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At least 919 records · Page 51Linked to original sources

PubMed2006

[Construction of the female subtractive cDNA library and screening of the specific expressing genes].

OBJECTIVE: To screen the Schistosoma japonicum female specific expressing genes. METHODS: S. japonicum adult worms were collected from the rabbits' vein after six-week infection by affusing method. The adult worms were stabilized by RNA-later liquid, the male and female worms were carefully separated with nipper. The high quality total RNA was extracted and mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Female subtractive (female as tester, male as driver) and male subtractive (male as tester, female as driver) cDNA libraries were constructed. The differentially expressed genes were further screened by dot-blot hybridization. The clones were selected and sequenced, which showed apparently higher signals when hybridizing with the female subtracting male probes, than those signals when hybridizing with the male subtracting female probes. The homology of these sequences was searched with BLAST program. The semi-quantitative PCR was applied to test the differential gene expression in female and male adult worms. RESULT: Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. After dot-blot hybridization, 50 clones were tested to be the potential female differentially expressed genes and were sequenced. 42 expressing sequence tags (ESTs) were received. After bioinformatics analysis, 17 fragments (about 40.5%) showed high identity with the S. japonicum egg-shell protein genes, 17 sequences (about 40.5%) were highly homologous to unknown S. japonicum genes and partly homologous to female specific 800 protein. 8 fragments (about 19.0%) showed high identity with other S. japonicum unknown genes. The fragments in clones of 577, 579, 668, 695, 720, and 708 were tested by RT-PCR to be the differentially expressed genes in female adult worms using S. japonicum actin gene as the internal standard. These fragments were highly homologous to S. japonicum egg shell protein gene AY222885, AY222895, AB017097, AF519182, M32281, and S. japonicum unknown gene AY813556 respectively. CONCLUSION: SSH is essential to screen the differentially expressed genes of S. japonicum female worms. A number of female specific genes have been found by this method.

Animals↗

PubMed2006

[Screen and identification of Streptocococcus pneumoniae genes specifically induced in host].

To identify in vivo-induced genes of S. pneumoniae and search new potential drug targets and vaccine candidates, a selection system was developed based on the in vivo expression technology (IVET). Promoter galU gene which is critical for the capsular polysaccharide biosynthesis and lacZY gene which encodes bea-galactosidase were employed as dual reporter genes. The galU-deficient mutant of S. pneumoniae is incapable of utilizing galactose and synthesizing capsular polysaccharide, therefore can't survival in the host. Firstly, the random pieces of S. pneumoniae chromosomal DNA (200-500 bp), obtained by partial Sau3Al restriction digestion, were subcloned into the Bgl II site of pEVP3-galU. Transformation by this plasmid library yielded promoter-trap library in S. pneumoniae. Then, the library was used to infect animals. Bacteria were harvested from lung tissue. White clones on TSA agar containing X-gal were used to reinfect animals. The sequence of infection and sorting was performed twice, 165 white clones harvested from the final round of infection were analyzed. A total of 15 unique sequences were obtained through in vivo screen. The bioinformatics analysis showed that these ivi genes involved in colonization and adherence, energy metabolism, nutrient substance transport, transcription regulation, DNA metabolism and cell wall synthesis. And there were some hypothetical proteins have unknown functions. Part of these genes may be related with virulence and can be used as vaccine candidates and drug targets.

Animals↗

PubMedDate not supplied

[Molecular cloning and characterization of a novel WRKY gene from Brassica chinensis].

A new WRKY gene was cloned from Brassica chinensis by rapid amplification of cDNA ends (RACE). The full-length cDNA of BcWRKY was 1175 bp long and contained a 924 bp open reading frame (ORF) encoding a putative W-box-binding protein of 308 amino acids. The predicted BcWRKY protein was found to have a potential bipartite nuclear localization sequence (NLS-BP) in its N-terminal region followed by a WRKY DNA-binding domain. Bioinformatic analysis revealed that BcWRKY resembled other WRKY domain-containing proteins from Arabidopsis (AtWRKY18), tobacco (WIZZ), parsley (PcWRKY4) and wild oat (ABF2). Expression of the BcWRKY gene could be induced by salicylic acid (SA), and influenced by Pseudomonas syringae pv. tomato strain DC3000 infection and wounding treatment. Our study implies that BcWRKY might have similar functions possessed by other WRKY genes such as inducing the expression of some defense-related genes and increasing plant's disease resistance ability.

