Comparative and functional genome analysis of fungi for development of the protein production host Trichoderma reesei

Filamentous fungi of the subphylum Pezizomycotina are well known as protein and secondary metabolite producers. Various industries take advantage of these capabilities. However, the molecular biology of yeasts, i.e. Saccharomycotina and especially that of Saccharomyces cerevisiae, the baker's yeast, is much better known. In an effort to explain fungal phenotypes through their genotypes we have compared protein coding gene contents of Pezizomycotina and Saccharomycotina. Only biomass degradation and secondary metabolism related protein families seem to have expanded recently in Pezizomycotina. Of the protein families clearly diverged between Pezizomycotina and Saccharomycotina, those related to mitochondrial functions emerge as the most prominent. However, the primary metabolism as described in S. cerevisiae is largely conserved in all fungi. Apart from the known secondary metabolism, Pezizomycotina have pathways that could link secondary metabolism to primary metabolism and a wealth of undescribed enzymes. Previous studies of individual Pezizomycotina genomes have shown that regardless of the difference in production efficiency and diversity of secreted proteins, the content of the known secretion machinery genes in Pezizomycotina and Saccharomycotina appears very similar. Genome wide analysis of gene products is therefore needed to better understand the efficient secretion of Pezizomycotina. We have developed methods applicable to transcriptome analysis of non-sequenced organisms. TRAC (Transcriptional profiling with the aid of affinity capture) has been previously developed at VTT for fast, focused transcription analysis. We introduce a version of TRAC that allows more powerful signal amplification and multiplexing. We also present computational optimisations of transcriptome analysis of non-sequenced organism and TRAC analysis in general. Trichoderma reesei is one of the most commonly used Pezizomycotina in the protein production industry. In order to understand its secretion system better and find clues for improvement of its industrial performance, we have analysed its transcriptomic response to protein secretion stress conditions. In comparison to S. cerevisiae, the response of T. reesei appears different, but still impacts on the same cellular functions. We also discovered in T. reesei interesting similarities to mammalian protein secretion stress response. Together these findings highlight targets for more detailed studies.

Metabolite and transcriptome analysis of Campylobacter jejuni in vitro growth reveals a stationary-phase physiological switch.

Campylobacter jejuni is a prevalent cause of food-borne diarrhoeal illness in humans. Understanding of the physiological and metabolic capabilities of the organism is limited. We report a detailed analysis of the C. jejuni growth cycle in batch culture. Combined transcriptomic, phenotypic and metabolic analysis demonstrates a highly dynamic 'stationary phase', characterized by a peak in motility, numerous gene expression changes and substrate switching, despite transcript changes that indicate a metabolic downshift upon the onset of stationary phase. Video tracking of bacterial motility identifies peak activity during stationary phase. Amino acid analysis of culture supernatants shows a preferential order of amino acid utilization. Proton NMR (1H-NMR) highlights an acetate switch mechanism whereby bacteria change from acetate excretion to acetate uptake, most probably in response to depletion of other substrates. Acetate production requires pta (Cj0688) and ackA (Cj0689), although the acs homologue (Cj1537c) is not required. Insertion mutants in Cj0688 and Cj0689 maintain viability less well during the stationary and decline phases of the growth cycle than wild-type C. jejuni, suggesting that these genes, and the acetate pathway, are important for survival.

Geometric optimization methods for the analysis of gene expression data

DNA microarrays provide such a huge amount of data that unsupervised methods are required to reduce the dimension of the data set and to extract meaningful biological information. This work shows that Independent Component Analysis (ICA) is a promising approach for the analysis of genome-wide transcriptomic data. The paper first presents an overview of the most popular algorithms to perform ICA. These algorithms are then applied on a microarray breast-cancer data set. Some issues about the application of ICA and the evaluation of biological relevance of the results are discussed. This study indicates that ICA significantly outperforms Principal Component Analysis (PCA).

Quantitative RNA-seq analysis of the campylobacter jejuni transcriptome

Campylobacter jejuni is the most common bacterial cause of foodborne disease in the developed world. Its general physiology and biochemistry, as well as the mechanisms enabling it to colonize and cause disease in various hosts, are not well understood, and new approaches are required to understand its basic biology. High-throughput sequencing technologies provide unprecedented opportunities for functional genomic research. Recent studies have shown that direct Illumina sequencing of cDNA (RNA-seq) is a useful technique for the quantitative and qualitative examination of transcriptomes. In this study we report RNA-seq analyses of the transcriptomes of C. jejuni (NCTC11168) and its rpoN mutant. This has allowed the identification of hitherto unknown transcriptional units, and further defines the regulon that is dependent on rpoN for expression. The analysis of the NCTC11168 transcriptome was supplemented by additional proteomic analysis using liquid chromatography-MS. The transcriptomic and proteomic datasets represent an important resource for the Campylobacter research community. © 2011 SGM.

