10 research outputs found
FANTOM5 CAGE profiles of human and mouse samples
In the FANTOM5 project, transcription initiation events across the human and
mouse genomes were mapped at a single base-pair resolution and their
frequencies were monitored by CAGE (Cap Analysis of Gene Expression) coupled
with single-molecule sequencing. Approximately three thousands of samples,
consisting of a variety of primary cells, tissues, cell lines, and time series
samples during cell activation and development, were subjected to a uniform
pipeline of CAGE data production. The analysis pipeline started by measuring
RNA extracts to assess their quality, and continued to CAGE library production
by using a robotic or a manual workflow, single molecule sequencing, and
computational processing to generate frequencies of transcription initiation.
Resulting data represents the consequence of transcriptional regulation in
each analyzed state of mammalian cells. Non-overlapping peaks over the CAGE
profiles, approximately 200,000 and 150,000 peaks for the human and mouse
genomes, were identified and annotated to provide precise location of known
promoters as well as novel ones, and to quantify their activities
Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing
Besides linear RNAs, pre-mRNA splicing generates three forms of RNAs: lariat introns, Y-structure introns from trans-splicing, and circular exons through exon skipping. To study the persistence of excised introns in total cellular RNA, we used three Escherichia coli 3′ to 5′ exoribonucleases. Ribonuclease R (RNase R) thoroughly degrades the abundant linear RNAs and the Y-structure RNA, while preserving the loop portion of a lariat RNA. Ribonuclease II (RNase II) and polynucleotide phosphorylase (PNPase) also preserve the lariat loop, but are less efficient in degrading linear RNAs. RNase R digestion of the total RNA from human skeletal muscle generates an RNA pool consisting of lariat and circular RNAs. RT–PCR across the branch sites confirmed lariat RNAs and circular RNAs in the pool generated by constitutive and alternative splicing of the dystrophin pre-mRNA. Our results indicate that RNase R treatment can be used to construct an intronic cDNA library, in which majority of the intron lariats are represented. The highly specific activity of RNase R implies its ability to screen for rare intragenic trans-splicing in any target gene with a large background of cis-splicing. Further analysis of the intronic RNA pool from a specific tissue or cell will provide insights into the global profile of alternative splicing
cRegions—a tool for detecting conserved cis-elements in multiple sequence alignment of diverged coding sequences
Identifying cis-acting elements and understanding regulatory mechanisms of a gene is crucial to fully understand the molecular biology of an organism. In general, it is difficult to identify previously uncharacterised cis-acting elements with an unknown consensus sequence. The task is especially problematic with viruses containing regions of limited or no similarity to other previously characterised sequences. Fortunately, the fast increase in the number of sequenced genomes allows us to detect some of these elusive cis-elements. In this work, we introduce a web-based tool called cRegions. It was developed to identify regions within a protein-coding sequence where the conservation in the amino acid sequence is caused by the conservation in the nucleotide sequence. The cRegion can be the first step in discovering novel cis-acting sequences from diverged protein-coding genes. The results can be used as a basis for future experimental analysis. We applied cRegions on the non-structural and structural polyproteins of alphaviruses as an example and successfully detected all known cis-acting elements. In this publication and in previous work, we have shown that cRegions is able to detect a wide variety of functional elements in DNA and RNA viruses. These functional elements include splice sites, stem-loops, overlapping reading frames, internal promoters, ribosome frameshifting signals and other embedded elements with yet unknown function. The cRegions web tool is available at http://bioinfo.ut.ee/cRegions/
Distributed Web Service Coordination for Collaboration Applications and Biological Workflows
In this dissertation work, we have investigated the main research thrust of decentralized coordination of workflows over web services. To address distributed workflow coordination, first we have developed “Web Coordination Bonds” as a capable set of dependency modeling primitives that enable each web service to manage its own dependencies. Web bond primitives are as powerful as extended Petri nets and have sufficient modeling and expressive capabilities to model workflow dependencies. We have designed and prototyped our “Web Service Coordination Management Middleware” (WSCMM) system that enhances current web services infrastructure to accommodate web bond enabled web services. Finally, based on core concepts of web coordination bonds and WSCMM, we have developed the “BondFlow” system that allows easy configuration distributed coordination of workflows. The footprint of the BonFlow runtime is 24KB and the additional third party software packages, SOAP client and XML parser, account for 115KB
Search for endotherm genes involved in early patterning of vertebrates.
