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ESTs
Selected AbstractsW(H)ITHER EMPIRICALLY SUPPORTED THERAPIES (ESTS)?ADDICTION, Issue 6 2007REPLY TO COMMENTARIES No abstract is available for this article. [source] ANALYSIS OF EXPRESSED SEQUENCE TAGS (ESTS) FROM THE POLAR DIATOM FRAGILARIOPSIS CYLINDRUS,JOURNAL OF PHYCOLOGY, Issue 1 2006Thomas Mock Analysis of expressed sequence tags (ESTs) was performed to gain insights into cold adaptation in the polar diatom Fragilariopsis cylindrus Grunow. The EST library was generated from RNA isolated 5 days after F. cylindrus cells were shifted from approximately +5° C to ,1.8°C. A total of 1376 ESTs were sequenced from a non-normalized cDNA library and assembled into 996 tentative unique sequences. About 27% of the ESTs displayed similarity (tBLASTX, e -value of ,10,4) to predicted proteins in the centric diatom Thalassiosira pseudonana Hasle & Heindal. Eleven additional algae and plant data bases were used for annotation of sequences not covered by Thalassiosira sequences (7%). Most of the ESTs were similar to genes encoding proteins responsible for translation, ribosomal structure, and biogenesis (3%), followed by genes encoding proteins for amino acid transport and metabolism and post-translational modifications. Interestingly, 66% of all the EST sequences from F. cylindrus displayed no similarity (e -value ,10,4) to sequences from the 12 non-redundant databases. Even 6 of the 10 strong to moderately expressed sequences in this EST library could not be identified. Adaptation of F. cylindrus to freezing temperatures of seawater may require a complex protein metabolism and possibly also genes, which were highly expressed but still unknown. However, it could also mean that due to low temperatures, there might have been a stronger pressure to adapt amino acid sequences, making it more difficult to identify these unknown sequences and/or that there are still few protist sequences available for comparison. [source] Transcriptional activity of paddy soil bacterial communitiesENVIRONMENTAL MICROBIOLOGY, Issue 4 2009Pravin Malla Shrestha Summary Bulk mRNA was used to explore the transcriptional activity of bacterial communities in oxic versus anoxic paddy soil. Two microbial cDNA libraries were constructed from composite samples using semi-randomly primed RT-PCR. cDNAs averaged 500,600 bp in length and were treated as expressed sequence tags (ESTs). Clustering analysis of 805 random cDNAs resulted in 179 and 155 different ESTs for the oxic and anoxic zones respectively. Using an E -value threshold of e,10, a total of 218 different ESTs could be assigned by blastx, while 116 ESTs were predicted novel. Both the proportion and significance of the EST assignments increased with cDNA length. Taxonomic assignment was more powerful in discriminating between the aerobic and anaerobic bacterial communities than functional inference, as most ESTs in both oxygen zones were putative indicators of similar housekeeping functions, in particular ABC-type transporters. A few ESTs were putative indicators for community function in a biogeochemical context, such as ,-oxidation of long-chain fatty acids specifically in the oxic zone. Expressed sequence tags assigned to Alpha- and Betaproteobacteria were predominantly found in the oxic zone, while those affiliated with Deltaproteobacteria were more frequently detected in the anoxic zone. At the genus level, multiple assignments to Bradyrhizobium and Geobacter were unique to the oxic and anoxic zones respectively. The phylum-level affiliations of 93 16S rRNA sequences corresponded well with two taxonomically distinct EST patterns. Expressed sequence tags affiliated with Acidobacteria and Chloroflexi were frequently detected in both oxygen zones. In summary, the soil metatranscriptome is accessible for global analysis and such studies have great potential in elucidating the taxonomic and functional status of soil bacterial communities, but study significance depends on the number and length of cDNAs being randomly analysed. [source] The antiepileptic drug levetiracetam selectively modifies kindling-induced alterations in gene expression in the temporal lobe of ratsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2004Jessie Gu Abstract Gene expression profiling by microarrays is a powerful tool for identification of genes that may encode key proteins involved in molecular mechanisms underlying epileptogenesis. Using the Affymetrix oligonucleotide microarray, we have surveyed the expression levels of more than 26,000 genes and expressed sequence tags (ESTs) in the amygdala-kindling model of temporal lobe epilepsy. Furthermore, the effect of the antiepileptic drug levetiracetam (LEV) on kindling-induced alterations of gene expression was studied. Treatment of rats with LEV during kindling acquisition significantly suppressed kindling development. For gene expression profiling, six groups of rats were included in the present study: (i) and (ii) sham-operated rats treated with saline or LEV; (iii) and (iv) electrode-implanted but non-kindled rats treated with saline or LEV; (v) and (vi) kindled rats treated with saline or LEV. Treatment was terminated after 11 or 12 daily amygdala stimulations, when all vehicle-treated rats had reached kindling criterion, i.e. a stage 5 seizure. Twenty-four hours later, the ipsilateral temporal lobe was dissected for mRNA preparation. Six temporal lobe preparations from each group were analysed for differential gene expression. In control (non-kindled) rats, LEV treatment was devoid of any significant effect on gene expression. In saline-treated kindled rats, a large number of genes were observed to display mRNA expression alterations compared with non-kindled