Meta-analysis of prostate cancer gene expression data identifies a novel discriminatory signature enriched for glycosylating enzymes

BACKGROUND: Tumorigenesis is characterised by changes in transcriptional control. Extensive transcript expression data have been acquired over the last decade and used to classify prostate cancers. Prostate cancer is, however, a heterogeneous multifocal cancer and this poses challenges in identifying robust transcript biomarkers.

METHODS: In this study, we have undertaken a meta-analysis of publicly available transcriptomic data spanning datasets and technologies from the last decade and encompassing laser capture microdissected and macrodissected sample sets.

RESULTS: We identified a 33 gene signature that can discriminate between benign tissue controls and localised prostate cancers irrespective of detection platform or dissection status. These genes were significantly overexpressed in localised prostate cancer versus benign tissue in at least three datasets within the Oncomine Compendium of Expression Array Data. In addition, they were also overexpressed in a recent exon-array dataset as well a prostate cancer RNA-seq dataset generated as part of the The Cancer Genomics Atlas (TCGA) initiative. Biologically, glycosylation was the single enriched process associated with this 33 gene signature, encompassing four glycosylating enzymes. We went on to evaluate the performance of this signature against three individual markers of prostate cancer, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) expression, prostate specific antigen (PSA) expression and androgen receptor (AR) expression in an additional independent dataset. Our signature had greater discriminatory power than these markers both for localised cancer and metastatic disease relative to benign tissue, or in the case of metastasis, also localised prostate cancer.

CONCLUSION: In conclusion, robust transcript biomarkers are present within datasets assembled over many years and cohorts and our study provides both examples and a strategy for refining and comparing datasets to obtain additional markers as more data are generated.

SNP discovery using Next Generation Transcriptomic Sequencing in Atlantic herring (Clupea harengus)

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild.

Characterizing the effects of ocean acidification in larval and juvenile Manila clam, Ruditapes philippinarum, using a transcriptomic approach

Ocean acidification as a result of anthropogenic carbon dioxide (CO2) emissions and global climate change poses a risk to the ecological landscape of intertidal and shallow subtidal communities. The organisms that inhabit these waters will have to cope with changing environmental conditions through the appropriate modulation of physiological processes. Calcifying organisms are particularly at risk, as increased atmospheric levels of CO2 in the atmosphere increase the partial pressure of CO2 (pCO2) in the oceans. Increased pCO2 reduces the saturation of carbonate minerals required to form calcified structures. Being able to cope with the increased energetic demand of maintaining these structures, in addition to other vital physiological processes, will be the key driver that determines which organisms will persist. Assessment of larval and juvenile Manila clam mortality and physiology in this study suggests that this species is capable of coping with elevated pCO2 conditions. The use of high throughput sequencing and RNA sequence analysis in larval clams revealed several physiological processes that play important roles in the Manila clam’s ability to tolerate elevated pCO2 conditions during this life stage. Exposure of juvenile Manila clams, acclimated to elevated pCO2 conditions, to a thermal stress revealed that this species might also be capable of coping with multiple stressors associated with global climate change. Manila clams could therefore represent a model for studying physiological mechanisms associated with successful acclimation of populations to ocean acidification.

Genomic, Epigenomic and Transcriptomic Molecular Characterization of Splenic Marginal Zone Lymphoma Reveals Recurrent Abnormalities in miR-182

Splenic marginal zone lymphoma (SMZL) is a low grade B-cell non-Hodgkin's lymphoma. The molecular pathology of this entity remains poorly understood. To characterise this lymphoma at the molecular level, we performed an integrated analysis of 1) genome wide genetic copy number alterations 2) gene expression profiles and 3) epigenetic DNA methylation profiles.We have previously shown that SMZL is characterised by recurrent alterations of chromosomes 7q, 6q, 3q, 9q and 18; however, gene resolution oligonucleotide array comparative genomic hybridisation did not reveal evidence of cryptic amplification or deletion in these regions. The most frequently lost 7q32 region contains a cluster of miRNAs. qRT-PCR revealed that three of these (miR-182/96/183) show underexpression in SMZL, and miR-182 is somatically mutated in >20% of cases of SMZL, as well as in >20% of cases of follicular lymphoma, and between 5-15% of cases of chronic lymphocytic leukaemia, MALT-lymphoma and hairy cell leukaemia. We conclude that miR-182 is a strong candidate novel tumour suppressor miRNA in lymphoma.The overall gene expression signature of SMZL was found to be strongly distinct fromthose of other lymphomas. Functional analysis of gene expression data revealed SMZL to be characterised by abnormalities in B-cell receptor signalling (especially through the CD19/21-PI3K/AKT pathway) and apoptotic pathways. In addition, genes involved in the response to viral infection appeared upregulated. SMZL shows a unique epigenetic profile, but analysis of differentially methylated genes showed few with methylation related transcriptional deregulation, suggesting that DNA methylation abnormalities are not a critical component of the SMZL malignant phenotype.

