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Suppression subtractive hybridization-mediated transcriptome analysis from multiple tissues of aspen (Populus tremuloides) altered in phenylpropanoid metabolism.

A PCR-based suppression subtractive hybridization (SSH) technique was used to identify differentially expressed genes in developing tissues of control and transgenic aspen (Populus tremuloides Michx.) with down-regulated 4CL1 (4-coumarate:coenzyme A ligase) expression and enhanced growth. A total of 11,308 expressed sequence tags (ESTs) representing 5,028 non-redundant transcripts encoding 4,224 unique proteins was obtained from shoot apex, young stem, young leaf and root tip SSH libraries. Putative functions can be assigned to 60% of these transcripts. Approximately 14% of the ESTs are not represented among the 111,000 entries already present in Populus EST databases. In general, ESTs of the metabolism class occurred at a higher frequency in control- than transgenic-enriched libraries of all tissues, whereas protein synthesis and protein fate ESTs were over-represented in meristematic tissues of transgenics where 4CL1 was relatively strongly suppressed. Among all tissues, leaves yielded the highest percentage of ESTs with either unknown protein function or insignificant similarity to other protein/DNA/EST sequences in existing databases. Of particular interest was a large number of ESTs (16%) associated with signal transduction in transgenic leaves. Among these were several leucine-rich-repeat receptor-like protein kinases with markedly elevated expression in transgenic leaves. We also identified homologs of transposable elements that were up-regulated in transgenic tissues, providing the first experimental data for active expression of DNA mobile elements in long-lived tree species.

DNA Transposable Elements↗

Transcriptome analysis of alfalfa glandular trichomes.

Glandular trichomes are a major site of plant natural product synthesis and accumulation for protection against insect predation. However, to date few studies have attempted to obtain a global view of trichome gene expression. Two contrasting approaches have been adopted to investigate genes expressed in glandular trichomes from alfalfa (Medicago sativa L.). In the first approach, 5,674 clones from an alfalfa glandular trichome cDNA library were sequenced. The most highly abundant expressed sequence tag (EST) corresponded to a lipid transfer protein. The presence of ESTs corresponding to enzymes for all steps in the biosynthesis of flavonoids suggests that these are important metabolites in alfalfa trichome biology, as confirmed by histochemistry and metabolite profiling. No ESTs corresponded to enzymes of cyclized terpenoid biosynthesis. In a second approach, microarray analysis was used to compare levels of alfalfa transcripts corresponding to 16,086 Medicago truncatula A17 genes in stems with and without trichomes. This revealed over 1,000 genes with strong preferential expression in the trichome fraction of the stem, 70% of which are of unknown function. These define a class of genes that are not trichome-specific, since M. truncatula A17 does not itself have glandular trichomes, but has potential importance for trichome function within the stem.

Base Sequence↗

Transcriptome analysis and molecular studies on sulfur metabolism in the human pathogenic fungus Paracoccidioides brasiliensis.

The dimorphic pathogenic fungus Paracoccidioides brasiliensis can grow as a prototroph for organic sulfur as a mycelial (non-pathogenic) form, but it is unable to assimilate inorganic sulfur as a yeast (pathogenic) form. Temperature and the inability to assimilate inorganic sulfur are the single conditions known to affect P. brasiliensis mycelium-to-yeast (M-Y) dimorphic transition. For a comprehensive evaluation of genes that have their expression modulated during the M-Y transition in different culture media, we performed a large-scale analysis of gene expression using a microarray hybridization approach. The results of the present work demonstrate the use of microarray hybridization analysis to examine gene expression during the M-Y transition in minimal medium and compare these results with the M-Y transition in complete medium. Our results showed that about 95% of the genes in our microarray are mainly responding to the temperature trigger, independently of the media where the M-Y transition took place. As a preliminary step to understand the inorganic sulfur inability in P. brasiliensis yeast form, we decided to characterize the mRNA accumulation of several genes involved in different aspects of both organic and inorganic sulfur assimilation. Our results suggest that although P. brasiliensis cannot use inorganic sulfur as a single sulfur source to initiate both M-Y transition and Y growth, the fungus can somehow use both organic and inorganic pathways during these growth processes.

Base Sequence↗

Transcriptome analysis of salinity stress responses in common wheat using a 22k oligo-DNA microarray.

