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Sample records for allele specific expression

  1. Allele-specific expression assays using Solexa

    PubMed Central

    Main, Bradley J; Bickel, Ryan D; McIntyre, Lauren M; Graze, Rita M; Calabrese, Peter P; Nuzhdin, Sergey V

    2009-01-01

    Background Allele-specific expression (ASE) assays can be used to identify cis, trans, and cis-by-trans regulatory variation. Understanding the source of expression variation has important implications for disease susceptibility, phenotypic diversity, and adaptation. While ASE is commonly measured via relative fluorescence at a SNP, next generation sequencing provides an opportunity to measure ASE in an accurate and high-throughput manner using read counts. Results We introduce a Solexa-based method to perform large numbers of ASE assays using only a single lane of a Solexa flowcell. In brief, transcripts of interest, which contain a known SNP, are PCR enriched and barcoded to enable multiplexing. Then high-throughput sequencing is used to estimate allele-specific expression using sequencing counts. To validate this method, we measured the allelic bias in a dilution series and found high correlations between measured and expected values (r>0.9, p < 0.001). We applied this method to a set of 5 genes in a Drosophila simulans parental mix, F1 and introgression and found that for these genes the majority of expression divergence can be explained by cis-regulatory variation. Conclusion We present a new method with the capacity to measure ASE for large numbers of assays using as little as one lane of a Solexa flowcell. This will be a valuable technique for molecular and population genetic studies, as well as for verification of genome-wide data sets. PMID:19740431

  2. Allele Workbench: transcriptome pipeline and interactive graphics for allele-specific expression.

    PubMed

    Soderlund, Carol A; Nelson, William M; Goff, Stephen A

    2014-01-01

    Sequencing the transcriptome can answer various questions such as determining the transcripts expressed in a given species for a specific tissue or condition, evaluating differential expression, discovering variants, and evaluating allele-specific expression. Differential expression evaluates the expression differences between different strains, tissues, and conditions. Allele-specific expression evaluates expression differences between parental alleles. Both differential expression and allele-specific expression have been studied for heterosis (hybrid vigor), where the hybrid has improved performance over the parents for one or more traits. The Allele Workbench software was developed for a heterosis study that evaluated allele-specific expression for a mouse F1 hybrid using libraries from multiple tissues with biological replicates. This software has been made into a distributable package, which includes a pipeline, a Java interface to build the database, and a Java interface for query and display of the results. The required input is a reference genome, annotation file, and one or more RNA-Seq libraries with optional replicates. It evaluates allelic imbalance at the SNP and transcript level and flags transcripts with significant opposite directional allele-specific expression. The Java interface allows the user to view data from libraries, replicates, genes, transcripts, exons, and variants, including queries on allele imbalance for selected libraries. To determine the impact of allele-specific SNPs on protein folding, variants are annotated with their effect (e.g., missense), and the parental protein sequences may be exported for protein folding analysis. The Allele Workbench processing results in transcript files and read counts that can be used as input to the previously published Transcriptome Computational Workbench, which has a new algorithm for determining a trimmed set of gene ontology terms. The software with demo files is available from https://code.google.com/p/allele

  3. Allele Workbench: Transcriptome Pipeline and Interactive Graphics for Allele-Specific Expression

    PubMed Central

    Soderlund, Carol A.; Nelson, William M.; Goff, Stephen A.

    2014-01-01

    Sequencing the transcriptome can answer various questions such as determining the transcripts expressed in a given species for a specific tissue or condition, evaluating differential expression, discovering variants, and evaluating allele-specific expression. Differential expression evaluates the expression differences between different strains, tissues, and conditions. Allele-specific expression evaluates expression differences between parental alleles. Both differential expression and allele-specific expression have been studied for heterosis (hybrid vigor), where the hybrid has improved performance over the parents for one or more traits. The Allele Workbench software was developed for a heterosis study that evaluated allele-specific expression for a mouse F1 hybrid using libraries from multiple tissues with biological replicates. This software has been made into a distributable package, which includes a pipeline, a Java interface to build the database, and a Java interface for query and display of the results. The required input is a reference genome, annotation file, and one or more RNA-Seq libraries with optional replicates. It evaluates allelic imbalance at the SNP and transcript level and flags transcripts with significant opposite directional allele-specific expression. The Java interface allows the user to view data from libraries, replicates, genes, transcripts, exons, and variants, including queries on allele imbalance for selected libraries. To determine the impact of allele-specific SNPs on protein folding, variants are annotated with their effect (e.g., missense), and the parental protein sequences may be exported for protein folding analysis. The Allele Workbench processing results in transcript files and read counts that can be used as input to the previously published Transcriptome Computational Workbench, which has a new algorithm for determining a trimmed set of gene ontology terms. The software with demo files is available from https://code.google.com/p/allele

  4. Dynamic variation in allele-specific gene expression of Paraoxonase-1 in murine and human tissues

    PubMed Central

    Parker-Katiraee, Layla; Bousiaki, Eleni; Monk, David; Moore, Gudrun E.; Nakabayashi, Kazuhiko; Scherer, Stephen W.

    2008-01-01

    Differential allelic expression has been shown to be common in mice, humans and maize, and variability in the expression of polymorphic alleles has been associated with human disease. Here, we describe the differential expression pattern of Paraoxonase-1, a gene involved in lipid metabolism and implicated in the formation of atherosclerotic lesions. We measured the expression of the murine Paraoxonase-1 gene (Pon1) in livers at different stages of embryonic development using F1 hybrid crosses and quantified the transcriptional level of both parental alleles. Using human foetal tissues, we analysed the expression of the human orthologue (PON1) and found monoallelic or preferential allelic expression in 6/7 and 4/4 samples from liver and pancreas, respectively. We observed that Pon1 does not show a parent-of-origin preference in its allelic expression, but has dramatic variations in allele-specific expression occurring throughout development. This study has important repercussions in the analysis of haplotypes at disease loci, since it implies that the expression of polymorphic alleles can be unequal and dynamic. PMID:18678600

  5. Known unknowns for allele-specific expression and genomic imprinting effects

    PubMed Central

    2014-01-01

    Recent studies have provided evidence for non-canonical imprinting effects that are associated with allele-specific expression biases at the tissue level in mice. These imprinting effects have features that are distinct from canonical imprinting effects that involve allele silencing. Here, I discuss some of the evidence for non-canonical imprinting effects in the context of random X-inactivation and epigenetic allele-specific expression effects on the autosomes. I propose several mechanisms that may underlie non-canonical imprinting effects and outline future directions and approaches to study these effects at the cellular level in vivo. The growing evidence for complex allele-specific expression effects that are cell- and developmental stage-specific has opened a new frontier for study. Currently, the function of these effects and the underlying regulatory mechanisms are largely unknown. PMID:25343032

  6. A uniform survey of allele-specific binding and expression over 1000-Genomes-Project individuals.

    PubMed

    Chen, Jieming; Rozowsky, Joel; Galeev, Timur R; Harmanci, Arif; Kitchen, Robert; Bedford, Jason; Abyzov, Alexej; Kong, Yong; Regan, Lynne; Gerstein, Mark

    2016-01-01

    Large-scale sequencing in the 1000 Genomes Project has revealed multitudes of single nucleotide variants (SNVs). Here, we provide insights into the functional effect of these variants using allele-specific behaviour. This can be assessed for an individual by mapping ChIP-seq and RNA-seq reads to a personal genome, and then measuring 'allelic imbalances' between the numbers of reads mapped to the paternal and maternal chromosomes. We annotate variants associated with allele-specific binding and expression in 382 individuals by uniformly processing 1,263 functional genomics data sets, developing approaches to reduce the heterogeneity between data sets due to overdispersion and mapping bias. Since many allelic variants are rare, aggregation across multiple individuals is necessary to identify broadly applicable 'allelic elements'. We also found SNVs for which we can anticipate allelic imbalance from the disruption of a binding motif. Our results serve as an allele-specific annotation for the 1000 Genomes variant catalogue and are distributed as an online resource (alleledb.gersteinlab.org). PMID:27089393

  7. A uniform survey of allele-specific binding and expression over 1000-Genomes-Project individuals

    PubMed Central

    Chen, Jieming; Rozowsky, Joel; Galeev, Timur R.; Harmanci, Arif; Kitchen, Robert; Bedford, Jason; Abyzov, Alexej; Kong, Yong; Regan, Lynne; Gerstein, Mark

    2016-01-01

    Large-scale sequencing in the 1000 Genomes Project has revealed multitudes of single nucleotide variants (SNVs). Here, we provide insights into the functional effect of these variants using allele-specific behaviour. This can be assessed for an individual by mapping ChIP-seq and RNA-seq reads to a personal genome, and then measuring ‘allelic imbalances' between the numbers of reads mapped to the paternal and maternal chromosomes. We annotate variants associated with allele-specific binding and expression in 382 individuals by uniformly processing 1,263 functional genomics data sets, developing approaches to reduce the heterogeneity between data sets due to overdispersion and mapping bias. Since many allelic variants are rare, aggregation across multiple individuals is necessary to identify broadly applicable ‘allelic elements'. We also found SNVs for which we can anticipate allelic imbalance from the disruption of a binding motif. Our results serve as an allele-specific annotation for the 1000 Genomes variant catalogue and are distributed as an online resource (alleledb.gersteinlab.org). PMID:27089393

  8. Allelic Specificity of Ube3a Expression in the Mouse Brain during Postnatal Development

    PubMed Central

    JUDSON, MATTHEW C.; SOSA-PAGAN, JASON O.; DEL CID, WILMER A.; HAN, JI EUN; PHILPOT, BENJAMIN D.

    2014-01-01

    Genetic alterations of the maternal UBE3A allele result in Angelman syndrome (AS), a neurodevelopmental disorder characterized by severe developmental delay, lack of speech, and difficulty with movement and balance. The combined effects of maternal UBE3A mutation and cell type-specific epigenetic silencing of paternal UBE3A are hypothesized to result in a complete loss of functional UBE3A protein in neurons. However, the allelic specificity of UBE3A expression in neurons and other cell types in the brain has yet to be characterized throughout development, including the early postnatal period when AS phenotypes emerge. Here we define maternal and paternal allele-specific Ube3a protein expression throughout postnatal brain development in the mouse, a species which exhibits orthologous epigenetic silencing of paternal Ube3a in neurons and AS-like behavioral phenotypes subsequent to maternal Ube3a deletion. We find that neurons downregulate paternal Ube3a protein expression as they mature and, with the exception of neurons born from postnatal stem cell niches, do not express detectable paternal Ube3a beyond the first postnatal week. By contrast, neurons express maternal Ube3a throughout postnatal development, during which time localization of the protein becomes increasingly nuclear. Unlike neurons, astrocytes and oligodendrotyes biallelically express Ube3a. Notably, mature oligodendrocytes emerge as the predominant Ube3a-expressing glial cell type in the cortex and white matter tracts during postnatal development. These findings demonstrate the spatiotemporal characteristics of allele-specific Ube3a expression in key brain cell types, thereby improving our understanding of the developmental parameters of paternal Ube3a silencing and the cellular basis of AS. PMID:24254964

  9. Allele-Specific Gene Expression Is Widespread Across the Genome and Biological Processes

    PubMed Central

    Goñi, Joaquín; Piedrafita, Gabriel; Fernando, Olga; Navarro, Arcadi; Villoslada, Pablo

    2009-01-01

    Allelic specific gene expression (ASGE) appears to be an important factor in human phenotypic variability and as a consequence, for the development of complex traits and diseases. In order to study ASGE across the human genome, we have performed a study in which genotyping was coupled with an analysis of ASGE by screening 11,500 SNPs using the Mapping 10 K Array to identify differential allelic expression. We found that from the 5,133 SNPs that were suitable for analysis (heterozygous in our sample and expressed in peripheral blood mononuclear cells), 2,934 (57%) SNPs had differential allelic expression. Such SNPs were equally distributed along human chromosomes and biological processes. We validated the presence or absence of ASGE in 18 out 20 SNPs (90%) randomly selected by real time PCR in 48 human subjects. In addition, we observed that SNPs close to -but not included in- segmental duplications had increased levels of ASGE. Finally, we found that transcripts of unknown function or non-coding RNAs, also display ASGE: from a total of 2,308 intronic SNPs, 1510 (65%) SNPs underwent differential allelic expression. In summary, ASGE is a widespread mechanism in the human genome whose regulation seems to be far more complex than expected. PMID:19127300

  10. Molecular genetic mechanisms of allelic specific regulation of murine Comt expression.

    PubMed

    Segall, Samantha K; Shabalina, Svetlana A; Meloto, Carolina B; Wen, Xia; Cunningham, Danielle; Tarantino, Lisa M; Wiltshire, Tim; Gauthier, Josée; Tohyama, Sarasa; Martin, Loren J; Mogil, Jeffrey S; Diatchenko, Luda

    2015-10-01

    A functional allele of the mouse catechol-O-methyltransferase (Comt) gene is defined by the insertion of a B2 short interspersed repeat element in its 3'-untranslated region (UTR). This allele has been associated with a number of phenotypes, such as pain and anxiety. In comparison with mice carrying the ancestral allele (Comt+), Comt B2i mice show higher Comt mRNA and enzymatic activity levels. Here, we investigated the molecular genetic mechanisms underlying this allelic specific regulation of Comt expression. Insertion of the B2 element introduces an early polyadenylation signal generating a shorter Comt transcript, in addition to the longer ancestral mRNA. Comparative analysis and in silico prediction of Comt mRNA potential targets within the transcript 3' to the B2 element was performed and allowed choosing microRNA (miRNA) candidates for experimental screening: mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667. Cell transfection with each miRNA downregulated the expression of the ancestral transcript and COMT enzymatic activity. Our in vivo experiments showed that mmu-miR-667-3p is strongly correlated with decreasing amounts of Comt mRNA in the brain, and lentiviral injections of mmu-miR-3470a, mmu-miR-3470b, and mmu-miR-667 increase hypersensitivity in the mouse formalin model, consistent with reduced COMT activity. In summary, our data demonstrate that the Comt+ transcript contains regulatory miRNA signals in its 3'-untranslated region leading to mRNA degradation; these signals, however, are absent in the shorter transcript, resulting in higher mRNA expression and activity levels. PMID:26067582

  11. Allele-specific gene expression in a wild nonhuman primate population

    PubMed Central

    Tung, J.; Akinyi, M. Y.; Mutura, S.; Altmann, J.; Wray, G. A.; Alberts, S. C.

    2015-01-01

    Natural populations hold enormous potential for evolutionary genetic studies, especially when phenotypic, genetic and environmental data are all available on the same individuals. However, untangling the genotype-phenotype relationship in natural populations remains a major challenge. Here, we describe results of an investigation of one class of phenotype, allele-specific gene expression (ASGE), in the well-studied natural population of baboons of the Amboseli basin, Kenya. ASGE measurements identify cases in which one allele of a gene is overexpressed relative to the alternative allele of the same gene, within individuals, thus providing a control for background genetic and environmental effects. Here, we characterize the incidence of ASGE in the Amboseli baboon population, focusing on the genetic and environmental contributions to ASGE in a set of eleven genes involved in immunity and defence. Within this set, we identify evidence for common ASGE in four genes. We also present examples of two relationships between cis-regulatory genetic variants and the ASGE phenotype. Finally, we identify one case in which this relationship is influenced by a novel gene-environment interaction. Specifically, the dominance rank of an individual’s mother during its early life (an aspect of that individual’s social environment) influences the expression of the gene CCL5 via an interaction with cis-regulatory genetic variation. These results illustrate how environmental and ecological data can be integrated into evolutionary genetic studies of functional variation in natural populations. They also highlight the potential importance of early life environmental variation in shaping the genetic architecture of complex traits in wild mammals. PMID:21226779

  12. Germline Allele-Specific Expression of DAPK1 in Chronic Lymphocytic Leukemia

    PubMed Central

    Hielscher, Thomas; Mertens, Daniel; Raval, Aparna; Oakes, Christopher C.; Tanner, Stephan M.; de la Chapelle, Albert; Byrd, John C.; Stilgenbauer, Stephan; Plass, Christoph

    2013-01-01

    We previously reported a rare germline variant (c.1-6531) that resulted in allele–specific expression (ASE) of death-associated protein kinase 1 (DAPK1) and predisposition to chronic lymphocytic leukemia (CLL). We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE) with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2%) CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5′ upstream regulatory region, within distinct exons or in the 3′-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL. PMID:23383130

  13. Nonsyntenic Genes Drive Tissue-Specific Dynamics of Differential, Nonadditive, and Allelic Expression Patterns in Maize Hybrids1[OPEN

    PubMed Central

    2016-01-01

    Distantly related maize (Zea mays) inbred lines display an exceptional degree of genomic diversity. F1 progeny of such inbred lines are often more vigorous than their parents, a phenomenon known as heterosis. In this study, we investigated how the genetic divergence of the maize inbred lines B73 and Mo17 and their F1 hybrid progeny is reflected in differential, nonadditive, and allelic expression patterns in primary root tissues. In pairwise comparisons of the four genotypes, the number of differentially expressed genes between the two parental inbred lines significantly exceeded those of parent versus hybrid comparisons in all four tissues under analysis. No differentially expressed genes were detected between reciprocal hybrids, which share the same nuclear genome. Moreover, hundreds of nonadditive and allelic expression ratios that were different from the expression ratios of the parents were observed in the reciprocal hybrids. The overlap of both nonadditive and allelic expression patterns in the reciprocal hybrids significantly exceeded the expected values. For all studied types of expression - differential, nonadditive, and allelic - substantial tissue-specific plasticity was observed. Significantly, nonsyntenic genes that evolved after the last whole genome duplication of a maize progenitor from genes with synteny to sorghum (Sorghum bicolor) were highly overrepresented among differential, nonadditive, and allelic expression patterns compared with the fraction of these genes among all expressed genes. This observation underscores the role of nonsyntenic genes in shaping the transcriptomic landscape of maize hybrids during the early developmental manifestation of heterosis in root tissues of maize hybrids. PMID:27208302

  14. Nonsyntenic Genes Drive Tissue-Specific Dynamics of Differential, Nonadditive, and Allelic Expression Patterns in Maize Hybrids.

    PubMed

    Baldauf, Jutta A; Marcon, Caroline; Paschold, Anja; Hochholdinger, Frank

    2016-06-01

    Distantly related maize (Zea mays) inbred lines display an exceptional degree of genomic diversity. F1 progeny of such inbred lines are often more vigorous than their parents, a phenomenon known as heterosis. In this study, we investigated how the genetic divergence of the maize inbred lines B73 and Mo17 and their F1 hybrid progeny is reflected in differential, nonadditive, and allelic expression patterns in primary root tissues. In pairwise comparisons of the four genotypes, the number of differentially expressed genes between the two parental inbred lines significantly exceeded those of parent versus hybrid comparisons in all four tissues under analysis. No differentially expressed genes were detected between reciprocal hybrids, which share the same nuclear genome. Moreover, hundreds of nonadditive and allelic expression ratios that were different from the expression ratios of the parents were observed in the reciprocal hybrids. The overlap of both nonadditive and allelic expression patterns in the reciprocal hybrids significantly exceeded the expected values. For all studied types of expression - differential, nonadditive, and allelic - substantial tissue-specific plasticity was observed. Significantly, nonsyntenic genes that evolved after the last whole genome duplication of a maize progenitor from genes with synteny to sorghum (Sorghum bicolor) were highly overrepresented among differential, nonadditive, and allelic expression patterns compared with the fraction of these genes among all expressed genes. This observation underscores the role of nonsyntenic genes in shaping the transcriptomic landscape of maize hybrids during the early developmental manifestation of heterosis in root tissues of maize hybrids. PMID:27208302

  15. High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta

    PubMed Central

    2010-01-01

    Background Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue. Results Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%). Conclusions Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term

  16. Organ-specific gene expression in maize: The P-wr allele. Final report, August 15, 1993--August 14, 1996

    SciTech Connect

    Peterson, T.A.

    1997-06-01

    The ultimate aim of our work is to understand how a regulatory gene produces a specific pattern of gene expression during plant development. Our model is the P-wr gene of maize, which produces a distinctive pattern of pigmentation of maize floral organs. We are investigating this system using a combination of classical genetic and molecular approaches. Mechanisms of organ-specific gene expression are a subject of intense research interest, as it is the operation of these mechanisms during eukaryotic development which determine the characteristics of each organism Allele-specific expression has been characterized in only a few other plant genes. In maize, organ-specific pigmentation regulated by the R, B, and Pl genes is achieved by differential transcription of functionally conserved protein coding sequences. Our studies point to a strikingly different mechanism of organ-specific gene expression, involving post-transcriptional regulation of the regulatory P gene. The novel pigmentation pattern of the P-wr allele is associated with differences in the encoded protein. Furthermore, the P-wr gene itself is present as a unique tandemly amplified structure, which may affect its transcriptional regulation.

  17. Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

    PubMed

    Chen, Ailu; Wang, Ruijia; Liu, Shikai; Peatman, Eric; Sun, Luyang; Bao, Lisui; Jiang, Chen; Li, Chao; Li, Yun; Zeng, Qifan; Liu, Zhanjiang

    2016-06-01

    Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8 % SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2 %) and 13,390 (7.5 %) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish. PMID:26747053

  18. Recommendations for Accurate Resolution of Gene and Isoform Allele-Specific Expression in RNA-Seq Data.

    PubMed

    Wood, David L A; Nones, Katia; Steptoe, Anita; Christ, Angelika; Harliwong, Ivon; Newell, Felicity; Bruxner, Timothy J C; Miller, David; Cloonan, Nicole; Grimmond, Sean M

    2015-01-01

    Genetic variation modulates gene expression transcriptionally or post-transcriptionally, and can profoundly alter an individual's phenotype. Measuring allelic differential expression at heterozygous loci within an individual, a phenomenon called allele-specific expression (ASE), can assist in identifying such factors. Massively parallel DNA and RNA sequencing and advances in bioinformatic methodologies provide an outstanding opportunity to measure ASE genome-wide. In this study, matched DNA and RNA sequencing, genotyping arrays and computationally phased haplotypes were integrated to comprehensively and conservatively quantify ASE in a single human brain and liver tissue sample. We describe a methodological evaluation and assessment of common bioinformatic steps for ASE quantification, and recommend a robust approach to accurately measure SNP, gene and isoform ASE through the use of personalized haplotype genome alignment, strict alignment quality control and intragenic SNP aggregation. Our results indicate that accurate ASE quantification requires careful bioinformatic analyses and is adversely affected by sample specific alignment confounders and random sampling even at moderate sequence depths. We identified multiple known and several novel ASE genes in liver, including WDR72, DSP and UBD, as well as genes that contained ASE SNPs with imbalance direction discordant with haplotype phase, explainable by annotated transcript structure, suggesting isoform derived ASE. The methods evaluated in this study will be of use to researchers performing highly conservative quantification of ASE, and the genes and isoforms identified as ASE of interest to researchers studying those loci. PMID:25965996

  19. Recommendations for Accurate Resolution of Gene and Isoform Allele-Specific Expression in RNA-Seq Data

    PubMed Central

    Wood, David L. A.; Nones, Katia; Steptoe, Anita; Christ, Angelika; Harliwong, Ivon; Newell, Felicity; Bruxner, Timothy J. C.; Miller, David; Cloonan, Nicole; Grimmond, Sean M.

    2015-01-01

    Genetic variation modulates gene expression transcriptionally or post-transcriptionally, and can profoundly alter an individual’s phenotype. Measuring allelic differential expression at heterozygous loci within an individual, a phenomenon called allele-specific expression (ASE), can assist in identifying such factors. Massively parallel DNA and RNA sequencing and advances in bioinformatic methodologies provide an outstanding opportunity to measure ASE genome-wide. In this study, matched DNA and RNA sequencing, genotyping arrays and computationally phased haplotypes were integrated to comprehensively and conservatively quantify ASE in a single human brain and liver tissue sample. We describe a methodological evaluation and assessment of common bioinformatic steps for ASE quantification, and recommend a robust approach to accurately measure SNP, gene and isoform ASE through the use of personalized haplotype genome alignment, strict alignment quality control and intragenic SNP aggregation. Our results indicate that accurate ASE quantification requires careful bioinformatic analyses and is adversely affected by sample specific alignment confounders and random sampling even at moderate sequence depths. We identified multiple known and several novel ASE genes in liver, including WDR72, DSP and UBD, as well as genes that contained ASE SNPs with imbalance direction discordant with haplotype phase, explainable by annotated transcript structure, suggesting isoform derived ASE. The methods evaluated in this study will be of use to researchers performing highly conservative quantification of ASE, and the genes and isoforms identified as ASE of interest to researchers studying those loci. PMID:25965996

  20. Analysis of LMNB1 Duplications in Autosomal Dominant Leukodystrophy Provides Insights into Duplication Mechanisms and Allele-Specific Expression

    PubMed Central

    Giorgio, Elisa; Rolyan, Harshvardhan; Kropp, Laura; Chakka, Anish Baswanth; Yatsenko, Svetlana; Gregorio, Eleonora Di; Lacerenza, Daniela; Vaula, Giovanna; Talarico, Flavia; Mandich, Paola; Toro, Camilo; Pierre, Eleonore Eymard; Labauge, Pierre; Capellari, Sabina; Cortelli, Pietro; Vairo, Filippo Pinto; Miguel, Diego; Stubbolo, Danielle; Marques, Lourenco Charles; Gahl, William; Boespflug-Tanguy, Odile; Melberg, Atle; Hassin-Baer, Sharon; Cohen, Oren S; Pjontek, Rastislav; Grau, Armin; Klopstock, Thomas; Fogel, Brent; Meijer, Inge; Rouleau, Guy; Bouchard, Jean-Pierre L; Ganapathiraju, Madhavi; Vanderver, Adeline; Dahl, Niklas; Hobson, Grace; Brusco, Alfredo; Brussino, Alessandro; Padiath, Quasar Saleem

    2013-01-01

    ABSTRACT Autosomal dominant leukodystrophy (ADLD) is an adult onset demyelinating disorder that is caused by duplications of the lamin B1 (LMNB1) gene. However, as only a few cases have been analyzed in detail, the mechanisms underlying LMNB1 duplications are unclear. We report the detailed molecular analysis of the largest collection of ADLD families studied, to date. We have identified the minimal duplicated region necessary for the disease, defined all the duplication junctions at the nucleotide level and identified the first inverted LMNB1 duplication. We have demonstrated that the duplications are not recurrent; patients with identical duplications share the same haplotype, likely inherited from a common founder and that the duplications originated from intrachromosomal events. The duplication junction sequences indicated that nonhomologous end joining or replication-based mechanisms such fork stalling and template switching or microhomology-mediated break induced repair are likely to be involved. LMNB1 expression was increased in patients’ fibroblasts both at mRNA and protein levels and the three LMNB1 alleles in ADLD patients show equal expression, suggesting that regulatory regions are maintained within the rearranged segment. These results have allowed us to elucidate duplication mechanisms and provide insights into allele-specific LMNB1 expression levels. PMID:23649844

  1. Delimiting Allelic Imbalance of TYMS by Allele-Specific Analysis

    PubMed Central

    Balboa-Beltrán, Emilia; Cruz, Raquel; Carracedo, Angel; Barros, Francisco

    2015-01-01

    Abstract Allelic imbalance of thymidylate synthase (TYMS) is attributed to polymorphisms in the 5′- and 3′-untranslated region (UTR). These polymorphisms have been related to the risk of suffering different cancers, for example leukemia, breast or gastric cancer, and response to different drugs, among which are methotrexate glutamates, stavudine, and specifically 5-fluorouracil (5-FU), as TYMS is its direct target. A vast literature has been published in relation to 5-FU, even suggesting the sole use of these polymorphisms to effectively manage 5-FU dosage. Estimates of the extent to which these polymorphisms influence in TYMS expression have in the past been based on functional analysis by luciferase assays and quantification of TYMS mRNA, but both these studies, as the association studies with cancer risk or with toxicity or response to 5-FU, are very contradictory. Regarding functional assays, the artificial genetic environment created in luciferase assay and the problems derived from quantitative polymerase chain reactions (qPCRs), for example the use of a reference gene, may have distorted the results. To avoid these sources of interference, we have analyzed the allelic imbalance of TYMS by allelic-specific analysis in peripheral blood mononuclear cells (PBMCs) from patients. Allelic imbalance in PBMCs, taken from 40 patients with suspected myeloproliferative haematological diseases, was determined by fluorescent fragment analysis (for the 3′-UTR polymorphism), Sanger sequencing and allelic-specific qPCR in multiplex (for the 5′-UTR polymorphisms). For neither the 3′- nor the 5′-UTR polymorphisms did the observed allelic imbalance exceed 1.5 fold. None of the TYMS polymorphisms is statistically associated with allelic imbalance. The results acquired allow us to deny the previously established assertion of an influence of 2 to 4 fold of the rs45445694 and rs2853542 polymorphisms in the expression of TYMS and narrow its allelic imbalance to 1.5 fold

  2. Transcriptome analysis revealed chimeric RNAs, single nucleotide polymorphisms and allele-specific expression in porcine prenatal skeletal muscle

    PubMed Central

    Yang, Yalan; Tang, Zhonglin; Fan, Xinhao; Xu, Kui; Mu, Yulian; Zhou, Rong; Li, Kui

    2016-01-01

    Prenatal skeletal muscle development genetically determines postnatal muscle characteristics such as growth and meat quality in pigs. However, the molecular mechanisms underlying prenatal skeletal muscle development remain unclear. Here, we performed the first genome-wide analysis of chimeric RNAs, single nuclear polymorphisms (SNPs) and allele-specific expression (ASE) in prenatal skeletal muscle in pigs. We identified 14,810 protein coding genes and 163 high-confidence chimeric RNAs expressed in prenatal skeletal muscle. More than 94.5% of the chimeric RNAs obeyed the canonical GT/AG splice rule and were trans-splicing events. Ten and two RNAs were aligned to human and mouse chimeric transcripts, respectively. We detected 106,457 high-quality SNPs (6,955 novel), which were mostly (89.09%) located within QTLs for production traits. The high proportion of non-exonic SNPs revealed the incomplete annotation status of the current swine reference genome. ASE analysis revealed that 11,300 heterozygous SNPs showed allelic imbalance, whereas 131 ASE variants were located in the chimeric RNAs. Moreover, 4 ASE variants were associated with various economically relevant traits of pigs. Taken together, our data provide a source for studies of chimeric RNAs and biomarkers for pig breeding, while illuminating the complex transcriptional events underlying prenatal skeletal muscle development in mammals. PMID:27352850

  3. Transcriptome analysis revealed chimeric RNAs, single nucleotide polymorphisms and allele-specific expression in porcine prenatal skeletal muscle.

    PubMed

    Yang, Yalan; Tang, Zhonglin; Fan, Xinhao; Xu, Kui; Mu, Yulian; Zhou, Rong; Li, Kui

    2016-01-01

    Prenatal skeletal muscle development genetically determines postnatal muscle characteristics such as growth and meat quality in pigs. However, the molecular mechanisms underlying prenatal skeletal muscle development remain unclear. Here, we performed the first genome-wide analysis of chimeric RNAs, single nuclear polymorphisms (SNPs) and allele-specific expression (ASE) in prenatal skeletal muscle in pigs. We identified 14,810 protein coding genes and 163 high-confidence chimeric RNAs expressed in prenatal skeletal muscle. More than 94.5% of the chimeric RNAs obeyed the canonical GT/AG splice rule and were trans-splicing events. Ten and two RNAs were aligned to human and mouse chimeric transcripts, respectively. We detected 106,457 high-quality SNPs (6,955 novel), which were mostly (89.09%) located within QTLs for production traits. The high proportion of non-exonic SNPs revealed the incomplete annotation status of the current swine reference genome. ASE analysis revealed that 11,300 heterozygous SNPs showed allelic imbalance, whereas 131 ASE variants were located in the chimeric RNAs. Moreover, 4 ASE variants were associated with various economically relevant traits of pigs. Taken together, our data provide a source for studies of chimeric RNAs and biomarkers for pig breeding, while illuminating the complex transcriptional events underlying prenatal skeletal muscle development in mammals. PMID:27352850

  4. A GWAS SNP for Schizophrenia Is Linked to the Internal MIR137 Promoter and Supports Differential Allele-Specific Expression

    PubMed Central

    Warburton, Alix; Breen, Gerome; Bubb, Vivien J.; Quinn, John P.

    2016-01-01

    Single nucleotide polymorphisms (SNPs) within the MIR137 gene locus have been shown to confer risk for schizophrenia through genome-wide association studies (GWAS). The expression levels of microRNA-137 (miR-137) and its validated gene targets have also been shown to be disrupted in several neuropsychiatric conditions, including schizophrenia. Regulation of miR-137 expression is thus imperative for normal neuronal functioning. We previously characterized an internal promoter domain within the MIR137 gene that contained a variable number tandem repeat (VNTR) polymorphism and could alter the in vitro levels of miR-137 in a stimulus-induced and allele-specific manner. We now demonstrate that haplotype tagging-SNP analysis linked the rs1625579 GWAS SNP for schizophrenia to this internal MIR137 promoter through a proxy SNP rs2660304 located at this domain. We postulated that the rs2660304 promoter SNP may act as predisposing factor for schizophrenia through altering the levels of miR-137 expression in a genotype-dependent manner. Reporter gene analysis of the internal MIR137 promoter containing the common VNTR variant demonstrated genotype-dependent differences in promoter activity with respect to rs2660304. In line with previous reports, the major allele of the rs2660304 proxy SNP, which has previously been linked with schizophrenia risk through genetic association, resulted in downregulation of reporter gene expression in a tissue culture model. The genetic influence of the rs2660304 proxy SNP on the transcriptional activity of the internal MIR137 promoter, and thus the levels of miR-137 expression, therefore offers a distinct regulatory mechanism to explain the functional significance of the rs1625579 GWAS SNP for schizophrenia risk. PMID:26429811

  5. A GWAS SNP for Schizophrenia Is Linked to the Internal MIR137 Promoter and Supports Differential Allele-Specific Expression.

    PubMed

    Warburton, Alix; Breen, Gerome; Bubb, Vivien J; Quinn, John P

    2016-07-01

    Single nucleotide polymorphisms (SNPs) within the MIR137 gene locus have been shown to confer risk for schizophrenia through genome-wide association studies (GWAS). The expression levels of microRNA-137 (miR-137) and its validated gene targets have also been shown to be disrupted in several neuropsychiatric conditions, including schizophrenia. Regulation of miR-137 expression is thus imperative for normal neuronal functioning. We previously characterized an internal promoter domain within the MIR137 gene that contained a variable number tandem repeat (VNTR) polymorphism and could alter the in vitro levels of miR-137 in a stimulus-induced and allele-specific manner. We now demonstrate that haplotype tagging-SNP analysis linked the rs1625579 GWAS SNP for schizophrenia to this internal MIR137 promoter through a proxy SNP rs2660304 located at this domain. We postulated that the rs2660304 promoter SNP may act as predisposing factor for schizophrenia through altering the levels of miR-137 expression in a genotype-dependent manner. Reporter gene analysis of the internal MIR137 promoter containing the common VNTR variant demonstrated genotype-dependent differences in promoter activity with respect to rs2660304. In line with previous reports, the major allele of the rs2660304 proxy SNP, which has previously been linked with schizophrenia risk through genetic association, resulted in downregulation of reporter gene expression in a tissue culture model. The genetic influence of the rs2660304 proxy SNP on the transcriptional activity of the internal MIR137 promoter, and thus the levels of miR-137 expression, therefore offers a distinct regulatory mechanism to explain the functional significance of the rs1625579 GWAS SNP for schizophrenia risk. PMID:26429811

  6. Comprehensively Evaluating cis-Regulatory Variation in the Human Prostate Transcriptome by Using Gene-Level Allele-Specific Expression

    PubMed Central

    Larson, Nicholas B.; McDonnell, Shannon; French, Amy J.; Fogarty, Zach; Cheville, John; Middha, Sumit; Riska, Shaun; Baheti, Saurabh; Nair, Asha A.; Wang, Liang; Schaid, Daniel J.; Thibodeau, Stephen N.

    2015-01-01

    The identification of cis-acting regulatory variation in primary tissues has the potential to elucidate the genetic basis of complex traits and further our understanding of transcriptomic diversity across cell types. Expression quantitative trait locus (eQTL) association analysis using RNA sequencing (RNA-seq) data can improve upon the detection of cis-acting regulatory variation by leveraging allele-specific expression (ASE) patterns in association analysis. Here, we present a comprehensive evaluation of cis-acting eQTLs by analyzing RNA-seq gene-expression data and genome-wide high-density genotypes from 471 samples of normal primary prostate tissue. Using statistical models that integrate ASE information, we identified extensive cis-eQTLs across the prostate transcriptome and found that approximately 70% of expressed genes corresponded to a significant eQTL at a gene-level false-discovery rate of 0.05. Overall, cis-eQTLs were heavily concentrated near the transcription start and stop sites of affected genes, and effects were negatively correlated with distance. We identified multiple instances of cis-acting co-regulation by using phased genotype data and discovered 233 SNPs as the most strongly associated eQTLs for more than one gene. We also noted significant enrichment (25/50, p = 2E−5) of previously reported prostate cancer risk SNPs in prostate eQTLs. Our results illustrate the benefit of assessing ASE data in cis-eQTL analyses by showing better reproducibility of prior eQTL findings than of eQTL mapping based on total expression alone. Altogether, our analysis provides extensive functional context of thousands of SNPs in prostate tissue, and these results will be of critical value in guiding studies examining disease of the human prostate. PMID:25983244

  7. Hybrid sterility and evolution in Hawaiian Drosophila: differential gene and allele-specific expression analysis of backcross males.

    PubMed

    Brill, E; Kang, L; Michalak, K; Michalak, P; Price, D K

    2016-08-01

    The Hawaiian Drosophila are an iconic example of sequential colonization, adaptive radiation and speciation on islands. Genetic and phenotypic analysis of closely related species pairs that exhibit incomplete reproductive isolation can provide insights into the mechanisms of speciation. Drosophila silvestris from Hawai'i Island and Drosophila planitibia from Maui are two closely related allopatric Hawaiian picture-winged Drosophila that produce sterile F1 males but fertile F1 females, a pattern consistent with Haldane's rule. Backcrossing F1 hybrid females between these two species to parental species gives rise to recombinant males with three distinct sperm phenotypes despite a similar genomic background: motile sperm, no sperm (sterile), and immotile sperm. We found that these three reproductive morphologies of backcross hybrid males produce divergent gene expression profiles in testes, as measured with RNA sequencing. There were a total of 71 genes significantly differentially expressed between backcross males with no sperm compared with those backcross males with motile sperm and immotile sperm, but no significant differential gene expression between backcross males with motile sperm and backcross males with immotile sperm. All of these genes were underexpressed in males with no sperm, including a number of genes with previously known activities in adult testis. An allele-specific expression analysis showed overwhelmingly more cis-divergent than trans-divergent genes, with no significant difference in the ratio of cis- and trans-divergent genes among the sperm phenotypes. Overall, the results indicate that the regulation of gene expression involved in sperm production likely diverged relatively rapidly between these two closely related species. PMID:27220308

  8. Presence of specific MHC Class II expressed alleles associates with clinical disease in ovine progressive pneumonia virus (OPPV) infected sheep

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A genetic tool hypothesized to predict which OPPV infected sheep will progress to debilitating clinical disease is MHC Class II Ovis aries (Ovar)-DRB1. Previously, fifteen Ovar-DRB1 beta 1 expressed alleles were identified in a ewe-lamb flock of 32 originating from an Idaho flock using RT-PCR, clon...

  9. Trans-species polymorphism and allele-specific expression in the CBF gene family of wild tomatoes.

    PubMed

    Mboup, Mamadou; Fischer, Iris; Lainer, Hilde; Stephan, Wolfgang

    2012-12-01

    Abiotic stresses such as drought, extreme temperatures, and salinity have a strong impact on plant adaptation. They act as selective forces on plant physiology and morphology. These selective pressures leave characteristic footprints that can be detected at the DNA sequence level using population genetic tools. On the basis of a candidate gene approach, we investigated signatures of adaptation in two wild tomato species, Solanum peruvianum and S. chilense. These species are native to western South America and constitute a model system for studying adaptation, due to their ability to colonize diverse habitats and the available genetic resources. We have determined the selective forces acting on the C-repeat binding factor (CBF) gene family, which consists of three genes, and is known to be involved in tolerance to abiotic stresses, in particular in cold tolerance. We also analyzed the expression pattern of these genes after drought and cold stresses. We found that CBF3 evolves under very strong purifying selection, CBF2 is under balancing selection in some populations of both species (S. peruvianum/Quicacha and S. chilense/Nazca) maintaining a trans-species polymorphism, and CBF1 is a pseudogene. In contrast to previous studies of cultivated tomatoes showing that only CBF1 was cold induced, we found that all three CBF genes are cold induced in wild tomatoes. All three genes are also drought induced. CBF2 exhibits an allele-specific expression pattern associated with the trans-species polymorphism. PMID:22787283

  10. Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression.

    PubMed

    Cavalli, Marco; Pan, Gang; Nord, Helena; Wallerman, Ola; Wallén Arzt, Emelie; Berggren, Olof; Elvers, Ingegerd; Eloranta, Maija-Leena; Rönnblom, Lars; Lindblad Toh, Kerstin; Wadelius, Claes

    2016-05-01

    Genome-wide association studies (GWAS) have identified a large number of disease-associated SNPs, but in few cases the functional variant and the gene it controls have been identified. To systematically identify candidate regulatory variants, we sequenced ENCODE cell lines and used public ChIP-seq data to look for transcription factors binding preferentially to one allele. We found 9962 candidate regulatory SNPs, of which 16 % were rare and showed evidence of larger functional effect than common ones. Functionally rare variants may explain divergent GWAS results between populations and are candidates for a partial explanation of the missing heritability. The majority of allele-specific variants (96 %) were specific to a cell type. Furthermore, by examining GWAS loci we found >400 allele-specific candidate SNPs, 141 of which were highly relevant in our cell types. Functionally validated SNPs support identification of an SNP in SYNGR1 which may expose to the risk of rheumatoid arthritis and primary biliary cirrhosis, as well as an SNP in the last intron of COG6 exposing to the risk of psoriasis. We propose that by repeating the ChIP-seq experiments of 20 selected transcription factors in three to ten people, the most common polymorphisms can be interrogated for allele-specific binding. Our strategy may help to remove the current bottleneck in functional annotation of the genome. PMID:26993500

  11. Identification of transcriptome SNPs between Xiphophorus lines and species for assessing allele specific gene expression within F1 interspecies hybrids☆

    PubMed Central

    Shen, Yingjia; Catchen, Julian; Garcia, Tzintzuni; Amores, Angel; Beldroth, Ion; Wagner, Jonathon R; Zhang, Ziping; Postlethwait, John; Warren, Wes; Schartl, Manfred; Walter, Ronald B.

    2011-01-01

    Variations in gene expression are essential for the evolution of novel phenotypes and for speciation. Studying allelic specific gene expression (ASGE) within interspecies hybrids provides a unique opportunity to reveal underlying mechanisms of genetic variation. Using Xiphophorus interspecies hybrid fishes and high-throughput next generation sequencing technology, we were able to assess variations between two closely related vertebrate species, X. maculatus and X. couchianus, and their F1 interspecies hybrids. We constructed transcriptome-wide SNP polymorphism sets between two highly inbred X. maculatus lines (JP 163 A and B), and between X. maculatus and a second species, X. couchianus. The X. maculatus JP 163 A and B parental lines have been separated in the laboratory for ≈ 70 years and we were able to identify SNPs at a resolution of 1 SNP per 49 kb of transcriptome. In contrast, SNP polymorphisms between X. couchianus and X. maculatus species, which diverged ≈ 5–10 million years ago, were identified about every 700 bp. Using 6,524 transcripts with identified SNPs between the two parental species (X. maculatus and X. couchianus), we mapped RNA-seq reads to determine ASGE within F1 interspecies hybrids. We developed an in silico X. couchianus transcriptome by replacing 90,788 SNP bases for X. maculatus transcriptome with the consensus X. couchianus SNP bases and provide evidence that this procedure overcomes read mapping biases. Employment of the insilico reference transcriptome and tolerating 5 mismatches during read mapping allow direct assessment of ASGE in the F1 interspecies hybrids. Overall, these results show that Xiphophorus is a tractable vertebrate experimental model to investigate how genetic variations that occur during speciation may affect gene interactions and the regulation of gene expression. PMID:21466860

  12. Effect of metallothionein 2A gene polymorphism on allele-specific gene expression and metal content in prostate cancer

    SciTech Connect

    Krześlak, Anna; Forma, Ewa; Jóźwiak, Paweł; Szymczyk, Agnieszka; Bryś, Magdalena

    2013-05-01

    Metallothioneins (MTs) are highly conserved, small molecular weight, cysteine rich proteins. The major physiological functions of metallothioneins include homeostasis of essential metals Zn and Cu and protection against cytotoxicity of heavy metals. The aim of this study was to determine whether there is an association between the − 5 A/G single nucleotide polymorphism (SNP; rs28366003) in core promoter region and expression of metallothionein 2A (MT2A) gene and metal concentration in prostate cancer tissues. MT2A polymorphism was determined by the polymerase chain reaction–restriction fragment length polymorphism technique (PCR–RFLP) using 412 prostate cancer tissue samples. MT2A gene expression analysis was performed by real-time RT-PCR method. A significant association between rs28366003 genotype and MT2A expression level was found. The average mRNA level was found to be lower among minor allele carriers (the risk allele) than average expression among homozygotes for the major allele. Metal levels were analyzed by flamed atomic absorption spectrometer system. Highly statistically significant associations were detected between the SNP and Cd, Zn, Cu and Pb levels. The results of Spearman's rank correlation showed that the expressions of MT2A and Cu, Pb and Ni concentrations were negatively correlated. On the basis of the results obtained in this study, we suggest that SNP polymorphism may affect the MT2A gene expression in prostate and this is associated with some metal accumulation. - Highlights: • MT2A gene expression and metal content in prostate cancer tissues • Association between SNP (rs28366003) and expression of MT2A • Significant associations between the SNP and Cd, Zn, Cu and Pb levels • Negative correlation between MT2A gene expression and Cu, Pb and Ni levels.

  13. Allele-specific expression of mutated in colorectal cancer (MCC) gene and alternative susceptibility to colorectal cancer in schizophrenia

    PubMed Central

    Wang, Yang; Cao, Yanfei; Huang, Xiaoye; Yu, Tao; Wei, Zhiyun; McGrath, John; Xu, Fei; Bi, Yan; Li, Xingwang; Yang, Fengping; Li, Weidong; Zou, Xia; Peng, Zhihai; Xiao, Yanzeng; Zhang, Yan; He, Lin; He, Guang

    2016-01-01

    Evidence has indicated that the incidence of colorectal cancer (CRC) among schizophrenia is lower than normal. To explore this potential protective effect, we employed an innovative strategy combining association study with allele-specific expression (ASE) analysis in MCC gene. We first genotyped four polymorphisms within MCC in 312 CRC patients, 270 schizophrenia patients and 270 controls. Using the MassArray technique, we performed ASE measurements in a second sample series consisting of 50 sporadic CRC patients, 50 schizophrenia patients and 52 controls. Rs2227947 showed significant differences between schizophrenia cases and controls, and haplotype analysis reported some significant discrepancies among these three subject groups. ASE values of rs2227948 and rs2227947 presented consistently differences between CRC (or schizophrenia) patients and controls. Of the three groups, highest frequencies of ASE in MCC were concordantly found in CRC group, whereas lowest frequencies of ASE were observed in schizophrenia group. Similar trends were confirmed in both haplotype frequencies and ASE frequencies (i.e. CRC > control > schizophrenia). We provide a first indication that MCC might confer alterative genetic susceptibility to CRC in individuals with schizophrenia promising to shed more light on the relationship between schizophrenia and cancer progression. PMID:27226254

  14. Allele-specific expression of mutated in colorectal cancer (MCC) gene and alternative susceptibility to colorectal cancer in schizophrenia.

    PubMed

    Wang, Yang; Cao, Yanfei; Huang, Xiaoye; Yu, Tao; Wei, Zhiyun; McGrath, John; Xu, Fei; Bi, Yan; Li, Xingwang; Yang, Fengping; Li, Weidong; Zou, Xia; Peng, Zhihai; Xiao, Yanzeng; Zhang, Yan; He, Lin; He, Guang

    2016-01-01

    Evidence has indicated that the incidence of colorectal cancer (CRC) among schizophrenia is lower than normal. To explore this potential protective effect, we employed an innovative strategy combining association study with allele-specific expression (ASE) analysis in MCC gene. We first genotyped four polymorphisms within MCC in 312 CRC patients, 270 schizophrenia patients and 270 controls. Using the MassArray technique, we performed ASE measurements in a second sample series consisting of 50 sporadic CRC patients, 50 schizophrenia patients and 52 controls. Rs2227947 showed significant differences between schizophrenia cases and controls, and haplotype analysis reported some significant discrepancies among these three subject groups. ASE values of rs2227948 and rs2227947 presented consistently differences between CRC (or schizophrenia) patients and controls. Of the three groups, highest frequencies of ASE in MCC were concordantly found in CRC group, whereas lowest frequencies of ASE were observed in schizophrenia group. Similar trends were confirmed in both haplotype frequencies and ASE frequencies (i.e. CRC > control > schizophrenia). We provide a first indication that MCC might confer alterative genetic susceptibility to CRC in individuals with schizophrenia promising to shed more light on the relationship between schizophrenia and cancer progression. PMID:27226254

  15. Bulk segregant RNA-seq reveals expression and positional candidate genes and allele-specific expression for disease resistance against enteric septicemia of catfish

    PubMed Central

    2013-01-01

    Background The application of RNA-seq has accelerated gene expression profiling and identification of gene-associated SNPs in many species. However, the integrated studies of gene expression along with SNP mapping have been lacking. Coupling of RNA-seq with bulked segregant analysis (BSA) should allow correlation of expression patterns and associated SNPs with the phenotypes. Results In this study, we demonstrated the use of bulked segregant RNA-seq (BSR-Seq) for the analysis of differentially expressed genes and associated SNPs with disease resistance against enteric septicemia of catfish (ESC). A total of 1,255 differentially expressed genes were found between resistant and susceptible fish. In addition, 56,419 SNPs residing on 4,304 unique genes were identified as significant SNPs between susceptible and resistant fish. Detailed analysis of these significant SNPs allowed differentiation of significant SNPs caused by genetic segregation and those caused by allele-specific expression. Mapping of the significant SNPs, along with analysis of differentially expressed genes, allowed identification of candidate genes underlining disease resistance against ESC disease. Conclusions This study demonstrated the use of BSR-Seq for the identification of genes involved in disease resistance against ESC through expression profiling and mapping of significantly associated SNPs. BSR-Seq is applicable to analysis of genes underlining various performance and production traits without significant investment in the development of large genotyping platforms such as SNP arrays. PMID:24373586

  16. Overall and allele-specific expression of the SMC1A gene in female Cornelia de Lange syndrome patients and healthy controls

    PubMed Central

    Parenti, Ilaria; Rovina, Davide; Masciadri, Maura; Cereda, Anna; Azzollini, Jacopo; Picinelli, Chiara; Limongelli, Giuseppe; Finelli, Palma; Selicorni, Angelo; Russo, Silvia; Gervasini, Cristina; Larizza, Lidia

    2014-01-01

    Cornelia de Lange syndrome (CdLS) is a rare multisystem disorder characterized by facial dysmorphisms, limb anomalies, and growth and cognitive deficits. Mutations in genes encoding subunits (SMC1A, SMC3, RAD21) or regulators (NIPBL, HDAC8) of the cohesin complex account for approximately 65% of clinically diagnosed CdLS cases. The SMC1A gene (Xp11.22), responsible for 5% of CdLS cases, partially escapes X chromosome inactivation in humans and the allele on the inactive X chromosome is variably expressed. In this study, we evaluated overall and allele-specific SMC1A expression. Real-time PCR analysis conducted on 17 controls showed that SMC1A expression in females is 50% higher than in males. Immunoblotting experiments confirmed a 44% higher protein level in healthy females than in males, and showed no significant differences in SMC1A protein levels between controls and patients. Pyrosequencing was used to assess the reciprocal level of allelic expression in six female carriers of different SMC1A mutations and 15 controls who were heterozygous at a polymorphic transcribed SMC1A locus. The two alleles were expressed at a 1:1 ratio in the control group and at a 2:1 ratio in favor of the wild type allele in the test group. Since a dominant negative effect is considered the pathogenic mechanism in SMC1A-defective female patients, the level of allelic preferential expression might be one of the factors contributing to the wide phenotypic variability observed in these patients. An extension of this study to a larger cohort containing mild to borderline cases could enhance our understanding of the clinical spectrum of SMC1A-linked CdLS. PMID:24756084

  17. Dissection of expression-quantitative trait locus and allele specificity using a haploid/diploid plant system - insights into compensatory evolution of transcriptional regulation within populations.

