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PubMed1993

Poly(GTG)5-associated profiles of Salmonella and Shigella genomic DNA.

DNA hybridization with oligonucleotide probes is a powerful technique to study population genetics and structures. The use of probes which recognize ubiquitously interspersed DNA sequences has a distinct advantage over other techniques (e.g. the analysis of patterns of restriction fragment length polymorphism) in that many independent loci can be detected simultaneously. In this communication, we investigated the use of a trinucleotide repetitive DNA oligonucleotide, poly(GTG)5, in Southern blot analysis of Salmonella serotypes and Shigella species. The strains in this study were isolated over several years from widely disparate geographic locations and can therefore be considered to represent the structure of part of the natural populations of these organisms. In most of the Salmonella serotypes, the poly(GTG)5-associated profile (GTG profile) phenotypes appeared to be clonally stable; in cases where only one isolate of a serotype was tested, the GTG profile was distinct from the others. On the other hand, when GTG profile analysis was applied to Shigella strains, each of the 12 isolates, belonging to the four Shigella species, produced a unique pattern phenotype of both the chromosome and plasmid DNA.

Autoradiography↗
PubMed2006

Detection of broadly expressed neuronal genes in C. elegans.

The genes that are expressed in most or all types of neurons define generic neuronal features and provide a window into the developmental origin and function of the nervous system. Few such genes (sometimes referred to as pan-neuronal or broadly expressed neuronal genes) have been defined to date and the mechanisms controlling their regulation are not well understood. As a first step in investigating their regulation, we used a computational approach to detect sequences overrepresented in their promoter elements. We identified a ten-nucleotide cis-regulatory motif shared by many broadly expressed neuronal genes and demonstrated that it is involved in control of neuronal expression. Our results further suggest that global and cell-type-specific controls likely act in concert to establish pan-neuronal gene expression. Using the newly discovered motif and genome-level gene expression data, we identified a set of 234 candidate broadly expressed genes. The known involvement of many of these genes in neurogenesis and physiology of the nervous system supports the utility of this set for future targeted analyses.

Animals↗
PubMed2003

Protein analysis on a proteomic scale.

The long-term challenge of proteomics is enormous: to define the identities, quantities, structures and functions of complete complements of proteins, and to characterize how these properties vary in different cellular contexts. One critical step in tackling this goal is the generation of sets of clones that express a representative of each protein of a proteome in a useful format, followed by the analysis of these sets on a genome-wide basis. Such studies enable genetic, biochemical and cell biological technologies to be applied on a systematic level, leading to the assignment of biochemical activities, the construction of protein arrays, the identification of interactions, and the localization of proteins within cellular compartments.

Animals↗
PubMed2004

MITOPRED: a genome-scale method for prediction of nucleus-encoded mitochondrial proteins.

MOTIVATION: Currently available methods for the prediction of subcellular location of mitochondrial proteins rely largely on the presence of mitochondrial targeting signals in the protein sequences. However, a large fraction of mitochondrial proteins lack such signals, making those tools ineffective for genome-scale prediction of mitochondria-targeted proteins. Here, we propose a method for genome-scale prediction of nucleus-encoded mitochondrial proteins. The new method, MITOPRED, is based on the Pfam domain occurrence patterns and the amino acid compositional differences between mitochondrial and non-mitochondrial proteins. RESULTS: MITOPRED could predict mitochondrial proteins with 100% specificity at a 44% sensitivity rate and with 67% specificity at 99% sensitivity. Additionally, it was sufficiently robust to predict mitochondrial proteins across different eukaryotic species with similar accuracy. Based on Matthews correlation coefficient measure, the prediction performance of MITOPRED is clearly superior (0.73) to those of the two popular methods TargetP (0.51) and PSORT (0.53). Using this method, we predicted the nucleus-encoded mitochondrial proteins from six complete genomes (three invertebrate, two vertebrate and one plant species) and estimated the total number in each genome. In human, our method estimated the existence of 1362 mitochondrial proteins corresponding to 4.8% of the total proteome. AVAILABILITY: MITOPRED program is freely accessible at http://mitopred.sdsc.edu. Source code is available on request from the authors. SUPPLEMENTARY INFORMATION: Training data sets are also available at http://mitopred.sdsc.edu

Algorithms↗
PubMed2005

Novel molecular features of the fibrolytic intestinal bacterium Fibrobacter intestinalis not shared with Fibrobacter succinogenes as determined by suppressive subtractive hybridization.