In this study, we constructed a 22k wheat oligo-DNA microarray. A total of 148,676 expressed sequence tags of common wheat were collected from the database of the Wheat Genomics Consortium of Japan. These were grouped into 34,064 contigs, which were then used to design an oligonucleotide DNA microarray. Following a multistep selection of the sense strand, 21,939 60-mer oligo-DNA probes were selected for attachment on the microarray slide. This 22k oligo-DNA microarray was used to examine the transcriptional response of wheat to salt stress. More than 95% of the probes gave reproducible hybridization signals when targeted with RNAs extracted from salt-treated wheat shoots and roots. With the microarray, we identified 1,811 genes whose expressions changed more than 2-fold in response to salt. These included genes known to mediate response to salt, as well as unknown genes, and they were classified into 12 major groups by hierarchical clustering. These gene expression patterns were also confirmed by real-time reverse transcription-PCR. Many of the genes with unknown function were clustered together with genes known to be involved in response to salt stress. Thus, analysis of gene expression patterns combined with gene ontology should help identify the function of the unknown genes. Also, functional analysis of these wheat genes should provide new insight into the response to salt stress. Finally, these results indicate that the 22k oligo-DNA microarray is a reliable method for monitoring global gene expression patterns in wheat.

Expressed Sequence Tags↗

Micro serial analysis of gene expression in normal human choroid and retinal pigment epithelial transcriptomes.

PURPOSE: To investigate the gene expression profile of the normal human choroid/retinal pigment epithelium(RPE) tissues. METHODS: Micro serial analysis of gene expression (Micro SAGE) was performed. A SAGE library was constructed from 110 microg of total RNA of normal human choroid/RPE tissue, and cloned tag concatemers transformed to E.coli were sequenced. The sequence data were analyzed by SAGE software and matched to GenBank and UniGene public databases. The sequence data were also compared with the choroid/RPE cDNA library of NEIBank. RESULTS: A total of 12 070 tags were sequenced; 3627 tags were unique. Of these 3627 tags, 2508 tags were encoded genes and 1119 tags were unknown tags in the UniGene database. The most frequently expressed tag was TCCCTATTAA, but the gene corresponding to this tag has not been identified yet. Other frequently expressed tags encoded a tissue inhibitor of matrix metalloproteinase 3, insulin-like growth factor binding protein-related protein 1, and transthyretin. These genes are notably different, with high expression frequencies when compared to the cDNA library of NEIBank. CONCLUSIONS: This gene expression profile of the normal human choroid/RPE tissue should provide further understanding of the biological function of the choroid and the pathogenesis of diseases in which the choroid and RPE play a role, such as choroidal neovascularization.

Aged↗

Gene-dosage effect on chromosome 21 transcriptome in trisomy 21: implication in Down syndrome cognitive disorders.

In the era of human functional genomics, the chromosome 21 has represented a prototype for pioneering global biotechnologies. Its relatively low gene content enabled studying Down syndrome at the chromosomal scale, for which the last years have seen intense research activity aiming at genotype-phenotype correlations. The global gene-dose dependent upregulation of gene expression seen in the context of trisomy and preliminary functional annotation of chromosome 21 genes points towards candidate genes and molecular pathways potentially associated with the cognitive defects observed in Down syndrome.

Chromosome Mapping↗

Transcriptome analysis reveals an osteoblast-like phenotype for human osteotropic breast cancer cells.

Metastatic breast cancer cells exhibit the selective ability to seed and grow in the skeleton. We and others have previously reported that human breast tumors which metastasize to the skeleton overexpress bone matrix extracellular proteins. In an attempt to reveal the osteoblast-like phenotype of osteotropic breast cancer cells, we performed a microarray study on a model of breast cancer bone metastasis consisting of the MDA-MB-231 human cell line and its variant B02 selected for its high capacity to form bone metastases in vivo. Analysis of B02 cells transcriptional profile revealed that 11 and 9 out of the 50 most up- and down-regulated mRNAs, respectively, corresponded to genes which expression has been previously associated with osteoblastic differentiation process. Thus, osteoblast specific cadherin 11 which mediates the differentiation of mesenchymal cells into osteoblastic cells is up-regulated in B02. While S100A4, recently described as a key negative regulator of osteoblast differentiation, is the most down-regulated gene in B02 cells. RT-PCR and western blotting experiments allowed the validation of the modulation of several genes of interest. Using immunohistochemistry, performed on human breast primary tumors and their matched liver and bone metastases, we were able to confirm that the osteoblast-like pattern of gene expression observed in our model holds true in vivo. This is the first report demonstrating a gene-expression pattern corresponding to the acquisition of an osteomimetic phenotype by bone metastatic breast cancer cells.

