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At least 1,081 records · Page 60Linked to original sources
PubMed2005

Phylogenetic shadowing of a histamine-gated chloride channel involved in insect vision.

A recently identified gene, hclA (synonym: ort), codes for an ionotrophic histamine receptor subunit in Drosophila melanogaster, and known hclA mutations lead to defects in the visual system, neurologic disorders and changed responsiveness to neurotoxins. To investigate whether this novel class of receptors is common across the Insecta, we analysed the genomes of 15 other insect species (Diptera, Hymenoptera, Coleoptera, Lepidoptera) and revealed orthologs of hclA in all of them. The predicted receptor domain of HCLA is extensively conserved (86-100% of identity) among the 16 proteins. Minor changes in the amino acid sequence that includes the putative transmembrane domains (TMs) 1-3 were found in non-drosophilid species only. Substantial amino acid variability was observed in the signal polypeptides, the intracellular loop domains and in TM4, in good accordance with known data on sequence variations in ligand-gated ion channels. Pairwise comparisons revealed three consensus sequences for N-glycosylation, conserved in HCLAs of all species studied, as well as a drosophilid-specific putative phosphorylation site. Real-time PCR analysis demonstrated that hclA-mRNA is abundant in heads of adult Drosophila. However, species- and sex-specific variations of the hclA expression levels were also observed.

Amino Acid Sequence↗
PubMed1999

Analysis of gene expression data using self-organizing maps.

DNA microarray technologies together with rapidly increasing genomic sequence information is leading to an explosion in available gene expression data. Currently there is a great need for efficient methods to analyze and visualize these massive data sets. A self-organizing map (SOM) is an unsupervised neural network learning algorithm which has been successfully used for the analysis and organization of large data files. We have here applied the SOM algorithm to analyze published data of yeast gene expression and show that SOM is an excellent tool for the analysis and visualization of gene expression profiles.

Algorithms↗
PubMed2002

Different structural organization of the encephalopsin gene in man and mouse.

Encephalopsin, also called Panopsin, is a recently discovered extraretinal photoreceptor, which may play a role in non-visual photic processes such as the entrainment of circadian rhythm or the regulation of pineal melatonin production. Based on RT-PCR data and comparative genomic sequence analysis, we show that the human OPN3 gene consists of six exons and expresses various splice variants, while the murine homologue contains four exons and produces just one splice form. Furthermore, the human OPN3 gene overlaps with the neighboring KMO gene on a genomic as well as on an RNA level, whereas the corresponding genes in mouse lie close together but do not overlap. This finding is of particular interest, since differences in gene organization between man and mouse, that have been reported so far, occur within gene clusters, i.e. the number of genes within a certain cluster may differ between man and mouse. OPN3 provides an exception to this rule, since it is positionally uncoupled from other genes of the opsin family.

3' Flanking Region↗
PubMed2026

Multiplexed RT-LAMP Assays in Handheld Devices for In-Situ Detection of Chikungunya, Dengue, Mayaro, and Zika Viruses.

Mosquito-borne viruses pose a significant global health challenge, particularly in resource-limited settings where multiple viruses often cause illnesses with similar symptoms that require different treatment. We introduce the first 7-plex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in a hand-held device capable of detecting the presence of Chikungunya virus (CHIKV), dengue virus serotypes (DENV 1-4), Mayaro virus (MAYV), and Zika virus (ZIKV) in a single test. If the result is positive from the single-plex device for the 7-plex assay, 3-plex and 4-plex devices are then used to identify the exact virus within a specimen. In-situ detection is achieved by integrating valve-enabled, paper-based sample preparation with fluorescence detection using a blue LED flashlight as a light source and a yellow plastic film as a filter, allowing visual discrimination between positive and negative samples by the naked eye or by recording images using a smart phone. The detection limits ranged from 2 genome equivalents (GE)/reaction (for ZIKV) to 92 GE/reaction (for DENV-3) across 7 types of viruses when 1 μL of viral RNA was used. We observed 90% overall agreement between the point-of-care (POC) device and lab-based reverse transcription polymerase chain reactions (RT-PCR) when blinded clinical specimens were tested. This assay and device have a potential to address critical surveillance gaps in endemic regions, enabling timely detection of multiple mosquito-borne viruses to guide appropriate clinical management and public health countermeasures in settings where laboratory resources are scarce.