    PubMed

    Verta, Jukka-Pekka; Landry, Christian R; MacKay, John

    2016-07-01

    Regulation of gene expression plays a central role in translating genotypic variation into phenotypic variation. Dissection of the genetic basis of expression variation is key to understanding how expression regulation evolves. Such analyses remain challenging in contexts where organisms are outbreeding, highly heterozygous and long-lived such as in the case of conifer trees. We developed an RNA sequencing (RNA-seq)-based approach for both expression-quantitative trait locus (eQTL) mapping and the detection of cis-acting (allele-specific) vs trans-acting (non-allele-specific) eQTLs. This method can be potentially applied to many conifers. We used haploid and diploid meiotic seed tissues of a single self-fertilized white spruce (Picea glauca) individual to dissect eQTLs according to linkage and allele specificity. The genetic architecture of local eQTLs linked to the expressed genes was particularly complex, consisting of cis-acting, trans-acting and, surprisingly, compensatory cis-trans effects. These compensatory effects influence expression in opposite directions and are neutral when combined in homozygotes. Nearly half of local eQTLs were under compensation, indicating that close linkage between compensatory cis-trans factors is common in spruce. Compensated genes were overrepresented in developmental and cell organization functions. Our haploid-diploid eQTL analysis in spruce revealed that compensatory cis-trans eQTLs segregate within populations and evolve in close genetic linkage. PMID:26891783

  18. The genetic association of RUNX3 with ankylosing spondylitis can be explained by allele-specific effects on IRF4 recruitment that alter gene expression

    PubMed Central

    Vecellio, Matteo; Roberts, Amity R; Cohen, Carla J; Cortes, Adrian; Knight, Julian C; Bowness, Paul; Wordsworth, B Paul

    2016-01-01

    Objectives To identify the functional basis for the genetic association of single nucleotide polymorphisms (SNP), upstream of the RUNX3 promoter, with ankylosing spondylitis (AS). Methods We performed conditional analysis of genetic association data and used ENCODE data on chromatin remodelling and transcription factor (TF) binding sites to identify the primary AS-associated regulatory SNP in the RUNX3 region. The functional effects of this SNP were tested in luciferase reporter assays. Its effects on TF binding were investigated by electrophoretic mobility gel shift assays and chromatin immunoprecipitation. RUNX3 mRNA levels were compared in primary CD8+ T cells of AS risk and protective genotypes by real-time PCR. Results The association of the RUNX3 SNP rs4648889 with AS (p<7.6×10−14) was robust to conditioning on all other SNPs in this region. We identified a 2 kb putative regulatory element, upstream of RUNX3, containing rs4648889. In reporter gene constructs, the protective rs4648889 ‘G’ allele increased luciferase activity ninefold but significantly less activity (4.3-fold) was seen with the AS risk ‘A’ allele (p≤0.01). The binding of Jurkat or CD8+ T-cell nuclear extracts to the risk allele was decreased and IRF4 recruitment was reduced. The AS-risk allele also affected H3K4Me1 histone methylation and associated with an allele-specific reduction in RUNX3 mRNA (p<0.05). Conclusion We identified a regulatory region upstream of RUNX3 that is modulated by rs4648889. The risk allele decreases TF binding (including IRF4) and reduces reporter activity and RUNX3 expression. These findings may have important implications for understanding the role of T cells and other immune cells in AS. PMID:26452539

  19. Brain Derived Neurotrophic Factor (BDNF) Expression Is Regulated by MicroRNAs miR-26a and miR-26b Allele-Specific Binding

    PubMed Central

    Caputo, Viviana; Parisi, Chiara; Catalanotto, Caterina; Pasini, Augusto; Cogoni, Carlo; Pizzuti, Antonio

    2011-01-01

    Brain-derived neurotrophic factor (BDNF) is a neurotrophin that plays an essential role in neuronal development and plasticity. MicroRNA (miRNAs) are small non-coding RNAs of about 22-nucleotides in length regulating gene expression at post-transcriptional level. In this study we explore the role of miRNAs as post-transcriptional inhibitors of BDNF and the effect of 3′UTR sequence variations on miRNAs binding capacity. Using an in silico approach we identified a group of miRNAs putatively regulating BDNF expression and binding to BDNF 3′UTR polymorphic sequences. Luciferase assays demonstrated that these miRNAs (miR-26a1/2 and miR-26b) downregulates BDNF expression and that the presence of the variant alleles of two single nucleotide polymorphisms (rs11030100 and rs11030099) mapping in BDNF 3′UTR specifically abrogates miRNAs targeting. Furthermore we found a high linkage disequilibrium rate between rs11030100, rs11030099 and the non-synonymous coding variant rs6265 (Val66Met), which modulates BDNF mRNA localization and protein intracellular trafficking. Such observation led to hypothesize that miR-26s mediated regulation could extend to rs6265 leading to an allelic imbalance with potentially functional effects, such as peptide's localization and activity-dependent secretion. Since rs6265 has been previously implicated in various neuropsychiatric disorders, we evaluated the distribution of rs11030100, rs11030099 and rs6265 both in a control and schizophrenic group, but no significant difference in allele frequencies emerged. In conclusion, in the present study we identified two novel miRNAs regulating BDNF expression and the first BDNF 3′UTR functional variants altering miRNAs-BDNF binding. PMID:22194877

  20. Biased Allelic Expression in Human Primary Fibroblast Single Cells

    PubMed Central

    Borel, Christelle; Ferreira, Pedro G.; Santoni, Federico; Delaneau, Olivier; Fort, Alexandre; Popadin, Konstantin Y.; Garieri, Marco; Falconnet, Emilie; Ribaux, Pascale; Guipponi, Michel; Padioleau, Ismael; Carninci, Piero; Dermitzakis, Emmanouil T.; Antonarakis, Stylianos E.

    2015-01-01

    The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability. PMID:25557783

  1. Differential and limited expression of mutant alleles in multiple myeloma

    PubMed Central

    Rashid, Naim U.; Sperling, Adam S.; Bolli, Niccolo; Wedge, David C.; Van Loo, Peter; Tai, Yu-Tzu; Shammas, Masood A.; Fulciniti, Mariateresa; Samur, Mehmet K.; Richardson, Paul G.; Magrangeas, Florence; Minvielle, Stephane; Futreal, P. Andrew; Anderson, Kenneth C.; Avet-Loiseau, Herve; Parmigiani, Giovanni

    2014-01-01

    Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications. PMID:25237203

  2. Differential and limited expression of mutant alleles in multiple myeloma.

    PubMed

    Rashid, Naim U; Sperling, Adam S; Bolli, Niccolo; Wedge, David C; Van Loo, Peter; Tai, Yu-Tzu; Shammas, Masood A; Fulciniti, Mariateresa; Samur, Mehmet K; Richardson, Paul G; Magrangeas, Florence; Minvielle, Stephane; Futreal, P Andrew; Anderson, Kenneth C; Avet-Loiseau, Herve; Campbell, Peter J; Parmigiani, Giovanni; Munshi, Nikhil C

    2014-11-13

    Recent work has delineated mutational profiles in multiple myeloma and reported a median of 52 mutations per patient, as well as a set of commonly mutated genes across multiple patients. In this study, we have used deep sequencing of RNA from a subset of these patients to evaluate the proportion of expressed mutations. We find that the majority of previously identified mutations occur within genes with very low or no detectable expression. On average, 27% (range, 11% to 47%) of mutated alleles are found to be expressed, and among mutated genes that are expressed, there often is allele-specific expression where either the mutant or wild-type allele is suppressed. Even in the absence of an overall change in gene expression, the presence of differential allelic expression within malignant cells highlights the important contribution of RNA-sequencing in identifying clinically significant mutational changes relevant to our understanding of myeloma biology and also for therapeutic applications. PMID:25237203

  3. Pyrosequencing for Accurate Imprinted Allele Expression Analysis

    PubMed Central

    Yang, Bing; Damaschke, Nathan; Yao, Tianyu; McCormick, Johnathon; Wagner, Jennifer; Jarrard, David

    2016-01-01

    Genomic imprinting is an epigenetic mechanism that restricts gene expression to one inherited allele. Improper maintenance of imprinting has been implicated in a number of human diseases and developmental syndromes. Assays are needed that can quantify the contribution of each paternal allele to a gene expression profile. We have developed a rapid, sensitive quantitative assay for the measurement of individual allelic ratios termed Pyrosequencing for Imprinted Expression (PIE). Advantages of PIE over other approaches include shorter experimental time, decreased labor, avoiding the need for restriction endonuclease enzymes at polymorphic sites, and prevent heteroduplex formation which is problematic in quantitative PCR-based methods. We demonstrate the improved sensitivity of PIE including the ability to detect differences in allelic expression down to 1%. The assay is capable of measuring genomic heterozygosity as well as imprinting in a single run. PIE is applied to determine the status of Insulin-like Growth Factor-2 (IGF2) imprinting in human and mouse tissues. PMID:25581900

  4. A functional polymorphism in the Eta-1 promoter is associated with allele specific binding to the transcription factor Sp1 and elevated gene expression.

    PubMed

    Hummelshoj, Tina; Ryder, Lars P; Madsen, Hans O; Odum, Niels; Svejgaard, Arne

    2006-03-01

    Early T lymphocyte activator 1 (Eta-1), also known as Osteopontin, is a cytokine produced by macrophages and T lymphocytes. It is involved in the regulation of IL-12 and IL-10 expression in macrophages and stimulates the polarization of T cells to the Th1 subset. Three promoter polymorphisms of the human Eta-1 gene, -443T/C, -156delG/G, -66T/G, were investigated for possible influence on gene expression. Electrophoretic mobility shift assays (EMSA) with nuclear extract from the human myeloid leukaemia premonocyte cell line, THP-1, revealed sequence specific binding of the transcription factor Sp1 to the -66T allele but not the -66G allele, and haplotype -443C/-156G/-66T showed a marked increase in promoter activity of a luciferase reporter gene. Thus, a substitution of the T-base with G at position -66 in the Eta-1 promoter modulates the promoter activity of the Eta-1 gene, which might influence the Th1 versus Th2 balance. These observations are discussed in relation to a recently reported related observation on the same gene, and it is argued that discrepancies between reporter gene assays in the two studies may be due to the use of different cell lines and may reflect requirements for different transcription factors in cells involved in immune responses compared with other cells. PMID:16009426

  5. Allele-specific MMP-3 transcription under in vivo conditions

    SciTech Connect

    Zhu Chaoyong; Odeberg, Jacob; Hamsten, Anders; Eriksson, Per . E-mail: Per.Eriksson@ki.se

    2006-09-29

    A common matrix metalloproteinases-3 (MMP-3) -1612 5A/6A promoter polymorphism is associated with risk for cardiovascular disease, rheumatoid arthritis, and other diseases. Here we used the haplotype chromatin immunoprecipitation method to study allele-specific MMP-3 expression under in vivo conditions in heterozygous THP-1 cells. Pyrosequencing was used to analyse the ratio of 5A-allele to 6A-allele after chromatin immunoprecipitation using an antibody against phosphorylated active RNA polymerase II. There was no allele-specific difference in transcriptional activity during basal conditions, i.e., in unstimulated monocytic THP-1 cells. However, after stimulation of MMP-3 expression by monocyte differentiation or incubation with IL-1{beta}, the haplotype containing the 5A-allele was associated with higher transcriptional activity compared with the 6A-containing haplotype. Electromobility shift assay demonstrated increased binding of nuclear proteins to the 5A-allele after monocyte differentiation. In conclusion, the common MMP-3 5A/6A promoter polymorphism appears to be functional only during specific environmental conditions involving inflammation.

  6. Mutated tumor alleles are expressed according to their DNA frequency

    PubMed Central

    Castle, John C.; Loewer, Martin; Boegel, Sebastian; Tadmor, Arbel D.; Boisguerin, Valesca; de Graaf, Jos; Paret, Claudia; Diken, Mustafa; Kreiter, Sebastian; Türeci, Özlem; Sahin, Ugur

    2014-01-01

    The transcription of tumor mutations from DNA into RNA has implications for biology, epigenetics and clinical practice. It is not clear if mutations are in general transcribed and, if so, at what proportion to the wild-type allele. Here, we examined the correlation between DNA mutation allele frequency and RNA mutation allele frequency. We sequenced the exome and transcriptome of tumor cell lines with large copy number variations, identified heterozygous single nucleotide mutations and absolute DNA copy number, and determined the corresponding DNA and RNA mutation allele fraction. We found that 99% of the DNA mutations in expressed genes are expressed as RNA. Moreover, we found a high correlation between the DNA and RNA mutation allele frequency. Exceptions are mutations that cause premature termination codons and therefore activate nonsense-mediated decay. Beyond this, we did not find evidence of any wide-scale mechanism, such as allele-specific epigenetic silencing, preferentially promoting mutated or wild-type alleles. In conclusion, our data strongly suggest that genes are equally transcribed from all alleles, mutated and wild-type, and thus transcribed in proportion to their DNA allele frequency. PMID:24752137

  7. Mutated tumor alleles are expressed according to their DNA frequency.

    PubMed

    Castle, John C; Loewer, Martin; Boegel, Sebastian; Tadmor, Arbel D; Boisguerin, Valesca; de Graaf, Jos; Paret, Claudia; Diken, Mustafa; Kreiter, Sebastian; Türeci, Özlem; Sahin, Ugur

    2014-01-01

    The transcription of tumor mutations from DNA into RNA has implications for biology, epigenetics and clinical practice. It is not clear if mutations are in general transcribed and, if so, at what proportion to the wild-type allele. Here, we examined the correlation between DNA mutation allele frequency and RNA mutation allele frequency. We sequenced the exome and transcriptome of tumor cell lines with large copy number variations, identified heterozygous single nucleotide mutations and absolute DNA copy number, and determined the corresponding DNA and RNA mutation allele fraction. We found that 99% of the DNA mutations in expressed genes are expressed as RNA. Moreover, we found a high correlation between the DNA and RNA mutation allele frequency. Exceptions are mutations that cause premature termination codons and therefore activate nonsense-mediated decay. Beyond this, we did not find evidence of any wide-scale mechanism, such as allele-specific epigenetic silencing, preferentially promoting mutated or wild-type alleles. In conclusion, our data strongly suggest that genes are equally transcribed from all alleles, mutated and wild-type, and thus transcribed in proportion to their DNA allele frequency. PMID:24752137

  8. Allelic variation contributes to bacterial host specificity

    SciTech Connect

    Yue, Min; Han, Xiangan; Masi, Leon De; Zhu, Chunhong; Ma, Xun; Zhang, Junjie; Wu, Renwei; Schmieder, Robert; Kaushik, Radhey S.; Fraser, George P.; Zhao, Shaohua; McDermott, Patrick F.; Weill, François-Xavier; Mainil, Jacques G.; Arze, Cesar; Fricke, W. Florian; Edwards, Robert A.; Brisson, Dustin; Zhang, Nancy R.; Rankin, Shelley C.; Schifferli, Dieter M.

    2015-10-30

    Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. In conclusion, together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts.

  9. Allelic variation contributes to bacterial host specificity

    DOE PAGESBeta

    Yue, Min; Han, Xiangan; Masi, Leon De; Zhu, Chunhong; Ma, Xun; Zhang, Junjie; Wu, Renwei; Schmieder, Robert; Kaushik, Radhey S.; Fraser, George P.; et al

    2015-10-30

    Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population andmore » functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. In conclusion, together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts.« less

  10. Allelic variation contributes to bacterial host specificity

    PubMed Central

    Yue, Min; Han, Xiangan; Masi, Leon De; Zhu, Chunhong; Ma, Xun; Zhang, Junjie; Wu, Renwei; Schmieder, Robert; Kaushik, Radhey S.; Fraser, George P.; Zhao, Shaohua; McDermott, Patrick F.; Weill, François-Xavier; Mainil, Jacques G.; Arze, Cesar; Fricke, W. Florian; Edwards, Robert A.; Brisson, Dustin; Zhang, Nancy R.; Rankin, Shelley C.; Schifferli, Dieter M.

    2015-01-01

    Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. Together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts. PMID:26515720

  11. Allelic variation contributes to bacterial host specificity.

    PubMed

    Yue, Min; Han, Xiangan; De Masi, Leon; Zhu, Chunhong; Ma, Xun; Zhang, Junjie; Wu, Renwei; Schmieder, Robert; Kaushik, Radhey S; Fraser, George P; Zhao, Shaohua; McDermott, Patrick F; Weill, François-Xavier; Mainil, Jacques G; Arze, Cesar; Fricke, W Florian; Edwards, Robert A; Brisson, Dustin; Zhang, Nancy R; Rankin, Shelley C; Schifferli, Dieter M

    2015-01-01

    Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. Together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts. PMID:26515720

  12. Proper Use of Allele-Specific Expression Improves Statistical Power for cis-eQTL Mapping with RNA-Seq Data

    PubMed Central

    HU, Yi-Juan; SUN, Wei; TZENG, Jung-Ying; PEROU, Charles M.

    2015-01-01

    Studies of expression quantitative trait loci (eQTLs) offer insight into the molecular mechanisms of loci that were found to be associated with complex diseases and the mechanisms can be classified into cis- and trans-acting regulation. At present, high-throughput RNA sequencing (RNA-seq) is rapidly replacing expression microarrays to assess gene expression abundance. Unlike microarrays that only measure the total expression of each gene, RNA-seq also provides information on allele-specific expression (ASE), which can be used to distinguish cis-eQTLs from trans-eQTLs and, more importantly, enhance cis-eQTL mapping. However, assessing the cis-effect of a candidate eQTL on a gene requires knowledge of the haplotypes connecting the candidate eQTL and the gene, which cannot be inferred with certainty. The existing two-stage approach that first phases the candidate eQTL against the gene and then treats the inferred phase as observed in the association analysis tends to attenuate the estimated cis-effect and reduce the power for detecting a cis-eQTL. In this article, we provide a maximum-likelihood framework for cis-eQTL mapping with RNA-seq data. Our approach integrates the inference of haplotypes and the association analysis into a single stage, and is thus unbiased and statistically powerful. We also develop a pipeline for performing a comprehensive scan of all local eQTLs for all genes in the genome by controlling for false discovery rate, and implement the methods in a computationally efficient software program. The advantages of the proposed methods over the existing ones are demonstrated through realistic simulation studies and an application to empirical breast cancer data from The Cancer Genome Atlas project. PMID:26568645

  13. Genome-wide identification and quantification of cis- and trans-regulated genes responding to Marek's disease virus infection via analysis of allele-specific expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background Marek’s disease (MD) is a commercially important neoplastic disease of chickens caused by the Marek’s disease virus (MDV), a naturally-occurring oncogenic alphaherpesvirus. We attempted to identify genes conferring MD resistance, by completing a genome-wide screen for allele-specific expr...

  14. Allele-specific DNA methylation reinforces PEAR1 enhancer activity.

    PubMed

    Izzi, Benedetta; Pistoni, Mariaelena; Cludts, Katrien; Akkor, Pinar; Lambrechts, Diether; Verfaillie, Catherine; Verhamme, Peter; Freson, Kathleen; Hoylaerts, Marc F

    2016-08-18

    Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation. PMID:27313330

  15. Fine mapping of QTL and genomic prediction using allele-specific expression SNPs demonstrates that the complex trait of genetic resistance to Marek’s disease is predominantly determined by transcriptional regulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The hypothesis that polymorphisms associated with transcriptional regulation are critical for viral disease resistance was tested by selecting birds using SNPs exhibiting allele-specific expression (ASE) in response to viral challenge. Analysis indicates ASE markers account for 83% of the disease re...

  16. Allele Specific p53 Mutant Reactivation

    PubMed Central

    Yu, Xin; Vazquez, Alexei; Levine, Arnold J.; Carpizo, Darren R.

    2012-01-01

    Summary Rescuing the function of mutant p53 protein is an attractive cancer therapeutic strategy. Using the NCI anticancer drug screen data, we identified two compounds from the thiosemicarbazone family that manifest increased growth inhibitory activity in mutant p53 cells, particularly for the p53R175 mutant. Mechanistic studies reveal that NSC319726 restores WT structure and function to the p53R175 mutant. This compound kills p53R172H knock-in mice with extensive apoptosis and inhibits xenograft tumor growth in a 175-allele specific mutant p53 dependent manner. This activity depends upon the zinc ion chelating properties of the compound as well as redox changes. These data identify NSC319726 as a p53R175 mutant reactivator and as a lead compound for p53 targeted drug development. PMID:22624712

  17. Schizophrenia susceptibility alleles are enriched for alleles that affect gene expression in adult human brain

    PubMed Central

    Richards, Alexander L; Jones, Lesley; Moskvina, Valentina; Kirov, George; Gejman, Pablo V; Levinson, Douglas F; Sanders, Alan R; Purcell, Shaun; Visscher, Peter M; Craddock, Nick; Owen, Michael J; Holmans, Peter; O’Donovan, Michael C

    2016-01-01

    It is widely thought that alleles that influence susceptibility to common diseases, including schizophrenia, will frequently do so through effects on gene expression. Since only a small proportion of the genetic variance for schizophrenia has been attributed to specific loci, this remains an unproven hypothesis. The International Schizophrenia Consortium (ISC) recently reported a substantial polygenic contribution to that disorder, and that schizophrenia risk alleles are enriched among SNPs selected for marginal evidence for association (p<0.5) from genome wide association studies (GWAS). It follows that if schizophrenia susceptibility alleles are enriched for those that affect gene expression, those marginally associated SNPs which are also eQTLs should carry more true association signals compared with SNPs which are not. To test this, we identified marginally associated (p<0.5) SNPs from two of the largest available schizophrenia GWAS datasets. We assigned eQTL status to those SNPs based upon an eQTL dataset derived from adult human brain. Using the polygenic score method of analysis reported by the ISC, we observed and replicated the observation that higher probability cis-eQTLs predicted schizophrenia better than those with a lower probability for being a cis-eQTL. Our data support the hypothesis that alleles conferring risk of schizophrenia are enriched among those that affect gene expression. Moreover, our data show that notwithstanding the likely developmental origin of schizophrenia, studies of adult brain tissue can in principle allow relevant susceptibility eQTLs to be identified. PMID:21339752

  18. Impriniting of human H19: Allele-specific CpG methylation, loss of the active allele in Wilms tumor, and potential for somatic allele switching

    SciTech Connect

    Zhang, Y.; Shields, T.; Crenshaw, T.; Hao, Y.; Moulton, T.; Tycko, B. )

    1993-07-01

    Genomic imprinting and monoallelic gene expression appear to play a role in human genetic disease and tumorigenesis. The human H19 gene, at chromosome 11p15, has previously been shown to be monoallelically expressed. Since CpG methylation has been implicated in imprinting, the authors analyzed methylation of H19 DNA. In fetal and adult organs the transcriptionally silent H19 allele was extensively hypermethylated through the entire gene and its promoter, and, consistent with a functional role for DNA methylation, expression of an H19 promoter-reporter construct was inhibited by in vitro methylation. Gynogenetic ovarian teratomas were found to contain only hypomethylated H19 DNA, suggesting that the expressed H19 allele might be maternal. This was confirmed by analysis of 11p15 polymorphisms in a patient with Wilms tumor. The tumor had lost the maternal 11p15, and H19 expression in the normal kidney was exclusively from this allele. Imprinting of human H19 appears to be susceptible to tissue-specific modulation in somatic development; in one individual, cerebellar cells were found to express only the otherwise silent allele. Implications of these findings for the role of DNA methylation in imprinting and for H19 as a candidate imprinted tumor-suppressor gene are discussed. 57 refs., 7 figs.

  19. Allele-specific disparity in breast cancer

    PubMed Central

    2011-01-01

    Background In a cancer cell the number of copies of a locus may vary due to amplification and deletion and these variations are denoted as copy number alterations (CNAs). We focus on the disparity of CNAs in tumour samples, which were compared to those in blood in order to identify the directional loss of heterozygosity. Methods We propose a numerical algorithm and apply it to data from the Illumina 109K-SNP array on 112 samples from breast cancer patients. B-allele frequency (BAF) and log R ratio (LRR) of Illumina were used to estimate Euclidian distances. For each locus, we compared genotypes in blood and tumour for subset of samples being heterozygous in blood. We identified loci showing preferential disparity from heterozygous toward either the A/B-allele homozygous (allelic disparity). The chi-squared and Cochran-Armitage trend tests were used to examine whether there is an association between high levels of disparity in single nucleotide polymorphisms (SNPs) and molecular, clinical and tumour-related parameters. To identify pathways and network functions over-represented within the resulting gene sets, we used Ingenuity Pathway Analysis (IPA). Results To identify loci with a high level of disparity, we selected SNPs 1) with a substantial degree of disparity and 2) with substantial frequency (at least 50% of the samples heterozygous for the respective locus). We report the overall difference in disparity in high-grade tumours compared to low-grade tumours (p-value < 0.001) and significant associations between disparity in multiple single loci and clinical parameters. The most significantly associated network functions within the genes represented in the loci of disparity were identified, including lipid metabolism, small-molecule biochemistry, and nervous system development and function. No evidence for over-representation of directional disparity in a list of stem cell genes was obtained, however genes appeared to be more often altered by deletion than by

  20. The − 5 A/G single-nucleotide polymorphism in the core promoter region of MT2A and its effect on allele-specific gene expression and Cd, Zn and Cu levels in laryngeal cancer

    SciTech Connect

    Starska, Katarzyna; Krześlak, Anna; Forma, Ewa; Morawiec-Sztandera, Alina; Aleksandrowicz, Paweł; Lewy-Trenda, Iwona; and others

    2014-10-15

    Metallothioneins (MTs) are low molecular weight, cysteine-rich heavy metal-binding proteins which participate in the mechanisms of Zn homeostasis, and protect against toxic metals. MTs contain metal-thiolate cluster groups and suppress metal toxicity by binding to them. The aim of this study was to determine the − 5 A/G (rs28366003) single-nucleotide polymorphism (SNP) in the core promoter region of the MT2A gene and to investigate its effect on allele-specific gene expression and Cd, Zn and Cu content in squamous cell laryngeal cancer (SCC) and non-cancerous laryngeal mucosa (NCM) as a control. The MT2A promoter region − 5 A/G SNP was determined by restriction fragment length polymorphism using 323 SCC and 116 NCM. MT2A gene analysis was performed by quantitative real-time PCR. The frequency of A allele carriage was 94.2% and 91.8% in SCC and NCM, respectively, while G allele carriage was detected in 5.8% and 8.2% of SCC and NCM samples, respectively. As a result, a significant association was identified between the − 5 A/G SNP in the MT2A gene with mRNA expression in both groups. Metal levels were analyzed by flame atomic absorption spectrometry. The significant differences were identified between A/A and both the A/G and G/G genotypes, with regard to the concentration of the contaminating metal. The Spearman rank correlation results showed that the MT2A expression and Cd, Zn, Cu levels were negatively correlated. Results obtained in this study suggest that − 5 A/G SNP in MT2A gene may have an effect on allele-specific gene expression and accumulation of metal levels in laryngeal cancer. - Highlights: • MT2A gene expression and metal content in laryngeal cancer tissues • Association between SNP (rs28366003) and expression of MT2A • Significant associations between the SNP and Cd, Zn and Cu levels • Negative correlation between MT2A gene expression and Cd, Zn and Cu levels.

  1. Inter- and Intra-Individual Variation in Allele-Specific DNA Methylation and Gene Expression in Children Conceived using Assisted Reproductive Technology

    PubMed Central

    Turan, Nahid; Katari, Sunita; Gerson, Leigh F.; Chalian, Raffi; Foster, Michael W.; Gaughan, John P.; Coutifaris, Christos; Sapienza, Carmen

    2010-01-01

    Epidemiological studies have reported a higher incidence of rare disorders involving imprinted genes among children conceived using assisted reproductive technology (ART), suggesting that ART procedures may be disruptive to imprinted gene methylation patterns. We examined intra- and inter-individual variation in DNA methylation at the differentially methylated regions (DMRs) of the IGF2/H19 and IGF2R loci in a population of children conceived in vitro or in vivo. We found substantial variation in allele-specific methylation at both loci in both groups. Aberrant methylation of the maternal IGF2/H19 DMR was more common in the in vitro group, and the overall variance was also significantly greater in the in vitro group. We estimated the number of trophoblast stem cells in each group based on approximation of the variance of the binomial distribution of IGF2/H19 methylation ratios, as well as the distribution of X chromosome inactivation scores in placenta. Both of these independent measures indicated that placentas of the in vitro group were derived from fewer stem cells than the in vivo conceived group. Both IGF2 and H19 mRNAs were significantly lower in placenta from the in vitro group. Although average birth weight was lower in the in vitro group, we found no correlation between birth weight and IGF2 or IGF2R transcript levels or the ratio of IGF2/IGF2R transcript levels. Our results show that in vitro conception is associated with aberrant methylation patterns at the IGF2/H19 locus. However, very little of the inter- or intra-individual variation in H19 or IGF2 mRNA levels can be explained by differences in maternal DMR DNA methylation, in contrast to the expectations of current transcriptional imprinting models. Extraembryonic tissues of embryos cultured in vitro appear to be derived from fewer trophoblast stem cells. It is possible that this developmental difference has an effect on placental and fetal growth. PMID:20661447

  2. The functional importance of sequence versus expression variability of MHC alleles in parasite resistance.

    PubMed

    Axtner, Jan; Sommer, Simone

    2012-12-01

    Understanding selection processes driving the pronounced allelic polymorphism of the major histocompatibility complex (MHC) genes and its functional associations to parasite load have been the focus of many recent wildlife studies. Two main selection scenarios are currently debated which explain the susceptibility or resistance to parasite infections either by the effects of (1) specific MHC alleles which are selected frequency-dependent in space and time or (2) a heterozygote or divergent allele advantage. So far, most studies have focused only on structural variance in co-evolutionary processes although this might not be the only trait subject to natural selection. In the present study, we analysed structural variance stretching from exon1 through exon3 of MHC class II DRB genes as well as genotypic expression variance in relation to the gastrointestinal helminth prevalence and infection intensity in wild yellow-necked mice (Apodemus flavicollis). We found support for the functional importance of specific alleles both on the sequence and expression level. By resampling a previously investigated study population we identified specific MHC alleles affected by temporal shifts in parasite pressure and recorded associated changes in allele frequencies. The allele Apfl-DRB*23 was associated with resistance to infections by the oxyurid nematode Syphacia stroma and at the same time with susceptibility to cestode infection intensity. In line with our expectation, MHC mRNA transcript levels tended to be higher in cestode-infected animals carrying the allele Apfl-DRB*23. However, no support for a heterozygote or divergent allele advantage on the sequence or expression level was detected. The individual amino acid distance of genotypes did not explain individual differences in parasite loads and the genetic distance had no effect on MHC genotype expression. For ongoing studies on the functional importance of expression variance in parasite resistance, allele-specific

  3. Preferential Allele Expression Analysis Identifies Shared Germline and Somatic Driver Genes in Advanced Ovarian Cancer

    PubMed Central

    Halabi, Najeeb M.; Martinez, Alejandra; Al-Farsi, Halema; Mery, Eliane; Puydenus, Laurence; Pujol, Pascal; Khalak, Hanif G.; McLurcan, Cameron; Ferron, Gwenael; Querleu, Denis; Al-Azwani, Iman; Al-Dous, Eman; Mohamoud, Yasmin A.; Malek, Joel A.; Rafii, Arash

    2016-01-01

    Identifying genes where a variant allele is preferentially expressed in tumors could lead to a better understanding of cancer biology and optimization of targeted therapy. However, tumor sample heterogeneity complicates standard approaches for detecting preferential allele expression. We therefore developed a novel approach combining genome and transcriptome sequencing data from the same sample that corrects for sample heterogeneity and identifies significant preferentially expressed alleles. We applied this analysis to epithelial ovarian cancer samples consisting of matched primary ovary and peritoneum and lymph node metastasis. We find that preferentially expressed variant alleles include germline and somatic variants, are shared at a relatively high frequency between patients, and are in gene networks known to be involved in cancer processes. Analysis at a patient level identifies patient-specific preferentially expressed alleles in genes that are targets for known drugs. Analysis at a site level identifies patterns of site specific preferential allele expression with similar pathways being impacted in the primary and metastasis sites. We conclude that genes with preferentially expressed variant alleles can act as cancer drivers and that targeting those genes could lead to new therapeutic strategies. PMID:26735499

  4. Preferential Allele Expression Analysis Identifies Shared Germline and Somatic Driver Genes in Advanced Ovarian Cancer.

    PubMed

    Halabi, Najeeb M; Martinez, Alejandra; Al-Farsi, Halema; Mery, Eliane; Puydenus, Laurence; Pujol, Pascal; Khalak, Hanif G; McLurcan, Cameron; Ferron, Gwenael; Querleu, Denis; Al-Azwani, Iman; Al-Dous, Eman; Mohamoud, Yasmin A; Malek, Joel A; Rafii, Arash

    2016-01-01

    Identifying genes where a variant allele is preferentially expressed in tumors could lead to a better understanding of cancer biology and optimization of targeted therapy. However, tumor sample heterogeneity complicates standard approaches for detecting preferential allele expression. We therefore developed a novel approach combining genome and transcriptome sequencing data from the same sample that corrects for sample heterogeneity and identifies significant preferentially expressed alleles. We applied this analysis to epithelial ovarian cancer samples consisting of matched primary ovary and peritoneum and lymph node metastasis. We find that preferentially expressed variant alleles include germline and somatic variants, are shared at a relatively high frequency between patients, and are in gene networks known to be involved in cancer processes. Analysis at a patient level identifies patient-specific preferentially expressed alleles in genes that are targets for known drugs. Analysis at a site level identifies patterns of site specific preferential allele expression with similar pathways being impacted in the primary and metastasis sites. We conclude that genes with preferentially expressed variant alleles can act as cancer drivers and that targeting those genes could lead to new therapeutic strategies. PMID:26735499

  5. Allele-Specific Induction of IL-1β Expression by C/EBPβ and PU.1 Contributes to Increased Tuberculosis Susceptibility

    PubMed Central

    Zhang, Guoliang; Zhou, Boping; Li, Shaoyuan; Yue, Jun; Yang, Hui; Wen, Yuxin; Zhan, Senlin; Wang, Wenfei; Liao, Mingfeng; Zhang, Mingxia; Zeng, Gucheng; Feng, Carl G.; Sassetti, Christopher M.; Chen, Xinchun

    2014-01-01

    Mycobacterium tuberculosis infection is associated with a spectrum of clinical outcomes, from long-term latent infection to different manifestations of progressive disease. Pro-inflammatory pathways, such as those controlled by IL-1β, have the contrasting potential both to prevent disease by restricting bacterial replication, and to promote disease by inflicting tissue damage. Thus, the ultimate contribution of individual inflammatory pathways to the outcome of M. tuberculosis infection remains ambiguous. In this study, we identified a naturally-occurring polymorphism in the human IL1B promoter region, which alters the association of the C/EBPβ and PU.1 transcription factors and controls Mtb-induced IL-1β production. The high-IL-1β expressing genotype was associated with the development of active tuberculosis, the severity of pulmonary disease and poor treatment outcome in TB patients. Higher IL-1β expression did not suppress the activity of IFN-γ-producing T cells, but instead correlated with neutrophil accumulation in the lung. These observations support a specific role for IL-1β and granulocytic inflammation as a driver of TB disease progression in humans, and suggest novel strategies for the prevention and treatment of tuberculosis. PMID:25329476

  6. Comparison and contrast of genes and biological pathways responding to Marek’s disease virus infection using allele-specific expression and differential expression in broiler and layer chickens

    PubMed Central

    2013-01-01

    Background Marek’s disease (MD) is a commercially important neoplastic disease of chickens caused by the Marek’s disease virus (MDV), a naturally occurring oncogenic alphaherpesvirus. Enhancing MD genetic resistance is desirable to augment current vaccines and other MD control measures. High throughput sequencing was used to profile splenic transcriptomes from individual F1 progeny infected with MDV at 4 days of age from both outbred broilers (meat-type) and inbred layer (egg-type) chicken lines that differed in MD genetic resistance. The resulting information was used to identify SNPs, genes, and biological pathways exhibiting allele-specific expression (ASE) in response to MDV infection in each type of chicken. In addition, we compared and contrasted the results of pathway analyses (ASE and differential expression (DE)) between chicken types to help inform on the biological response to MDV infection. Results With 7 individuals per line and treatment group providing high power, we identified 6,132 single nucleotide polymorphisms (SNPs) in 4,768 genes and 4,528 SNPs in 3,718 genes in broilers and layers, respectively, that exhibited ASE in response to MDV infection. Furthermore, 548 and 434 genes in broilers and layers, respectively, were found to show DE following MDV infection. Comparing the datasets, only 72 SNPs and 850 genes for ASE and 20 genes for DE were common between the two bird types. Although the chicken types used in this study were genetically different, at the pathway level, both TLR receptor and JAK/STAT signaling pathways were enriched as well as exhibiting a high proportion of ASE genes, especially at the beginning of both above mentioned regulatory pathways. Conclusions RNA sequencing with adequate biological replicates is a powerful approach to identify high confidence SNPs, genes, and pathways that are associated with transcriptional response to MDV infection. In addition, the SNPs exhibiting ASE in response to MDV infection provide a

  7. The Non-coding Mammary Carcinoma Susceptibility Locus, Mcs5c, Regulates Pappa Expression via Age-Specific Chromatin Folding and Allele-Dependent DNA Methylation

    PubMed Central

    Henning, Amanda N.; Haag, Jill D.; Smits, Bart M. G.; Gould, Michael N.

    2016-01-01

    In understanding the etiology of breast cancer, the contributions of both genetic and environmental risk factors are further complicated by the impact of breast developmental stage. Specifically, the time period ranging from childhood to young adulthood represents a critical developmental window in a woman’s life when she is more susceptible to environmental hazards that may affect future breast cancer risk. Although the effects of environmental exposures during particular developmental Windows of Susceptibility (WOS) are well documented, the genetic mechanisms governing these interactions are largely unknown. Functional characterization of the Mammary Carcinoma Susceptibility 5c, Mcs5c, congenic rat model of breast cancer at various stages of mammary gland development was conducted to gain insight into the interplay between genetic risk factors and WOS. Using quantitative real-time PCR, chromosome conformation capture, and bisulfite pyrosequencing we have found that Mcs5c acts within the mammary gland to regulate expression of the neighboring gene Pappa during a critical mammary developmental time period in the rat, corresponding to the human young adult WOS. Pappa has been shown to positively regulate the IGF signaling pathway, which is required for proper mammary gland/breast development and is of increasing interest in breast cancer pathogenesis. Mcs5c-mediated regulation of Pappa appears to occur through age-dependent and mammary gland-specific chromatin looping, as well as genotype-dependent CpG island shore methylation. This represents, to our knowledge, the first insight into cellular mechanisms underlying the WOS phenomenon and demonstrates the influence developmental stage can have on risk locus functionality. Additionally, this work represents a novel model for further investigation into how environmental factors, together with genetic factors, modulate breast cancer risk in the context of breast developmental stage. PMID:27537370

  8. The Non-coding Mammary Carcinoma Susceptibility Locus, Mcs5c, Regulates Pappa Expression via Age-Specific Chromatin Folding and Allele-Dependent DNA Methylation.

    PubMed

    Henning, Amanda N; Haag, Jill D; Smits, Bart M G; Gould, Michael N

    2016-08-01

    In understanding the etiology of breast cancer, the contributions of both genetic and environmental risk factors are further complicated by the impact of breast developmental stage. Specifically, the time period ranging from childhood to young adulthood represents a critical developmental window in a woman's life when she is more susceptible to environmental hazards that may affect future breast cancer risk. Although the effects of environmental exposures during particular developmental Windows of Susceptibility (WOS) are well documented, the genetic mechanisms governing these interactions are largely unknown. Functional characterization of the Mammary Carcinoma Susceptibility 5c, Mcs5c, congenic rat model of breast cancer at various stages of mammary gland development was conducted to gain insight into the interplay between genetic risk factors and WOS. Using quantitative real-time PCR, chromosome conformation capture, and bisulfite pyrosequencing we have found that Mcs5c acts within the mammary gland to regulate expression of the neighboring gene Pappa during a critical mammary developmental time period in the rat, corresponding to the human young adult WOS. Pappa has been shown to positively regulate the IGF signaling pathway, which is required for proper mammary gland/breast development and is of increasing interest in breast cancer pathogenesis. Mcs5c-mediated regulation of Pappa appears to occur through age-dependent and mammary gland-specific chromatin looping, as well as genotype-dependent CpG island shore methylation. This represents, to our knowledge, the first insight into cellular mechanisms underlying the WOS phenomenon and demonstrates the influence developmental stage can have on risk locus functionality. Additionally, this work represents a novel model for further investigation into how environmental factors, together with genetic factors, modulate breast cancer risk in the context of breast developmental stage. PMID:27537370

  9. MICB Allele Genotyping on Microarrays by Improving the Specificity of Extension Primers

    PubMed Central

    Baek, In-Cheol; Jang, Jung-Pil; Choi, Eun-Jeong; Kim, Tai-Gyu

    2015-01-01

    Major histocompatibility complex (MHC) class I chain-related gene B (MICB) encodes a ligand for activating NKG2D that expressed in natural killer cells, γδ T cells, and αβ CD8+ T cells, which is associated with autoimmune diseases, cancer, and infectious diseases. Here, we have established a system for genotyping MICB alleles using allele-specific primer extension (ASPE) on microarrays. Thirty-six high quality, allele-specific extension primers were evaluated using strict and reliable cut-off values using mean fluorescence intensity (MFI), whereby an MFI >30,000 represented a positive signal and an MFI <10,000 represented a negative signal. Eight allele-specific extension primers were found to be false positives, five of which were improved by adjusting their length, and three of which were optimized by refractory modification. The MICB alleles (*002:01, *003, *005:02/*010, *005:03, *008, *009N, *018, and *024) present in the quality control panel could be exactly defined by 22 allele-specific extension primers. MICB genotypes that were identified by ASPE on microarrays were in full concordance with those identified by PCR-sequence-based typing. In conclusion, we have developed a method for genotyping MICB alleles using ASPE on microarrays; which can be applicable for large-scale single nucleotide polymorphism typing studies of population and disease associations. PMID:26569110

  10. Distinct allelic patterns of nanog expression impart embryonic stem cell population heterogeneity.

    PubMed

    Wu, Jincheng; Tzanakakis, Emmanuel S

    2013-01-01

    Nanog is a principal pluripotency regulator exhibiting a disperse distribution within stem cell populations in vivo and in vitro. Increasing evidence points to a functional role of Nanog heterogeneity on stem cell fate decisions. Allelic control of Nanog gene expression was reported recently in mouse embryonic stem cells. To better understand how this mode of regulation influences the observed heterogeneity of NANOG in stem cell populations, we assembled a multiscale stochastic population balance equation framework. In addition to allelic control, gene expression noise and random partitioning at cell division were considered. As a result of allelic Nanog expression, the distribution of Nanog exhibited three distinct states but when combined with transcriptional noise the profile became bimodal. Regardless of their allelic expression pattern, initially uniform populations of stem cells gave rise to the same Nanog heterogeneity within ten cell cycles. Depletion of NANOG content in cells switching off both gene alleles was slower than the accumulation of intracellular NANOG after cells turned on at least one of their Nanog gene copies pointing to Nanog state-dependent dynamics. Allelic transcription of Nanog also raises issues regarding the use of stem cell lines with reporter genes knocked in a single allelic locus. Indeed, significant divergence was observed in the reporter and native protein profiles depending on the difference in their half-lives and insertion of the reporter gene in one or both alleles. In stem cell populations with restricted Nanog expression, allelic regulation facilitates the maintenance of fractions of self-renewing cells with sufficient Nanog content to prevent aberrant loss of pluripotency. Our findings underline the role of allelic control of Nanog expression as a prime determinant of stem cell population heterogeneity and warrant further investigation in the contexts of stem cell specification and cell reprogramming. PMID:23874182

  11. Tissue-specific patterns of allelically-skewed DNA methylation

    PubMed Central

    Marzi, Sarah J.; Meaburn, Emma L.; Dempster, Emma L.; Lunnon, Katie; Paya-Cano, Jose L.; Smith, Rebecca G.; Volta, Manuela; Troakes, Claire; Schalkwyk, Leonard C.; Mill, Jonathan

    2016-01-01

    ABSTRACT While DNA methylation is usually thought to be symmetrical across both alleles, there are some notable exceptions. Genomic imprinting and X chromosome inactivation are two well-studied sources of allele-specific methylation (ASM), but recent research has indicated a more complex pattern in which genotypic variation can be associated with allelically-skewed DNA methylation in cis. Given the known heterogeneity of DNA methylation across tissues and cell types we explored inter- and intra-individual variation in ASM across several regions of the human brain and whole blood from multiple individuals. Consistent with previous studies, we find widespread ASM with > 4% of the ∼220,000 loci interrogated showing evidence of allelically-skewed DNA methylation. We identify ASM flanking known imprinted regions, and show that ASM sites are enriched in DNase I hypersensitivity sites and often located in an extended genomic context of intermediate DNA methylation. We also detect examples of genotype-driven ASM, some of which are tissue-specific. These findings contribute to our understanding of the nature of differential DNA methylation across tissues and have important implications for genetic studies of complex disease. As a resource to the community, ASM patterns across each of the tissues studied are available in a searchable online database: http://epigenetics.essex.ac.uk/ASMBrainBlood. PMID:26786711

  12. Evidence of Allelic Suppression for Transcripts Expressed in Day 30 Pig Embryos by SNP Genotyping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genomic imprinting results in alleles being differentially expressed in a parent-of-origin specific manner. Parthenogenetic and biparental pig embryo gene expression profiles were compared using three cDNA microarray platforms. Comparison of the profiles of the two tissue types indicated different...

  13. Origins, distribution and expression of the Duarte-2 (D2) allele of galactose-1-phosphate uridylyltransferase

    PubMed Central

    Carney, Amanda E.; Sanders, Rebecca D.; Garza, Kerry R.; McGaha, Lee Anne; Bean, Lora J. H.; Coffee, Bradford W.; Thomas, James W.; Cutler, David J.; Kurtkaya, Natalie L.; Fridovich-Keil, Judith L.

    2009-01-01

    Duarte galactosemia is a mild to asymptomatic condition that results from partial impairment of galactose-1-phosphate uridylyltransferase (GALT). Patients with Duarte galactosemia demonstrate reduced GALT activity and carry one profoundly impaired GALT allele (G) along with a second, partially impaired GALT allele (Duarte-2, D2). Molecular studies reveal at least five sequence changes on D2 alleles: a p.N314D missense substitution, three intronic base changes and a 4 bp deletion in the 5′ proximal sequence. The four non-coding sequence changes are unique to D2. The p.N314D substitution, however, is not; it is found together with a silent polymorphism, p.L218(TTA), on functionally normal Duarte-1 alleles (D1, also called Los Angeles or LA alleles). The HapMap database reveals that p.N314D is a common human variant, and cross-species comparisons implicate D314 as the ancestral allele. The p.N314D substitution is also functionally neutral in mammalian cell and yeast expression studies. In contrast, the 4 bp 5′ deletion characteristic of D2 alleles appears to be functionally impaired in reporter gene transfection studies. Here we present allele-specific qRT–PCR evidence that D2 alleles express less mRNA in vivo than their wild-type counterparts; the difference is small but statistically significant. Furthermore, we characterize the prevalence of the 4 bp deletion in GG, NN and DG populations; the deletion appears exclusive to D2 alleles. Combined, these data strongly implicate the 4 bp 5′ deletion as a causal mutation in Duarte galactosemia and suggest that direct tests for this deletion, as proposed here, could enhance or supplant current tests, which define D2 alleles on the basis of the presence and absence of linked coding sequence polymorphisms. PMID:19224951

  14. How the Number of Alleles Influences Gene Expression

    NASA Astrophysics Data System (ADS)

    Hat, Beata; Paszek, Pawel; Kimmel, Marek; Piechor, Kazimierz; Lipniacki, Tomasz

    2007-07-01

    The higher organisms, eukaryotes, are diploid and most of their genes have two homological copies (alleles). However, the number of alleles in a cell is not constant. In the S phase of the cell cycle all the genome is duplicated and then in the G2 phase and mitosis, which together last for several hours, most of the genes have four copies instead of two. Cancer development is, in many cases, associated with a change in allele number. Several genetic diseases are caused by haploinsufficiency: Lack of one of the alleles or its improper functioning. In the paper we consider the stochastic expression of a gene having a variable number of copies. We applied our previously developed method in which the reaction channels are split into slow (connected with change of gene state) and fast (connected with mRNA/protein synthesis/decay), the later being approximated by deterministic reaction rate equations. As a result we represent gene expression as a piecewise deterministic time-continuous Markov process, which is further related with a system of partial differential hyperbolic equations for probability density functions (pdfs) of protein distribution. The stationary pdfs are calculated analytically for haploidal gene or numerically for diploidal and tetraploidal ones. We distinguished nine classes of simultaneous activation of haploid, diploid and tetraploid genes. This allows for analysis of potential consequences of gene duplication or allele loss. We show that when gene activity is autoregulated by a positive feedback, the change in number of gene alleles may have dramatic consequences for its regulation and may not be compensated by the change of efficiency of mRNA synthesis per allele.

  15. Allele-specific tumor spectrum in pten knockin mice.

    PubMed

    Wang, Hui; Karikomi, Matt; Naidu, Shan; Rajmohan, Ravi; Caserta, Enrico; Chen, Hui-Zi; Rawahneh, Maysoon; Moffitt, Julie; Stephens, Julie A; Fernandez, Soledad A; Weinstein, Michael; Wang, Danxin; Sadee, Wolfgang; La Perle, Krista; Stromberg, Paul; Rosol, Thomas J; Eng, Charis; Ostrowski, Michael C; Leone, Gustavo

    2010-03-16

    Germline mutations in the tumor suppressor gene PTEN (phosphatase and tensin homology deleted on chromosome 10) cause Cowden and Bannayan-Riley-Ruvalcaba (BRR) syndromes, two dominantly inherited disorders characterized by mental retardation, multiple hamartomas, and variable cancer risk. Here, we modeled three sentinel mutant alleles of PTEN identified in patients with Cowden syndrome and show that the nonsense Pten(4-5) and missense Pten(C124R) and Pten(G129E) alleles lacking lipid phosphatase activity cause similar developmental abnormalities but distinct tumor spectra with varying severity and age of onset. Allele-specific differences may be accounted for by loss of function for Pten(4-5), hypomorphic function for Pten(C124R), and gain of function for Pten(G129E). These data demonstrate that the variable tumor phenotypes observed in patients with Cowden and BRR syndromes can be attributed to specific mutations in PTEN that alter protein function through distinct mechanisms. PMID:20194734

  16. Characterizing noise structure in single-cell RNA-seq distinguishes genuine from technical stochastic allelic expression.

    PubMed

    Kim, Jong Kyoung; Kolodziejczyk, Aleksandra A; Ilicic, Tomislav; Illicic, Tomislav; Teichmann, Sarah A; Marioni, John C

    2015-01-01

    Single-cell RNA-sequencing (scRNA-seq) facilitates identification of new cell types and gene regulatory networks as well as dissection of the kinetics of gene expression and patterns of allele-specific expression. However, to facilitate such analyses, separating biological variability from the high level of technical noise that affects scRNA-seq protocols is vital. Here we describe and validate a generative statistical model that accurately quantifies technical noise with the help of external RNA spike-ins. Applying our approach to investigate stochastic allele-specific expression in individual cells, we demonstrate that a large fraction of stochastic allele-specific expression can be explained by technical noise, especially for lowly and moderately expressed genes: we predict that only 17.8% of stochastic allele-specific expression patterns are attributable to biological noise with the remainder due to technical noise. PMID:26489834

  17. SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes

    PubMed Central

    Krueger, Felix; Andrews, Simon R.