N-nitroso compound exposure-associated transcriptomic profiles are indicative of an increased risk for colorectal cancer

Endogenous formation of N-nitroso compounds (NOCs), which are known animal carcinogens, could contribute to human carcinogenesis but definitive evidence is still lacking. To investigate the relevance of NOCs in human colorectal cancer (CRC) development, we analyzed whole genome gene expression modifications in human colon biopsies in relation to fecal NOC exposure. We had a particular interest in patients suffering from intestinal inflammation as this may stimulate endogenous NOC formation, and consequently predispose to CRC risk. Inflammatory bowel disease (IBD) patients diagnosed with ulcerative colitis and irritable bowel syndrome patients without inflammation, serving as controls, were therefore recruited. Fecal NOC were demonstrated in the majority of subjects. By associating gene expression levels of all subjects to fecal NOC levels, we identified a NOC exposure-associated transcriptomic response that suggests that physiological NOC concentrations may potentially induce genotoxic responses and chromatin modifications in human colon tissue, both of which are linked to carcinogenicity. In a network analysis, chromatin modifications were linked to 11 significantly modulated histone genes, pointing towards a possible epigenetic mechanism that may be relevant in comprehending NOC-induced carcinogenesis. In addition, pro-inflammatory transcriptomic modifications were identified in visually non-inflamed regions of the IBD colon. However, fecal NOC levels were slightly but not significantly increased in IBD patients, suggesting that inflammation did not strongly stimulate NOC formation. We conclude that NOC exposure is associated with gene expression modifications in the human colon that may suggest a potential role of these compounds in CRC development.

Optimising the analysis of transcript data using high density oligonucleotide arrays and genomic DNA-based probe selection

Background: Affymetrix GeneChip arrays are widely used for transcriptomic studies in a diverse range of species. Each gene is represented on a GeneChip array by a probe- set, consisting of up to 16 probe-pairs. Signal intensities across probe- pairs within a probe-set vary in part due to different physical hybridisation characteristics of individual probes with their target labelled transcripts. We have previously developed a technique to study the transcriptomes of heterologous species based on hybridising genomic DNA (gDNA) to a GeneChip array designed for a different species, and subsequently using only those probes with good homology. Results: Here we have investigated the effects of hybridising homologous species gDNA to study the transcriptomes of species for which the arrays have been designed. Genomic DNA from Arabidopsis thaliana and rice (Oryza sativa) were hybridised to the Affymetrix Arabidopsis ATH1 and Rice Genome GeneChip arrays respectively. Probe selection based on gDNA hybridisation intensity increased the number of genes identified as significantly differentially expressed in two published studies of Arabidopsis development, and optimised the analysis of technical replicates obtained from pooled samples of RNA from rice. Conclusion: This mixed physical and bioinformatics approach can be used to optimise estimates of gene expression when using GeneChip arrays.

A Transcriptomic View of the Proteome Variability of Newborn and Adult Bothrops jararaca Snake Venoms

Background: Snake bite is a neglected public health problem in communities in rural areas of several countries. Bothrops jararaca causes many snake bites in Brazil and previous studies have demonstrated that the pharmacological activities displayed by its venom undergo a significant ontogenetic shift. Similarly, the venom proteome of B. jararaca exhibits a considerable variation upon neonate to adult transition, which is associated with changes in diet from ectothermic prey in early life to endothermic prey in adulthood. Moreover, it has been shown that the Brazilian commercial antibothropic antivenom, which is produced by immunization with adult venom, is less effective in neutralizing newborn venom effects. On the other hand, venom gland transcripts of newborn snakes are poorly known since all transcriptomic studies have been carried out using mRNA from adult specimens. Methods/Principal Findings: Here we analyzed venom gland cDNA libraries of newborn and adult B. jararaca in order to evaluate whether the variability demonstrated for its venom proteome and pharmacological activities was correlated with differences in the structure of toxin transcripts. The analysis revealed that the variability in B. jararaca venom gland transcriptomes is quantitative, as illustrated by the very high content of metalloproteinases in the newborn venom glands. Moreover, the variability is also characterized by the structural diversity of SVMP precursors found in newborn and adult transcriptomes. In the adult transcriptome, however, the content of metalloproteinase precursors considerably diminishes and the number of transcripts of serine proteinases, C-type lectins and bradykinin-potentiating peptides increase. Moreover, the comparison of the content of ESTs encoding toxins in adult male and female venom glands showed some genderrelated differences. Conclusions/Significance: We demonstrate a substantial shift in toxin transcripts upon snake development and a marked decrease in the metalloproteinase P-III/P-I class ratio which are correlated with changes in the venom proteome complexity and pharmacological activities.