rats. LEV treatment induced marked effects on gene expression from kindled rats. Previously described epilepsy-related genes, such as neuropeptide Y (NPY), thyrotropin-releasing hormone (TRH) and glial fibrillary acidic protein (GFAP) were confirmed to be up-regulated by kindling and partially normalized by LEV treatment. Real-time quantitative polymerase chain reaction confirmed NPY, TRH and GFAP expression data from chip experiments. Furthermore, a number of novel genes were identified from the gene chip experiments. A subgroup of these genes demonstrated correlation between expression changes and kindled phenotype measurements. In summary, this study identified many genes with potentially important roles in epileptogenesis and highlighted several important issues in using the gene chip technology for the study of animal models of CNS disorders. [source] TNFAIP3 is the target gene of chromosome band 6q23.3-q24.1 loss in ocular adnexal marginal zone B cell lymphomaGENES, CHROMOSOMES AND CANCER, Issue 1 2008Keiichiro Honma The genomic aberrations in extra nodal marginal zone B cell lymphoma vary according to their anatomical origin. This polarization is a reflection of the participation of different genes in the lymphomagenesis of marginal zone B cell lymphoma. We previously demonstrated by means of genome-wide array comparative genomic hybridization (CGH) that the genomic profile of ocular adnexal marginal zone B cell lymphoma is distinct from that of pulmonary or nodal marginal zone B cell lymphoma. The novel finding was a recurrent deletion of a 2.9-Mb region at chromosome band 6q23.3-q24.1, including homozygous loss, in ocular adnexal marginal zone B cell lymphoma. For a more detailed examination of the deletions of 6q23.3-24.1, we used contig bacterial artificial chromosome (BAC) array CGH, containing 24 BAC clones covering the 2.9-Mb region, to analyze nine cases with 6q23.3-q24.1 loss. We narrowed the minimal common region down to a length of 586 kb with two genes and four expressed sequence tags (ESTs). All of these genes and ESTs were subjected to RT-PCR and real-time quantitative RT-PCR. Correlation between genomic loss and expression level was found only for TNFAIP3, demonstrating that TNFAIP3 is a target gene of 6q deletion in ocular adnexal marginal zone B cell lymphoma. TNFAIP3 is an inhibitor of NF-kB signaling so that loss of this gene may play an important role in lymphomagenesis and suggests that TNFAIP3 may act as a tumor suppressor gene in ocular adnexal marginal zone B cell lymphoma. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. © 2007 Wiley-Liss, Inc. [source] Multiple forms of genetic instability within a 2-Mb chromosomal segment of 3q26.3,q27 are associated with development of esophageal adenocarcinomaGENES, CHROMOSOMES AND CANCER, Issue 4 2006Lin Lin Gene amplification is one of the mechanisms to activate oncogenes in many cancers, including esophageal adenocarcinoma (EA). In the present study, we used two-dimensional restriction landmark genome scanning to clone a NotI/DpnII fragment that showed increased genomic dosage in 1 of 44 EAs analyzed. This fragment maps to 3q26.3,q27, and subsequent experiments identified two intrachromosomal amplicons within a 10-Mb DNA segment in 7 of 75 (9%) EAs. The distal amplified-core region maps centromeric to the PIK3CA locus, and a microsatellite (D3S1754) within this region exhibited significant instability (MSI), in stark contrast to the genomewide microsatellite stability found in EA. D3S1754-MSI arises in premalignant Barrett's dysplastic cells and preceded amplification of the nascent MSI allele in the corresponding EA. Seven ESTs within the amplified-core were overexpressed in amplicon-containing EAs. One of these, EST AW513672, represents a chimeric transcript that initiated from an antisense promoter sequence in the 5,UTR of a full-length LINE-1 element (L1-5,ASP). Similar chimeric transcripts encoding portions of the MET oncogene and the BCAS3 gene also were overexpressed in EAs, suggesting that L1-5,ASP activation may occur at a broad level in primary EAs. Thus, the fine dissection of a 2-Mb amplified DNA segment in 3q26.3,q27 in EA revealed multiple genetic alterations that had occurred sequentially and/or concurrently during EA development. This article has supplementary material, available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. © 2005 Wiley-Liss, Inc. [source] Pyrosequencing and characterization of immune response genes from the American dog tick, Dermacentor variabilis (L.)INSECT MOLECULAR BIOLOGY, Issue 5 2010D. C. Jaworski Abstract Ticks continue to be a threat to animal and human health, and new and novel control strategies are needed for ticks and tick-borne pathogens. The characterization of the tick,pathogen interface and the tick immune response to microbial infections is fundamental toward the formulation of new control strategies for ticks and the pathogens they transmit. Our overall hypothesis for this research is that the tick immune system manages the maintenance of pathogens. Therefore, discovery of tick immune response genes may provide targets for novel control strategies directed toward reducing vector competency and pathogen transmission. In these studies, 454 pyrosequencing, a high-throughput genomic sequencing method was used to discover tick genes expressed in response to bacterial and fungal infections. Expressed sequence tags (ESTs) were analysed from Dermacentor variabilis ticks that had been injected with bacteria (Escherichia coli, Bacillus subtilis, Micrococcus luteus) or fungi (Saccharomyces cerevisiae and Candida albicans) and ticks that were naturally infected with the intracellular bacterium, Anaplasma marginale. By this approach, ESTs were