    2016-01-01

    Sequencing reads overlapping polymorphic sites in diploid mammalian genomes may be assigned to one allele or the other. This holds the potential to detect gene expression, chromatin modifications, DNA methylation or nuclear interactions in an allele-specific fashion. SNPsplit is an allele-specific alignment sorter designed to read files in SAM/BAM format and determine the allelic origin of reads or read-pairs that cover known single nucleotide polymorphic (SNP) positions. For this to work libraries must have been aligned to a genome in which all known SNP positions were masked with the ambiguity base 'N' and aligned using a suitable mapping program such as Bowtie2, TopHat, STAR, HISAT2, HiCUP or Bismark. SNPsplit also provides an automated solution to generate N-masked reference genomes for hybrid mouse strains based on the variant call information provided by the Mouse Genomes Project. The unique ability of SNPsplit to work with various different kinds of sequencing data including RNA-Seq, ChIP-Seq, Bisulfite-Seq or Hi-C opens new avenues for the integrative exploration of allele-specific data. PMID:27429743

  18. SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes.

    PubMed

    Krueger, Felix; Andrews, Simon R

    2016-01-01

    Sequencing reads overlapping polymorphic sites in diploid mammalian genomes may be assigned to one allele or the other. This holds the potential to detect gene expression, chromatin modifications, DNA methylation or nuclear interactions in an allele-specific fashion. SNPsplit is an allele-specific alignment sorter designed to read files in SAM/BAM format and determine the allelic origin of reads or read-pairs that cover known single nucleotide polymorphic (SNP) positions. For this to work libraries must have been aligned to a genome in which all known SNP positions were masked with the ambiguity base 'N' and aligned using a suitable mapping program such as Bowtie2, TopHat, STAR, HISAT2, HiCUP or Bismark. SNPsplit also provides an automated solution to generate N-masked reference genomes for hybrid mouse strains based on the variant call information provided by the Mouse Genomes Project. The unique ability of SNPsplit to work with various different kinds of sequencing data including RNA-Seq, ChIP-Seq, Bisulfite-Seq or Hi-C opens new avenues for the integrative exploration of allele-specific data. PMID:27429743

  19. Multiple Avirulence Loci and Allele-Specific Effector Recognition Control the Pm3 Race-Specific Resistance of Wheat to Powdery Mildew[OPEN

    PubMed Central

    Roffler, Stefan; Stirnweis, Daniel; Treier, Georges; Herren, Gerhard; Korol, Abraham B.; Wicker, Thomas

    2015-01-01

    In cereals, several mildew resistance genes occur as large allelic series; for example, in wheat (Triticum aestivum and Triticum turgidum), 17 functional Pm3 alleles confer agronomically important race-specific resistance to powdery mildew (Blumeria graminis). The molecular basis of race specificity has been characterized in wheat, but little is known about the corresponding avirulence genes in powdery mildew. Here, we dissected the genetics of avirulence for six Pm3 alleles and found that three major Avr loci affect avirulence, with a common locus_1 involved in all AvrPm3-Pm3 interactions. We cloned the effector gene AvrPm3a2/f2 from locus_2, which is recognized by the Pm3a and Pm3f alleles. Induction of a Pm3 allele-dependent hypersensitive response in transient assays in Nicotiana benthamiana and in wheat demonstrated specificity. Gene expression analysis of Bcg1 (encoded by locus_1) and AvrPm3 a2/f2 revealed significant differences between isolates, indicating that in addition to protein polymorphisms, expression levels play a role in avirulence. We propose a model for race specificity involving three components: an allele-specific avirulence effector, a resistance gene allele, and a pathogen-encoded suppressor of avirulence. Thus, whereas a genetically simple allelic series controls specificity in the plant host, recognition on the pathogen side is more complex, allowing flexible evolutionary responses and adaptation to resistance genes. PMID:26452600

  20. Correction of Hair Shaft Defects through Allele-Specific Silencing of Mutant Krt75.

    PubMed

    Liu, Ying; Snedecor, Elizabeth R; Zhang, Xu; Xu, Yanfeng; Huang, Lan; Jones, Evan C; Zhang, Lianfeng; Clark, Richard A; Roop, Dennis R; Qin, Chuan; Chen, Jiang

    2016-01-01

    Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele-specific small interfering RNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific short hairpin RNA (shRNA) persistently suppressed this phenotype. The phenotypic correction was associated with a significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of mutant keratin genes. PMID:26763422

  1. Correction of hair shaft defects through allele-specific silencing of mutant Krt75

    PubMed Central

    Liu, Ying; Snedecor, Elizabeth R.; Zhang, Xu; Xu, Yan-Feng; Huang, Lan; Jones, Evan; Zhang, Lianfeng; Clark, Richard A.; Roop, Dennis R.; Qin, Chuan; Chen, Jiang

    2015-01-01

    Dominant mutations in keratin genes can cause a number of inheritable skin disorders characterized by intraepidermal blistering, epidermal hyperkeratosis, or abnormalities in skin appendages, such as nail plate dystrophy and structural defects in hair. Allele-specific silencing of mutant keratins through RNA interference is a promising therapeutic approach for suppressing the expression of mutant keratins and related phenotypes in the epidermis. However, its effectiveness on skin appendages remains to be confirmed in vivo. In this study, we developed allele specific siRNAs capable of selectively suppressing the expression of a mutant Krt75, which causes hair shaft structural defects characterized by the development of blebs along the hair shaft in mice. Hair regenerated from epidermal keratinocyte progenitor cells isolated from mutant Krt75 mouse models reproduced the blebbing phenotype when grafted in vivo. In contrast, mutant cells manipulated with a lentiviral vector expressing mutant Krt75-specific shRNA persistently suppressed this phenotype. The phenotypic correction was associated with significant reduction of mutant Krt75 mRNA in the skin grafts. Thus, data obtained from this study demonstrated the feasibility of utilizing RNA interference to achieve durable correction of hair structural phenotypes through allele-specific silencing of the mutant keratin genes. PMID:26763422

  2. Regulatory Divergence between Parental Alleles Determines Gene Expression Patterns in Hybrids

    PubMed Central

    Combes, Marie-Christine; Hueber, Yann; Dereeper, Alexis; Rialle, Stéphanie; Herrera, Juan-Carlos; Lashermes, Philippe

    2015-01-01

    Both hybridization and allopolyploidization generate novel phenotypes by conciliating divergent genomes and regulatory networks in the same cellular context. To understand the rewiring of gene expression in hybrids, the total expression of 21,025 genes and the allele-specific expression of over 11,000 genes were quantified in interspecific hybrids and their parental species, Coffea canephora and Coffea eugenioides using RNA-seq technology. Between parental species, cis- and trans-regulatory divergences affected around 32% and 35% of analyzed genes, respectively, with nearly 17% of them showing both. The relative importance of trans-regulatory divergences between both species could be related to their low genetic divergence and perennial habit. In hybrids, among divergently expressed genes between parental species and hybrids, 77% was expressed like one parent (expression level dominance), including 65% like C. eugenioides. Gene expression was shown to result from the expression of both alleles affected by intertwined parental trans-regulatory factors. A strong impact of C. eugenioides trans-regulatory factors on the upregulation of C. canephora alleles was revealed. The gene expression patterns appeared determined by complex combinations of cis- and trans-regulatory divergences. In particular, the observed biased expression level dominance seemed to be derived from the asymmetric effects of trans-regulatory parental factors on regulation of alleles. More generally, this study illustrates the effects of divergent trans-regulatory parental factors on the gene expression pattern in hybrids. The characteristics of the transcriptional response to hybridization appear to be determined by the compatibility of gene regulatory networks and therefore depend on genetic divergences between the parental species and their evolutionary history. PMID:25819221

  3. Allelic Imbalance Is a Prevalent and Tissue-Specific Feature of the Mouse Transcriptome

    PubMed Central

    Pinter, Stefan F.; Colognori, David; Beliveau, Brian J.; Sadreyev, Ruslan I.; Payer, Bernhard; Yildirim, Eda; Wu, Chao-ting; Lee, Jeannie T.

    2015-01-01

    In mammals, several classes of monoallelic genes have been identified, including those subject to X-chromosome inactivation (XCI), genomic imprinting, and random monoallelic expression (RMAE). However, the extent to which these epigenetic phenomena are influenced by underlying genetic variation is unknown. Here we perform a systematic classification of allelic imbalance in mouse hybrids derived from reciprocal crosses of divergent strains. We observe that deviation from balanced biallelic expression is common, occurring in ∼20% of the mouse transcriptome in a given tissue. Allelic imbalance attributed to genotypic variation is by far the most prevalent class and typically is tissue-specific. However, some genotype-based imbalance is maintained across tissues and is associated with greater genetic variation, especially in 5′ and 3′ termini of transcripts. We further identify novel random monoallelic and imprinted genes and find that genotype can modify penetrance of parental origin even in the setting of large imprinted regions. Examination of nascent transcripts in single cells from inbred parental strains reveals that genes showing genotype-based imbalance in hybrids can also exhibit monoallelic expression in isogenic backgrounds. This surprising observation may suggest a competition between alleles and/or reflect the combined impact of cis- and trans-acting variation on expression of a given gene. Our findings provide novel insights into gene regulation and may be relevant to human genetic variation and disease. PMID:25858912

  4. Naturally occurring ERAP1 haplotypes encode functionally distinct alleles with fine substrate specificity.

    PubMed

    Reeves, Emma; Edwards, Christopher J; Elliott, Tim; James, Edward

    2013-07-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims peptides for MHC class I presentation, influencing the degree and specificity of CD8(+) T cell responses. Single-nucleotide polymorphisms within the exons encoding ERAP1 are associated with autoimmune diseases and cervical carcinoma, but it is not known whether they act independently or as disease-associated haplotypes. We sequenced ERAP1 from 20 individuals and show that single-nucleotide polymorphisms occur as distinct haplotypes in the human population and that these haplotypes encode functionally distinct ERAP1 alleles. Using a wide range of substrates, we are able to demonstrate that for any given substrate distinct ERAP1 alleles can be "normal," "hypofunctional," or "hyperfunctional" and that each allele has a trend bias toward one of these three activities. Thus, the repertoire of peptides presented at the cell surface for recognition by CTL is likely to depend on the precise combination of both MHC class I and ERAP1 alleles expressed within an individual, and has important implications for predisposition to disease. PMID:23733883

  5. Extensive allele-specific translational regulation in hybrid mice

    PubMed Central

    Hou, Jingyi; Wang, Xi; McShane, Erik; Zauber, Henrik; Sun, Wei; Selbach, Matthias; Chen, Wei

    2015-01-01

    Translational regulation is mediated through the interaction between diffusible trans-factors and cis-elements residing within mRNA transcripts. In contrast to extensively studied transcriptional regulation, cis-regulation on translation remains underexplored. Using deep sequencing-based transcriptome and polysome profiling, we globally profiled allele-specific translational efficiency for the first time in an F1 hybrid mouse. Out of 7,156 genes with reliable quantification of both alleles, we found 1,008 (14.1%) exhibiting significant allelic divergence in translational efficiency. Systematic analysis of sequence features of the genes with biased allelic translation revealed that local RNA secondary structure surrounding the start codon and proximal out-of-frame upstream AUGs could affect translational efficiency. Finally, we observed that the cis-effect was quantitatively comparable between transcriptional and translational regulation. Such effects in the two regulatory processes were more frequently compensatory, suggesting that the regulation at the two levels could be coordinated in maintaining robustness of protein expression. PMID:26253569

  6. Tissue-specific imprinting of the mouse insulin-like growth factor II receptor gene correlates with differential allele-specific DNA methylation.

    PubMed

    Hu, J F; Oruganti, H; Vu, T H; Hoffman, A R

    1998-02-01

    Imprinted genes may be expressed uniparentally in a tissue- and development-specific manner. The insulin-like growth factor II receptor gene (Igf2r), one of the first imprinted genes to be identified, is an attractive candidate for studying the molecular mechanism of genomic imprinting because it is transcribed monoallelically in the mouse but biallelically in humans. To identify the factors that control genomic imprinting, we examined allelic expression of Igf2r at different ages in interspecific mice. We found that Igf2r is not always monoallelically expressed. Paternal imprinting of Igf2r is maintained in peripheral tissues, including liver, kidney, heart, spleen, intestine, bladder, skin, bone, and skeletal muscle. However, in central nervous system (CNS), Igf2r is expressed from both parental alleles. Southern analysis of the Igf2r promoter (region 1) revealed that, outside of the CNS where Igf2r is monoallelically expressed, the suppressed paternal allele is fully methylated while the expressed maternal allele is completely unmethylated. In CNS, however, both parental alleles are unmethylated in region 1. The importance of DNA methylation in the maintenance of the genomic imprint was also confirmed by the finding that Igf2r imprinting was relaxed by 5-azacytidine treatment. The correlation between genomic imprinting and allelic Igf2r methylation in CNS and other tissues thus suggests that the epigenetic modification in the promoter region may function as one of the major factors in maintaining the monoallelic expression of Igf2r. PMID:9482664

  7. Allelic divergence and cultivar-specific SSR alleles revealed by capillary electrophoresis using fluorescence-labeled SSR markers in sugarcane

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Though sugarcane cultivars (Saccharum spp. hybrids) are complex aneu-polyploid hybrids, genetic evaluation and tracking of clone- or cultivar-specific alleles become possible due to capillary electrophoregrams (CE) using fluorescence-labeled SSR primer pairs. Twenty-four sugarcane cultivars, 12 each...

  8. Allele-specific enzymatic amplification of. beta. -globin genomic DNA for diagnosis of sickle cell anemia

    SciTech Connect

    Wu, D.Y.; Ugozzoli, L.; Pal, B.K.; Wallace, B. )

    1989-04-01

    A rapid nonradioactive approach to the diagnosis of sickle cell anemia is described based on an allele-specific polymerase chain reaction (ASPCR). This method allows direct detection of the normal or the sickle cell {beta}-globin allele in genomic DNA without additional steps of probe hybridization, ligation, or restriction enzyme cleavage. Two allele-specific oligonucleotide primers, one specific for the sickle cell allele and one specific for the normal allele, together with another primer complementary to both alleles were used in the polymerase chain reaction with genomic DNA templates. The allele-specific primers differed from each other in their terminal 3{prime} nucleotide. Under the proper annealing temperature and polymerase chain reaction conditions, these primers only directed amplification on their complementary allele. In a single blind study of DNA samples from 12 individuals, this method correctly and unambiguously allowed for the determination of the genotypes with no false negatives or positives. If ASPCR is able to discriminate all allelic variation (both transition and transversion mutations), this method has the potential to be a powerful approach for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing.

  9. Allele-specific silencing of mutant Ataxin-7 in SCA7 patient-derived fibroblasts.

    PubMed

    Scholefield, Janine; Watson, Lauren; Smith, Danielle; Greenberg, Jacquie; Wood, Matthew J A

    2014-12-01

    Polyglutamine (polyQ) disorders are inherited neurodegenerative conditions defined by a common pathogenic CAG repeat expansion leading to a toxic gain-of-function of the mutant protein. Consequences of this toxicity include activation of heat-shock proteins (HSPs), impairment of the ubiquitin-proteasome pathway and transcriptional dysregulation. Several studies in animal models have shown that reducing levels of toxic protein using small RNAs would be an ideal therapeutic approach for such disorders, including spinocerebellar ataxia-7 (SCA7). However, testing such RNA interference (RNAi) effectors in genetically appropriate patient cell lines with a disease-relevant phenotype has yet to be explored. Here, we have used primary adult dermal fibroblasts from SCA7 patients and controls to assess the endogenous allele-specific silencing of ataxin-7 by two distinct siRNAs. We further identified altered expression of two disease-relevant transcripts in SCA7 patient cells: a twofold increase in levels of the HSP DNAJA1 and a twofold decrease in levels of the de-ubiquitinating enzyme, UCHL1. After siRNA treatment, the expression of both genes was restored towards normal levels. To our knowledge, this is the first time that allele-specific silencing of mutant ataxin-7, targeting a common SNP, has been demonstrated in patient cells. These findings highlight the advantage of an allele-specific RNAi-based therapeutic approach, and indicate the value of primary patient-derived cells as useful models for mechanistic studies and for measuring efficacy of RNAi effectors on a patient-to-patient basis in the polyQ diseases. PMID:24667781

  10. Comparative Anatomy of Chromosomal Domains with Imprinted and Non-Imprinted Allele-Specific DNA Methylation

    PubMed Central

    Kerkel, Kristi; Yale, Alexander; Yotova, Iveta; Drost, Natalia; Lax, Simon; Nhan-Chang, Chia-Ling; Powell, Charles; Borczuk, Alain; Aviv, Abraham; Wapner, Ronald; Chen, Xiaowei; Nagy, Peter L.; Schork, Nicholas; Do, Catherine; Torkamani, Ali; Tycko, Benjamin

    2013-01-01

    Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM. PMID:24009515

  11. Comparative anatomy of chromosomal domains with imprinted and non-imprinted allele-specific DNA methylation.

    PubMed

    Paliwal, Anupam; Temkin, Alexis M; Kerkel, Kristi; Yale, Alexander; Yotova, Iveta; Drost, Natalia; Lax, Simon; Nhan-Chang, Chia-Ling; Powell, Charles; Borczuk, Alain; Aviv, Abraham; Wapner, Ronald; Chen, Xiaowei; Nagy, Peter L; Schork, Nicholas; Do, Catherine; Torkamani, Ali; Tycko, Benjamin

    2013-08-01

    Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM. PMID:24009515

  12. Determination of allelic expression of h19 in pre- and peri-implantation mouse embryos.

    PubMed

    Negrón-Pérez, Verónica M; Echevarría, Franklin D; Huffman, Sarah R; Rivera, Rocío Melissa

    2013-04-01

    H19 is a maternally expressed, imprinted, noncoding RNA with tumor-suppressor activity. During mouse preimplantation development, H19 is primarily expressed in the trophectoderm cells. The purpose of this project was to determine allelic expression of H19 in pre- and peri-implantation mouse embryos. We were further interested in determining if loss of imprinted H19 expression during blastocyst development occurred as a result of superovulation and/or culture. Our last goal was to ascertain if differential H19 allelic expression occurred between the inner cell mass (ICM)-containing half and the primary trophoblast giant cell (PTGC)-containing half of the embryo. C57BL/6J((Cast-7))xC57BL/6J F1 embryos were collected from the uterus at 84, 96, and 108 h following natural ovulation or superovulation. In vitro-cultured F1 embryos were harvested from the oviduct at the 2-cell stage and cultured in KSOM + aa supplemented with amino acids or Whitten media and collected at the above-mentioned times. Allele-specific H19 expression in single embryos was determined by qRT-PCR followed by fluorescence resonance electron transfer or RT-PCR followed by restriction fragment length polymorphism and polyacrylamide gel electrophoresis (RFLP-PAGE). Peri-implantation embryos were microdissected into two sections, one containing the ICM and the other containing the PTGC. TaqMan probes for Dek, Pou5f1, Itga7, H19, and Igf2 were used to ascertain gene expression enrichment in each section. Allele-specific H19 expression in embryo sections was determined by RFLP-PAGE. We found that as embryos advance through preimplantation development they start expressing H19 in a biallelic manner and this phenomenon was observed in the cultured and the in vivo-developed embryos. The PTGC-containing half of the embryo had greater expression of H19 when compared to the ICM-containing half of the embryo, as determined by qRT-PCR. In conclusion, loss of imprinting of H19 occurs in the PTGC

  13. THE MAIZE UNSTABLE FACTOR FOR ORANGE1 IS A DOMINANT EPIGENETIC MODIFIER OF A TISSUE SPECIFICALLY SILENT ALLELE OF PERICARP COLOR1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have characterized Ufo1 (unstable factor for orange1), a dominant, allele-specific modifier of expression of the maize pericarp color1 (p1) gene. The p1 gene encodes a Myb-homologous transcriptional activator of genes required for biosynthesis of red phlobaphene pigments. The P1-wr allele speci...

  14. Transvection in the Drosophila Ultrabithorax Gene: A Cbx(1) Mutant Allele Induces Ectopic Expression of a Normal Allele in Trans

    PubMed Central

    Castelli-Gair, J. E.; Micol, J. L.; Garcia-Bellido, A.

    1990-01-01

    In wild-type Drosophila melanogaster larvae, the Ultrabithorax (Ubx) gene is expressed in the haltere imaginal discs but not in the majority of cells of the wing imaginal discs. Ectopic expression of the Ubx gene in wing discs can be elicited by the presence of Contrabithorax (Cbx) gain-of-function alleles of the Ubx gene or by loss-of-function mutations in Polycomb (Pc) or in other trans-regulatory genes which behave as repressors of Ubx gene activity. Several Ubx loss-of-function alleles cause the absence of detectable Ubx proteins (UBX) or the presence of truncated UBX lacking the homeodomain. We have compared adult wing phenotypes with larval wing disc UBX patterns in genotypes involving double mutant chromosomes carrying in cis one of those Ubx mutations and the Cbx(1) mutation. We show that such double mutant genes are (1) active in the same cells in which the single mutant Cbx(1) is expressed, although they are unable to yield functional proteins, and (2) able to induce ectopic expression of a normal homologous Ubx allele in a part of the cells in which the single mutant Cbx(1) is active. That induction is conditional upon pairing of the homologous chromosomes (the phenomenon known as transvection), and it is not mediated by UBX. Depletion of Pc gene products by Pc(3) mutation strongly enhances the induction phenomenon, as shown by (1) the increase of the number of wing disc cells in which induction of the homologous allele is detectable, and (2) the induction of not only a paired normal allele but also an unpaired one. PMID:2121595

  15. Biased Allele Expression and Aggression in Hybrid Honeybees may be Influenced by Inappropriate Nuclear-Cytoplasmic Signaling

    PubMed Central

    Gibson, Joshua D.; Arechavaleta-Velasco, Miguel E.; Tsuruda, Jennifer M.; Hunt, Greg J.

    2015-01-01

    Hybrid effects are often exhibited asymmetrically between reciprocal families. One way this could happen is if silencing of one parent’s allele occurs in one lineage but not the other, which could affect the phenotypes of the hybrids asymmetrically by silencing that allele in only one of the hybrid families. We have previously tested for allele-specific expression biases in hybrids of European and Africanized honeybees and we found that there was an asymmetric overabundance of genes showing a maternal bias in the family with a European mother. Here, we further analyze allelic bias in these hybrids to ascertain whether they may underlie previously described asymmetries in metabolism and aggression in similar hybrid families and we speculate on what mechanisms may produce this biased allele usage. We find that there are over 500 genes that have some form of biased allele usage and over 200 of these are biased toward the maternal allele but only in the family with European maternity, mirroring the pattern observed for aggression and metabolic rate. This asymmetrically biased set is enriched for genes in loci associated with aggressive behavior and also for mitochondrial-localizing proteins. It contains many genes that play important roles in metabolic regulation. Moreover we find genes relating to the piwi-interacting RNA (piRNA) pathway, which is involved in chromatin modifications and epigenetic regulation and may help explain the mechanism underlying this asymmetric allele use. Based on these findings and previous work investigating aggression and metabolism in bees, we propose a novel hypothesis; that the asymmetric pattern of biased allele usage in these hybrids is a result of inappropriate use of piRNA-mediated nuclear-cytoplasmic signaling that is normally used to modulate aggression in honeybees. This is the first report of widespread asymmetric effects on allelic expression in hybrids and may represent a novel mechanism for gene regulation. PMID:26648977

  16. Identification of genes escaping X inactivation by allelic expression analysis in a novel hybrid mouse model

    PubMed Central

    Berletch, Joel B.; Ma, Wenxiu; Yang, Fan; Shendure, Jay; Noble, William S.; Disteche, Christine M.; Deng, Xinxian

    2015-01-01

    X chromosome inactivation (XCI) is a female-specific mechanism that serves to balance gene dosage between the sexes whereby one X chromosome in females is inactivated during early development. Despite this silencing, a small portion of genes escape inactivation and remain expressed from the inactive X (Xi). Little is known about the distribution of escape from XCI in different tissues in vivo and about the mechanisms that control tissue-specific differences. Using a new binomial model in conjunction with a mouse model with identifiable alleles and skewed X inactivation we are able to survey genes that escape XCI in vivo. We show that escape from X inactivation can be a common feature of some genes, whereas others escape in a tissue specific manner. Furthermore, we characterize the chromatin environment of escape genes and show that expression from the Xi correlates with factors associated with open chromatin and that CTCF co-localizes with escape genes. Here, we provide a detailed description of the experimental design and data analysis pipeline we used to assay allele-specific expression and epigenetic characteristics of genes escaping X inactivation. The data is publicly available through the GEO database under ascension numbers GSM1014171, GSE44255, and GSE59779. Interpretation and discussion of these data are included in a previously published study (Berletch et al., 2015) [1]. PMID:26693509

  17. Use of allele-specific FAIRE to determine functional regulatory polymorphism using large-scale genotyping arrays.

    PubMed

    Smith, Andrew J P; Howard, Philip; Shah, Sonia; Eriksson, Per; Stender, Stefan; Giambartolomei, Claudia; Folkersen, Lasse; Tybjærg-Hansen, Anne; Kumari, Meena; Palmen, Jutta; Hingorani, Aroon D; Talmud, Philippa J; Humphries, Steve E

    2012-01-01

    Following the widespread use of genome-wide association studies (GWAS), focus is turning towards identification of causal variants rather than simply genetic markers of diseases and traits. As a step towards a high-throughput method to identify genome-wide, non-coding, functional regulatory variants, we describe the technique of allele-specific FAIRE, utilising large-scale genotyping technology (FAIRE-gen) to determine allelic effects on chromatin accessibility and regulatory potential. FAIRE-gen was explored using lymphoblastoid cells and the 50,000 SNP Illumina CVD BeadChip. The technique identified an allele-specific regulatory polymorphism within NR1H3 (coding for LXR-α), rs7120118, coinciding with a previously GWAS-identified SNP for HDL-C levels. This finding was confirmed using FAIRE-gen with the 200,000 SNP Illumina Metabochip and verified with the established method of TaqMan allelic discrimination. Examination of this SNP in two prospective Caucasian cohorts comprising 15,000 individuals confirmed the association with HDL-C levels (combined beta = 0.016; p = 0.0006), and analysis of gene expression identified an allelic association with LXR-α expression in heart tissue. Using increasingly comprehensive genotyping chips and distinct tissues for examination, FAIRE-gen has the potential to aid the identification of many causal SNPs associated with disease from GWAS. PMID:22916038

  18. PAP-LMPCR for improved, allele-specific footprinting and automated chromatin fine structure analysis

    PubMed Central

    Ingram, R.; Gao, C.; LeBon, J.; Liu, Q.; Mayoral, R. J.; Sommer, S. S.; Hoogenkamp, M.; Riggs, A. D.; Bonifer, C.

    2008-01-01

    The analysis of chromatin fine structure and transcription factor occupancy of differentially expressed genes by in vivo footprinting and ligation-mediated-PCR (LMPCR) is a powerful tool to understand the impact of chromatin on gene expression. However, as with all PCR-based techniques, the accuracy of the experiments has often been reduced by sequence similarities and the presence of GC-rich or repeat sequences, and some sequences are completely refractory to analysis. Here we describe a novel method, pyrophosphorolysis activated polymerization LMPCR or PAP-LMPCR, which is capable of generating accurate and reproducible footprints specific for individual alleles and can read through sequences previously not accessible for analysis. In addition, we have adapted this technique for automation, thus enabling the simultaneous and rapid analysis of chromatin structure at many different genes. PMID:18208840

  19. Allelic imbalance modulates surface expression of the tolerance-inducing HLA-G molecule on primary trophoblast cells.

    PubMed

    Djurisic, S; Teiblum, S; Tolstrup, C K; Christiansen, O B; Hviid, T V F

    2015-03-01

    The HLA-G molecule is expressed on trophoblast cells at the feto-maternal interface, where it interacts with local immune cells, and upholds tolerance against the semi-allogeneic fetus. Aberrant HLA-G expression in the placenta and reduced soluble HLA-G levels are observed in pregnancy complications, partly explained by HLA-G polymorphisms which are associated with differences in the alternative splicing pattern and of the stability of HLA-G mRNA. Of special importance is a 14 bp insertion/deletion polymorphism located in the 3'-untranslated region of the HLA-G gene. In the current study, we present novel evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, using a very accurate and sensitive Digital droplet PCR technique. Allelic imbalance in heterozygous samples was observed as differential expression levels of 14 bp insertion/deletion allele-specific mRNA transcripts, which was further associated with low levels of HLA-G surface expression on primary trophoblast cells. Full gene sequencing of HLA-G allowed us to study correlations between HLA-G extended haplotypes and single-nucleotide polymorphisms and HLA-G surface expression. We found that a 1:1 expression (allelic balance) of the 14 bp insertion/deletion mRNA alleles was associated with high surface expression of HLA-G and with a specific HLA-G extended haplotype. The 14 bp del/del genotype was associated with a significantly lower abundance of the G1 mRNA isoform, and a higher abundance of the G3 mRNA isoform. Overall, the present study provides original evidence for allelic imbalance of the 14 bp insertion/deletion polymorphism, which influences HLA-G surface expression on primary trophoblast cells, considered to be important in the pathogenesis of pre-eclampsia and other pregnancy complications. PMID:25425608

  20. Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA Methylation in Nasonia.

    PubMed

    Wang, Xu; Werren, John H; Clark, Andrew G

    2016-07-01

    Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia. PMID

  1. Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA Methylation in Nasonia

    PubMed Central

    Wang, Xu; Clark, Andrew G.

    2016-01-01

    Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia. PMID

  2. Hypervariable Domains of Self-Incompatibility RNases Mediate Allele-Specific Pollen Recognition.

    PubMed Central

    Matton, D. P.; Maes, O.; Laublin, G.; Xike, Q.; Bertrand, C.; Morse, D.; Cappadocia, M.

    1997-01-01

    Self-incompatibility (SI) in angiosperms is a genetic mechanism that promotes outcrossing through rejection of self-pollen. In the Solanaceae, SI is determined by a multiallelic S locus whose only known product is an S RNase. S RNases show a characteristic pattern of five conserved and two hypervariable regions. These are thought to be involved in the catalytic function and in allelic specificity, respectively. When the Solanum chacoense S12S14 genotype is transformed with an S11 RNase, the styles of plants expressing significant levels of the transgene reject S11 pollen. A previously characterized S RNase, S13, differs from the S11 RNase by only 10 amino acids, four of which are located in the hypervariable regions. When S12S14 plants were transformed with a chimeric S11 gene in which these four residues were substituted with those present in the S13 RNase, the transgenic plants acquired the S13 phenotype. This result demonstrates that the S RNase hypervariable regions control allelic specificity. PMID:12237346

  3. Allele-specific RNAi Mitigates Phenotypic Progression in a Transgenic Model of Alzheimer's Disease

    PubMed Central

    Rodríguez-Lebrón, Edgardo; Gouvion, Cynthia M; Moore, Steven A; Davidson, Beverly L; Paulson, Henry L

    2009-01-01

    Despite recent advances suggesting new therapeutic targets, Alzheimer's disease (AD) remains incurable. Aberrant production and accumulation of the Aβ peptide resulting from altered processing of the amyloid precursor protein (APP) is central to the pathogenesis of disease, particularly in dominantly inherited forms of AD. Thus, modulating the production of APP is a potential route to effective AD therapy. Here, we describe the successful use of an allele-specific RNA interference (RNAi) approach targeting the Swedish variant of APP (APPsw) in a transgenic mouse model of AD. Using recombinant adeno-associated virus (rAAV), we delivered an anti-APPsw short-hairpin RNA (shRNA) to the hippocampus of AD transgenic mice (APP/PS1). In short- and long-term transduction experiments, reduced levels of APPsw transprotein were observed throughout targeted regions of the hippocampus while levels of wild-type murine APP remained unaltered. Moreover, intracellular production of transfer RNA (tRNA)-valine promoter–driven shRNAs did not lead to detectable neuronal toxicity. Finally, long-term bilateral hippocampal expression of anti-APPsw shRNA mitigated abnormal behaviors in this mouse model of AD. The difference in phenotype progression was associated with reduced levels of soluble Aβ but not with a reduced number of amyloid plaques. Our results support the development of allele-specific RNAi strategies to treat familial AD and other dominantly inherited neurodegenerative diseases. PMID:19532137

  4. Quantitative and functional interrogation of parent-of-origin allelic expression biases in the brain.

    PubMed

    Perez, Julio D; Rubinstein, Nimrod D; Fernandez, Daniel E; Santoro, Stephen W; Needleman, Leigh A; Ho-Shing, Olivia; Choi, John J; Zirlinger, Mariela; Chen, Shau-Kwaun; Liu, Jun S; Dulac, Catherine

    2015-01-01

    The maternal and paternal genomes play different roles in mammalian brains as a result of genomic imprinting, an epigenetic regulation leading to differential expression of the parental alleles of some genes. Here we investigate genomic imprinting in the cerebellum using a newly developed Bayesian statistical model that provides unprecedented transcript-level resolution. We uncover 160 imprinted transcripts, including 41 novel and independently validated imprinted genes. Strikingly, many genes exhibit parentally biased--rather than monoallelic--expression, with different magnitudes according to age, organ, and brain region. Developmental changes in parental bias and overall gene expression are strongly correlated, suggesting combined roles in regulating gene dosage. Finally, brain-specific deletion of the paternal, but not maternal, allele of the paternally-biased Bcl-x, (Bcl2l1) results in loss of specific neuron types, supporting the functional significance of parental biases. These findings reveal the remarkable complexity of genomic imprinting, with important implications for understanding the normal and diseased brain. PMID:26140685

  5. Quantitative and functional interrogation of parent-of-origin allelic expression biases in the brain

    PubMed Central

    Perez, Julio D; Rubinstein, Nimrod D; Fernandez, Daniel E; Santoro, Stephen W; Needleman, Leigh A; Ho-Shing, Olivia; Choi, John J; Zirlinger, Mariela; Chen, Shau-Kwaun; Liu, Jun S; Dulac, Catherine

    2015-01-01

    The maternal and paternal genomes play different roles in mammalian brains as a result of genomic imprinting, an epigenetic regulation leading to differential expression of the parental alleles of some genes. Here we investigate genomic imprinting in the cerebellum using a newly developed Bayesian statistical model that provides unprecedented transcript-level resolution. We uncover 160 imprinted transcripts, including 41 novel and independently validated imprinted genes. Strikingly, many genes exhibit parentally biased—rather than monoallelic—expression, with different magnitudes according to age, organ, and brain region. Developmental changes in parental bias and overall gene expression are strongly correlated, suggesting combined roles in regulating gene dosage. Finally, brain-specific deletion of the paternal, but not maternal, allele of the paternally-biased Bcl-x, (Bcl2l1) results in loss of specific neuron types, supporting the functional significance of parental biases. These findings reveal the remarkable complexity of genomic imprinting, with important implications for understanding the normal and diseased brain. DOI: http://dx.doi.org/10.7554/eLife.07860.001 PMID:26140685

  6. Site- and allele-specific polycomb dysregulation in T-cell leukaemia.

    PubMed

    Navarro, Jean-Marc; Touzart, Aurore; Pradel, Lydie C; Loosveld, Marie; Koubi, Myriam; Fenouil, Romain; Le Noir, Sandrine; Maqbool, Muhammad Ahmad; Morgado, Ester; Gregoire, Claude; Jaeger, Sebastien; Mamessier, Emilie; Pignon, Charles; Hacein-Bey-Abina, Salima; Malissen, Bernard; Gut, Marta; Gut, Ivo G; Dombret, Hervé; Macintyre, Elizabeth A; Howe, Steven J; Gaspar, H Bobby; Thrasher, Adrian J; Ifrah, Norbert; Payet-Bornet, Dominique; Duprez, Estelle; Andrau, Jean-Christophe; Asnafi, Vahid; Nadel, Bertrand

    2015-01-01

    T-cell acute lymphoblastic leukaemias (T-ALL) are aggressive malignant proliferations characterized by high relapse rates and great genetic heterogeneity. TAL1 is amongst the most frequently deregulated oncogenes. Yet, over half of the TAL1(+) cases lack TAL1 lesions, suggesting unrecognized (epi)genetic deregulation mechanisms. Here we show that TAL1 is normally silenced in the T-cell lineage, and that the polycomb H3K27me3-repressive mark is focally diminished in TAL1(+) T-ALLs. Sequencing reveals that >20% of monoallelic TAL1(+) patients without previously known alterations display microinsertions or RAG1/2-mediated episomal reintegration in a single site 5' to TAL1. Using 'allelic-ChIP' and CrispR assays, we demonstrate that such insertions induce a selective switch from H3K27me3 to H3K27ac at the inserted but not the germline allele. We also show that, despite a considerable mechanistic diversity, the mode of oncogenic TAL1 activation, rather than expression levels, impact on clinical outcome. Altogether, these studies establish site-specific epigenetic desilencing as a mechanism of oncogenic activation. PMID:25615415

  7. Direct Fluorescence Detection of Allele-Specific PCR Products Using Novel Energy-Transfer Labeled Primers.

    PubMed

    Winn-Deen

    1998-12-01

    Background: Currently analysis of point mutations can be done by allele-specific polymerase chain reaction (PCR) followed by gel analysis or by gene-specific PCR followed by hybridization with an allele-specific probe. Both of these mutation detection methods require post-PCR laboratory time and run the risk of contaminating subsequent experiments with the PCR product liberated during the detection step. The author has combined the PCR amplification and detection steps into a single procedure suitable for closed-tube analysis. Methods and Results: Allele-specific PCR primers were designed as Sunrise energy-transfer primers and contained a 3' terminal mismatch to distinguish between normal and mutant DNA. Cloned normal (W64) and mutant (R64) templates of the beta3-adrenergic receptor gene were tested to verify amplification specificity and yield. A no-target negative control was also run with each reaction. After PCR, each reaction was tested for fluorescence yield by measuring fluorescence on a spectrofluorimeter or fluorescent microtitreplate reader. The cloned controls and 24 patient samples were tested for the W64R mutation by two methods. The direct fluorescence results with the Sunrise allele-specific PCR method gave comparable genotypes to those obtained with the PCR/ restriction digest/gel electrophoresis control method. No PCR artifacts were observed in the negative controls or in the PCR reactions run with the mismatched target. Conclusions: The results of this pilot study indicate good PCR product and fluorescence yield from allele-specific energy-transfer labeled primers, and the capability of distinguishing between normal and mutant alleles based on fluorescence alone, without the need for restriction digestion, gel electrophoresis, or hybridization with an allele-specific probe. PMID:10089280

  8. Paternal-specific S-allele transmission in sweet cherry (Prunus avium L.): the potential for sexual selection.

    PubMed

    Hedhly, A; Wünsch, A; Kartal, Ö; Herrero, M; Hormaza, J I

    2016-03-01

    Homomorphic self-incompatibility is a well-studied example of a physiological process that is thought to increase population diversity and reduce the expression of inbreeding depression. Whereas theoretical models predict the presence of a large number of S-haplotypes with equal frequencies at equilibrium, unequal allele frequencies have been repeatedly reported and attributed to sampling effects, population structure, demographic perturbation, sheltered deleterious mutations or selection pressure on linked genes. However, it is unclear to what extent unequal segregations are the results of gametophytic or sexual selection. Although these two forces are difficult to disentangle, testing S-alleles in the offspring of controlled crosses provides an opportunity to separate these two phenomena. In this work, segregation and transmission of S-alleles have been characterized in progenies of mixed donors and fully compatible pollinations under field conditions in Prunus avium. Seed set patterns and pollen performance have also been characterized. The results reveal paternal-specific distorted transmission of S-alleles in most of the crosses. Interestingly, S-allele segregation within any given paternal or maternal S-locus was random. Observations on pollen germination, pollen tube growth rate, pollen tube cohort size, seed set dynamics and transmission patterns strongly suggest post-pollination, prezygotic sexual selection, with male-male competition as the most likely mechanism. According to these results, post-pollination sexual selection takes precedence over frequency-dependent selection in explaining unequal S-haplotype frequencies. PMID:26559165

  9. Generation of Shox2-Cre allele for tissue specific manipulation of genes in the developing heart, palate, and limb.

    PubMed

    Sun, Cheng; Zhang, Tao; Liu, Chao; Gu, Shuping; Chen, YiPing

    2013-07-01

    Shox2 is expressed in several developing organs in a tissue specific manner in both mice and humans, including the heart, palate, limb, and nervous system. To better understand the spatial and temporal expression patterns of Shox2 and to systematically dissect the genetic cascade regulated by Shox2, we created Shox2-LacZ and Shox2-Cre knock-in mouse lines. We show that the Shox2-LacZ allele expresses beta-galactosidase reporter gene in a fashion that recapitulates the endogenous Shox2 expression pattern in developing organs, including the sinoatrial node (SAN), the anterior portion of the palate, and the proximal region of the limb bud. Conditional deletion of Shox2 in mice carrying the Shox2-Cre allele yielded SAN phenotypes that resemble conventional Shox2 knockout mice. Our results indicate that the Shox2-Cre allele offer a useful tool for tissue specific manipulation of genes in a number of developing organs, particularly in the developing SAN. PMID:23620086

  10. Allele-Specific Deletions in Mouse Tumors Identify Fbxw7 as Germline Modifier of Tumor Susceptibility

    PubMed Central

    Perez-Losada, Jesus; Wu, Di; DelRosario, Reyno; Balmain, Allan; Mao, Jian-Hua

    2012-01-01

    Genome-wide association studies (GWAS) have been successful in finding associations between specific genetic variants and cancer susceptibility in human populations. These studies have identified a range of highly statistically significant associations between single nucleotide polymorphisms (SNPs) and susceptibility to development of a range of human tumors. However, the effect of each SNP in isolation is very small, and all of the SNPs combined only account for a relatively minor proportion of the total genetic risk (5–10%). There is therefore a major requirement for alternative routes to the discovery of genetic risk factors for cancer. We have previously shown using mouse models that chromosomal regions harboring susceptibility genes identified by linkage analysis frequently exhibit allele-specific genetic alterations in tumors. We demonstrate here that the Fbxw7 gene, a commonly mutated gene in a wide range of mouse and human cancers, shows allele-specific deletions in mouse lymphomas and skin tumors. Lymphomas from three different F1 hybrids show 100% allele-specificity in the patterns of allelic loss. Parental alleles from 129/Sv or Spretus/Gla mice are lost in tumors from F1 hybrids with C57BL/6 animals, due to the presence of a specific non-synonymous coding sequence polymorphism at the N-terminal portion of the gene. A specific genetic test of association between this SNP and lymphoma susceptibility in interspecific backcross mice showed a significant linkage (p = 0.001), but only in animals with a functional p53 gene. These data therefore identify Fbxw7 as a p53-dependent tumor susceptibility gene. Increased p53-dependent tumor susceptibility and allele-specific losses were also seen in a mouse skin model of skin tumor development. We propose that analysis of preferential allelic imbalances in tumors may provide an efficient means of uncovering genetic variants that affect mouse and human tumor susceptibility. PMID:22348067

  11. Effects of sequence variation on differential allelic transcription factor occupancy and gene expression.

    PubMed

    Reddy, Timothy E; Gertz, Jason; Pauli, Florencia; Kucera, Katerina S; Varley, Katherine E; Newberry, Kimberly M; Marinov, Georgi K; Mortazavi, Ali; Williams, Brian A; Song, Lingyun; Crawford, Gregory E; Wold, Barbara; Willard, Huntington F; Myers, Richard M

    2012-05-01

    A complex interplay between transcription factors (TFs) and the genome regulates transcription. However, connecting variation in genome sequence with variation in TF binding and gene expression is challenging due to environmental differences between individuals and cell types. To address this problem, we measured genome-wide differential allelic occupancy of 24 TFs and EP300 in a human lymphoblastoid cell line GM12878. Overall, 5% of human TF binding sites have an allelic imbalance in occupancy. At many sites, TFs clustered in TF-binding hubs on the same homolog in especially open chromatin. While genetic variation in core TF binding motifs generally resulted in large allelic differences in TF occupancy, most allelic differences in occupancy were subtle and associated with disruption of weak or noncanonical motifs. We also measured genome-wide differential allelic expression of genes with and without heterozygous exonic variants in the same cells. We found that genes with differential allelic expression were overall less expressed both in GM12878 cells and in unrelated human cell lines. Comparing TF occupancy with expression, we found strong association between allelic occupancy and expression within 100 bp of transcription start sites (TSSs), and weak association up to 100 kb from TSSs. Sites of differential allelic occupancy were significantly enriched for variants associated with disease, particularly autoimmune disease, suggesting that allelic differences in TF occupancy give functional insights into intergenic variants associated with disease. Our results have the potential to increase the power and interpretability of association studies by targeting functional intergenic variants in addition to protein coding sequences. PMID:22300769

  12. Probe-free allele-specific copy number detection and analysis of tumors.

    PubMed

    Zhu, Ailin; Guan, Xiaowei; Gu, Xinbin; Xie, Guiqin

    2016-03-15

    Cancer development and progression frequently involve nucleotide mutations as well as amplifications and deletions of genomic segments. Quantification of allele-specific copy number is an important step in characterizing tumor genomes for precision medicine. Despite advances in approaches to high-throughput genomic DNA analysis, inexpensive and simple methods for analyzing complex nucleotide and copy number variants are still needed. Real-time polymerase chain reaction (PCR) methods for discovering and genotyping single nucleotide polymorphisms are becoming increasingly important in genetic analysis. In this study, we describe a simple, single-tube, probe-free method that combines SYBR Green I-based quantitative real-time PCR and quantitative melting curve analysis both to detect specific nucleotide variants and to quantify allele-specific copy number variants of tumors. The approach is based on the quantification of the targets of interest and the relative abundance of two alleles in a single tube. The specificity, sensitivity, and utility of the assay were demonstrated in detecting allele-specific copy number changes critical for carcinogenesis and therapeutic intervention. Our approach would be useful for allele-specific copy number analysis or precise genotyping. PMID:26743720

  13. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping.

    PubMed

    Lee, Han B; Schwab, Tanya L; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L; Cervera, Roberto Lopez; McNulty, Melissa S; Bostwick, Hannah S; Clark, Karl J

    2016-06-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98-100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  14. Allele-Specific Quantitative PCR for Accurate, Rapid, and Cost-Effective Genotyping

    PubMed Central

    Lee, Han B.; Schwab, Tanya L.; Koleilat, Alaa; Ata, Hirotaka; Daby, Camden L.; Cervera, Roberto Lopez; McNulty, Melissa S.; Bostwick, Hannah S.; Clark, Karl J.

    2016-01-01

    Customizable endonucleases such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) enable rapid generation of mutant strains at genomic loci of interest in animal models and cell lines. With the accelerated pace of generating mutant alleles, genotyping has become a rate-limiting step to understanding the effects of genetic perturbation. Unless mutated alleles result in distinct morphological phenotypes, mutant strains need to be genotyped using standard methods in molecular biology. Classic restriction fragment length polymorphism (RFLP) or sequencing is labor-intensive and expensive. Although simpler than RFLP, current versions of allele-specific PCR may still require post-polymerase chain reaction (PCR) handling such as sequencing, or they are more expensive if allele-specific fluorescent probes are used. Commercial genotyping solutions can take weeks from assay design to result, and are often more expensive than assembling reactions in-house. Key components of commercial assay systems are often proprietary, which limits further customization. Therefore, we developed a one-step open-source genotyping method based on quantitative PCR. The allele-specific qPCR (ASQ) does not require post-PCR processing and can genotype germline mutants through either threshold cycle (Ct) or end-point fluorescence reading. ASQ utilizes allele-specific primers, a locus-specific reverse primer, universal fluorescent probes and quenchers, and hot start DNA polymerase. Individual laboratories can further optimize this open-source system as we completely disclose the sequences, reagents, and thermal cycling protocol. We have tested the ASQ protocol to genotype alleles in five different genes. ASQ showed a 98–100% concordance in genotype scoring with RFLP or Sanger sequencing outcomes. ASQ is time-saving because a single qPCR without post-PCR handling suffices to score

  15. Allele-specific copy-number discovery from whole-genome and whole-exome sequencing

    PubMed Central

    Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J.; Szatkiewicz, Jin P.

    2015-01-01

    Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. PMID:25883151

  16. Allele-specific copy-number discovery from whole-genome and whole-exome sequencing.

    PubMed

    Wang, WeiBo; Wang, Wei; Sun, Wei; Crowley, James J; Szatkiewicz, Jin P

    2015-08-18

    Copy-number variants (CNVs) are a major form of genetic variation and a risk factor for various human diseases, so it is crucial to accurately detect and characterize them. It is conceivable that allele-specific reads from high-throughput sequencing data could be leveraged to both enhance CNV detection and produce allele-specific copy number (ASCN) calls. Although statistical methods have been developed to detect CNVs using whole-genome sequence (WGS) and/or whole-exome sequence (WES) data, information from allele-specific read counts has not yet been adequately exploited. In this paper, we develop an integrated method, called AS-GENSENG, which incorporates allele-specific read counts in CNV detection and estimates ASCN using either WGS or WES data. To evaluate the performance of AS-GENSENG, we conducted extensive simulations, generated empirical data using existing WGS and WES data sets and validated predicted CNVs using an independent methodology. We conclude that AS-GENSENG not only predicts accurate ASCN calls but also improves the accuracy of total copy number calls, owing to its unique ability to exploit information from both total and allele-specific read counts while accounting for various experimental biases in sequence data. Our novel, user-friendly and computationally efficient method and a complete analytic protocol is freely available at https://sourceforge.net/projects/asgenseng/. PMID:25883151

  17. Differential Allelic Expression in the Human Genome: A Robust Approach To Identify Genetic and Epigenetic Cis-Acting Mechanisms Regulating Gene Expression

    PubMed Central

    Serre, David; Gurd, Scott; Ge, Bing; Sladek, Robert; Sinnett, Donna; Harmsen, Eef; Bibikova, Marina; Chudin, Eugene; Barker, David L.; Dickinson, Todd; Fan, Jian-Bing; Hudson, Thomas J.

    2008-01-01

    The recent development of whole genome association studies has lead to the robust identification of several loci involved in different common human diseases. Interestingly, some of the strongest signals of association observed in these studies arise from non-coding regions located in very large introns or far away from any annotated genes, raising the possibility that these regions are involved in the etiology of the disease through some unidentified regulatory mechanisms. These findings highlight the importance of better understanding the mechanisms leading to inter-individual differences in gene expression in humans. Most of the existing approaches developed to identify common regulatory polymorphisms are based on linkage/association mapping of gene expression to genotypes. However, these methods have some limitations, notably their cost and the requirement of extensive genotyping information from all the individuals studied which limits their applications to a specific cohort or tissue. Here we describe a robust and high-throughput method to directly measure differences in allelic expression for a large number of genes using the Illumina Allele-Specific Expression BeadArray platform and quantitative sequencing of RT-PCR products. We show that this approach allows reliable identification of differences in the relative expression of the two alleles larger than 1.5-fold (i.e., deviations of the allelic ratio larger than 60∶40) and offers several advantages over the mapping of total gene expression, particularly for studying humans or outbred populations. Our analysis of more than 80 individuals for 2,968 SNPs located in 1,380 genes confirms that differential allelic expression is a widespread phenomenon affecting the expression of 20% of human genes and shows that our method successfully captures expression differences resulting from both genetic and epigenetic cis-acting mechanisms. PMID:18454203

  18. Analysis of FBN1 allele expression by dermal fibroblasts from Marfan syndrome patients

    SciTech Connect

    Putman, E.A.; Cao, S.N.; Milewicz, D.M.

    1994-09-01

    Screening for mutations in the FBN1 cDNA from Marfan patient cell strains has detected mutations in only 10-15% of patients. In an attempt to explain this poor detection rate, we examined FBN1 allele expression and fibrillin synthesis by 26 cell strains from Marfan patients. DNA from the patients and 10 controls was assessed for the presence of a polymorphic Rsa I restriction site in the 3{prime} untranslated region of the FBN1 gene. Twelve of 26 patient and 5 of 10 control DNAs were heterozygous. Fibroblast RNA from the heterozygous cell strains was reverse-transcribed and subsequently PCR amplified using a [{sup 32}P]-labelled primer, digested with Rsa I and analyzed. Although 3 samples showed no transcript from one allele by ethidium bromide staining, a Betagen scanner detected low levels (10-15%) of that allele. In addition, there was unequal expression of the two alleles in three other patients; for example, only 30% expression from one allele. The remaining patients and the controls had equal expression of each allele. Fibrillin protein synthesis by fibroblasts from these heterozygous patients was also examined. After a 30 minute pulse with [{sup 35}S]-cysteine, cell lysates were collected and proteins analyzed by SDS-PAGE. The amount of fibrillin produced relative to a reference protein was determined using a Betagen scanner. Fibrillin protein synthesis was reduced in 2 of the 3 patients with very low RNA production from one of the FBN1 alleles. All other Marfan and control cell strains showed normal amounts of fibrillin synthesized. The low expression levels from one allele may contribute to, but not fully account for, the low detection rate of FBN1 mutations. Interestingly, protein synthesis levels were not affected in 4 of 6 cell strains demonstrating low levels of RNA expression.

