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A transcriptome analysis of isoamyl alcohol-induced filamentation in yeast reveals a novel role for Gre2p as isovaleraldehyde reductase.

A transcriptome analysis was performed of Saccharomyces cerevisiae undergoing isoamyl alcohol-induced filament formation. In the crucial first 5 h of this process, only four mRNA species displayed strong and statistically significant increases in their levels of more than 10-fold. Two of these (YEL071w/DLD3 and YOL151w/GRE2) appear to play important roles in filamentation. The biochemical activities ascribed to these two genes (d-lactate dehydrogenase and methylglyoxal reductase, respectively) displayed similarly timed increases to those of their respective mRNAs. Mutants carrying dld3 mutations displayed reduced filamentation in 0.5% isoamyl alcohol and needed a higher concentration of isoamyl alcohol to effect more complete filament formation. Hence, DLD3 seems to be required for a full response to isoamyl alcohol, but is not absolutely essential for it. Mutants carrying gre2 mutations were derepressed for filament formation and formed large, invasive filaments even in the absence of isoamyl alcohol. These results indicate a previously unsuspected and novel role for the GRE2 gene product as a suppressor of filamentation by virtue of encoding isovaleraldehyde reductase activity.

Aldehydes↗

Analysing the platelet transcriptome.

Historical studies, using Northern blot hybridization, RT-PCR and cDNA library construction have demonstrated the presence of a variety of mRNA molecules in platelets. The development of microarray technology has allowed further characterization of the transcripts represented in the platelet transcriptome. In this review, these studies will be summarized and their findings in relation to the study of platelet function and the identification of disease risk genes discussed.

Blood Platelets↗

The human transcriptome map: clustering of highly expressed genes in chromosomal domains.

The chromosomal position of human genes is rapidly being established. We integrated these mapping data with genome-wide messenger RNA expression profiles as provided by SAGE (serial analysis of gene expression). Over 2.45 million SAGE transcript tags, including 160,000 tags of neuroblastomas, are presently known for 12 tissue types. We developed algorithms to assign these tags to UniGene clusters and their chromosomal position. The resulting Human Transcriptome Map generates gene expression profiles for any chromosomal region in 12 normal and pathologic tissue types. The map reveals a clustering of highly expressed genes to specific chromosomal regions. It provides a tool to search for genes that are overexpressed or silenced in cancer.

Algorithms↗

Sex-dependent gene expression and evolution of the Drosophila transcriptome.

Comparison of the gene-expression profiles between adults of Drosophila melanogaster and Drosophila simulans has uncovered the evolution of genes that exhibit sex-dependent regulation. Approximately half the genes showed differences in expression between the species, and among these, approximately 83% involved a gain, loss, increase, decrease, or reversal of sex-biased expression. Most of the interspecific differences in messenger RNA abundance affect male-biased genes. Genes that differ in expression between the species showed functional clustering only if they were sex-biased. Our results suggest that sex-dependent selection may drive changes in expression of many of the most rapidly evolving genes in the Drosophila transcriptome.

Animals↗

Antisense transcription in the mammalian transcriptome.

Antisense transcription (transcription from the opposite strand to a protein-coding or sense strand) has been ascribed roles in gene regulation involving degradation of the corresponding sense transcripts (RNA interference), as well as gene silencing at the chromatin level. Global transcriptome analysis provides evidence that a large proportion of the genome can produce transcripts from both strands, and that antisense transcripts commonly link neighboring "genes" in complex loci into chains of linked transcriptional units. Expression profiling reveals frequent concordant regulation of sense/antisense pairs. We present experimental evidence that perturbation of an antisense RNA can alter the expression of sense messenger RNAs, suggesting that antisense transcription contributes to control of transcriptional outputs in mammals.

Animals↗

Transcriptomic and functional analysis of an autolysis-deficient, teicoplanin-resistant derivative of methicillin-resistant Staphylococcus aureus.