assembled into 5995 contigs. Contigs fell into the five main functional categories of metabolism, genetic information processing, environmental information processing, cellular processes and human diseases. We identified more than 30 genes that are likely to encode for proteins involved in tick immune function. We further analysed by reverse transcriptase PCR (RT-PCR) the expression of 22 of these genes in each of our bacterial or fungal treatment groups and found that seven were up-regulated. Up-regulation of these seven genes was confirmed for bacterial, but not fungal treatment by quantitative PCR (qPCR). One of these products was novel, encoding a new tick defensin. Our results clearly demonstrate the complexities of the tick immune system and mark new directions for further study and characterization of proteins that modulate microbial infections in the American dog tick. [source] Analysis of expressed sequence tags for Frankliniella occidentalis, the western flower thripsINSECT MOLECULAR BIOLOGY, Issue 4 2010D. Rotenberg Abstract Thrips are members of the insect order Thysanoptera and Frankliniella occidentalis (the western flower thrips) is the most economically important pest within this order. F. occidentalis is both a direct pest of crops and an efficient vector of plant viruses, including Tomato spotted wilt virus (TSWV). Despite the world-wide importance of thrips in agriculture, there is little knowledge of the F. occidentalis genome or gene functions at this time. A normalized cDNA library was constructed from first instar thrips and 13 839 expressed sequence tags (ESTs) were obtained. Our EST data assembled into 894 contigs and 11 806 singletons (12 700 nonredundant sequences). We found that 31% of these sequences had significant similarity (E, 10,10) to protein sequences in the National Center for Biotechnology Information nonredundant (nr) protein database, and 25% were functionally annotated using Blast 2GO. We identified 74 sequences with putative homology to proteins associated with insect innate immunity. Sixteen sequences had significant similarity to proteins associated with small RNA-mediated gene silencing pathways (RNA interference; RNAi), including the antiviral pathway (short interfering RNA-mediated pathway). Our EST collection provides new sequence resources for characterizing gene functions in F. occidentalis and other thrips species with regards to vital biological processes, studying the mechanism of interactions with the viruses harboured and transmitted by the vector, and identifying new insect gene-centred targets for plant disease and insect control. [source] Transcriptome analysis of the salivary glands of the female tick Amblyomma americanum (Acari: Ixodidae)INSECT MOLECULAR BIOLOGY, Issue 2 2009M. N. Aljamali Abstract Ticks infest a wide range of hosts while bypassing their immune, inflammatory and haemostatic responses during their extended feeding, which may last for more than two weeks. Here, we present a transcriptome analysis of 3868 expressed sequence tags (ESTs) from three cDNA libraries generated from the salivary glands of adult female Ambyomma americanum ticks at different stages of feeding. We applied a normalization step for one library, significantly decreasing the abundance of mitochondrial sequences amongst the 2292 sequences from the normalized library. Our ESTs include homologues that may modulate haemostatic, immune and inflammatory responses of the hosts. Other ESTs probably represent important components of the highly efficient secretory pathways for salivary proteins and concomitantly transmitted pathogens. [source] Mining an Ostrinia nubilalis midgut expressed sequence tag (EST) library for candidate genes and single nucleotide polymorphisms (SNPs)INSECT MOLECULAR BIOLOGY, Issue 6 2008B. S. Coates Abstract Genes expressed in lepidopteran midgut tissues are involved in digestion and Bacillus thuringiensis (Bt) toxin resistance traits. Five hundred and thirty five unique transcripts were annotated from 1745 high quality O. nubilalis larval midgut expressed sequence tags (ESTs). Full-length cDNA sequence of 12 putative serine proteinase genes and 3 partial O. nubilalis aminopeptidase N protein genes, apn1, apn3, and apn4, were obtained, and genes may have roles in plant feeding and Bt toxin resistance traits of Ostrinia larvae. The EST library was not normalized and insert frequencies reflect transcript levels under the initial treatment conditions and redundancy of inserts from highly expressed transcripts allowed prediction of putative single nucleotide polymorphisms (SNPs). Ten di-, tri- or tetranucleotide repeat unit microsatellite loci were identified, and minisatellite repeats were observed within the C-termini of two encoded serine proteinases. Molecular markers showed polymorphism at 28 SNP loci and one microsatellite locus, and Mendelian inheritance indicated that markers were applicable to genome mapping applications. This O. nubilalis larval midgut EST collection is a resource for gene discovery, expression information, and allelic variation for use in genetic marker development. [source] Analysis of the structure and expression of the 30K protein genes in silkworm, Bombyx moriINSECT SCIENCE, Issue 1 2007QUAN SUN Abstract A group of lipoproteins with molecular sizes of approximately 30 kDa, referred to as 30K proteins, are synthesized in fat body cells in the fifth instar larvae of silkworm, Bombyx mori. Analyzing the silkworm genome and its expressed sequence tags (ESTs), we found 10 genes encoding 30K proteins, which are mainly distributed in three subfamilies. Of these, seven coding proteins were found to harbor the degrading sites of 30kP protease A, although the number of degrading sites may