  19. Allele-specific copy number profiling by next-generation DNA sequencing.

    PubMed

    Chen, Hao; Bell, John M; Zavala, Nicolas A; Ji, Hanlee P; Zhang, Nancy R

    2015-02-27

    The progression and clonal development of tumors often involve amplifications and deletions of genomic DNA. Estimation of allele-specific copy number, which quantifies the number of copies of each allele at each variant loci rather than the total number of chromosome copies, is an important step in the characterization of tumor genomes and the inference of their clonal history. We describe a new method, falcon, for finding somatic allele-specific copy number changes by next generation sequencing of tumors with matched normals. falcon is based on a change-point model on a bivariate mixed Binomial process, which explicitly models the copy numbers of the two chromosome haplotypes and corrects for local allele-specific coverage biases. By using the Binomial distribution rather than a normal approximation, falcon more effectively pools evidence from sites with low coverage. A modified Bayesian information criterion is used to guide model selection for determining the number of copy number events. Falcon is evaluated on in silico spike-in data and applied to the analysis of a pre-malignant colon tumor sample and late-stage colorectal adenocarcinoma from the same individual. The allele-specific copy number estimates obtained by falcon allows us to draw detailed conclusions regarding the clonal history of the individual's colon cancer. PMID:25477383

  20. Genome Destabilizing Mutator Alleles Drive Specific Mutational Trajectories in Saccharomyces cerevisiae

    PubMed Central

    Stirling, Peter C.; Shen, Yaoqing; Corbett, Richard; Jones, Steven J. M.; Hieter, Philip

    2014-01-01

    In addition to environmental factors and intrinsic variations in base substitution rates, specific genome-destabilizing mutations can shape the mutational trajectory of genomes. How specific alleles influence the nature and position of accumulated mutations in a genomic context is largely unknown. Understanding the impact of genome-destabilizing alleles is particularly relevant to cancer genomes where biased mutational signatures are identifiable. We first created a more complete picture of cellular pathways that impact mutation rate using a primary screen to identify essential Saccharomyces cerevisiae gene mutations that cause mutator phenotypes. Drawing primarily on new alleles identified in this resource, we measure the impact of diverse mutator alleles on mutation patterns directly by whole-genome sequencing of 68 mutation-accumulation strains derived from wild-type and 11 parental mutator genotypes. The accumulated mutations differ across mutator strains, displaying base-substitution biases, allele-specific mutation hotspots, and break-associated mutation clustering. For example, in mutants of POLα and the Cdc13–Stn1–Ten1 complex, we find a distinct subtelomeric bias for mutations that we show is independent of the target sequence. Together our data suggest that specific genome-instability mutations are sufficient to drive discrete mutational signatures, some of which share properties with mutation patterns seen in tumors. Thus, in a population of cells, genome-instability mutations could influence clonal evolution by establishing discrete mutational trajectories for genomes. PMID:24336748

  1. A novel type 2 diabetes risk allele increases the promoter activity of the muscle-specific small ankyrin 1 gene.

    PubMed

    Yan, Rengna; Lai, Shanshan; Yang, Yang; Shi, Hongfei; Cai, Zhenming; Sorrentino, Vincenzo; Du, Hong; Chen, Huimei

    2016-01-01

    Genome-wide association studies have identified Ankyrin-1 (ANK1) as a common type 2 diabetes (T2D) susceptibility locus. However, the underlying causal variants and functional mechanisms remain unknown. We screened for 8 tag single nucleotide polymorphisms (SNPs) in ANK1 between 2 case-control studies. Genotype analysis revealed significant associations of 3 SNPs, rs508419 (first identified here), rs515071, and rs516946 with T2D (P < 0.001). These SNPs were in linkage disequilibrium (r(2) > 0.80); subsequent analysis indicated that the CCC haplotype associated with increased T2D susceptibility (OR 1.447, P < 0.001). Further mapping showed that rs508419 resides in the muscle-specific ANK1 gene promoter. Allele-specific mRNA and protein level measurements confirmed association of the C allele with increased small ANK1 (sAnk1) expression in human skeletal muscle (P = 0.018 and P < 0.001, respectively). Luciferase assays showed increased rs508419-C allele transcriptional activity in murine skeletal muscle C2C12 myoblasts, and electrophoretic mobility-shift assays demonstrated altered rs508419 DNA-protein complex formation. Glucose uptake was decreased with excess sAnk1 expression upon insulin stimulation. Thus, the ANK1 rs508419-C T2D-risk allele alters DNA-protein complex binding leading to increased promoter activity and sAnk1 expression; thus, increased sAnk1 expression in skeletal muscle might contribute to T2D susceptibility. PMID:27121283

  2. A novel type 2 diabetes risk allele increases the promoter activity of the muscle-specific small ankyrin 1 gene

    PubMed Central

    Yan, Rengna; Lai, Shanshan; Yang, Yang; Shi, Hongfei; Cai, Zhenming; Sorrentino, Vincenzo; Du, Hong; Chen, Huimei

    2016-01-01

    Genome-wide association studies have identified Ankyrin-1 (ANK1) as a common type 2 diabetes (T2D) susceptibility locus. However, the underlying causal variants and functional mechanisms remain unknown. We screened for 8 tag single nucleotide polymorphisms (SNPs) in ANK1 between 2 case-control studies. Genotype analysis revealed significant associations of 3 SNPs, rs508419 (first identified here), rs515071, and rs516946 with T2D (P < 0.001). These SNPs were in linkage disequilibrium (r2 > 0.80); subsequent analysis indicated that the CCC haplotype associated with increased T2D susceptibility (OR 1.447, P < 0.001). Further mapping showed that rs508419 resides in the muscle-specific ANK1 gene promoter. Allele-specific mRNA and protein level measurements confirmed association of the C allele with increased small ANK1 (sAnk1) expression in human skeletal muscle (P = 0.018 and P < 0.001, respectively). Luciferase assays showed increased rs508419-C allele transcriptional activity in murine skeletal muscle C2C12 myoblasts, and electrophoretic mobility-shift assays demonstrated altered rs508419 DNA-protein complex formation. Glucose uptake was decreased with excess sAnk1 expression upon insulin stimulation. Thus, the ANK1 rs508419-C T2D-risk allele alters DNA-protein complex binding leading to increased promoter activity and sAnk1 expression; thus, increased sAnk1 expression in skeletal muscle might contribute to T2D susceptibility. PMID:27121283

  3. Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection.

    PubMed

    Kc, R; Srivastava, A; Wilkowski, J M; Richter, C E; Shavit, J A; Burke, D T; Bielas, S L

    2016-01-01

    CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies. PMID:27557703

  4. Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection

    PubMed Central

    KC, R.; Srivastava, A.; Wilkowski, J. M.; Richter, C. E.; Shavit, J. A.; Burke, D. T.; Bielas, S. L.

    2016-01-01

    CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies. PMID:27557703

  5. Inactive allele-specific methylation and chromatin structure of the imprinted gene U2af1-rs1 on mouse chromosome 11

    SciTech Connect

    Shibata, Hideo; Yoshino, Kiyoshi; Kamiya, Mamoru

    1996-07-01

    The imprinted U2Af1-rs1 gene that maps to mouse chromosome 11 is predominately expressed from the paternal allele. We examined the methylation of genomic sequences in and around the U2af1-rs1 locus to establish the extent of sequence modifications that accompanied the silencing of the maternal allele. The analysis of HapII or HhaI sites showed that the silent maternal allele was hypermethylated in a block of CpG sequences that covered more than 10 kb. By comparison, the expressed paternal allele was unmethylated from a CpG island upstream of the transcribed region through 2 kb. An analysis of DNaseI hypersensitivity of a putative promoter of U2af1-rs1 showed an open chromatin conformation only on the unmethylated, expressed paternal allele. These results suggest that allele-specific hypermethylation covering the gene and its upstream CpG island plays a role in maternal allele repression of U2af1-rs1, which is reflected in altered chromatin conformation of DNaseI hypersensitive sites. 9 refs., 2 figs.

  6. Specific HLA-DQB and HLA-DRB1 alleles confer susceptibility to pemphigus vulgaris.

    PubMed Central

    Scharf, S J; Freidmann, A; Steinman, L; Brautbar, C; Erlich, H A

    1989-01-01

    The autoimmune dermatologic disease pemphigus vulgaris (PV) is associated with the serotypes HLA-DR4 and HLA-DRw6. Based on nucleotide sequence and oligonucleotide probe analysis of enzymatically amplified DNA encoding HLA-DR beta chain (HLA-DRB) and HLA-DQ beta chain (HLA-DQB; henceforth HLA is omitted from designations), we showed previously that the DR4 susceptibility was associated with the Dw10 DRB1 allele [encoding the mixed lymphocyte culture (MLC)-defined Dw10 specificity]. The DRw6 susceptibility similarly was shown to be associated with a rare DQB allele (DQB1.3), which differed from another nonsusceptible allele by only a valine-to-aspartic acid substitution at position 57. Given the linkage disequilibrium that characterizes HLA haplotypes, it is difficult to assign disease susceptibility to a specific locus rather than to a closely linked gene(s) on the same haplotype. To address this problem, we have analyzed all of the polymorphic loci of the class II HLA region (DRB1, DRB3, DQA, DQB, and DPB) on the DRw6 haplotypes in patients and controls. In 22 PV patients, 4 different DRw6 haplotypes were found that encode the same DQ beta chain (DQB1.3) but contained silent nucleotide differences at the DQB locus as well as coding sequence differences in the DQA and DRB loci. These results, obtained by using a method for allele-specific polymerase chain reaction amplification, strongly support the hypothesis that the allele DQB1.3 confers susceptibility. This DQB allele is correlated with the MLC-defined Dw9 specificity and is associated with two different DRB1 alleles (the common "6A" associated with DRw13 and the rare "6B" associated with DRw14). Since 86% (19 of 22) of DRw6+ patients contain the DQB1.3 allele (vs. 3% of controls), whereas 64% (14 of 22) contain the DRB1 allele 6B (vs. 6% of the controls), we conclude that most of the DRw6 susceptibility to PV can be accounted for by the DQ beta chain. Images PMID:2503828

  7. Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2.

    PubMed

    Redis, Roxana S; Vela, Luz E; Lu, Weiqin; Ferreira de Oliveira, Juliana; Ivan, Cristina; Rodriguez-Aguayo, Cristian; Adamoski, Douglas; Pasculli, Barbara; Taguchi, Ayumu; Chen, Yunyun; Fernandez, Agustin F; Valledor, Luis; Van Roosbroeck, Katrien; Chang, Samuel; Shah, Maitri; Kinnebrew, Garrett; Han, Leng; Atlasi, Yaser; Cheung, Lawrence H; Huang, Gilbert Y; Monroig, Paloma; Ramirez, Marc S; Catela Ivkovic, Tina; Van, Long; Ling, Hui; Gafà, Roberta; Kapitanovic, Sanja; Lanza, Giovanni; Bankson, James A; Huang, Peng; Lai, Stephen Y; Bast, Robert C; Rosenblum, Michael G; Radovich, Milan; Ivan, Mircea; Bartholomeusz, Geoffrey; Liang, Han; Fraga, Mario F; Widger, William R; Hanash, Samir; Berindan-Neagoe, Ioana; Lopez-Berestein, Gabriel; Ambrosio, Andre L B; Gomes Dias, Sandra M; Calin, George A

    2016-02-18

    Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA. PMID:26853146

  8. DNMT1 and AIM1 Imprinting in human placenta revealed through a genome-wide screen for allele-specific DNA methylation

    PubMed Central

    2013-01-01

    Background Genomic imprinting is an epigenetically regulated process wherein genes are expressed in a parent-of-origin specific manner. Many imprinted genes were initially identified in mice; some of these were subsequently shown not to be imprinted in humans. Such discrepancy reflects developmental, morphological and physiological differences between mouse and human tissues. This is particularly relevant for the placenta. Study of genomic imprinting thus needs to be carried out in a species and developmental stage-specific manner. We describe here a new strategy to study allele-specific DNA methylation in the human placenta for the discovery of novel imprinted genes. Results Using this methodology, we confirmed 16 differentially methylated regions (DMRs) associated with known imprinted genes. We chose 28 genomic regions for further testing and identified two imprinted genes (DNMT1 and AIM1). Both genes showed maternal allele-specific methylation and paternal allele-specific transcription. Imprinted expression for AIM1 was conserved in the cynomolgus macaque placenta, but not in other macaque tissues or in the mouse. Conclusions Our study indicates that while there are many genomic regions with allele-specific methylation in tissues like the placenta, only a small sub-set of them are associated with allele-specific transcription, suggesting alternative functions for such genomic regions. Nonetheless, novel tissue-specific imprinted genes remain to be discovered in humans. Their identification may help us better understand embryonic and fetal development. PMID:24094292

  9. Myotonic Dystrophy: Increased expression of the normal allele in CDM infants muscle

    SciTech Connect

    Radvanyi, H.H.; Gourdon, G.; Junien, C. |

    1994-09-01

    Myotonic dystrophy (DM) is an autosomal dominant multisystemic disorder characterized by a highly variable clinical phenotype. The mutation has been identified as an unstable trinucleotide CTG repeat in the 3{prime} untranslated region of the myotonin-protein kinase (MT-PK) gene. Congenital myotonic dystrophy (CDM), which represents the most severe phenotype, is exclusively maternally inherited. Recent studies, analysis by Northern blots and RT-PCR provided apparently conflicting results on the mutated allele expression in samples from congenitally affected children. The level of expression of the mutant allele depends on the extent of the repeat in the adult form and is no longer expressed when over 800-1300 repeats, whether in adult forms or in CDM. Could this decrease account for the late onset forms? However, the differences between the two phenotypes cannot be explained by the same mechanism. Alternatively, these differences could be due to differences in expression of the normal allele. We analyzed by quantitative RT-PCR the expression of the MT-PK gene in muscle samples from four CDM infants and two aged-matched normal controls. In two of these, the mutant allele (3.3 and 8 kb) was undetectable on Northern blots. We observed an increased expression of the MT-PK gene (10- to 20-fold) in tissues of severely affected congenital patients which can be attributed to the normal allele. Since expression of the normal allele is either normal or slightly decreased in the adult form, the dramatic increase in the congenital form could reflect a disturbance in muscle differentiation. Expression studies of MT-PK at different stages of development and, especially after the 20th week, are therefore required.

  10. Novel interactions of fission yeast kinesin 8 revealed through in vivo expression of truncation alleles.

    PubMed

    West, Robert R; McIntosh, J Richard

    2008-08-01

    Fission yeast expresses two kinesin 8s, klp5+ and klp6+, which are important for diverse cellular functions: mitosis, meiosis, and the maintenance of normal cell morphology. During vegetative growth these motors display complex localization patterns, moving from the cytoplasm during interphase to the kinetochores in early mitosis, the interpolar spindle in anaphase B, and then back into the cytoplasm. We have expressed GFP-tagged alleles of domains from these motors, seeking the signals required for their localizations. The tail of Klp5p localized to the interphase nucleus, more specifically to telomeres. Addition of the neck re-directed this fragment to microtubules in the cytoplasm. Klp6-tail and the neck-tail domains of both motors localized at microtubule ends. Klp6-neck-tail localized to the spindle in early mitosis but to the pole-proximal ends of the spindle in anaphase B. The Klp5-motor and motor-neck localized to microtubules, often causing them to bundle. Over-expression of Klp6-motor or motor-neck resulted in shorter microtubules. These localization patterns were no different when constructs were expressed in strains lacking either or both of the endogenous, full-length proteins. Our results indicate that the localization signals for these kinesins are not derived from simple amino acid sequences but from complex interactions among multiple domains of each motor. PMID:18553361

  11. Identification and Evolution of Functional Alleles of the Previously Described Pollen Specific Myrosinase Pseudogene AtTGG6 in Arabidopsis thaliana

    PubMed Central

    Fu, Lili; Han, Bingying; Tan, Deguan; Wang, Meng; Ding, Mei; Zhang, Jiaming

    2016-01-01

    Myrosinases are β-thioglucoside glucohydrolases and serve as defense mechanisms against insect pests and pathogens by producing toxic compounds. AtTGG6 in Arabidopsis thaliana was previously reported to be a myrosinase pseudogene but specifically expressed in pollen. However, we found that AlTGG6, an ortholog to AtTGG6 in A. lyrata (an outcrossing relative of A. thaliana) was functional, suggesting that functional AtTGG6 alleles may still exist in A. thaliana. AtTGG6 alleles in 29 A. thaliana ecotypes were cloned and sequenced. Results indicate that ten alleles were functional and encoded Myr II type myrosinase of 512 amino acids, and myrosinase activity was confirmed by overexpressing AtTGG6 in Pichia pastoris. However, the 19 other ecotypes had disabled alleles with highly polymorphic frame-shift mutations and diversified sequences. Thirteen frame-shift mutation types were identified, which occurred independently many times in the evolutionary history within a few thousand years. The functional allele was expressed specifically in pollen similar to the disabled alleles but at a higher expression level, suggesting its role in defense of pollen against insect pests such as pollen beetles. However, the defense function may have become less critical after A. thaliana evolved to self-fertilization, and thus resulted in loss of function in most ecotypes. PMID:26907263

  12. Allele-Specific Suppression of Mutant Huntingtin Using Antisense Oligonucleotides: Providing a Therapeutic Option for All Huntington Disease Patients

    PubMed Central

    Skotte, Niels H.; Southwell, Amber L.; Østergaard, Michael E.; Carroll, Jeffrey B.; Warby, Simon C.; Doty, Crystal N.; Petoukhov, Eugenia; Vaid, Kuljeet; Kordasiewicz, Holly; Watt, Andrew T.; Freier, Susan M.; Hung, Gene; Seth, Punit P.; Bennett, C. Frank; Swayze, Eric E.; Hayden, Michael R.

    2014-01-01

    Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder. PMID:25207939

  13. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing.

    PubMed

    Sharma, Divya; Lather, Manila; Dykes, Cherry L; Dang, Amita S; Adak, Tridibes; Singh, Om P

    2016-01-01

    The rapid spread of antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective malaria control strategy. P. falciparum dihydropteroate synthase (Pfdhps) and P. falciparum dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the antimalarial drugs sulphadoxine and pyrimethamine, respectively. Resistance to pyrimethamine which is used as a partner drug in artemisinin combination therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's DNA sequencing method to detect minor alleles present in multiple clone infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor drug-resistant alleles present in multiple clonal infections. PMID:26407876

  14. Multiplex Allele-Specific Amplification from Whole Blood for Detecting Multiple Polymorphisms Simultaneously

    PubMed Central

    Zhu, Jianjie; Chen, Lanxin; Mao, Yong; Zhou, Huan

    2013-01-01

    Allele-specific amplification on the basis of polymerase chain reaction (PCR) has been widely used for single-nucleotide polymorphism (SNP) genotyping. However, the extraction of PCR-compatible genomic DNA from whole blood is usually required. This process is complicated and tedious, and is prone to cause cross-contamination between samples. To facilitate direct PCR amplification from whole blood without the extraction of genomic DNA, we optimized the pH value of PCR solution and the concentrations of magnesium ions and facilitator glycerol. Then, we developed multiplex allele-specific amplifications from whole blood and applied them to a case–control study. In this study, we successfully established triplex, five-plex, and eight-plex allele-specific amplifications from whole blood for determining the distribution of genotypes and alleles of 14 polymorphisms in 97 gastric cancer patients and 141 healthy controls. Statistical analysis results showed significant association of SNPs rs9344, rs1799931, and rs1800629 with the risk of gastric cancer. This method is accurate, time-saving, cost-effective, and easy-to-do, especially suitable for clinical prediction of disease susceptibility. PMID:23072573

  15. Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities

    PubMed Central

    Iyer, Swathi V.; Parrales, Alejandro; Begani, Priya; Narkar, Akshay; Adhikari, Amit S.; Martinez, Luis A.; Iwakuma, Tomoo

    2016-01-01

    Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53-specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53mut) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53-specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies. PMID:26700961

  16. Human-specific derived alleles of CD33 and other genes protect against postreproductive cognitive decline

    PubMed Central

    Schwarz, Flavio; Springer, Stevan A.; Altheide, Tasha K.; Varki, Nissi M.; Gagneux, Pascal; Varki, Ajit

    2016-01-01

    The individuals of most vertebrate species die when they can no longer reproduce. Humans are a rare exception, having evolved a prolonged postreproductive lifespan. Elders contribute to cooperative offspring care, assist in foraging, and communicate important ecological and cultural knowledge, increasing the survival of younger individuals. Age-related deterioration of cognitive capacity in humans compromises these benefits and also burdens the group with socially costly members. We investigated the contribution of the immunoregulatory receptor CD33 to a uniquely human postreproductive disease, Alzheimer’s dementia. Surprisingly, even though selection at advanced age is expected to be weak, a CD33 allele protective against Alzheimer’s disease is derived and unique to humans and favors a functional molecular state of CD33 resembling that of the chimpanzee. Thus, derived alleles may be compensatory and restore interactions altered as a consequence of human-specific brain evolution. We found several other examples of derived alleles at other human loci that protect against age-related cognitive deterioration arising from neurodegenerative disease or cerebrovascular insufficiency. Selection by inclusive fitness may be strong enough to favor alleles protecting specifically against cognitive decline in postreproductive humans. Such selection would operate by maximizing the contributions of postreproductive individuals to the fitness of younger kin. PMID:26621708

  17. Human-specific derived alleles of CD33 and other genes protect against postreproductive cognitive decline.

    PubMed

    Schwarz, Flavio; Springer, Stevan A; Altheide, Tasha K; Varki, Nissi M; Gagneux, Pascal; Varki, Ajit

    2016-01-01

    The individuals of most vertebrate species die when they can no longer reproduce. Humans are a rare exception, having evolved a prolonged postreproductive lifespan. Elders contribute to cooperative offspring care, assist in foraging, and communicate important ecological and cultural knowledge, increasing the survival of younger individuals. Age-related deterioration of cognitive capacity in humans compromises these benefits and also burdens the group with socially costly members. We investigated the contribution of the immunoregulatory receptor CD33 to a uniquely human postreproductive disease, Alzheimer's dementia. Surprisingly, even though selection at advanced age is expected to be weak, a CD33 allele protective against Alzheimer's disease is derived and unique to humans and favors a functional molecular state of CD33 resembling that of the chimpanzee. Thus, derived alleles may be compensatory and restore interactions altered as a consequence of human-specific brain evolution. We found several other examples of derived alleles at other human loci that protect against age-related cognitive deterioration arising from neurodegenerative disease or cerebrovascular insufficiency. Selection by inclusive fitness may be strong enough to favor alleles protecting specifically against cognitive decline in postreproductive humans. Such selection would operate by maximizing the contributions of postreproductive individuals to the fitness of younger kin. PMID:26621708

  18. TP53 allele loss, mutations and expression in malignant melanoma.

    PubMed Central

    Flørenes, V. A.; Oyjord, T.; Holm, R.; Skrede, M.; Børresen, A. L.; Nesland, J. M.; Fodstad, O.

    1994-01-01

    p53 alterations at the DNA, mRNA and protein levels were studied in tumour metastases sampled from 30 patients with malignant melanoma. Paraffin-embedded sections from these and an additional 12 patients were examined for the presence of p53 protein. TP53 gene aberrations were found in 7 of 30 (23%) of the patients, six of which showed loss of heterozygosity (LOH). Point mutations were detected in only two cases, one of which had LOH whereas the other was non-informative. Increased levels of p53 mRNA were present in only one tumour with, but in six cases without, detectable DNA abnormalities. Four of the latter and six tumours with normal transcript levels had immunohistochemically detectable levels of p53 protein. In 25 cases in which corresponding primary and metastatic lesions could be compared, closely similar immunoreactivity patterns were observed. Increased expression of the MDM2 gene was found in only one tumour in parallel with overexpression of p53. Altogether, the data indicate that inactivation of the p53 regulatory pathway is not of major significance in the tumorigenesis of malignant melanoma. However, a significant association was found between p53 immunoreactivity and the relapse-free period in patients with superficial spreading melanoma. That increased protein expression was predominantly found in tumours without DNA alterations might suggest a role for the wild-type p53 protein in restricting malignant cell proliferation in these cases. Images Figure 2 PMID:7905277

  19. HLA-B allele dropout in PCR sequence-specific oligonucleotide probe typing due to intronic polymorphism in the novel B*58:01:01:02 allele.

    PubMed

    He, Y; Wang, W; Han, Z; He, J; Chen, N; Dong, L; Tao, S; Zhang, W; He, J; Zhu, F; Lv, H

    2016-06-01

    Currently, Luminex technology based on the PCR sequence-specific oligonucleotide (SSO) probe method has been widely used for HLA genotyping in the immunogenetics laboratories. Here, we reported a case with HLA-B allele dropout by Luminex technology. The initial HLA-B result of the Luminex method with a commercial agent kit was inconclusive, and then, the result of PCR-SBT technology indicated the dropout as a HLA-B*58 allele. Subsequently, the full-length sequence of HLA-B allele was determined by TOPO-TA cloning, and a novel allele B*58:01:01:02 was identified in the individual. Compared with HLA-B*58:01:01:01, the novel allele showed some nucleotides difference at 509 C>T, 521 T>G and CCC insertion in position 503 of intron 2. According to the full-length sequence, the new mutations of intron 2 were contributed to HLA-B locus allele dropout in the sample. Our results indicated multiplatform should be used to improve the HLA typing accuracy when a conclusive HLA genotype cannot be determined. PMID:27016176

  20. Differential alleleic expression of the type II collagen gene (COL2A2) in osteoarthritic cartilage

    SciTech Connect

    Loughlin, J.; Irven, C.; Sykes, B.; Athanasou, N.; Carr, A.

    1995-05-01

    Osteoarthritis (OA) is a common debilitating disease resulting from the degeneration of articular cartilage. The major protein of cartilage is type II collagen, which is encoded by the COL2A1 gene. Mutations at this locus have been discovered in several individuals with inherited disorders of cartilage. We have identified 27 primary OA patients who are heterozygous for sequence dimorphisms located in the coding region of COL2A1. These dimorphisms were used to distinguish the mRNA output from each of the two COL2A1 alleles in articular cartilage obtained from each patient. Three patients demonstrated differential allelic expression and produced <12% of the normal level of mRNA from one of their COL2A1 alleles. The same allele shows reduced expression in a well-defined OA population than in a control group, suggesting the possible existence of a rare COL2A1 allele that predisposes to OA. 31 refs., 4 figs., 3 tabs.

  1. Detection of mutation by allele-specific loop-mediated isothermal amplification (AS-LAMP).

    PubMed

    Aonuma, Hiroka; Badolo, Athanase; Okado, Kiyoshi; Kanuka, Hirotaka

    2013-01-01

    For effective control of pathogen-transmitting mosquitoes, precise surveillance data of mosquito distribution are essential. Recently, an increase of insecticide resistance due to the kdr mutation in Anopheles gambiae, a mosquito that transmits the malaria parasite, has been reported. With the aim of developing a simple and effective method for surveying resistant mosquitoes, LAMP was applied to the allele-specific detection of the kdr gene in An. gambiae. Allele-specific LAMP (AS-LAMP) method successfully distinguished the kdr homozygote from the heterozygote and the wild type. The robustness of AS-LAMP suggests its usefulness for routine identification of insects, not only mosquitoes but also other vectors and agricultural pests. Here we describe the method of AS-LAMP to detect mutation in Anopheles mosquitoes. PMID:24026691

  2. Swine Leukocyte Antigen (SLA) class I allele typing of Danish swine herds and identification of commonly occurring haplotypes using sequence specific low and high resolution primers.

    PubMed

    Pedersen, Lasse Eggers; Jungersen, Gregers; Sorensen, Maria Rathmann; Ho, Chak-Sum; Vadekær, Dorte Fink

    2014-12-15

    The swine major histocompatibility complex (MHC) genomic region (SLA) is extremely polymorphic comprising high numbers of different alleles, many encoding a distinct MHC class I molecule, which binds and presents endogenous peptides to circulating T cells of the immune system. Upon recognition of such peptide-MHC complexes (pMHC) naïve T cells can become activated and respond to a given pathogen leading to its elimination and the generation of memory cells. Hence SLA plays a crucial role in maintaining overall adaptive immunologic resistance to pathogens. Knowing which SLA alleles that are commonly occurring can be of great importance in regard to future vaccine development and the establishment of immune protection in swine through broad coverage, highly specific, subunit based vaccination against viruses such as swine influenza, porcine reproductive and respiratory syndrome virus, vesicular stomatitis virus, foot-and-mouth-disease virus and others. Here we present the use of low- and high-resolution PCR-based typing methods to identify individual and commonly occurring SLA class I alleles in Danish swine. A total of 101 animals from seven different herds were tested, and by low resolution typing the top four most frequent SLA class I alleles were those of the allele groups SLA-3*04XX, SLA-1*08XX, SLA-2*02XX, and SLA-1*07XX, respectively. Customised high resolution primers were used to identify specific alleles within the above mentioned allele groups as well as within the SLA-2*05XX allele group. Our studies also suggest the most common haplotype in Danish pigs to be Lr-4.0 expressing the SLA-1*04XX, SLA-2*04XX, and SLA-3*04XX allele combination. PMID:25457547

  3. Analysis by transposable element mutagenesis of tissue-specific R alleles in maize

    SciTech Connect

    Not Available

    1984-04-01

    Transposition of the mobile sequence Dissociation to an R allele which confers seed but not plant color (Sc) destabilizes expression giving spotted kernels. Removal of the corresponding regulator from the genome immobilizes Ds at R. The resulting (Sc)Ds derivatives are colorless or pale seeded and stable as homozygotes. Strong seed color is rescued through recombination in heterozygotes of (Sc)Ds with an allele which confers only plant color, (P). Such crossovers provide a means of (1) mapping the position of Ds insertions within (Sc) relative to the site which distinguishes (Sc) from (P) action, and (2) systematically mutagenizing (P) and other such R elements by transferring Ds from (Sc) to homologous sites by recombination.

  4. Upstream Transcription Factor 1 (USF1) allelic variants regulate lipoprotein metabolism in women and USF1 expression in atherosclerotic plaque

    PubMed Central

    Fan, Yue-Mei; Hernesniemi, Jussi; Oksala, Niku; Levula, Mari; Raitoharju, Emma; Collings, Auni; Hutri-Kähönen, Nina; Juonala, Markus; Marniemi, Jukka; Lyytikäinen, Leo-Pekka; Seppälä, Ilkka; Mennander, Ari; Tarkka, Matti; Kangas, Antti J.; Soininen, Pasi; Salenius, Juha Pekka; Klopp, Norman; Illig, Thomas; Laitinen, Tomi; Ala-Korpela, Mika; Laaksonen, Reijo; Viikari, Jorma; Kähönen, Mika; Raitakari, Olli T.; Lehtimäki, Terho

    2014-01-01

    Upstream transcription factor 1 (USF1) allelic variants significantly influence future risk of cardiovascular disease and overall mortality in females. We investigated sex-specific effects of USF1 gene allelic variants on serum indices of lipoprotein metabolism, early markers of asymptomatic atherosclerosis and their changes during six years of follow-up. In addition, we investigated the cis-regulatory role of these USF1 variants in artery wall tissues in Caucasians. In the Cardiovascular Risk in Young Finns Study, 1,608 participants (56% women, aged 31.9 ± 4.9) with lipids and cIMT data were included. For functional study, whole genome mRNA expression profiling was performed in 91 histologically classified atherosclerotic samples. In females, serum total, LDL cholesterol and apoB levels increased gradually according to USF1 rs2516839 genotypes TT < CT < CC and rs1556259 AA < AG < GG as well as according to USF1 H3 (GCCCGG) copy number 0 < 1 < 2. Furthermore, the carriers of minor alleles of rs2516839 (C) and rs1556259 (G) of USF1 gene had decreased USF1 expression in atherosclerotic plaques (P = 0.028 and 0.08, respectively) as compared to non-carriers. The genetic variation in USF1 influence USF1 transcript expression in advanced atherosclerosis and regulates levels and metabolism of circulating apoB and apoB-containing lipoprotein particles in sex-dependent manner, but is not a major determinant of early markers of atherosclerosis. PMID:24722012

  5. Allele-specific silencing of EEC p63 mutant R304W restores p63 transcriptional activity

    PubMed Central

    Novelli, F; Lena, A M; Panatta, E; Nasser, W; Shalom-Feuerstein, R; Candi, E; Melino, G

    2016-01-01

    EEC (ectrodactily-ectodermal dysplasia and cleft lip/palate) syndrome is a rare genetic disease, autosomal dominant inherited. It is part of the ectodermal dysplasia disorders caused by heterozygous mutations in TP63 gene. EEC patients present limb malformations, orofacial clefting, skin and skin's appendages defects, ocular abnormalities. The transcription factor p63, encoded by TP63, is a master gene for the commitment of ectodermal-derived tissues, being expressed in the apical ectodermal ridge is critical for vertebrate limb formation and, at a later stage, for skin and skin's appendages development. The ΔNp63α isoform is predominantly expressed in epithelial cells and it is indispensable for preserving the self-renewal capacity of adult stem cells and to engage specific epithelial differentiation programs. Small interfering RNA (siRNA) offers a potential therapy approach for EEC patients by selectively silencing the mutant allele. Here, using a systemic screening based on a dual-luciferase reported gene assay, we have successfully identified specific siRNAs for repressing the EEC-causing p63 mutant, R304W. Upon siRNA treatment, we were able to restore ΔNp63-WT allele transcriptional function in induced pluripotent stem cells that were derived from EEC patient biopsy. This study demonstrates that siRNAs approach is promising and, may pave the way for curing/delaying major symptoms, such as cornea degeneration and skin erosions in young EEC patients. PMID:27195674

  6. A new mib allele with a chromosomal deletion covering foxc1a exhibits anterior somite specification defect

    PubMed Central

    Hsu, Chia-Hao; Lin, Ji-Sheng; Po Lai, Keng; Li, Jing-Woei; Chan, Ting-Fung; You, May-Su; Tse, William Ka Fai; Jiang, Yun-Jin

    2015-01-01

    mibnn2002, found from an allele screen, showed early segmentation defect and severe cell death phenotypes, which are different from previously known mib mutants. Despite distinct morphological phenotypes, the typical mib molecular phenotypes: her4 down-regulation, neurogenic phenotype and cold sensitive dlc expression pattern, still remained. The linkage analysis also indicated that mibnn2002 is a new mib allele. Failure of specification in anterior 7-10 somites is likely due to lack of foxc1a expression in mibnn2002 homozygotes. Somites and somite markers gradually appeared after 7-10 somite stage, suggesting that foxc1a is only essential for the formation of anterior 7-10 somites. Apoptosis began around 16-somite stage with p53 up-regulation. To find the possible links of mib, foxc1a and apoptosis, transcriptome analysis was employed. About 140 genes, including wnt3a, foxc1a and mib, were not detected in the homozygotes. Overexpression of foxc1a mRNA in mibnn2002 homozygotes partially rescued the anterior somite specification. In the process of characterizing mibnn2002 mutation, we integrated the scaffolds containing mib locus into chromosome 2 (or linkage group 2, LG2) based on synteny comparison and transcriptome results. Genomic PCR analysis further supported the conclusion and showed that mibnn2002 has a chromosomal deletion with the size of about 9.6 Mbp. PMID:26039894

  7. Allele-specific silencing of EEC p63 mutant R304W restores p63 transcriptional activity.

    PubMed

    Novelli, F; Lena, A M; Panatta, E; Nasser, W; Shalom-Feuerstein, R; Candi, E; Melino, G

    2016-01-01

    EEC (ectrodactily-ectodermal dysplasia and cleft lip/palate) syndrome is a rare genetic disease, autosomal dominant inherited. It is part of the ectodermal dysplasia disorders caused by heterozygous mutations in TP63 gene. EEC patients present limb malformations, orofacial clefting, skin and skin's appendages defects, ocular abnormalities. The transcription factor p63, encoded by TP63, is a master gene for the commitment of ectodermal-derived tissues, being expressed in the apical ectodermal ridge is critical for vertebrate limb formation and, at a later stage, for skin and skin's appendages development. The ΔNp63α isoform is predominantly expressed in epithelial cells and it is indispensable for preserving the self-renewal capacity of adult stem cells and to engage specific epithelial differentiation programs. Small interfering RNA (siRNA) offers a potential therapy approach for EEC patients by selectively silencing the mutant allele. Here, using a systemic screening based on a dual-luciferase reported gene assay, we have successfully identified specific siRNAs for repressing the EEC-causing p63 mutant, R304W. Upon siRNA treatment, we were able to restore ΔNp63-WT allele transcriptional function in induced pluripotent stem cells that were derived from EEC patient biopsy. This study demonstrates that siRNAs approach is promising and, may pave the way for curing/delaying major symptoms, such as cornea degeneration and skin erosions in young EEC patients. PMID:27195674

  8. Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference

    PubMed Central

    Sierant, Malgorzata; Paduszynska, Alina; Kazmierczak-Baranska, Julia; Nacmias, Benedetta; Sorbi, Sandro; Bagnoli, Silvia; Sochacka, Elzbieta; Nawrot, Barbara

    2011-01-01

    RNA interference (RNAi) technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs) are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G) alleles of human Presenilin1 gene (PSEN1). This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimer's disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2-Thiouridine chemical modification introduced at the 3′-end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide. PMID:21559198

  9. Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus

    PubMed Central

    Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for a wide range of biological applications including genome editing. In some cases, dissection of genome functions requires allele-specific genome editing, but the use of CRISPR for this purpose has not been studied in detail. In this study, using the p16INK4a gene in HCT116 as a model locus, we investigated whether chromatin states, such as CpG methylation, or a single-nucleotide gap form in a target site can be exploited for allele-specific locus binding and genome editing by CRISPR in vivo. First, we showed that allele-specific locus binding and genome editing could be achieved by targeting allele-specific CpG-methylated regions, which was successful for one, but not all guide RNAs. In this regard, molecular basis underlying the success remains elusive at this stage. Next, we demonstrated that an allele-specific single-nucleotide gap form could be employed for allele-specific locus binding and genome editing by CRISPR, although it was important to avoid CRISPR tolerance of a single nucleotide mismatch brought about by mismatched base skipping. Our results provide information that might be useful for applications of CRISPR in studies of allele-specific functions in the genomes. PMID:27465215

  10. Allele-specific deposition of macroH2A1 in Imprinting Control Regions

    SciTech Connect

    Choo, J H; Kim, J D; Chung, J H; Stubbs, L; Kim, J

    2006-01-13

    In the current study, we analyzed the deposition patterns of macroH2A1 at a number of different genomic loci located in X chromosome and autosomes. MacroH2A1 is preferentially deposited at methylated CpG CpG-rich regions located close to promoters. The macroH2A1 deposition patterns at the methylated CpG islands of several imprinted domains, including the Imprinting Control Regions (ICRs) of Xist, Peg3, H19/Igf2 Igf2, Gtl2/Dlk1, and Gnas domains, show consistent allele-specificity towards inactive, methylated alleles. The macroH2A1 deposition levels at the ICRs and other Differentially Methylated Regions (DMRs) of these domains are also either higher or comparable to those observed at the inactive X chromosome of female mammals. Overall, our results indicate that besides DNA methylation macroH2A1 is another epigenetic component in the chromatin of ICRs displaying differential association with two parental alleles.

  11. Allele-Specific Amplification in Cancer Revealed by SNP Array Analysis

    PubMed Central

    2005-01-01

    Amplification, deletion, and loss of heterozygosity of genomic DNA are hallmarks of cancer. In recent years a variety of studies have emerged measuring total chromosomal copy number at increasingly high resolution. Similarly, loss-of-heterozygosity events have been finely mapped using high-throughput genotyping technologies. We have developed a probe-level allele-specific quantitation procedure that extracts both copy number and allelotype information from single nucleotide polymorphism (SNP) array data to arrive at allele-specific copy number across the genome. Our approach applies an expectation-maximization algorithm to a model derived from a novel classification of SNP array probes. This method is the first to our knowledge that is able to (a) determine the generalized genotype of aberrant samples at each SNP site (e.g., CCCCT at an amplified site), and (b) infer the copy number of each parental chromosome across the genome. With this method, we are able to determine not just where amplifications and deletions occur, but also the haplotype of the region being amplified or deleted. The merit of our model and general approach is demonstrated by very precise genotyping of normal samples, and our allele-specific copy number inferences are validated using PCR experiments. Applying our method to a collection of lung cancer samples, we are able to conclude that amplification is essentially monoallelic, as would be expected under the mechanisms currently believed responsible for gene amplification. This suggests that a specific parental chromosome may be targeted for amplification, whether because of germ line or somatic variation. An R software package containing the methods described in this paper is freely available at http://genome.dfci.harvard.edu/~tlaframb/PLASQ. PMID:16322765

  12. Defining a personal, allele-specific, and single-molecule long-read transcriptome.

    PubMed

    Tilgner, Hagen; Grubert, Fabian; Sharon, Donald; Snyder, Michael P

    2014-07-01

    Personal transcriptomes in which all of an individual's genetic variants (e.g., single nucleotide variants) and transcript isoforms (transcription start sites, splice sites, and polyA sites) are defined and quantified for full-length transcripts are expected to be important for understanding individual biology and disease, but have not been described previously. To obtain such transcriptomes, we sequenced the lymphoblastoid transcriptomes of three family members (GM12878 and the parents GM12891 and GM12892) by using a Pacific Biosciences long-read approach complemented with Illumina 101-bp sequencing and made the following observations. First, we found that reads representing all splice sites of a transcript are evident for most sufficiently expressed genes ≤3 kb and often for genes longer than that. Second, we added and quantified previously unidentified splicing isoforms to an existing annotation, thus creating the first personalized annotation to our knowledge. Third, we determined SNVs in a de novo manner and connected them to RNA haplotypes, including HLA haplotypes, thereby assigning single full-length RNA molecules to their transcribed allele, and demonstrated Mendelian inheritance of RNA molecules. Fourth, we show how RNA molecules can be linked to personal variants on a one-by-one basis, which allows us to assess differential allelic expression (DAE) and differential allelic isoforms (DAI) from the phased full-length isoform reads. The DAI method is largely independent of the distance between exon and SNV--in contrast to fragmentation-based methods. Overall, in addition to improving eukaryotic transcriptome annotation, these results describe, to our knowledge, the first large-scale and full-length personal transcriptome. PMID:24961374

  13. Short alleles, bigger smiles? The effect of 5-HTTLPR on positive emotional expressions.

    PubMed

    Haase, Claudia M; Beermann, Ursula; Saslow, Laura R; Shiota, Michelle N; Saturn, Sarina R; Lwi, Sandy J; Casey, James J; Nguyen, Nguyen K; Whalen, Patrick K; Keltner, Dacher; Levenson, Robert W

    2015-08-01

    The present research examined the effect of the 5-HTTLPR polymorphism in the serotonin transporter gene on objectively coded positive emotional expressions (i.e., laughing and smiling behavior objectively coded using the Facial Action Coding System). Three studies with independent samples of participants were conducted. Study 1 examined young adults watching still cartoons. Study 2 examined young, middle-aged, and older adults watching a thematically ambiguous yet subtly amusing film clip. Study 3 examined middle-aged and older spouses discussing an area of marital conflict (that typically produces both positive and negative emotion). Aggregating data across studies, results showed that the short allele of 5-HTTLPR predicted heightened positive emotional expressions. Results remained stable when controlling for age, gender, ethnicity, and depressive symptoms. These findings are consistent with the notion that the short allele of 5-HTTLPR functions as an emotion amplifier, which may confer heightened susceptibility to environmental conditions. PMID:26029940

  14. Short Alleles, Bigger Smiles? The Effect of 5-HTTLPR on Positive Emotional Expressions

    PubMed Central

    Haase, Claudia M.; Beermann, Ursula; Saslow, Laura R.; Shiota, Michelle N.; Saturn, Sarina R.; Lwi, Sandy J.; Casey, James J.; Nguyen, Nguyen K.; Whalen, Patrick K.; Keltner, Dacher J.; Levenson, Robert W.

    2015-01-01

    The present research examined the effect of the 5-HTTLPR polymorphism in the serotonin transporter gene on objectively coded positive emotional expressions (i.e., laughing and smiling behavior objectively coded using the Facial Action Coding System). Three studies with independent samples of participants were conducted. Study 1 examined young adults watching still cartoons. Study 2 examined young, middle-aged, and older adults watching a thematically ambiguous yet subtly amusing film clip. Study 3 examined middle-aged and older spouses discussing an area of marital conflict (which typically produces both positive and negative emotion). Aggregating data across studies, results showed that the short allele of 5-HTTLPR predicted heightened positive emotional expressions. Results remained stable when controlling for age, gender, ethnicity, and depressive symptoms. These findings are consistent with the notion that the short allele of 5-HTTLPR functions as an emotion amplifier, which may confer heightened susceptibility to environmental conditions. PMID:26029940

  15. siRNA-mediated Allele-specific Silencing of a COL6A3 Mutation in a Cellular Model of Dominant Ullrich Muscular Dystrophy

    PubMed Central

    Bolduc, Véronique; Zou, Yaqun; Ko, Dayoung; Bönnemann, Carsten G

    2014-01-01

    Congenital muscular dystrophy type Ullrich (UCMD) is a severe disorder of early childhood onset for which currently there is no effective treatment. UCMD commonly is caused by dominant-negative mutations in the genes coding for collagen type VI, a major microfibrillar component of the extracellular matrix surrounding the muscle fibers. To explore RNA interference (RNAi) as a potential therapy for UCMD, we designed a series of small interfering RNA (siRNA) oligos that specifically target the most common mutations resulting in skipping of exon 16 in the COL6A3 gene and tested them in UCMD-derived dermal fibroblasts. Transcript analysis by semiquantitative and quantitative reverse transcriptase PCR showed that two of these siRNAs were the most allele-specific, i.e., they efficiently knocked down the expression from the mutant allele, without affecting the normal allele. In HEK293T cells, these siRNAs selectively suppressed protein expression from a reporter construct carrying the mutation, with no or minimal suppression of the wild-type (WT) construct, suggesting that collagen VI protein levels are as also reduced in an allele-specific manner. Furthermore, we found that treating UCMD fibroblasts with these siRNAs considerably improved the quantity and quality of the collagen VI matrix, as assessed by confocal microscopy. Our current study establishes RNAi as a promising molecular approach for treating dominant COL6-related dystrophies. PMID:24518369

  16. Allele-Specific Behavior of Molecular Networks: Understanding Small-Molecule Drug Response in Yeast

    PubMed Central

    Li, Chunquan; Hao, Dapeng; Zhang, Shaojun; Zhou, Meng; Su, Fei; Chen, Xi; Zhi, Hui; Li, Xia

    2013-01-01

    The study of systems genetics is changing the way the genetic and molecular basis of phenotypic variation, such as disease susceptibility and drug response, is being analyzed. Moreover, systems genetics aids in the translation of insights from systems biology into genetics. The use of systems genetics enables greater attention to be focused on the potential impact of genetic perturbations on the molecular states of networks that in turn affects complex traits. In this study, we developed models to detect allele-specific perturbations on interactions, in which a genetic locus with alternative alleles exerted a differing influence on an interaction. We utilized the models to investigate the dynamic behavior of an integrated molecular network undergoing genetic perturbations in yeast. Our results revealed the complexity of regulatory relationships between genetic loci and networks, in which different genetic loci perturb specific network modules. In addition, significant within-module functional coherence was found. We then used the network perturbation model to elucidate the underlying molecular mechanisms of individual differences in response to 100 diverse small molecule drugs. As a result, we identified sub-networks in the integrated network that responded to variations in DNA associated with response to diverse compounds and were significantly enriched for known drug targets. Literature mining results provided strong independent evidence for the effectiveness of these genetic perturbing networks in the elucidation of small-molecule responses in yeast. PMID:23308257

  17. In Vivo Evaluation of Candidate Allele-specific Mutant Huntingtin Gene Silencing Antisense Oligonucleotides

    PubMed Central

    Southwell, Amber L; Skotte, Niels H; Kordasiewicz, Holly B; Østergaard, Michael E; Watt, Andrew T; Carroll, Jeffrey B; Doty, Crystal N; Villanueva, Erika B; Petoukhov, Eugenia; Vaid, Kuljeet; Xie, Yuanyun; Freier, Susan M; Swayze, Eric E; Seth, Punit P; Bennett, Clarence Frank; Hayden, Michael R

    2014-01-01

    Huntington disease (HD) is a dominant, genetic neurodegenerative disease characterized by progressive loss of voluntary motor control, psychiatric disturbance, and cognitive decline, for which there is currently no disease-modifying therapy. HD is caused by the expansion of a CAG tract in the huntingtin (HTT) gene. The mutant HTT protein (muHTT) acquires toxic functions, and there is significant evidence that muHTT lowering would be therapeutically efficacious. However, the wild-type HTT protein (wtHTT) serves vital functions, making allele-specific muHTT lowering strategies potentially safer than nonselective strategies. CAG tract expansion is associated with single nucleotide polymorphisms (SNPs) that can be targeted by gene silencing reagents such as antisense oligonucleotides (ASOs) to accomplish allele-specific muHTT lowering. Here we evaluate ASOs targeted to HD-associated SNPs in acute in vivo studies including screening, distribution, duration of action and dosing, using a humanized mouse model of HD, Hu97/18, that is heterozygous for the targeted SNPs. We have identified four well-tolerated lead ASOs that potently and selectively silence muHTT at a broad range of doses throughout the central nervous system for 16 weeks or more after a single intracerebroventricular (ICV) injection. With further validation, these ASOs could provide a therapeutic option for individuals afflicted with HD. PMID:25101598

  18. A rapid and efficient strategy to generate allele-specific anti-HLA monoclonal antibodies.

    PubMed

    Yamazaki, Satoshi; Suzuki, Nao; Saito, Tsuneyoshi; Ishii, Yumiko; Takiguchi, Masafumi; Nakauchi, Hiromitsu; Watanabe, Nobukazu

    2009-03-31

    That generation of allele-specific anti-human leukocyte antigen (HLA) monoclonal antibodies (ASHmAb) is very difficult is well known. This is thought to be due to the unique epitope structure, an assemblage of amino acid residues that lie separately in the amino acid sequence of human HLA, and to its low antigenicity compared with that of common epitopes recognized as xenogeneic determinants by mice. Here we report a rapid and efficient strategy to generate ASHmAb. Different from usual immunization methods is that we suppressed the production of non-allele-specific anti-HLA antibodies against xenogeneic determinants of HLA molecules by immunizing human HLA-B51 transgenic mice against non-HLA-B51 HLA tetramers. In addition, HLA-coated beads enabled rapid and efficient screening for ASHmAb. ASHmAb generated by this strategy will be useful for HLA typing and for clinical diagnosis, such as flow cytometry-based chimerism analysis for early detection of graft failure and relapse of leukemia after HLA-mismatched hematopoietic stem cell transplantation. PMID:19187783

  19. Allelic expression of mammalian imprinted genes in a matrotrophic lizard, Pseudemoia entrecasteauxii.

    PubMed

    Griffith, Oliver W; Brandley, Matthew C; Belov, Katherine; Thompson, Michael B

    2016-03-01

    Genomic imprinting is a process that results in the differential expression of genes depending on their parent of origin. It occurs in both plants and live-bearing mammals, with imprinted genes typically regulating the ability of an embryo to manipulate the maternal provision of nutrients. Genomic imprinting increases the potential for selection to act separately on paternally and maternally expressed genes, which increases the number of opportunities that selection can facilitate embryonic control over maternal nutrient provision. By looking for imprinting in an independent matrotrophic lineage, the viviparous lizard Pseudemoia entrecasteauxii (Scincidae), we test the hypothesis that genomic imprinting facilitates the evolution of substantial placental nutrient transport to embryos (matrotrophy). We sequenced transcriptomes from the embryonic component of lizard placentae to determine whether there are parent-of-origin differences in expression of genes that are imprinted in mammals. Of these genes, 19 had sufficiently high expression in the lizard to identify polymorphisms in transcribed sequences. We identified bi-allelic expression in 17 genes (including insulin-like growth factor 2), indicating that neither allele was imprinted. These data suggest that either genomic imprinting has not evolved in this matrotrophic skink or, if it has, it has evolved in different genes to mammals. We outline how these hypotheses can be tested. This study highlights important differences between mammalian and reptile pregnancy and the absence of any shared imprinting genes reflects fundamental differences in the way that pregnancy has evolved in these two lineages. PMID:26943808

  20. Cellular Adhesion Gene SELP Is Associated with Rheumatoid Arthritis and Displays Differential Allelic Expression

    PubMed Central

    Petit-Teixeira, Elisabeth; Hugo Teixeira, Vitor; Steiner, Anke; Quente, Elfi; Wolfram, Grit; Scholz, Markus; Pierlot, Céline; Migliorini, Paola; Bombardieri, Stefano; Balsa, Alejandro; Westhovens, René; Barrera, Pilar; Radstake, Timothy R. D. J.; Alves, Helena; Bardin, Thomas; Prum, Bernard; Emmrich, Frank; Cornelis, François

    2014-01-01

    In rheumatoid arthritis (RA), a key event is infiltration of inflammatory immune cells into the synovial lining, possibly aggravated by dysregulation of cellular adhesion molecules. Therefore, single nucleotide polymorphisms of 14 genes involved in cellular adhesion processes (CAST, ITGA4, ITGB1, ITGB2, PECAM1, PTEN, PTPN11, PTPRC, PXN, SELE, SELP, SRC, TYK2, and VCAM1) were analyzed for association with RA. Association analysis was performed consecutively in three European RA family sample groups (Nfamilies = 407). Additionally, we investigated differential allelic expression, a possible functional consequence of genetic variants. SELP (selectin P, CD62P) SNP-allele rs6136-T was associated with risk for RA in two RA family sample groups as well as in global analysis of all three groups (ptotal = 0.003). This allele was also expressed preferentially (p<10−6) with a two- fold average increase in regulated samples. Differential expression is supported by data from Genevar MuTHER (p1 = 0.004; p2 = 0.0177). Evidence for influence of rs6136 on transcription factor binding was also found in silico and in public datasets reporting in vitro data. In summary, we found SELP rs6136-T to be associated with RA and with increased expression of SELP mRNA. SELP is located on the surface of endothelial cells and crucial for recruitment, adhesion, and migration of inflammatory cells into the joint. Genetically determined increased SELP expression levels might thus be a novel additional risk factor for RA. PMID:25147926

  1. Geographically Distinct and Domain-Specific Sequence Variations in the Alleles of Rice Blast Resistance Gene Pib

    PubMed Central

    Vasudevan, Kumar; Vera Cruz, Casiana M.; Gruissem, Wilhelm; Bhullar, Navreet K.

