The mammalian transcriptome and the cellular complexity of the brain.
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Animals↗
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A comprehensive survey of the Plasmodium life cycle by genomic, transcriptomic, and proteomic analyses.
Plasmodium berghei and Plasmodium chabaudi are widely used model malaria species. Comparison of their genomes, integrated with proteomic and microarray data, with the genomes of Plasmodium falciparum and Plasmodium yoelii revealed a conserved core of 4500 Plasmodium genes in the central regions of the 14 chromosomes and highlighted genes evolving rapidly because of stage-specific selective pressures. Four strategies for gene expression are apparent during the parasites' life cycle: (i) housekeeping; (ii) host-related; (iii) strategy-specific related to invasion, asexual replication, and sexual development; and (iv) stage-specific. We observed posttranscriptional gene silencing through translational repression of messenger RNA during sexual development, and a 47-base 3' untranslated region motif is implicated in this process.
3' Untranslated Regions↗
Parallel patterns of evolution in the genomes and transcriptomes of humans and chimpanzees.
The determination of the chimpanzee genome sequence provides a means to study both structural and functional aspects of the evolution of the human genome. Here we compare humans and chimpanzees with respect to differences in expression levels and protein-coding sequences for genes active in brain, heart, liver, kidney, and testis. We find that the patterns of differences in gene expression and gene sequences are markedly similar. In particular, there is a gradation of selective constraints among the tissues so that the brain shows the least differences between the species whereas liver shows the most. Furthermore, expression levels as well as amino acid sequences of genes active in more tissues have diverged less between the species than have genes active in fewer tissues. In general, these patterns are consistent with a model of neutral evolution with negative selection. However, for X-chromosomal genes expressed in testis, patterns suggestive of positive selection on sequence changes as well as expression changes are seen. Furthermore, although genes expressed in the brain have changed less than have genes expressed in other tissues, in agreement with previous work we find that genes active in brain have accumulated more changes on the human than on the chimpanzee lineage.
Adult↗
Elucidation of the small RNA component of the transcriptome.
Small RNAs play important regulatory roles in most eukaryotes, but only a small proportion of these molecules have been identified. We sequenced more than two million small RNAs from seedlings and the inflorescence of the model plant Arabidopsis thaliana. Known and new microRNAs (miRNAs) were among the most abundant of the nonredundant set of more than 75,000 sequences, whereas more than half represented lower abundance small interfering RNAs (siRNAs) that match repetitive sequences, intergenic regions, and genes. Individual or clusters of highly regulated small RNAs were readily observed. Targets of antisense RNA or miRNA did not appear to be preferentially associated with siRNAs. Many genomic regions previously considered featureless were found to be sites of numerous small RNAs.
Arabidopsis↗
The transcriptome of the sea urchin embryo.
The sea urchin Strongylocentrotus purpuratus is a model organism for study of the genomic control circuitry underlying embryonic development. We examined the complete repertoire of genes expressed in the S. purpuratus embryo, up to late gastrula stage, by means of high-resolution custom tiling arrays covering the whole genome. We detected complete spliced structures even for genes known to be expressed at low levels in only a few cells. At least 11,000 to 12,000 genes are used in embryogenesis. These include most of the genes encoding transcription factors and signaling proteins, as well as some classes of general cytoskeletal and metabolic proteins, but only a minor fraction of genes encoding immune functions and sensory receptors. Thousands of small asymmetric transcripts of unknown function were also detected in intergenic regions throughout the genome. The tiling array data were used to correct and authenticate several thousand gene models during the genome annotation process.
Animals↗
Essay: Amersham Pharmacia Biotech & Science prize. Tantalizing transcriptomes--SAGE and its use in global gene expression analysis.
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Awards and Prizes↗
How unique is the rice transcriptome?
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Amino Acid Sequence↗
Global transcriptome analysis of the responses of a fluoroquinolone-resistant Streptococcus pneumoniae mutant and its parent to ciprofloxacin.
Streptococcus pneumoniae M22 is a multidrug-resistant mutant selected after exposure of capsulated wild-type S. pneumoniae NCTC 7465 (strain M4) to ciprofloxacin. DNA microarray analysis comparing the gene expression profiles of strain M22 with those of strain M4 showed that strain M22 constitutively expressed 22 genes at levels higher than those observed in strain M4 under all conditions studied. These included the genes encoding the enzymes involved in branched-chain amino acid biosynthesis and two genes (patA and patB) with sequences suggestive of ABC transporter proteins. Expression of the patA and patB genes was induced by ciprofloxacin in both strains, but in strain M4 it only reached the levels observed in strain M22 after long incubation with high concentrations of ciprofloxacin. The altered expression profile observed with strain M22 suggested that the mutation or mutations acquired during resistance selection bring the cell into a state in which the expression of critical genes is preemptively altered to correct for the potential effects of ciprofloxacin on gene expression in the parent strain.
Ciprofloxacin↗
Transcriptome of a Nitrosomonas europaea mutant with a disrupted nitrite reductase gene (nirK).