be different. As some potential core promoters and regulatory elements were supposed to be essential for gene transcription, the expression profiles of these genes were examined by semi-quantitative reverse transcription polymerase chain reaction. Eight 30K protein genes were detected to express luxuriantly in the fat body, while two were hardly expressed. Such results suggest that these 30K proteins may have different functions, and their adjacent regulatory elements play a crucial role in regulating their transcription. [source] Porcine ESTs detected by differential display representing possible candidates for the trait ,eye muscle area'JOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 1 2000By S. Ponsuksili In order to identify ESTs which represent possible candidates for carcass traits in pigs, the differential display approach was used. F2 animals of a resource population and pure-bred German Landrace (DL) pigs were selected for the trait ,eye muscle area' in order to build up groups of three high and three low performing individuals within each population. To increase the probability that differentially expressed DNA fragments were not found due to the genetic background but due to differences in a few genes affecting the trait of interest, siblings were included in the high and in the low performing groups. RNA was isolated from M. longissimus dorsi and four ,intra-litter constrasting pools' were prepared: high performing F2, low performing F2, high performing DL and low performing DL. Differential display banding patterns were produced using (d)T11VA (V:A,C,G) and 20 arbitrary primers. Comparing the banding patterns of the four RNA pools revealed 27 nonshared bands. Here we report on the analysis of seven of these bands, including sequencing, search for homology and mapping using a somatic cell hybrid panel. Two clones showed high homology to known genes, two were homologous to an EST and a SINE sequence. Three clones did not show any homology. Differential expression was tested by semiquantitative reverse transcription,polymerase chain reaction (RT,PCR) and could be confirmed for six clones. [source] Identification of Novel Regulators Associated With Early-Phase Osteoblast Differentiation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2004Diana S de Jong Abstract Key regulatory components of the BMP-induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2-induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP-induced osteoblast differentiation. Introduction: Elucidation of the genetic cascade guiding mesenchymal stem cells to become osteoblasts is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify regulators of the early phases of bone morphogenetic protein (BMP)2-induced osteoblast differentiation. Materials and Methods: Osteoblast differentiation of mouse C2C12 cells was induced by treatment with BMP2, and regulation of gene expression was studied during the subsequent 24 h using high-density microarrays. The regulated genes were grouped by means of model-based clustering, and protein functions were assigned. Real-time quantitative RT-PCR analysis was used to validate BMP2-induced gene expression patterns in C2C12 cells. Osteoblast specificity was studied by comparing these expression patterns with those in C3H10T1/2 and NIH3T3 cells under similar conditions. In situ hybridization of mRNA in embryos at embryonic day (E)14.5 and E16.5 of gestation and on newborn mouse tails were used to study in vivo expression patterns. Cells constitutively expressing the regulated gene Tcf7 were used to investigate its influence on BMP-induced osteoblast differentiation. Results and Conclusions: A total of 184 genes and expressed sequence tags (ESTs) were differentially expressed in the first 24 h after BMP2 treatment and grouped in subsets of immediate early, intermediate early, and late early response genes. Signal transduction regulatory factors mainly represented the subset of immediate early genes. Regulation of expression of these genes was direct, independent of de novo protein synthesis and independent of the cell type studied. The intermediate early and late early genes consisted primarily of genes related to processes that modulate morphology, basement membrane formation, and synthesis of extracellular calcified matrix. The late early genes require de novo protein synthesis and show osteoblast specificity. In vivo and in vitro experiments showed that the transcription factors Hey1 and Tcf7 exhibited expression characteristics and cell type specificity very similar to those of the osteoblast specific transcription factor Cbfa1, and constitutive expression of Tcf7 in C2C12 cells differentially regulated osteoblast differentiation marker genes. [source] Potentialities of multivariate approaches in genome-based cancer research: identification of candidate genes for new diagnostics by PLS discriminant analysis,JOURNAL OF CHEMOMETRICS, Issue 3-4 2004G. Musumarra Abstract Partial least squares discriminant analysis (PLS-DA) provides a sound statistical basis for the selection, from an original 9605-data set, of a limited number of gene transcripts most effective in discriminating different tumour histotypes. The potentialities of the PLS-DA approach are pointed out by its ability to identify genes which, according to current knowledge, are associated with cancer development. Moreover, PLS-DA was able to identify MUC 13 and S100P proteins as candidates for the development of new colon cancer diagnostics. Various genes with unknown function and ESTs (expressed sequence tags), found to be important in discriminating genes for colon, leukaemia, renal and central nervous system tumour cells, are indicated as deserving high priority in future molecular