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1.
Biotechnol Biofuels ; 11: 170, 2018.
Article in English | MEDLINE | ID: mdl-29951114

ABSTRACT

BACKGROUND: Switchgrass (Panicum virgatum L.) is an important bioenergy crop widely used for lignocellulosic research. While extensive transcriptomic analyses have been conducted on this species using short read-based sequencing techniques, very little has been reliably derived regarding alternatively spliced (AS) transcripts. RESULTS: We present an analysis of transcriptomes of six switchgrass tissue types pooled together, sequenced using Pacific Biosciences (PacBio) single-molecular long-read technology. Our analysis identified 105,419 unique transcripts covering 43,570 known genes and 8795 previously unknown genes. 45,168 are novel transcripts of known genes. A total of 60,096 AS transcripts are identified, 45,628 being novel. We have also predicted 1549 transcripts of genes involved in cell wall construction and remodeling, 639 being novel transcripts of known cell wall genes. Most of the predicted transcripts are validated against Illumina-based short reads. Specifically, 96% of the splice junction sites in all the unique transcripts are validated by at least five Illumina reads. Comparisons between genes derived from our identified transcripts and the current genome annotation revealed that among the gene set predicted by both analyses, 16,640 have different exon-intron structures. CONCLUSIONS: Overall, substantial amount of new information is derived from the PacBio RNA data regarding both the transcriptome and the genome of switchgrass.

2.
BMC Genomics ; 14 Suppl 5: S13, 2013.
Article in English | MEDLINE | ID: mdl-24564380

ABSTRACT

BACKGROUND: Small bioinformatics databases, unlike institutionally funded large databases, are vulnerable to discontinuation and many reported in publications are no longer accessible. This leads to irreproducible scientific work and redundant effort, impeding the pace of scientific progress. RESULTS: We describe a Web-accessible system, available online at http://biodb100.apbionet.org, for archival and future on demand re-instantiation of small databases within minutes. Depositors can rebuild their databases by downloading a Linux live operating system (http://www.bioslax.com), preinstalled with bioinformatics and UNIX tools. The database and its dependencies can be compressed into an ".lzm" file for deposition. End-users can search for archived databases and activate them on dynamically re-instantiated BioSlax instances, run as virtual machines over the two popular full virtualization standard cloud-computing platforms, Xen Hypervisor or vSphere. The system is adaptable to increasing demand for disk storage or computational load and allows database developers to use the re-instantiated databases for integration and development of new databases. CONCLUSIONS: Herein, we demonstrate that a relatively inexpensive solution can be implemented for archival of bioinformatics databases and their rapid re-instantiation should the live databases disappear.


Subject(s)
Computational Biology/methods , Databases, Factual , Internet , Archives , Software , User-Computer Interface
3.
BMC Med Genomics ; 4: 41, 2011 May 16.
Article in English | MEDLINE | ID: mdl-21575170

ABSTRACT

BACKGROUND: The zebrafish is recognized as a versatile cancer and drug screening model. However, it is not known whether the estrogen-responsive genes and signaling pathways that are involved in estrogen-dependent carcinogenesis and human cancer are operating in zebrafish. In order to determine the potential of zebrafish model for estrogen-related cancer research, we investigated the molecular conservation of estrogen responses operating in both zebrafish and human cancer cell lines. METHODS: Microarray experiment was performed on zebrafish exposed to estrogen (17ß-estradiol; a classified carcinogen) and an anti-estrogen (ICI 182,780). Zebrafish estrogen-responsive genes sensitive to both estrogen and anti-estrogen were identified and validated using real-time PCR. Human homolog mapping and knowledge-based data mining were performed on zebrafish estrogen responsive genes followed by estrogen receptor binding site analysis and comparative transcriptome analysis with estrogen-responsive human cancer cell lines (MCF7, T47D and Ishikawa). RESULTS: Our transcriptome analysis captured multiple estrogen-responsive genes and signaling pathways that increased cell proliferation, promoted DNA damage and genome instability, and decreased tumor suppressing effects, suggesting a common mechanism for estrogen-induced carcinogenesis. Comparative analysis revealed a core set of conserved estrogen-responsive genes that demonstrate enrichment of estrogen receptor binding sites and cell cycle signaling pathways. Knowledge-based and network analysis led us to propose that the mechanism involving estrogen-activated estrogen receptor mediated down-regulation of human homolog HES1 followed by up-regulation cell cycle-related genes (human homologs E2F4, CDK2, CCNA, CCNB, CCNE), is highly conserved, and this mechanism may involve novel crosstalk with basal AHR. We also identified mitotic roles of polo-like kinase as a conserved signaling pathway with multiple entry points for estrogen regulation. CONCLUSION: The findings demonstrate the use of zebrafish for characterizing estrogen-like environmental carcinogens and anti-estrogen drug screening. From an evolutionary perspective, our findings suggest that estrogen regulation of cell cycle is perhaps one of the earliest forms of steroidal-receptor controlled cellular processes. Our study provides first evidence of molecular conservation of estrogen-responsiveness between zebrafish and human cancer cell lines, hence demonstrating the potential of zebrafish for estrogen-related cancer research.


