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GeneExpress: a computer system for description, analysis, and recognition of regulatory sequences in eukaryotic genome.

GeneExpress system has been designed to integrate description, analysis, and recognition of eukaryotic regulatory sequences. The system includes 5 basic units: (1) GeneNet contains an object-oriented database for accumulation of data on gene networks and signal transduction pathways and a Java-based viewer that allows an exploration and visualization of the GeneNet information; (2) Transcription Regulation combines the database on transcription regulatory regions of eukaryotic genes (TRRD) and TRRD Viewer; (3) Transcription Factor Binding Site Recognition contains a compilation of transcription factor binding sites (TFBSC) and programs for their analysis and recognition; (4) mRNA Translation is designed for analysis of structural and contextual features of mRNA 5'UTRs and prediction of their translation efficiency; and (5) ACTIVITY is the module for analysis and site activity prediction of a given nucleotide sequence. Integration of the databases in the GeneExpress is based on the Sequence Retrieval System (SRS) created in the European Bioinformatics Institute.

Artificial Intelligence↗

The effects of nuclear DNA content (C-value) on the quality and utility of AFLP fingerprints.

BACKGROUND AND AIMS: Nuclear DNA content (C-value) varies approximately 1000-fold across the angiosperms, and this variation has been reported to have an effect on the quality of AFLP fingerprints. Various methods have been proposed for circumventing the problems associated with small and large genomes. Here we investigate the range of nuclear DNA contents across which the standard AFLP protocol can be used. METHODS: AFLP fingerprinting was conducted on an automated platform using the standard protocol (with 3 + 3 selective bases) in which DNA fragments are visualized as bands. Species with nuclear DNA contents ranging from 1C = 0.2 to 32.35 pg were included, and the total number of bands and the number of polymorphic bands were counted. For the species with the smallest C-value (Bixa orellana) and for one of the species with a large C-value (Damasonium alisma), alternative protocols using 2 + 3 and 3 + 4 selective bases, respectively, were also used. KEY RESULTS: Acceptable AFLP traces were obtained using the standard protocol with 1C-values of 0.30-8.43 pg. Below this range, the quality was improved by using 2 + 3 selective bases. Above this range, the traces were generally characterized by a few strongly amplifying bands and noisy baselines. Damasonium alisma, however, gave more even traces, probably due to it being a tetraploid. CONCLUSIONS: We propose that for known polyploids, genome size is a more useful indicator than the 1C-value in deciding which AFLP protocol to use. Thus, knowledge of ploidy (allowing estimation of genome size) and C-value are both important. For small genomes, the number of interpretable bands can be increased by decreasing the number of selective bases. For larger genomes, increasing the number of bases does not necessarily decrease the number of bands as predicted. The presence of a small number of strongly amplifying bands is likely to be linked to the presence of repetitive DNA sequences in high copy number in taxa with large genomes.

Cell Nucleus↗

Novel de novo mutation in a patient with Best macular dystrophy.

OBJECTIVE: To report a novel de novo vitelliform macular dystrophy (VMD2) mutation in a patient with Best macular dystrophy. METHODS: Best-corrected visual acuity, dilated fundus examination, and electro-oculography were performed in a patient with Best macular dystrophy and his parents. Both the patient and his parents also had blood samples drawn, and their DNA was analyzed by direct genomic sequencing. RESULTS: A heterozygous VMD2 gene missense mutation in exon 2 (Thr6Ala [ACA>GCA]) was identified in the proband. This mutation was not present in his clinically unaffected parents. CONCLUSIONS: A novel de novo mutation in the VMD2 gene was found in a patient whose phenotype and electro-oculographic findings were characteristic of Best macular dystrophy, whereas both parents were phenotypically and genetically unaffected. The findings in this family document that a de novo mutation needs to be considered when an isolated family member is found to have a Best disease phenotype.

Bestrophins↗

TICO: a tool for postprocessing the predictions of prokaryotic translation initiation sites.

Exact localization of the translation initiation sites (TIS) in prokaryotic genomes is difficult to achieve using conventional gene finders. We recently introduced the program TICO for postprocessing TIS predictions based on a completely unsupervised learning algorithm. The program can be utilized through our web interface at http://tico.gobics.de/ and it is also freely available as a commandline version for Linux and Windows. The latest version of our program provides a tool for visualization of the resulting TIS model. Although the underlying method is not based on any specific assumptions about characteristic sequence features of prokaryotic TIS the prediction rates of our tool are competitive on experimentally verified test data.