Nucleic Acid Amplification Techniques↗
PubMed2005

Repetitive shuttling of a motor protein on DNA.

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.

Adenosine Triphosphate↗
PubMed2005

Conservative extraction of over-represented extensible motifs.

MOTIVATION: The discovery of motifs in biosequences is frequently torn between the rigidity of the model on the one hand and the abundance of candidates on the other. In particular, the variety of motifs described by strings that include 'don't care' (dot) patterns escalates exponentially with the length of the motif, and this gets only worse if a dot is allowed to stretch up to some prescribed maximum length. This circumstance tends to generate daunting computational burdens, and often gives rise to tables that are impossible to visualize and digest. This is unfortunate, as it seems to preclude precisely those massive analyses that have become conceivable with the increasing availability of massive genomic and protein data. Although a part of the problem is endemic, another part of it seems rooted in the various characterizations offered for the notion of a motif, that are typically based either on syntax or on statistics alone. It seems worthwhile to consider alternatives that result from a prudent combination of these two aspects in the model. RESULTS: We introduce and study a notion of extensible motif in a sequence which tightly combines the structure of the motif pattern, as described by its syntactic specification, with the statistical measure of its occurrence count. We show that a combination of appropriate saturation conditions (expressed in terms of minimum number of dots compatible with a given list of occurrences) and the monotonicity of probabilistic scores over regions of constant frequency afford us significant parsimony in the generation and testing of candidate over-represented motifs. The merits of the method are documented by the results obtained in implementation, which specifically targeted protein sequence families. In all cases tested, the motif reported in PROSITE as the most important in terms of functional/structural relevance emerges among the top 30 extensible motifs returned by our algorithm, often right at the top. Of equal importance seems the fact that the sets of all surprising motifs returned in each experiment are extracted faster and come in much more manageable sizes than would be obtained in the absence of saturation constrains. AVAILABILITY: This software will be available for use with the suite of tools at www.research.ibm.com/bioinformatics.

Algorithms↗
PubMed2003

Molecular biology of myopia.

Experiments in animal models of myopia have emphasised the importance of visual input in emmetropisation but it is also evident that the development of human myopia is influenced to some degree by genetic factors. Molecular genetic approaches can help to identify both the genes involved in the control of ocular development and the potential targets for pharmacological intervention. This review covers a variety of techniques that are being used to study the molecular biology of myopia. In the first part, we describe techniques used to analyse visually induced changes in gene expression: Northern Blot, polymerase chain reaction (PCR) and real-time PCR to obtain semi-quantitative and quantitative measures of changes in transcription level of a known gene, differential display reverse transcription PCR (DD-RT-PCR) to search for new genes that are controlled by visual input, rapid amplification of 5' cDNA (5'-RACE) to extend the 5' end of sequences that are regulated by visual input, in situ hybridisation to localise the expression of a given gene in a tissue and oligonucleotide microarray assays to simultaneously test visually induced changes in thousands of transcripts in single experiments. In the second part, we describe techniques that are used to localise regions in the genome that contain genes that are involved in the control of eye growth and refractive errors in mice and humans. These include quantitative trait loci (QTL) mapping, exploiting experimental test crosses of mice and transmission disequilibrium tests (TDT) in humans to find chromosomal intervals that harbour genes involved in myopia development. We review several successful applications of this battery of techniques in myopia research.

Animals↗
PubMed1993

Detection of hepatitis C virus RNA in the liver by in situ hybridization.

To examine HCV infection histologically, we attempted non-radioactive in situ hybridization of HCV-RNA in the liver. We amplified cDNA probe (360 base pairs) by PCR using the primers deduced from the core region of the HCV genome. The probe was labelled with digoxigenin by PCR and used for in situ hybridization on paraformaldehyde-fixed frozen liver sections. The hybrids were visualized immunohistochemically with alkaline-phosphatase-conjugated anti-digoxigenin and alkaline-phosphatase substrates. HCV-RNA-cDNA hybrids were detected in 21 of 24 patients with positive serum HCV markers, whereas there were no positive signals in the liver of 12 cases without HCV infection. The signal intensity of HCV-RNA-cDNA hybrids was abolished after RNase treatment. Various other specificity experiments also verified specific hybridization of HCV-RNA-cDNA. HCV-RNA was visualized in liver cells and most of them were regarded as hepatocytes from their characteristic features. The infected hepatocytes were frequently associated with mononuclear cell infiltration. Hepatocytes positive for HCV-RNA were sometimes binuclear and distributed in various patterns among cases tested. The present in situ hybridization of HCV RNA is highly sensitive and specific and the results suggest the host immune response to HCV-infected cells.