Blotting, Western↗

Spinal cord transcriptome analysis using suppression subtractive hybridization and mirror orientation selection.

Comparison of cDNA libraries derived from the spinal cord with those derived from the visual cortex by means of forward and reverse subtractive hybridization resulted in the cataloguing of 60 genes differentially expressed in the spinal cord. 1. The differentially expressed genes represent a mixture of novel and known sequences with known and unknown protein products. 2. The possibility that the subtraction process was simply overwhelmed by background sequences was significantly reduced by several observations including comparisons between suppression subtractive hybridization (SSH) and mirror orientation selection (MOS). 3. Nearly half of all genes up-regulated in the spinal cord are of myelin origin. 4. Twenty-five percent of all up-regulated clones in the spinal cord versus the visual cortex are for proteolipid protein. 5. Ten percent of all up-regulated clones in spinal cord versus visual cortex are for ferretin heavy chain, which is known to be produced in oligodendroglial cells in the CNS. 6. Two of the up-regulated sequences, proteolipid protein and N-myc down-regulated gene 4, are identified with genes known to directly affect neuron survival. 7. Two of the up-regulated genes, ferritin and transferrin, are indirectly associated with apoptosis through their ability to sequester iron and reduce free radical formation.

Animals↗

Repeat structure of the catfish genome: a genomic and transcriptomic assessment of Tc1-like transposon elements in channel catfish (Ictalurus punctatus).

We have assessed the distribution and diversity of members of the Tc1/mariner superfamily of transposable elements in the channel catfish (Ictalurus punctatus) genome as well as evaluating the extent of transcription of Tc1 transposases in the species. Through use of PCR amplification and sequencing, assessment of random BAC end sequences (BES) equivalent to 1.2% genome coverage, and screening of over 45,000 catfish ESTs, a significant proportion of Tc1-like elements and their associated transcripts were captured. Up to 4.2% of the catfish genome in base pairs appears to be composed of Tc1-like transposon-related sequences and a significant fraction of the catfish cellular mRNA, approximately 0.6%, was transcribed from transposon-related sequences in both sense and antisense orientations. Based on results of repeat-masking, as much as 10% of BAC end sequences from catfish, which is a random survey of the genome, contain some remnant of Tc1 elements, suggesting that these elements are present in the catfish genome as numerous, small remnants of the transposons. Phylogenetic analysis allowed comparison of catfish Tc1 transposase types with those found in other vertebrate and invertebrate species. In spite of the existence of many types of Tc1-like sequences that are not yet able to be placed in clades with strong statistical support, it is clear that multiple families of Tc1-like elements exist in channel catfish.

Animals↗

Transcriptome expression profiles in prenatal pigs in relation to myogenesis.

Myogenesis, the formation of muscle fibers, is a complex process. Pigs have been selected for efficient muscle growth for the past decades making them interesting to study myogenesis. We studied expression profiles of genes known to affect myogenesis, muscle structural proteins, and energy metabolism in prenatal pigs from 14 to 91 days of gestation. Primary and secondary muscle fiber formation takes place during days 30-60 and 54-90 of gestation, respectively. Differential expression and expression levels of the genes were studied using microarray technology. Gene activation and repression profiles were studied counting the number of spots with detectable signal. The number of spots for muscle tissue structural protein genes showing upregulated expression increased constantly from day 14 until day 91 of gestation indicating continued activation of genes during this period. The mRNA expression level of the genes showed a peak around day 35 of gestation. The expression levels of genes affecting myogenic differentiation (stimulating and inhibiting) showed a peak at day 35 of gestation. The number of spots for differentiation-stimulating genes showing differential expression reaches a first peak around day 35 of gestation and a nadir at day 49 of gestation while the number of spots for differentiation-inhibiting genes reaches a nadir at day 35 of gestation. Myogenic differentiation seems less a matter of the expression level of genes affecting differentiation, but depends on the balance between the number of significantly activated genes for stimulating and inhibiting differentiation. Genes stimulating myoblast proliferation showed a small peak expression prior to day 35 of gestation indicating myoblast proliferation before differentiation. The number of spots and the expression levels of genes for glycolysis and ATP-metabolism are at a nadir around days 35 and 49-63 of gestation suggesting that the energy metabolism is low during fusion of myoblasts into multinucleated muscle fibers.

Animals↗

Effect of herbal polyphenols on atherogenic transcriptome.