A quantitative proteomic and transcriptomic comparison of human mesenchymal stem cells from bone marrow and umbilical cord vein

Human mesenchymal stem cells (hMSCs) are adult multipotent cells that have high therapeutic potential due to their immunological properties. They can be isolated from several different tissues with bone marrow (BM) being the most common source. Because the isolation procedure is invasive, other tissues such as human umbilical cord vein (UCV) have been considered. However, their interchangeability remains unclear. In the present study, total protein extracts of BM-hMSCs and UCV-hMSCs were quantitatively compared using gel-LC-MS/MS. Previous SAGE analysis of the same cells was re-annotated to enable comparison and combination of these two data sets. We observed a more than 63% correlation between proteomic and transcriptomic data. In silico analysis of highly expressed genes in cells of both origins suggests that they can be modulated by microRNA, which can change protein abundance. Our results showed that MSCs from both tissues shared high similarity in metabolic and functional processes relevant to their therapeutic potential, especially in the immune system process, response to stimuli, and processes related to the delivery of the hMSCs to a given tissue, such as migration and adhesion. Hence, our results support the idea that the more accessible UCV could be a potentially less invasive source of MSCs.

Proteomic analysis of the Theileria annulata schizont

The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites.

Whole transcriptome analysis of the coral Acropora millepora reveals complex responses to CO2-driven acidification during the initiation of calcification

The impact of ocean acidification (OA) on coral calcification, a subject of intense current interest, is poorly understood in part because of the presence of symbionts in adult corals. Early life history stages of Acropora spp. provide an opportunity to study the effects of elevated CO(2) on coral calcification without the complication of symbiont metabolism. Therefore, we used the Illumina RNAseq approach to study the effects of acute exposure to elevated CO(2) on gene expression in primary polyps of Acropora millepora, using as reference a novel comprehensive transcriptome assembly developed for this study. Gene ontology analysis of this whole transcriptome data set indicated that CO(2) -driven acidification strongly suppressed metabolism but enhanced extracellular organic matrix synthesis, whereas targeted analyses revealed complex effects on genes implicated in calcification. Unexpectedly, expression of most ion transport proteins was unaffected, while many membrane-associated or secreted carbonic anhydrases were expressed at lower levels. The most dramatic effect of CO(2) -driven acidification, however, was on genes encoding candidate and known components of the skeletal organic matrix that controls CaCO(3) deposition. The skeletal organic matrix effects included elevated expression of adult-type galaxins and some secreted acidic proteins, but down-regulation of other galaxins, secreted acidic proteins, SCRiPs and other coral-specific genes, suggesting specialized roles for the members of these protein families and complex impacts of OA on mineral deposition. This study is the first exhaustive exploration of the transcriptomic response of a scleractinian coral to acidification and provides an unbiased perspective on its effects during the early stages of calcification.

Distinct and conserved transcriptomic changes during nematode-induced giant cell development in tomato compared with Arabidopsis: a functional role for gene repression

Root-knot nematodes (RKNs) induce giant cells (GCs) from root vascular cells inside the galls. Accompanying molecular changes as a function of infection time and across different species, and their functional impact, are still poorly understood. Thus, the transcriptomes of tomato galls and laser capture microdissected (LCM) GCs over the course of parasitism were compared with those of Arabidopsis, and functional analysis of a repressed gene was performed. Microarray hybridization with RNA from galls and LCM GCs, infection-reproduction tests and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) transcriptional profiles in susceptible and resistant (Mi-1) lines were performed in tomato. Tomato GC-induced genes include some possibly contributing to the epigenetic control of GC identity. GC-repressed genes are conserved between tomato and Arabidopsis, notably those involved in lignin deposition. However, genes related to the regulation of gene expression diverge, suggesting that diverse transcriptional regulators mediate common responses leading to GC formation in different plant species. TPX1, a cell wall peroxidase specifically involved in lignification, was strongly repressed in GCs/galls, but induced in a nearly isogenic Mi-1 resistant line on nematode infection. TPX1 overexpression in susceptible plants hindered nematode reproduction and GC expansion. Time-course and cross-species comparisons of gall and GC transcriptomes provide novel insights pointing to the relevance of gene repression during RKN establishment.