965 resultados para Genomic Imprinting


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The advent of experimental techniques capable of probing biomolecules and cells at high levels of resolution has led to a rapid change in the methods used for the analysis of experimental molecular biology data. In this article we give an overview over visualization techniques and methods that can be used to assess various aspects of genomic data.

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The last few years have seen the advent of high-throughput technologies to analyze various properties of the transcriptome and proteome of several organisms. The congruency of these different data sources, or lack thereof, can shed light on the mechanisms that govern cellular function. A central challenge for bioinformatics research is to develop a unified framework for combining the multiple sources of functional genomics information and testing associations between them, thus obtaining a robust and integrated view of the underlying biology. We present a graph theoretic approach to test the significance of the association between multiple disparate sources of functional genomics data by proposing two statistical tests, namely edge permutation and node label permutation tests. We demonstrate the use of the proposed tests by finding significant association between a Gene Ontology-derived "predictome" and data obtained from mRNA expression and phenotypic experiments for Saccharomyces cerevisiae. Moreover, we employ the graph theoretic framework to recast a surprising discrepancy presented in Giaever et al. (2002) between gene expression and knockout phenotype, using expression data from a different set of experiments.

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DNA sequence copy number has been shown to be associated with cancer development and progression. Array-based Comparative Genomic Hybridization (aCGH) is a recent development that seeks to identify the copy number ratio at large numbers of markers across the genome. Due to experimental and biological variations across chromosomes and across hybridizations, current methods are limited to analyses of single chromosomes. We propose a more powerful approach that borrows strength across chromosomes and across hybridizations. We assume a Gaussian mixture model, with a hidden Markov dependence structure, and with random effects to allow for intertumoral variation, as well as intratumoral clonal variation. For ease of computation, we base estimation on a pseudolikelihood function. The method produces quantitative assessments of the likelihood of genetic alterations at each clone, along with a graphical display for simple visual interpretation. We assess the characteristics of the method through simulation studies and through analysis of a brain tumor aCGH data set. We show that the pseudolikelihood approach is superior to existing methods both in detecting small regions of copy number alteration and in accurately classifying regions of change when intratumoral clonal variation is present.

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Thirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coli AG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 microg/ml). The phenotype was linked to insertion sequence (IS) insertions in marR or acrR or unstable large tandem genomic amplifications which included acrAB and which were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lon mutations. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7x10(-4)) of lon::IS186 mutants, (ii) new lon mutants appeared during the selection on antibiotic-containing agar plates, (iii) lon mutants could slowly grow in the presence of low amounts (about 2x MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.

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A key energy-saving adaptation to chronic hypoxia that enables cardiomyocytes to withstand severe ischemic insults is hibernation, i.e., a reversible arrest of contractile function. Whereas hibernating cardiomyocytes represent the critical reserve of dysfunctional cells that can be potentially rescued, a lack of a suitable animal model has hampered insights on this medically important condition. We developed a transgenic mouse system for conditional induction of long-term hibernation and a system to rescue hibernating cardiomyocytes at will. Via myocardium-specific induction (and, in turn, deinduction) of a VEGF-sequestering soluble receptor, we show that VEGF is indispensable for adjusting the coronary vasculature to match increased oxygen consumption and exploit this finding to generate a hypoperfused heart. Importantly, ensuing ischemia is tunable to a level at which large cohorts of cardiomyocytes are driven to enter a hibernation mode, without cardiac cell death. Relieving the VEGF blockade even months later resulted in rapid revascularization and full recovery of contractile function. Furthermore, we show that left ventricular remodeling associated with hibernation is also fully reversible. The unique opportunity to uncouple hibernation from other ischemic heart phenotypes (e.g., infarction) was used to determine the genetic program of hibernation; uncovering hypoxia-inducible factor target genes associated with metabolic adjustments and induced expression of several cardioprotective genes. Autophagy, specifically self-digestion of mitochondria, was identified as a key prosurvival mechanism in hibernating cardiomyocytes. This system may lend itself for examining the potential utility of treatments to rescue dysfunctional cardiomyocytes and reverse maladaptive remodeling.

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Mutations in the FBN1 gene are the major cause of Marfan syndrome (MFS), an autosomal dominant connective tissue disorder, which displays variable manifestations in the cardiovascular, ocular, and skeletal systems. Current molecular genetic testing of FBN1 may miss mutations in the promoter region or in other noncoding sequences as well as partial or complete gene deletions and duplications. In this study, we tested for copy number variations by successively applying multiplex ligation-dependent probe amplification (MLPA) and the Affymetrix Human Mapping 500 K Array Set, which contains probes for approximately 500,000 single-nucleotide polymorphisms (SNPs) across the genome. By analyzing genomic DNA of 101 unrelated individuals with MFS or related phenotypes in whom standard genetic testing detected no mutation, we identified FBN1 deletions in two patients with MFS. Our high-resolution approach narrowed down the deletion breakpoints. Subsequent sequencing of the junctional fragments revealed the deletion sizes of 26,887 and 302,580 bp, respectively. Surprisingly, both deletions affect the putative regulatory and promoter region of the FBN1 gene, strongly indicating that they abolish transcription of the deleted allele. This expectation of complete loss of function of one allele, i.e. true haploinsufficiency, was confirmed by transcript analyses. Our findings not only emphasize the importance of screening for large genomic rearrangements in comprehensive genetic testing of FBN1 but, importantly, also extend the molecular etiology of MFS by providing hitherto unreported evidence that true haploinsufficiency is sufficient to cause MFS.

