AMOD: a morpholino oligonucleotide selection tool

AMOD is a web-based program that aids in the functional evaluation of nucleotide sequences through sequence characterization and antisense morpholino oligonucleotide (target site) selection. Submitted sequences are analyzed by translation initiation site prediction algorithms and sequence-to-sequence comparisons; results are used to characterize sequence features required for morpholino design. Within a defined subsequence, base composition and homodimerization values are computed for all putative morpholino oligonucleotides. Using these properties, morpholino candidates are selected and compared with genomic and transcriptome databases with the goal to identify target-specific enriched morpholinos. AMOD has been used at the University of Minnesota to design ∼200 morpholinos for a functional genomics screen in zebrafish. The AMOD web server and a tutorial are freely available to both academic and commercial users at

ESTpass: a web-based server for processing and annotating expressed sequence tag (EST) sequences

We present a web-based server, called ESTpass, for processing and annotating sequence data from expressed sequence tag (EST) projects. ESTpass accepts a FASTA-formatted EST file and its quality file as inputs, and it then executes a back-end EST analysis pipeline consisting of three consecutive steps. The first is cleansing the input EST sequences. The second is clustering and assembling the cleansed EST sequences using d2_cluster and CAP3 programs and producing putative transcripts. From the CAP3 output, ESTpass detects chimeric EST sequences which are confirmed through comparison with the nr database. The last step is annotating the putative transcript sequences using RefSeq, InterPro, GO and KEGG gene databases according to user-specified options. The major advantages of ESTpass are the integration of cleansing and annotating processes, rigorous chimeric EST detection, exhaustive annotation, and email reporting to inform the user about the progress and to send the analysis results. The ESTpass results include three reports (summary, cleansing and annotation) and download function, as well as graphic statistics. They can be retrieved and downloaded using a standard web browser. The server is available at http://estpass.kobic.re.kr/

Genome sequences analysis using neural networks

วิทยานิพนธ์ (วท.ม. (วิทยาการคอมพิวเตอร์))--มหาวิทยาลัยสงขลานครินทร์, 255

Identification of novel target genes for the plasticity-related transcription factor Zif268

Activity based alterations in synaptic connectivity are thought to underlie the processes involved in learning and memory. Measurable changes in neuronal activation by long-term potentiation (LTP) are widely investigated as a possible cellular correlate of this phenomenon, as it can be induced quickly to elicit long-lasting modifications. These long-term changes in the activity of neuronal circuits are sustained by an altered pattern of gene expression and protein synthesis. The inducible transcription factor Zif268 has been implicated in almost all models of neuronal plasticity. Downstream targets of zif268 are widely believed to contribute to the duration and stabilisation of NMDA receptor dependent LTP which, in turn, can be linked to various models of learning and memory. However, these downstream targets are only just starting to receive attention. By utilising a wide range of contemporary neuroscience techniques covering molecular & cell biology approaches, this thesis proposes two known proteins, gephyrin and ubiquilin, as well as a novel gene (urma), as potential downstream targets of Zif268. Both gephyrin and ubiquilin are associated with GABAA receptors at inhibitory synapses. Gephyrin is thought to cluster and anchor GABAA receptors at postsynaptic sites whilst ubiquilin is reported to regulate receptor surface expression. We found that gephyrin mRNA and protein expression levels were downregulated in response to increased levels of zif268 by transient transfection in PC-12 cells and NMDA stimulation in primary cultured cortical neurones. In addition, ubiquilin mRNA and protein levels were also downregulated within the same experimental paradigms, implying that both gephyrin and ubiquilin are downstream transcriptional targets of this plasticity-related gene. A previously reported microarray experiment (James et al. 2005) contained 144 ESTs significantly affected by the transient transfection of Zif268 in PC-12 cells compared to control. Bioinformatic analyses of these tags revealed interesting genomic areas pertaining to little published information. After further investigation, EST AI169020 revealed a novel transcript that was downregulated in response to NMDA treatment of primary cortical neurones. Additional data mining suggests that urma may be a rarely expressed transcription factor. Basal levels of urma mRNA were also decreased in the Zif268 knockout mouse, as were ubiquilin mRNA levels. Zif268 is a regulatory immediate early gene, activating or suppressing downstream targets that play a role in the duration and stabilisation of LTP. These results indicate that gephyrin and ubiquilin are potential mediators of NMDA receptor-dependent plasticity, by modifying inhibitory-signalling. In addition, Zif268 may actively suppress a novel plasticity-related transcription factor, urma. These findings may underlie important processes in learning and memory

