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PubMed2005

Genomic analysis of bacteriophage PhiJL001: insights into its interaction with a sponge-associated alpha-proteobacterium.

Bacteriophage PhiJL001 infects a novel marine bacterium in the alpha subclass of the Proteobacteria isolated from the marine sponge Ircinia strobilina. PhiJL001 is a siphovirus and forms turbid plaques on its host. The genome sequence of PhiJL001 was determined in order to better understand the interaction between the marine phage and its sponge-associated host bacterium. The complete genome sequence of PhiJL001 comprised 63,469 bp with an overall G+C content of 62%. The genome has 91 predicted open reading frames (ORFs), and 17 ORFs have been assigned putative functions. PhiJL001 appears to be a temperate phage, and the integrase gene was identified in the genome. DNA hybridization analysis showed that the PhiJL001 genome does not integrate into the host chromosome under the conditions tested. DNA hybridization experiments therefore suggested that PhiJL001 has some pseudolysogenic characteristics. The genome of PhiJL001 contains many putative genes involved in phage DNA replication (e.g., helicase, DNA polymerase, and thymidylate synthase genes) and also contains a putative integrase gene associated with the lysogenic cycle. Phylogeny based on DNA polymerase gene sequences indicates that PhiJL001 is related to a group of siphoviruses that infect mycobacteria. Designation of PhiJL001 as a siphovirus is consistent with the morphology of the phage visualized by transmission electron microscopy. The unique marine phage-host system described here provides a model system for studying the role of phages in sponge microbial communities.

Alphaproteobacteria↗

PubMed2006

IsoSVM--distinguishing isoforms and paralogs on the protein level.

BACKGROUND: Recent progress in cDNA and EST sequencing is yielding a deluge of sequence data. Like database search results and proteome databases, this data gives rise to inferred protein sequences without ready access to the underlying genomic data. Analysis of this information (e.g. for EST clustering or phylogenetic reconstruction from proteome data) is hampered because it is not known if two protein sequences are isoforms (splice variants) or not (i.e. paralogs/orthologs). However, even without knowing the intron/exon structure, visual analysis of the pattern of similarity across the alignment of the two protein sequences is usually helpful since paralogs and orthologs feature substitutions with respect to each other, as opposed to isoforms, which do not. RESULTS: The IsoSVM tool introduces an automated approach to identifying isoforms on the protein level using a support vector machine (SVM) classifier. Based on three specific features used as input of the SVM classifier, it is possible to automatically identify isoforms with little effort and with an accuracy of more than 97%. We show that the SVM is superior to a radial basis function network and to a linear classifier. As an example application we use IsoSVM to estimate that a set of Xenopus laevis EST clusters consists of approximately 81% cases where sequences are each other's paralogs and 19% cases where sequences are each other's isoforms. The number of isoforms and paralogs in this allotetraploid species is of interest in the study of evolution. CONCLUSION: We developed an SVM classifier that can be used to distinguish isoforms from paralogs with high accuracy and without access to the genomic data. It can be used to analyze, for example, EST data and database search results. Our software is freely available on the Web, under the name IsoSVM.

Algorithms↗

PubMed2004

Biomedical literature mining: challenges and solutions in the 'omics' era.

It is now obvious that the rate-limiting step in high throughput experimentation is neither data acquisition nor analysis, but rather our ability to interpret data on a genome-wide scale. Indeed, the explosion of data sampling capacity combined with increasing publication rates greatly impairs our ability to find meaning in vast collections of data. In order to support data interpretation, bioinformatic tools are needed to identify critical information contained in large bodies of literature. However, extracting knowledge embedded in free text is an arduous task, compounded in the biomedical field by an inconsistent gene nomenclature, domain-specific language and restricted access to full text articles. This paper presents a selection of currently available biomedical literature mining software. These tools rely on statistic and, more recently, semantic analyses (Natural Language Processing) to automatically extract information from the literature. In addition, a literature mining strategy has been developed to explore patterns of term occurrences in abstracts. This method automatically identifies relevant keywords in collections of abstracts, and uses a pattern discovery algorithm to generate a visual interface for exploring functional associations among genes. Term occurrence heatmaps can also be combined with gene expression profiles to provide valuable functional annotations. Furthermore, as demonstrated with tumor cell line literature profiling results, this approach can be applied to a variety of themes beyond genomic data analysis. Altogether, these examples illustrate how literature analysis can be employed to support knowledge discovery in biomedical research.