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Hardwoods comprise about half of the biomass of forestlands in North America and present many uses including economic, ecological and aesthetic functions. Forest trees rely on the genetic variation within tree populations to overcome the many biotic, abiotic, anthropogenic factors which are further worsened by climate change, that threaten their continued survival and functionality. To harness these inherent genetic variations of tree populations, informed knowledge of the genomic resources and techniques, which are currently lacking or very limited, are imperative for forest managers. The current study therefore aimed to develop genomic microsatellite markers for the leguminous tree species, honey locust, Gleditsia triacanthos L. and test their applicability in assessing genetic variation, estimation of gene flow patterns and identification of a full-sib mapping population. We also aimed to test the usefulness of already developed nuclear and gene-based microsatellite markers in delineation of species and taxonomic relationships between four of the taxonomically difficult Section Lobatae species (Quercus coccinea, Q. ellipsoidalis, Q. rubra and Q. velutina. We recorded 100% amplification of G. triacanthos genomic microsatellites developed using Illumina sequencing techniques in a panel of seven unrelated individuals with 14 of these showing high polymorphism and reproducibility. When characterized in 36 natural population samples, we recorded 20 alleles per locus with no indication for null alleles at 13 of the 14 microsatellites. This is the first report of genomic microsatellites for this species. Honey locust trees occur in fragmented populations of abandoned farmlands and pastures and is described as essentially dioecious. Pollen dispersal if the main source of gene flow within and between populations with the ability to offset the effects of random genetic drift. Factors known to influence gene include fragmentation and degree of isolation, which make the patterns gene flow in fragmented populations of honey locust a necessity for their sustainable management. In this follow-up study, we used a subset of nine of the 14 developed gSSRs to estimate gene flow and identify a full-sib mapping population in two isolated fragments of honey locust. Our analyses indicated that the majority of the seedlings (65-100% - at both strict and relaxed assignment thresholds) were sired by pollen from outside the two fragment populations. Only one selfing event was recorded confirming the functional dioeciousness of honey locust and that the seed parents are almost completely outcrossed. From the Butternut Valley, TN population, pollen donor genotypes were reconstructed and used in paternity assignment analyses to identify a relatively large full-sib family comprised of 149 individuals, proving the usefulness of isolated forest fragments in identification of full-sib families. In the Ames Plantation stand, contemporary pollen dispersal followed a fat-tailed exponential-power distribution, an indication of effective gene flow. Our estimate of δ was 4,282.28 m, suggesting that insect pollinators of honey locust disperse pollen over very long distances. The high proportion of pollen influx into our sampled population implies that our fragment population forms part of a large effectively reproducing population. The high tendency of oak species to hybridize while still maintaining their species identity make it difficult to resolve their taxonomic relationships. Oaks of the section Lobatae are famous in this regard and remain unresolved at both morphological and genetic markers. We applied 28 microsatellite markers including outlier loci with potential roles in reproductive isolation and adaptive divergence between species to natural populations of four known interfertile red oaks, Q. coccinea, Q. ellpsoidalis, Q. rubra and Q. velutina. To better resolve the taxonomic relationships in this difficult clade, we assigned individual samples to species, identified hybrids and introgressive forms and reconstructed phylogenetic relationships among the four species after exclusion of genetically intermediate individuals. Genetic assignment analyses identified four distinct species clusters, with Q. rubra most differentiated from the three other species, but also with a comparatively large number of misclassified individuals (7.14%), hybrids (7.14%) and introgressive forms (18.83%) between Q. ellipsoidalis and Q. velutina. After the exclusion of genetically intermediate individuals, Q. ellipsoidalis grouped as sister species to the largely parapatric Q. coccinea with high bootstrap support (91 %). Genetically intermediate forms in a mixed species stand were located proximate to both potential parental species, which supports recent hybridization of Q. velutina with both Q. ellipsoidalis and Q. rubra. Analyses of genome-wide patterns of interspecific differentiation can provide a better understanding of speciation processes and taxonomic relationships in this taxonomically difficult group of red oak species.