Suppressive subtractive hybridization was conducted to identify unique genes coding for plant cell wall hydrolytic enzymes and other properties of the gastrointestinal bacterium Fibrobacter intestinalis DR7 not shared by Fibrobacter succinogenes S85. Subtractive clones from F. intestinalis were sequenced and assembled to form 712 nonredundant contigs with an average length of 525 bp. Of these, 55 sequences were unique to F. intestinalis. The remaining contigs contained 764 genes with BLASTX similarities to other proteins; of these, 80% had the highest similarities to proteins in F. succinogenes, including 30 that coded for carbohydrate active enzymes. The expression of 17 of these genes was verified by Northern dot blot analysis. Of genes not exhibiting BLASTX similarity to F. succinogenes, 30 encoded putative transposases, 6 encoded restriction modification genes, and 45% had highest similarities to proteins in other species of gastrointestinal bacteria, a finding suggestive of either horizontal gene transfer to F. intestinalis or gene loss from F. succinogenes. Analysis of contigs containing segments of two or more adjacent genes revealed that only 35% exhibited BLASTX similarity and were in the same orientation as those of F. succinogenes, indicating extensive chromosomal rearrangement. The expression of eight transposases, and three restriction-modification genes was confirmed by Northern dot blot analysis. These data clearly document the maintenance of carbohydrate active enzymes in F. intestinalis necessitated by the preponderance of polysaccharide substrates available in the ruminal environment. It also documents substantive changes in the genome from that of F. succinogenes, which may be related to the introduction of the array of transposase and restriction-modification genes.

Bacterial Proteins↗
PubMed2001

Recent advances in DNA microarrays.

The structure of the human genome is almost completely elucidated and the life sciences will now aim for a general and integrated study of gene expressions and the functional elucidation of proteins. In such a study, various new techniques have been developed, and DNA microarray technology is the most representative one. As for the DNA microarray techniques, several thousands to tens of thousands of gene segments are immobilized on a glass slide at high density, and cDNA probes prepared from specific cells or tissues are hybridized on the slides from which gene expression profiles are obtained at one sweep in a short time. The present development of this technique and its possible application to medicine-related fields are described.

Cell Cycle↗
PubMed2005

Proteomic analysis of Korean ginseng (Panax ginseng C.A. Meyer).

Although many reports have been published regarding the pharmacological effects of ginseng, little is known about the biochemical pathways operant in ginsenoside biosynthesis, or the genes involved therein. Proteomics analysis is an approach to elucidate the physiological characteristics and biosynthetic pathways of ginsenosides, main components of ginseng. In this review, we introduced the recent progress in proteomics studies of ginseng (Panax ginseng C.A. Meyer). We briefly reference the genomic analyses of P. ginseng, without which proteomics approaches would have been impossible. Functional genomics studies regarding secondary metabolism in P. ginseng are also introduced here, in order to introduce possible future prospects for further study.

Databases, Genetic↗
PubMed2005

Identification of a novel group of evolutionarily conserved members within the rapidly diverging murine Cea family.

The carcinoembryonic antigen (CEA) family comprises a still actively evolving populous group of proteins that are involved in controlling tissue homeostasis, immune responses, and host/pathogen interactions. The genes identified to date in rodents and primates exhibit low sequence similarity and an extremely variable domain composition. Among the 22 murine Cea-related genes, only for Ceacam1 has an ortholog been assigned. To identify all CEA-related genes in mouse, rat, and human we undertook genome-wide analyses. Eight of 9 new expressible genes (Ceacam12-Ceacam20) could be located within the approximately 6.5-Mb murine Cea locus. Five of the genes were rodent-specific (Ceacam12-Ceacam15 and Ceacam17). Surprisingly, for the remaining 4 (Ceacam16 and Ceacam18-Ceacam20) orthologs could be detected in all three genomes at syntenic locations. Gene-specific reverse transcription/PCR analyses of total RNA from 31 murine adult, placental, and embryonic tissues as well as tumors revealed very distinct expression patterns, suggesting diversified functions within the CEA family.

Amino Acid Sequence↗
PubMed2003

OligoArray 2.0: design of oligonucleotide probes for DNA microarrays using a thermodynamic approach.

There is a substantial interest in implementing bioinformatics technologies that allow the design of oligonucleotides to support the development of microarrays made from short synthetic DNA fragments spotted or in situ synthesized on slides. Ideally, such oligonucleotides should be totally specific to their respective targets to avoid any cross-hybridization and should not form stable secondary structures that may interfere with the labeled probes during hybridization. We have developed OligoArray 2.0, a program that designs specific oligonucleotides at the genomic scale. It uses a thermodynamic approach to predict secondary structures and to calculate the specificity of targets on chips for a unique probe in a mixture of labeled probes. Furthermore, OligoArray 2.0 can adjust the oligonucleotide length, according to user input, to fit a narrow T(m) range compatible with hybridization requirements. Combined with on chip oligonucleotide synthesis, this program makes it feasible to perform expression analysis on a genomic scale for any organism for which the genome sequence is known. This is without relying on cDNA or oligonucleotide libraries. OligoArray 2.0 was used to design 75 764 oligonucleotides representing 26 140 transcripts from Arabidopsis thaliana. Among this set, we provide at least one specific oligonucleotide for 93% of these transcripts.