Hepacivirus↗
PubMed2006

Laser microdissection of plant tissue: what you see is what you get.

Laser microdissection (LM) utilizes a cutting or harvesting laser to isolate specific cells from histological sections; the process is guided by microscopy. This provides a means of removing selected cells from complex tissues, based only on their identification by microscopic appearance, location, or staining properties (e.g., immunohistochemistry, reporter gene expression, etc.). Cells isolated by LM can be a source of cell-specific DNA, RNA, protein or metabolites for subsequent evaluation of DNA modifications, transcript/protein/metabolite profiling, or other cell-specific properties that would be averaged with those of neighboring cell types during analysis of undissected complex tissues. Plants are particularly amenable to the application of LM; the highly regular tissue organization and stable cell walls of plants facilitate the visual identification of most cell types even in unstained tissue sections. Plant cells isolated by LM have been the starting point for a variety of genomic and metabolite studies of specific cell types.

Chromosomes, Plant↗
PubMed1994

Increased FISH efficiency using APC probes generated by direct incorporation of labeled nucleotides by PCR.

Probes of various sizes from the adenomatous polyposis coli gene (APC) were directly biotinylated by polymerase chain reaction (PCR) from genomic DNA. PCR labeling gave high efficiency in detection of fluorescence in situ hybridization (FISH) signals. Probes as small as 250 base pairs could be visualized through a fluorescence microscope without any image processing.

Adenomatous Polyposis Coli↗
PubMed1995

Chromosome painting with human chromosome-specific DNA libraries reveals the extent and distribution of conserved segments in bovine chromosomes.

Commercially available human chromosome-specific DNA libraries covering the whole karyotype were hybridized to normal bovine metaphase spreads to characterize the conserved chromosomal segments between man and cattle. All chromosome libraries except the Y chromosome library displayed a signal on at least part of one or more bovine chromosomes. The labeling was clearly visualized and permitted precise delineation of the hybridized bovine chromosomal segments. This study indicates that the reorganization of the genetic material between human and bovine genomes is not as great as expected from classical comparative cytogenetics based on banding patterns. However, apart from interchromosomal rearrangements between ancestral forms of human and bovine chromosomes, a majority of intrachromosomal rearrangements must have occurred in these species during evolution to explain the differences in the banding patterns of their chromosomes. These results show that chromosome painting with heterologous chromosome-specific DNA libraries can provide useful information in comparative studies on karyotypes and gene maps of distantly related mammalian species. The observations are discussed in relation to published data on gene mapping in man and cattle.

Animals↗
PubMed2005

Genetic analysis of early endocrine pancreas formation in zebrafish.

Endocrine pancreas of zebrafish consist of at least four different cell types that function similarly to mammalian pancreatic islet. No mutants specifically affecting formation of the endocrine pancreas have been identified during the previous large-scale mutagenesis screens in zebrafish due to invisibility of a pancreatic islet. We combined in situ hybridization method to visualize pancreatic islet with an ethyl-nitroso-urea mutagenesis screen to identify novel genes involved in pancreatic islet formation in zebrafish. We screened 900 genomes and identified 11 mutations belonging to nine different complementation groups. These mutants fall into three major phenotypic classes displaying severely reduced insulin expression, reduced insulin expression with abnormal islet morphology, or abnormal islet morphology with relatively normal number of insulin expressing cells. Seven of these mutants do not have any other visible phenotypes associated. These mutations affect different processes in pancreatic islet development. Additional analysis on glucagon and somatostatin cell specification revealed that somatostatin cells are specified at a separate domain from insulin cells whereas glucagon cells are specified adjacent to insulin cells. Furthermore, glucagon cells and somatostatin cells are always associated with insulin cells in mutants that have scattered insulin expression. These data indicate that there are separate mechanisms regulating endocrine cell migration, proliferation, and differentiation. Further study on these mutants will reveal important information on novel genes involved in pancreatic islet cell specification and morphogenesis.