    2016-01-01

    Rice blast is caused by Magnaporthe oryzae, which is the most destructive fungal pathogen affecting rice growing regions worldwide. The rice blast resistance gene Pib confers broad-spectrum resistance against Southeast Asian M. oryzae races. We investigated the allelic diversity of Pib in rice germplasm originating from 12 major rice growing countries. Twenty-five new Pib alleles were identified that have unique single nucleotide polymorphisms (SNPs), insertions and/or deletions, in addition to the polymorphic nucleotides that are shared between the different alleles. These partially or completely shared polymorphic nucleotides indicate frequent sequence exchange events between the Pib alleles. In some of the new Pib alleles, nucleotide diversity is high in the LRR domain, whereas, in others it is distributed among the NB-ARC and LRR domains. Most of the polymorphic amino acids in LRR and NB-ARC2 domains are predicted as solvent-exposed. Several of the alleles and the unique SNPs are country specific, suggesting a diversifying selection of alleles in various geographical locations in response to the locally prevalent M. oryzae population. Together, the new Pib alleles are an important genetic resource for rice blast resistance breeding programs and provide new information on rice-M. oryzae interactions at the molecular level. PMID:27446145

  2. H pylori iceA alleles are disease-specific virulence factors

    PubMed Central

    Caner, Vildan; Yilmaz, Mustafa; Yonetci, Nadir; Zencir, Sevil; Karagenc, Nedim; Kaleli, Ilknur; Bagci, Huseyin

    2007-01-01

    AIM: To characterize and compare genotype profiles of H pylori strains isolated from patients with chronic gastritis and duodenal ulcer in western part of Turkey. METHODS: A total of 46 patients [30 chronic gastritis (CG) and 16 duodenal ulcer (DU)] who had undergone endoscopy because of dyspeptic complaints were studied. The antral biopsy specimens were evaluated for the presence of H pylori by rapid urease test and culture, and the genotype profiles were determined by real-time PCR. RESULTS: The cagA gene was observed in 43 (93.5%) isolates. The vacA s1m2 genotype was the predominant subtype, found in 63.3% and 68.7% of isolates in patients with CG and DU, respectively. Twenty (66.6%) isolates from patients with CG were iceA2 positive while the iceA1 was predominant in those with DU (68.8%). In terms of the association of the iceA alleles to other genes, both alleles were significantly associated with the cagA vacA s1m2 genotype. CONCLUSION: The prevalent circulating genotypes in CG and DU were cagA vacA s1m2 iceA2 and cagA vacA s1m2 iceA1 genotype, respectively. It was found that cagA vacA s1m2 genotype seems to be common virulence factors in both CG and DU while iceA alleles show specificity for gastroduodenal pathologies in this study. PMID:17552005

  3. The maize unstable factor for orange1 is a dominant epigenetic modifier of a tissue specifically silent allele of pericarp color1.

    PubMed Central

    Chopra, Surinder; Cocciolone, Suzy M; Bushman, Shaun; Sangar, Vineet; McMullen, Michael D; Peterson, Thomas

    2003-01-01

    We have characterized Unstable factor for orange1 (Ufo1), a dominant, allele-specific modifier of expression of the maize pericarp color1 (p1) gene. The p1 gene encodes an Myb-homologous transcriptional activator of genes required for biosynthesis of red phlobaphene pigments. The P1-wr allele specifies colorless kernel pericarp and red cobs, whereas Ufo1 modifies P1-wr expression to confer pigmentation in kernel pericarp, as well as vegetative tissues, which normally do not accumulate significant amounts of phlobaphene pigments. In the presence of Ufo1, P1-wr transcript levels and transcription rate are increased in kernel pericarp. The P1-wr allele contains approximately six p1 gene copies present in a hypermethylated and multicopy tandem array. In P1-wr Ufo1 plants, methylation of P1-wr DNA sequences is reduced, whereas the methylation state of other repetitive genomic sequences was not detectably affected. The phenotypes produced by the interaction of P1-wr and Ufo1 are unstable, exhibiting somatic mosaicism and variable penetrance. Moreover, the changes in P1-wr expression and methylation are not heritable: meiotic segregants that lack Ufo1 revert to the normal P1-wr expression and methylation patterns. These results demonstrate the existence of a class of modifiers of gene expression whose effects are associated with transient changes in DNA methylation of specific loci. PMID:12663550

  4. Allele-specific distribution of RNA polymerase II on female X chromosomes.

    PubMed

    Kucera, Katerina S; Reddy, Timothy E; Pauli, Florencia; Gertz, Jason; Logan, Jenae E; Myers, Richard M; Willard, Huntington F

    2011-10-15

    While the distribution of RNA polymerase II (PolII) in a variety of complex genomes is correlated with gene expression, the presence of PolII at a gene does not necessarily indicate active expression. Various patterns of PolII binding have been described genome wide; however, whether or not PolII binds at transcriptionally inactive sites remains uncertain. The two X chromosomes in female cells in mammals present an opportunity to examine each of the two alleles of a given locus in both active and inactive states, depending on which X chromosome is silenced by X chromosome inactivation. Here, we investigated PolII occupancy and expression of the associated genes across the active (Xa) and inactive (Xi) X chromosomes in human female cells to elucidate the relationship of gene expression and PolII binding. We find that, while PolII in the pseudoautosomal region occupies both chromosomes at similar levels, it is significantly biased toward the Xa throughout the rest of the chromosome. The general paucity of PolII on the Xi notwithstanding, detectable (albeit significantly reduced) binding can be observed, especially on the evolutionarily younger short arm of the X. PolII levels at genes that escape inactivation correlate with the levels of their expression; however, additional PolII sites can be found at apparently silenced regions, suggesting the possibility of a subset of genes on the Xi that are poised for expression. Consistent with this hypothesis, we show that a high proportion of genes associated with PolII-accessible sites, while silenced in GM12878, are expressed in other female cell lines. PMID:21791549

  5. A knock-in allele of En1 expressing dre recombinase.

    PubMed

    Plummer, Nicholas W; de Marchena, Jacqueline; Jensen, Patricia

    2016-08-01

    Engrailed 1 (En1) is a homeobox-containing transcription factor expressed during development in diverse tissues, including the embryonic midbrain and anterior hindbrain. To facilitate investigation of genetic and developmental heterogeneity among cells with a history of En1 expression, we have generated En1(Dre) , a knock-in allele expressing Dre recombinase. En1(Dre) can be used with existing Cre and Flp recombinase lines for genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by transient expression of En1. To avoid disrupting En1 function, the Dre cDNA is inserted at the 3' end of the En1 coding sequence, together with a viral 2A peptide to mediate translation of separate EN1 and Dre proteins. Consequently, viable and fertile En1(Dre) homozygotes can be used to increase the proportion of useful genotypes produced in complex crosses. The pattern of Dre expression from En1(Dre) is indistinguishable from wild-type En1 expression in mid-gestation mouse embryos, and En1(Dre) controls Dre-responsive indicator alleles by efficiently recombining rox sites in vivo. Through the application of genetic tools that allow manipulation of cells based on combinatorial expression of multiple distinct recombinases, En1(Dre) will significantly extend the ability to target important subpopulations of neurons and other cells within the broader En1 expression domain. genesis 54:447-454, 2016. Published 2016. This article is a US Government work and is in the public domain in the USA. PMID:27313055

  6. Maternal and paternal genomes function independently in mouse ova in establishing expression of the imprinted genes Snrpn and Igf2r: no evidence for allelic trans-sensing and counting mechanisms.

    PubMed Central

    Szabó, P E; Mann, J R

    1996-01-01

    It has often been suggested that the parental-specific expression of mammalian imprinted genes might be dependent on maternal-paternal intergenomic or interallelic interactions. Using quantitative allele-specific RT-PCR single nucleotide primer extension assays developed for two imprinted genes, Snrpn and Igf2r, we demonstrate: (i) No role for maternal-paternal allelic interactions: the modes of parental-specific expression of Snrpn and Igf2r in normal ova were unchanged in gynogenetic and androgenetic ova; the latter contain two maternal and two paternal genomes respectively, and cannot undergo maternal-paternal interactions. (ii) No role for allelic counting or exclusion mechanisms: in individual blastomeres of androgenetic ova, both paternal Snrpn alleles were active (Snrpn was not expressed in gynogenetic ova), and in individual gynogenetic and androgenetic blastomeres, both maternal and paternal Igf2r alleles, respectively, were active. (iii) No role for ploidy: the mode of parental-specific expression of Snrpn and Igf2r in normal diploid ova was unchanged in individual blastomeres of triploid and tetraploid ova. Thus, the maternal and paternal genomes function independently in establishing the pre-implantation mode of parental-specific expression of Snrpn and Igf2r, with no role for trans-allelic/genomic interaction phenomena. In addition, the results show that inactive and biallelic modes of expression of imprinted genes are potential mechanisms for the death of gynogenones and androgenones at the peri-implantation stage. Images PMID:8947024

  7. [Study on identification of cistanche hebra and its adulterants by PCR amplification of specific alleles based on ITS sequences].

    PubMed

    Li, Zhen-Hua; Long, Ping; Zou, De-Zhi; Li, Yue; Cui, Zhan-Hu; Li, Min-Hui

    2014-10-01

    To explore the new method of discriminating Cistanche deserticola, Cynomorium songaricum and Orobanche pycnostachya by using PCR amplification of specific alleles. 30 samples of the different C. deserticola, 21 samples of C. songaricum and O. pycnostachya were collected. The total DNA of the samples were extracted, the ITS sequences from C. deserticola, C. songaricum and O. pycnostachya were amplified by PCR and sequenced unidirectionally. These sequences were aligned by using ClustulW. Specific primer was designed according to the ITS sequences of specific alleles, and PCR reaction system was optimized. Additionally, compare with the identification of specific PCR method and DNA sequence analysis method. The result showed that the 331 bp identification band for C. deserticola and the adulterants not amplified bands by a single PCR reaction, which showed good identification ability to the three species. PCR amplification of specific alleles can be used to identify C. deserticola, C. songaricum and O. pycnostachya successfully. PMID:25612421

  8. Utilizing ethnic-specific differences in minor allele frequency to recategorize reported pathogenic deafness variants.

    PubMed

    Shearer, A Eliot; Eppsteiner, Robert W; Booth, Kevin T; Ephraim, Sean S; Gurrola, José; Simpson, Allen; Black-Ziegelbein, E Ann; Joshi, Swati; Ravi, Harini; Giuffre, Angelica C; Happe, Scott; Hildebrand, Michael S; Azaiez, Hela; Bayazit, Yildirim A; Erdal, Mehmet Emin; Lopez-Escamez, Jose A; Gazquez, Irene; Tamayo, Marta L; Gelvez, Nancy Y; Leal, Greizy Lopez; Jalas, Chaim; Ekstein, Josef; Yang, Tao; Usami, Shin-ichi; Kahrizi, Kimia; Bazazzadegan, Niloofar; Najmabadi, Hossein; Scheetz, Todd E; Braun, Terry A; Casavant, Thomas L; LeProust, Emily M; Smith, Richard J H

    2014-10-01

    Ethnic-specific differences in minor allele frequency impact variant categorization for genetic screening of nonsyndromic hearing loss (NSHL) and other genetic disorders. We sought to evaluate all previously reported pathogenic NSHL variants in the context of a large number of controls from ethnically distinct populations sequenced with orthogonal massively parallel sequencing methods. We used HGMD, ClinVar, and dbSNP to generate a comprehensive list of reported pathogenic NSHL variants and re-evaluated these variants in the context of 8,595 individuals from 12 populations and 6 ethnically distinct major human evolutionary phylogenetic groups from three sources (Exome Variant Server, 1000 Genomes project, and a control set of individuals created for this study, the OtoDB). Of the 2,197 reported pathogenic deafness variants, 325 (14.8%) were present in at least one of the 8,595 controls, indicating a minor allele frequency (MAF) > 0.00006. MAFs ranged as high as 0.72, a level incompatible with pathogenicity for a fully penetrant disease like NSHL. Based on these data, we established MAF thresholds of 0.005 for autosomal-recessive variants (excluding specific variants in GJB2) and 0.0005 for autosomal-dominant variants. Using these thresholds, we recategorized 93 (4.2%) of reported pathogenic variants as benign. Our data show that evaluation of reported pathogenic deafness variants using variant MAFs from multiple distinct ethnicities and sequenced by orthogonal methods provides a powerful filter for determining pathogenicity. The proposed MAF thresholds will facilitate clinical interpretation of variants identified in genetic testing for NSHL. All data are publicly available to facilitate interpretation of genetic variants causing deafness. PMID:25262649

  9. Utilizing Ethnic-Specific Differences in Minor Allele Frequency to Recategorize Reported Pathogenic Deafness Variants

    PubMed Central

    Shearer, A. Eliot; Eppsteiner, Robert W.; Booth, Kevin T.; Ephraim, Sean S.; Gurrola, José; Simpson, Allen; Black-Ziegelbein, E. Ann; Joshi, Swati; Ravi, Harini; Giuffre, Angelica C.; Happe, Scott; Hildebrand, Michael S.; Azaiez, Hela; Bayazit, Yildirim A.; Erdal, Mehmet Emin; Lopez-Escamez, Jose A.; Gazquez, Irene; Tamayo, Marta L.; Gelvez, Nancy Y.; Leal, Greizy Lopez; Jalas, Chaim; Ekstein, Josef; Yang, Tao; Usami, Shin-ichi; Kahrizi, Kimia; Bazazzadegan, Niloofar; Najmabadi, Hossein; Scheetz, Todd E.; Braun, Terry A.; Casavant, Thomas L.; LeProust, Emily M.; Smith, Richard J.H.

    2014-01-01

    Ethnic-specific differences in minor allele frequency impact variant categorization for genetic screening of nonsyndromic hearing loss (NSHL) and other genetic disorders. We sought to evaluate all previously reported pathogenic NSHL variants in the context of a large number of controls from ethnically distinct populations sequenced with orthogonal massively parallel sequencing methods. We used HGMD, ClinVar, and dbSNP to generate a comprehensive list of reported pathogenic NSHL variants and re-evaluated these variants in the context of 8,595 individuals from 12 populations and 6 ethnically distinct major human evolutionary phylogenetic groups from three sources (Exome Variant Server, 1000 Genomes project, and a control set of individuals created for this study, the OtoDB). Of the 2,197 reported pathogenic deafness variants, 325 (14.8%) were present in at least one of the 8,595 controls, indicating a minor allele frequency (MAF) >0.00006. MAFs ranged as high as 0.72, a level incompatible with pathogenicity for a fully penetrant disease like NSHL. Based on these data, we established MAF thresholds of 0.005 for autosomal-recessive variants (excluding specific variants in GJB2) and 0.0005 for autosomal-dominant variants. Using these thresholds, we recategorized 93 (4.2%) of reported pathogenic variants as benign. Our data show that evaluation of reported pathogenic deafness variants using variant MAFs from multiple distinct ethnicities and sequenced by orthogonal methods provides a powerful filter for determining pathogenicity. The proposed MAF thresholds will facilitate clinical interpretation of variants identified in genetic testing for NSHL. All data are publicly available to facilitate interpretation of genetic variants causing deafness. PMID:25262649

  10. Mechanisms and Disease Associations of Haplotype-Dependent Allele-Specific DNA Methylation.

    PubMed

    Do, Catherine; Lang, Charles F; Lin, John; Darbary, Huferesh; Krupska, Izabela; Gaba, Aulona; Petukhova, Lynn; Vonsattel, Jean-Paul; Gallagher, Mary P; Goland, Robin S; Clynes, Raphael A; Dwork, Andrew; Kral, John G; Monk, Catherine; Christiano, Angela M; Tycko, Benjamin

    2016-05-01

    Haplotype-dependent allele-specific methylation (hap-ASM) can impact disease susceptibility, but maps of this phenomenon using stringent criteria in disease-relevant tissues remain sparse. Here we apply array-based and Methyl-Seq approaches to multiple human tissues and cell types, including brain, purified neurons and glia, T lymphocytes, and placenta, and identify 795 hap-ASM differentially methylated regions (DMRs) and 3,082 strong methylation quantitative trait loci (mQTLs), most not previously reported. More than half of these DMRs have cell type-restricted ASM, and among them are 188 hap-ASM DMRs and 933 mQTLs located near GWAS signals for immune and neurological disorders. Targeted bis-seq confirmed hap-ASM in 12/13 loci tested, including CCDC155, CD69, FRMD1, IRF1, KBTBD11, and S100A(∗)-ILF2, associated with immune phenotypes, MYT1L, PTPRN2, CMTM8 and CELF2, associated with neurological disorders, NGFR and HLA-DRB6, associated with both immunological and brain disorders, and ZFP57, a trans-acting regulator of genomic imprinting. Polymorphic CTCF and transcription factor (TF) binding sites were over-represented among hap-ASM DMRs and mQTLs, and analysis of the human data, supplemented by cross-species comparisons to macaques, indicated that CTCF and TF binding likelihood predicts the strength and direction of the allelic methylation asymmetry. These results show that hap-ASM is highly tissue specific; an important trans-acting regulator of genomic imprinting is regulated by this phenomenon; and variation in CTCF and TF binding sites is an underlying mechanism, and maps of hap-ASM and mQTLs reveal regulatory sequences underlying supra- and sub-threshold GWAS peaks in immunological and neurological disorders. PMID:27153397

  11. Allele-specific chromatin remodeling in the ZPBP2/GSDMB/ORMDL3 locus associated with the risk of asthma and autoimmune disease.

    PubMed

    Verlaan, Dominique J; Berlivet, Soizik; Hunninghake, Gary M; Madore, Anne-Marie; Larivière, Mathieu; Moussette, Sanny; Grundberg, Elin; Kwan, Tony; Ouimet, Manon; Ge, Bing; Hoberman, Rose; Swiatek, Marcin; Dias, Joana; Lam, Kevin C L; Koka, Vonda; Harmsen, Eef; Soto-Quiros, Manuel; Avila, Lydiana; Celedón, Juan C; Weiss, Scott T; Dewar, Ken; Sinnett, Daniel; Laprise, Catherine; Raby, Benjamin A; Pastinen, Tomi; Naumova, Anna K

    2009-09-01

    Common SNPs in the chromosome 17q12-q21 region alter the risk for asthma, type 1 diabetes, primary biliary cirrhosis, and Crohn disease. Previous reports by us and others have linked the disease-associated genetic variants with changes in expression of GSDMB and ORMDL3 transcripts in human lymphoblastoid cell lines (LCLs). The variants also alter regulation of other transcripts, and this domain-wide cis-regulatory effect suggests a mechanism involving long-range chromatin interactions. Here, we further dissect the disease-linked haplotype and identify putative causal DNA variants via a combination of genetic and functional analyses. First, high-throughput resequencing of the region and genotyping of potential candidate variants were performed. Next, additional mapping of allelic expression differences in Yoruba HapMap LCLs allowed us to fine-map the basis of the cis-regulatory differences to a handful of candidate functional variants. Functional assays identified allele-specific differences in nucleosome distribution, an allele-specific association with the insulator protein CTCF, as well as a weak promoter activity for rs12936231. Overall, this study shows a common disease allele linked to changes in CTCF binding and nucleosome occupancy leading to altered domain-wide cis-regulation. Finally, a strong association between asthma and cis-regulatory haplotypes was observed in three independent family-based cohorts (p = 1.78 x 10(-8)). This study demonstrates the requirement of multiple parallel allele-specific tools for the investigation of noncoding disease variants and functional fine-mapping of human disease-associated haplotypes. PMID:19732864

  12. Textpresso Site-Specific Recombinases: a text-mining server for the recombinase literature including Cre mice and conditional alleles

    PubMed Central

    Urbanski, William M.; Condie, Brian G.

    2016-01-01

    Textpresso Site Specific Recombinases (http://ssrc.genetics.uga.edu/) is a text-mining web server for searching a database of over 9000 full-text publications. The papers and abstracts in this database represent a wide range of topics related to site-specific recombinase (SSR) research tools. Included in the database are most of the papers that report the characterization or use of mouse strains that express Cre recombinase as well as papers that describe or analyze mouse lines that carry conditional (floxed) alleles or SSR-activated transgenes/knockins. The database also includes reports describing SSR-based cloning methods such as the Gateway or the Creator systems, papers reporting the development or use of SSR-based tools in systems such as Drosophila, bacteria, parasites, stem cells, yeast, plants, zebrafish and Xenopus as well as publications that describe the biochemistry, genetics or molecular structure of the SSRs themselves. Textpresso Site Specific Recombinases is the only comprehensive text-mining resource available for the literature describing the biology and technical applications of site-specific recombinases. PMID:19882667

  13. DNA methylation and mRNA expression of HLA-DQA1 alleles in type 1 diabetes mellitus.

    PubMed

    Cepek, Pavel; Zajacova, Marta; Kotrbova-Kozak, Anna; Silhova, Elena; Cerna, Marie

    2016-06-01

    Type 1 diabetes (T1D) belongs among polygenic multifactorial autoimmune diseases. The highest risk is associated with human leucocyte antigen (HLA) class II genes, including HLA-DQA1 gene. Our aim was to investigate DNA methylation of HLA-DQA1 promoter alleles (QAP) and correlate methylation status with individual HLA-DQA1 allele expression of patients with T1D and healthy controls. DNA methylation is one of the epigenetic modifications that regulate gene expression and is known to be shaped by the environment.Sixty one patients with T1D and 39 healthy controls were involved in this study. Isolated DNA was treated with sodium bisulphite and HLA-DQA1 promoter sequence was amplified using nested PCR. After sequencing, DNA methylation of HLA-DQA1 promoter alleles was analysed. Individual mRNA HLA-DQA1 relative allele expression was assessed using two different endogenous controls (PPIA, DRA). We have found statistically significant differences in HLA-DQA1 allele 02:01 expression (PPIA normalization, Pcorr = 0·041; DRA normalization, Pcorr = 0·052) between healthy controls and patients with T1D. The complete methylation profile of the HLA-DQA1 promoter was gained with the most methylated allele DQA1*02:01 and the least methylated DQA1*05:01 in both studied groups. Methylation profile observed in patients with T1D and healthy controls was similar, and no correlation between HLA-DQA1 allele expression and DNA methylation was found. Although we have not proved significant methylation differences between the two groups, detailed DNA methylation status and its correlation with expression of each HLA-DQA1 allele in patients with T1D have been described for the first time. PMID:26854762

  14. Mono-allelic expression of variegating transgene locus in the mouse.

    PubMed

    Opsahl, Margaret L; Springbett, Anthea; Lathe, Richard; Colman, Alan; McClenaghan, Margaret; Whitelaw, C Bruce A

    2003-12-01

    We have generated transgenic mice which express an ovine beta-lactoglobulin transgene during lactation. In two transgenic lines, BLG/7 and BLG/45, beta-lactoglobulin protein levels vary between siblings, reflected at the cellular level by a mosaic transgene expression pattern in the mammary tissue that is reminiscent of position effect variegation. To investigate whether this variegating expression profile can be affected by the introduction of an identical variegating locus on the homologous chromosome, we compared the beta-lactoglobulin expression profiles in mice hemizygous or homozygous for the transgene locus. In BLG/45 mice, milk protein analysis revealed that transgene expression was effectively doubled in homozygous compared to hemizygous mice. In contrast, beta-lactoglobulin protein in hemizygous and homozygous BLG/7 mice displayed a similar range; although minimum expression levels were doubled in the homozygous population, the maximum level of expression was indistinguishable between the two populations. Fluorescent in situ hybridisation (FISH) for transgene mRNA indicated that for a given protein level, the extent of cellular expression is similar in both BLG/7 populations. In homozygous mice genomic DNA and nuclear RNA FISH demonstrated that only one of the two BLG/7 loci is active in expressing cells, while two transcription foci were present in BLG/45 homozygous mice. This mono-allelic transgene expression pattern is not inherited through the germline, as hemizygous mice bred from homozygous parents expressed at the expected hemizygous population level. We discuss these observations in the context of known epigenetic events such as imprinting and trans-inactivation. PMID:14713195

  15. Allelic Diversity of the Plasmodium falciparum Erythrocyte Membrane Protein 1 Entails Variant-Specific Red Cell Surface Epitopes

    PubMed Central

    Vigan-Womas, Inès; Guillotte, Micheline; Juillerat, Alexandre; Vallieres, Cindy; Lewit-Bentley, Anita; Tall, Adama; Baril, Laurence; Bentley, Graham A.; Mercereau-Puijalon, Odile

    2011-01-01

    The clonally variant Plasmodium falciparum PfEMP1 adhesin is a virulence factor and a prime target of humoral immunity. It is encoded by a repertoire of functionally differentiated var genes, which display architectural diversity and allelic polymorphism. Their serological relationship is key to understanding the evolutionary constraints on this gene family and rational vaccine design. Here, we investigated the Palo Alto/VarO and IT4/R29 and 3D7/PF13_003 parasites lines. VarO and R29 form rosettes with uninfected erythrocytes, a phenotype associated with severe malaria. They express an allelic Cys2/group A NTS-DBL1α1 PfEMP1 domain implicated in rosetting, whose 3D7 ortholog is encoded by PF13_0003. Using these three recombinant NTS-DBL1α1 domains, we elicited antibodies in mice that were used to develop monovariant cultures by panning selection. The 3D7/PF13_0003 parasites formed rosettes, revealing a correlation between sequence identity and virulence phenotype. The antibodies cross-reacted with the allelic domains in ELISA but only minimally with the Cys4/group B/C PFL1955w NTS-DBL1α. By contrast, they were variant-specific in surface seroreactivity of the monovariant-infected red cells by FACS analysis and in rosette-disruption assays. Thus, while ELISA can differentiate serogroups, surface reactivity assays define the more restrictive serotypes. Irrespective of cumulated exposure to infection, antibodies acquired by humans living in a malaria-endemic area also displayed a variant-specific surface reactivity. Although seroprevalence exceeded 90% for each rosetting line, the kinetics of acquistion of surface-reactive antibodies differed in the younger age groups. These data indicate that humans acquire an antibody repertoire to non-overlapping serotypes within a serogroup, consistent with an antibody-driven diversification pressure at the population level. In addition, the data provide important information for vaccine design, as production of a vaccine

  16. First example of an FY*01 allele associated with weakened expression of Fya on red blood cells.

    PubMed

    Arndt, Patricia A; Horn, Trina; Keller, Jessica A; Heri, Suzanne M; Keller, Margaret A

    2015-01-01

    Duffy antigens are important in immunohematology. the reference allele for the Duffy gene (FY) is FY*02, which encodes Fy(b). An A>G single nucleotide polymorphism (SNP) at coding nucleotide (c.) 125 in exon 2 defines the FY*01 allele, which encodes the antithetical Fy(a). A C>T SNP at c.265 in the FY*02 allele is associated with weakening of Fy(b) expression on red blood cells (R BCs) (called Fy(x)). until recently, this latter change had not been described on a FY*01 background allele. Phenotype-matched units were desired for a multi-transfused Vietnamese fetus with α-thalassemia. Genotyping of the fetus using a microarray assay that interrogates three SNPs (c.1-67, c.125, and c.265) in FY yielded indeterminate results for the predicted Duffy phenotype. Genomic sequencing of FY exon 2 showed that the fetal sample had one wild-type FY*01 allele and one new FY*01 allele with the c.265C>T SNP, which until recently had only been found on the FY*02 allele. Genotyping performed on samples from the proband's parents indicated that the father had the same FY genotype as the fetus. Flow cytometry, which has been previously demonstrated as a useful method to study antigen strength on cells, was used to determine if this new FY*01 allele was associated with reduced Fy(a) expression on the father's RBCs. Median fluorescence intensity of the father's RBCs (after incubation with anti-FY(a) and fluorescein-labeled anti-IgG) was similar to known FY*01 heterozygotes. and significantly weaker than known FY*01 homozygotes. In conclusion, the fetus and father both had one normal FY*01 allele and one new FY*01W.01, is associated with weakened expression of Fy(a) on RBCs. PMID:26829175

  17. Evidence of the phenotypic expression of a lethal recessive allele under inbreeding in a wild population of conservation concern.

    PubMed

    Trask, Amanda E; Bignal, Eric M; McCracken, Davy I; Monaghan, Pat; Piertney, Stuart B; Reid, Jane M

    2016-07-01

    Deleterious recessive alleles that are masked in outbred populations are predicted to be expressed in small, inbred populations, reducing both individual fitness and population viability. However, there are few definitive examples of phenotypic expression of lethal recessive alleles under inbreeding conditions in wild populations. Studies that demonstrate the action of such alleles, and infer their distribution and dynamics, are required to understand their potential impact on population viability and inform management responses. The Scottish population of red-billed choughs (Pyrrhocorax pyrrhocorax), which currently totals <60 breeding pairs and is of major conservation concern, has recently been affected by lethal blindness in nestlings. We used family data to show that the pattern of occurrence of blindness within and across affected families that produced blind nestlings was exactly 0·25, matching that expected given a single-locus autosomal lethal recessive allele. Furthermore, the observed distribution of blind nestlings within affected families did not differ from that expected given Mendelian inheritance of such an allele. Relatedness estimates showed that individuals from affected families were not more closely related to each other than they were to individuals from unaffected families that did not produce blind nestlings. Blind individuals tended to be less heterozygous than non-blind individuals, as expected if blindness was caused by the expression of a recessive allele under inbreeding. However, there was no difference in the variance in heterozygosity estimates, suggesting that some blind individuals were relatively outbred. These results suggest carriers of the blindness allele may be widely distributed across contemporary families rather than restricted to a single family lineage, implying that the allele has persisted across multiple generations. Blindness occurred at low frequency (affecting 1·6% of observed nestlings since 1981). However

  18. Nested Allele-Specific PCR Primers Distinguish Genetic Groups of Uncinula necator

    PubMed Central

    Délye, Christophe; Ronchi, Valérie; Laigret, Frédéric; Corio-Costet, Marie-France

    1999-01-01

    Isolates of the obligately biotrophic fungus Uncinula necator cluster in three distinct genetic groups (groups I, II, and III). We designed PCR primers specific for these groups in order to monitor field populations of U. necator. We used the nucleotide sequences of the gene that encodes eburicol 14α-demethylase (CYP51) and of the ribosomal DNA internal transcribed spacer 1 (ITS1), ITS2, and 5.8S regions. We identified four point mutations (three in CYP51 and one in ITS1) that distinguished groups I and II from group III based on a sample of 132 single-spore isolates originating from Europe, Tunisia, Israel, India, and Australia. We developed a nested allele-specific PCR assay in which the CYP51 point mutations were used to detect and distinguish groups I and II from group III in crude mildewed samples from vineyards. In a preliminary study performed with samples from French vineyards in which isolates belonging to genetic groups I and III were present, we found that a shift from a population composed primarily of group I isolates to a population composed primarily of group III isolates occurred during the grapevine growing season. PMID:10473400

  19. Allelic diversity of a beer haze active protein gene in cultivated and Tibetan wild barley and development of allelic specific markers.

    PubMed

    Ye, Lingzhen; Dai, Fei; Qiu, Long; Sun, Dongfa; Zhang, Guoping

    2011-07-13

    The formation of haze is a serious quality problem in beer production. It has been shown that the use of silica elute (SE)-ve malt (absence of molecular weight (MW) ∼14000 Da) for brewing can improve haze stability in the resultant beer, and the protein was identified as a barley trypsin inhibitor of the chloroform/methanol type (BTI-CMe). The objectives of this study were to determine (1) the allelic diversity of the gene controlling BTI-CMe in cultivated and Tibetan wild barley and (2) allele-specific (AS) markers for screening SE protein type. A survey of 172 Tibetan annual wild barley accessions and 71 cultivated barley genotypes was conducted, and 104 wild accessions and 35 cultivated genotypes were identified as SE+ve and 68 wild accessions and 36 cultivated genotypes as SE-ve. The allelic diversity of the gene controlling BTI-CMe was investigated by cloning, alignment, and association analysis. It was found that there were significant differences between the SE+ve and SE-ve types in single-nucleotide polymorphisms at 234 (SNP(234)), SNP(313), and SNP(385.) Furthermore, two sets of AS markers were developed to screen SE protein type based on SNP(313). AS-PCR had results very similar to those obtained by immunoblot method. Mapping analysis showed that the gene controlling the MW∼14 kDa band was located on the short arm of chromosome 3H, at the position of marker BPB-0527 (33.302 cM) in the Franklin/Yerong DH population. PMID:21608526

  20. Allelic family-specific humoral responses to merozoite surface protein 2 (MSP2) in Gabonese residents with Plasmodium falciparum infections

    PubMed Central

    EKALA, M-T; JOUIN, H; LEKOULOU, F; MERCEREAU-PUIJALON, O; NTOUMI, F

    2002-01-01

    Merozoite surface protein 2 (MSP2) expressed by Plasmodium falciparum asexual blood stages has been identified as a promising vaccine candidate. In order to explore allelic family-specific humoral responses which may be responsible for parasite neutralization during natural infections, isolates from individuals with either asymptomatic infections or uncomplicated malaria and residing in a Central African area where Plasmodium transmission is high and perennial, were analysed using MSP2 as polymorphic marker. The family-specific antibody responses were assessed by ELISA using MSP2 synthetic peptides. We observed an age-dependence of P. falciparum infection complexity. The decrease of infection complexity around 15 years of age was observed simultaneously with an increase in the mean number of MSP2 variants recognized. No significant difference in the P. falciparum genetic diversity and infection complexity was found in isolates from asymptomatic subjects and patients with uncomplicated malaria. The longitudinal follow-up showed a rapid development of immune responses to various regions of MSP2 variants within one week. Comparing humoral responses obtained with the other major antigen on the merozoite surface, MSP1, our findings suggest that different pathways of responsiveness are involved in antibody production to merozoite surface antigens. PMID:12165090

  1. Expression of the PlA2 allele of glycoprotein IIIa and its impact on platelet function

    PubMed Central

    Ferro, Albert; Warner, Timothy D

    2015-01-01

    Background The platelet fibrinogen receptor represents the final common pathway of platelet activation, and is formed from two glycoprotein (GP) subunits (GPIIb/IIIa). Carriage of the mutant PlA2 allele of GPIIIa has been shown to confer an increased risk of cardiovascular events, but published studies have disagreed as to the mechanism for this association. Objectives To assess whether carriage of the PlA2 allele conforms to Mendelian patterns of expression and to identify whether carriage of the mutant allele modulates platelet function. Methods Expression of the PlA2 allele was assessed in both healthy subjects (n = 25) and patients with known coronary artery disease (n = 90) through the development and validation of a liquid chromatography, tandem mass spectrometry (LC-MS/MS) assay. Platelet function was assessed in the patient cohort in response to multiple agonists, and these data were analysed in the context of the proteomic data. Results Expression of the wild-type PlA1 allele and mutant PlA2 alleles was readily quantifiable and conformed to Mendelian patterns in both healthy and patient cohorts. Patients who were homozygous for the mutant PlA2 allele had an increased aggregatory response to adenosine diphosphate, collagen, adrenaline, ristocetin, thrombin receptor-activating peptide 6 and U46619, when assessed using agonist-concentration response curves. Conclusions These findings support the hypothesis that carriage of the mutant PlA2 allele mediates an increased risk of cardiovascular events through the modulation of platelet reactivity. PMID:26858830

  2. Quantitative genotyping of single-nucleotide polymorphisms by allele-specific oligonucleotide hybridization on DNA microarrays.

    PubMed

    Rickert, Andreas M; Ballvora, Agim; Matzner, Ulrich; Klemm, Manfred; Gebhardt, Christiane

    2005-08-01

    Genotyping of SNPs (single-nucleotide polymorphisms) has challenged the development of several novel techniques. Most of these methods have been introduced to discriminate binary SNPs in diploid species. In the present study, the quantitative genotyping of SNPs in natural DNA pools of a polyploid organism via DNA microarrays was analysed. Three randomly selected SNP loci were genotyped in the tetraploid species potato (Solanum tuberosum). For each SNP, 24 oligomers were designed, 12 with forward and 12 with reverse orientation. They contained the polymorphic site at one of the positions 11, 14 and 17. Several steps of optimizations were performed, including the 'materials' used and the establishment of hybridization conditions. Glass surfaces were either epoxy- or aldehyde-modified, and allele-specific oligonucleotides contained either SH or NH2 groups. Hybridization stringency conditions were established by varying the concentration of formamide in the hybridization buffer. For SNP BA213c14t7/403, the quantitative discrimination between all four different naturally occurring genotypes could be demonstrated. PMID:15847606

  3. Analysis of common mitochondrial DNA mutations by allele-specific oligonucleotide and Southern blot hybridization.

    PubMed

    Tang, Sha; Halberg, Michelle C; Floyd, Kristen C; Wang, Jing

    2012-01-01

    Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described. PMID:22215554

  4. Allele-specific analysis of DNA replication origins in mammalian cells.

    PubMed

    Bartholdy, Boris; Mukhopadhyay, Rituparna; Lajugie, Julien; Aladjem, Mirit I; Bouhassira, Eric E

    2015-01-01

    The mechanisms that control the location and timing of firing of replication origins are poorly understood. Using a novel functional genomic approach based on the analysis of SNPs and indels in phased human genomes, we observe that replication asynchrony is associated with small cumulative variations in the initiation efficiency of multiple origins between the chromosome homologues, rather than with the activation of dormant origins. Allele-specific measurements demonstrate that the presence of G-quadruplex-forming sequences does not correlate with the efficiency of initiation. Sequence analysis reveals that the origins are highly enriched in sequences with profoundly asymmetric G/C and A/T nucleotide distributions and are almost completely depleted of antiparallel triplex-forming sequences. We therefore propose that although G4-forming sequences are abundant in replication origins, an asymmetry in nucleotide distribution, which increases the propensity of origins to unwind and adopt non-B DNA structure, rather than the ability to form G4, is directly associated with origin activity. PMID:25987481

  5. Allele-specific FKBP5 DNA demethylation mediates gene–childhood trauma interactions

    PubMed Central

    Klengel, Torsten; Mehta, Divya; Anacker, Christoph; Rex-Haffner, Monika; Pruessner, Jens C; Pariante, Carmine M; Pace, Thaddeus W W; Mercer, Kristina B; Mayberg, Helen S; Bradley, Bekh; Nemeroff, Charles B; Holsboer, Florian; Heim, Christine M; Ressler, Kerry J; Rein, Theo; Binder, Elisabeth B

    2014-01-01

    Although the fact that genetic predisposition and environmental exposures interact to shape development and function of the human brain and, ultimately, the risk of psychiatric disorders has drawn wide interest, the corresponding molecular mechanisms have not yet been elucidated. We found that a functional polymorphism altering chromatin interaction between the transcription start site and long-range enhancers in the FK506 binding protein 5 (FKBP5) gene, an important regulator of the stress hormone system, increased the risk of developing stress-related psychiatric disorders in adulthood by allele-specific, childhood trauma–dependent DNA demethylation in functional glucocorticoid response elements of FKBP5. This demethylation was linked to increased stress-dependent gene transcription followed by a long-term dysregulation of the stress hormone system and a global effect on the function of immune cells and brain areas associated with stress regulation. This identification of molecular mechanisms of genotype-directed long-term environmental reactivity will be useful for designing more effective treatment strategies for stress-related disorders. PMID:23201972

  6. Allele-specific analysis of DNA replication origins in mammalian cells

    PubMed Central

    Bartholdy, Boris; Mukhopadhyay, Rituparna; Lajugie, Julien; Aladjem, Mirit I.; Bouhassira, Eric E.

    2015-01-01

    The mechanisms that control the location and timing of firing of replication origins are poorly understood. Using a novel functional genomic approach based on the analysis of SNPs and indels in phased human genomes, we observe that replication asynchrony is associated with small cumulative variations in the initiation efficiency of multiple origins between the chromosome homologues, rather than with the activation of dormant origins. Allele-specific measurements demonstrate that the presence of G-quadruplex-forming sequences does not correlate with the efficiency of initiation. Sequence analysis reveals that the origins are highly enriched in sequences with profoundly asymmetric G/C and A/T nucleotide distributions and are almost completely depleted of antiparallel triplex-forming sequences. We therefore propose that although G4-forming sequences are abundant in replication origins, an asymmetry in nucleotide distribution, which increases the propensity of origins to unwind and adopt non-B DNA structure, rather than the ability to form G4, is directly associated with origin activity. PMID:25987481

  7. Allele-Specific Network Reveals Combinatorial Interaction That Transcends Small Effects in Psoriasis GWAS

    PubMed Central

    Climer, Sharlee; Templeton, Alan R.; Zhang, Weixiong

    2014-01-01

    Hundreds of genetic markers have shown associations with various complex diseases, yet the “missing heritability” remains alarmingly elusive. Combinatorial interactions may account for a substantial portion of this missing heritability, but their discoveries have been impeded by computational complexity and genetic heterogeneity. We present BlocBuster, a novel systems-level approach that efficiently constructs genome-wide, allele-specific networks that accurately segregate homogenous combinations of genetic factors, tests the associations of these combinations with the given phenotype, and rigorously validates the results using a series of unbiased validation methods. BlocBuster employs a correlation measure that is customized for single nucleotide polymorphisms and returns a multi-faceted collection of values that captures genetic heterogeneity. We applied BlocBuster to analyze psoriasis, discovering a combinatorial pattern with an odds ratio of 3.64 and Bonferroni-corrected p-value of 5.01×10−16. This pattern was replicated in independent data, reflecting robustness of the method. In addition to improving prediction of disease susceptibility and broadening our understanding of the pathogenesis underlying psoriasis, these results demonstrate BlocBuster's potential for discovering combinatorial genetic associations within heterogeneous genome-wide data, thereby transcending the limiting “small effects” produced by individual markers examined in isolation. PMID:25233071

  8. Allele-Specific Methylation Occurs at Genetic Variants Associated with Complex Disease

    PubMed Central

    Hutchinson, John N.; Raj, Towfique; Fagerness, Jes; Stahl, Eli; Viloria, Fernando T.; Gimelbrant, Alexander; Seddon, Johanna; Daly, Mark; Chess, Andrew; Plenge, Robert

    2014-01-01

    We hypothesize that the phenomenon of allele-specific methylation (ASM) may underlie the phenotypic effects of multiple variants identified by Genome-Wide Association studies (GWAS). We evaluate ASM in a human population and document its genome-wide patterns in an initial screen at up to 380,678 sites within the genome, or up to 5% of the total genomic CpGs. We show that while substantial inter-individual variation exists, 5% of assessed sites show evidence of ASM in at least six samples; the majority of these events (81%) are under genetic influence. Many of these cis-regulated ASM variants are also eQTLs in peripheral blood mononuclear cells and monocytes and/or in high linkage-disequilibrium with variants linked to complex disease. Finally, focusing on autoimmune phenotypes, we extend this initial screen to confirm the association of cis-regulated ASM with multiple complex disease-associated variants in an independent population using next-generation bisulfite sequencing. These four variants are implicated in complex phenotypes such as ulcerative colitis and AIDS progression disease (rs10491434), Celiac disease (rs2762051), Crohn's disease, IgA nephropathy and early-onset inflammatory bowel disease (rs713875) and height (rs6569648). Our results suggest cis-regulated ASM may provide a mechanistic link between the non-coding genetic changes and phenotypic variation observed in these diseases and further suggests a route to integrating DNA methylation status with GWAS results. PMID:24911414

  9. Allele-specific FKBP5 DNA demethylation mediates gene-childhood trauma interactions.

    PubMed

    Klengel, Torsten; Mehta, Divya; Anacker, Christoph; Rex-Haffner, Monika; Pruessner, Jens C; Pariante, Carmine M; Pace, Thaddeus W W; Mercer, Kristina B; Mayberg, Helen S; Bradley, Bekh; Nemeroff, Charles B; Holsboer, Florian; Heim, Christine M; Ressler, Kerry J; Rein, Theo; Binder, Elisabeth B

    2013-01-01

    Although the fact that genetic predisposition and environmental exposures interact to shape development and function of the human brain and, ultimately, the risk of psychiatric disorders has drawn wide interest, the corresponding molecular mechanisms have not yet been elucidated. We found that a functional polymorphism altering chromatin interaction between the transcription start site and long-range enhancers in the FK506 binding protein 5 (FKBP5) gene, an important regulator of the stress hormone system, increased the risk of developing stress-related psychiatric disorders in adulthood by allele-specific, childhood trauma-dependent DNA demethylation in functional glucocorticoid response elements of FKBP5. This demethylation was linked to increased stress-dependent gene transcription followed by a long-term dysregulation of the stress hormone system and a global effect on the function of immune cells and brain areas associated with stress regulation. This identification of molecular mechanisms of genotype-directed long-term environmental reactivity will be useful for designing more effective treatment strategies for stress-related disorders. PMID:23201972

  10. Preferential Binding to Elk-1 by SLE-Associated IL10 Risk Allele Upregulates IL10 Expression

    PubMed Central

    Kelly, Jennifer A.; Brown, Elizabeth E.; Harley, John B.; Bae, Sang-Cheol; Alarcόn-Riquelme, Marta E.; Edberg, Jeffrey C.; Kimberly, Robert P.; Ramsey-Goldman, Rosalind; Petri, Michelle A.; Reveille, John D.; Vilá, Luis M.; Alarcón, Graciela S.; Kaufman, Kenneth M.; Vyse, Timothy J.; Jacob, Chaim O.; Gaffney, Patrick M.; Sivils, Kathy Moser; James, Judith A.; Kamen, Diane L.; Gilkeson, Gary S.; Niewold, Timothy B.; Merrill, Joan T.; Scofield, R. Hal; Criswell, Lindsey A.; Stevens, Anne M.; Boackle, Susan A.; Kim, Jae-Hoon; Choi, Jiyoung; Pons-Estel, Bernardo A.; Freedman, Barry I.; Anaya, Juan-Manuel; Martin, Javier; Yu, C. Yung; Chang, Deh-Ming; Song, Yeong Wook; Langefeld, Carl D.; Chen, Weiling; Grossman, Jennifer M.; Cantor, Rita M.; Hahn, Bevra H.; Tsao, Betty P.

    2013-01-01

    Immunoregulatory cytokine interleukin-10 (IL-10) is elevated in sera from patients with systemic lupus erythematosus (SLE) correlating with disease activity. The established association of IL10 with SLE and other autoimmune diseases led us to fine map causal variant(s) and to explore underlying mechanisms. We assessed 19 tag SNPs, covering the IL10 gene cluster including IL19, IL20 and IL24, for association with SLE in 15,533 case and control subjects from four ancestries. The previously reported IL10 variant, rs3024505 located at 1 kb downstream of IL10, exhibited the strongest association signal and was confirmed for association with SLE in European American (EA) (P = 2.7×10−8, OR = 1.30), but not in non-EA ancestries. SNP imputation conducted in EA dataset identified three additional SLE-associated SNPs tagged by rs3024505 (rs3122605, rs3024493 and rs3024495 located at 9.2 kb upstream, intron 3 and 4 of IL10, respectively), and SLE-risk alleles of these SNPs were dose-dependently associated with elevated levels of IL10 mRNA in PBMCs and circulating IL-10 protein in SLE patients and controls. Using nuclear extracts of peripheral blood cells from SLE patients for electrophoretic mobility shift assays, we identified specific binding of transcription factor Elk-1 to oligodeoxynucleotides containing the risk (G) allele of rs3122605, suggesting rs3122605 as the most likely causal variant regulating IL10 expression. Elk-1 is known to be activated by phosphorylation and nuclear localization to induce transcription. Of interest, phosphorylated Elk-1 (p-Elk-1) detected only in nuclear extracts of SLE PBMCs appeared to increase with disease activity. Co-expression levels of p-Elk-1 and IL-10 were elevated in SLE T, B cells and monocytes, associated with increased disease activity in SLE B cells, and were best downregulated by ERK inhibitor. Taken together, our data suggest that preferential binding of activated Elk-1 to the IL10 rs3122605-G allele upregulates IL

  11. Quantification of factors influencing fluorescent protein expression using RMCE to generate an allelic series in the ROSA26 locus in mice

    PubMed Central

    Chen, Sara X.; Osipovich, Anna B.; Ustione, Alessandro; Potter, Leah A.; Hipkens, Susan; Gangula, Rama; Yuan, Weiping; Piston, David W.; Magnuson, Mark A.

    2011-01-01

    SUMMARY Fluorescent proteins (FPs) have great utility in identifying specific cell populations and in studying cellular dynamics in the mouse. To quantify the factors that determine both the expression and relative brightness of FPs in mouse embryonic stem cells (mESCs) and in mice, we generated eight different FP-expressing ROSA26 alleles using recombinase-mediated cassette exchange (RMCE). These alleles enabled us to analyze the effects on FP expression of a translational enhancer and different 3′-intronic and/or polyadenylation sequences, as well as the relative brightness of five different FPs, without the confounding position and copy number effects that are typically associated with randomly inserted transgenes. We found that the expression of a given FP can vary threefold or more depending on the genetic features present in the allele. The optimal FP expression cassette contained both a translational enhancer sequence in the 5′-untranslated region (UTR) and an intron-containing rabbit β-globin sequence within the 3′-UTR. The relative expressed brightness of individual FPs varied up to tenfold. Of the five different monomeric FPs tested, Citrine (YFP) was the brightest, followed by Apple, eGFP, Cerulean (CFP) and Cherry. Generation of a line of Cherry-expressing mice showed that there was a 30-fold variation of Cherry expression among different tissues and that there was a punctate expression pattern within cells of all tissues examined. This study should help investigators make better-informed design choices when expressing FPs in mESCs and mice. PMID:21324933

  12. Recognition and Activation Domains Contribute to Allele-Specific Responses of an Arabidopsis NLR Receptor to an Oomycete Effector Protein

    PubMed Central

    Steinbrenner, Adam D.; Goritschnig, Sandra; Staskawicz, Brian J.