Global gene expression was compared between the Nitrosomonas europaea wild type and a nitrite reductase-deficient mutant using a genomic microarray. Forty-one genes were differentially regulated between the wild type and the nirK mutant, including the nirK operon, genes for cytochrome c oxidase, and seven iron uptake genes. Relationships of differentially regulated genes to the nirK mutant phenotype are discussed.
Electron Transport Complex IV↗
Development of Sinorhizobium meliloti pilot macroarrays for transcriptome analysis.
In order to prepare for whole-genome expression analysis in Sinorhizobium meliloti, pilot DNA macroarrays were designed for 34 genes of known regulation. The experimental parameters assessed were the length of the PCR products, the influence of a tag at the 5' end of the primers, and the method of RNA labeling. Variance and principal-component analysis showed that the most important nonbiological parameter was the labeling method. The sizes of PCR products were also found to be important, whereas the influence of 5' tags was minimal. The variability between replicated spots on a membrane was found to be low. These experimental procedures were validated by analyzing the effects of microaerobic conditions on gene expression.
Analysis of Variance↗
Adaptation of Corynebacterium glutamicum to ammonium limitation: a global analysis using transcriptome and proteome techniques.
Theresponse of Corynebacterium glutamicum to ammonium limitation was studied by transcriptional and proteome profiling of cells grown in a chemostat. Our results show that ammonium-limited growth of C. glutamicum results in a rearrangement of the cellular transport capacity, changes in metabolic pathways for nitrogen assimilation, amino acid biosynthesis, and carbon metabolism, as well as a decreased cell division. Since transcription at different growth rates was studied, it was possible to distinguish specific responses to ammonium limitation and more general, growth rate-dependent alterations in gene expression. The latter include a number of genes encoding ribosomal proteins and genes for F(o)F(1)-ATP synthase subunits.
Adaptation, Physiological↗
Transcriptome analysis of recombinant protein secretion by Aspergillus nidulans and the unfolded-protein response in vivo.
Filamentous fungi have a high capacity for producing large amounts of secreted proteins, a property that has been exploited for commercial production of recombinant proteins. However, the secretory pathway, which is key to the production of extracellular proteins, is rather poorly characterized in filamentous fungi compared to yeast. We report the effects of recombinant protein secretion on gene expression levels in Aspergillus nidulans by directly comparing a bovine chymosin-producing strain with its parental wild-type strain in continuous culture by using expressed sequence tag microarrays. This approach demonstrated more subtle and specific changes in gene expression than those observed when mimicking the effects of protein overproduction by using a secretion blocker. The impact of overexpressing a secreted recombinant protein more closely resembles the unfolded-protein response in vivo.
Aspergillus nidulans↗
Long serial analysis of gene expression for gene discovery and transcriptome profiling in the widespread marine coccolithophore Emiliania huxleyi.
The abundant and widespread coccolithophore Emiliania huxleyi plays an important role in mediating CO2 exchange between the ocean and the atmosphere through its impact on marine photosynthesis and calcification. Here, we use long serial analysis of gene expression (SAGE) to identify E. huxleyi genes responsive to nitrogen (N) or phosphorus (P) starvation. Long SAGE is an elegant approach for examining quantitative and comprehensive gene expression patterns without a priori knowledge of gene sequences via the detection of 21-bp nucleotide sequence tags. E. huxleyi appears to have a robust transcriptional-level response to macronutrient deficiency, with 42 tags uniquely present or up-regulated twofold or greater in the N-starved library and 128 tags uniquely present or up-regulated twofold or greater in the P-starved library. The expression patterns of several tags were validated with reverse transcriptase PCR. Roughly 48% of these differentially expressed tags could be mapped to publicly available genomic or expressed sequence tag (EST) sequence data. For example, in the P-starved library a number of the tags mapped to genes with a role in P scavenging, including a putative phosphate-repressible permease and a putative polyphosphate synthetase. In short, the long SAGE analyses have (i) identified many new differentially regulated gene sequences, (ii) assigned regulation data to EST sequences with no database homology and unknown function, and (iii) highlighted previously uncharacterized aspects of E. huxleyi N and P physiology. To this end, our long SAGE libraries provide a new public resource for gene discovery and transcriptional analysis in this biogeochemically important marine organism.
Base Sequence↗
Transcriptomic analysis of extensive changes in metabolic regulation in Kluyveromyces lactis strains.