Algorithms↗

Limitations of in situ hybridization with total genomic DNA in routine screening for alien introgressions in wheat.

In situ hybridization with total genomic DNA (GISH) has become a powerful tool in characterization of alien introgressions in wheat. With recent simplification it can now be used in large scale screening for new chromosome constructs. Its level of resolution in routine applications was tested on sets of recombined wheat-rye chromosomes with genetically determined positions of the translocation breakpoints. The resolution level of GISH visualized by an enzymatic color reaction was much lower than that of GISH with fluorescent probes but both techniques failed to reveal the presence of some distally located breakpoints. The limits of resolution for the two methods were at least 9.8 and 3.5 cM of the relative genetic lengths of chromosome arms, respectively, in configurations with proximal rye and terminal wheat segments when rye DNA was used as a probe. When wheat DNA was used as a probe, a terminal wheat segment estimated to be ca. 1.6 cM in length could not be visualized. An example of induced recombination between a chromosome of Agropyron elongatum and wheat illustrates that these resolution limits of GISH may hamper isolation of critical translocation breakpoints in a chromosome engineering effort.

Chromosome Mapping↗

Identification and characterization of Lactobacillus helveticus PepO2, an endopeptidase with post-proline specificity.

A post-proline endopeptidase (PepO2) was detected in cell extracts from a genomic library of Lactobacillus helveticus CNRZ32 by using the synthetic substrate N-acetyl-beta-casein-(f203-209)-rho-nitroanilide in a coupled reaction with aminopeptidase N. Isolates with activity for this substrate contained plasmids with visually indistinguishable restriction profiles. Nucleotide sequence analysis revealed a 1,947-bp open reading frame, designated pepO2, encoding a putative 71.4-kDa protein. Analysis of the predicted peptide sequence revealed that L. helveticus PepO2 contained the zinc-dependent metalloprotease motif HEXXH and exhibited levels of amino acid sequence similarity of 72, 61, 59, and 53% to L. helveticus PepO, Lactococcus lactis PepO2, L. lactis PepO, and Lactobacillus rhamnosus PepO, respectively. Northern hybridization results indicated that the transcript containing pepO2 was monocistronic. Despite the high degrees of amino acid similarity to PepO proteins from other lactic acid bacteria, the specificity of the L. helveticus PepO2 for post-proline bonds distinguishes it from other PepO-type endopeptidases characterized to date. The specificity for post-proline bonds also suggests that this enzyme may play a central role in the hydrolysis of casein-derived bitter peptides, such as beta-casein(f193-209).

Amino Acid Sequence↗

RiceGAAS: an automated annotation system and database for rice genome sequence.

An extensive effort of the International Rice Genome Sequencing Project (IRGSP) has resulted in rapid accumulation of genome sequence, and >137 Mb has already been made available to the public domain as of August 2001. This requires a high-throughput annotation scheme to extract biologically useful and timely information from the sequence data on a regular basis. A new automated annotation system and database called Rice Genome Automated Annotation System (RiceGAAS) has been developed to execute a reliable and up-to-date analysis of the genome sequence as well as to store and retrieve the results of annotation. The system has the following functional features: (i) collection of rice genome sequences from GenBank; (ii) execution of gene prediction and homology search programs; (iii) integration of results from various analyses and automatic interpretation of coding regions; (iv) re-execution of analysis, integration and automatic interpretation with the latest entries in reference databases; (v) integrated visualization of the stored data using web-based graphical view. RiceGAAS also has a data submission mechanism that allows public users to perform fully automated annotation of their own sequences. The system can be accessed at http://RiceGAAS.dna.affrc.go.jp/.

Automation↗

Individualized nutritional recommendations: do we have the measurements needed to assess risk and make dietary recommendations?