Subject(s)
Cell Cycle/drug effects , Cell Cycle/genetics , Estrogens/pharmacology , Neoplasms/genetics , Precancerous Conditions/genetics , Zebrafish/genetics , Animals , Binding Sites , Cell Line, Tumor , Cluster Analysis , Down-Regulation/drug effects , Estradiol/pharmacology , Gene Expression Profiling , Gene Regulatory Networks/genetics , Humans , Male , Oligonucleotide Array Sequence Analysis , Reproducibility of Results , Response Elements/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Nucleic Acid , Signal Transduction/drug effects , Signal Transduction/genetics , Up-Regulation/drug effects , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolism
4.
BMC Bioinformatics ; 11 Suppl 7: S4, 2010 Oct 15.
Article in English | MEDLINE | ID: mdl-21106126

ABSTRACT

BACKGROUND: Effectors of Type III Secretion System (T3SS) play a pivotal role in establishing and maintaining pathogenicity in the host and therefore the identification of these effectors is important in understanding virulence. However, the effectors display high level of sequence diversity, therefore making the identification a difficult process. There is a need to collate and annotate existing effector sequences in public databases to enable systematic analyses of these sequences for development of models for screening and selection of putative novel effectors from bacterial genomes that can be validated by a smaller number of key experiments. RESULTS: Herein, we present T3SEdb http://effectors.bic.nus.edu.sg/T3SEdb, a specialized database of annotated T3SS effector (T3SE) sequences containing 1089 records from 46 bacterial species compiled from the literature and public protein databases. Procedures have been defined for i) comprehensive annotation of experimental status of effectors, ii) submission and curation review of records by users of the database, and iii) the regular update of T3SEdb existing and new records. Keyword fielded and sequence searches (BLAST, regular expression) are supported for both experimentally verified and hypothetical T3SEs. More than 171 clusters of T3SEs were detected based on sequence identity comparisons (intra-cluster difference up to ~60%). Owing to this high level of sequence diversity of T3SEs, the T3SEdb provides a large number of experimentally known effector sequences with wide species representation for creation of effector predictors. We created a reliable effector prediction tool, integrated into the database, to demonstrate the application of the database for such endeavours. CONCLUSIONS: T3SEdb is the first specialised database reported for T3SS effectors, enriched with manual annotations that facilitated systematic construction of a reliable prediction model for identification of novel effectors. The T3SEdb represents a platform for inclusion of additional annotations of metadata for future developments of sophisticated effector prediction models for screening and selection of putative novel effectors from bacterial genomes/proteomes that can be validated by a small number of key experiments.


Subject(s)
Bacteria/genetics , Bacteria/pathogenicity , Bacterial Secretion Systems/genetics , Databases, Protein , Virulence Factors/genetics , Algorithms , Genetic Variation
5.
J Clin Invest ; 119(8): 2171-83, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19620787