Animals↗
PubMed2002

Putative histamine-gated chloride channel subunits of the insect visual system and thoracic ganglion.

Histamine-gated chloride channels, members of the ligand-gated ion channel superfamily, are thought to be peculiar for arthropods. Their cognate ligand, histamine, is the transmitter of all arthropod photoreceptors and of thoracic mechanoreceptors. To identify putative histamine-gated chloride channel subunits we scanned the Drosophila genome for putative ligand-gated chloride channel subunits and found 12 candidate genes. We found four groups of transcripts based on their expression pattern. Only members of the last group show an expression pattern that is consistent with our knowledge about histamine-gated chloride channels in insects. In the brain these transcripts (Dm HA-Cl I and II) are exclusively present in interneurones postsynaptic to photoreceptors. Within the lamina (the first visual ganglion) only the L1-L3 neurones are labelled. The lack of non-photoreceptor dependent staining in the brain indicates that mechanosensory transmission differs between the head and the thorax/abdomen, and that the receptors responding to brain-intrinsic histaminergic cells use different signalling pathways. The putative histamine-gated chloride channels show the greatest homology mammalian glycine receptors. These ion-channels are the first specific molecular markers for postsynaptic cells in the insect visual system, thus representing ideal tools to study its physiology and development.

Animals↗
PubMed1999

Heritability of event-related brain potentials in families with a history of alcoholism.

Event-related brain potentials (ERPs) are altered in patients with a variety of psychiatric disorders and may represent quantitative correlates of disease liability that are more amenable to genetic analysis than disease status itself. Estimates of heritability are presented for amplitude and latency of the N1 and P3 components of the ERP measured at 19 scalp locations in response to visual and auditory stimuli for 604 individuals in 100 pedigrees ascertained as part of the Collaborative Study on the Genetics of Alcoholism. Significant heritabilities were found for visual P3 amplitude in response to all stimuli and for visual P3 latency in response to target and novel, but not non-target, stimuli. Heritability of visual N1 latencies was uniformly low, whereas heritability of visual N1 amplitude was significant for all electrodes in response to the non-target stimuli but only for posterior electrodes in the other two stimulus conditions. Heritabilities for auditory target P3 were similar to those of the visual stimuli, with auditory target P3 amplitudes and latencies both demonstrating significant heritability. For auditory P2 in response to non-target stimuli, peak amplitude was heritable, but latency was not. Auditory N1 amplitude and latency were significantly heritable for both target and non-target conditions and did not demonstrate the anterior/posterior patterning obtained for visual N1 amplitude. This study represents the first systematic assessment of heritability of these potential neurophysiological markers in families with a history of alcoholism and suggests that many of these ERP phenotypes have heritabilities strong enough to justify genomic screening for loci jointly influencing ERP abnormalities and liability to alcoholism.

Adolescent↗
PubMed2007

Rescue of the albino phenotype by introducing a functional tyrosinase minigene into Kunming albino mice.

AIM: To use the tyrosinase minigene as a visual marker to perform microinjection training and improve the techniques related with transgene to greatly elevate the efficiency of gene transfer. METHODS: A mouse tyrosinase minigene, i.e., TyBS, in which the 2.25-kb authentic genomic 5' non-coding flanking sequence of mouse tyrosinase was fused to a mouse tyrosinase cDNA, was introduced into the fertilized eggs of outbred Kunming albino mice. RESULTS: Of the 11 animals that developed from the injected eggs, two mice (P1 and #8) exhibited pigmented hair (P1) and eyes (P1 and #8), as confirmed by PCR analysis for the tyrosinase minigene integrated into the genome. When founder P1 was bred to Kunming male mouse, six progeny out of 11 offspring inherited the transgene and the pigmented-eye phenotype. CONCLUSION: Taken together, these results suggest that this minigene encodes the active tyrosinase protein and that its 5' flanking region contains the sequences regulating the expression of mouse tyrosinase gene as expected. We have rescued the albino phenotype by introduction and expression of a functional tyrosinase minigene in the Kunming albino mouse and the transgene can be passed to subsequent generation. These findings also indicate that TyBS can be a useful visual marker gene in the co-transgenic experiments.

Animals↗
PubMed2005

Power-law rheology of isolated nuclei with deformation mapping of nuclear substructures.