Amino Acid Sequence↗

PubMed2006

Screening of genes for proteins interacting with the PS1TP5 protein of hepatitis B virus: probing a human leukocyte cDNA library using the yeast two-hybrid system.

BACKGROUND: The hepatitis B virus (HBV) genome includes S, C, P and X regions. The S region is divided into four subregions of pre-pre-S, pre-S1, pre-S2 and S. PS1TP5 (human gene 5 transactivated by pre-S1 protein of HBV) is a novel target gene transactivated by the pre-S1 protein that has been screened with a suppression subtractive hybridization technique in our laboratory (GenBank accession: AY427953). In order to investigate the biological function of the PS1TP5 protein, we performed a yeast two-hybrid system 3 to screen proteins from a human leukocyte cDNA library interacting with the PS1TP5 protein. METHODS: The reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the gene of PS1TP5 from the mRNA of HepG2 cells and the gene was then cloned into the pGEM-T vector. After being sequenced and analyzed with Vector NTI 9.1 and NCBI BLAST software, the target gene of PS1TP5 was cut from the pGEM-T vector and cloned into a yeast expression plasmid pGBKT7, then "bait" plasmid pGBKT7-PS1TP5 was transformed into the yeast strain AH109. The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization. After expression of the pGBKT7-PS1TP5 fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening was performed by mating AH109 with Y187 containing a leukocyte cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for alpha-gal activity. The interaction between the PS1TP5 protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybrid screen. After extracting and sequencing of plasmids from blue colonies we carried out a bioinformatic analysis. RESULTS: Forty true positive colonies were selected and sequenced, full length sequences were obtained and we searched for homologous DNA sequences from GenBank. Among the 40 positive colonies, 23 coding genes with known functions were obtained, including Homo sapien leukocyte adhesion protein p150, 95, interleukin 2 receptor gamma chain, PALM2-AKAP2 protein (PALM2-AKAP2), eukaryotic translation initiation factor 4A, beta-2-microglobin, solute carrier family 9 (sodium/hydrogen exchanger), calreticulin, asialoglycoprotein receptor 1 (ASGR1), MHC class II lymphocyte antigen, cytochrome c oxidase subunit 1, lymphocyte antigen 86 (LY86) and lymphocyte cytosolic protein 1. One novel gene with unknown function was found and named as PS1TP5BP1. After being electronically spliced, it was deposited in GenBank (accession number: DQ471327). CONCLUSIONS: Genes of proteins interacting with PS1TP5 were successfully screened from leukocyte cDNA library. These results suggested that PS1TP5 was closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, the formation of hepatic fibrosis and initiation and development of tumors and also brought some new clues for further studying the biological functions of the pre-S1 protein.

Amino Acid Sequence↗

PubMed2006

The MPI Bioinformatics Toolkit for protein sequence analysis.

The MPI Bioinformatics Toolkit is an interactive web service which offers access to a great variety of public and in-house bioinformatics tools. They are grouped into different sections that support sequence searches, multiple alignment, secondary and tertiary structure prediction and classification. Several public tools are offered in customized versions that extend their functionality. For example, PSI-BLAST can be run against regularly updated standard databases, customized user databases or selectable sets of genomes. Another tool, Quick2D, integrates the results of various secondary structure, transmembrane and disorder prediction programs into one view. The Toolkit provides a friendly and intuitive user interface with an online help facility. As a key feature, various tools are interconnected so that the results of one tool can be forwarded to other tools. One could run PSI-BLAST, parse out a multiple alignment of selected hits and send the results to a cluster analysis tool. The Toolkit framework and the tools developed in-house will be packaged and freely available under the GNU Lesser General Public Licence (LGPL). The Toolkit can be accessed at http://toolkit.tuebingen.mpg.de.

Computational Biology↗

PubMed2006

Bioinformatics for comprehensive finding and analysis of glycosyltransferases.

Bioinformatics is a very powerful tool in the field of glycoproteomics as well as genomics and proteomics. As a part of the Glycogene Project (GG project), we have developed a novel bioinformatics system for the comprehensive identification and in silico cloning of human glycogenes. Using our system, a total of 105 candidate human glycogenes were identified and then engineered for heterologous expression. Of these candidates, 38 recombinant proteins were successfully identified for their enzyme activity and substrate specificity. We also classified 47 out of 60 carbohydrate-active enzyme glycosyltransferase families into 4 superfamilies using the profile Hidden Markov Model method. On the basis of our classification and the relationship between glycosylation pathways and superfamilies, we propose the evolution of glycosyltransferases.