Algorithms↗

PubMed2026

In vitro fertilization-conceived offspring exhibit altered Long Interspersed Nuclear Elements-1 retrotransposition dynamics associated with long-term disease risks.

BACKGROUND: In vitro fertilization has transformed reproductive medicine, yet offspring conceived through in vitro fertilization display elevated risks for diverse long-term health conditions, with underlying mechanisms unclear. Long Interspersed Nuclear Elements-1, a mobile genetic element responsive to environmental stress, represents a potential mediator. OBJECTIVE: This study aimed to test the hypothesis that in vitro fertilization procedures may act as an embryonic stressor that alters Long Interspersed Nuclear Elements-1 dynamics, potentially contributing to genomic instability associated with long-term disease susceptibility. STUDY DESIGN: Umbilical cord blood or peripheral blood from 33 in vitro fertilization and 42 naturally conceived neonates were collected for whole-genome sequencing. Total Long Interspersed Nuclear Elements-1 proportion in individual genome was counted with Bowtie2 software. De novo Long Interspersed Nuclear Elements-1 insertion and Long Interspersed Nuclear Elements-1 deletion were detected with Mobile Element Locator Tool. Three parent-matched in vitro fertilization-naturally conceived sibling pairs were included to control for genetic background. Disease association analysis was performed for genes within 500 kb of differential Long Interspersed Nuclear Elements-1 sites in The Database for Annotation, Visualization and Integrated Discovery (DAVID). Statistical analysis was performed using the R language. RESULTS: In vitro fertilization offspring demonstrate elevated global Long Interspersed Nuclear Elements-1 content compared to naturally conceived controls (P=.04). This finding was corroborated in 3 sibling pairs from identical genetic backgrounds, where in vitro fertilization-conceived children consistently exhibited higher Long Interspersed Nuclear Elements-1 levels than their naturally conceived siblings. Eleven genomic loci with differential Long Interspersed Nuclear Elements-1 insertion frequencies and 14 loci with differential Long Interspersed Nuclear Elements-1 deletion frequencies between in vitro fertilization offspring and naturally conceived controls were identified. Notably, these differential Long Interspersed Nuclear Elements-1 sites demonstrated significant enrichment near genes implicated in metabolic, cardiovascular, neuropsychiatric, and neoplastic diseases, conditions associated with in vitro fertilization conception. CONCLUSION: These findings provide preliminary evidence that in vitro fertilization conception is associated with increased Long Interspersed Nuclear Elements-1 content and altered genomic distribution of Long Interspersed Nuclear Elements-1 elements. The proximity of these differential Long Interspersed Nuclear Elements-1 sites to disease-associated genes suggests a plausible genomic mechanism linking in vitro fertilization-associated embryonic stress to elevated disease risk. This work provides valuable molecular insights that may inform the ongoing discussion about assisted reproductive technology safety and suggests that continued attention to genomic integrity in in vitro fertilization-conceived individuals would be beneficial.

Humans↗

PubMed2004

The world of geography: visualizing a knowledge domain with cartographic means.

From an informed critique of existing methods to the development of original tools, cartographic engagement can provide a unique perspective on knowledge domain visualization. Along with a discussion of some principles underlying a cartographically informed visualization methodology, results of experiments involving several thousand conference abstracts will be sketched and their plausibility reflected on.

Chromosome Mapping↗

PubMed2025

Permutation tests to assess sex differences in omics data.