Algorithms↗
PubMed2004

Development of a micro-array to detect human and mouse microRNAs and characterization of expression in human organs.

MicroRNAs (miRNAs) are believed to play important roles in developmental and other cellular processes by hybridizing to complementary target mRNA transcripts. This results in either cleavage of the hybridized transcript or negative regulation of translation. Little is known about the regulation or pattern of miRNA expression. The predicted presence of numerous miRNA sequences in higher eukaryotes makes it highly likely that the expression levels of individual miRNA molecules themselves should play an important role in regulating multiple cellular processes. Therefore, determining the pattern of global miRNA expression levels in mammals and other higher eukaryotes is essential to help understand both the mechanism of miRNA transcriptional regulation as well as to help identify miRNA regulated gene expression. Here, we describe a novel method to detect global processed miRNA expression levels in higher eukaryotes, including human, mouse and rats, by using a high-density oligonucleotide array. Array results have been validated by subsequent confirmation of mir expression using northern-blot analysis. Major differences in mir expression have been detected in samples from diverse sources, suggesting highly regulated mir expression, and specific gene regulatory functions for individual miRNA transcripts. For example, five different miRNAs were found to be preferentially expressed in human kidney compared with other human tissues. Comparative analysis of surrounding genomic sequences of the kidney-specific miRNA clusters revealed the occurrence of specific transcription factor binding sites located in conserved phylogenetic foot prints, suggesting that these may be involved in regulating mir expression in kidney.

Animals↗
PubMed2000

Enterobacter cloacae sepsis outbreak in a newborn unit caused by contaminated total parenteral nutrition solution.

OBJECTIVE: The study aimed to investigate an outbreak caused by Enterobacter cloacae in a neonate intensive care unit. DESIGN: A descriptive study of an outbreak of sepsis in high-risk neonates was used. SETTING: The study was set in a tertiary care university teaching hospital. PATIENTS: The patients were 11 neonates infected with Enterobacter cloacae whose symptoms and signs of sepsis developed during a 16-hour period. All but one neonate received parenteral nutrition. Isolates from blood cultures, in-use parenteral nutrition solutions, and control aliquots of parenteral nutrition solution were typed by pulsed-field gel electrophoresis. RESULTS: Enterobacter cloacae was found in the refrigerated aliquots of parenteral nutrition solution, in blood cultures from infected newborns, and from in-use parenteral nutrition solutions. All these strains of Enterobacter cloacae had the same antibiotic susceptibility pattern and the same genomic DNA profile. The strain isolated from the one patient who did not receive parenteral nutrition presented a different susceptibility profile and genotype. CONCLUSION: The source of the nosocomial sepsis was the parenteral nutrition solution in 10 neonates. This contamination apparently occurred during preparation of the parenteral solution.

Disease Outbreaks↗
PubMed2003

Scaling up accurate phylogenetic reconstruction from gene-order data.

MOTIVATION: Phylogenetic reconstruction from gene-order data has attracted increasing attention from both biologists and computer scientists over the last few years. Methods used in reconstruction include distance-based methods (such as neighbor-joining), parsimony methods using sequence-based encodings, Bayesian approaches, and direct optimization. The latter, pioneered by Sankoff and extended by us with the software suite GRAPPA, is the most accurate approach, but cannot handle more than about 15 genomes of limited size (e.g. organelles). RESULTS: We report here on our successful efforts to scale up direct optimization through a two-step approach: the first step decomposes the dataset into smaller pieces and runs the direct optimization (GRAPPA) on the smaller pieces, while the second step builds a tree from the results obtained on the smaller pieces. We used the sophisticated disk-covering method (DCM) pioneered by Warnow and her group, suitably modified to take into account the computational limitations of GRAPPA. We find that DCM-GRAPPA scales gracefully to at least 1000 genomes of a few hundred genes each and retains surprisingly high accuracy throughout the range: in our experiments, the topological error rate rarely exceeded a few percent. Thus, reconstruction based on gene-order data can now be accomplished with high accuracy on datasets of significant size.

Algorithms↗
PubMed2005

WebArray: an online platform for microarray data analysis.