In addition to giving rise to the gut, the endoderm plays a crucial role in embryonic axis determination. The murine extra-embryonic endoderm is thought to provide an early positional cue defining the antero-posterior axis of the embryo. The axial mesendoderm, which emanates from the gastrula organizer, populates the midline of the embryo and patterns it in all three axes. Later, maintenance and refinement of the antero-posterior axis of the brain requires the embryonic endoderm (reviewed in Martinez-Barbera and Beddington, 2001). Genes expressed in the endoderm are responsible for imparting it with its patterning properties. It is therefore useful to identify the expression profile of the endoderm. To this end, a cDNA library was made from 7.5 days post-coitum mouse endoderm (Harrison et al., 1995). Many clones from this library were sequenced and constitute a set of expression sequence tags (ESTs). I screened these ESTs in silico for non- essential molecules whose role in embryonic patterning had not been determined. I then screened clones obeying these criteria by whole-mount in situ hybridisation on 6.5 - 9.5 dpc mouse embryos. Restricted expression was displayed by 18% of the clones (from the total of my work and that of two other students). The restricted expression patterns encountered are presented. One of the restricted genes I encountered in the mouse in situ hybridisation screen was that encoding the serum and glucocorticoid-regulated kinase (Sgk). I was very interested in the expression pattern of Sgk since it was asymmetric in the visceral endoderm at the onset of gastrulation. Sgk expression presented other interesting features, such as being exclusively expressed in angioblasts at 9.5 dpc. I constructed a targeting vector in order to analyse Sgk function in mouse by a loss-of-function approach. I targeted embryonic stem (ES) cells with this construct and recovered neomycin-resistant clones. One of these is possibly a clone where homologous recombination took place at the Sgk locus. I cloned zebrafish orthologues of some of the restrictedly expressed endoderm genes. I functionally screened these genes in zebrafish by a loss-of-function approach, using antisense morpholino oligonucleotides (MOs). I uncovered several molecules required for proper early embryonic development, one of which I studied in more detail. This was Nop seven-associated protein 2 (Nsa2), a eukaryotic protein involved in ribosome biogenesis (Harnpicharnchai et al., 2001). Zebrafish nsa2 morphants have slowed epiboly and early patterning defects. Furthermore, nsa2 morphant cells gradually die by apoptosis. A smaller embryo develops from surviving nsa2 morphant cells during the first day of development, after which presumably all cells die. The phenotype of nsa2 zebrafish morphants is analogous to that of morphants for the ribosomal proteins RpL19 and RpS5, which when mutated in fly cause the Minute phenotype. I describe the zebrafish Minute phenotype and hypothesize that nsa2 is likely to be a Minute
Identification and characterization of photomedins: novel olfactomedin-domain-containing proteins with chondroitin sulphate-E-binding activity
We screened more than 60000 RIKEN mouse cDNAs for novel ECM (extracellular matrix) proteins by extensive computational screening followed by recombinant expression and immunohistochemical characterization. We identified two novel olfactomedin-family proteins characterized by the presence of tandem CXCXCX(9)C motifs in the N-terminal region, a coiled-coil domain and an olfactomedin domain in the C-terminal region. These proteins, named photomedin-1 and photomedin-2, were secreted as disulphide-bonded dimers (photomedin-1) or oligomers/multimers (photomedin-2) with O-linked carbohydrate chains, although photomedin-1 was proteolytically processed in the middle of the molecule after secretion. In the retina, photomedin-1 was selectively expressed in the outer segment of photoreceptor cells and photomedin-2 was expressed in all retinal neurons. Among a panel of ECM components, including glycosaminoglycans, photomedins preferentially bound to chondroitin sulphate-E and heparin. These results, together, indicate that photomedins are novel olfactomedin-domain-containing extracellular proteins capable of binding to proteoglycans containing these glycosaminoglycan chains
Initial sequencing and comparative analysis of the mouse genome
The sequence of the mouse genome is a key informational tool for understanding the contents of the human genome and a key experimental tool for biomedical research. Here, we report the results of an international collaboration to produce a high-quality draft sequence of the mouse genome. We also present an initial comparative analysis of the mouse and human genomes, describing some of the insights that can be gleaned from the two sequences. We discuss topics including the analysis of the evolutionary forces shaping the size, structure and sequence of the genomes; the conservation of large-scale synteny across most of the genomes; the much lower extent of sequence orthology covering less than half of the genomes; the proportions of the genomes under selection; the number of protein-coding genes; the expansion of gene families related to reproduction and immunity; the evolution of proteins; and the identification of intraspecies polymorphism