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Maternal antibodies protect newborns whilst they are immunologically immature. This study shows that maternal antibodies can also shape the B cell repertoire of the offspring long after the maternal antibodies themselves become undetectable. V(H)DJ(H) gene-targeted (VI10) mice expressing a heavy chain specific for vesicular stomatitis virus (VSV) produce a 20-fold increased spontaneous titer of VSV-neutralizing antibodies. When transferred from mother to offspring, these antibodies prevented accumulation of Ag-specific transitional type 2 and marginal zone B cells with an activated phenotype and favored selection to the B cell follicles. This effect was B cell-intrinsic and lasted up to adulthood. The pups nursed by mothers producing specific antibodies developed higher endogenous antibody titers of this specificity which perpetuated the effects of specific B cell selection into the mature follicular compartment, presumably by blocking auto-Ag-dependent development of transitional type 2 B cells in the spleen. This repertoire change was functional, as following infection of adult mice with VSV, those pups that had received specific maternal antibodies as neonates had increased pre-immune titers and mounted strong early IgG neutralizing antibodies.

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Unique and shared cytogenetic abnormalities have been documented for marginal zone lymphomas (MZLs) arising at different sites. Recently, homozygous deletions of the chromosomal band 6q23, involving the tumor necrosis factor alpha-induced protein 3 (TNFAIP3, A20) gene, a negative regulator of NF-kappaB, were described in ocular adnexal MZL, suggesting a role for A20 as a tumor suppressor in this disease. Here, we investigated inactivation of A20 by DNA mutations or deletions in a panel of extranodal MZL (EMZL), nodal MZL (NMZL), and splenic MZL (SMZL). Inactivating mutations encoding truncated A20 proteins were identified in 6 (19%) of 32 MZLs, including 2 (18%) of 11 EMZLs, 3 (33%) of 9 NMZLs, and 1 (8%) of 12 SMZLs. Two additional unmutated nonsplenic MZLs also showed monoallelic or biallelic A20 deletions by fluorescent in situ hybridization (FISH) and/or SNP-arrays. Thus, A20 inactivation by either somatic mutation and/or deletion represents a common genetic aberration across all MZL subtypes, which may contribute to lymphomagenesis by inducing constitutive NF-kappaB activation.

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Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.

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Highland cattle with congenital crop ears have notches of variable size on the tips of both ears. In some cases, cartilage deformation can be seen and occasionally the external ears are shortened. We collected 40 cases and 80 controls across Switzerland. Pedigree data analysis confirmed a monogenic autosomal dominant mode of inheritance with variable expressivity. All affected animals could be traced back to a single common ancestor. A genome-wide association study was performed and the causative mutation was mapped to a 4 Mb interval on bovine chromosome 6. The H6 family homeobox 1 (HMX1) gene was selected as a positional and functional candidate gene. By whole genome re-sequencing of an affected Highland cattle, we detected 6 non-synonymous coding sequence variants and two variants in an ultra-conserved element at the HMX1 locus with respect to the reference genome. Of these 8 variants, only a non-coding 76 bp genomic duplication (g.106720058_106720133dup) located in the conserved region was perfectly associated with crop ears. The identified copy number variation probably results in HMX1 misregulation and possible gain-of-function. Our findings confirm the role of HMX1 during the development of the external ear. As it is sometimes difficult to phenotypically diagnose Highland cattle with slight ear notches, genetic testing can now be used to improve selection against this undesired trait.

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Background: Speciation reversal: the erosion of species differentiation via an increase in introgressive hybridization due to the weakening of previously divergent selection regimes, is thought to be an important, yet poorly understood, driver of biodiversity loss. Our study system, the Alpine whitefish (Coregonus spp.) species complex is a classic example of a recent postglacial adaptive radiation: forming an array of endemic lake flocks, with the independent origination of similar ecotypes among flocks. However, many of the lakes of the Alpine radiation have been seriously impacted by anthropogenic nutrient enrichment, resulting in a collapse in neutral genetic and phenotypic differentiation within the most polluted lakes. Here we investigate the effects of eutrophication on the selective forces that have shaped this radiation, using population genomics. We studied eight sympatric species assemblages belonging to five independent parallel adaptive radiations, and one species pair in secondary contact. We used AFLP markers, and applied FST outlier (BAYESCAN, DFDIST) and logistic regression analyses (MATSAM), to identify candidate regions for disruptive selection in the genome and their associations with adaptive traits within each lake flock. The number of outlier and adaptive trait associated loci identified per lake were then regressed against two variables (historical phosphorus concentration and contemporary oxygen concentration) representing the strength of eutrophication. Results: Whilst we identify disruptive selection candidate regions in all lake flocks, we find similar trends, across analysis methods, towards fewer disruptive selection candidate regions and fewer adaptive trait/candidate loci associations in the more polluted lakes. Conclusions: Weakened disruptive selection and a concomitant breakdown in reproductive isolating mechanisms in more polluted lakes has lead to increased gene flow between coexisting Alpine whitefish species. We hypothesize that the resulting higher rates of interspecific recombination reduce either the number or extent of genomic islands of divergence surrounding loci evolving under disruptive natural selection. This produces the negative trend seen in the number of selection candidate loci recovered during genome scans of whitefish species flocks, with increasing levels of anthropogenic eutrophication: as the likelihood decreases that AFLP restriction sites will fall within regions of heightened genomic divergence and therefore be classified as FST outlier loci. This study explores for the first time the potential effects of human-mediated relaxation of disruptive selection on heterogeneous genomic divergence between coexisting species.