The molecular basis of glycopeptide-intermediate S. aureus (GISA) isolates is not well defined though frequently involves phenotypes such as thickened cell walls and decreased autolysis. We have exploited an isogenic pair of teicoplanin-susceptible (strain MRGR3) and teicoplanin-resistant (strain 14-4) methicillin-resistant S. aureus strains for detailed transcriptomic profiling and analysis of altered autolytic properties. Strain 14-4 displayed markedly deficient Triton X-100-triggered autolysis compared to its teicoplanin-susceptible parent, although microarray analysis paradoxically did not reveal significant reductions in expression levels of major autolytic genes atl, lytM, and lytN, except for sle1, which showed a slight decrease. The most important paradox was a more-than-twofold increase in expression of the cidABC operon in 14-4 compared to MRGR3, which was correlated with decreased expression of autolysis negative regulators lytSR and lrgAB. In contrast, the autolysis-deficient phenotype of 14-4 was correlated with both increased expression of negative autolysis regulators (arlRS, mgrA, and sarA) and decreased expression of positive regulators (agr RNAII and RNAIII). Quantitative bacteriolytic assays and zymographic analysis of concentrated culture supernatants showed a striking reduction in Atl-derived, extracellular bacteriolytic hydrolase activities in 14-4 compared to MRGR3. This observed difference was independent of the source of cell wall substrate (MRGR3 or 14-4) used for analysis. Collectively, our results suggest that altered autolytic properties in 14-4 are apparently not driven by significant changes in the transcription of key autolytic effectors. Instead, our analysis points to alternate regulatory mechanisms that impact autolysis effectors which may include changes in posttranscriptional processing or export.

Bacteriolysis↗

Transcriptome analysis reveals mechanisms by which Lactococcus lactis acquires nisin resistance.

Nisin, a posttranslationally modified antimicrobial peptide produced by Lactococcus lactis, is widely used as a food preservative. Yet, the mechanisms leading to the development of nisin resistance in bacteria are poorly understood. We used whole-genome DNA microarrays of L. lactis IL1403 to identify the factors underlying acquired nisin resistance mechanisms. The transcriptomes of L. lactis IL1403 and L. lactis IL1403 Nis(r), which reached a 75-fold higher nisin resistance level, were compared. Differential expression was observed in genes encoding proteins that are involved in cell wall biosynthesis, energy metabolism, fatty acid and phospholipid metabolism, regulatory functions, and metal and/or peptide transport and binding. These results were further substantiated by showing that several knockout and overexpression mutants of these genes had strongly altered nisin resistance levels and that some knockout strains could no longer become resistant to the same level of nisin as that of the wild-type strain. The acquired nisin resistance mechanism in L. lactis is complex, involving various different mechanisms. The four major mechanisms are (i) preventing nisin from reaching the cytoplasmic membrane, (ii) reducing the acidity of the extracellular medium, thereby stimulating the binding of nisin to the cell wall, (iii) preventing the insertion of nisin into the membrane, and (iv) possibly transporting nisin across the membrane or extruding nisin out of the membrane.

Anti-Bacterial Agents↗

Temporal transcriptomic analysis as Desulfovibrio vulgaris Hildenborough transitions into stationary phase during electron donor depletion.

Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the stationary phase during electron donor depletion. In addition to temporal transcriptomics, total protein, carbohydrate, lactate, acetate, and sulfate levels were measured. The microarray data were examined for statistically significant expression changes, hierarchical cluster analysis, and promoter element prediction and were validated by quantitative PCR. As the cells transitioned from the exponential phase to the stationary phase, a majority of the down-expressed genes were involved in translation and transcription, and this trend continued at the remaining times. There were general increases in relative expression for intracellular trafficking and secretion, ion transport, and coenzyme metabolism as the cells entered the stationary phase. As expected, the DNA replication machinery was down-expressed, and the expression of genes involved in DNA repair increased during the stationary phase. Genes involved in amino acid acquisition, carbohydrate metabolism, energy production, and cell envelope biogenesis did not exhibit uniform transcriptional responses. Interestingly, most phage-related genes were up-expressed at the onset of the stationary phase. This result suggested that nutrient depletion may affect community dynamics and DNA transfer mechanisms of sulfate-reducing bacteria via the phage cycle. The putative feoAB system (in addition to other presumptive iron metabolism genes) was significantly up-expressed, and this suggested the possible importance of Fe2+ acquisition under metal-reducing conditions. The expression of a large subset of carbohydrate-related genes was altered, and the total cellular carbohydrate levels declined during the growth phase transition. Interestingly, the D. vulgaris genome does not contain a putative rpoS gene, a common attribute of the delta-Proteobacteria genomes sequenced to date, and the transcription profiles of other putative rpo genes were not significantly altered. Our results indicated that in addition to expected changes (e.g., energy conversion, protein turnover, translation, transcription, and DNA replication and repair), genes related to phage, stress response, carbohydrate flux, the outer envelope, and iron homeostasis played important roles as D. vulgaris cells experienced electron donor depletion.

Bacterial Proteins↗

Transcriptome-based identification of the Sinorhizobium meliloti NodD1 regulon.

The NodD1 regulon of Sinorhizobium meliloti was determined through the analysis of the S. meliloti transcriptome in response to the plant flavone luteolin and the overexpression of nodD1. Nine new genes regulated by both NodD1 and luteolin were identified, demonstrating that NodD1 controls few functions behind nodulation in S. meliloti.

Bacterial Proteins↗

Transcriptomic and proteomic approach for understanding the molecular basis of adaptation of Saccharomyces cerevisiae to wine fermentation.

Throughout alcoholic fermentation, Saccharomyces cerevisiae cells have to cope with several stress conditions that could affect their growth and viability. In addition, the metabolic activity of yeast cells during this process leads to the production of secondary compounds that contribute to the organoleptic properties of the resulting wine. Commercial strains have been selected during the last decades for inoculation into the must to carry out the alcoholic fermentation on the basis of physiological traits, but little is known about the molecular basis of the fermentative behavior of these strains. In this work, we present the first transcriptomic and proteomic comparison between two commercial strains with different fermentative behaviors. Our results indicate that some physiological differences between the fermentative behaviors of these two strains could be related to differences in the mRNA and protein profiles. In this sense, at the level of gene expression, we have found differences related to carbohydrate metabolism, nitrogen catabolite repression, and response to stimuli, among other factors. In addition, we have detected a relative increase in the abundance of proteins involved in stress responses (the heat shock protein Hsp26p, for instance) and in fermentation (in particular, the major cytosolic aldehyde dehydrogenase Ald6p) in the strain with better behavior during vinification. Moreover, in the case of the other strain, higher levels of enzymes required for sulfur metabolism (Cys4p, Hom6p, and Met22p) are observed, which could be related to the production of particular organoleptic compounds or to detoxification processes.

Adaptation, Physiological↗

Description of the transcriptomes of immune response-activated hemocytes from the mosquito vectors Aedes aegypti and Armigeres subalbatus.