Algorithms↗

PubMed2003

Structural bioinformatics and QSAR analysis applied to the acetylcholinesterase and bispyridinium aldoximes.

The methods of bioinformatics, molecular modelling, and quantitative structure-activity relationships (QSARs) using regression and artificial neural network (ANN) analyses were applied to develop safer aldoxime antidotes against poisoning by organophosphorus (OP) agents with high, mean, and low aging rates. We start here from a molecular modelling of the mouse AChE at an atomistic level. Aim is to predict qualitatively the structural requirements of an aldoxime that shows an unique reactivating activity against the three classes of OPs. An antidotal action should occur by a three-site mechanism: the aldoxime groups of the first pyridinium ring should point towards the catalytic site, and the second pyridinium ring and its substituents should be anchored at the peripherical and anionic subsites. Based on this model, it is predicted that a suitable substituent is based on an arginine-like moiety. Then, an ANN-based QSAR analysis using a training set of aldoximes with known structure and activities was applied. Its input layer consisted of seven nodes: the group-membership descriptors that parameterize the type of the OP, the logarithms of the distribution coefficients at pH 7.4 and their squared term, the lowest unoccupied molecular orbital (LUMO) energies, the scaled molar refractions of the substituents, and their squared term. It was shown that the qualitative prediction made by molecular modelling can be quantified by an ANN prediction.

Acetylcholinesterase↗

PubMed2025

A Comprehensive Bioinformatics Approach to Analysis of Variants: Variant Calling, Annotation, and Prioritization.

Next-Generation Sequencing (NGS), also known as high-throughput sequencing technologies, has enabled rapid and efficient sequencing of large amounts of DNA and RNA. These technologies have revolutionized the field of genomics, transcriptomics, and proteomics and have been widely used in cancer research, leading to advances in clinical diagnosis and treatment. Improvements in the NGS technologies enabled millions of fragments to be sequenced simultaneously in a time- and cost-effective manner and resulted in large amount of genomic data which require efficient analysis methods. Analysis of the genomic data requires both efficient computer resources and bioinformatics approaches. This chapter details a comprehensive computational approach and analysis steps for genomic data analysis.

Computational Biology↗

PubMed2005

Workflows in bioinformatics: meta-analysis and prototype implementation of a workflow generator.

BACKGROUND: Computational methods for problem solving need to interleave information access and algorithm execution in a problem-specific workflow. The structures of these workflows are defined by a scaffold of syntactic, semantic and algebraic objects capable of representing them. Despite the proliferation of GUIs (Graphic User Interfaces) in bioinformatics, only some of them provide workflow capabilities; surprisingly, no meta-analysis of workflow operators and components in bioinformatics has been reported. RESULTS: We present a set of syntactic components and algebraic operators capable of representing analytical workflows in bioinformatics. Iteration, recursion, the use of conditional statements, and management of suspend/resume tasks have traditionally been implemented on an ad hoc basis and hard-coded; by having these operators properly defined it is possible to use and parameterize them as generic re-usable components. To illustrate how these operations can be orchestrated, we present GPIPE, a prototype graphic pipeline generator for PISE that allows the definition of a pipeline, parameterization of its component methods, and storage of metadata in XML formats. This implementation goes beyond the macro capacities currently in PISE. As the entire analysis protocol is defined in XML, a complete bioinformatic experiment (linked sets of methods, parameters and results) can be reproduced or shared among users. AVAILABILITY: http://if-web1.imb.uq.edu.au/Pise/5.a/gpipe.html (interactive), ftp://ftp.pasteur.fr/pub/GenSoft/unix/misc/Pise/ (download). CONCLUSION: From our meta-analysis we have identified syntactic structures and algebraic operators common to many workflows in bioinformatics. The workflow components and algebraic operators can be assimilated into re-usable software components. GPIPE, a prototype implementation of this framework, provides a GUI builder to facilitate the generation of workflows and integration of heterogeneous analytical tools.

Algorithms↗

PubMed1999

Genome-wide bioinformatic and molecular analysis of introns in Saccharomyces cerevisiae.