The ancient Indian system of medicine supports the antiatherogenic properties of some herbs. The crosstalk amongst the genes coding for LDLR, LXRalpha, PPARs (alpha,gamma), CD-36 and c-myc may be important in atherogenesis because these genes control lipid metabolism, cytokine production and cellular activity within the arterial wall. Hence, we attempted for the first time to explore whether or not the polyphenols extracted from medicinal herbs had any effect on the transcription of these genes. Normal human mononuclear cells were cultured in the presence of polyphenols (and their HPLC purified sub-fractions) extracted from Green tea (Camellia sinensis), Neem (Azadirachta indica) and Tulsi (Ocimum sanctum). Transcriptional expression of these genes was measured by using RT-PCR and SCION IMAGE analysis software. These polyphenolic extracts were found to have the inherent capacity to inhibit the transcriptional expression of genes having direct involvement in atherogenic process. On the basis of these results, we propose for the first time that HPLC purified polyphenolic fraction IV of Tulsi may have a profound antiatherogenic effect.

Atherosclerosis↗

Importance of LXR-alpha transcriptome in the modulation of innate immunity.

Liver-X-Receptor alpha (LXR-alpha) that belongs to nuclear receptor/transcriptional factor family has been recognized to play crucial role in the regulation of lipid metabolism and inflammation. Consequently, the present study was addressed to explore the functional genomics of LXR-alpha within human blood immunomodulatory cells. The results of such a study, which involved LXR-alpha gene silencing through siRNA approach, revealed that: (a) the mRNA expression of genes coding for IL-8, IL-4, CX3CR1, LDLR, hTERT and c-myc was significantly elevated in response to LXR-alpha gene silencing whereas mRNA expression of genes coding for PPARs(alpha, gamma), CD36 and Dicer could not be detected; (b) the expression of Receptor C( k ) protein remained unaffected; (c) the mRNA expression of IFN-gamma gene was down regulated in LXR-alpha knockdown cells. Based upon these results we propose that LXR-alpha gene plays a crucial role in the regulation of innate immunity at the genomic level.

CD36 Antigens↗

Functional characterization of AATF transcriptome in human leukemic cells.

The study, addressed to explore the transcriptional expression and regulation of Apoptosis-antagonizing transcription factor (AATF) gene within various types of human leukemic cell lines, revealed that AATF gene was overexpressed ubiquitously in all the leukemic cell lines studied and this upregulation was accompanied by c-myc gene overamplification in these cells. Downregulation of AATF gene transcription within leukemic cells not only resulted in the downregulation of c-myc gene and vice-versa but also contributed to apoptosis leading to cell death. Further, the link between AATF expression and leukemic cellular apoptosis involved PI3K/Akt pathway. Based on these results we propose that AATF gene may be of crucial importance in maintaining the leukemic state of a cell compartment through its ability to initiate cell proliferation coupled with repression of cellular apoptosis.

Androstadienes↗

Sorghum bicolor's transcriptome response to dehydration, high salinity and ABA.

Genome wide changes in gene expression were monitored in the drought tolerant C4 cereal Sorghum bicolor, following exposure of seedlings to high salinity (150 mM NaCl), osmotic stress (20% polyethylene glycol) or abscisic acid (125 microM ABA). A sorghum cDNA microarray providing data on 12,982 unique gene clusters was used to examine gene expression in roots and shoots at 3- and 27-h post-treatment. Expression of approximately 2200 genes, including 174 genes with currently unknown functions, of which a subset appear unique to monocots and/or sorghum, was altered in response to dehydration, high salinity or ABA. The modulated sorghum genes had homology to proteins involved in regulation, growth, transport, membrane/protein turnover/repair, metabolism, dehydration protection, reactive oxygen scavenging, and plant defense. Real-time PCR was used to quantify changes in relative mRNA abundance for 333 genes that responded to ABA, NaCl or osmotic stress. Osmotic stress inducible sorghum genes identified for the first time included a beta-expansin expressed in shoots, actin depolymerization factor, inositol-3-phosphate synthase, a non-C4 NADP-malic enzyme, oleosin, and three genes homologous to 9-cis-epoxycarotenoid dioxygenase that may be involved in ABA biosynthesis. Analysis of response profiles demonstrated the existence of a complex gene regulatory network that differentially modulates gene expression in a tissue- and kinetic-specific manner in response to ABA, high salinity and water deficit. Modulation of genes involved in signal transduction, chromatin structure, transcription, translation and RNA metabolism contributes to sorghum's overlapping but nonetheless distinct responses to ABA, high salinity, and osmotic stress. Overall, this study provides a foundation of information on sorghum's osmotic stress responsive gene complement that will accelerate follow up biochemical, QTL and comparative studies.