ABSTRACT

Aggressive forms of cancer are often defined by recurrent chromosomal alterations, yet in most cases, the causal or contributing genetic components remain poorly understood. Here, we utilized microarray informatics to identify candidate oncogenes potentially contributing to aggressive breast cancer behavior. We identified the Rab-coupling protein RCP (also known as RAB11FIP1), which is located at a chromosomal region frequently amplified in breast cancer (8p11-12) as a potential candidate. Overexpression of RCP in MCF10A normal human mammary epithelial cells resulted in acquisition of tumorigenic properties such as loss of contact inhibition, growth-factor independence, and anchorage-independent growth. Conversely, knockdown of RCP in human breast cancer cell lines inhibited colony formation, invasion, and migration in vitro and markedly reduced tumor formation and metastasis in mouse xenograft models. Overexpression of RCP enhanced ERK phosphorylation and increased Ras activation in vitro. As these results indicate that RCP is a multifunctional gene frequently amplified in breast cancer that encodes a protein with Ras-activating function, we suggest it has potential importance as a therapeutic target. Furthermore, these studies provide new insight into the emerging role of the Rab family of small G proteins and their interacting partners in carcinogenesis.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Breast Neoplasms/genetics , Genes, ras , Membrane Proteins/genetics , Oncogenes , Adaptor Proteins, Signal Transducing/analysis , Adaptor Proteins, Signal Transducing/antagonists & inhibitors , Adaptor Proteins, Signal Transducing/physiology , Animals , Breast Neoplasms/etiology , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Transformation, Neoplastic , Computational Biology , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Gene Dosage , Gene Expression Profiling , Genes, erbB-2 , Humans , Immunohistochemistry , Membrane Proteins/analysis , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/physiology , Mice , Mice, Inbred BALB C , Phosphorylation , RNA Interference , rab GTP-Binding Proteins/physiology
6.
Cell ; 133(6): 1106-17, 2008 Jun 13.
Article in English | MEDLINE | ID: mdl-18555785

ABSTRACT

Transcription factors (TFs) and their specific interactions with targets are crucial for specifying gene-expression programs. To gain insights into the transcriptional regulatory networks in embryonic stem (ES) cells, we use chromatin immunoprecipitation coupled with ultra-high-throughput DNA sequencing (ChIP-seq) to map the locations of 13 sequence-specific TFs (Nanog, Oct4, STAT3, Smad1, Sox2, Zfx, c-Myc, n-Myc, Klf4, Esrrb, Tcfcp2l1, E2f1, and CTCF) and 2 transcription regulators (p300 and Suz12). These factors are known to play different roles in ES-cell biology as components of the LIF and BMP signaling pathways, self-renewal regulators, and key reprogramming factors. Our study provides insights into the integration of the signaling pathways into the ES-cell-specific transcription circuitries. Intriguingly, we find specific genomic regions extensively targeted by different TFs. Collectively, the comprehensive mapping of TF-binding sites identifies important features of the transcriptional regulatory networks that define ES-cell identity.


Subject(s)
Embryonic Stem Cells/metabolism , Gene Regulatory Networks , Signal Transduction , Animals , Base Sequence , Binding Sites , Chromatin Immunoprecipitation , Genome , Kruppel-Like Factor 4 , Mice , Multiprotein Complexes , Transcription Factors/metabolism
7.
PLoS One ; 3(1): e1479, 2008 Jan 23.
Article in English | MEDLINE | ID: mdl-18213387

ABSTRACT

BACKGROUND: Notch signaling is an evolutionarily conserved developmental pathway. Zebrafish mind bomb (mib) mutants carry mutations on mib gene, which encodes a RING E3 ligase required for Notch activation via Delta/Jagged ubiquitylation and internalization. METHODOLOGY/PRINCIPAL FINDINGS: We examined the mib mutants for defects in pancreas development using in situ hybridization and GFP expression analysis of pancreas-specific GFP lines, carried out the global gene expression profile analysis of three different mib mutant alleles and validated the microarray data using real-time PCR and fluorescent double in situ hybridization. Our study showed that the mib mutants have diminished exocrine pancreas and this defect was most severe in mib(ta52b) followed by mib(m132) and then mib(tfi91), which is consistent with the compromised Notch activity found in corresponding mib mutant alleles. Global expression profile analysis of mib mutants showed that there is a significant difference in gene expression profile of wt and three mib mutant alleles. There are 91 differentially expressed genes that are common to all three mib alleles. Through detailed analysis of microarray data, we have identified several previously characterized genes and some putative Notch-responsive genes involved in pancreas development. Moreover, results from real-time PCR and fluorescent double in situ hybridization were largely consistent with microarray data. CONCLUSIONS/SIGNIFICANCE: This study provides, for the first time, a global gene expression profile in mib mutants generating useful genomic resources and providing an opportunity to identify the function of novel genes involved in Notch signaling and Notch-regulated developmental processes.