Mosquito-borne diseases, including dengue, malaria, and lymphatic filariasis, exact a devastating toll on global health and economics, killing or debilitating millions every year (54). Mosquito innate immune responses are at the forefront of concerted research efforts aimed at defining potential target genes that could be manipulated to engineer pathogen resistance in vector populations. We aimed to describe the pivotal role that circulating blood cells (called hemocytes) play in immunity by generating a total of 11,952 Aedes aegypti and 12,790 Armigeres subalbatus expressed sequence tag (EST) sequences from immune response-activated hemocyte libraries. These ESTs collapsed into 2,686 and 2,107 EST clusters, respectively. The clusters were used to adapt the web-based interface for annotating bacterial genomes called A Systematic Annotation Package for Community Analysis of Genomes (ASAP) for analysis of ESTs. Each cluster was categorically characterized and annotated in ASAP based on sequence similarity to five sequence databases. The sequence data and annotations can be viewed in ASAP at https://asap.ahabs.wisc.edu/annotation/php/ASAP1.htm. The data presented here represent the results of the first high-throughput in vivo analysis of the transcriptome of immunocytes from an invertebrate. Among the sequences are those for numerous immunity-related genes, many of which parallel those employed in vertebrate innate immunity, that have never been described for these mosquitoes. The sequences and annotations presented in this paper have been submitted to GenBank under accession numbers AY 431103 to AY 433788 (Aedes aegypti) and AY 439334 to AY 441440 (Armigeres subalbatus).

Aedes↗

Global transcriptome analysis of Tropheryma whipplei in response to temperature stresses.

Tropheryma whipplei, the agent responsible for Whipple disease, is a poorly known pathogen suspected to have an environmental origin. The availability of the sequence of the 0.92-Mb genome of this organism made a global gene expression analysis in response to thermal stresses feasible, which resulted in unique transcription profiles. A few genes were differentially transcribed after 15 min of exposure at 43 degrees C. The effects observed included up-regulation of the dnaK regulon, which is composed of six genes and is likely to be under control of two HspR-associated inverted repeats (HAIR motifs) found in the 5' region. Putative virulence factors, like the RibC and IspDF proteins, were also overexpressed. While it was not affected much by heat shock, the T. whipplei transcriptome was strongly modified following cold shock at 4 degrees C. For the 149 genes that were differentially transcribed, eight regulons were identified, and one of them was composed of five genes exhibiting similarity with genes encoding ABC transporters. Up-regulation of these genes suggested that there was an increase in nutrient uptake when the bacterium was exposed to cold stress. As observed for other bacterial species, the major classes of differentially transcribed genes encode membrane proteins and enzymes involved in fatty acid biosynthesis, indicating that membrane modifications are critical. Paradoxically, the heat shock proteins GroEL2 and ClpP1 were up-regulated. Altogether, the data show that despite the lack of classical regulation pathways, T. whipplei exhibits an adaptive response to thermal stresses which is consistent with its specific environmental origin and could allow survival under cold conditions.

Actinomycetales↗

Transcriptomic analysis reveals a bifurcated terephthalate degradation pathway in Rhodococcus sp. strain RHA1.

Phthalate isomers and their esters are important pollutants whose biodegradation is not well understood. Rhodococcus sp. strain RHA1 is notable for its ability to degrade a wide range of aromatic compounds. RHA1 was previously shown to degrade phthalate (PTH) and to have genes putatively encoding terephthalate (TPA) degradation. Transcriptomic analysis of 8,213 genes indicated that 150 were up-regulated during growth on PTH and that 521 were up-regulated during growth on TPA. Distinct ring cleavage dioxygenase systems were differentially expressed during growth on PTH and TPA. Genes encoding the protocatechuate (PCA) pathway were induced on both substrates, while genes encoding the catechol branch of the PCA pathway were additionally induced only on TPA. Accordingly, protocatechuate-3,4-dioxygenase activity was induced in cells grown on both substrates, while catechol-1,2-dioxygenase activity was induced only in cells grown on TPA. Knockout analysis indicated that pcaL, encoding 3-oxoadipate enol-lactone hydrolase and 4-carboxymuconolactone decarboxylase, was required for growth on both substrates but that pcaB, encoding beta-carboxy-cis,cis-muconate lactonizing enzyme, was required for growth on PTH only. These results indicate that PTH is degraded solely via the PCA pathway, whereas TPA is degraded via a bifurcated pathway that additionally includes the catechol branch of the PCA pathway.

Gene Expression Regulation, Bacterial↗

Correlation between Bacillus subtilis scoC phenotype and gene expression determined using microarrays for transcriptome analysis.