studies. Copyright © 2004 John Wiley & Sons, Ltd. [source] Evidence-based practice in clinical psychology: What it is, why it matters; what you need to knowJOURNAL OF CLINICAL PSYCHOLOGY, Issue 7 2007Bonnie Spring The history and meaning of evidence-based practice (EBP) in the health disciplines was described to the Council of University Directors of Clinical Psychology (CUDCP) training programs. Evidence-based practice designates a process of clinical decision-making that integrates research evidence, clinical expertise, and patient preferences and characteristics. Evidence-based practice is a transdisciplinary, idiographic approach that promotes lifelong learning. Empirically supported treatments (ESTs) are an important component of EBP, but EBP cannot be reduced to ESTs. Psychologists need additional skills to act as creators, synthesizers, and consumers of research evidence, who act within their scope of clinical expertise and engage patients in shared decision-making. Training needs are identified in the areas of clinical trial methodology and reporting, systematic reviews, search strategies, measuring patient preferences, and acquisition of clinical skills to perform ESTs. © 2007 Wiley Periodicals, Inc. J Clin Psychol 63: 611,631, 2007. [source] Noxp20 and Noxp70, two new markers of early neuronal differentiation, detected in teratocarcinoma-derived neuroectodermic precursor cellsJOURNAL OF NEUROCHEMISTRY, Issue 2 2006M. Boucquey Abstract The murine 1C11 cell line, derived from F9 pluripotent teratocarcinoma cells, exhibits features of a bipotential neuronal precursor as it converts into serotonergic or catecholaminergic neurons under appropriate induction. In order to point out molecular markers expressed in this early neuroectodermic commitment, we used a cDNA subtractive hybridization method. The 105 different isolated cDNAs represented 75 known genes, expressed sequence tags (EST) or genomic fragments. A majority of known proteins encoded by these sequences are involved in cellular mobility or migration. We characterized two sequences showing identities with ESTs and we called them Noxp20 and Noxp70. The Noxp20 transcript encodes a putative protein with a predicted caspase recruitment domain and the Noxp70 transcript encodes a putative protein displaying a Zn-finger domain. Consistent with their roles in neuronal cell development, in situ hybridization showed that Noxp20 and Noxp70 are over-expressed in brain. At embryonic days 12 and 15, Noxp20 is strongly expressed in the ventricular and intermediate zones of the brain and of the spinal cord. At embryonic day 15, Noxp70 was found to be strongly expressed in the ventricular zone around the telencephalic ventricle, and to a lower extent in the thalamus and hypothalamus. At post-natal day 10, Noxp20 mRNA was detected in the dentate gyrus, the hippocampus, the cerebellum and the olfactory bulb. [source] Generation and analysis of 5318 expressed sequence tags from the filamentous sporophyte of Porphyra haitanensis (Rhodophyta),JOURNAL OF PHYCOLOGY, Issue 6 2007Fan Xiaolei Porphyra haitanensis T. J. Chang et B. F. Zheng (Bangiales, Rhodophyta) is cultivated in China and widely consumed in Asia. To gain more insight into its physiological and biochemical properties, we generated 5318 expressed sequence tags (ESTs) from the sporophyte of P. haitanensis, and upon assembling into a nonredundant set, 2535 sequences were obtained, among which only 32.2% (816) shared certain similarity with published sequences (Nr and KOG). Functional classification of such ESTs revealed that most of the transcripts were related to its conservative biological metabolism, and P. haitanensis most likely possesses cyanide-resistant respiration and a C4-like carbon-fixation pathway, both of which have never been reported in a rhodophyte before. Twenty-eight percent of the nonredundant gene clusters exhibited significant similarity to those from P. yezoensis Ueda sporophytes, and 16 genes up-regulated in P. yezoensis sporophytes were also expressed abundantly in P. haitanensis. Codon usage analysis indicated that exposure to high GC pressure might occur during evolution of P. haitanensis. These findings represent the most extensive collection of ESTs from P. haitanensis to date, and all the ESTs in this study have been submitted to GenBank (accession nos. DN604790,DN608469, EG016226,EG018540). [source] CHARACTERIZATION OF A DINOFLAGELLATE CRYPTOCHROME BLUE-LIGHT RECEPTOR WITH A POSSIBLE ROLE IN CIRCADIAN CONTROL OF THE CELL CYCLE,JOURNAL OF PHYCOLOGY, Issue 3 2007Stephanie A. Brunelle Karenia brevis (C. C. Davis) G. Hansen et Moestrup is a dinoflagellate responsible for red tides in the Gulf of Mexico. The signaling pathways regulating its cell cycle are of interest because they are the key to the formation of toxic blooms that cause mass marine animal die-offs and human illness. Karenia brevis displays phased cell division, in which cells enter S phase at precise times relative to the onset of light. Here, we demonstrate that a circadian rhythm underlies this behavior and that light quality affects the rate of cell-cycle progression: in blue light, K. brevis entered the S phase early relative to its behavior in white light of similar intensity, whereas in red light, K. brevis was not affected. A data base of 25,000 K. brevis expressed sequence tags (ESTs) revealed several sequences with similarity to cryptochrome blue-light receptors, but none related to known red-light receptors. We characterized the K. brevis cryptochrome (Kb CRY) and modeled its three-dimensional protein structure. Phylogenetic analysis of the photolyase/CRY gene family showed that Kb CRY is a member of the cryptochrome DASH (CRY DASH) clade. Western blotting with an