Is the information currently available to adjust nutritional recommendations and develop individualized nutrition? No. There is not even the information needed for setting dietary recommendations with confidence now at the group level. Will it be available soon? The answer to this question depends on the drive and will of the nutritional community, the success in recruiting funding to the area, the education of nutritionists and the spawning of great ideas and approaches. The emerging tools of genomics, proteomics and metabolomics are enabling the in-depth study of relationships between diet, genetics and metabolism. The advent of technologies can be compared with the discovery of the microscope and the new dimensions of scientific visualization enabled by that discovery. Nutritionists stand at the crest of new waves of data that can be generated, and new methods for their digestion will be required. To date, the study of dietary requirements has been based largely on a black box approach. Subjects are supplemented or depleted and clinical outcomes are observed. Few recommendations are based on metabolic outcomes. Metabolomics and nutrigenomics promise tools with which recommendations can be refined to meet individual requirements and the potential of individualized nutrition can be explored. As yet, these tools are not being widely applied in nutritional research and are rarely being applied by nutritionists. The result is often interesting research that is frequently nutritionally flawed, resulting in inappropriate conclusions. Nutritional education is needed to put nutritionists at the forefront of the development of applications for these technologies, creating a generation of nutrigenomicists. A new generation of nutritionists should be working interdisciplinarily with geneticists, molecular biologists and bioinformaticians in the development of research strategies. The present paper reviews the current status of nutrigenomic research, the current controversies and limitations, and developments needed to advance nutrigenomics and explore fully the promise of individualized nutritional recommendations.

Diet↗

JVirGel: Calculation of virtual two-dimensional protein gels.

We developed JVirGel, a collection of tools for the simulation and analysis of proteomics data. The software creates and visualizes virtual two-dimensional (2D) protein gels based on the migration behaviour of proteins in dependence of their theoretical molecular weights in combination with their calculated isoelectric points. The utilization of all proteins of an organism of interest deduced from genes of the corresponding genome project in combination with the elimination of obvious membrane proteins permits the creation of an optimized calculated proteome map. The electrophoretic separation behaviour of single proteins is accessible interactively in a Java(TM) applet (small application in a web browser) by selecting a pI/MW range and an electrophoretic timescale of interest. The calculated pattern of protein spots helps to identify unknown proteins and to localize known proteins during experimental proteomics approaches. Differences between the experimentally observed and the calculated migration behaviour of certain proteins provide first indications for potential protein modification events. When possible, the protein spots are directly linked via a mouse click to the public databases SWISS-PROT and PRODORIC. Additionally, we provide tools for the serial calculation and visualization of specific protein properties like pH dependent charge curves and hydrophobicity profiles. These values are helpful for the rational establishment of protein purification procedures. The proteomics tools are available on the World Wide Web at http://prodoric.tu-bs.de/proteomics.php.

Computer Graphics↗

Detection and subtyping of swine influenza H1N1, H1N2 and H3N2 viruses in clinical samples using two multiplex RT-PCR assays.

A total of 360 type A swine influenza virus-positive samples including cell culture isolates, nasal swabs or lung tissues along with 30 virus-negative samples were tested for the detection and subtyping of H1N1, H1N2 or H3N2 by two multiplex reverse transcription (RT)-PCR assays. The positive samples had been collected between 1999 and 2001 from pigs with respiratory diseases, and type A influenza virus was isolated and subtyped by hemagglutination inhibition (HI) test at the Minnesota Veterinary Diagnostic Laboratory (MVDL). Two multiplex RT-PCR assays specific for H1 and H3, and N1 and N2 were developed. RT-PCR products with unique sizes characteristic of each subtype of influenza A virus were sequenced, and the sequences were demonstrated to be specific for H1N1, H1N2 or H3N2. Genomic RNAs or DNAs from 12 common swine pathogens other than type A influenza viruses were not amplified when the PCR assays were performed with these primer sets. Positive amplification reaction could be visualized with RNA extracted from up to 10(-5) dilution of each reference virus with original infectivity titer of 10(5) TCID(50)/ml. Of the 360 samples tested, swine influenza virus H1N1, H1N2 and H3N2 were identified in 200, 13 and 139 samples, respectively. The remaining eight samples were positive for both H1N1 and H3N2 viruses. The results of multiplex RT-PCR were 100% in agreement with those of virus isolation. These results demonstrate the usefulness of multiplex RT-PCR for detection and identification of influenza A virus subtypes. The results also indicate an increased occurrence of H1N2 in US swine population.

Animals↗

The neuropeptide Y (Y4) receptor is highly expressed in neurones of the rat dorsal vagal complex.