Investigation of the Dimerization of the Nitric Oxide-Sensitive Guanylyl Cyclase

Die Stickstoffmonoxid-sensitive Guanylyl-Cyclase ist ein wichtiges Ziel für Arzneimittel zur Behandlung von Herzkreislauferkrankungen. Das Enzym besteht aus einer α1- und einer β1 Untereinheit. Im Rahmen der vorliegenden Arbeit konnten wir eine neue besonders effiziente Anreinigung dieses Heterodimers etablieren. Mit Hilfe dieser neuen Methode konnten wir nachweisen, dass eine humane Spleißvariante der α1 Untereinheit mit der β1 Untereinheit ein katalytisch aktives Heterodimer bildet. Darüber hinaus zeigte sich in der konfokalen Fluoreszenzmikroskopie, dass die aminoterminal um 258 Aminosäuren verkürzte α1-Variante eine spezifische Lokalisation am endoplasmatischen Retikulum aufweist. Dieser Befund deckt sich mit Ergebnissen zur subzellulären Verteilung der Spleißvariante bei Differenzierungsprozessen in embryonalen Stammzellen. In einem zweiten Teil der Arbeit wurden die Bedingungen der Dimerisierung und Expression der Stickstoffmonoxid-sensitiven Guanylyl-Cyclase näher untersucht. Dabei konnten wir zeigen, dass eine weitergehende aminoterminale Deletion der α1 Untereinheit bei Koexpression mit der β1 Untereinheit zu einem Verlust der Aktivierbarkeit durch NO führt. Die Bindung von Häm und die Dimerisierung blieben hierbei erhalten. Dagegen führte die analoge aminoterminale Deletion der β1 Untereinheit nach Koexpression mit der α1 Untereinheit zu einem hämfreien Enzymkomplex. In zellfreien Expressionsystemen konnten wir die Untereinheiten des Enzyms erfolgreich exprimieren. Es kam allerdings nicht zur Bildung von funktionell aktiven Heterodimeren. Die Ergebnisse deuten auf eine wichtige Rolle von zellulären Bestandteilen für die Bildung eines intakten heterodimeren Enzyms hin.The nitric oxide-sensitive guanylyl cyclase is an important target for drugs to treat cardiovascular diseases. The enzyme consists of an α1-and β1 subunit. In the present study we were able to establish a new efficient purification method of this heterodimer. Using this new protocol we were able to demonstrate that a splice variant of human α1 subunit is able to build a catalytically active heterodimer with β1 subunit. Moreover, we could show that this truncated α1-variant has a specific localization at the endoplasmic reticulum. This finding is consistent with results of the subcellular distribution of the splice variant in differentiation processes in embryonic stem cells. In a second part, the terms of the dimerization and expression of nitric oxide-sensitive guanylyl cyclase were examined. We have shown that a more extensive amino-terminal deletion of the α1 subunit leads to a loss of activation by NO. The binding of heme and dimerization were obtained here. In contrast, the analogous amino-terminal deletion of the β1 subunit coexpression led to a heme free enzyme complex. In cell-free expression systems we were able to express the subunits of the enzyme successfully. However, the formation of functionally active heterodimers could not be shown. The results suggest an important role of cellular components for the formation of an intact heterodimeric enzyme

Development of a comprehensive annotation and curation framework for analysis of Glossina Morsitans Morsitans expresses sequence tags

Philosophiae Doctor - PhDThis study has successfully identified transcripts differentially expressed in the salivary gland and midgut and provides candidate genes that are critical to response to parasite invasion. Furthermore, an open-source Glossina resource (G-ESTMAP) was developed that provides interactive features and browsing of functional genomics data for researchers working in the field of Trypanosomiasis on the African continent.South Afric