The availability of the complete sequence of the Bacillus subtilis chromosome (F. Kunst et al., Nature 390:249-256, 1997) makes possible the construction of genome-wide DNA arrays and the study of this organism on a global scale. Because we have a long-standing interest in the effects of scoC on late-stage developmental phenomena as they relate to aprE expression, we studied the genome-wide effects of a scoC null mutant with the goal of furthering the understanding of the role of scoC in growth and developmental processes. In the present work we compared the expression patterns of isogenic B. subtilis strains, one of which carries a null mutation in the scoC locus (scoC4). The results obtained indicate that scoC regulates, either directly or indirectly, the expression of at least 560 genes in the B. subtilis genome. ScoC appeared to repress as well as activate gene expression. Changes in expression were observed in genes encoding transport and binding proteins, those involved in amino acid, carbohydrate, and nucleotide and/or nucleoside metabolism, and those associated with motility, sporulation, and adaptation to atypical conditions. Changes in gene expression were also observed for transcriptional regulators, along with sigma factors, regulatory phosphatases and kinases, and members of sensor regulator systems. In this report, we discuss some of the phenotypes associated with the scoC mutant in light of the transcriptome changes observed.

Bacillus subtilis↗

Proteome and transcriptome analysis of the virulence genes regulated by the VirR/VirS system in Clostridium perfringens.

The proteins under the control of the two-component system VirR/VirS in Clostridium perfringens were analyzed by using two-dimensional gel electrophoresis of the culture supernatant from the wild type and the virR mutant. Based on matrix-assisted laser desorption ionization-time of flight/mass spectrometry, seven positively regulated proteins and eight negatively regulated proteins were identified. Transcriptome analysis confirmed that 7 of the 15 proteins were regulated by the VirR/VirS system at the transcriptional level, but the remaining proteins were modified with a VirR/VirS-directed protease at the posttranslation and secretion levels. We purified and characterized the VirR/VirS-directed protease from the culture supernatant and identified it as a kind of clostripain. Because this proteolytic activity was strongly inhibited by leupeptin and antipain, it was concluded that this protease was a member of the family of cysteine proteases of C. perfringens.

Bacterial Proteins↗

Transcriptome and proteome analysis of Bacillus subtilis gene expression modulated by amino acid availability.

A comprehensive study of Bacillus subtilis gene expression patterns in response to amino acid availability was performed by means of proteomics and transcriptomics. The methods of two-dimensional protein gel electrophoresis and DNA macroarray technology were combined to analyze cells exponentially grown in minimal medium with and without 0.2% Casamino Acids (CAA). This approach revealed about 120 genes predominantly involved in amino acid biosynthesis, sporulation, and competence, which were downregulated in CAA-containing medium. Determination of sporulation frequencies confirmed the physiological relevance of the expression data.

Amino Acids↗

Transcriptome analysis of Neisseria meningitidis during infection.

Neisseria meningitidis is the cause of septicemia and meningococcal meningitis. During the course of infection, N. meningitidis encounters multiple environments within its host, which makes rapid adaptation to environmental changes a crucial factor for neisserial pathogenicity. Employing oligonucleotide-based DNA microarrays, we analyzed the transcriptome of N. meningitidis during two key steps of meningococcal infection, i.e., the interaction with epithelial cells (HeLa cells) and endothelial cells (human brain microvascular endothelial cells). Seventy-two genes were differentially regulated after contact with epithelial cells, and 48 genes were differentially regulated after contact with endothelial cells, including a considerable proportion of well-known virulence genes. While a considerable number of genes were in concordance between bacteria adherent to both cell types, we identified several open reading frames that were differentially regulated in only one system. The data obtained with this novel approach may provide insight into the pathogenicity mechanisms of N. meningitidis and could demonstrate the importance of gene regulation on the transcriptional level during different stages of meningococcal infection.

Bacterial Adhesion↗