Subject(s)
Alleles , Pancreas/abnormalities , Receptors, Notch/physiology , Ubiquitin-Protein Ligases/genetics , Zebrafish Proteins/genetics , Zebrafish/genetics , Animals , Gene Expression Profiling , In Situ Hybridization, Fluorescence , Mutation , Polymerase Chain Reaction
8.
Genetics ; 174(2): 735-52, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16888327

ABSTRACT

Hedgehog proteins play critical roles in organizing the embryonic development of animals, largely through modulation of target gene expression. Little is currently known, however, about the kinds and numbers of genes whose expression is controlled, directly or indirectly, by Hedgehog activity. Using techniques to globally repress or activate Hedgehog signaling in zebrafish embryos followed by microarray-based expression profiling, we have discovered a cohort of genes whose expression responds significantly to loss or gain of Hedgehog function. We have confirmed the Hedgehog responsiveness of a representative set of these genes with whole-mount in situ hybridization as well as real time PCR. In addition, we show that the consensus Gli-binding motif is enriched within the putative regulatory elements of a sizeable proportion of genes that showed positive regulation in our assay, indicating that their expression is directly induced by Hedgehog. Finally, we provide evidence that the Hedgehog-dependent spatially restricted transcription of one such gene, nkx2.9, is indeed mediated by Gli1 through a single Gli recognition site located within an evolutionarily conserved enhancer fragment. Taken together, this study represents the first comprehensive survey of target genes regulated by the Hedgehog pathway during vertebrate development. Our data also demonstrate for the first time the functionality of the Gli-binding motif in the control of Hedgehog signaling-induced gene expression in the zebrafish embryo.


Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Developmental/physiology , Genome/physiology , Hedgehog Proteins/physiology , Signal Transduction/genetics , Zebrafish/genetics , Animals , Base Sequence , Molecular Sequence Data , Oncogene Proteins/chemistry , Oncogene Proteins/physiology , Trans-Activators/chemistry , Trans-Activators/physiology , Zebrafish/embryology , Zinc Finger Protein GLI1
9.
Nat Biotechnol ; 24(1): 73-5, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16327811

ABSTRACT

The zebrafish (Danio rerio) has been long advocated as a model for cancer research, but little is known about the real molecular similarities between zebrafish and human tumors. Comparative analysis of microarray data from zebrafish liver tumors with those from four human tumor types revealed molecular conservation at various levels between fish and human tumors. This approach provides a useful strategy for identifying an expression signature that is strongly associated with a disease phenotype.


Subject(s)
Gene Expression Regulation, Neoplastic , Liver Neoplasms, Experimental/genetics , Liver Neoplasms, Experimental/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Zebrafish/genetics , Zebrafish/metabolism , Amino Acid Sequence , Animals , Carcinogens , Conserved Sequence , Disease Progression , Gene Expression Profiling , Liver Neoplasms, Experimental/chemically induced , Molecular Sequence Data , Sequence Homology, Amino Acid , Species Specificity
10.
PLoS Genet ; 1(2): 260-76, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16132083

ABSTRACT

Zebrafish (Danio rerio) is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula) revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as co-regulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html).

11.
Bioinformatics ; 19(2): 309-10, 2003 Jan 22.
Article in English | MEDLINE | ID: mdl-12538264

ABSTRACT

SUMMARY: Binding of short antigenic peptides to Major histocompatibility complex (MHC) proteins is the first step in T-cell mediated immune response. To understand the structural principles governing MHC-specific peptide recognition and binding, we have developed the MHC-Peptide Interaction Database (MPID), containing sequence-structure-function information. MPID (version 1.2) contains curated x-ray crystallographic data on 86 MHC peptide complexes, with precomputed interaction parameters (solvent accessibility, hydrogen bonds, gap volume and gap index). A user-friendly web interface and query tools will facilitate the development of predictive algorithms for MHC-peptide binding from a structural viewpoint. AVAILABILITY: Freely accessible from http://surya.bic.nus.edu.sg/mpid.


Subject(s)
Database Management Systems , Databases, Protein , Major Histocompatibility Complex , Peptides/chemistry , Structure-Activity Relationship , Binding Sites , Histocompatibility Antigens/chemistry , Information Storage and Retrieval/methods , Macromolecular Substances , Peptide Fragments/chemistry , Protein Binding , Protein Conformation , User-Computer Interface
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