Introns have typically been discovered in an ad hoc fashion: introns are found as a gene is characterized for other reasons. As complete eukaryotic genome sequences become available, better methods for predicting RNA processing signals in raw sequence will be necessary in order to discover genes and predict their expression. Here we present a catalog of 228 yeast introns, arrived at through a combination of bioinformatic and molecular analysis. Introns annotated in the Saccharomyces Genome Database (SGD) were evaluated, questionable introns were removed after failing a test for splicing in vivo, and known introns absent from the SGD annotation were added. A novel branchpoint sequence, AAUUAAC, was identified within an annotated intron that lacks a six-of-seven match to the highly conserved branchpoint consensus UACUAAC. Analysis of the database corroborates many conclusions about pre-mRNA substrate requirements for splicing derived from experimental studies, but indicates that splicing in yeast may not be as rigidly determined by splice-site conservation as had previously been thought. Using this database and a molecular technique that directly displays the lariat intron products of spliced transcripts (intron display), we suggest that the current set of 228 introns is still not complete, and that additional intron-containing genes remain to be discovered in yeast. The database can be accessed at http://www.cse.ucsc.edu/research/compbi o/yeast_introns.html.

Computational Biology↗

PubMed2003

Bioinformatic and molecular analysis of hydroxymethylbutenyl diphosphate synthase (GCPE) gene expression during carotenoid accumulation in ripening tomato fruit.

Carotenoids are plastidic isoprenoid pigments of great biological and biotechnological interest. The precursors for carotenoid production are synthesized through the recently elucidated methylerythritol phosphate (MEP) pathway. Here we have identified a tomato ( Lycopersicon esculentum Mill.) cDNA sequence encoding a full-length protein with homology to the MEP pathway enzyme hydroxymethylbutenyl 4-diphosphate synthase (HDS, also called GCPE). Comparison with other plant and bacterial HDS sequences showed that the plant enzymes contain a plastid-targeting N-terminal sequence and two highly conserved plant-specific domains in the mature protein with no homology to any other sequence in the databases. The ubiquitous distribution of HDS-encoding expressed sequence tags (ESTs) in the tomato collections suggests that the corresponding gene is likely expressed throughout the plant. The role of HDS in controlling the supply of precursors for carotenoid biosynthesis was estimated from the bioinformatic and molecular analysis of transcript abundance in different stages of fruit development. No significant changes in HDS gene expression were deduced from the statistical analysis of EST distribution during fruit ripening, when an active MEP pathway is required to support a massive accumulation of carotenoids. RNA blot experiments confirmed that similar transcript levels were present in both the wild-type and carotenoid-depleted yellow ripe ( r) mutant fruit independent of the stage of development and the carotenoid composition of the fruit. Together, our results are consistent with a non-limiting role for HDS in carotenoid biosynthesis during tomato fruit ripening.

Amino Acid Sequence↗

PubMed2004

Serial analysis of gene expression (SAGE): unraveling the bioinformatics tools.

Serial analysis of gene expression (SAGE) is a powerful technique that can be used for global analysis of gene expression. Its chief advantage over other methods is that it does not require prior knowledge of the genes of interest and provides qualitative and quantitative data of potentially every transcribed sequence in a particular cell or tissue type. This is a technique of expression profiling, which permits simultaneous, comparative and quantitative analysis of gene-specific, 9- to 13-basepair sequences. These short sequences, called SAGE tags, are linked together for efficient sequencing. The sequencing data are then analyzed to identify each gene expressed in the cell and the levels at which each gene is expressed. The main benefit of SAGE includes the digital output and the identification of novel genes. In this review, we present an outline of the method, various bioinformatics methods for data analysis and general applications of this important technology.

Animals↗

PubMed2005

Using bioinformatics and genome analysis for new therapeutic interventions.

The genome era provides two sources of knowledge to investigators whose goal is to discover new cancer therapies: first, information on the 20,000 to 40,000 genes that comprise the human genome, the proteins they encode, and the variation in these genes and proteins in human populations that place individuals at risk or that occur in disease; second, genome-wide analysis of cancer cells and tissues leads to the identification of new drug targets and the design of new therapeutic interventions. Using genome resources requires the storage and analysis of large amounts of diverse information on genetic variation, gene and protein functions, and interactions in regulatory processes and biochemical pathways. Cancer bioinformatics deals with organizing and analyzing the data so that important trends and patterns can be identified. Specific gene and protein targets on which cancer cells depend can be identified. Therapeutic agents directed against these targets can then be developed and evaluated. Finally, molecular and genetic variation within a population may become the basis of individualized treatment.

Computational Biology↗

PubMed2006

CAPweb: a bioinformatics CGH array Analysis Platform.