Abscisic Acid↗

Distinct developmental defense activations in barley embryos identified by transcriptome profiling.

Proper embryo development is crucial for normal growth and development of barley. Numerous related aspects of this process--for example how the embryo establishes and sustains disease resistance for extended periods during dormancy--remain largely unknown. Here we report the results of microarray analyses of >22,000 genes, which together with measurements of jasmonic acid and salicylic acid during embryo development provide new information on the initiation in the developing barley embryo of at least two distinct types of developmental defense activation (DDA). Early DDA is characterized by the up-regulation of a specific set of genes around 20 days after flowering, including co-regulation of those for encoding 9-lipoxygenase and several oxylipin-generating enzymes, possibly leading to the formation of alpha-ketols. The same developmental phase includes an up-regulation of several defense genes, and indications of co-regulation of those for enzymes involved in the generation of phenylpropanoid phytoalexins. Late DDA is initiated prior to grain desiccation, around 37 days after flowering, with up-regulation of several genes encoding proteins with roles in antioxidant responses as well as a simultaneous up-regulation of several PR genes is notable. Throughout barley embryo development, there are no indications of an increased biosynthesis of either jasmonic acid or salicylic acid. Collectively, the results help explain how the proposed DDA enables protection of the developing barley embryo and grain for purposes of disease resistance.

Cell Cycle↗

Transcriptomic adaptations in rice suspension cells under sucrose starvation.

Sugar is an important resource for energy generation and developmental regulation in plants, and sucrose starvation causes enormous changes in cellular morphology, enzyme activities and gene expression. Genome-wide gene expression profiling provides a comprehensive knowledge of gene expression under nutrient depletion and senescence; however, that of a monocot model plant, rice, under sucrose depletion is still under investigation. Here, the time-course monitoring of gene expression profiles in sucrose-starved rice (Oryza sativa cv Tainung67) suspension cells was investigated by 21495 probes contained in Agilent rice chip. In sucrose-starved cells, the induced vacuolar biogenesis coincided with significantly upregulated transcripts of H+-pyrophosphatase, delta-TIP, one putative alpha-TIP, several vacuolar proteases and proteinase inhibitors, and one OsATG3. To survey the overall metabolic adaptations under sucrose depletion, the genes with significantly altered expression level were incorporated into multiple metabolic pathways. Most genes encoding enzymes involved in biosynthesis and degradation pathways of various macromolecules were comprehensively down-and upregulated, respectively, with sucrose starvation. Transcriptional regulation of gene expression is important for physiological adaptations to environmental stress, and many transcription factors, including bZIPs, NACs, and WRKY, showed significant increase in transcriptional level under sucrose starvation. Concurrently, statistical analysis revealed that their corresponding consensus cis-elements, such as ABA-responsive element, CACG, ACI, ACII and CTTATCC, were frequently found in the promoter regions of many sucrose starvation-upregulated genes. Particle bombardment-mediated and luciferase activity-based transient promoter assays revealed the CTTATCC, derived form TATCCA, and the AC motifs to be promising sucrose-starvation responsive activators in rice suspension cells.

Base Sequence↗

A combined strategy of "in silico" transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies.

Traditionally housekeeping genes have been employed as endogenous reference (internal control) genes for normalization in gene expression studies. Since the utilization of single housekeepers cannot assure an unbiased result, new normalization methods involving multiple housekeeping genes and normalizing using their mean expression have been recently proposed. Moreover, since a gold standard gene suitable for every experimental condition does not exist, it is also necessary to validate the expression stability of every putative control gene on the specific requirements of the planned experiment. As a consequence, finding a good set of reference genes is for sure a non-trivial problem requiring quite a lot of lab-based experimental testing. In this work we identified novel candidate barley reference genes suitable for normalization in gene expression studies. An advanced web search approach aimed to collect, from publicly available web resources, the most interesting information regarding the expression profiling of candidate housekeepers on a specific experimental basis has been set up and applied, as an example, on stress conditions. A complementary lab-based analysis has been carried out to verify the expression profile of the selected genes in different tissues and during heat shock response. This combined dry/wet approach can be applied to any species and physiological condition of interest and can be considered very helpful to identify putative reference genes to be shortlisted every time a new experimental design has to be set up.

Base Sequence↗