Nascent RNA sequencing reveals mechanisms of gene regulation in the human malaria parasite Plasmodium falciparum.

Gene expression in Plasmodium falciparum is tightly regulated to ensure successful propagation of the parasite throughout its complex life cycle. The earliest transcriptomics studies in P. falciparum suggested a cascade of transcriptional activity over the course of the 48-hour intraerythrocytic developmental cycle (IDC); however, the just-in-time transcriptional model has recently been challenged by findings that show the importance of post-transcriptional regulation. To further explore the role of transcriptional regulation, we performed the first genome-wide nascent RNA profiling in P. falciparum. Our findings indicate that the majority of genes are transcribed simultaneously during the trophozoite stage of the IDC and that only a small subset of genes is subject to differential transcriptional timing. RNA polymerase II is engaged with promoter regions prior to this transcriptional burst, suggesting that Pol II pausing plays a dominant role in gene regulation. In addition, we found that the overall transcriptional program during gametocyte differentiation is surprisingly similar to the IDC, with the exception of relatively small subsets of genes. Results from this study suggest that further characterization of the molecular players that regulate stage-specific gene expression and Pol II pausing will contribute to our continuous search for novel antimalarial drug targets

Novel subtractive transcription-based amplification of mRNA (STAR) method and its application in search of rare and differentially expressed genes in AD brains

BACKGROUND: Alzheimer's disease (AD) is a complex disorder that involves multiple biological processes. Many genes implicated in these processes may be present in low abundance in the human brain. DNA microarray analysis identifies changed genes that are expressed at high or moderate levels. Complementary to this approach, we described here a novel technology designed specifically to isolate rare and novel genes previously undetectable by other methods. We have used this method to identify differentially expressed genes in brains affected by AD. Our method, termed Subtractive Transcription-based Amplification of mRNA (STAR), is a combination of subtractive RNA/DNA hybridization and RNA amplification, which allows the removal of non-differentially expressed transcripts and the linear amplification of the differentially expressed genes. RESULTS: Using the STAR technology we have identified over 800 differentially expressed sequences in AD brains, both up- and down- regulated, compared to age-matched controls. Over 55% of the sequences represent genes of unknown function and roughly half of them were novel and rare discoveries in the human brain. The expression changes of nearly 80 unique genes were further confirmed by qRT-PCR and the association of additional genes with AD and/or neurodegeneration was established using an in-house literature mining tool (LitMiner). CONCLUSION: The STAR process significantly amplifies unique and rare sequences relative to abundant housekeeping genes and, as a consequence, identifies genes not previously linked to AD. This method also offers new opportunities to study the subtle changes in gene expression that potentially contribute to the development and/or progression of AD

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

<p>Abstract</p> <p>Background</p> <p>Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor <it>KISS1</it>, lack of αvβ3-integrin and low levels of <it>RHOC</it>.</p> <p>Methods</p> <p>Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified.</p> <p>Results</p> <p>We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library.</p> <p>Conclusion</p> <p>This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.</p

Analysis of Differential Gene Expression by Boolean Selection: Identification of Transcripts Associated with the Formation and Reorganization of Neuronal Networks.

The quantitative comparison of gene expression represents one of the major approaches for the analysis of gene function. Recently, the discovery of unknown genes based only upon their expression pattern has become possible through the application of various differential cloning methods, including subtractive hybridization, differential display, and expressed sequence tag analysis. Due to numerous technical and procedural limitations, however, none of these technologies is currently capable of rapidly identifying differentially regulated transcripts based on multiple expression-related criteria. Therefore, an alternative approach was devised which allows for the sequential application of numerous selection criteria during the initial screening process, thereby enabling the rapid identification of transcripts with highly specific expression patterns. Because of its use of multiple positive and negative selection steps, this approach is called Boolean selection. One of the first applications of this new approach was the search for novel genes associated with the formation and reorganization of neuronal networks in the mammalian brain. An ideal model system for this analysis is the retinocollicular system of the rat, where numerous aspects of neurite outgrowth, axon guidance, synapse formation and reorganization, and regulation of neuronal survival can easily be studied. Boolean selection was applied for the identification of transcripts preferentially expressed in the superior colliculus during late prenatal development as well as after the loss of afferent input in the adult animal. An initial analysis of several of these selected transcripts revealed that all satisfied at least one selection criterion and that over half satisfied both, indicating the usefulness of Boolean selection for the rapid identification of differentially regulated genes. Although additional analysis by in situ hybridization will still be necessary to verify the extent of differential expression and to provide information on the cellular localization of the transcripts identified so far, it was possible with this preliminary screen to gain new insight into some of the molecular and cellular activities in the superior colliculus during periods associated with the formation and reorganization of synaptic connections. Some of the identified transcripts encode proteins associated with post-transcriptional protein synthesis and modification events, including ribophorin (an essential subunit of the protein glycosylation complex ligosaccharyltransferase), calnexin (a membrane-associated molecular chaperone of the endoplasmic reticulum), and translation initiation factor, which together suggest enhanced levels of protein production and secretion. Other transcipts include rSec6 (part of the protein complex involved in the docking and fusion of synaptic vesicles to the plasma membrane) and Fyn (a nonreceptor tyrosine kinase found at high concentrations in axonal growth cones and also known be involved in synaptic plasticity), Fas-associated protein factor (a recently identified molecule potentially involved in Fas-mediated signal transduction during program m ed cell death), and a novel member of the EOF superfamily of transmembrane growth factors (which presently includes the EGF/TGFa family and the neuregulins). Further analysis of these molecules may provide additional information about the signaling mechanisms and cell-cell interactions at work during the establishment and modification of neuronal networks

Transcriptomic Analyses Reveal Novel Genes with Sexually Dimorphic Expression in the Zebrafish Gonad and Brain