    2015-01-01

    In plants, specific recognition of pathogen effector proteins by nucleotide-binding leucine-rich repeat (NLR) receptors leads to activation of immune responses. RPP1, an NLR from Arabidopsis thaliana, recognizes the effector ATR1, from the oomycete pathogen Hyaloperonospora arabidopsidis, by direct association via C-terminal leucine-rich repeats (LRRs). Two RPP1 alleles, RPP1-NdA and RPP1-WsB, have narrow and broad recognition spectra, respectively, with RPP1-NdA recognizing a subset of the ATR1 variants recognized by RPP1-WsB. In this work, we further characterized direct effector recognition through random mutagenesis of an unrecognized ATR1 allele, ATR1-Cala2, screening for gain-of-recognition phenotypes in a tobacco hypersensitive response assay. We identified ATR1 mutants that a) confirm surface-exposed residues contribute to recognition by RPP1, and b) are recognized by and activate the narrow-spectrum allele RPP1-NdA, but not RPP1-WsB, in co-immunoprecipitation and bacterial growth inhibition assays. Thus, RPP1 alleles have distinct recognition specificities, rather than simply different sensitivity to activation. Using chimeric RPP1 constructs, we showed that RPP1-NdA LRRs were sufficient for allele-specific recognition (association with ATR1), but insufficient for receptor activation in the form of HR. Additional inclusion of the RPP1-NdA ARC2 subdomain, from the central NB-ARC domain, was required for a full range of activation specificity. Thus, cooperation between recognition and activation domains seems to be essential for NLR function. PMID:25671309

  13. Analysis of the vomeronasal receptor repertoire, expression and allelic diversity in swine.

    PubMed

    Dinka, Hunduma; Le, Minh Thong; Ha, Heekyun; Cho, Hyesun; Choi, Min-Kyeung; Choi, Hojun; Kim, Jin-Hoi; Soundarajan, Nagasundarapandian; Park, Jin-Ki; Park, Chankyu

    2016-05-01

    Here we report a comprehensive analysis of the vomeronasal receptor repertoire in pigs. We identified a total of 25 V1R sequences consisting of 10 functional genes, 3 pseudogenes, and 12 partial genes, while functional V2R and FPR genes were not present in the pig genome. Pig V1Rs were classified into three subfamilies, D, F, and J. Using direct high resolution sequencing-based typing of all functional V1Rs from 10 individuals of 5 different breeds, a total of 24 SNPs were identified, indicating that the allelic diversity of V1Rs is much lower than that of the olfactory receptors. A high expression level of V1Rs was detected in the vomeronasal organ (VNO) and testes, while a low expression level of V1Rs was observed in all other tissues examined. Our results showed that pigs could serve as an interesting large animal model system to study pheromone-related neurobiology because of their genetic simplicity. PMID:26482471

  14. The MS Risk Allele of CD40 Is Associated with Reduced Cell-Membrane Bound Expression in Antigen Presenting Cells: Implications for Gene Function

    PubMed Central

    Field, Judith; Shahijanian, Fernando; Schibeci, Stephen; Johnson, Laura; Gresle, Melissa; Laverick, Louise; Parnell, Grant; Stewart, Graeme; McKay, Fiona; Kilpatrick, Trevor; Butzkueven, Helmut; Booth, David

    2015-01-01

    Human genetic and animal studies have implicated the costimulatory molecule CD40 in the development of multiple sclerosis (MS). We investigated the cell specific gene and protein expression variation controlled by the CD40 genetic variant(s) associated with MS, i.e. the T-allele at rs1883832. Previously we had shown that the risk allele is expressed at a lower level in whole blood, especially in people with MS. Here, we have defined the immune cell subsets responsible for genotype and disease effects on CD40 expression at the mRNA and protein level. In cell subsets in which CD40 is most highly expressed, B lymphocytes and dendritic cells, the MS-associated risk variant is associated with reduced CD40 cell-surface protein expression. In monocytes and dendritic cells, the risk allele additionally reduces the ratio of expression of full-length versus truncated CD40 mRNA, the latter encoding secreted CD40. We additionally show that MS patients, regardless of genotype, express significantly lower levels of CD40 cell-surface protein compared to unaffected controls in B lymphocytes. Thus, both genotype-dependent and independent down-regulation of cell-surface CD40 is a feature of MS. Lower expression of a co-stimulator of T cell activation, CD40, is therefore associated with increased MS risk despite the same CD40 variant being associated with reduced risk of other inflammatory autoimmune diseases. Our results highlight the complexity and likely individuality of autoimmune pathogenesis, and could be consistent with antiviral and/or immunoregulatory functions of CD40 playing an important role in protection from MS. PMID:26068105

  15. Differential allelic expression of dopamine D1 receptor gene (DRD1) is modulated by microRNA miR-504

    PubMed Central

    Huang, Weihua; Li, Ming D.

    2009-01-01

    Background Previously, we not only reported that dopamine D1 receptor gene (DRD1) is associated with nicotine dependence (ND), but demonstrated that two alleles (A and G) of polymorphism rs686 in the 3′-untranslated region (3′UTR) of DRD1 are expressed differentially. However, the mechanism underlying the differential expression remains to be determined. We hypothesize that it is caused by miRNA targeting. Methods We first used the MicroInspector algorithm to identify microRNAs (miRNAs) potentially targeting the rs686 polymorphism in the DRD1 3′UTR, and then employed a luciferase reporter assay combined with site-directed mutagenesis to test the predicted miRNA targeting. We also examined the miRNA targeting of DRD1 with a gene expression assay. Results Of two miRNAs predicted by computational analyses, we found that miR-504, not miR-296, up-regulated reporter luciferase activity and increased DRD1 expression by targeting the DRD1 3′UTR, whereas inhibition of miR-504, not miR-296, had the opposite effect. Furthermore, we revealed that the direct binding of miR-504 to the DRD1 3′UTR, verified by site-directed mutagenesis, caused a significant expression difference between the two alleles. Conclusion miR-504 up-regulates DRD1 expression by direct binding to the 3′UTR, which leads to differential allele-specific expression of DRD1. PMID:19135651

  16. Allele-specific recognition by LILRB3 and LILRA6 of a cytokeratin 8 - associated ligand on necrotic glandular epithelial cells

    PubMed Central

    López-Álvarez, María R.; Jahnke, Martin; Russell, Alasdair I.; Radjabova, Valeria; Trowsdale, Alice R.Z.; Trowsdale, John

    2016-01-01

    The LILRs are a family of receptors that regulate the activities of myelomonocytic cells. We found that specific allelic variants of two related members of the LILR family, LILRB3 and LILRA6, interact with a ligand exposed on necrotic glandular epithelial cells. The extracellular domains of LILRB3 and LILRA6 are very similar and their genes are highly polymorphic. A commonly occurring allele, LILRB3*12, displayed particularly strong binding of these necrotic cells and further screening of the products of LILRB3 alleles identified motifs that correlated with binding. Immunoprecipitation of the ligand from epithelial cell lysates using recombinant LILRB3*12, identified cytokeratins 8, 18 and 19. Purified proteins obtained from epithelial cell lysates, using anti-cytokeratin 8 antibodies, were able to activate LILRB3*12 reporter cells. Knock-down of cytokeratin 8 in epithelial cells abrogated expression of the LILRB3 ligand, while staining with recombinant LILRB3*12 showed co-localisation with cytokeratin 8 and 18 in permeabilised breast cancer cells. Necrosis is a common feature of tumours. The finding of a necrosis-associated ligand for these two receptors raises the possibility of a novel interaction that alters immune responses within the tumour microenvironment. Since LILRB3 and LILRA6 genes are highly polymorphic the interaction may influence an individual's immune response to tumours. PMID:26769854

  17. Allelic Expression Imbalance of JAK2 V617F Mutation in BCR-ABL Negative Myeloproliferative Neoplasms

    PubMed Central

    Kim, Yeo-Kyeoung; Kim, Hyeoung-Joon; Shin, Jong-Hee; Suh, Soon-Pal; Ryang, Dong-Wook; Shin, Myung-Geun

    2013-01-01

    The discovery of a single point mutation in the JAK2 gene in patients with BCR/ABL-negative myeloproliferative neoplasms (MPNs) has not only brought new insights and pathogenesis, but also has made the diagnosis of MPNs much easier. Although, to date, several mechanisms for the contribution of single JAK2V617F point mutation to phenotypic diversity of MPNs have been suggested in multiple studies, but it is not clear how a unique mutation can cause the phenotypic diversity of MPNs. In this study, our results show that allelic expression imbalance of JAK2 V617F mutant frequently occurs and contributes to phenotypic diversity of BCR-ABL-negative MPNs. The proportion of JAK2 V617F mutant allele was significantly augmented in RNA levels as compared with genomic DNA differently by distinct MPNs subtypes. In detail, preferential expression of JAK2 mutant allele showed threefold increase from the cDNA compared with the genomic DNA from patients with essential thrombocythemia and twofold increase in polycythemia vera. In conclusion, allelic expression imbalance of JAK2 V617F mutant proposes another plausible mechanism for the contribution of single JAK2 point mutation to phenotypic diversity of MPNs. PMID:23349688

  18. [Study on identification of "Digeda" raw materials in Mongolian patent medicine by PCR amplification of specific alleles].

    PubMed

    Cui, Zhan-hu; Huang, Xian-zhang; Long, Ping; Zhang, Le; Zhao, Dong-dong; Wang, Ying-li; Li, Min-hui

    2015-03-01

    To explore a new method for identification of Mongolian patent medicine (MPM) by PCR amplification of specific alleles. Eight kinds of MPM were used to study the identification of "Digeda" raw materials. The total DNA of Lomatogonium rotatum and Corydalis bungeana samples were extracted through modified CTAB method, psbA-trnH sequence was amplified by PCR and sequenced directionally. Specific primer was designed. The DNA of 8 kinds of MPM also was extracted and purified by the commercial DNA purification kits. The rbcL and two pair of specific primers sequences were amplified. The specific amplified products were sequenced in forward directions. All specific sequences were aligned and were analyzed. The results indicated that L rotatum can be identified by specific primers from Digeda-4 Tang, Digeda-8 San, Digeda-4 San, and C. bungeana medicinal materials can be identified by specific primers from Li Dan Ba Wei San, Yi He Ha Ri-12 and A Ga Ri-35. PCR amplification of specific alleles can stably and accurately distinguish raw medicinal materials in MPM. PMID:26087535

  19. MicroRNA-3148 modulates allelic expression of toll-like receptor 7 variant associated with systemic lupus erythematosus.

    PubMed

    Deng, Yun; Zhao, Jian; Sakurai, Daisuke; Kaufman, Kenneth M; Edberg, Jeffrey C; Kimberly, Robert P; Kamen, Diane L; Gilkeson, Gary S; Jacob, Chaim O; Scofield, R Hal; Langefeld, Carl D; Kelly, Jennifer A; Ramsey-Goldman, Rosalind; Petri, Michelle A; Reveille, John D; Vilá, Luis M; Alarcón, Graciela S; Vyse, Timothy J; Pons-Estel, Bernardo A; Freedman, Barry I; Gaffney, Patrick M; Sivils, Kathy Moser; James, Judith A; Gregersen, Peter K; Anaya, Juan-Manuel; Niewold, Timothy B; Merrill, Joan T; Criswell, Lindsey A; Stevens, Anne M; Boackle, Susan A; Cantor, Rita M; Chen, Weiling; Grossman, Jeniffer M; Hahn, Bevra H; Harley, John B; Alarcόn-Riquelme, Marta E; Brown, Elizabeth E; Tsao, Betty P

    2013-01-01

    We previously reported that the G allele of rs3853839 at 3'untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10(-10), odds ratio (OR) (95%CI) = 1.27 (1.17-1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10(-11), OR = 1.24 [1.18-1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3'UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R(2) = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3'UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta  = 2.0×10(-19), OR = 1.25 [1.20-1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor

  20. MicroRNA-3148 Modulates Allelic Expression of Toll-Like Receptor 7 Variant Associated with Systemic Lupus Erythematosus

    PubMed Central

    Sakurai, Daisuke; Kaufman, Kenneth M.; Edberg, Jeffrey C.; Kimberly, Robert P.; Kamen, Diane L.; Gilkeson, Gary S.; Jacob, Chaim O.; Scofield, R. Hal; Langefeld, Carl D.; Kelly, Jennifer A.; Ramsey-Goldman, Rosalind; Petri, Michelle A.; Reveille, John D.; Vilá, Luis M.; Alarcón, Graciela S.; Vyse, Timothy J.; Pons-Estel, Bernardo A.; Freedman, Barry I.; Gaffney, Patrick M.; Sivils, Kathy Moser; James, Judith A.; Gregersen, Peter K.; Anaya, Juan-Manuel; Niewold, Timothy B.; Merrill, Joan T.; Criswell, Lindsey A.; Stevens, Anne M.; Boackle, Susan A.; Cantor, Rita M.; Chen, Weiling; Grossman, Jeniffer M.; Hahn, Bevra H.; Harley, John B.; Alarcόn-Riquelme, Marta E.; Brown, Elizabeth E.; Tsao, Betty P.

    2013-01-01

    We previously reported that the G allele of rs3853839 at 3′untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10−10, odds ratio (OR) (95%CI) = 1.27 (1.17–1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10−11, OR = 1.24 [1.18–1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3′UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R2 = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3′UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta = 2.0×10−19, OR = 1.25 [1.20–1.32]), which confers allelic effect on transcript turnover via differential binding to the

  1. Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1) / Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele

    PubMed Central

    Davis, Melissa B.; Walens, Andrea; Hire, Rupali; Mumin, Kauthar; Brown, Andrea M.; Ford, DeJuana; Howerth, Elizabeth W.; Monteil, Michele

    2015-01-01

    The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups. PMID:26473357

  2. Cardiac-Restricted Expression of VCP/TER94 RNAi or Disease Alleles Perturbs Drosophila Heart Structure and Impairs Function

    PubMed Central

    Viswanathan, Meera C.; Blice-Baum, Anna C.; Sang, Tzu-Kang; Cammarato, Anthony

    2016-01-01

    Valosin-containing protein (VCP) is a highly conserved mechanoenzyme that helps maintain protein homeostasis in all cells and serves specialized functions in distinct cell types. In skeletal muscle, it is critical for myofibrillogenesis and atrophy. However, little is known about VCP's role(s) in the heart. Its functional diversity is determined by differential binding of distinct cofactors/adapters, which is likely disrupted during disease. VCP mutations cause multisystem proteinopathy (MSP), a pleiotropic degenerative disorder that involves inclusion body myopathy. MSP patients display progressive muscle weakness. They also exhibit cardiomyopathy and die from cardiac and respiratory failure, which are consistent with critical myocardial roles for the enzyme. Nonetheless, efficient models to interrogate VCP in cardiac muscle remain underdeveloped and poorly studied. Here, we investigated the significance of VCP and mutant VCP in the Drosophila heart. Cardiac-restricted RNAi-mediated knockdown of TER94, the Drosophila VCP homolog, severely perturbed myofibrillar organization and heart function in adult flies. Furthermore, expression of MSP disease-causing alleles engendered cardiomyopathy in adults and structural defects in embryonic hearts. Drosophila may therefore serve as a valuable model for examining role(s) of VCP in cardiogenesis and for identifying novel heart-specific VCP interactions, which when disrupted via mutation, contribute to or elicit cardiac pathology. PMID:27500162

  3. N-Glycosylation of Asparagine 8 Regulates Surface Expression of Major Histocompatibility Complex Class I Chain-related Protein A (MICA) Alleles Dependent on Threonine 24*

    PubMed Central

    Mellergaard, Maiken; Skovbakke, Sarah Line; Schneider, Christine L.; Lauridsen, Felicia; Andresen, Lars; Jensen, Helle; Skov, Søren

    2014-01-01

    NKG2D is an activating receptor expressed on several types of human lymphocytes. NKG2D ligands can be induced upon cell stress and are frequently targeted post-translationally in infected or transformed cells to avoid immune recognition. Virus infection and inflammation alter protein N-glycosylation, and we have previously shown that changes in cellular N-glycosylation are involved in regulation of NKG2D ligand surface expression. The specific mode of regulation through N-glycosylation is, however, unknown. Here we investigated whether direct N-glycosylation of the NKG2D ligand MICA itself is critical for cell surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (Asn8) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N-glycosylation site. Mutational analysis revealed that a single amino acid (Thr24) in the extracellular domain of MICA018 was essential for the N-glycosylation dependence, whereas the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N-glycosylation, and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018, and we pinpoint the residues essential for this N-glycosylation dependence. In addition, we show that this regulatory mechanism of MICA surface expression is likely targeted during different pathological conditions. PMID:24872415

  4. Comparison of immunohistochemistry, DNA sequencing and allele-specific PCR for the detection of IDH1 mutations in gliomas.

    PubMed

    Loussouarn, Delphine; Le Loupp, Anne-Gaëlle; Frenel, Jean-Sébastien; Leclair, François; Von Deimling, Andreas; Aumont, Maud; Martin, Stéphane; Campone, Mario; Denis, Marc G

    2012-06-01

    Previous studies have identified mutations of the isocitrate dehydrogenase 1 (IDH1) gene in more than 70% of World Health Organization (WHO) grade II and III gliomas. The most frequent mutation leads to a specific amino acid change from arginine to histidine at codon 132 (c.395G>A, p.R132H). IDH1 mutated tumors have a better prognosis than IDH1 non-mutated tumors. The aim of our study was to evaluate and compare the methods of mIDH1 R132H immunohistochemistry, allele-specific PCR and DNA sequencing for determination of IDH1 status. We performed a retrospective study of 91 patients with WHO grade II (n=43) and III (n=48) oligodendrogliomas. A fragment of exon 4 spanning the sequence encoding the catalytic domain of IDH1, including codon 132, was amplified and sequenced using standard conditions. Allele-specific amplification was performed using two forward primers with variations in their 3' nucleotides such that each was specific for the wild-type or the mutated variant, and one reverse primer. Immunohistochemistry was performed with mouse monoclonal mIDH1 R132H. DNA was extracted from FFPE sections following macrodissection. IDH1 mutations were found in 55/90 patients (61.1%) by direct sequencing. R132H mutations were found in 47/55 patients (85.4%). The results of the allele-specific PCR positively correlated with those from DNA sequencing. Other mutations (p.R132C, p.R132S and pR132G) were found by DNA sequencing in 3, 3 and 2 tumors, respectively (8/55 patients, 14.6%). mIDH1 R132H immunostaining was found in the 47 patients presenting the R132H mutation (sensitivity 47/47, 100% for this mutation). None of the tumors presenting a wild-type IDH1 gene were stained (specificity 35/35, 100%). Our results demonstrate that immunohistochemistry using the mIDH1 R132H antibody and allele-specific amplification are highly sensitive techniques to detect the most frequent mutation of the IDH1 gene. PMID:22447191

  5. incurvata13, a Novel Allele of AUXIN RESISTANT6, Reveals a Specific Role for Auxin and the SCF Complex in Arabidopsis Embryogenesis, Vascular Specification, and Leaf Flatness1[W][OA

    PubMed Central

    Esteve-Bruna, David; Pérez-Pérez, José Manuel; Ponce, María Rosa; Micol, José Luis

    2013-01-01

    Auxin plays a pivotal role in plant development by modulating the activity of SCF ubiquitin ligase complexes. Here, we positionally cloned Arabidopsis (Arabidopsis thaliana) incurvata13 (icu13), a mutation that causes leaf hyponasty and reduces leaf venation pattern complexity and auxin responsiveness. We found that icu13 is a novel recessive allele of AUXIN RESISTANT6 (AXR6), which encodes CULLIN1, an invariable component of the SCF complex. Consistent with a role for auxin in vascular specification, the vascular defects in the icu13 mutant were accompanied by reduced expression of auxin transport and auxin perception markers in provascular cells. This observation is consistent with the expression pattern of AXR6, which we found to be restricted to vascular precursors and hydathodes in wild-type leaf primordia. AXR1, RELATED TO UBIQUITIN1-CONJUGATING ENZYME1, CONSTITUTIVE PHOTOMORPHOGENIC9 SIGNALOSOME5A, and CULLIN-ASSOCIATED NEDD8-DISSOCIATED1 participate in the covalent modification of CULLIN1 by RELATED TO UBIQUITIN. Hypomorphic alleles of these genes also display simple venation patterns, and their double mutant combinations with icu13 exhibited a synergistic, rootless phenotype reminiscent of that caused by loss of function of MONOPTEROS (MP), which forms an auxin-signaling module with BODENLOS (BDL). The phenotypes of double mutant combinations of icu13 with either a gain-of-function allele of BDL or a loss-of-function allele of MP were synergistic. In addition, a BDL:green fluorescent protein fusion protein accumulated in icu13, and BDL loss of function or MP overexpression suppressed the phenotype of icu13. Our results demonstrate that the MP-BDL module is required not only for root specification in embryogenesis and vascular postembryonic development but also for leaf flatness. PMID:23319550

  6. Parent-of-origin specific allelic associations among 106 genomic loci for age at menarche

    PubMed Central

    Thompson, Deborah J; Ferreira, Teresa; He, Chunyan; Chasman, Daniel I; Esko, Tõnu; Thorleifsson, Gudmar; Albrecht, Eva; Ang, Wei Q; Corre, Tanguy; Cousminer, Diana L; Feenstra, Bjarke; Franceschini, Nora; Ganna, Andrea; Johnson, Andrew D; Kjellqvist, Sanela; Lunetta, Kathryn L; McMahon, George; Nolte, Ilja M; Paternoster, Lavinia; Porcu, Eleonora; Smith, Albert V; Stolk, Lisette; Teumer, Alexander; Tšernikova, Natalia; Tikkanen, Emmi; Ulivi, Sheila; Wagner, Erin K; Amin, Najaf; Bierut, Laura J; Byrne, Enda M; Hottenga, Jouke-Jan; Koller, Daniel L; Mangino, Massimo; Pers, Tune H; Yerges-Armstrong, Laura M; Zhao, Jing Hua; Andrulis, Irene L; Anton-Culver, Hoda; Atsma, Femke; Bandinelli, Stefania; Beckmann, Matthias W; Benitez, Javier; Blomqvist, Carl; Bojesen, Stig E; Bolla, Manjeet K; Bonanni, Bernardo; Brauch, Hiltrud; Brenner, Hermann; Buring, Julie E; Chang-Claude, Jenny; Chanock, Stephen; Chen, Jinhui; Chenevix-Trench, Georgia; Collée, J. Margriet; Couch, Fergus J; Couper, David; Coveillo, Andrea D; Cox, Angela; Czene, Kamila; D’adamo, Adamo Pio; Smith, George Davey; De Vivo, Immaculata; Demerath, Ellen W; Dennis, Joe; Devilee, Peter; Dieffenbach, Aida K; Dunning, Alison M; Eiriksdottir, Gudny; Eriksson, Johan G; Fasching, Peter A; Ferrucci, Luigi; Flesch-Janys, Dieter; Flyger, Henrik; Foroud, Tatiana; Franke, Lude; Garcia, Melissa E; García-Closas, Montserrat; Geller, Frank; de Geus, Eco EJ; Giles, Graham G; Gudbjartsson, Daniel F; Gudnason, Vilmundur; Guénel, Pascal; Guo, Suiqun; Hall, Per; Hamann, Ute; Haring, Robin; Hartman, Catharina A; Heath, Andrew C; Hofman, Albert; Hooning, Maartje J; Hopper, John L; Hu, Frank B; Hunter, David J; Karasik, David; Kiel, Douglas P; Knight, Julia A; Kosma, Veli-Matti; Kutalik, Zoltan; Lai, Sandra; Lambrechts, Diether; Lindblom, Annika; Mägi, Reedik; Magnusson, Patrik K; Mannermaa, Arto; Martin, Nicholas G; Masson, Gisli; McArdle, Patrick F; McArdle, Wendy L; Melbye, Mads; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L; Nevanlinna, Heli; Neven, Patrick; Nohr, Ellen A; Oldehinkel, Albertine J; Oostra, Ben A; Palotie, Aarno; Peacock, Munro; Pedersen, Nancy L; Peterlongo, Paolo; Peto, Julian; Pharoah, Paul DP; Postma, Dirkje S; Pouta, Anneli; Pylkäs, Katri; Radice, Paolo; Ring, Susan; Rivadeneira, Fernando; Robino, Antonietta; Rose, Lynda M; Rudolph, Anja; Salomaa, Veikko; Sanna, Serena; Schlessinger, David; Schmidt, Marjanka K; Southey, Mellissa C; Sovio, Ulla; Stampfer, Meir J; Stöckl, Doris; Storniolo, Anna M; Timpson, Nicholas J; Tyrer, Jonathan; Visser, Jenny A; Vollenweider, Peter; Völzke, Henry; Waeber, Gerard; Waldenberger, Melanie; Wallaschofski, Henri; Wang, Qin; Willemsen, Gonneke; Winqvist, Robert; Wolffenbuttel, Bruce HR; Wright, Margaret J; Boomsma, Dorret I; Econs, Michael J; Khaw, Kay-Tee; Loos, Ruth JF; McCarthy, Mark I; Montgomery, Grant W; Rice, John P; Streeten, Elizabeth A; Thorsteinsdottir, Unnur; van Duijn, Cornelia M; Alizadeh, Behrooz Z; Bergmann, Sven; Boerwinkle, Eric; Boyd, Heather A; Crisponi, Laura; Gasparini, Paolo; Gieger, Christian; Harris, Tamara B; Ingelsson, Erik; Järvelin, Marjo-Riitta; Kraft, Peter; Lawlor, Debbie; Metspalu, Andres; Pennell, Craig E; Ridker, Paul M; Snieder, Harold; Sørensen, Thorkild IA; Spector, Tim D; Strachan, David P; Uitterlinden, André G; Wareham, Nicholas J; Widen, Elisabeth; Zygmunt, Marek; Murray, Anna; Easton, Douglas F

    2014-01-01

    Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality1. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation2,3, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P<5×10−8) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1/WDR25, MKRN3/MAGEL2 and KCNK9) demonstrating parent-of-origin specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and gamma-aminobutyric acid-B2 receptor signaling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition. PMID:25231870

  7. Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche.

    PubMed

    Perry, John R B; Day, Felix; Elks, Cathy E; Sulem, Patrick; Thompson, Deborah J; Ferreira, Teresa; He, Chunyan; Chasman, Daniel I; Esko, Tõnu; Thorleifsson, Gudmar; Albrecht, Eva; Ang, Wei Q; Corre, Tanguy; Cousminer, Diana L; Feenstra, Bjarke; Franceschini, Nora; Ganna, Andrea; Johnson, Andrew D; Kjellqvist, Sanela; Lunetta, Kathryn L; McMahon, George; Nolte, Ilja M; Paternoster, Lavinia; Porcu, Eleonora; Smith, Albert V; Stolk, Lisette; Teumer, Alexander; Tšernikova, Natalia; Tikkanen, Emmi; Ulivi, Sheila; Wagner, Erin K; Amin, Najaf; Bierut, Laura J; Byrne, Enda M; Hottenga, Jouke-Jan; Koller, Daniel L; Mangino, Massimo; Pers, Tune H; Yerges-Armstrong, Laura M; Hua Zhao, Jing; Andrulis, Irene L; Anton-Culver, Hoda; Atsma, Femke; Bandinelli, Stefania; Beckmann, Matthias W; Benitez, Javier; Blomqvist, Carl; Bojesen, Stig E; Bolla, Manjeet K; Bonanni, Bernardo; Brauch, Hiltrud; Brenner, Hermann; Buring, Julie E; Chang-Claude, Jenny; Chanock, Stephen; Chen, Jinhui; Chenevix-Trench, Georgia; Collée, J Margriet; Couch, Fergus J; Couper, David; Coviello, Andrea D; Cox, Angela; Czene, Kamila; D'adamo, Adamo Pio; Davey Smith, George; De Vivo, Immaculata; Demerath, Ellen W; Dennis, Joe; Devilee, Peter; Dieffenbach, Aida K; Dunning, Alison M; Eiriksdottir, Gudny; Eriksson, Johan G; Fasching, Peter A; Ferrucci, Luigi; Flesch-Janys, Dieter; Flyger, Henrik; Foroud, Tatiana; Franke, Lude; Garcia, Melissa E; García-Closas, Montserrat; Geller, Frank; de Geus, Eco E J; Giles, Graham G; Gudbjartsson, Daniel F; Gudnason, Vilmundur; Guénel, Pascal; Guo, Suiqun; Hall, Per; Hamann, Ute; Haring, Robin; Hartman, Catharina A; Heath, Andrew C; Hofman, Albert; Hooning, Maartje J; Hopper, John L; Hu, Frank B; Hunter, David J; Karasik, David; Kiel, Douglas P; Knight, Julia A; Kosma, Veli-Matti; Kutalik, Zoltan; Lai, Sandra; Lambrechts, Diether; Lindblom, Annika; Mägi, Reedik; Magnusson, Patrik K; Mannermaa, Arto; Martin, Nicholas G; Masson, Gisli; McArdle, Patrick F; McArdle, Wendy L; Melbye, Mads; Michailidou, Kyriaki; Mihailov, Evelin; Milani, Lili; Milne, Roger L; Nevanlinna, Heli; Neven, Patrick; Nohr, Ellen A; Oldehinkel, Albertine J; Oostra, Ben A; Palotie, Aarno; Peacock, Munro; Pedersen, Nancy L; Peterlongo, Paolo; Peto, Julian; Pharoah, Paul D P; Postma, Dirkje S; Pouta, Anneli; Pylkäs, Katri; Radice, Paolo; Ring, Susan; Rivadeneira, Fernando; Robino, Antonietta; Rose, Lynda M; Rudolph, Anja; Salomaa, Veikko; Sanna, Serena; Schlessinger, David; Schmidt, Marjanka K; Southey, Mellissa C; Sovio, Ulla; Stampfer, Meir J; Stöckl, Doris; Storniolo, Anna M; Timpson, Nicholas J; Tyrer, Jonathan; Visser, Jenny A; Vollenweider, Peter; Völzke, Henry; Waeber, Gerard; Waldenberger, Melanie; Wallaschofski, Henri; Wang, Qin; Willemsen, Gonneke; Winqvist, Robert; Wolffenbuttel, Bruce H R; Wright, Margaret J; Boomsma, Dorret I; Econs, Michael J; Khaw, Kay-Tee; Loos, Ruth J F; McCarthy, Mark I; Montgomery, Grant W; Rice, John P; Streeten, Elizabeth A; Thorsteinsdottir, Unnur; van Duijn, Cornelia M; Alizadeh, Behrooz Z; Bergmann, Sven; Boerwinkle, Eric; Boyd, Heather A; Crisponi, Laura; Gasparini, Paolo; Gieger, Christian; Harris, Tamara B; Ingelsson, Erik; Järvelin, Marjo-Riitta; Kraft, Peter; Lawlor, Debbie; Metspalu, Andres; Pennell, Craig E; Ridker, Paul M; Snieder, Harold; Sørensen, Thorkild I A; Spector, Tim D; Strachan, David P; Uitterlinden, André G; Wareham, Nicholas J; Widen, Elisabeth; Zygmunt, Marek; Murray, Anna; Easton, Douglas F; Stefansson, Kari; Murabito, Joanne M; Ong, Ken K

    2014-10-01

    Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P < 5 × 10(-8)) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1-WDR25, MKRN3-MAGEL2 and KCNK9) demonstrating parent-of-origin-specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and γ-aminobutyric acid-B2 receptor signalling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition. PMID:25231870

  8. Population-ethnic group specific genome variation allele frequency data: a querying and visualization journey.

    PubMed

    Viennas, Emmanouil; Gkantouna, Vassiliki; Ioannou, Marina; Georgitsi, Marianthi; Rigou, Maria; Poulas, Konstantinos; Patrinos, George P; Tzimas, Giannis

    2012-08-01

    National/ethnic mutation databases aim to document the genetic heterogeneity in various populations and ethnic groups worldwide. We have previously reported the development and upgrade of FINDbase (www.findbase.org), a database recording causative mutations and pharmacogenomic marker allele frequencies in various populations around the globe. Although this database has recently been upgraded, we continuously try to enhance its functionality by providing more advanced visualization tools that would further assist effective data querying and comparisons. We are currently experimenting in various visualization techniques on the existing FINDbase causative mutation data collection aiming to provide a dynamic research tool for the worldwide scientific community. We have developed an interactive web-based application for population-based mutation data retrieval. It supports sophisticated data exploration allowing users to apply advanced filtering criteria upon a set of multiple views of the underlying data collection and enables browsing the relationships between individual datasets in a novel and meaningful way. PMID:22659238

  9. A Saccharomyces Cerevisiae Rad52 Allele Expressing a C-Terminal Truncation Protein: Activities and Intragenic Complementation of Missense Mutations

    PubMed Central

    Boundy-Mills, K. L.; Livingston, D. M.

    1993-01-01

    A nonsense allele of the yeast RAD52 gene, rad52-327, which expresses the N-terminal 65% of the protein was compared to two missense alleles, rad52-1 and rad52-2, and to a deletion allele. While the rad52-1 and the deletion mutants have severe defects in DNA repair, recombination and sporulation, the rad52-327 and rad52-2 mutants retain either partial or complete capabilities in repair and recombination. These two mutants behave similarly in most tests of repair and recombination during mitotic growth. One difference between these two alleles is that a homozygous rad52-2 diploid fails to sporulate, whereas the homozygous rad52-327 diploid sporulates weakly. The low level of sporulation by the rad52-327 diploid is accompanied by a low percentage of spore viability. Among these viable spores the frequency of crossing over for markers along chromosome VII is the same as that found in wild-type spores. rad52-327 complements rad52-2 for repair and sporulation. Weaker intragenic complementation occurs between rad52-327 and rad52-1. PMID:8417987

  10. Modulation of the phenotype in dominant erythropoietic protoporphyria by a low expression of the normal ferrochelatase allele.

    PubMed Central

    Gouya, L.; Deybach, J. C.; Lamoril, J.; Da Silva, V.; Beaumont, C.; Grandchamp, B.; Nordmann, Y.

    1996-01-01

    Erythropoietic protoporphyria (EPP) is a monogenic inherited disorder of the heme biosynthetic pathway due to ferrochelatase (FC) deficiency. EPP is generally considered to be transmitted as an autosomal dominant disease with incomplete penetrance, although autosomal recessive inheritance has been documented at the enzymatic and molecular level in some families. In the dominant form of EPP, statistical analysis of FC activities documented a significantly lower mean value in patients than in asymptomatic carriers, suggesting a more complex mode of inheritance. To account for these findings, we tested a multiallelic inheritance model in one EPP family in which the enzymatic data were compatible with this hypothesis. In this EPP family, the specific FC gene mutation was an exon 10 skipping (delta Ex10), resulting from a G deletion within the exon 10 consensus splice donor site. The segregation of all FC alleles within the family was followed using the delta Ex10 mutation and a new intragenic dimorphism (1520 C/T). mRNAs transcribed from each FC allele were then subjected to relative quantification by a primer extension assay and to absolute quantification by a ribonuclease protection assay. The data support the hypothesis that in this family the EPP phenotype results from the coinheritance of a low output normal FC allele and a mutant delta Ex10 allele. Images Figure 2 Figure 4 PMID:8571955

  11. Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations.

    PubMed

    Ding, Yu; Xia, Bo-Hou; Liu, Qi; Li, Mei-Ya; Huang, Shui-Xian; Zhuo, Guang-Chao

    2016-10-10

    Mutations in mitochondrial 12S rRNA (MT-RNR1) are the important causes of sensorineural hearing loss. Of these mutations, the homoplasmic m.1555A>G or m.1494C>T mutation in the highly conserved A-site of MT-RNR1 gene has been found to be associated with both aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. Since the m.1555A>G and m.1494C>T mutations are sensitive to ototoxic drugs, therefore, screening for the presence of these mutations is important for early diagnosis and prevention of deafness. For this purpose, we recently developed a novel allele-specific PCR (AS-PCR) which is able to simultaneously detect these mutations. To assess its accuracy, in this study, we employed this method to screen the frequency of m.1555A>G and m.1494C>T mutations in 200 deafness patients and 120 healthy subjects. Consequently, four m.1555A>G and four m.1494C>T mutations were identified; among these, only one patient with the m.1494C>T mutation had an obvious family history of hearing loss. Strikingly, clinical evaluation showed that this family exhibited a high penetrance of hearing loss. In particular, the penetrances of hearing loss were 80% with the aminoglycoside included and 20% when excluded. PCR-Sanger sequencing of the mitochondrial genomes confirmed the presence of the m.1494C>T mutation and identified a set of polymorphisms belonging to mitochondrial haplogroup A. However, the lack of functional variants in mitochondrial and nuclear modified genes (GJB2 and TRMU) in this family indicated that mitochondrial haplogroup and nuclear genes may not play important roles in the phenotypic expression of the m.1494C>T mutation. Thus, other modification factors, such as environmental factor, aminoglycosides or epigenetic modification may have contributed to the high penetrance of hearing loss in this family. Taken together, our data showed that this assay is an effective approach that could be used for detection the deafness-associated MT-RNR1

  12. New primer for specific amplification of the CAG repeat in Huntington disease alleles

    SciTech Connect

    Bond, C.E.; Hodes, M.E.

    1994-09-01

    Huntington disease is an autosomal dominant neurodegenerative disorder caused by an expansion of a CAG trinucleotide repeat near the 5{prime} end of the gene for Huntington disease (IT15). The CAG repeat is flanked by a variable-length CCG repeat that is included in the amplification product obtained with most currently used primer sets and PCR protocols. Inclusion of this adjacent CCG repeat complicates the accurate assessment of CAG repeat length and interferes with the genotype determination of those individuals carrying alleles in the intermediate range between normal and expanded sized. Due to the GC-rich nature of this region, attempts at designing a protocol for amplification of only the CAG repeat have proved unreliable and difficult to execute. We report here the development of a compatible primer set and PCR protocol that yields consistent amplification of the CAG-repeat region. PCR products can be visualized in ethidium bromide-stained agarose gels for rapid screening or in 6% polyacrylamide gels for determination of exact repeat length. This assay produces bands that can be sized accurately, while eliminating most nonspecific products. Fifty-five specimens examined showed consistency with another well-known method, but one that amplifies the CCG repeats as well. The results we obtained also matched the known carrier status of the donors.

  13. Molecular and classical genetic analyses of his-3 mutants of Neurospora crassa. I. Tests for allelic complementation and specific revertibility.

    PubMed

    Overton, L K; Dubins, J S; de Serres, F J

    1989-10-01

    A collection of 81 his-3 mutants of Neurospora crassa was analyzed in assays for allelic complementation and specific revertibility. In these studies, the linearity of the complementation map of the his-3 cistron (Webber, 1965) was confirmed and mutants were classified as complementing with non-polarized or polarized complementation patterns, or non-complementing. In the assays for spontaneous or induced revertibility, 89% (71/80) of the mutants reverted either spontaneously or after treatment with the chemical mutagens N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methanesulfonate, 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino) acridine dihydrochloride, nitrous acid or hydroxylamine. The frequency of revertible mutants among the non-polarized complementing mutants was 96% (45/47), and 79% (15/19) for the polarized complementing and 79% (11/14) for the non-complementing mutants. The results of these classical genetic assays for allelic complementation and specific revertibility suggest a correlation between complementation pattern and presumptive genetic alterations at the molecular level among his-3 mutants similar to that found with ad-3B mutants induced by nitrous acid (Malling and de Serres, 1967), ethyl methanesulfonate (Malling and de Serres, 1968), or ultraviolet (Kilbey et al., 1971). PMID:2529437

  14. Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in α-spectrin–deficient red cells

    PubMed Central

    Robledo, Raymond F.; Lambert, Amy J.; Birkenmeier, Connie S.; Cirlan, Marius V.; Cirlan, Andreea Flavia M.; Campagna, Dean R.; Lux, Samuel E.

    2010-01-01

    Five spontaneous, allelic mutations in the α-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph1J, sph2J, sph2BC, sphDem). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph3J, a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sphIhj, a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent β-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph4J, a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, β-adducin. The severity of anemia in sph4J indicates that the highly conserved cysteine residue at the C-terminus of α-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3. PMID:20056793

  15. Human Platelet Antigen Alleles in 998 Taiwanese Blood Donors Determined by Sequence-Specific Primer Polymerase Chain Reaction

    PubMed Central

    Burnouf, Thierry; Chen, Jen-Wei; Lin, Liang-In

    2013-01-01

    Polymorphism of human platelet antigens (HPAs) leads to alloimmunizations and immune-mediated platelet disorders including fetal-neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura (PTP), and platelet transfusion refractoriness (PTR). HPA typing and knowledge of antigen frequency in a population are important in particular for the provision of HPA-matched blood components for patients with PTR. We have performed allele genotyping for HPA-1 through -6 and -15 among 998 platelet donors from 6 blood centers in Taiwan using sequence-specific primer polymerase chain reaction. The HPA allele frequency was 99.55, and 0.45% for HPA-1a and -1b; 96.49, and 3.51% for HPA-2a and -2b; 55.81, and 44.19% for HPA-3a and -3b; 99.75, and 0.25% for HPA-4a and -4b; 98.50, and 1.50% for HPA-5a and -5b; 97.75 and 2.25% for HPA-6a and -6b; 53.71 and 46.29% for HPA-15a and -15b. HPA-15b and HPA-3a, may be considered the most important, followed by HPA-2, -6, -1, -5, and -4 systems, as a cause of FNAIT, PTP, and PTR based on allele frequency. HPA-4b and HPA-5b role cannot be excluded based on their immunogenicity. A larger-scale study will now be conducted to confirm these hypotheses and to establish an apheresis donor database for the procurement of HPA-matched apheresis platelets for patients with PTR. PMID:23865077

  16. Competitive allele-specific TaqMan PCR (Cast-PCR) is a sensitive, specific and fast method for BRAF V600 mutation detection in Melanoma patients

    PubMed Central

    Barbano, Raffaela; Pasculli, Barbara; Coco, Michelina; Fontana, Andrea; Copetti, Massimiliano; Rendina, Michelina; Valori, Vanna Maria; Graziano, Paolo; Maiello, Evaristo; Fazio, Vito Michele; Parrella, Paola

    2015-01-01

    BRAF codon 600 mutation testing of melanoma patients is mandatory for the choice of the most appropriate therapy in the clinical setting. Competitive allele specific TaqMan PCR (Cast-PCR) technology allows not only the selective amplification of minor alleles, but it also blocks the amplification of non-mutant allele. We genotyped codon 600 of the BRAF gene in 54 patients’ samples by Cast-PCR and bidirectional direct sequence analysis. All the mutations detected by sequencing were also identified by Cast-PCR. In addition, Cast-PCR assay detected four samples carrying mutations and was able to clearly identify two mutations of uncertain interpretation by Sanger sequencing. The limit of detection of Cast-PCR was evaluated by constructing dilution curves of BRAFV600E and BRAFV600K mutated clinical samples mixed with a not-mutated specimens. Both mutations could be detected until a 1:100 mutated/not mutated ratio. Cloning and sequencing of the clones was used to confirm mutations on representative discrepant cases. Cast PCR performances were not affected by intratumour heterogeneity, and less affected by melanin content. Our results indicate that Cast-PCR is a reliable diagnostic tool for the identification of melanoma patients as eligible to be treated with TKIs and might be implemented in the clinical setting as elective screening method. PMID:26690267

  17. Genome-wide allelic methylation analysis reveals disease-specific susceptibility to multiple methylation defects in imprinting syndromes.

    PubMed

    Court, Franck; Martin-Trujillo, Alex; Romanelli, Valeria; Garin, Intza; Iglesias-Platas, Isabel; Salafsky, Ira; Guitart, Miriam; Perez de Nanclares, Guiomar; Lapunzina, Pablo; Monk, David

    2013-04-01

    Genomic imprinting is the parent-of-origin-specific allelic transcriptional silencing observed in mammals, which is governed by DNA methylation established in the gametes and maintained throughout the development. The frequency and extent of epimutations associated with the nine reported imprinting syndromes varies because it is evident that aberrant preimplantation maintenance of imprinted differentially methylated regions (DMRs) may affect multiple loci. Using a custom Illumina GoldenGate array targeting 27 imprinted DMRs, we profiled allelic methylation in 65 imprinting defect patients. We identify multilocus hypomethylation in numerous Beckwith-Wiedemann syndrome, transient neonatal diabetes mellitus (TNDM), and pseudohypoparathyroidism 1B patients, and an individual with Silver-Russell syndrome. Our data reveal a broad range of epimutations exist in certain imprinting syndromes, with the exception of Prader-Willi syndrome and Angelman syndrome patients that are associated with solitary SNRPN-DMR defects. A mutation analysis identified a 1 bp deletion in the ZFP57 gene in a TNDM patient with methylation defects at multiple maternal DMRs. In addition, we observe missense variants in ZFP57, NLRP2, and NLRP7 that are not consistent with maternal effect and aberrant establishment or methylation maintenance, and are likely benign. This work illustrates that further extensive molecular characterization of these rare patients is required to fully understand the mechanism underlying the etiology of imprint establishment and maintenance. PMID:23335487

  18. Bivariate segmentation of SNP-array data for allele-specific copy number analysis in tumour samples

    PubMed Central

    2013-01-01

    Background SNP arrays output two signals that reflect the total genomic copy number (LRR) and the allelic ratio (BAF), which in combination allow the characterisation of allele-specific copy numbers (ASCNs). While methods based on hidden Markov models (HMMs) have been extended from array comparative genomic hybridisation (aCGH) to jointly handle the two signals, only one method based on change-point detection, ASCAT, performs bivariate segmentation. Results In the present work, we introduce a generic framework for bivariate segmentation of SNP array data for ASCN analysis. For the matter, we discuss the characteristics of the typically applied BAF transformation and how they affect segmentation, introduce concepts of multivariate time series analysis that are of concern in this field and discuss the appropriate formulation of the problem. The framework is implemented in a method named CnaStruct, the bivariate form of the structural change model (SCM), which has been successfully applied to transcriptome mapping and aCGH. Conclusions On a comprehensive synthetic dataset, we show that CnaStruct outperforms the segmentation of existing ASCN analysis methods. Furthermore, CnaStruct can be integrated into the workflows of several ASCN analysis tools in order to improve their performance, specially on tumour samples highly contaminated by normal cells. PMID:23497144

  19. RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression

    PubMed Central

    Liu, Jing; Pendergraff, Hannah; Narayanannair, K. Jayaprakash; Lackey, Jeremy G.; Kuchimanchi, Satya; Rajeev, Kallanthottathil G.; Manoharan, Muthiah; Hu, Jiaxin; Corey, David R.

    2013-01-01

    Abasic substitutions within DNA or RNA are tools for evaluating the impact of absent nucleobases. Because of the importance of abasic sites in genetic damage, most research has involved DNA. Little information is available on the impact of abasic substitutions within RNA or on RNA interference (RNAi). Here, we examine the effect of abasic substitutions on RNAi and allele-selective gene silencing. Huntington's disease (HD) and Machado Joseph Disease (MJD) are severe neurological disorders that currently have no cure. HD and MJD are caused by an expansion of CAG repeats within one mRNA allele encoding huntingtin (HTT) and ataxin-3 (ATX-3) proteins. Agents that silence mutant HTT or ATX-3 expression would remove the cause of HD or MJD and provide an option for therapeutic development. We describe flexible syntheses for abasic substitutions and show that abasic RNA duplexes allele-selectively inhibit both mutant HTT and mutant ATX-3. Inhibition involves the RNAi protein argonaute 2, even though the abasic substitution disrupts the catalytic cleavage of RNA target by argonaute 2. Several different abasic duplexes achieve potent and selective inhibition, providing a broad platform for subsequent development. These findings introduce abasic substitutions as a tool for tailoring RNA duplexes for gene silencing. PMID:23887934

  20. Utilization of a ts-sacB selection system for the generation of a Mycobacterium avium serovar-8 specific glycopeptidolipid allelic exchange mutant

    PubMed Central

    Irani, Vida R; Lee, Sun-Hwa; Eckstein, Torsten M; Inamine, Julia M; Belisle, John T; Maslow, Joel N

    2004-01-01

    Background Mycobacterium avium are ubiquitous environmental organisms and a cause of disseminated infection in patients with end-stage AIDS. The glycopeptidolipids (GPL) of M. avium are proposed to participate in the pathogenesis of this organism, however, establishment of a clear role for GPL in disease production has been limited by the inability to genetically manipulate M. avium. Methods To be able to study the role of the GPL in M. avium pathogenesis, a ts-sacB selection system, not previously used in M. avium, was employed as a means to achieve homologous recombination for the rhamnosyltransferase (rtfA) gene of a pathogenic serovar 8 strain of M. avium to prevent addition of serovar-specific sugars to rhamnose of the fatty acyl-peptide backbone of GPL. The genotype of the resultant rtfA mutant was confirmed by polymerase chain reaction and southern hybridization. Disruption in the proximal sugar of the haptenic oligosaccharide resulted in the loss of serovar specific GPL with no change in the pattern of non-serovar specific GPL moieties as shown by thin layer chromatography and gas chromatography/mass spectrometry. Complementation of wild type (wt) rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wt serovar 8 parent strain. Results In this study, we affirm our results that rtfA encodes an enzyme responsible for the transfer of Rha to 6d-Tal and provide evidence of a second allelic exchange mutagenesis system suitable for M. avium. Conclusion We report the second allelic exchange system for M. avium utilizing ts-sacB as double-negative and xylE as positive counter-selection markers, respectively. This system of allelic exchange would be especially useful for M. avium strains that demonstrate significant isoniazid (INH) resistance despite transformation with katG. Through the construction of mutants in GPL or other mycobacterial components, their roles in M. avium pathogenesis, biosynthesis, or drug

  1. A novel allele of Alx4 results in reduced Fgf10 expression and failure of eyelid fusion in mice.

    PubMed

    Curtain, Michelle; Heffner, Caleb S; Maddox, Dennis M; Gudis, Polyxeni; Donahue, Leah Rae; Murray, Stephen A

    2015-04-01

    Normal fusion of developing eyelids requires coordination of inductive signals from the eyelid mesenchyme with migration of the periderm cell layer and constriction of the eyelids across the eye. Failure of this process results in an eyelids open at birth (EOB) phenotype in mice. We have identified a novel spontaneous allele of Alx4 that displays EOB, in addition to polydactyly and cranial malformations. Alx4 is expressed in the eyelid mesenchyme prior to and during eyelid fusion in a domain overlapping the expression of genes that also play a role in normal eyelid development. We show that Alx4 mutant mice have reduced expression of Fgf10, a key factor expressed in the mesenchyme that is required for initiation of eyelid fusion by the periderm. This is accompanied by a reduced number of periderm cells expressing phosphorylated c-Jun, consistent with the incomplete ablation of Fgf10 expression. Together, these data demonstrate that eyelid fusion in mice requires the expression of Alx4, accompanied by the loss of normal expression of essential components of the eyelid fusion pathway. PMID:25673119

  2. Enhanced specificity of TPMT*2 genotyping using unidirectional wild-type and mutant allele-specific scorpion primers in a single tube.