It is common to sex-stratify analyses of omics data and to report effects as 'sex-specific' when they are significant in only one sex. However, when analysing hundreds or thousands of molecules, this approach will yield many spurious 'sex-specific' effects if not supported by significant interactions. I illustrate this problem using an RNA sequencing dataset showing almost no significant sex by treatment interactions, but where sex-stratified analyses yield hundreds of 'sex-specific' effects of treatment. These 'sex-specific' effects could be spurious or could be real but not show interactions due to low statistical power. To distinguish these possibilities, I describe permutation tests, which provide an intuitive way to determine if a pattern of observations differs from what would be expected due to chance. For this dataset, assigning sex at random often generates more 'sex-specific' effects than the real data, demonstrating that there is little evidence of sex differences. Next, I simulate an RNA sequencing dataset that includes genes modelled to have sex-specific effects of a condition. As expected, analysis of this simulated dataset yields both significant interactions and sex-specific effects in sex-stratified analyses. While stratified analyses detect a higher number of sex-specific effects than the analysis of interactions, they erroneously identify genes not modelled to show sex-specific effects more often than interactions. A permutation test confirms that the number of sex-specific effects observed in the simulated dataset is greater than expected due to chance. Permutation tests can be applied to omics studies of sex differences, simultaneously providing (i) a clear and simple demonstration of the problems of sex-stratified analyses, and (ii) additional evidence of sex-specific effects where these are present. R code is provided for permutations, simulations, and plots to visualize potential sex-specific effects, which can be adapted to other types of data.

Female↗

PubMed1997

Apoptotic condensations in M-phase cells.

BACKGROUND: Apoptosis is a morphologically distinctive form of programmed cell death/cell suicide in which genomic DNA degradation/fragmentation and variegated dense chromatin aggregates are characteristic hallmarks that have never been demonstrated in mitotic cells. Perceptions of mutual exclusivity between apoptosis and mitosis imply that M-phase cells cannot be apoptotic. However, in the present study we show apoptotic morphologies in M-phase cells after an acute oxidative stress and endonuclease digestion. METHODS: Degradation of genomic DNA in human Chang liver cells (American Type Culture Collection, ATCC CCL13) was demonstrated by flow cytometric cell-by-cell evaluation of (a) propidium iodide intercalative binding to DNA and (b) terminal deoxynucleotidyl transferase (TdT)-mediated 3'OH nick end labeling (TUNEL) of fragmented DNA. Oxidative stress was imposed by a 30-min prepulse with 200 microM vanadyl(4), which produces hydroxyl free radicals (OH*), the most reactive of the free radical species. Oxidative stress in the cells was demonstrated by evaluating glutathione-S-transferase (GST)-mediated monochlorobimane-glutathione adduct fluorescence for glutathione content, the main reducing agent of a cell, and methylene blue redox metachromasia, which is a deep color when oxidized and colorless when reduced. Cells with DNA fragmentation were highlighted by TUNEL. Apoptotic morphologies were visualized by staining with Giemsa and neutral red dyes and by DNA-propidium iodide binding to chromatin. Direct endonuclease induction of apoptotic morphologies in permeabilized M-phase cells was produced by 1 hr incubation (37 degrees C) with 16 units/ml of micrococcal nuclease. RESULTS: The genomic DNA of proliferative cells, namely in G2/M phase of the cell cycle, was degraded by vanadyl(4) prepulsing and by micrococcal nuclease digestion, concomitantly with DNA fragmentation shown by TUNEL. Cytological profiles showed GSH depletion and M-phase cells with particularly high oxidative reactivity indicated by methylene blue redox metachromasia. DNA fragmentation in M-phase cells was highlighted by TUNEL. Characteristic apoptotic condensations, ranging from single-ball condensations to "pulverized" aggregates of a mitotic catastrophe, buddings, and "apoptotic bodies," were found in prophase, metaphase, anaphase, and telophase mitotic cells. The observed separation of condensed chromatin aggregates from the main chromosome mass in prophase and metaphase cells could explain micronuclei, linking it with apoptosis. Direct endonuclease digestion readily produced apoptotic morphologies in interphase and in M-phase cells. CONCLUSION: Apoptotic morphologies in M-phase cells can be induced indirectly via oxidative stress or directly via endonuclease activity, which has long been established as a pervading hallmark of apoptosis.

Apoptosis↗

PubMed1993

Targeted transfection and expression of hepatitis B viral DNA in human hepatoma cells.