Genome-wide analysis of transcriptional regulation is generally carried out on well-characterized reference laboratory strains; hence, the characteristics of industrial isolates are therefore overlooked. In a previous study on the major cheese yeast Kluyveromyces lactis, we have shown that the reference strain and an industrial strain used in cheese making display a differential gene expression when grown on a single carbon source. Here, we have used more controlled conditions, i.e., growth in a fermentor with pH and oxygen maintained constant, to study how these two isolates grown in glucose reacted to an addition of lactose. The observed differences between sugar consumption and the production of various metabolites, ethanol, acetate, and glycerol, correlated with the response were monitored by the analysis of the expression of 482 genes. Extensive differences in gene expression between the strains were revealed in sugar transport, glucose repression, ethanol metabolism, and amino acid import. These differences were partly due to repression by glucose and another, yet-unknown regulation mechanism. Our results bring to light a new type of K. lactis strain with respect to hexose transport gene content and repression by glucose. We found that a combination of point mutations and variation in gene regulation generates a biodiversity within the K. lactis species that was not anticipated. In contrast to S. cerevisiae, in which there is a massive increase in the number of sugar transporter and fermentation genes, in K. lactis, interstrain diversity in adaptation to a changing environment is based on small changes at the level of key genes and cell growth control.
Acetates↗
Metabolic-state-dependent remodeling of the transcriptome in response to anoxia and subsequent reoxygenation in Saccharomyces cerevisiae.
We conducted a comprehensive genomic analysis of the temporal response of yeast to anaerobiosis (six generations) and subsequent aerobic recovery ( approximately 2 generations) to reveal metabolic-state (galactose versus glucose)-dependent differences in gene network activity and function. Analysis of variance showed that far fewer genes responded (raw P value of or=2 generations) with the activation of Upc2- and Mot3-regulated networks involved in sterol and cell wall homeostasis. The response to reoxygenation was rapid (<5 min) and similar in both media, dominated by Yap1 networks involved in oxidative stress/redox regulation and the concomitant activation of heme-regulated ones. Our analyses revealed extensive networks of genes subject to combinatorial regulation by both heme-dependent (e.g., Hap1, Hap2/3/4/5, Rox1, Mot3, and Upc2) and heme-independent (e.g., Yap1, Skn7, and Puf3) factors under these conditions. We also uncover novel functions for several cis-regulatory sites and trans-acting factors and define functional regulons involved in the physiological acclimatization to changes in oxygen availability.
Anaerobiosis↗
Transcriptome analysis of Aspergillus nidulans exposed to camptothecin-induced DNA damage.
We have used an Aspergillus nidulans macroarray carrying sequences of 2,787 genes from this fungus to monitor gene expression of both wild-type and uvsB(ATR) (the homologue of the ATR gene) deletion mutant strains in a time course exposure to camptothecin (CPT). The results revealed a total of 1,512 and 1,700 genes in the wild-type and uvsB(ATR) deletion mutant strains that displayed a statistically significant difference at at least one experimental time point. We characterized six genes that have increased mRNA expression in the presence of CPT in the wild-type strain relative to the uvsB(ATR) mutant strain: fhdA (encoding a forkhead-associated domain protein), tprA (encoding a hypothetical protein that contains a tetratrico peptide repeat), mshA (encoding a MutS homologue involved in mismatch repair), phbA (encoding a prohibitin homologue), uvsC(RAD51) (the homologue of the RAD51 gene), and cshA (encoding a homologue of the excision repair protein ERCC-6 [Cockayne's syndrome protein]). The induced transcript levels of these genes in the presence of CPT require uvsB(ATR). These genes were deleted, and surprisingly, only the DeltauvsC mutant strain was sensitive to CPT; however, the others displayed sensitivity to a range of DNA-damaging and oxidative stress agents. These results indicate that the selected genes when inactivated display very complex and heterogeneous sensitivity behavior during growth in the presence of agents that directly or indirectly cause DNA damage. Moreover, with the exception of UvsC, deletion of each of these genes partially suppressed the sensitivity of the DeltauvsB strain to menadione and paraquat. Our results provide the first insight into the overall complexity of the response to DNA damage in filamentous fungi and suggest that multiple pathways may act in parallel to mediate DNA repair.
Algorithms↗
Transcriptome analysis of Paracoccidioides brasiliensis cells undergoing mycelium-to-yeast transition.
Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). The results revealed a total of 2,583 genes that displayed statistically significant modulation in at least one experimental time point. Among the identified gene homologues, some encoded enzymes involved in amino acid catabolism, signal transduction, protein synthesis, cell wall metabolism, genome structure, oxidative stress response, growth control, and development. The expression pattern of 20 genes was independently verified by real-time reverse transcription-PCR, revealing a high degree of correlation between the data obtained with the two methodologies. One gene, encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and the use of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], a specific inhibitor of 4-HPPD activity, as well as that of NTBC derivatives, was able to inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. These data set the stage for further studies involving NTBC and its derivatives as new chemotherapeutic agents against PCM and confirm the potential of array-based approaches to identify new targets for the development of alternative treatments against pathogenic microorganisms.
4-Hydroxyphenylpyruvate Dioxygenase↗
Transcriptome analysis of Pseudomonas aeruginosa after interaction with human airway epithelial cells.
The transcriptional profile of Pseudomonas aeruginosa after interactions with primary normal human airway epithelial cells was determined using Affymetrix GeneChip technology. Gene expression profiles indicated that various genes involved in phosphate acquisition and iron scavenging were differentially regulated.
Bacterial Proteins↗