Assessing variations in DNA copy number is crucial for understanding constitutional or somatic diseases, particularly cancers. The recently developed array-CGH (comparative genomic hybridization) technology allows this to be investigated at the genomic level. We report the availability of a web tool for analysing array-CGH data. CAPweb (CGH array Analysis Platform on the Web) is intended as a user-friendly tool enabling biologists to completely analyse CGH arrays from the raw data to the visualization and biological interpretation. The user typically performs the following bioinformatics steps of a CGH array project within CAPweb: the secure upload of the results of CGH array image analysis and of the array annotation (genomic position of the probes); first level analysis of each array, including automatic normalization of the data (for correcting experimental biases), breakpoint detection and status assignment (gain, loss or normal); validation or deletion of the analysis based on a summary report and quality criteria; visualization and biological analysis of the genomic profiles and results through a user-friendly interface. CAPweb is accessible at http://bioinfo.curie.fr/CAPweb.

Chromosome Breakage↗

PubMed2006

Bioinformatic and expression analysis of novel porcine beta-defensins.

Beta-defensins are a major group of mammalian antimicrobial peptides. Although more than 30 beta-defensins have been identified in humans, only one porcine beta-defensin has been reported. In this article we report the identification and initial characterization of 11 novel porcine beta-defensins (pBD). Using bioinformatic approaches, we screened 287,821 porcine expressed sequence tags for similarity of their predicted peptides to known human beta-defensins and identified full-length or partial sequences for the 11 novel pBDs. Similar to the previously identified pBD1, all of these peptides have a consensus beta-defensin motif. A differential expression pattern for these newly identified genes was found. For example, unlike most beta-defensins, pBD2 and pBD3 were expressed in bone marrow and in other lymphoid tissues including thymus, spleen, lymph nodes, duodenum, and liver. Including pBD2 and pBD3, six porcine beta-defensins were expressed in lung and skin. Several newly identified porcine beta-defensins, including pBD123, pBD125, and pBD129, were expressed in male reproductive tissues, including lobuli testis and some segments of the epididymis. Phylogenetic analysis indicates that in most cases the evolutionary relationship between individual porcine beta-defensins and their human orthologs is closer than the relationship among beta-defensins in the same species. These findings establish the existence of multiple porcine beta-defensins and suggest that the pig may be an ideal model for the characterization of beta-defensin diversity and function.

Amino Acid Sequence↗

PubMed2001

[Transcriptome analysis in cancerology: bioinformatics aspects].

Recent technological advances (e.g. various DNA arrays and chips) allow the measurement of expression level (mRNA abundance) for thousand of genes simultaneously, over multiple conditions or time. Initially developed and tested on model systems such as yeast or in vitro cell line cultures, these techniques have recently begun to be applied to the analysis of human cancers. Initial results are promising, and large-scale gene expression profiling is now expected to become a clinical tool for better tumour identification, prognosis, and optimal treatment design. It is thus important that clinicians become familiar with the theoretical principles underlying the interpretation of gene expression profiles as used in three different contexts: gene discovery, tumour class prediction, and molecular diagnosis. This is the purpose of the present article.

Forecasting↗

PubMed2004

Expression analysis of members of the neuronal calcium sensor protein family: combining bioinformatics and Western blot analysis.

We have used in silico mining of public databases (NCBI UniGene and NCI SAGE Anatomic Viewer) as a tool to obtain the tissue distribution pattern of three members of the neuronal calcium sensor protein family, namely VILIP-1, hippocalcin, and NCS-1 in humans. The theoretical human mRNA expression profile of the calcium sensor proteins derived from expressed sequence tag (EST) and serial analysis of gene expression (SAGE) data was compared with expression data from human tissues obtained by Western blot analysis. Since the EST databank searches do not yet give comparable results for rat which is often used as model animal, we have also analyzed the protein expression in rat tissues. Similar to the human expression profile in rat tissues calcium sensor proteins are mainly detected in the nervous system, but the data consistently implicated the additional expression in peripheral tissues with remarkable differences between the calcium sensor proteins.

Animals↗

PubMed2005

Bioinformatic and expression analysis of the putative gliotoxin biosynthetic gene cluster of Aspergillus fumigatus.

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic animal pathogen Aspergillus fumigatus. It is a member of the epipolythiodioxopiperazine (ETP) class of toxins characterised by a disulphide bridged cyclic dipeptide. A putative cluster of 12 genes involved in gliotoxin biosynthesis has been identified in A. fumigatus by a comparative genomics approach based on homology to genes from the sirodesmin (another ETP) biosynthetic gene cluster of Leptosphaeria maculans. The physical limits of the cluster in A. fumigatus have been defined by bioinformatics and by identifying the genes that are co-regulated and whose timing of expression correlates with the production of gliotoxin in culture.

Aspergillus fumigatus↗