A soluble DNA carrier system consisting of an asialoglycoprotein covalently linked to poly-L-lysine was used to bind DNA and deliver hepatitis B virus (HBV) DNA constructs to asialoglycoprotein receptor-positive human hepatoma cells. 4 d after transfection with surface or core gene expression constructs, HBsAg and HBeAg in the media were measured to be 16 ng/ml and 32 U/ml per 10(7) cells, respectively. Antigen production was completely inhibited by the addition of an excess of asialoorosomucoid. On the other hand, asialoglycoprotein receptor-negative human hepatoma cells, SK-Hep1, did not produce any viral antigens under identical conditions after incubation with HBV DNA complexed to a conjugate composed of asialoorosomucoid and poly-L-lysine. Using a complete HBV genome construct, HBsAg and HBeAg levels reached 16 ng/ml and 16 U/ml per 10(7) cells, respectively. Northern blots revealed characteristic HBV RNA transcripts including 3.5-, 2.4-, and 2.1-kb fragments. Intracellular and extracellular HBV DNA sequences including relaxed circular, linear and single stranded forms were detected by Southern blot hybridization. Finally, 42-nm Dane particles purified from the spent cultures medium were visualized by electron microscopy. This study demonstrates that a targetable DNA carrier system can transfect HBV DNA in vitro resulting in the production of complete HBV virions.

Carcinoma, Hepatocellular↗

PubMed2001

Mapping sites where replication initiates in mammalian cells using DNA fibers.

DNA replication in mammalian chromosomes takes place as a unit of replicon clusters. Here we show a powerful method to detect replication origins and fork movement on DNA fibers from mammalian cells. Cells were loaded with nucleotide analogs, DNA fibers were prepared, and replicated DNA was detected. Using this approach, we could detect origins as close as 10 kb apart and found that the average size of replicon is smaller ( approximately 46 kb) than previously estimated. In addition, the procedure visualizes the complex structure of replicon clusters, e.g. sequential activation of origins in a cluster and flexible initiation sites in different cell cycles. Combined with fluorescence in situ hybridization, replication origins can be mapped in genomic loci including repetitive DNA and a single-copy gene.

Animals↗

PubMed1999

Visualization of a conditionally dispensable chromosome in the filamentous ascomycete Nectria haematococca by fluorescence in situ hybridization.

Supernumerary chromosomes, termed "conditionally dispensable" (CD) chromosomes, are known in Nectria haematococca. Because these CD chromosomes had been revealed solely by pulsed-field gel electrophoresis, their morphological properties were unknown. In this study, we visualized a 1.6-Mb CD chromosome of this fungus by three different types of fluorescence in situ hybridization. The CD chromosome at mitotic metaphase was similar in its appearance to the other chromosomes in the genome. Heterochromatic condensation was not distinct in the CD chromosome, suggesting that it is primarily euchromatic. It was also evident that the CD chromosome is unique and not a duplicate of other chromosomes in the genome. At interphase and prophase, the CD chromosome was not dispersed throughout the nucleus, but occupied a limited domain. Occasionally, occurrence of two distinct unattached copies of the CD chromosome were observed during interphase and metaphase.

Ascomycota↗

PubMed2002

Molecular characterization and mapping of ATOH7, a human atonal homolog with a predicted role in retinal ganglion cell development.

The human ATOH7 gene encodes a basic helix-loop-helix (bHLH) transcription factor that is highly similar to Drosophila Atonal within the conserved bHLH domain. The ATOH7 coding region is contained within a single exon. We mapped ATOH7 to Chromosome (Chr) 10q21.3-22.1, a region syntenic to the segment of mouse Chr 10 where Atoh7 (formerly Math5) is located. The evolutionary relationship between ATOH7 and other atonal homologs was investigated using parsimony analysis. A direct comparison of ATH5/7 and ATH1 protein subgroups to Atonal also revealed a nonrandom distribution of amino acid changes across the bHLH domain, which may be related to their separate visual and proprioceptive sensory functions. Among bHLH genes, ATOH7 is most closely related to Atoh7. This sequence conservation extends significantly beyond the coding region. We define blocks of strong homology in flanking human and mouse genomic DNA, which are likely to include cis regulatory elements. Because targeted deletion of Atoh7 causes optic nerve agenesis in mice, we propose ATOH7 as a candidate for human optic nerve aplasia and related clinical syndromes.

Amino Acid Sequence↗

PubMed1996

trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry.

