Results of our integrative tripartite study strongly suggest that evolution under constant cold has resulted in dramatic genomic expansions of specific protein gene families, augmenting gene expression and gene functions contributing to physiological fitness of Antarctic notothenioids in freezing polar conditions.

It has been reported that the disaccharide trehalose is capable of increasing the thermostability and thermoactivity of reverse transcriptase, and therefore improving the length of cDNA synthesis. However, no test has been done on how the disaccharide trehalose performs in the context of the entire cDNA synthesis processes, or whether it can seamlessly integrate into the commercially available cDNA synthesis kit. In this report, we optimized a protocol to incorporate trehalose in the Stratagene's cDNA library construction kit in order to demonstrate great improvement in cDNA's length (average length of 1.8 kb in the trehalose group versus 1.0 kb in the control). Sequence analysis of the cDNA clones showed that the addition of trehalose did not increase the error rate of the RT products but greatly increase the quantity of full-length in cDNA library.

Background Large-scale sequencing efforts produced millions of Expressed Sequence Tags (ESTs) collectively representing differentiated biochemical and functional states. Analysis of these EST libraries reveals differential gene expressions, and therefore EST data sets constitute valuable resources for comparative transcriptomics. To translate differentially expressed genes into a better understanding of the underlying biological phenomena, existing microarray analysis approaches usually involve the integration of gene expression with Gene Ontology (GO) databases to derive comparable functional profiles. However, methods are not available yet to process EST-derived transcription maps to enable GO-based global functional profiling for comparative transcriptomics in a high throughput manner. Results Here we present GO-Diff, a GO-based functional profiling approach towards high throughput EST-based gene expression analysis and comparative transcriptomics. Utilizing holistic gene expression information, the software converts EST frequencies into EST Coverage Ratios of GO Terms. The ratios are then tested for statistical significances to uncover differentially represented GO terms between the compared transcriptomes, and functional differences are thus inferred. We demonstrated the validity and the utility of this software by identifying differentially represented GO terms in three application cases: intra-species comparison; meta-analysis to test a specific hypothesis; inter-species comparison. GO-Diff findings were consistent with previous knowledge and provided new clues for further discoveries. A comprehensive test on the GO-Diff results using series of comparisons between EST libraries of human and mouse tissues showed acceptable levels of consistency: 61% for human-human; 69% for mouse-mouse; 47% for human-mouse. Conclusion GO-Diff is the first software integrating EST profiles with GO knowledge databases to mine functional differentiation between biological systems, e.g. tissues of the same species or the same tissue cross species. With rapid accumulation of EST resources in the public domain and expanding sequencing effort in individual laboratories, GO-Diff is useful as a screening tool before undertaking serious expression studies.