antibody designed to bind a conserved peptide within Kb CRY identified a single band at ,55 kDa. Immunolocalization showed that Kb CRY, like CRY DASH in Arabidopsis, is localized to the chloroplast. This is the first blue-light receptor to be characterized in a dinoflagellate. As the Kb CRY appears to be the only blue-light receptor expressed, it is a likely candidate for circadian entrainment of the cell cycle. [source] ANALYSIS OF EXPRESSED SEQUENCE TAGS (ESTS) FROM THE POLAR DIATOM FRAGILARIOPSIS CYLINDRUS,JOURNAL OF PHYCOLOGY, Issue 1 2006Thomas Mock Analysis of expressed sequence tags (ESTs) was performed to gain insights into cold adaptation in the polar diatom Fragilariopsis cylindrus Grunow. The EST library was generated from RNA isolated 5 days after F. cylindrus cells were shifted from approximately +5° C to ,1.8°C. A total of 1376 ESTs were sequenced from a non-normalized cDNA library and assembled into 996 tentative unique sequences. About 27% of the ESTs displayed similarity (tBLASTX, e -value of ,10,4) to predicted proteins in the centric diatom Thalassiosira pseudonana Hasle & Heindal. Eleven additional algae and plant data bases were used for annotation of sequences not covered by Thalassiosira sequences (7%). Most of the ESTs were similar to genes encoding proteins responsible for translation, ribosomal structure, and biogenesis (3%), followed by genes encoding proteins for amino acid transport and metabolism and post-translational modifications. Interestingly, 66% of all the EST sequences from F. cylindrus displayed no similarity (e -value ,10,4) to sequences from the 12 non-redundant databases. Even 6 of the 10 strong to moderately expressed sequences in this EST library could not be identified. Adaptation of F. cylindrus to freezing temperatures of seawater may require a complex protein metabolism and possibly also genes, which were highly expressed but still unknown. However, it could also mean that due to low temperatures, there might have been a stronger pressure to adapt amino acid sequences, making it more difficult to identify these unknown sequences and/or that there are still few protist sequences available for comparison. [source] ANALYSIS OF EXPRESSED SEQUENCE TAGS FROM THE GREEN ALGA ULVA LINZA (CHLOROPHYTA),JOURNAL OF PHYCOLOGY, Issue 6 2005Michele S. Stanley There is a general lack of genomic information available for chlorophyte seaweed genera such as Ulva, and in particular there is no information concerning the genes that contribute to adhesion and cell wall biosynthesis for this organism. Partial sequencing of cDNA libraries to generate expressed sequence tags (ESTs) is an effective means of gene discovery and characterization of expression patterns. In this study, a cDNA library was created from sporulating tissue of Ulva linza L. Initially, 650 ESTs were randomly selected from a cDNA library and sequenced from their 5, ends to obtain an indication of the level of redundancy of the library (21%). The library was normalized to enrich for rarer sequences, and a further 1920 ESTs were sequenced. These sequences were subjected to contig assembly that resulted in a unigene set of approximately 1104 ESTs. Forty-eight percent of these sequences exhibited significant similarity to sequences in the databases. Phylogenetic comparisons are made between selected sequences with similarity in the databases to proteins involved in aspects of extracellular matrix/cell wall assembly and adhesion. [source] Expression Profile During the Development of Appressoria Induced by Hydrophobic Surfaces in Magnaporthe grisea Y34JOURNAL OF PHYTOPATHOLOGY, Issue 3 2010Qingchao Jin Abstract To study the gene expression profile during appressorium developmental process of Magnaphorthe grisea strain Y34 isolated from the rich area of Asia cultivated rice resources, expressed sequence tags (ESTs) and cDNA array analysis were performed. A total of 4756 tentative unique transcripts (TUTs) were obtained from 13 057 ESTs of the 3, ends of the strain, which was approximately 25% of the total M. grisea EST sequences deposited in the GenBank database. Approximately 84% of these TUTs matched with the published draft genome sequences of strain 70-15. Southern analyses with 12 TUT probes revealed no obvious DNA polymorphism among strains 70-15, Guy11 and Y34. A cDNA array with 4108 TUTs was used to monitor gene expression patterns during appressorium development of M. grisea. Compared with ungerminated conidia, the number of up-regulated and down-regulated genes was almost consistent at any time-points of 2, 8, 20 and 30 h during appressorium development. More genes were differentially expressed during appressorium maturation (20 and 30 h) than during appressorium induction (2 h) and formation (8 h). During appressorium maturation (20,30 h), genes generally seemed to be most actively expressed. [source] Accumulation of Defence Response-related and Unique Expressed Sequence Tags during the Incompatible Interaction in the Oryza sativa,Magnaporthe oryzae PathosystemJOURNAL OF PHYTOPATHOLOGY, Issue 7-8 2009Rekha Dixit Abstract Resistance gene-dependent accumulation of expressed sequence tags (ESTs) was studied in a blast resistant, Oryza sativa ssp. indica cv. Tetep after challenge inoculation with an incompatible race of Magnaporthe oryzae. The nucleotide sequence of 287 randomly selected cDNA clones from the rice cDNA library constructed from the RNA isolated after challenge inoculation of the host was obtained and submitted in NCBI Genbank (Accession Nos. DN475717,DN475431). Of these, 184 (63%) ESTs were highly representative of the rice transcriptomes. A set of 178 unique transcripts was identified after assembly of 287 ESTs into unigenes. These unigenes were categorized