SUMMARY: Capacitative calcium entry (CCE) describes CA2+ influx into cells that replenishes CA2+ stores emptied through the action of IP3 and other agents. It is an essential component of cellular responses to many hormones and growth factors. The molecular basis of this form of Ca2+ entry is complex and may involve more than one type of channel. Studies on visual signal transduction in Drosophila led to the hypothesis that a protein encoded in trp may be a component of CCE channels. We reported the existence of six trp-related genes in the mouse genome. Expression in L cells of small portions of these genes in antisense orientation suppressed CCE. Expression in COS cells of two full-length cDNAs encoding human trp homologs, Htrp1 and Htrp3, increased CCE. This identifies mammalian gene products that participate in CCE. We propose that trp homologs are subunits of CCE channels, not unlike those of classical voltage-gated ion channels.

Amino Acid Sequence↗

PubMed1997

Arsenic alters cytosine methylation patterns of the promoter of the tumor suppressor gene p53 in human lung cells: a model for a mechanism of carcinogenesis.

Arsenic is a potent human carcinogen to which there is significant worldwide exposure through natural contamination of food and drinking water sources. Because arsenic is detoxified via methylation using a methyltransferase (MTase) and S-adenosylmethionine (SAM) as the methyl donor, we hypothesized that a mechanism of carcinogenesis of arsenic could involve alterations of MTase/SAM-dependent DNA methylation of a tumor suppressor gene. We found that exposure of human lung adenocarcinoma A549 cells to sodium arsenite (0.08-2 microM) or sodium arsenate (30-300 microM), but not dimethylarsenic acid (2-2000 microM), produced significant dose-responsive hypermethylation within a 341-base pair fragment of the promoter of p53. This was determined by quantitative PCR/HpaII restriction site analysis to analyze methylation status of two CCGG sites. In experiments with arsenite, DNA sequencing using bisulfite to visualize 5-methylcytosine (5-MeC) over the entire promoter region confirmed data obtained by restriction analysis. Limited data using SssI methylase also suggested that over-methylation of CpG sequences may exist over the entire genome in response to arsenite exposure. We propose that alteration of DNA methylation by arsenic offers a plausible, unified hypothesis for the carcinogenic mechanism of action of arsenic, and we present a model for arsenic carcinogenesis that utilizes perturbations of DNA methylation as the basis for the carcinogenic effects of arsenic.

Adenocarcinoma↗

PubMed1998

Zomit: biological data visualization and browsing.

MOTIVATION: The problems caused by the difficulty in visualizing and browsing biological databases have become crucial. Scientists can no longer interact directly with the huge amount of available data. However, future breakthroughs in biology depend on this interaction. We propose a new metaphor for biological data visualization and browsing that allows navigation in very large databases in an intuitive way. The concepts underlying our approach are based on navigation and visualization with zooming, semantic zooming and portals; and on data transformation via magic lenses. We think that these new visualization and navigation techniques should be applied globally to a federation of biological databases. RESULTS: We have implemented a generic tool, called Zomit, that provides an application programming interface for developing servers for such navigation and visualization, and a generic architecture-independent client (Javatrade mark applet) that queries such servers. As an illustration of the capabilities of our approach, we have developed ZoomMap, a prototype browser for the HuGeMap human genome map database. AVAILABILITY: Zomit and ZoomMap are available at the URL http://www.infobiogen.fr/services/zomit.

Biological Science Disciplines↗

PubMed2003

A genetic screen for synaptic transmission mutants mapping to the right arm of chromosome 3 in Drosophila.

Neuronal function depends upon the proper formation of synaptic connections and rapid communication at these sites, primarily through the regulated exocytosis of chemical neurotransmitters. Recent biochemical and genomic studies have identified a large number of candidate molecules that may function in these processes. To complement these studies, we are pursuing a genetic approach to identify genes affecting synaptic transmission in the Drosophila visual system. Our screening approach involves a recently described genetic method allowing efficient production of mosaic flies whose eyes are entirely homozygous for a mutagenized chromosome arm. From a screen of 42,500 mutagenized flies, 32 mutations on chromosome 3R that confer synaptic transmission defects in the visual system were recovered. These mutations represent 14 complementation groups, of which at least 9 also appear to perform functional roles outside of the eye. Three of these complementation groups disrupt photoreceptor axonal projection, whereas the remaining complementation groups confer presynaptic defects in synaptic transmission without detectably altering photoreceptor structure. Mapping and complementation testing with candidate mutations revealed new alleles of the neuronal fate determinant svp and the synaptic vesicle trafficking component lap among the collection of mutants recovered in this screen. Given the tools available for investigation of synaptic function in Drosophila, these mutants represent a valuable resource for future analysis of synapse development and function.