Force-induced changes in genome expression as well as remodeling of nuclear architecture in development and disease motivate a deeper understanding of nuclear mechanics. Chromatin and green fluorescent protein-lamin B dynamics were visualized in a micropipette aspiration of isolated nuclei, and both were shown to contribute to viscoelastic properties of the somatic cell nucleus. Reversible swelling by almost 200% in volume, with changes in salt, demonstrates the resilience and large dilational capacity of the nuclear envelope, nucleoli, and chromatin. Swelling also proves an effective way to separate the mechanical contributions of nuclear elements. In unswollen nuclei, chromatin is a primary force-bearing element, whereas swollen nuclei are an order of magnitude softer, with the lamina sustaining much of the load. In both cases, nuclear deformability increases with time, scaling as a power law-thus lacking any characteristic timescale-when nuclei are either aspirated or indented by atomic force microscopy. The nucleus is stiff and resists distortion at short times, but it softens and deforms more readily at longer times. Such results indicate an essentially infinite spectrum of timescales for structural reorganization, with implications for regulating genome expression kinetics.

Animals↗
PubMed2003

Sequence determinants in human polyadenylation site selection.

BACKGROUND: Differential polyadenylation is a widespread mechanism in higher eukaryotes producing mRNAs with different 3' ends in different contexts. This involves several alternative polyadenylation sites in the 3' UTR, each with its specific strength. Here, we analyze the vicinity of human polyadenylation signals in search of patterns that would help discriminate strong and weak polyadenylation sites, or true sites from randomly occurring signals. RESULTS: We used human genomic sequences to retrieve the region downstream of polyadenylation signals, usually absent from cDNA or mRNA databases. Analyzing 4956 EST-validated polyadenylation sites and their -300/+300 nt flanking regions, we clearly visualized the upstream (USE) and downstream (DSE) sequence elements, both characterized by U-rich (not GU-rich) segments. The presence of a USE and a DSE is the main feature distinguishing true polyadenylation sites from randomly occurring A(A/U)UAAA hexamers. While USEs are indifferently associated with strong and weak poly(A) sites, DSEs are more conspicuous near strong poly(A) sites. We then used the region encompassing the hexamer and DSE as a training set for poly(A) site identification by the ERPIN program and achieved a prediction specificity of 69 to 85% for a sensitivity of 56%. CONCLUSION: The availability of complete genomes and large EST sequence databases now permit large-scale observation of polyadenylation sites. Both U-rich sequences flanking both sides of poly(A) signals contribute to the definition of "true" sites. However, the downstream U-rich sequences may also play an enhancing role. Based on this information, poly(A) site prediction accuracy was moderately but consistently improved compared to the best previously available algorithm.

3' Flanking Region↗
PubMed2004

Mulan: multiple-sequence local alignment and visualization for studying function and evolution.

Multiple-sequence alignment analysis is a powerful approach for understanding phylogenetic relationships, annotating genes, and detecting functional regulatory elements. With a growing number of partly or fully sequenced vertebrate genomes, effective tools for performing multiple comparisons are required to accurately and efficiently assist biological discoveries. Here we introduce Mulan (http://mulan.dcode.org/), a novel method and a network server for comparing multiple draft and finished-quality sequences to identify functional elements conserved over evolutionary time. Mulan brings together several novel algorithms: the TBA multi-aligner program for rapid identification of local sequence conservation, and the multiTF program for detecting evolutionarily conserved transcription factor binding sites in multiple alignments. In addition, Mulan supports two-way communication with the GALA database; alignments of multiple species dynamically generated in GALA can be viewed in Mulan, and conserved transcription factor binding sites identified with Mulan/multiTF can be integrated and overlaid with extensive genome annotation data using GALA. Local multiple alignments computed by Mulan ensure reliable representation of short- and large-scale genomic rearrangements in distant organisms. Mulan allows for interactive modification of critical conservation parameters to differentially predict conserved regions in comparisons of both closely and distantly related species. We illustrate the uses and applications of the Mulan tool through multispecies comparisons of the GATA3 gene locus and the identification of elements that are conserved in a different way in avians than in other genomes, allowing speculation on the evolution of birds. Source code for the aligners and the aligner-evaluation software can be freely downloaded from http://www.bx.psu.edu/miller_lab/.

Animals↗