Recently, the cDNA encoding the Y4 neuropeptide Y (NPY) receptor cDNA was cloned from a rat genomic library. The Y4 receptor is characterized by having a high affinity for pancreatic polypeptide (PP) and peptide YY (PYY). By using in situ hybridization histochemistry with 35S-labelled riboprobes, we have visualized the cellular expression of mRNA encoding the Y4 receptor protein in the rat dorsal vagal complex at the light microscopical level. High densities of silver grains were observed over neurones of the dorsal vagal motor nucleus, and over neurones of a subregion of the nucleus of the solitary tract known as the subnucleus gelatinosus. Furthermore, cells within the ventral margin of the area postrema expressed high levels of Y4 mRNA. These observations indicate that circulating PP and/or NPY/PYY via the blood-brain barrier-free area postrema and subpostremal area could influence neurones of the dorsal vagal complex with profound influence on numerous homeostatic mechanisms governed by this nuclear complex.

Animals↗

How to describe protein motion without amino acid sequence and atomic coordinates.

This paper reports a computational method, the quantized elastic deformational model, that can reliably describe the conformational flexibility of a protein in the absence of the amino acid sequence and atomic coordinates. The essence of this method lies in the fact that, in modeling the functionally important conformational changes such as domain movements, it is possible to abandon the traditional concepts of protein structure (bonds, angles, dihedrals, etc.) and treat the protein as an elastic object. The shape and mass distribution of the object are described by the electron density maps, at various resolutions, from methods such as x-ray diffraction or cryo-electron microscopy. The amplitudes and directionality of the elastic deformational modes of a protein, whose patterns match the biologically relevant conformational changes, can then be derived solely based on the electron density map. The method yields an accurate description of protein dynamics over a wide range of resolutions even as low as 15-20 A at which there is nearly no visually distinguishable internal structures. Therefore, this method dramatically enhances the capability of studying protein motions in structural biology. It is also expected to have ample applications in related fields such as bioinformatics, structural genomics, and proteomics, in which one's ability to extract functional information from the not-so-well-defined structural models is vitally important.

Amino Acid Sequence↗

Detection of specific DNA sequences by fluorescence amplification: a color complementation assay.

We have developed a color complementation assay that allows rapid screening of specific genomic DNA sequences. It is based on the simultaneous amplification of two or more DNA segments with fluorescent oligonucleotide primers such that the generation of a color, or combination of colors, can be visualized and used for diagnosis. Color complementation assay obviates the need for gel electrophoresis and has been applied to the detection of a large and small gene deletion, a chromosomal translocation, an infectious agent, and a single-base substitution. DNA amplification with fluorescent oligonucleotide primers has also been used to multiplex and discriminate five different amplified DNA loci simultaneously. Each primer set is conjugated to a different dye, and the fluorescence of each dye respective to its amplified DNA locus is scored on a fluorometer. This method is valuable for DNA diagnostics of genetic, acquired, and infectious diseases, as well as in DNA forensics. It also lends itself to complete automation.

Base Sequence↗

Twenty Drosophila visual system cDNA clones: one is a homolog of human arrestin.

From a group of 436 Drosophila melanogaster cDNA clones, we selected 39 that are expressed exclusively or predominantly in the adult visual system. By sequence analysis, 20 of the clones appear to represent previously unreported distinct cDNAs. The corresponding transcripts are detected in the retina and optic lobes. The genes are scattered throughout the genome, some near mutations known to affect eye function. One of these clones has been identified, by sequence analysis, as the structural gene (Arr) for a Drosophila homolog of human arrestin. Vertebrate arrestin interacts with rhodopsin in phototransduction and has been associated with an autoimmune form of uveitis in primates. The presence of an arrestin homolog in Drosophila suggests that both the vertebrate and invertebrate phototransduction cascades are regulated in a similar manner.

Amino Acid Sequence↗

DiffTool: building, visualizing and querying protein clusters.

UNLABELLED: DiffTool is a resource to build and visualize protein clusters computed from a sequence database. The package provides a clustering tool to construct protein families according to sequence similarities and a web interface to query the corresponding clusters. A subtractive genome analysis tool selects protein families specific for a genome or a group of genomes. For each protein cluster, DiffTool includes access to sequences, coloured multiple alignments and phylogenetic trees. AVAILABILITY: A cluster database built from yeast and complete prokaryotic genomes is queryable at http://bioweb.pasteur.fr/seqanal/difftool. All the Perl sources are freely available to non-profit organizations upon request.