    PubMed

    Chen, Dong; Yang, Zhao; Xia, Han; Huang, Jun-Fu; Zhang, Yang; Jiang, Tian-Nun; Wang, Gui-Yu; Chuai, Zheng-Ran; Fu, Wei-Ling; Huang, Qing

    2014-01-01

    Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques. PMID:24705376

  3. Enhanced Specificity of TPMT*2 Genotyping Using Unidirectional Wild-Type and Mutant Allele-Specific Scorpion Primers in a Single Tube

    PubMed Central

    Chen, Dong; Yang, Zhao; Xia, Han; Huang, Jun-Fu; Zhang, Yang; Jiang, Tian-Nun; Wang, Gui-Yu; Chuai, Zheng-Ran; Fu, Wei-Ling; Huang, Qing

    2014-01-01

    Genotyping of thiopurine S-methyltransferase (TPMT) is recommended for predicting the adverse drug response of thiopurines. In the current study, a novel version of allele-specific PCR (AS-PCR), termed competitive real-time fluorescent AS-PCR (CRAS-PCR) was developed to analyze the TPMT*2 genotype in ethnic Chinese. This technique simultaneously uses wild-type and mutant allele-specific scorpion primers in a single reaction. To determine the optimal conditions for both traditional AS-PCR and CRAS-PCR, we used the Taguchi method, an engineering optimization process that balances the concentrations of all components using an orthogonal array rather than a factorial array. Instead of running up to 264 experiments with the conventional factorial method, the Taguchi method achieved the same optimization using only 16 experiments. The optimized CRAS-PCR system completely avoided non-specific amplification occurring in traditional AS-PCR and could be performed at much more relaxed reaction conditions at 1% sensitivity, similar to traditional AS-PCR. TPMT*2 genotyping of 240 clinical samples was consistent with published data. In conclusion, CRAS-PCR is a novel and robust genotyping method, and the Taguchi method is an effective tool for the optimization of molecular analysis techniques. PMID:24705376

  4. Citrus (Rutaceae) SNP markers based on Competitive Allele-Specific PCR; transferability across the Aurantioideae subfamily1

    PubMed Central

    Garcia-Lor, Andres; Ancillo, Gema; Navarro, Luis; Ollitrault, Patrick

    2013-01-01

    • Premise of the study: Single nucleotide polymorphism (SNP) markers based on Competitive Allele-Specific PCR (KASPar) were developed from sequences of three Citrus species. Their transferability was tested in 63 Citrus genotypes and 19 relative genera of the subfamily Aurantioideae to estimate the potential of SNP markers, selected from a limited intrageneric discovery panel, for ongoing broader diversity analysis at the intra- and intergeneric levels and systematic germplasm bank characterization. • Methods and Results: Forty-two SNP markers were developed using KASPar technology. Forty-one were successfully genotyped in all of the Citrus germplasm, where intra- and interspecific polymorphisms were observed. The transferability and diversity decreased with increasing taxonomic distance. • Conclusions: SNP markers based on the KASPar method developed from sequence data of a limited intrageneric discovery panel provide a valuable molecular resource for genetic diversity analysis of germplasm within a genus and should be useful for germplasm fingerprinting at a much broader diversity level. PMID:25202535

  5. Genotyping by Sequencing Using Specific Allelic Capture to Build a High-Density Genetic Map of Durum Wheat

    PubMed Central

    Holtz, Yan; Ardisson, Morgane; Ranwez, Vincent; Besnard, Alban; Leroy, Philippe; Poux, Gérard; Roumet, Pierre; Viader, Véronique; Santoni, Sylvain; David, Jacques

    2016-01-01

    Targeted sequence capture is a promising technology which helps reduce costs for sequencing and genotyping numerous genomic regions in large sets of individuals. Bait sequences are designed to capture specific alleles previously discovered in parents or reference populations. We studied a set of 135 RILs originating from a cross between an emmer cultivar (Dic2) and a recent durum elite cultivar (Silur). Six thousand sequence baits were designed to target Dic2 vs. Silur polymorphisms discovered in a previous RNAseq study. These baits were exposed to genomic DNA of the RIL population. Eighty percent of the targeted SNPs were recovered, 65% of which were of high quality and coverage. The final high density genetic map consisted of more than 3,000 markers, whose genetic and physical mapping were consistent with those obtained with large arrays. PMID:27171472

  6. SNP Detection in mRNA in Living Cells Using Allele Specific FRET Probes

    PubMed Central

    Dahan, Liya; Huang, Lingyan; Kedmi, Ranit; Behlke, Mark A.; Peer, Dan

    2013-01-01

    Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET) probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP) in murine Hras mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS) bonds, 2′OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes in vivo molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies. PMID:24039756

  7. Allele-specific polymerase chain reaction for the detection of Alzheimer’s disease-related single nucleotide polymorphisms

    PubMed Central

    2013-01-01

    Background The incidence of Alzheimer’s disease, particularly in developing countries, is expected to increase exponentially as the population ages. Continuing research in this area is essential in order to better understand this disease and develop strategies for treatment and prevention. Genome-wide association studies have identified several loci as genetic risk factors of AD aside from apolipoprotein E such as bridging integrator (BIN1), clusterin (CLU), ATP-binding cassette sub-family A member 7 (ABCA7), complement receptor 1 (CR1) and phosphatidylinositol binding clathrin assembly protein (PICALM). However genetic research in developing countries is often limited by lack of funding and expertise. This study therefore developed and validated a simple, cost effective polymerase chain reaction based technique to determine these single nucleotide polymorphisms. Methods An allele-specific PCR method was developed to detect single nucleotide polymorphisms of BIN1 rs744373, CLU rs11136000, ABCA7 rs3764650, CR1 rs3818361 and PICALM rs3851179 in human DNA samples. Allele-specific primers were designed by using appropriate software to permit the PCR amplification only if the nucleotide at the 3’-end of the primer complemented the base at the wild-type or variant-type DNA sample. The primers were then searched for uniqueness using the Basic Local Alignment Search Tool search engine. Results The assay was tested on a hundred samples and accurately detected the homozygous wild-type, homozygous variant-type and heterozygous of each SNP. Validation was by direct DNA sequencing. Conclusion This method will enable researchers to carry out genetic polymorphism studies for genetic risk factors associated with late-onset Alzheimer’s disease (BIN1, CLU, ABCA7, CR1 and PICALM) without the use of expensive instrumentation and reagents. PMID:23419238

  8. Allele Mining in Barley Genetic Resources Reveals Genes of Race-Non-Specific Powdery Mildew Resistance

    PubMed Central

    Spies, Annika; Korzun, Viktor; Bayles, Rosemary; Rajaraman, Jeyaraman; Himmelbach, Axel; Hedley, Pete E.; Schweizer, Patrick

    2012-01-01

    Race-non-specific, or quantitative, pathogen resistance is of high importance to plant breeders due to its expected durability. However, it is usually controlled by multiple quantitative trait loci (QTL) and therefore difficult to handle in practice. Knowing the genes that underlie race-non-specific resistance (NR) would allow its exploitation in a more targeted manner. Here, we performed an association-genetic study in a customized worldwide collection of spring barley accessions for candidate genes of race-NR to the powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) and combined data with results from QTL mapping as well as functional-genomics approaches. This led to the identification of 11 associated genes with converging evidence for an important role in race-NR in the presence of the Mlo gene for basal susceptibility. Outstanding in this respect was the gene encoding the transcription factor WRKY2. The results suggest that unlocking plant genetic resources and integrating functional-genomic with genetic approaches can accelerate the discovery of genes underlying race-NR in barley and other crop plants. PMID:22629270

  9. Insulin Like Growth Factor 2 Expression in the Rat Brain Both in Basal Condition and following Learning Predominantly Derives from the Maternal Allele

    PubMed Central

    Ye, Xiaojing; Kohtz, Amy; Pollonini, Gabriella; Riccio, Andrea; Alberini, Cristina M.

    2015-01-01

    Insulin like growth factor 2 (Igf2) is known as a maternally imprinted gene involved in growth and development. Recently, Igf2 was found to also be regulated and required in the adult rat hippocampus for long-term memory formation, raising the question of its allelic regulation in adult brain regions following experience and in cognitive processes. We show that, in adult rats, Igf2 is abundantly expressed in brain regions involved in cognitive functions, like hippocampus and prefrontal cortex, compared to the peripheral tissues. In contrast to its maternal imprinting in peripheral tissues, Igf2 is mainly expressed from the maternal allele in these brain regions. The training-dependent increase in Igf2 expression derives proportionally from both parental alleles, and, hence, is mostly maternal. Thus, Igf2 parental expression in the adult rat brain does not follow the imprinting rules found in peripheral tissues, suggesting differential expression regulation and functions of imprinted genes in the brain. PMID:26495851

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

    SciTech Connect

    Wilson, R.C.; Wei, J.Q.; Cheng, K.C.

    1995-05-01

    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 radio active 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. 26 refs., 3 figs., 2 tabs.

  11. Haplotype and isoform specific expression estimation using multi-mapping RNA-seq reads

    PubMed Central

    2011-01-01

    We present a novel pipeline and methodology for simultaneously estimating isoform expression and allelic imbalance in diploid organisms using RNA-seq data. We achieve this by modeling the expression of haplotype-specific isoforms. If unknown, the two parental isoform sequences can be individually reconstructed. A new statistical method, MMSEQ, deconvolves the mapping of reads to multiple transcripts (isoforms or haplotype-specific isoforms). Our software can take into account non-uniform read generation and works with paired-end reads. PMID:21310039

  12. A and MdMYB1 allele-specific markers controlling apple (Malus x domestica Borkh.) skin color and suitability for marker-assisted selection.

    PubMed

    Zhang, X J; Wang, L X; Chen, X X; Liu, Y L; Meng, R; Wang, Y J; Zhao, Z Y

    2014-01-01

    Pre-selection for fruit skin color at the seedling stage would be highly advantageous, with marker-assisted selection offering a potential method for apple pre-selection. A and MdMYB1 alleles are allele-specific DNA markers that are potentially associated with apple skin color, and co-segregate with the Rf and Rni loci, respectively. Here, we assessed the potential application of these 2 alleles for marker-assisted breeding across 30 diverse cultivars and 2 apple seedling progenies. The red skin color phenotype was usually associated with the MdMYB1-1 allele and A(1) allele, respectively, while the 2 molecular markers provided approximately 91% predictability in the 'Fuji' x 'Cripps Pink' and 'Fuji' x 'Gala' progenies. The results obtained from the 30 cultivars and 2 progenies were consistent for the 2 molecular markers. Hence, the results supported that Rf and Rni could be located in a gene cluster, or even correspond to alleles of the same gene. Our results are consistent with the hypothesis that red/yellow dimorphism is controlled by a monogenic system, with the presence of the red anthocyanin pigmentation being dominant. In addition, our results supported that the practical utilization of the 2 function markers to efficiently and accurately select red-skinned apple cultivars in apple scion breeding programs. PMID:25366802

  13. Diurnal Expression Pattern, Allelic Variation, and Association Analysis Reveal Functional Features of the E1 Gene in Control of Photoperiodic Flowering in Soybean

    PubMed Central

    Wu, Hongyan; Zhang, Yupeng; Zhang, Xingzheng; Yang, Jiayin; Wang, Yaying; Yang, Guang; Qiu, Hongmei; Cui, Tingting; Xia, Zhengjun

    2015-01-01

    Although four maturity genes, E1 to E4, in soybean have been successfully cloned, their functional mechanisms and the regulatory network of photoperiodic flowering remain to be elucidated. In this study, we investigated how the diurnal expression pattern of the E1 gene is related to photoperiodic length; and to what extent allelic variation in the B3-like domain of the E1 gene is associated with flowering time phenotype. The bimodal expression of the E1 gene peaked first at around 2 hours after dawn in long-day condition. The basal expression level of E1 was enhanced by the long light phase, and decreased by duration of dark. We identified a 5bp (3 SNP and 2-bp deletion) mutation, referred to an e1-b3a, which occurs in the middle of B3 domain of the E1 gene in the early flowering cultivar Yanhuang 3. Subcellular localization analysis showed that the putative truncated e1-b3a protein was predominately distributed in nuclei, indicating the distribution pattern of e1-b3a was similar to that of E1, but not to that of e1-as. Furthermore, genetic analysis demonstrated allelic variations at the E1 locus significantly underlay flowering time in three F2 populations. Taken together, we can conclude the legume specific E1 gene confers some special features in photoperiodic control of flowering in soybean. Further characterization of the E1 gene will extend our understanding of the soybean flowering pathway in soybean. PMID:26275311

  14. Transcriptome and Allele Specificity Associated with a 3BL Locus for Fusarium Crown Rot Resistance in Bread Wheat

    PubMed Central

    Ma, Jian; Stiller, Jiri; Zhao, Qiang; Feng, Qi; Cavanagh, Colin; Wang, Penghao; Gardiner, Donald; Choulet, Frédéric; Feuillet, Catherine; Zheng, You-Liang; Wei, Yuming; Yan, Guijun; Han, Bin; Manners, John M.; Liu, Chunji

    2014-01-01

    Fusarium pathogens cause two major diseases in cereals, Fusarium crown rot (FCR) and head blight (FHB). A large-effect locus conferring resistance to FCR disease was previously located to chromosome arm 3BL (designated as Qcrs-3B) and several independent sets of near isogenic lines (NILs) have been developed for this locus. In this study, five sets of the NILs were used to examine transcriptional changes associated with the Qcrs-3B locus and to identify genes linked to the resistance locus as a step towards the isolation of the causative gene(s). Of the differentially expressed genes (DEGs) detected between the NILs, 12.7% was located on the single chromosome 3B. Of the expressed genes containing SNP (SNP-EGs) detected, 23.5% was mapped to this chromosome. Several of the DEGs and SNP-EGs are known to be involved in host-pathogen interactions, and a large number of the DEGs were among those detected for FHB in previous studies. Of the DEGs detected, 22 were mapped in the Qcrs-3B interval and they included eight which were detected in the resistant isolines only. The enrichment of DEG, and not necessarily those containing SNPs between the resistant and susceptible isolines, around the Qcrs-3B locus is suggestive of local regulation of this region by the resistance allele. Functions for 13 of these DEGs are known. Of the SNP-EGs, 28 were mapped in the Qcrs-3B interval and biological functions for 16 of them are known. These results provide insights into responses regulated by the 3BL locus and identify a tractable number of target genes for fine mapping and functional testing to identify the causative gene(s) at this QTL. PMID:25405461

  15. Transcriptome and allele specificity associated with a 3BL locus for Fusarium crown rot resistance in bread wheat.

    PubMed

    Ma, Jian; Stiller, Jiri; Zhao, Qiang; Feng, Qi; Cavanagh, Colin; Wang, Penghao; Gardiner, Donald; Choulet, Frédéric; Feuillet, Catherine; Zheng, You-Liang; Wei, Yuming; Yan, Guijun; Han, Bin; Manners, John M; Liu, Chunji

    2014-01-01

    Fusarium pathogens cause two major diseases in cereals, Fusarium crown rot (FCR) and head blight (FHB). A large-effect locus conferring resistance to FCR disease was previously located to chromosome arm 3BL (designated as Qcrs-3B) and several independent sets of near isogenic lines (NILs) have been developed for this locus. In this study, five sets of the NILs were used to examine transcriptional changes associated with the Qcrs-3B locus and to identify genes linked to the resistance locus as a step towards the isolation of the causative gene(s). Of the differentially expressed genes (DEGs) detected between the NILs, 12.7% was located on the single chromosome 3B. Of the expressed genes containing SNP (SNP-EGs) detected, 23.5% was mapped to this chromosome. Several of the DEGs and SNP-EGs are known to be involved in host-pathogen interactions, and a large number of the DEGs were among those detected for FHB in previous studies. Of the DEGs detected, 22 were mapped in the Qcrs-3B interval and they included eight which were detected in the resistant isolines only. The enrichment of DEG, and not necessarily those containing SNPs between the resistant and susceptible isolines, around the Qcrs-3B locus is suggestive of local regulation of this region by the resistance allele. Functions for 13 of these DEGs are known. Of the SNP-EGs, 28 were mapped in the Qcrs-3B interval and biological functions for 16 of them are known. These results provide insights into responses regulated by the 3BL locus and identify a tractable number of target genes for fine mapping and functional testing to identify the causative gene(s) at this QTL. PMID:25405461

  16. Contribution of allelic variability in prostate specific antigen (PSA) & androgen receptor (AR) genes to serum PSA levels in men with prostate cancer

    PubMed Central

    Chavan, Sushant V.; Maitra, Anurupa; Roy, Nobhojit; Chavan, Padma R.

    2014-01-01

    Background & objectives: Wide variability in serum prostate specific antigen (PSA) levels exists in malignant conditions of the prostate. PSA is expressed in normal range in 20 to 25 per cent of prostate cancer cases even in presence of high grade Gleason score. This study was aimed to assess the influence of genetic variants exhibited by PSA and androgen receptor (AR) genes towards the variable expression of PSA in prostate cancer. Methods: Pre-treatment serum PSA levels from 101 prostate cancer cases were retrieved from medical record. PSA genotype analysis in promoter region and AR gene microsatellite Cytosine/Adenine/Guanine (CAG) repeat analysis in exon 1 region was performed using DNA sequencing and fragment analysis techniques. Results: A total of seven single nucleotide polymorphisms (SNPs) in the PSA promoter region were noted. Only two SNPs viz., 158G/A (P<0.001) in the proximal promoter region and -3845G/A (P<0.001) in enhancer region showed significant association with serum PSA levels. The carriers of homozygous GG genotype (P<0.001) at both of these polymorphic sites showed higher expression of PSA whereas homozygous AA genotype (P<0.001) carriers demonstrated lower PSA levels. The combination effect of PSA genotypes along with stratified AR CAG repeats lengths (long, intermediate and short) was also studied. The homozygous GG genotype along with AR long CAG repeats and homozygous AA genotype along with AR short CAG repeats at position -3845 and -158 showed strong interaction and thus influenced serum PSA levels. Interpretation & conclusions: The genetic variants exhibited by PSA gene at positions -3845G/A and -158G/A may be accountable towards wide variability of serum PSA levels in prostate cancer. Also the preferential binding of G and A alleles at these polymorphic sites along with AR long and short CAG repeats may contribute towards PSA expression. PMID:24820830

  17. The Sp1-mediaded allelic regulation of MMP13 expression by an ESCC susceptibility SNP rs2252070

    PubMed Central

    Shi, Meng; Xia, Jianhong; Xing, Huaixin; Yang, Wenjun; Xiong, Xiangyu; Pan, Wenting; Han, Sichong; Shang, Jinhua; Zhou, Changchun; Zhou, Liqing; Yang, Ming

    2016-01-01

    Metallopeptidase 13 (MMP13), a well-known and highly regulated zinc-dependent MMP collagenase, plays a crucial part in development and progression of esophageal squamous cell carcinoma (ESCC). Therefore, we examined associations between ESCC susceptibility and four haplotype-tagging single nucleotide polymorphisms (htSNPs) using a two stage case-control strategy. Odds ratios (OR) and 95% confidence intervals (95% CI) were computed by logistic regression model. After analyzing 1588 ESCC patients and frequency-matched 1600 unaffected controls, we found that MMP13 rs2252070 G > A genetic polymorphism is significantly associated with ESCC risk in Chinese Han populations (GA: OR = 0.63, 95% CI = 0.54–0.74, P = 1.7 × 10−6, AA: OR = 0.73, 95% CI = 0.66–0.81, P = 1.8 × 10−6). Interestingly, the rs2252070 G-to-A change was shown to diminish a Sp1-binding site in ESCC cells. Reporter gene assays indicated that the rs2252070 A allele locating in a potential MMP13 promoter has low promoter activities. After measuring MMP13 gene expression in sixty-six pairs of esophageal cancer and normal tissues, we observed that the rs2252070 A protective allele carriers showed decreased oncogene MMP13 expression. Results of these analyses underline the support of the notion that MMP13 might function as a key oncogene in esophageal carcinogenesis. PMID:27245877

  18. The homeologous Zea mays gigantea genes: characterization of expression and novel mutant alleles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The two homeologous Zea mays gigantea (gi) genes, gi1 and gi2, arose from the last genome duplication event in the maize lineage. Homologs of these genes in other species are required for correct circadian rhythms and proper regulation of growth and development. Here we characterized the expression ...

  19. Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay.

    PubMed

    Zhang, Guoqing; Cai, Fangping; de Rivera, Ivette Lorenzana; Zhou, Zhiyong; Zhang, Jing; Nkengasong, John; Gao, Feng; Yang, Chunfu

    2016-01-01

    A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing. PMID:26560533

  20. Detection of Fusarium oxysporum f. sp. vasinfectum race 3 by single-base extension method and allele-specific polymerase chain reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We developed allele specific (AS) SNP primers for rapid detection of Fusarium oxysporum f.sp vasinfectum (FOV) race 3. FOV_BT_SNP_R3 and FOV_BT_AS_R3 primers were designed based on single nucleotide polymorphisms of partial sequence alignment of the ß-tubulin (BT) gene from several FOV races. These ...

  1. Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay

    PubMed Central

    Zhang, Guoqing; Cai, Fangping; de Rivera, Ivette Lorenzana; Zhou, Zhiyong; Zhang, Jing; Nkengasong, John

    2015-01-01

    A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing. PMID:26560533

  2. Analysis of S-locus and expression of S-alleles of self-compatible rapid-cycling Brassica oleracea 'TO1000DH3'.

    PubMed

    Hee-Jeong, Jung; Nasar Uddin, Ahmed; Jong-In, Park; Senthil Kumar, Thamilarasan; Hye-Ran, Kim; Yong-Gu, Cho; Ill-Sup, Nou

    2014-10-01

    Brassica oleracea is a strictly self-incompatible (SI) plant, but rapid-cycling B. oleracea 'TO1000DH3' is self-compatible (SC). Self-incompatibility in Brassicaceae is controlled by multiple alleles of the S-locus. Three S-locus genes, S-locus glycoprotein (SLG), S-locus receptor kinase (SRK) and S-locus protein 11 or S-locus cysteine-rich (SP11/SCR), have been reported to date, all of which are classified into class I and II. In this study, we investigated the molecular mechanism behind alterations of SI to SC in rapid-cycling B. olerace 'TO1000DH3'. Class I SRK were identified by genomic DNA PCR and PCR-RFLP analysis using SRK specific markers and found to be homozygous. Cloning and sequencing of class I SRK revealed a normal kinase domain without any S-domain/transmembrane domain. Moreover, S-locus sequencing analysis revealed only an SLG sequence, but no SP11/SCR. Expression analysis showed no SRK expression in the stigma, although other genes involved in the SI recognition reaction (SLG, MLPK, ARC1, THL) were found to have normal expression in the stigma. Taken together, the above results suggest that structural aberrations such as deletion of the SI recognition genes may be responsible for the breakdown of SI in rapid-cycling B. oleracea 'TO1000DH3'. PMID:24969488

  3. The Length Distribution of Class I-Restricted T Cell Epitopes Is Determined by Both Peptide Supply and MHC Allele-Specific Binding Preference.

    PubMed

    Trolle, Thomas; McMurtrey, Curtis P; Sidney, John; Bardet, Wilfried; Osborn, Sean C; Kaever, Thomas; Sette, Alessandro; Hildebrand, William H; Nielsen, Morten; Peters, Bjoern

    2016-02-15

    HLA class I-binding predictions are widely used to identify candidate peptide targets of human CD8(+) T cell responses. Many such approaches focus exclusively on a limited range of peptide lengths, typically 9 aa and sometimes 9-10 aa, despite multiple examples of dominant epitopes of other lengths. In this study, we examined whether epitope predictions can be improved by incorporating the natural length distribution of HLA class I ligands. We found that, although different HLA alleles have diverse length-binding preferences, the length profiles of ligands that are naturally presented by these alleles are much more homogeneous. We hypothesized that this is due to a defined length profile of peptides available for HLA binding in the endoplasmic reticulum. Based on this, we created a model of HLA allele-specific ligand length profiles and demonstrate how this model, in combination with HLA-binding predictions, greatly improves comprehensive identification of CD8(+) T cell epitopes. PMID:26783342

  4. Generation and Characterization of Mice Expressing a Conditional Allele of the Interleukin-1 Receptor Type 1

    PubMed Central

    Robson, Matthew J.; Zhu, Chong-Bin; Quinlan, Meagan A.; Botschner, David A.; Baganz, Nicole L.; Lindler, Kathryn M.; Thome, Jason G.; Hewlett, William A.; Blakely, Randy D.

    2016-01-01

    The cytokines IL-1α and IL-1β exert powerful pro-inflammatory actions throughout the body, mediated primarily by the intracellular signaling capacity of the interleukin-1 receptor (IL-1R1). Although Il1r1 knockout mice have been informative with respect to a requirement for IL-1R1 signaling in inflammatory events, the constitutive nature of gene elimination has limited their utility in the assessment of temporal and spatial patterns of cytokine action. To pursue such questions, we have generated C57Bl/6J mice containing a floxed Il1r1 gene (Il1r1loxP/loxP), with loxP sites positioned to flank exons 3 and 4 and thereby the ability to spatially and temporally eliminate Il1r1 expression and signaling. We found that Il1r1loxP/loxP mice breed normally and exhibit no gross physical or behavioral phenotypes. Moreover, Il1r1loxP/loxP mice exhibit normal IL-1R1 receptor expression in brain and spleen, as well as normal IL-1R1-dependent increases in serum IL-6 following IL-1α injections. Breeding of Il1r1loxP/loxP mice to animals expressing a cytomegalovirus (CMV)-driven Cre recombinase afforded efficient excision at the Il1r1 locus. The Il1r1loxP/loxP line should be a valuable tool for the assessment of contributions made by IL-1R1 signaling in diverse cell types across development. PMID:26930558

  5. Evaluation of a blood-specific DNA methylated region and trial for allele-specific blood identification from mixed body fluid DNA.

    PubMed

    Watanabe, Ken; Akutsu, Tomoko; Takamura, Ayari; Sakurada, Koichi

    2016-09-01

    The identification of blood samples obtained from crime scenes has been an important step in forensic investigation. Recently, a novel approach using the blood-specific methylated CpG site cg06379435 has been reported. In this study, we developed a real-time polymerase-chain-reaction-based method that can simply and rapidly quantitate the methylation ratio of cg06379435 and its neighboring CpGs and set the threshold ratios for blood identification by analyzing various body fluid samples. Blood identification using the thresholds was successfully performed in the analysis of a small amount (1ng) of DNA from blood and various aged blood samples, including 29-year-old stains. We also demonstrated a test for allele-specific blood identification from a mixed DNA sample by bisulfite sequencing analysis of these CpG sites and their neighboring single nucleotide polymorphism, rs7359943 (A/G), which is of relevance in cases where mixed samples are obtained from crime scenes. The stability of DNA methylation in aged samples and the usefulness of neighboring genetic information shown in this study suggest that DNA-methylation-based body fluid identification will play a major role in future forensic investigations. PMID:27591539

  6. Genome-wide Association Study of Subtype-Specific Epithelial Ovarian Cancer Risk Alleles Using Pooled DNA

    PubMed Central

    Earp, Madalene A.; Kelemen, Linda E.; Magliocco, Anthony M.; Swenerton, Kenneth D.; Chenevix–Trench, Georgia; Lu, Yi; Hein, Alexander; Ekici, Arif B.; Beckmann, Matthias W.; Fasching, Peter A.; Lambrechts, Diether; Despierre, Evelyn; Vergote, Ignace; Lambrechts, Sandrina; Doherty, Jennifer A.; Rossing, Mary Anne; Chang-Claude, Jenny; Rudolph, Anja; Friel, Grace; Moysich, Kirsten B.; Odunsi, Kunle; Sucheston-Campbell, Lara; Lurie, Galina; Goodman, Marc T.; Carney, Michael E.; Thompson, Pamela J.; Runnebaum, Ingo B.; Dürst, Matthias; Hillemanns, Peter; Dörk, Thilo; Antonenkova, Natalia; Bogdanova, Natalia; Leminen, Arto; Nevanlinna, Heli; Pelttari, Liisa M.; Butzow, Ralf; Bunker, Clareann H.; Modugno, Francesmary; Edwards, Robert P.; Ness, Roberta B.; du Bois, Andreas; Heitz, Florian; Schwaab, Ira; Harter, Philipp; Karlan, Beth Y.; Walsh, Christine; Lester, Jenny; Jensen, Allan; Kjær, Susanne K.; Høgdall, Claus K.; Høgdall, Estrid; Lundvall, Lene; Sellers, Thomas A.; Fridley, Brooke L.; Goode, Ellen L.; Cunningham, Julie M.; Vierkant, Robert A.; Giles, Graham G.; Baglietto, Laura; Severi, Gianluca; Southey, Melissa C.; Liang, Dong; Wu, Xifeng; Lu, Karen; Hildebrandt, Michelle A.T.; Levine, Douglas A.; Bisogna, Maria; Schildkraut, Joellen M.; Iversen, Edwin S.; Weber, Rachel Palmieri; Berchuck, Andrew; Cramer, Daniel W.; Terry, Kathryn L.; Poole, Elizabeth M.; Tworoger, Shelley S.; Bandera, Elisa V.; Chandran, Urmila; Orlow, Irene; Olson, Sara H.; Wik, Elisabeth; Salvesen, Helga B.; Bjorge, Line; Halle, Mari K.; van Altena, Anne M.; Aben, Katja K.H.; Kiemeney, Lambertus A.; Massuger, Leon F.A.G.; Pejovic, Tanja; Bean, Yukie T.; Cybulski, Cezary; Gronwald, Jacek; Lubinski, Jan; Wentzensen, Nicolas; Brinton, Louise A.; Lissowska, Jolanta; Garcia–Closas, Montserrat; Dicks, Ed; Dennis, Joe; Easton, Douglas F.; Song, Honglin; Tyrer, Jonathan P.; Pharoah, Paul D. P.; Eccles, Diana; Campbell, Ian G.; Whittemore, Alice S.; McGuire, Valerie; Sieh, Weiva; Rothstein, Joseph H.; Flanagan, James M.; Paul, James; Brown, Robert; Phelan, Catherine M.; Risch, Harvey A.; McLaughlin, John R.; Narod, Steven A.; Ziogas, Argyrios; Anton-Culver, Hoda; Gentry-Maharaj, Aleksandra; Menon, Usha; Gayther, Simon A.; Ramus, Susan J.; Wu, Anna H.; Pearce, Celeste L.; Pike, Malcolm C.; Dansonka-Mieszkowska, Agnieszka; Rzepecka, Iwona K; Szafron, Lukasz M; Kupryjanczyk, Jolanta; Cook, Linda S.; Le, Nhu D.; Brooks–Wilson, Angela

    2014-01-01

    Epithelial ovarian cancer (EOC) is a heterogeneous cancer with both genetic and environmental risk factors. Variants influencing the risk of developing the less-common EOC subtypes have not been fully investigated. We performed a genome-wide association study (GWAS) of EOC according to subtype by pooling genomic DNA from 545 cases and 398 controls of European descent, and testing for allelic associations. We evaluated for replication 188 variants from the GWAS (56 variants for mucinous, 55 for endometrioid and clear cell, 53 for low malignant potential (LMP) serous, and 24 for invasive serous EOC), selected using pre-defined criteria. Genotypes from 13,188 cases and 23,164 controls of European descent were used to perform unconditional logistic regression under the log-additive genetic model; odds ratios (OR) and 95% confidence intervals are reported. Nine variants tagging 6 loci were associated with subtype-specific EOC risk at P<0.05, and had an OR that agreed in direction of effect with the GWAS results. Several of these variants are in or near genes with a biological rationale for conferring EOC risk, including ZFP36L1 and RAD51B for mucinous EOC (rs17106154, OR=1.17, P=0.029, n=1,483 cases), GRB10 for endometrioid and clear cell EOC (rs2190503, P=0.014, n=2,903 cases), and C22orf26/BPIL2 for LMP serous EOC (rs9609538, OR=0.86, P=0.0043, n=892 cases). In analyses that included the 75 GWAS samples, the association between rs9609538 (OR=0.84, P=0.0007) and LMP serous EOC risk remained statistically significant at P<0.0012 adjusted for multiple testing. Replication in additional samples will be important to verify these results for the less-common EOC subtypes. PMID:24190013

  7. The specificity of long noncoding RNA expression.

    PubMed

    Gloss, Brian S; Dinger, Marcel E

    2016-01-01

    Over the last decade, long noncoding RNAs (lncRNAs) have emerged as a fundamental molecular class whose members play pivotal roles in the regulation of the genome. The observation of pervasive transcription of mammalian genomes in the early 2000s sparked a revolution in the understanding of information flow in eukaryotic cells and the incredible flexibility and dynamic nature of the transcriptome. As a molecular class, distinct loci yielding lncRNAs are set to outnumber those yielding mRNAs. However, like many important discoveries, the road leading to uncovering this diverse class of molecules that act through a remarkable repertoire of mechanisms, was not a straight one. The same characteristic that most distinguishes lncRNAs from mRNAs, i.e. their developmental-stage, tissue-, and cell-specific expression, was one of the major impediments to their discovery and recognition as potentially functional regulatory molecules. With growing numbers of lncRNAs being assigned to biological functions, the specificity of lncRNA expression is now increasingly recognized as a characteristic that imbues lncRNAs with great potential as biomarkers and for the development of highly targeted therapeutics. Here we review the history of lncRNA research and how technological advances and insight into biological complexity have gone hand-in-hand in shaping this revolution. We anticipate that as increasing numbers of these molecules, often described as the dark matter of the genome, are characterized and the structure-function relationship of lncRNAs becomes better understood, it may ultimately be feasible to decipher what these non-(protein)-coding genes encode. This article is part of a Special Issue entitled: Clues to long noncoding RNA taxonomy1, edited by Dr. Tetsuro Hirose and Dr. Shinichi Nakagawa. PMID:26297315

  8. Carbapenem-resistant Acinetobacter baumannii from Brazil: role of carO alleles expression and blaOXA-23 gene

    PubMed Central

    2013-01-01

    Background Carbapenems are the antibiotics of choice to treat infections caused by Acinetobacter baumannii, and resistance to this class can be determined by loss of membrane permeability and enzymatic mechanisms. Here, we analyzed the basis of carbapenem resistance in clinical A. baumannii isolates from different Brazilian regions. Results The analyses addressed the carbapenemase activity of OXA-23, CarO expression and alterations in its primary structure. Susceptibility test revealed that the strains presented the COS (Colistin-Only-Sensitive) profile. PCR and sequencing showed the presence of the chromosomally-encoded blaOXA-51 in all isolates. The majority of strains (53%) carried the carbapenemase blaOXA-23 gene associated with ISAba1. The Hodge test indicated that these strains are carbapenemase producers. PFGE revealed 14 genotypes among strains from Rio de Janeiro and Maranhão. The influence of carO on imipenem resistance was evaluated considering two aspects: the composition of the primary amino acid sequence; and the expression level of this porin. Sequencing and in silico analyses showed the occurrence of CarOa, CarOb and undefined CarO types, and Real Time RT-PCR revealed basal and reduced carO transcription levels among isolates. Conclusions We concluded that, in general, for these Brazilian isolates, the major carbapenem resistance mechanism was due to OXA-23 carbapenemase activity and that loss of CarO porin plays a minor role in this phenotype. However, it was possible to associate the carO alleles and their expression with imipenem resistance. Therefore, these findings underline the complexity in addressing the role of different mechanisms in carbapenem resistance and highlight the possible influence of CarO type in this phenotype. PMID:24195496

  9. Exploring the Effect of Sequence Length and Composition on Allele-Selective Inhibition of Human Huntingtin Expression by Single-Stranded Silencing RNAs

    PubMed Central

    Hu, Jiaxin; Liu, Jing; Yu, Dongbo; Aiba, Yuichiro; Lee, Suheung; Pendergraff, Hannah; Boubaker, Jihane; Artates, Jonathan W.; Lagier-Tourenne, Clotilde; Lima, Walt F.; Swayze, Eric E.; Prakash, Thazha P.

    2014-01-01

    Mutant huntingtin (HTT) protein is the cause of Huntington's disease (HD), an incurable neurological disorder. Almost all patients are heterozygous for mutant HTT and approaches that reduce levels of mutant HTT while leaving expression of wild-type HTT intact might be ideal options for therapeutic development. We have developed several allele-selective strategies for silencing HTT, including single-stranded silencing RNAs (ss-siRNAs). ss-siRNAs are oligonucleotides containing chemical modifications that permit action through the RNA interference (RNAi) pathway. Modified ss-siRNAs chosen to test the effects of varying oligomer length, lipid modification, the introduction of mismatched bases, and variation of chemical modification. We find that several modified ss-siRNA are potent and allele-selective inhibitors of HTT expression. An ss-siRNA with three mismatched bases relative to the CAG repeat was an allele-selective inhibitor of HTT expression in the HdhQ175 mouse model. Multiple allele-selective ss-siRNAs provide a wide platform of modifications to draw on for further optimization and therapeutic development. Our data provide insights into how ss-siRNAs can be modified to improve their properties and facilitate the discovery of the lead compounds necessary for further development. PMID:24694346

  10. Exploring the effect of sequence length and composition on allele-selective inhibition of human huntingtin expression by single-stranded silencing RNAs.

    PubMed

    Hu, Jiaxin; Liu, Jing; Yu, Dongbo; Aiba, Yuichiro; Lee, Suheung; Pendergraff, Hannah; Boubaker, Jihane; Artates, Jonathan W; Lagier-Tourenne, Clotilde; Lima, Walt F; Swayze, Eric E; Prakash, Thazha P; Corey, David R

    2014-06-01

    Mutant huntingtin (HTT) protein is the cause of Huntington's disease (HD), an incurable neurological disorder. Almost all patients are heterozygous for mutant HTT and approaches that reduce levels of mutant HTT while leaving expression of wild-type HTT intact might be ideal options for therapeutic development. We have developed several allele-selective strategies for silencing HTT, including single-stranded silencing RNAs (ss-siRNAs). ss-siRNAs are oligonucleotides containing chemical modifications that permit action through the RNA interference (RNAi) pathway. Modified ss-siRNAs chosen to test the effects of varying oligomer length, lipid modification, the introduction of mismatched bases, and variation of chemical modification. We find that several modified ss-siRNA are potent and allele-selective inhibitors of HTT expression. An ss-siRNA with three mismatched bases relative to the CAG repeat was an allele-selective inhibitor of HTT expression in the HdhQ175 mouse model. Multiple allele-selective ss-siRNAs provide a wide platform of modifications to draw on for further optimization and therapeutic development. Our data provide insights into how ss-siRNAs can be modified to improve their properties and facilitate the discovery of the lead compounds necessary for further development. PMID:24694346

  11. Selective alleviation of Mitomycin C sensitivity in lexA3 strains of Escherichia coli demands allele specificity of rif-nal mutations: a pivotal role for rpoB87-gyrA87 mutations.

    PubMed

    Shanmughapriya, Vinod; Meenakshi, Shanmugaraja; Munavar, M Hussain

    2014-01-01

    Very recently, we have reported about an unconventional mode of elicitation of Mitomycin C (MMC) specific resistance in lexA3 (SOS repair deficient) mutants due to a combination of Rif-Nal mutations (rpoB87-gyrA87). We have clearly shown that UvrB is mandatory for this unconventional MMC resistance in rpoB87-gyrA87-lexA3 strains and uvrB is expressed more even without DNA damage induction from its LexA dependent promoter despite the uncleavable LexA3 repressor. The rpoB87 allele is same as the rpoB3595 which is known to give rise to a fast moving RNA Polymerase and gyrA87 is a hitherto unreported Nal(R) allele. Thus, it is proposed that the RNA Polymerase with higher elongation rate with the mutant DNA Gyrase is able to overcome the repressional hurdle posed by LexA3 to express uvrB. In this study we have systematically analysed the effect of three other rpoB (rif) mutations-two known to give rise to fast moving RNAP (rpoB2 and rpoB111) and one to a slow moving RNAP (rpoB8) and four different alleles of gyrA Nal(R) mutations (gyrA199, gyrA247, gyrA250, gyrA259) isolated spontaneously, on elicitation of MMC resistance in lexA3 strains. Our results indicate that in order to acquire resistance to 0.5 µg/ml MMC cells require both rpoB87 and gyrA87 but resistance to 0.25 µg/ml of MMC can be brought about by either rpoB87, gyrA87, fast moving rpoB mutations or other nal mutations also. We have also depicted increased constitutive uvrB expression in strains carrying fast moving RNAP (rpoB2 and rpoB111) with gyrA87 and another nal mutation with rpoB87 and expression level in these strains is lesser than rpoB87-gyrA87 strain. These results evidently suggest an allele specific role for the rif-nal mutations to acquire MMC resistance in lexA3 strains via increased constitutive uvrB expression and a pivotal role for rpoB87-gyrA87 combination to elicit higher levels of resistance. PMID:24498357

  12. TRANSGENE INTEGRATION INTO THE SAME CHROMOSOME LOCATION CAN PRODUCE ALLELES THAT EXPRESS AT A PREDICTABLE LEVEL, OR ALLELES THAT ARE DIFFERENTIALLY SILENCED

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In an effort to control the variability of transgene expression in plants, we used Cre-lox mediated recombination to insert a gus reporter gene precisely and reproducibly into different target loci. Each integrant line chosen for analysis harbors a single-copy of the transgene at the designated targ...

  13. The Regulation of White Locus Expression: A Dominant Mutant Allele at the White Locus of DROSOPHILA MELANOGASTER

    PubMed Central

    Bingham, Paul M.

    1980-01-01

    A new mutant allele (wDZL) at the white locus of Drosophila melanogaster is dominant to the wild-type allele, but apparently only when the two alleles are synapsed. When chromosomal rearrangements prevent somatic pairing between the two white alleles, wDZL is rendered recessive to wild type. This observation suggests that the dominance of wDZL is sensitive to a synapsis (transvection) effect. On the basis of this and other properties, it is proposed that wDZL causes the repression of transcription of a synapsed w+ allele, but not of a w+ allele elsewhere in the same nucleus. One model to account for this supposes that wDZL produces a repressor of white-locus transcription. This repressor is presumed to be so unstable that other white genes, removed from wDZL but in the same nucleus, are not detectably repressed. These properties may be simply understood if it is assumed that the repressor produced by the wDZL allele is an RNA molecule. PMID:17249039

  14. Tissue specificity and variability of imprinted IGF2 expression in humans

    SciTech Connect

    Giannoukakis, N.; Rouleau, G.; Polychronakos, C.

    1994-09-01

    Parental genomic imprinting refers to the phenomenon where expression of a gene copy depends on the sex of the parent from which it is derived. The human insulin-like growth factor II gene, IGF2, is parentally imprinted with the paternal gene copy exclusively expressed in fetal and term placenta as well as in fetal kidney. In mice, imprinted IGF2 expression is tissue-specific. In a preliminary approach to investigate tissue-specific IGF2 imprinting in humans, we evaluated allele-specific expression in four samples of umbilical cord blood leukocytes of fetuses found to imprint IGF2 in placenta. IGF2 mRNA transcripts from the gene copy transmitted from each parent were distinguished using a transcribed ApaI polymorphism by performing reverse transcription-PCR on total RNA from cord blood leukocytes. Postnatal peripheral blood was examined using the same method. Of 77 informative individuals, 68 expressed both IGF2 copies, but 9 individuals showed unambiguous monoallelic expression. Two individuals from each category were screened again and the results were identical. These data indicate that imprinted IGF2 expression is tissue-specific and show variability of IGF2 imprinting among individuals. This variability may be genetic. We are in the process of screening large pedigrees to test this hypothesis.

  15. Ethylnitrosourea Mutagenesis and the Isolation of Mutant Alleles for Specific Genes Located in the t Region of Mouse Chromosome 17

    PubMed Central

    Bode, Vernon C.

    1984-01-01

    Ethylnitrosourea mutagenesis of spermatogonia in male mice is very efficient and makes it practical to isolate new desired mutant alleles by subsequent progeny screening. This is demonstrated for three genes in the t region of chromosome 17. The first, a mutation designated t-int, interacts with the dominant mutation, T (Brachyury), to produce a tailless mouse. Previously, mutant alleles of the t-int gene were available only in t haplotypes, where they are part of a t chromatin block within which recombination with wild-type chromosomes is inhibited. In addition to t-int, new mutations at the quaking and tufted loci were obtained, as well as at several loci not on chromosome 17, e.g., an X-linked lethal that causes a mottled phenotype in the heterozygote and four new mutant W alleles on chromosome 5. In the experiment, an average of one fertilizing spermatozoan in 1500 was mutant at a given locus and an average of one male in five was able to sire mutants at that locus. PMID:6500258

  16. Expression of the Drosophila retrovirus gypsy as ultrastructurally detectable particles in the ovaries of flies carrying a permissive flamenco allele.

    PubMed

    Lécher, P; Bucheton, A; Pélisson, A

    1997-09-01

    The endogenous retrovirus gypsy is controlled by the Drosophila gene flamenco (flam). New insertions of gypsy occur in any individual Drosophila if its mother is homozygous for the flam1 permissive allele and contains functional gypsy proviruses. The ovaries of flam1 females also contain high amounts of gypsy RNAs. Unexpectedly however, gypsy derepression does not occur in the flam1 female germ-line proper but in the somatic follicular epithelium of the ovary. Since extracts from these females are able to efficiently infect the germ-line of a strain devoid of active gypsy proviruses, we assume that a similar kind of germ-line infection, which would occur inside the flam1 females themselves, could be required for gypsy insertions to occur in their progeny. This hypothesis was confirmed by electron microscopy observations showing that non-enveloped intracytoplasmic particles containing gypsy RNAs accumulate in the apical region of the flam1 follicle cells, close to specific membrane domains to which the gypsy envelope proteins are targeted, whereas both are absent in the flam+ controls. Low amounts of similar virus-like particles were also observed in flam1 oocytes, but it is not yet known whether they entered passively or as a result of membrane fusion. This is the first report of the beginning of a retrovirus cycle in invertebrates and these observations should be taken into account when explaining the maternal effect of the flamenco gene on the multiplication of gypsy proviruses. PMID:9292028

  17. Expression of Overdominance for Specific Activity at the Phosphoglucomutase-2 Locus in the Pacific Oyster, Crassostrea Gigas

    PubMed Central

    Pogson, G. H.

    1991-01-01

    Environmental and genetic components of specific activity variation at the phosphoglucomutase-2 locus in the Pacific oyster, Crassostrea gigas, were examined to assess the direct role played by this polymorphism in a heterozygosity/growth relationship. Both environmental variables studied, season and intertidal position, exerted highly significant effects on phosphoglucomutase specific activity but no interactions occurred between these factors and Pgm-2 genotype. Highly significant differences were also detected between Pgm-2 genotypes. The three most common heterozygotes (Pgm-2(92/100), Pgm-2(96/100) and Pgm-2(100/104)) consistently expressed greater specific activities than the Pgm-2(92/92), Pgm-2(96/96), Pgm-2(100/100) and Pgm-2(104/104) homozygotes. Overall, the specific activities of heterozygotes for the Pgm-2(100) allele exceeded heterozygotes by 24% and 20% in the mantle and adductor muscle tissues, respectively. Heterozygotes formed between the three less frequent Pgm-2(92), Pgm-2(96) and Pgm-2(104) alleles differed sharply from those possessing the Pgm-2(100) allele in being indistinguishable from homozygotes. The possibility of these patterns arising from the undetected presence of an inactive Pgm-2 allele was examined and found to be inconsistent with all of its predicted effects on the specific activity data. Genuine overdominance was shown to be capable of explaining the specific activities of ten structural locus genotypes, allelic frequency distributions in natural populations, and the maintenance of the enzyme polymorphism in a balanced state. The results provide evidence favoring the overdominance explanation for one locus involved in a heterozygosity/growth relationship and suggest that the reported effects of this locus on adult body weight may have been caused by the greater flux capacities of heterozygotes for the Pgm-2(100) allele. PMID:1829426

  18. Characterization of the peptide binding specificity of the HLA class I alleles B*38:01 and B*39:06.

    PubMed

    Sidney, John; Schloss, Jennifer; Moore, Carrie; Lindvall, Mikaela; Wriston, Amanda; Hunt, Donald F; Shabanowitz, Jeffrey; DiLorenzo, Teresa P; Sette, Alessandro

    2016-03-01

    B*38:01 and B*39:06 are present with phenotypic frequencies <2% in the general population, but are of interest as B*39:06 is the B allele most associated with type 1 diabetes susceptibility and 38:01 is most protective. A previous study derived putative main anchor motifs for both alleles based on peptide elution data. The present study has utilized panels of single amino acid substitution peptide libraries to derive detailed quantitative motifs accounting for both primary and secondary influences on peptide binding. From these analyses, both alleles were confirmed to utilize the canonical position 2/C-terminus main anchor spacing. B*38:01 preferentially bound peptides with the positively charged or polar residues H, R, and Q in position 2 and the large hydrophobic residues I, F, L, W, and M at the C-terminus. B*39:06 had a similar preference for R in position 2, but also well-tolerated M, Q, and K. A more dramatic contrast between the two alleles was noted at the C-terminus, where the specificity of B*39:06 was clearly for small residues, with A as most preferred, followed by G, V, S, T, and I. Detailed position-by-position and residue-by-residue coefficient values were generated from the panels to provide detailed quantitative B*38:01 and B*39:06 motifs. It is hoped that these detailed motifs will facilitate the identification of T cell epitopes recognized in the context of two class I alleles associated with dramatically different dispositions towards type 1 diabetes, offering potential avenues for the investigation of the role of CD8 T cells in this disease. PMID:26754738

  19. Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood.

    PubMed

    Chen, Chia-Hsiang

    2016-01-01

    Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer's disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories. PMID:27078154

  20. Development of a Melting Curve-Based Allele-Specific PCR of Apolipoprotein E (APOE) Genotyping Method for Genomic DNA, Guthrie Blood Spot, and Whole Blood

    PubMed Central

    Chen, Chia-Hsiang

    2016-01-01

    Genetic polymorphisms of apolipoprotein E (APOE) are associated with various health conditions and diseases, such as Alzheimer’s disease, cardiovascular diseases, type 2 diabetes, etc. Hence, genotyping of APOE has broad applications in biomedical research and clinical settings, particularly in the era of precision medicine. The study aimed to develop a convenient and accurate method with flexible throughput to genotype the APOE polymorphisms. A melting curve-based allele-specific PCR method was developed to genotype two single nucleotide polymorphisms (SNPs) of APOE, i.e. rs429358 at codon 112 and rs7412 at codon 158. These two SNPs determine the genotype of APOE2, E3, and E4. PCR-based Sanger sequencing was used as the reference method for APOE genotyping. A 100% concordance rate was obtained in 300 subjects between the melting curve-based allele-specific PCR method and the Sanger sequencing method. This method was applied to a genetic association analysis of APOE and schizophrenia consisting of 711 patients with schizophrenia and 665 control subjects from Taiwan. However, no significant differences in the allele and genotype frequencies were detected between these two groups. Further experiments showed that DNA dissolved from blood collected on Guthrie filter paper and total blood cell lysate without DNA extraction can be used in the melting curve-based allele-specific PCR method. Thus, we suggest that this is a fast, accurate and robust APOE genotyping method with a flexible throughput and suitable for DNA template from different preparations. This convenient method shall meet the different needs of various research and clinical laboratories. PMID:27078154

  1. Vip3A Resistance Alleles Exist at High Levels in Australian Targets before Release of Cotton Expressing This Toxin

    PubMed Central

    Mahon, Rod J.; Downes, Sharon J.; James, Bill

    2012-01-01

    Crops engineered to produce insecticidal crystal (Cry) proteins from the soil bacterium Bacillus thuringiensis (Bt) have revolutionised pest control in agriculture. However field-level resistance to Bt has developed in some targets. Utilising novel vegetative insecticidal proteins (Vips), also derived from Bt but genetically distinct from Cry toxins, is a possible solution that biotechnical companies intend to employ. Using data collected over two seasons we determined that, before deployment of Vip-expressing plants in Australia, resistance alleles exist in key targets as polymorphisms at frequencies of 0.027 (n = 273 lines, 95% CI = 0.019–0.038) in H. armigera and 0.008 (n = 248 lines, 0.004–0.015) in H. punctigera. These frequencies are above mutation rates normally encountered. Homozygous resistant neonates survived doses of Vip3A higher than those estimated in field-grown plants. Fortunately the resistance is largely, if not completely, recessive and does not confer resistance to the Bt toxins Cry1Ac or Cry2Ab already deployed in cotton crops. These later characteristics are favourable for resistance management; however the robustness of Vip3A inclusive varieties will depend on resistance frequencies to the Cry toxins when it is released (anticipated 2016) and the efficacy of Vip3A throughout the season. It is appropriate to pre-emptively screen key targets of Bt crops elsewhere, especially those such as H. zea in the USA, which is not only closely related to H. armigera but also will be exposed to Vip in several varieties of cotton and corn. PMID:22761737

  2. Allele-specific extension allows base-pair neutral homozygotes to be discriminated by high-resolution melting of small amplicons.

    PubMed

    Cai, Yanning; Yuan, Yanpeng; Lin, Qingling; Chan, Piu

    2010-11-01

    Not all single-nucleotide polymorphisms (SNPs) can be determined using high-resolution melting (HRM) of small amplicons, especially class 3 and 4 SNPs. This is due mainly to the small shift in the melting temperature (Tm) between two types of homozygote. Choosing rs1869458 (a class 4 SNP) as a sample, we developed a modified small amplicon HRM assay. An allele-specific extension (ASE) primer, which ended at an SNP site and matched only one of the alleles, was added to the reaction as well as additional thermal steps for ASE. Following asymmetric polymerase chain reaction and melting curve analysis, heterozygotes were easily identified. Two types of homozygote were also distinguishable, indicating that extension primers 11 to 13 bases in length worked efficiently in an allele-specific way. Modification of the limiting amplification primer with locked nucleic acid increased the Tm difference between extension and amplification peaks and facilitated subsequent genotyping. In addition, 194 human genomic DNA samples were genotyped with the developed assay and by direct sequencing, with the different methods providing identical genotyping results. In conclusion, ASE-HRM is a simple, inexpensive, closed-tube genotyping method that can be used to examine all types of SNP. PMID:20599636

  3. Dissemination of the highly expressed Bx7 glutenin subunit (Glu-B1al allele) in wheat as revealed by novel PCR markers and RP-HPLC.