into 17 functional groups. Analysis of this EST library illustrated a broad functional representation. Twenty-one unigenes were identified as putative homologues of the genes that were up regulated during host,pathogen interaction. Similarity search of 178 unigenes with NCBI database of 14 plants unigenes showed similarity ranging from 29,100%. The unigenes obtained in this study were physically located on the pseudomolecules of rice genome. This information can be used for determining the arrays of genes being expressed during Oryza sativa,M. oryzae interactions, which will be helpful in understanding the molecular basis of disease resistance. [source] Giant Axonal Neuropathy Locus Refinement To A < 590 KB Critical IntervalJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2001L Cavalier Giant axonal neuropathy (GAN) is a rare autosomal recessive neurodegenerative disorder, characterised clinically by the development of chronic distal polyneuropathy during childhood, mental retardation, kinky or curly hair, skeletal abnormalities and, ultrastructurally, by axons in the central and peripheral nervous systems distended by masses of tightly woven neurofilaments. We recently localised the CAN locus in 16q24.1 to a 5-cM interval between the D16S507 and D16S511 markers by homozygosity mapping in three consanguineous Tunisian families. We have now established a contig-based physical map of the region comprising YACs and BACs where we have placed four genes, ten ESTs, three STSs and two additional microsatellite markers, and where we have identified six new SSCP polymorphisms and six new microsatellite markers. Using these markers, we have refined the position of our previous flanking recombinants. We also identified a shared haplotype between two Tunisian families and a small region of homozygosity in a Turkish family with distant consanguinity, both suggesting the occurrence of historic recombinations and supporting the conclusions based on the phase-known recombinations. Taken together, these results allow us to establish a transcription map of the region, and to narrow down the GAN position to a < 590 kb critical interval, an important step toward the identification of the defective gene. [source] Differences in lymphocyte gene expression between tolerant and syngeneic liver grafted ratsLIVER TRANSPLANTATION, Issue 3 2004Masayuki Fujino Induction of tolerance to allogeneic donor grafts is a clinically desirable goal in bone marrow and solid organ transplantation. We have taken the advantage of DNA microarray technology to investigate gene expression mechanism in regulatory cells. In the present study, using a tacrolimus (FK506) induced tolerance of the fully mismatched liver allograft rat model, we demonstrated that, in contrast with peripheral blood lymphocytes (PBLs) from syngeneic recipients, PBLs taken from tolerant recipients 100 days after transplantation were able to suppress the in vitro proliferation of allogeneic PBLs and to prolong the survival of second syngeneic recipients. We also compared messenger RNA profiles in PBLs from tolerant recipients with those from syngeneic recipients using a DNA microarray with probe sets corresponding to more than 8000 rat genes. There were 96 up-regulated and 103 down-regulated genes in the tolerant recipients. In the up-regulated group, there were 76 known genes and 20 expressed sequence tags (ESTs). In the down-regulated groups, there were 87 known genes and 16 ESTs. Our data indicated that FK506 treatment induced tolerance and expansion of regulatory cells and the DNA microarray technology was useful for this application and provided many informative insights into the mechanism of lymphocyte regulation. (Liver Transpl 2004;10:379,391.) [source] Identification of distinct and common gene expression changes after oxidative stress and gamma and ultraviolet radiation,MOLECULAR CARCINOGENESIS, Issue 2 2003Alexandra N. Heinloth Abstract The human genome is exposed to many different kinds of DNA-damaging agents. While most damage is detected and repaired through complex damage recognition and repair machineries, some damage has the potential to escape these mechanisms. Unrepaired DNA damage can give rise to alterations and mutations in the genome in an individual cell, which can result in malignant transformation, especially when critical genes are deregulated. In this study, we investigated gene expression changes in response to oxidative stress, gamma (,) radiation, and ultraviolet (UV) radiation and their potential implications in cancer development. Doses were selected for each of the three treatments, based on their ability to cause a similar G1 checkpoint induction and slow down in early S-phase progression, as reflected by a comparable reduction in cyclin E,associated kinase activity of at least 75% in logarithmically growing human dermal diploid fibroblasts. To investigate gene expression changes, logarithmically growing dermal diploid fibroblasts were exposed to either , radiation (5 Gy), oxidative stress (75 ,M of tert-butyl hydroperoxide (t -butyl-OOH)), or UV radiation (UVC) (7.5 J/m2) and RNA was harvested 6 h after treatment. Gene expression was analyzed using the NIEHS Human ToxChip 2.0 with approximately 1901 cDNA clones representing known genes and expressed sequence tags (ESTs). We were able to identify common and distinct responses in dermal diploid fibroblasts to the three different stimuli used. Within our analysis, gene expression profiles in response to , radiation and oxidative stress appeared to be more similar than profiles expressed after UV radiation. Interestingly, equivalent cyclin E,associated kinase activity reduction with all the three treatments was associated with greater transcriptional