Background Our knowledge on zebrafish reproduction is very limited. We generated a gonad-derived cDNA microarray from zebrafish and used it to analyze large-scale gene expression profiles in adult gonads and other organs. Methodology/Principal Findings We have identified 116638 gonad-derived zebrafish expressed sequence tags (ESTs), 21% of which were isolated in our lab. Following in silico normalization, we constructed a gonad-derived microarray comprising 6370 unique, full-length cDNAs from differentiating and adult gonads. Labeled targets from adult gonad, brain, kidney and ‘rest-of-body’ from both sexes were hybridized onto the microarray. Our analyses revealed 1366, 881 and 656 differentially expressed transcripts (34.7% novel) that showed highest expression in ovary, testis and both gonads respectively. Hierarchical clustering showed correlation of the two gonadal transcriptomes and their similarities to those of the brains. In addition, we have identified 276 genes showing sexually dimorphic expression both between the brains and between the gonads. By in situ hybridization, we showed that the gonadal transcripts with the strongest array signal intensities were germline-expressed. We found that five members of the GTP-binding septin gene family, from which only one member (septin 4) has previously been implicated in reproduction in mice, were all strongly expressed in the gonads. Conclusions/Significance We have generated a gonad-derived zebrafish cDNA microarray and demonstrated its usefulness in identifying genes with sexually dimorphic co-expression in both the gonads and the brains. We have also provided the first evidence of large-scale differential gene expression between female and male brains of a teleost. Our microarray would be useful for studying gonad development, differentiation and function not only in zebrafish but also in related teleosts via cross-species hybridizations. Since several genes have been shown to play similar roles in gonadogenesis in zebrafish and other vertebrates, our array may even provide information on genetic disorders affecting gonadal phenotypes and fertility in mammals

Zygote gene expression and plasmodial development in Didymium iridis

Didymium iridis is a cosmopolitan species of plasmodial slime mold consisting of two distinct life stages. Haploid amoebae and diploid plasmodia feed on microscopic organisms such as bacteria and fungi through phagocytosis. Sexually compatible haploid amoebae act as gametes which when fused embark on an irreversible developmental change resulting in a diploid zygote. The zygote can undergo closed mitosis resulting in a multinucleated plasmodium. Little is known about changes in gene expression during this developmental transition. Our principal goal in this study was to provide a comprehensive list of genes likely to be involved in plasmodial development. We performed suppressive subtractive hybridization to create cDNA libraries enriched for zygote or plasmodial specific genes. The cDNA libraries were then cloned and sequenced. The sequences were used to search against GenBank gene databases to identify related sequences and characterized proteins. We have compiled a list of candidate genes likely to be involved in the amoebae-zygote transition and have arranged them by their known or predicted function. Genes related to cytoskeletal structure, cell signaling, ubiquitin-proteasome pathways, mitochondrial inheritance, and DNA binding proteins were of particular interest due their possible role in this developmental transition. Selected gene sequences were also tested for differential expression by dot blot and RT-PCR

Diversity Arrays Technology (DArT) for Pan-Genomic Evolutionary Studies of Non-Model Organisms

Background: High-throughput tools for pan-genomic study, especially the DNA microarray platform, have sparked a remarkable increase in data production and enabled a shift in the scale at which biological investigation is possible. The use of microarrays to examine evolutionary relationships and processes, however, is predominantly restricted to model or near-model organisms. Methodology/Principal Findings: This study explores the utility of Diversity Arrays Technology (DArT) in evolutionary studies of non-model organisms. DArT is a hybridization-based genotyping method that uses microarray technology to identify and type DNA polymorphism. Theoretically applicable to any organism (even one for which no prior genetic data are available), DArT has not yet been explored in exclusively wild sample sets, nor extensively examined in a phylogenetic framework. DArT recovered 1349 markers of largely low copy-number loci in two lineages of seed-free land plants: the diploid fern Asplenium viride and the haploid moss Garovaglia elegans. Direct sequencing of 148 of these DArT markers identified 30 putative loci including four routinely sequenced for evolutionary studies in plants. Phylogenetic analyses of DArT genotypes reveal phylogeographic and substrate specificity patterns in A. viride, a lack of phylogeographic pattern in Australian G. elegans, and additive variation in hybrid or mixed samples. Conclusions/Significance: These results enable methodological recommendations including procedures for detecting and analysing DArT markers tailored specifically to evolutionary investigations and practical factors informing the decision to use DArT, and raise evolutionary hypotheses concerning substrate specificity and biogeographic patterns. Thus DArT is a demonstrably valuable addition to the set of existing molecular approaches used to infer biological phenomena such as adaptive radiations, population dynamics, hybridization, introgression, ecological differentiation and phylogeography

PHLPP1 counter-regulates STAT1-mediated inflammatory signaling.

Inflammation is an essential aspect of innate immunity but also contributes to diverse human diseases. Although much is known about the kinases that control inflammatory signaling, less is known about the opposing phosphatases. Here we report that deletion of the gene encoding PH domain Leucine-rich repeat Protein Phosphatase 1 (PHLPP1) protects mice from lethal lipopolysaccharide (LPS) challenge and live Escherichia coli infection. Investigation of PHLPP1 function in macrophages reveals that it controls the magnitude and duration of inflammatory signaling by dephosphorylating the transcription factor STAT1 on Ser727 to inhibit its activity, reduce its promoter residency, and reduce the expression of target genes involved in innate immunity and cytokine signaling. This previously undescribed function of PHLPP1 depends on a bipartite nuclear localization signal in its unique N-terminal extension. Our data support a model in which nuclear PHLPP1 dephosphorylates STAT1 to control the magnitude and duration of inflammatory signaling in macrophages