BACKGROUND: Many cutting-edge microarray analysis tools and algorithms, including commonly used limma and affy packages in Bioconductor, need sophisticated knowledge of mathematics, statistics and computer skills for implementation. Commercially available software can provide a user-friendly interface at considerable cost. To facilitate the use of these tools for microarray data analysis on an open platform we developed an online microarray data analysis platform, WebArray, for bench biologists to utilize these tools to explore data from single/dual color microarray experiments. RESULTS: The currently implemented functions were based on limma and affy package from Bioconductor, the spacings LOESS histogram (SPLOSH) method, PCA-assisted normalization method and genome mapping method. WebArray incorporates these packages and provides a user-friendly interface for accessing a wide range of key functions of limma and others, such as spot quality weight, background correction, graphical plotting, normalization, linear modeling, empirical bayes statistical analysis, false discovery rate (FDR) estimation, chromosomal mapping for genome comparison. CONCLUSION: WebArray offers a convenient platform for bench biologists to access several cutting-edge microarray data analysis tools. The website is freely available at http://bioinformatics.skcc.org/webarray/. It runs on a Linux server with Apache and MySQL.

Algorithms↗
PubMed2004

Iconix Pharmaceuticals, Inc.--removing barriers to efficient drug discovery through chemogenomics.

Iconix Pharmaceuticals, Inc. is pioneering the new field of chemogenomics--the integration of chemistry and genomics to profile, optimize and monitor clinical candidates and drug treatment. Iconix's chemogenomic capabilities enable pharmaceutical companies to increase the odds of advancing the right compounds to the clinic and managing the development of those compounds in human testing, thus reducing attrition rates and the costs of drug discovery.

Clinical Trials as Topic↗
PubMed2003

MICAS: a fully automated web server for microsatellite extraction and analysis from prokaryote and viral genomic sequences.

MICAS is a web server for extracting microsatellite information from completely sequenced prokaryote and viral genomes, or user-submitted sequences. This server provides an integrated platform for MICdb (database of prokaryote and viral microsatellites), W-SSRF (simple sequence repeat finding program) and Autoprimer (primer design software). MICAS, through dynamic HTML page generation, helps in the systematic extraction of microsatellite information from selected genomes hosted on MICdb or from user-submitted sequences. Further, it assists in the design of primers with the help of Autoprimer, for sequences containing selected microsatellite tracts.

Algorithms↗
PubMed2005

Identification of microRNAs of the herpesvirus family.

Epstein-Barr virus (EBV or HHV4), a member of the human herpesvirus (HHV) family, has recently been shown to encode microRNAs (miRNAs). In contrast to most eukaryotic miRNAs, these viral miRNAs do not have close homologs in other viral genomes or in the genome of the human host. To identify other miRNA genes in pathogenic viruses, we combined a new miRNA gene prediction method with small-RNA cloning from several virus-infected cell types. We cloned ten miRNAs in the Kaposi sarcoma-associated virus (KSHV or HHV8), nine miRNAs in the mouse gammaherpesvirus 68 (MHV68) and nine miRNAs in the human cytomegalovirus (HCMV or HHV5). These miRNA genes are expressed individually or in clusters from either polymerase (pol) II or pol III promoters, and share no substantial sequence homology with one another or with the known human miRNAs. Generally, we predicted miRNAs in several large DNA viruses, and we could neither predict nor experimentally identify miRNAs in the genomes of small RNA viruses or retroviruses.

Chromosome Mapping↗
PubMed2003

Sequence-based cancer genomics: progress, lessons and opportunities.

Technologies that provide a genome-wide view offer an unprecedented opportunity to scrutinize the molecular biology of the cancer cell. The information that is derived from these technologies is well suited to the development of public databases of alterations in the cancer genome and its expression. Here, we describe the synergistic efforts of research programmes in Brazil, the United Kingdom and the United States towards building integrated databases that are widely accessible to the research community, to enable basic and applied applications in cancer research.

Brazil↗
PubMed2003

A comparative analysis of HGSC and Celera human genome assemblies and gene sets.

MOTIVATION: Since the simultaneous publication of the human genome assembly by the International Human Genome Sequencing Consortium (HGSC) and Celera Genomics, several comparisons have been made of various aspects of these two assemblies. In this work, we set out to provide a more comprehensive comparative analysis of the two assemblies and their associated gene sets. RESULTS: The local sequence content for both draft genome assemblies has been similar since the early releases, however it took a year for the quality of the Celera assembly to approach that of HGSC, suggesting an advantage of HGSC's hierarchical shotgun (HS) sequencing strategy over Celera's whole genome shotgun (WGS) approach. While similar numbers of ab initio predicted genes can be derived from both assemblies, Celera's Otto approach consistently generated larger, more varied gene sets than the Ensembl gene build system. The presence of a non-overlapping gene set has persisted with successive data releases from both groups. Since most of the unique genes from either genome assembly could be mapped back to the other assembly, we conclude that the gene set discrepancies do not reflect differences in local sequence content but rather in the assemblies and especially the different gene-prediction methodologies.

Databases, Protein↗