Cluster Analysis↗

Genetic and ultrastructural characterization of a European isolate of the fatal endotheliotropic elephant herpesvirus.

A male Asian elephant (Elephas maximus) died at the Berlin zoological gardens in August 1998 of systemic infection with the novel endotheliotropic elephant herpesvirus (ElHV-1). This virus causes a fatal haemorrhagic disease in Asian elephants, the so-called endothelial inclusion body disease, as reported from North American zoological gardens. In the present work, ElHV-1 was visualized ultrastructurally in affected organ material. Furthermore, a gene block comprising the complete glycoprotein B (gB) and DNA polymerase (DPOL) genes as well as two partial genes was amplified by PCR-based genome walking and sequenced. The gene content and arrangement were similar to those of members of the Betaherpesvirinae. However, phylogenetic analysis with gB and DPOL consistently revealed a very distant relationship to the betaherpesviruses. Therefore, ElHV-1 may be a member of a new genus or even a new herpesvirus subfamily. The sequence information generated was used to set up a nested-PCR assay for diagnosis of suspected cases of endothelial inclusion body disease. Furthermore, it will aid in the development of antibody-based detection methods and of vaccination strategies against this fatal herpesvirus infection in the endangered Asian elephant.

Animals↗

Assessment of genetic variability of haploids extracted from tetraploid (2n = 4x = 48) Solanum tuberosum.

The objectives of this study were to assess the genetic variability of haploids (2n = 2x = 24) extracted from tetraploid Solanum tuberosum through 4x x 2x crosses with Solanum phureja. Molecular and phenotypic analyses were performed to fingerprint the genotypes used and to evaluate their potential use in breeding programs. AFLP analysis revealed the presence of specific bands derived from the tetraploid seed parent S. phureja, as well as ex novo originated bands. On average, 210 bands were visualized per genotype, 149 (70%) of which were common to both parental genotypes. The percentage of S. tuberosum specific bands ranged from 25.1% to 18.6%, with an average of 22%. The fraction of genome coming from S. phureja ranged from 1.9% to 6.5%, with an average value of 4%. The percentage of ex novo bands varied from 1.9% to 9.0%. The presence of S. phureja DNA is very interesting because it indicated that S. phureja pollinator is involved in the mechanism of haploid formation. The characterization for resistance to Erwinia carotovora subsp. carotovora and potato virus X (PVX) provided evidence that haploids may express traits that are lacking in the tetraploids they come from, which can be useful for both genetic studies and breeding purposes. It is noteworthy that genotypes combining resistance to both diseases and good pollen stainability were identified. Other possible breeding implications owing to the presence of S. phureja genome in the haploids analyzed are discussed.

Breeding↗

Haplotype tagging single nucleotide polymorphisms and association studies.

OBJECTIVES: Discrete blocks of low haplotype diversity exist within the human genome. The non-redundant subset of 'haplotype tagging' single nucleotide polymorphisms (htSNPs) in such blocks can distinguish a majority of the haplotypes. Several approaches have been proposed to determine htSNPs, ranging from visual inspection to formal analytic procedures. Optimal htSNPs can be estimated using a small subgroup of an association study population that have been genotyped for a dense SNP map, and it is just these htSNPs that are genotyped in the remainder of the samples. We investigated by simulation how the size of the subsample affects the power of association studies, and what type of subjects it should include. METHODS: We used the program tagSNPs [Stram et al., Hum Hered 2003;55:27-36], which selects htSNPs to minimize the uncertainty in predicting common haplotypes for individuals with unphased genotype data. RESULTS: On average, 27% of the SNPs were designated as htSNPs. Genotyping as few as 25 unphased individuals to select the htSNPs did not appear to reduce the power of an association study, as compared with using all SNPs. For the disease models considered, selecting htSNPs based on cases, controls, or a mixture of both gave similar results. CONCLUSIONS: These results suggest that the genotyping effort in an association study can be substantially reduced with little loss of power by identifying htSNPs in a small subsample of individuals.

Chromosome Mapping↗