Animals↗

PubMed2004

Rapid detection of human HLA transgenes in pigs by fluorescence in situ hybridization (FISH) for adjuvant study of human xenotransplantation.

OBJECTIVES: We have recently generated several lines of transgenic pigs for HLA-DP and -DQ to elucidate the role of HLA-II antigens in the modulation of cell-mediated rejection of xenotransplantation. Using fluorescence in situ hybridization (FISH) analysis, the aim of this study was to determine integration sites and to test zygosity of these transgenes in the piglets after cross mating. METHODS: Blood lymphocytes of transgenic pigs for HLA-DP and -DQ were collected and cultured. Chromosome spreads were prepared by standard methodology. Gene constructs of HLA-DP A1+B1, -DQ A1 & B1 were labeled with fluorescein isothiocyanate or Texas Red by nick-translation. Hybridization was based on a standard FISH protocol. RESULTS: FISH analysis revealed that the HLA-DP probe hybridized to porcine chromosome 6, while both HLA-DQ A1 and B1 probes hybridized to porcine chromosome 11 at the same site. There was no cross-hybridization of HLA transgenes to the swine leukocyte antigen complex. Mosaic integration of HLA-DQ transgenes in the genome of F0, but full penetrance in F1 after selective breeding was observed. Both HLA-DP and HLA-DQ lines were determined to be heterozygous at the integration site. CONCLUSION: By FISH, we have detected specific integration sites of the HLA-DP and -DQ transgenes in pig genome and determined mosaic levels and zygosity types of these transgenes. We conclude that FISH is both sensitive and labor-efficient in confirming and differentiating transgenic pigs for multiple rejection-regulatory genes by visualizing individual integration sites in chromosomes or interphase nuclei.

Animals↗

PubMed1995

[Ultrasound blood flow measurement in malignant tumors is a possible new prognostic factor].

OBJECTIVE: The aim of the present study was to find a possible relationship between biological behavior of breast cancer and sonographically detectable blood flow. METHODS: 157 patients with ductal invasive breast cancer were examined by means of a new sonographic procedure, the MEM (Maximum Entropy Method), able to detect considerably lower blood flow velocities than doppler sonography. In the absence of objectifyable quantification methods, findings were allotted to 3 classes, depending on the visual color information obtained. Blood flow was correlated to tumor size, lymph node and receptor status, ploidy and S-phase-fraction. RESULTS: Most patients with small tumors, no lymph node metatases, positive receptors, diploid genome and low S-phase found to have low blood flow. CONCLUSION: This close relation between established prognostic factors and results of sonographic blood flow examination with the MEM might indicate a new preoperative prognostic factor, which, however, will have to be proven by larger studies.

Biomarkers, Tumor↗

PubMed1995

Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene.

Rapid DNA analysis based on allele-specific polymerase chain reaction (PCR) using mutation site-specific primers was developed to detect mutations in the CYP21 gene known to cause steroid 21-hydroxylase deficiency. In contrast to the previous method, in which PCR of genomic DNA was followed by dot blot analysis with radioactive probes and multiple rounds of stripping and reprobing for each of the 8 most common mutation sites, the results using this new method were immediately visualized after the PCR run by ethidium bromide-stained agarose gel electrophoresis. Using allele-specific PCR, mutation(s) were identified on 148 affected chromosomes out of 160 tested. Although mutation(s) were identified on only one chromosome of 11 of these patients, their parents showed a consistent pattern on DNA analysis. The only exception was that in one family, in which the parents each had a detectable mutation, a mutation was detected on only one allele of the patient. Most likely there is a mutation in the patient's other allele that could have arisen de novo or was inherited from the parent and was not evident in the transmitting parent's phenotype. When compared with the dot blot procedure, allele-specific PCR is more rapid, less labor-intensive, and avoids the use of radioactivity.

Alleles↗