changes after UV radiation than after , radiation and oxidative stress. While samples treated with UV radiation displayed modulations of their mitogen activated protein kinase (MAPK) pathway, , radiation had its major influence on cell-cycle progression in S-phase and mitosis. In addition, cell cultures from different individuals displayed significant differences in their gene expression responses to DNA damage. Published 2003 Wiley-Liss, Inc. [source] Genomic tools and cDNA derived markers for butterfliesMOLECULAR ECOLOGY, Issue 9 2005ALEXIE PAPANICOLAOU Abstract The Lepidoptera have long been used as examples in the study of evolution, but some questions remain difficult to resolve due to a lack of molecular genetic data. However, as technology improves, genomic tools are becoming increasingly available to tackle unanswered evolutionary questions. Here we have used expressed sequence tags (ESTs) to develop genetic markers for two Müllerian mimic species, Heliconius melpomene and Heliconius erato. In total 1363 ESTs were generated, representing 330 gene objects in H. melpomene and 431 in H. erato. User-friendly bioinformatic tools were used to construct a nonredundant database of these putative genes (available at http://www.heliconius.org), and annotate them with blast similarity searches, InterPro matches and Gene Ontology terms. This database will be continually updated with EST sequences for the Papilionideae as they become publicly available, providing a tool for gene finding in the butterflies. Alignments of the Heliconius sequences with putative homologues derived from Bombyx mori or other public data sets were used to identify conserved PCR priming sites, and develop 55 markers that can be amplified from genomic DNA in both H. erato and H. melpomene. These markers will be used for comparative linkage mapping in Heliconius and will have applications in other phylogenetic and genomic studies in the Lepidoptera. [source] EST-derived polymorphic microsatellites from cultivated strawberry (Fragaria×ananassa) are useful for diversity studies and varietal identification among Fragaria speciesMOLECULAR ECOLOGY RESOURCES, Issue 4 2006D. J. GIL-ARIZA Abstract Microsatellite or simple sequence repeat markers derived from expressed sequence tags (ESTs) provide genetic markers within potentially functional genes, which could be very useful for breeding programs. To date, the development of microsatellite markers in the genus Fragaria has focused mainly on Fragaria vesca. However, most of the interests of breeding programs relate to specific characteristics of cultivated strawberry. Here, we describe a set of 10 EST-derived microsatellites from Fragaria × ananassa. These markers showed high levels of polymorphism within strawberry cultivars and among different Fragaria species, indicating their potential for genetic studies not only on strawberry but also in other species within the genus. [source] Development of EST-SSRs in Cucumis sativus from sequence databaseMOLECULAR ECOLOGY RESOURCES, Issue 4 2006Q. KONG Abstract Simple sequence repeat markers derived from expressed sequence tags (EST-SSR) are potentially valuable tools for plant breeding and germplasm collection conservation, and increasingly, efforts have been made for developing this type of marker. We have identified 20 polymorphic SSR markers from cucumber ESTs deposited in public sequence database. The average allele number was 3.3 per locus, ranging from two to six alleles during screening 20 cucumber genotypes with the mean expected heterozygosity of 0.477. Amplification products were also detected by 13 pairs of primer in Cucumis melo. These informative EST-SSR markers can be used in cucumber genetic improvement projects. [source] Fifteen polymorphic simple sequence repeat markers from expressed sequence tags of Liriodendron tulipiferaMOLECULAR ECOLOGY RESOURCES, Issue 3 2006MENG XU Abstract We developed and evaluated simple sequence repeat (SSR) markers derived from expressed sequence tags (ESTs) of Liriodendron tulipifera. Characteristics of 15 EST-SSR loci were investigated using 33 L. tulipifera individuals. The number of alleles per locus ranged from two to five. The expected and observed heterozygosities ranged from 0.216 to 0.751 and from 0.182 to 0.97, respectively. These loci were further tested for their cross-species transferability to Liriodendron Chinense. Because of their high level of polymorphism and transferability, our 15 single-locus EST-SSR markers will be valuable tools for research on mating system, population genetics and systemic evolution of Liriodendron. [source] A bioinformatic tool for analysis of EST transcript abundance during infection-related development by Magnaporthe griseaMOLECULAR PLANT PATHOLOGY, Issue 5 2005DARREN M. SOANES SUMMARY Information regarding the levels of mRNA transcript abundance under different conditions, or in specific tissue types, can be obtained by analysis of the frequency of EST sequences in randomly sequenced cDNA libraries. Here we report a bioinformatics tool, which provides a means of identifying genes that are differentially expressed during pathogenesis-related development by the rice blast fungus Magnaporthe grisea. A total of 31 534 M. grisea ESTs were obtained from dbEST at NCBI, clustered into 8821 unique sequences (unisequences) and manually annotated. Transcript profiles were then calculated for 958 unigenes identified from eight different cDNA libraries. The data were integrated into the Consortium for Functional Genomics of Microbial Eukaryotes (COGEME) database (http://cogeme.ex.ac.uk/) and a web-based front end was designed to allow users to access and interrogate the generated datasets. [source] | |||||||||||||||||||||||||||||||||||||