    PubMed

    Butow, B J; Gale, K R; Ikea, J; Juhász, A; Bedö, Z; Tamás, L; Gianibelli, M C

    2004-11-01

    Increased expression of the high molecular weight glutenin subunit (HMW-GS) Bx7 is associated with improved dough strength of wheat (Triticum aestivum L.) flour. Several cultivars and landraces of widely different genetic backgrounds from around the world have now been found to contain this so-called 'over-expressing' allelic form of the Bx7 subunit encoded by Glu-B1al. Using three methods of identification, SDS-PAGE, RP-HPLC and PCR marker analysis, as well as pedigree information, we have traced the distribution and source of this allele from a Uruguayan landrace, Americano 44D, in the mid-nineteenth century. Results are supported by knowledge of the movement of wheat lines with migrants. All cultivars possessing the Glu-B1al allele can be identified by the following attributes: (1) the elution of the By sub-unit peak before the Dx sub-unit peak by RP-HPLC, (2) high expression levels of Bx7 (>39% Mol% Bx), (3) a 43 bp insertion in the matrix-attachment region (MAR) upstream of the gene promoter relative to Bx7 and an 18 bp nucleotide duplication in the coding region of the gene. Evidence is presented indicating that these 18 and 43 bp sequence insertions are not causal for the high expression levels of Bx7 as they were also found to be present in a small number of hexaploid species, including Chinese Spring, and species expressing Glu-B1ak and Glu-B1a alleles. In addition, these sequence inserts were found in different isolates of the tetraploid wheat, T. turgidum, indicating that these insertion/deletion events occurred prior to hexaploidization. PMID:15340686

  4. [The Use of Specific DNA Markers for the Identification of Alleles of the FAD3 Genes in Rape (Brassica napus L.)].

    PubMed

    Lemesh, V A; Mozgova, G V; Grushetskaya, Z E; Sidorenko, E V; Pilyuk, Ya E; Bakanovskaya, A V

    2015-08-01

    A search was conducted for the alleles responsible for the quality of food-grade rapeseed oil in a collection of 21 samples of spring and winter oilseed rape of Belarusian and Russian breeding. We also developed A- and C-gene-specific DNA markers to assess the genomic polymorphisms of rape for FAD3 genes and selected plants with a low content of linolenic acid for use in the selection process. The development of a method for identifying FAD3 alleles, which control the level of linolenic acid in rapeseed oil, as well as of the design for new dCAPS markers, enabled the identification of plants homozygous for individual FAD3A and/or FAD3C genes in the F2-generation. These plants are currently involved in the selection process of new varieties with a reduced content of linolenic acid in rapeseed oil. PMID:26601489

  5. Genetic variation in the functional ENG allele inherited from the non-affected parent associates with presence of pulmonary arteriovenous malformation in hereditary hemorrhagic telangiectasia 1 (HHT1) and may influence expression of PTPN14

    PubMed Central

    Letteboer, Tom G. W.; Benzinou, Michael; Merrick, Christopher B.; Quigley, David A.; Zhau, Kechen; Kim, Il-Jin; To, Minh D.; Jablons, David M.; van Amstel, Johannes K. P.; Westermann, Cornelius J. J.; Giraud, Sophie; Dupuis-Girod, Sophie; Lesca, Gaetan; Berg, Jonathan H.; Balmain, Allan; Akhurst, Rosemary J.

    2015-01-01

    HHT shows clinical variability within and between families. Organ site and prevalence of arteriovenous malformations (AVMs) depend on the HHT causative gene and on environmental and genetic modifiers. We tested whether variation in the functional ENG allele, inherited from the unaffected parent, alters risk for pulmonary AVM in HHT1 mutation carriers who are ENG haploinsufficient. Genetic association was found between rs10987746 of the wild type ENG allele and presence of pulmonary AVM [relative risk = 1.3 (1.0018–1.7424)]. The rs10987746-C at-risk allele associated with lower expression of ENG RNA in a panel of human lymphoblastoid cell lines (P = 0.004). Moreover, in angiogenically active human lung adenocarcinoma tissue, but not in uninvolved quiescent lung, rs10987746-C was correlated with expression of PTPN14 (P = 0.004), another modifier of HHT. Quantitative TAQMAN expression analysis in a panel of normal lung tissues from 69 genetically heterogeneous inter-specific backcross mice, demonstrated strong correlation between expression levels of Eng, Acvrl1, and Ptpn14 (r2 = 0.75–0.9, P < 1 × 10−12), further suggesting a direct or indirect interaction between these three genes in lung in vivo. Our data indicate that genetic variation within the single functional ENG gene influences quantitative and/or qualitative differences in ENG expression that contribute to risk of pulmonary AVM in HHT1, and provide correlative support for PTPN14 involvement in endoglin/ALK1 lung biology in vivo. PTPN14 has been shown to be a negative regulator of Yap/Taz signaling, which is implicated in mechanotransduction, providing a possible molecular link between endoglin/ALK1 signaling and mechanical stress. EMILIN2, which showed suggestive genetic association with pulmonary AVM, is also reported to interact with Taz in angiogenesis. Elucidation of the molecular mechanisms regulating these interactions in endothelial cells may ultimately provide more rational choices for HHT

  6. The Minor Allele of rs7574865 in the STAT4 Gene Is Associated with Increased mRNA and Protein Expression

    PubMed Central

    Lamana, Amalia; López-Santalla, Mercedes; Castillo-González, Raquel; Ortiz, Ana María; Martín, Javier; García-Vicuña, Rosario; González-Álvaro, Isidoro

    2015-01-01

    Objective The T allele of rs7574865 in STAT4 confers risk of developing autoimmune disorders. However, its functional significance remains unclear. Here we analyze how rs7574865 affects the transcription of STAT4 and its protein expression. Methods We studied 201 patients (80% female; median age, 54 years; median disease duration, 5.4 months) from PEARL study. Demographic, clinical, laboratory and therapeutic data were collected at each visit. IL-6 serum levels were measured by enzyme immune assay. The rs7574865 was genotyped using TaqMan probes. The expression levels of STAT4 mRNA were determined at 182 visits from 69 patients using quantitative real-time polymerase chain reaction. STAT4 protein was assessed by western blot in 62 samples from 34 patients. To determine the effect of different variables on the expression of STAT4 mRNA and protein, we performed multivariate longitudinal analyses using generalized linear models. Results After adjustment for age, disease activity and glucocorticoid dose as confounders, the presence of at least one copy of the T allele of rs7574865 was significantly associated with higher levels of STAT4 mRNA. Similarly, TT patients showed significantly higher levels of STAT4 protein than GG patients. IL-6 induced STAT4 and STAT5 phosphorylation in peripheral blood lymphocytes. Patients carrying at least one T allele of rs7574865 displayed lower levels of serum IL-6 compared to GG homozygous; by contrast the production of C-reactive protein was similar in both populations. Conclusion Our data suggest that the presence of the rs7574865 T allele enhances STAT4 mRNA transcription and protein expression. It may enhance the signaling of molecules depending on the STAT4 pathway. PMID:26569609

  7. Molecular cloning of a full-length cDNA for dentatorubral-pallidoluysian atrophy and regional expressions of the expanded alleles in the CNS

    SciTech Connect

    Onodera, Osamu; Oyake, Mutsuo; Takano, Hiroki

    1995-11-01

    Dentatorubral-pallidoluysian atrophy (DRPLA) is an autosomal dominant neurodegenerative disorder characterized by genetic anticipation and variable combinations of symptoms including myoclonus, epilepsy, cerebellar ataxia, choreoathetosis, and dementia. Recently, we discovered that DRPLA is caused by unstable expansion of a CAG repeat of a gene on the short arm of chromosome 12. We determined the consensus DRPLA cDNA sequence containing the complete coding region for 1,185 amino acids. The CAG repeat, which is expanded in DRPLA, is located 1,462 bp downstream from the putative methionine initiation codon and encodes a poly-glutamine tract. Although poly-serine and proline tracts exist near the CAG repeats, these poly-serine or proline tracts did not show any polymorphisms, which is in strong contrast to the high heterogeneity in the length of the CAG repeat. Northern blot analysis revealed a 4.7-kb transcript that is widely expressed in various tissues including heart, lung, kidney, placenta, skeletal muscle, and brain. Reverse transcription-PCR analysis revealed that the expanded alleles are transcribed to levels comparable to those of normal alleles. These results indicate that there is no difference in transcriptional efficiency between expanded and normal alleles. Furthermore, mRNA from cerebellar hemispheres of DRPLA patients showed smaller sizes of CAG repeats compared with other regions of the brain, which reflects somatic mosaicism of the expanded alleles of the DRPLA gene. 49 refs., 6 figs.

  8. From genes to phenotypes - evaluation of two methods for the SNP analysis in archaeological remains: pyrosequencing and competitive allele specific PCR (KASPar).

    PubMed

    Pruvost, Melanie; Reissmann, Monika; Benecke, Norbert; Ludwig, Arne

    2012-01-20

    The amplification length of the DNA fragments is one major limitation of most paleogenetic analyses. Routinely, only fragments below 200 bp can be amplified, significantly reducing the content of genetic information. Although overlapping PCR strategies and next generation sequencing techniques have strongly improved data mining recently, these methods are still expensive and time consuming. In contrast, SNP analyses are easy to handle, fast and cheap. In this study, we compare two methods of SNP detection as to efficiency, cost and reliability for their use in ancient DNA applications: pyrosequencing and competitive allele specific PCR (KASPar). Our sample set consisted of 16 horse bones from two Scythian graves (600-800 BC). In conclusion, both approaches produced reliable results for most allelic patterns. But an indel of 11 bp (ASIP) could not be detected in the KASPar approach and produced problems in the pyrosequencing method (70% success rate). In such cases, we recommend checking allelic distribution using a gel approach or capillary sequencing. Overall, in comparison with the traditional mode of ancient DNA investigations (PCR, cloning, capillary sequencing), both approaches are superior for SNP analyses especially of large sample sets. PMID:22154270

  9. A simple and rapid method for HLA-DQA1 genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases.

    PubMed

    Maeda, M; Murayama, N; Ishii, H; Uryu, N; Ota, M; Tsuji, K; Inoko, H

    1989-11-01

    The second exon of the HLA-DQA1 genes was selectively amplified from genomic DNAs of 72 HLA-homozygous B cell lines by the polymerase chain reaction (PCR). Amplified DNAs were digested with HaeIII, Ddel, ScrFI, FokI and RsaI, which recognize allelic sequence variations in the polymorphic segments of the DQA1 second exon, and then subjected to electrophoresis in polyacrylamide gels. Eight different polymorphic patterns of restriction fragments were obtained, and seven were identical to patterns predicted from the known DNA sequences, correlating with each HLA-DQw type defined by serological typing. The remaining one pattern cannot be explained from the sequence data, suggesting the presence of a novel DQA1 allele at the nucleotide level. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DQ types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes and can be extended and applied to HLA-DR, -Dw DP typing. PMID:2576477

  10. Susceptibility allele-specific loss of miR-1324-mediated silencing of the INO80B chromatin-assembly complex gene in pre-eclampsia.

    PubMed

    Oudejans, Cees B M; Michel, Omar J; Janssen, Rob; Habets, Rob; Poutsma, Ankie; Sistermans, Erik A; Weiss, Marjan M; Incarnato, Danny; Oliviero, Salvatore; Kleiverda, Gunilla; Van Dijk, Marie; Arngrímsson, Reynir

    2015-01-01

    In humans, the elucidation of the genetics underlying multifactorial diseases such as pre-eclampsia remains complex. Given the current day availability of genome-wide linkage- and expression data pools, we applied pathway-guided genome-wide meta-analysis guided by the premise that the functional network underlying these multifactorial syndromes is under selective genetic pressure. This approach drastically reduced the genomic region of interest, i.e. 2p13 linked with pre-eclampsia in Icelandic families, from 8 679 641 bp (region with linkage) to 45 264 bp (coding exons of prioritized genes) (0.83%). Mutation screening of the candidate genes (n = 13) rapidly reduced the minimal critical region and showed the INO80B gene, encoding a novel winged helix domain (pfam14465) and part of the chromatin-remodeling complex, to be linked to pre-eclampsia. The functional defect in placental cells involved a susceptibility allele-dependent loss-of-gene silencing due to increased INO80B RNA stability as a consequence of differential binding of miR-1324 to the susceptibility allele of rs34174194. This risk allele is located at position 1 in an absolutely conserved 7-mer (UUGUCUG) in the 3-UTR of INO80B immediately downstream of a variant Pumillio Recognition Element (UGUANAAG). These data support that pre-eclampsia genes affect a conserved fundamental mechanism that evolved as a consequence of hemochorial placentation. Functionally, this involves founder-dependent, placentally expressed paralogous genes that regulate an essential trophoblast differentiation pathway but act at different entry points. PMID:25143393

  11. Methylation similarities of two CpG sites within exon 5 of human H19 between normal tissues and testicular germ cell tumours of adolescents and adults, without correlation with allelic and total level of expression.

    PubMed Central

    Gillis, A. J.; Verkerk, A. J.; Dekker, M. C.; van Gurp, R. J.; Oosterhuis, J. W.; Looijenga, L. H.

    1997-01-01

    Testicular germ cell tumours (TGCTs) of adolescents and adults morphologically mimic different stages of embryogenesis. Established cell lines of these cancers are used as informative models to study early development. We found that, in contrast to normal development, TGCTs show a consistent biallelic expression of imprinted genes, including H19, irrespective of histology. Methylation of particular cytosine residues of H19 correlates with inhibition of expression, which has not been studied in TGCTs thus far. We investigated the methylation status of two CpG sites within the 3' region of H19 (exon 5: positions 3321 and 3324) both in normal tissues as well as in TGCTs. To obtain quantitative data of these specific sites, the ligation-mediated polymerase chain reaction technique, instead of Southern blot analysis, was applied. The results were compared with the allelic status and the total level of expression of this gene. Additionally, the undifferentiated cells and differentiated derivatives of the TGCT-derived cell line NT2-D1 were analysed. While peripheral blood showed no H19 expression and complete methylation, a heterogeneous but consistent pattern of methylation and level of expression was found in the other normal tissues, without a correlation between the two. The separate histological entities of TGCTs resembled the pattern of their nonmalignant tissues. While the CpG sites remained completely methylated in NT2-D1, H19 expression was induced upon differentiation. These data indicate that methylation of the CpG sites within exon 5 of H19 is tissue dependent, without regulating allelic status and/or total level of expression. Of special note is the finding that, also regarding methylation of these particular sites of H19, TGCTs mimic their non-malignant counterparts, in spite of their consistent biallelic expression. Images Figure 1 Figure 3 Figure 4 PMID:9310237

  12. Cloning of the pleiotropic T locus in soybean and two recessive alleles that differentially affect structure and expression of the encoded flavonoid 3' hydroxylase.

    PubMed Central

    Zabala, Gracia; Vodkin, Lila

    2003-01-01

    Three loci (I, R, and T) control pigmentation of the seed coats in Glycine max and are genetically distinct from those controlling flower color. The T locus also controls color of the trichome hairs. We report the identification and isolation of a flavonoid 3' hydroxylase gene from G. max (GmF3'H) and the linkage of this gene to the T locus. This GmF3'H gene was highly expressed in early stages of seed coat development and was expressed at very low levels or not at all in other tissues. Evidence that the GmF3'H gene is linked to the T locus came from the occurrence of multiple RFLPs in lines with varying alleles of the T locus, as well as in a population of plants segregating at that locus. GmF3'H genomic and cDNA sequence analysis of color mutant lines with varying t alleles revealed a frameshift mutation in one of the alleles. In another line derived from a mutable genetic stock, the abundance of the mRNAs for GmF3'H was dramatically reduced. Isolation of the GmF3'H gene and its identification as the T locus will enable investigation of the pleiotropic effects of the T locus on cell wall integrity and its involvement in the regulation of the multiple branches of the flavonoid pathway in soybean. PMID:12586717

  13. Rapid Origin Determination of the Northern Mauxia Shrimp (Acetes chinensis) Based on Allele Specific Polymerase Chain Reaction of Partial Mitochondrial 16S rRNA Gene

    PubMed Central

    Kang, Jung-Ha; Noh, Eun-Soo; Park, Jung-Youn; An, Chel-Min; Choi, Jung-Hwa; Kim, Jin-Koo

    2015-01-01

    Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis′ economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis′ origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3′-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China. PMID:25656197

  14. Rapid Origin Determination of the Northern Mauxia Shrimp (Acetes chinensis) Based on Allele Specific Polymerase Chain Reaction of Partial Mitochondrial 16S rRNA Gene.

    PubMed

    Kang, Jung-Ha; Noh, Eun-Soo; Park, Jung-Youn; An, Chel-Min; Choi, Jung-Hwa; Kim, Jin-Koo

    2015-04-01

    Acetes chinensis is an economically important shrimp that belongs to the Sergestidae family; following fermentation, A. chinensis' economic value, however, is low in China, and much of the catch in China is exported to Korea at a low price, thus leading to potential false labeling. For this reason, we developed a simple method to identify A. chinensis' origin using allele-specific polymerase chain reaction (PCR). Ten single nucleotide polymorphisms (SNPs) were identified from partial (i.e., 570 bp) DNA sequence analysis of the mitochondrial 16s rRNA gene in 96 Korean and 96 Chinese individual shrimp. Among 10 SNP sites, four sites were observed in populations from both countries, and two sites located in the middle with SNP sites at their 3'-ends were used to design allele-specific primers. Among the eight internal primers, the C220F primer specific to the Chinese A. chinensis population amplified a DNA fragment of 364 bp only from that population. We were able to identify the A. chinensis population origin with 100% accuracy using multiplex PCR performed with two external primers and C220F primers. These results show that the 16S rRNA gene that is generally used for the identification of species can be used for the identification of the origin within species of A. chinensis, which is an important finding for the fair trade of the species between Korea and China. PMID:25656197

  15. Imprinting in rice: the role of DNA and histone methylation in modulating parent-of-origin specific expression and determining transcript start sites.

    PubMed

    Du, Miru; Luo, Ming; Zhang, Ruofang; Finnegan, E Jean; Koltunow, Anna M G

    2014-07-01

    Over 200 imprinted genes in rice endosperm are known, but the mechanisms modulating their parental allele-specific expression are poorly understood. Here we use three imprinted genes, OsYUCCA11, yellow2-like and ubiquitin hydrolase, to show that differential DNA methylation and tri-methylation of histone H3 lysine 27 (H3K27me3 ) in the promoter and/or gene body influences allele-specific expression or the site of transcript initiation. Paternal expression of OsYUCCA11 required DNA methylation in the gene body whereas the gene body of the silenced maternal allele was hypomethylated and marked with H3K27me3 . These differential markings mirror those proposed to modulate paternal expression of two Arabidopsis genes, PHERES1 and a YUCCA homolog, indicating conservation of imprinting mechanisms. At yellow2-like, DNA hypomethylation in the upstream flanking region resulted in maternal transcripts that were longer than paternal transcripts; the maternal transcript initiation site was marked by DNA methylation in the paternal allele, and transcription initiated ~700 bp downstream. The paternal allele of an ubiquitin hydrolase gene exhibited gene body DNA methylation and produced full-length transcripts, while the maternal allele was hypomethylated in the 5' gene body and transcripts initiated from a downstream promoter. Inhibition of DNA methylation by 5-azacytidine or zebularine activated the long transcripts from yellow2-like and enhanced expression of the short transcripts from the ubiquitin hydrolase in seedlings, indicating that DNA methylation prevents transcript initiation from cryptic promoters. These observations suggest a paradigm whereby maternal genome hypomethylation is associated with the production of distinct transcripts, potentially diversifying the gene products from the two alleles. PMID:24819479

  16. p16(INK4a) promoter methylation and 9p21 allelic loss in colorectal carcinomas: relation with immunohistochemical p16(INK4a) expression and with tumor budding.

    PubMed

    Prall, Friedrich; Ostwald, Christiane; Weirich, Volker; Nizze, Horst

    2006-05-01

    In colorectal carcinomas, p16(INK4a) inactivation is known to occur by allelic loss and by promoter methylation, but mutations are rare. p16(INK4a) is up-regulated in tumor buds, and the consequent shutdown of proliferation may be a prerequisite for tumor budding. Fifty-seven colorectal carcinomas from a consecutive series were investigated. Using DNA from tissue homogenates, p16(INK4a) promoter methylation was seen in 17 of 57 tumors by methylation-specific polymerase chain reaction, and this could be confirmed using DNA from laser-capture microdissected material in 16 of these cases. A total loss of immunohistochemical p16(INK4a) expression was seen in 6 of 17 tumors with promoter methylation. Quantification of immunohistochemical p16(INK4a) expression for the remaining 11 cases revealed statistically lower frequencies of expression as compared with cases without p16(INK4a) promoter methylation. 9p21 allelic loss was observed in 9 cases, but p16(INK4a) expression in these carcinomas was not reduced. Attempted linear regression of p16(INK4a) expression in tumor buds on the degree of tumor budding, as counted on pan-cytokeratin immunostains, did not show a correlation. p16(INK4a) promoter methylation can completely abrogate p16(INK4a) expression in colorectal carcinomas. In many cases, however, it has an appreciable but only modulatory influence on p16(INK4a) expression. Possibly, methylations are heterozygous, and/or mosaic in colorectal carcinomas and/or methylations are not totally stable but can be lost between carcinoma cell replication cycles. Up-regulation of p16(INK4a) does not seem to be a strict requirement for tumor budding, hence, the absence of a correlation. PMID:16647956

  17. SAAS-CNV: A Joint Segmentation Approach on Aggregated and Allele Specific Signals for the Identification of Somatic Copy Number Alterations with Next-Generation Sequencing Data

    PubMed Central

    Zhang, Zhongyang; Hao, Ke

    2015-01-01

    Cancer genomes exhibit profound somatic copy number alterations (SCNAs). Studying tumor SCNAs using massively parallel sequencing provides unprecedented resolution and meanwhile gives rise to new challenges in data analysis, complicated by tumor aneuploidy and heterogeneity as well as normal cell contamination. While the majority of read depth based methods utilize total sequencing depth alone for SCNA inference, the allele specific signals are undervalued. We proposed a joint segmentation and inference approach using both signals to meet some of the challenges. Our method consists of four major steps: 1) extracting read depth supporting reference and alternative alleles at each SNP/Indel locus and comparing the total read depth and alternative allele proportion between tumor and matched normal sample; 2) performing joint segmentation on the two signal dimensions; 3) correcting the copy number baseline from which the SCNA state is determined; 4) calling SCNA state for each segment based on both signal dimensions. The method is applicable to whole exome/genome sequencing (WES/WGS) as well as SNP array data in a tumor-control study. We applied the method to a dataset containing no SCNAs to test the specificity, created by pairing sequencing replicates of a single HapMap sample as normal/tumor pairs, as well as a large-scale WGS dataset consisting of 88 liver tumors along with adjacent normal tissues. Compared with representative methods, our method demonstrated improved accuracy, scalability to large cancer studies, capability in handling both sequencing and SNP array data, and the potential to improve the estimation of tumor ploidy and purity. PMID:26583378

  18. Allelic Variation in a Simple Sequence Repeat Element of Neisserial pglB2 and Its Consequences for Protein Expression and Protein Glycosylation

    PubMed Central

    Viburiene, Raimonda; Vik, Åshild; Koomey, Michael

    2013-01-01

    Neisseria species express an O-linked glycosylation system in which functionally distinct proteins are elaborated with variable glycans. A major source of glycan diversity in N. meningitidis results from two distinct pglB alleles responsible for the synthesis of either N,N′-diacetylbacillosamine or glyceramido-acetamido trideoxyhexose that occupy the reducing end of the oligosaccharides. Alternative modifications at C-4 of the precursor UDP-4-amino are attributable to distinct C-terminal domains that dictate either acetyltransferase or glyceramidotransferase activity, encoded by pglB and pglB2, respectively. Naturally occurring alleles of pglB2 have homopolymeric tracts of either 7 or 8 adenosines (As) bridging the C-terminal open reading frame (ORF) and the ORF encompassing the conserved N-terminal domain associated with phosphoglycosyltransferase activity. In the work presented here, we explored the consequences of such pglB2 allele variation and found that, although both alleles are functional vis-à-vis glycosylation, the 7A form results in the expression of a single, multidomain protein, while the 8A variant elicits two single-domain proteins. We also found that the glyceramidotransferase activity-encoding domain is essential to protein glycosylation, showing the critical role of the C-4 modification of the precursor UDP-4-amino in the pathway. These findings were further extended and confirmed by examining the phenotypic consequences of extended poly(A) tract length variation. Although ORFs related to those of pglB2 are broadly distributed in eubacteria, they are primarily found as two distinct, juxtaposed ORFs. Thus, the neisserial pglB2 system provides novel insights into the potential influence of hypermutability on modular evolution of proteins by providing a unique snapshot of the progression of ongoing gene fusion. PMID:23729645

  19. Allelic variation in a simple sequence repeat element of neisserial pglB2 and its consequences for protein expression and protein glycosylation.

    PubMed

    Viburiene, Raimonda; Vik, Åshild; Koomey, Michael; Børud, Bente

    2013-08-01

    Neisseria species express an O-linked glycosylation system in which functionally distinct proteins are elaborated with variable glycans. A major source of glycan diversity in N. meningitidis results from two distinct pglB alleles responsible for the synthesis of either N,N'-diacetylbacillosamine or glyceramido-acetamido trideoxyhexose that occupy the reducing end of the oligosaccharides. Alternative modifications at C-4 of the precursor UDP-4-amino are attributable to distinct C-terminal domains that dictate either acetyltransferase or glyceramidotransferase activity, encoded by pglB and pglB2, respectively. Naturally occurring alleles of pglB2 have homopolymeric tracts of either 7 or 8 adenosines (As) bridging the C-terminal open reading frame (ORF) and the ORF encompassing the conserved N-terminal domain associated with phosphoglycosyltransferase activity. In the work presented here, we explored the consequences of such pglB2 allele variation and found that, although both alleles are functional vis-à-vis glycosylation, the 7A form results in the expression of a single, multidomain protein, while the 8A variant elicits two single-domain proteins. We also found that the glyceramidotransferase activity-encoding domain is essential to protein glycosylation, showing the critical role of the C-4 modification of the precursor UDP-4-amino in the pathway. These findings were further extended and confirmed by examining the phenotypic consequences of extended poly(A) tract length variation. Although ORFs related to those of pglB2 are broadly distributed in eubacteria, they are primarily found as two distinct, juxtaposed ORFs. Thus, the neisserial pglB2 system provides novel insights into the potential influence of hypermutability on modular evolution of proteins by providing a unique snapshot of the progression of ongoing gene fusion. PMID:23729645

  20. Specific Arabidopsis HSP90.2 alleles recapitulate RAR1 cochaperone function in plant NB-LRR disease resistance protein regulation

    PubMed Central

    Hubert, David A.; He, Yijian; McNulty, Brian C.; Tornero, Pablo; Dangl, Jeffery L.

    2009-01-01

    Both plants and animals require the activity of proteins containing nucleotide binding (NB) domain and leucine-rich repeat (LRR) domains for proper immune system function. NB-LRR proteins in plants (NLR proteins in animals) also require conserved regulation via the proteins SGT1 and cytosolic HSP90. RAR1, a protein specifically required for plant innate immunity, interacts with SGT1 and HSP90 to maintain proper NB-LRR protein steady-state levels. Here, we present the identification and characterization of specific mutations in Arabidopsis HSP90.2 that suppress all known phenotypes of rar1. These mutations are unique with respect to the many mutant alleles of HSP90 identified in all systems in that they can bypass the requirement for a cochaperone and result in the recovery of client protein accumulation and function. Additionally, these mutations separate HSP90 ATP hydrolysis from HSP90 function in client protein folding and/or accumulation. By recapitulating the activity of RAR1, these novel hsp90 alleles allow us to propose that RAR1 regulates the physical open–close cycling of a known “lid structure” that is used as a dynamic regulatory HSP90 mechanism. Thus, in rar1, lid cycling is locked into a conformation favoring NB-LRR client degradation, likely via SGT1 and the proteasome. PMID:19487680

  1. Identification of Stmm3 locus Conferring Resistance to Late-stage Chemically Induced Skin Papillomas on Mouse Chromosome 4 by Congenic Mappingand Allele-specific Alteration Analysis

    PubMed Central

    Saito, Megumi; Okumura, Kazuhiro; Miura, Ikuo; Wakana, Shigeharu; Kominami, Ryo; Wakabayashi, Yuichi

    2014-01-01

    Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to chemically induced skin papillomas on chromosome 4 and 7 with a large number of [(FVB/N × MSM/Ms) F1 × FVB/N] backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 4. We used linkage analysis and a congenic mouse strain, FVB.MSM-Stmm3 to refine the location of Stmm3 (Skin tumor modifier of MSM 3) locus within a physical interval of about 34 Mb on distal chromosome 4. In addition, we used patterns of allele-specific imbalances in tumors from N2 and N10 congenic mice to narrow down further the region of Stmm3 locus to a physical distance of about 25 Mb. Furthermore, immunohistochemical analysis showed papillomas from congenic mice had less proliferative activity. These results suggest that Stmm3 responsible genes may have an influence on papilloma formation in the two-stage skin carcinogenesis by regulating papilloma growth rather than development. PMID:25077764

  2. Co-Occurrence of Two Allelic Variants of CYP51 in Erysiphe necator and Their Correlation with Over-Expression for DMI Resistance

    PubMed Central

    Rallos, Lynn Esther E.; Baudoin, Anton B.

    2016-01-01

    Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other. PMID:26839970

  3. Co-Occurrence of Two Allelic Variants of CYP51 in Erysiphe necator and Their Correlation with Over-Expression for DMI Resistance.

    PubMed

    Rallos, Lynn Esther E; Baudoin, Anton B

    2016-01-01

    Demethylation inhibitors (DMIs) have been an important tool in the management of grapevine powdery mildew caused by Erysiphe necator. Long-term, intensive use of DMIs has resulted in reduced sensitivity in field populations. To further characterize DMI resistance and understand resistance mechanisms in this pathogen, we investigated the cyp51 sequence of 24 single-spored isolates from Virginia and surrounding states and analyzed gene expression in isolates representing a wide range of sensitivity. Two cyp51 alleles were found with respect to the 136th codon of the predicted EnCYP51 sequence: the wild-type (TAT) and the mutant (TTT), which results in the known Y136F amino acid change. Some isolates possessed both alleles, demonstrating gene duplication or increased gene copy number and possibly a requirement for at least one mutant copy of CYP51 for resistance. Cyp51 was over-expressed 1.4- to 19-fold in Y136F-mutant isolates. However, the Y136F mutation was absent in one isolate with moderate to high resistance factor. Two additional synonymous mutations were detected as well, one of which, A1119C was present only in isolates with high cyp51 expression. Overall, our results indicate that at least two mechanisms, cyp51 over-expression and the known target-site mutation in CYP51, contribute to resistance in E. necator, and may be working in conjunction with each other. PMID:26839970

  4. Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function.

    PubMed

    Barbaro, Vanessa; Nasti, Annamaria A; Del Vecchio, Claudia; Ferrari, Stefano; Migliorati, Angelo; Raffa, Paolo; Lariccia, Vincenzo; Nespeca, Patrizia; Biasolo, Mariangela; Willoughby, Colin E; Ponzin, Diego; Palù, Giorgio; Parolin, Cristina; Di Iorio, Enzo

    2016-06-01

    Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-ΔNp63α-RFP and R279H-ΔNp63α-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome. Stem Cells 2016;34:1588-1600. PMID:26891374

  5. Detection and molecular characterization of two FAD3 genes controlling linolenic acid content and development of allele-specific markers in yellow mustard (Sinapis alba).

    PubMed

    Tian, Entang; Zeng, Fangqin; MacKay, Kimberly; Roslinsky, Vicky; Cheng, Bifang

    2014-01-01

    Development of yellow mustard (Sinapis alba L.) with superior quality traits (low erucic and linolenic acid contents, and low glucosinolate content) can make this species as a potential oilseed crop. We have recently isolated three inbred lines Y1127, Y514 and Y1035 with low (3.8%), medium (12.3%) and high (20.8%) linolenic acid (C18∶3) content, respectively, in this species. Inheritance studies detected two fatty acid desaturase 3 (FAD3) gene loci controlling the variation of C18∶3 content. QTL mapping revealed that the two FAD3 gene loci responsible for 73.0% and 23.4% of the total variation and were located on the linkage groups Sal02 and Sal10, respectively. The FAD3 gene on Sal02 was referred to as SalFAD3.LA1 and that on Sal10 as SalFAD3.LA2. The dominant and recessive alleles were designated as LA1 and la1 for SalFAD3.LA1, and LA2 and la2 for SalFAD3.LA2. Cloning and alignment of the coding and genomic DNA sequences revealed that the SalFAD3.LA1 and SalFAD3.LA2 genes each contained 8 exons and 7 introns. LA1 had a coding DNA sequence (CDS) of 1143 bp encoding a polypeptide of 380 amino acids, whereas la1 was a loss-of-function allele due to an insertion of 584 bp in exon 3. Both LA2 and la2 had a CDS of 1152 bp encoding a polypeptide of 383 amino acids. Allele-specific markers for LA1, la1, LA2 and la2 co-segregated with the C18∶3 content in the F2 populations and will be useful for improving fatty acid composition through marker assisted selection in yellow mustard breeding. PMID:24823372

  6. Allelic differences between Europeans and Chinese for CREB1 SNPs and their implications in gene expression regulation, hippocampal structure and function, and bipolar disorder susceptibility.

    PubMed

    Li, M; Luo, X-J; Rietschel, M; Lewis, C M; Mattheisen, M; Müller-Myhsok, B; Jamain, S; Leboyer, M; Landén, M; Thompson, P M; Cichon, S; Nöthen, M M; Schulze, T G; Sullivan, P F; Bergen, S E; Donohoe, G; Morris, D W; Hargreaves, A; Gill, M; Corvin, A; Hultman, C; Toga, A W; Shi, L; Lin, Q; Shi, H; Gan, L; Meyer-Lindenberg, A; Czamara, D; Henry, C; Etain, B; Bis, J C; Ikram, M A; Fornage, M; Debette, S; Launer, L J; Seshadri, S; Erk, S; Walter, H; Heinz, A; Bellivier, F; Stein, J L; Medland, S E; Arias Vasquez, A; Hibar, D P; Franke, B; Martin, N G; Wright, M J; Su, B

    2014-04-01

    Bipolar disorder (BD) is a polygenic disorder that shares substantial genetic risk factors with major depressive disorder (MDD). Genetic analyses have reported numerous BD susceptibility genes, while some variants, such as single-nucleotide polymorphisms (SNPs) in CACNA1C have been successfully replicated, many others have not and subsequently their effects on the intermediate phenotypes cannot be verified. Here, we studied the MDD-related gene CREB1 in a set of independent BD sample groups of European ancestry (a total of 64,888 subjects) and identified multiple SNPs significantly associated with BD (the most significant being SNP rs6785[A], P=6.32 × 10(-5), odds ratio (OR)=1.090). Risk SNPs were then subjected to further analyses in healthy Europeans for intermediate phenotypes of BD, including hippocampal volume, hippocampal function and cognitive performance. Our results showed that the risk SNPs were significantly associated with hippocampal volume and hippocampal function, with the risk alleles showing a decreased hippocampal volume and diminished activation of the left hippocampus, adding further evidence for their involvement in BD susceptibility. We also found the risk SNPs were strongly associated with CREB1 expression in lymphoblastoid cells (P<0.005) and the prefrontal cortex (P<1.0 × 10(-6)). Remarkably, population genetic analysis indicated that CREB1 displayed striking differences in allele frequencies between continental populations, and the risk alleles were completely absent in East Asian populations. We demonstrated that the regional prevalence of the CREB1 risk alleles in Europeans is likely caused by genetic hitchhiking due to natural selection acting on a nearby gene. Our results suggest that differential population histories due to natural selection on regional populations may lead to genetic heterogeneity of susceptibility to complex diseases, such as BD, and explain inconsistencies in detecting the genetic markers of these diseases among

  7. The A Allele at rs13419896 of EPAS1 Is Associated with Enhanced Expression and Poor Prognosis for Non-Small Cell Lung Cancer

    PubMed Central

    Lee, Kian Leong; Yamane, Yuko; Gustine, Ewita; Isobe, Takeshi; Nishiyama, Masahiko; Hiyama, Keiko; Poellinger, Lorenz; Tanimoto, Keiji

    2015-01-01

    Hypoxia-inducible factor-2α (HIF-2α, or EPAS1) is important for cancer progression, and is a putative biomarker for poor prognosis for non-small cell lung cancer (NSCLC). However, molecular mechanisms underlying the EPAS1 overexpression are not still fully understood. We explored a role of a single nucleotide polymorphism (SNP), rs13419896 located within intron 1 of the EPAS1 gene in regulation of its expression. Bioinformatic analyses suggested that a region including the rs13419896 SNP plays a role in regulation of the EPAS1 gene expression and the SNP alters the binding activity of transcription factors. In vitro analyses demonstrated that a fragment containing the SNP locus function as a regulatory region and that a fragment with A allele showed higher transactivation activity than one with G, especially in the presence of overexpressed c-Fos or c-Jun. Moreover, NSCLC patients with the A allele showed poorer prognosis than those with G at the SNP even after adjustment with various variables. In conclusion, the genetic polymorphism of the EPAS1 gene may lead to variation of its gene expression levels to drive progression of the cancer and serve as a prognostic marker for NSCLC. PMID:26263511

  8. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons

    PubMed Central

    Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia

    2016-01-01

    The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson’s disease. PMID:26886559

  9. Tissue Specific Expression of Cre in Rat Tyrosine Hydroxylase and Dopamine Active Transporter-Positive Neurons.

    PubMed

    Liu, Zhenyi; Brown, Andrew; Fisher, Dan; Wu, Yumei; Warren, Joe; Cui, Xiaoxia

    2016-01-01

    The rat is a preferred model system over the mouse for neurological studies, and cell type-specific Cre expression in the rat enables precise ablation of gene function in neurons of interest, which is especially valuable for neurodegenerative disease modeling and optogenetics. Yet, few such Cre rats are available. Here we report the characterization of two Cre rats, tyrosine hydroxylase (TH)-Cre and dopamine active transporter (DAT or Slc6a3)-Cre, by using a combination of immunohistochemistry (IHC) and mRNA fluorescence in situ hybridization (FISH) as well as a fluorescent reporter for Cre activity. We detected Cre expression in expected neurons in both Cre lines. Interestingly, we also found that in Th-Cre rats, but not DAT-Cre rats, Cre is expressed in female germ cells, allowing germline excision of the floxed allele and hence the generation of whole-body knockout rats. In summary, our data demonstrate that targeted integration of Cre cassette lead to faithful recapitulation of expression pattern of the endogenous promoter, and mRNA FISH, in addition to IHC, is an effective method for the analysis of the spatiotemporal gene expression patterns in the rat brain, alleviating the dependence on high quality antibodies that are often not available against rat proteins. The Th-Cre and the DAT-Cre rat lines express Cre in selective subsets of dopaminergic neurons and should be particularly useful for researches on Parkinson's disease. PMID:26886559

  10. Strain-specific vulnerability to alcohol exposure in utero via hippocampal parent-of-origin expression of deiodinase-III

    PubMed Central

    Sittig, Laura J.; Shukla, Pradeep K.; Herzing, Laura B. K.; Redei, Eva E.

    2011-01-01

    Prenatal exposure to alcohol is thought to be the most prevalent nongenetic cause of a wide range of neurodevelopmental deficits. Insufficient thyroid hormone levels are one mechanism that hampers development of the alcohol-exposed brain, and we hypothesized that altered dosage of the imprinted thyroid hormone-inactivating gene deiodinase-III (Dio3) is responsible. To follow parent-of-origin allelic expression of Dio3 in the fetal and adult offspring of alcohol-consuming and control dams, we reciprocally crossed 2 polymorphic rat strains. In the frontal cortex, prenatal alcohol exposure altered imprinting patterns and total expression of Dio3 in the fetus and produced a permanent hypothyroid milieu in the adult. In the hippocampus, alcohol affected the paternal and total expression of Dio3 in the fetus and in the adult male, where thyroid hormone levels were concomitantly increased. Hippocampus-dependent behavioral deficits were identified exclusively in males, suggesting they are dependent on aberrant allelic Dio3 expression. None of these effects were observed in offspring of the reciprocal cross. Thus, genetic background and sex modify vulnerability to prenatal alcohol via brain region-specific expression of Dio3. This finding implies that phenotypic heterogeneity in human fetal alcohol spectrum disorder can be linked to genetic vulnerability in affected brain regions.—Sittig, L. J., Shukla, P. K., Herzing, L. B. K., Redei, E. E. Strain-specific vulnerability to alcohol exposure in utero via hippocampal parent-of-origin expression of deiodinase-III. PMID:21429942

  11. Allele-specific malE mutations that restore interactions between maltose-binding protein and the inner-membrane components of the maltose transport system.

    PubMed

    Treptow, N A; Shuman, H A

    1988-08-20

    Active accumulation of maltose and maltodextrins by Escherichia coli depends on an outer-membrane protein. LamB, a periplasmic maltose-binding protein (MalE, MBP) and three inner-membrane proteins, MalF, MalG and MalK. MalF and MalG are integral transmembrane proteins, while MalK is associated with the inner aspect of the cytoplasmic membrane via an interaction with MalG. Previously we have shown that MBP is essential for movement of maltose across the inner membrane. We have taken advantage of malF and malG mutants in which MBP interacts improperly with the membrane proteins. We describe the properties of malE mutations in which a proper interaction between MBP and defective MalF and MalG proteins has been restored. We found that these malE suppressor mutations are able to restore transport activity in an allele-specific manner. That is, a given malE mutation restores transport activity to different extents in different malF and malG mutants. Since both malF and malG mutations could be suppressed by allele-specific malE suppressors, we propose that, in wild-type bacteria, MBP interacts with sites on both MalF and MalG during active transport. The locations of different malE suppressor mutations indicate specific regions on MBP that are important for interacting with MalF and MalG. PMID:3050132

  12. Recognition of AvrBs3-Like Proteins Is Mediated by Specific Binding to Promoters of Matching Pepper Bs3 Alleles1[W

    PubMed Central

    Römer, Patrick; Strauss, Tina; Hahn, Simone; Scholze, Heidi; Morbitzer, Robert; Grau, Jan; Bonas, Ulla; Lahaye, Thomas

    2009-01-01

    The pepper (Capsicum annuum) bacterial spot (Bs) resistance gene Bs3 and its allelic variant Bs3-E mediate recognition of the Xanthomonas campestris pv vesicatoria type III effector protein AvrBs3 and its deletion derivative AvrBs3Δrep16. Recognition specificity resides in the Bs3 and Bs3-E promoters and is determined by a defined promoter region, the UPA (for up-regulated by AvrBs3) box. Using site-directed mutagenesis, we defined the exact boundaries of the UPAAvrBs3 box of the Bs3 promoter and the UPAAvrBs3Δrep16 box of the Bs3-E promoter and show that both boxes overlap by at least 11 nucleotides. Despite partial sequence identity, the UPAAvrBs3 box and the UPAAvrBs3Δrep16 box were bound specifically by the corresponding AvrBs3 and AvrBs3Δrep16 proteins, respectively, suggesting that selective promoter binding of AvrBs3-like proteins is the basis for promoter activation specificity. We also demonstrate that the UPAAvrBs3 box retains its functionality at different positions within the pepper Bs3 promoter and confers AvrBs3 inducibility in a novel promoter context. Notably, the transfer of the UPAAvrBs3 box to different promoter locations is always correlated with a new transcriptional start site. The analysis of naturally occurring Bs3 alleles revealed many pepper accessions that encode a nonfunctional Bs3 variant. These accessions showed no apparent abnormalities, supporting the supposition that Bs3 functions only in disease resistance and not in other developmental or physiological processes. PMID:19448036

  13. Repressor-mediated tissue-specific gene expression in plants

    DOEpatents

    Meagher, Richard B.; Balish, Rebecca S.; Tehryung, Kim; McKinney, Elizabeth C.

    2009-02-17

    Plant tissue specific gene expression by way of repressor-operator complexes, has enabled outcomes including, without limitation, male sterility and engineered plants having root-specific gene expression of relevant proteins to clean environmental pollutants from soil and water. A mercury hyperaccumulation strategy requires that mercuric ion reductase coding sequence is strongly expressed. The actin promoter vector, A2pot, engineered to contain bacterial lac operator sequences, directed strong expression in all plant vegetative organs and tissues. In contrast, the expression from the A2pot construct was restricted primarily to root tissues when a modified bacterial repressor (LacIn) was coexpressed from the light-regulated rubisco small subunit promoter in above-ground tissues. Also provided are analogous repressor operator complexes for selective expression in other plant tissues, for example, to produce male sterile plants.

  14. Surface expression, peptide repertoire, and thermostability of chicken class I molecules correlate with peptide transporter specificity.

    PubMed

    Tregaskes, Clive A; Harrison, Michael; Sowa, Anna K; van Hateren, Andy; Hunt, Lawrence G; Vainio, Olli; Kaufman, Jim

    2016-01-19

    The chicken major histocompatibility complex (MHC) has strong genetic associations with resistance and susceptibility to certain infectious pathogens. The cell surface expression level of MHC class I molecules varies as much as 10-fold between chicken haplotypes and is inversely correlated with diversity of peptide repertoire and with resistance to Marek's disease caused by an oncogenic herpesvirus. Here we show that the average thermostability of class I molecules isolated from cells also varies, being higher for high-expressing MHC haplotypes. However, we find roughly the same amount of class I protein synthesized by high- and low-expressing MHC haplotypes, with movement to the cell surface responsible for the difference in expression. Previous data show that chicken TAP genes have high allelic polymorphism, with peptide translocation specific for each MHC haplotype. Here we use assembly assays with peptide libraries to show that high-expressing B15 class I molecules can bind a much wider variety of peptides than are found on the cell surface, with the B15 TAPs restricting the peptides available. In contrast, the translocation specificity of TAPs from the low-expressing B21 haplotype is even more permissive than the promiscuous binding shown by the dominantly expressed class I molecule. B15/B21 heterozygote cells show much greater expression of B15 class I molecules than B15/B15 homozygote cells, presumably as a result of receiving additional peptides from the B21 TAPs. Thus, chicken MHC haplotypes vary in several correlated attributes, with the most obvious candidate linking all these properties being molecular interactions within the peptide-loading complex (PLC). PMID:26699458

  15. Surface expression, peptide repertoire, and thermostability of chicken class I molecules correlate with peptide transporter specificity

    PubMed Central

    Tregaskes, Clive A.; Harrison, Michael; Sowa, Anna K.; van Hateren, Andy; Hunt, Lawrence G.; Vainio, Olli; Kaufman, Jim

    2016-01-01

    The chicken major histocompatibility complex (MHC) has strong genetic associations with resistance and susceptibility to certain infectious pathogens. The cell surface expression level of MHC class I molecules varies as much as 10-fold between chicken haplotypes and is inversely correlated with diversity of peptide repertoire and with resistance to Marek’s disease caused by an oncogenic herpesvirus. Here we show that the average thermostability of class I molecules isolated from cells also varies, being higher for high-expressing MHC haplotypes. However, we find roughly the same amount of class I protein synthesized by high- and low-expressing MHC haplotypes, with movement to the cell surface responsible for the difference in expression. Previous data show that chicken TAP genes have high allelic polymorphism, with peptide translocation specific for each MHC haplotype. Here we use assembly assays with peptide libraries to show that high-expressing B15 class I molecules can bind a much wider variety of peptides than are found on the cell surface, with the B15 TAPs restricting the peptides available. In contrast, the translocation specificity of TAPs from the low-expressing B21 haplotype is even more permissive than the promiscuous binding shown by the dominantly expressed class I molecule. B15/B21 heterozygote cells show much greater expression of B15 class I molecules than B15/B15 homozygote cells, presumably as a result of receiving additional peptides from the B21 TAPs. Thus, chicken MHC haplotypes vary in several correlated attributes, with the most obvious candidate linking all these properties being molecular interactions within the peptide-loading complex (PLC). PMID:26699458

  16. Sequence variation at the rice blast resistance gene Pi-km locus: Implications for the development of allele specific markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The recently cloned blast resistance (R) gene Pi-km protects rice crops against specific races of the fungal pathogen Magnaporthe oryzae in a gene-for-gene manner. The use of blast R genes remains the most cost-effective method for an integrated disease management strategy. To facilitate rice breed...

  17. Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways1[OPEN

    PubMed Central

    Lu, Xunli; Dittgen, Jan; Piślewska-Bednarek, Mariola; Molina, Antonio; Schneider, Bernd; Doubský, Jan; Schneeberger, Korbinian; Schulze-Lefert, Paul

    2015-01-01

    Arabidopsis (Arabidopsis thaliana) PENETRATION (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-β-d-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes. PMID:26023163

  18. Expressive versus Receptive Language Skills in Specific Reading Disorder

    ERIC Educational Resources Information Center

    Stojanovik, Vesna; Riddell, Patricia

    2008-01-01

    Despite ample research into the language skills of children with specific reading disorder no studies so far have investigated whether there may be a difference between expressive and receptive language skills in this population. Yet, neuro-anatomical models would predict that children who have specific reading disorder which is not associated…

  19. Detection of EGFR Mutations by TaqMan Mutation Detection Assays Powered by Competitive Allele-Specific TaqMan PCR Technology

    PubMed Central

    Roma, Cristin; Esposito, Claudia; Rachiglio, Anna Maria; Pasquale, Raffaella; Chicchinelli, Nicoletta; Mancini, Rita; Pisconti, Salvatore; Botti, Gerardo; Morabito, Alessandro

    2013-01-01

    Epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are predictive of response to treatment with tyrosine kinase inhibitors. Competitive Allele-Specific TaqMan PCR (castPCR) is a highly sensitive and specific technology. EGFR mutations were assessed by TaqMan Mutation Detection Assays (TMDA) based on castPCR technology in 64 tumor samples: a training set of 30 NSCLC and 6 colorectal carcinoma (CRC) samples and a validation set of 28 NSCLC cases. The sensitivity and specificity of this method were compared with routine diagnostic techniques including direct sequencing and the EGFR Therascreen RGQ kit. Analysis of the training set allowed the identification of the threshold value for data analysis (0.2); the maximum cycle threshold (Ct = 37); and the cut-off ΔCt value (7) for the EGFR TMDA. By using these parameters, castPCR technology identified both training and validation set EGFR mutations with similar frequency as compared with the Therascreen kit. Sequencing detected rare mutations that are not identified by either castPCR or Therascreen, but in samples with low tumor cell content it failed to detect common mutations that were revealed by real-time PCR based methods. In conclusion, our data suggest that castPCR is highly sensitive and specific to detect EGFR mutations in NSCLC clinical samples. PMID:24364033

  20. Deletion endpoint allele-specificity in the developmentally regulated elimination of an internal sequence (IES) in Paramecium.

    PubMed

    Dubrana, K; Le Mouël, A; Amar, L

    1997-06-15

    Ciliated protozoa undergo thousands of site-specific DNA deletion events during the programmed development of micronuclear genomes to macronuclear genomes. Two deletion elements, W1 and W2, were identified in the Paramecium primaurelia wild-type 156 strain. Here, we report the characterization of both elements in wild-type strain 168 and show that they display variant deletion patterns when compared with those of strain 156. The W1 ( 168 ) element is defective for deletion. The W2 ( 168 ) element is excised utilizing two alternative boundaries on one side, both are different from the boundary utilized to excise the W2156 element. By crossing the 156 and 168 strains, we demonstrate that the definition of all deletion endpoints are each controlled by cis -acting determinant(s) rather than by strain-specific trans-acting factor(s). Sequence comparison of all deleted DNA segments indicates that the 5'-TA-3'terminal sequence is strictly required at their ends. Furthermore the identity of the first eight base pairs of these ends to a previously established consensus sequence correlates with the frequency of the corresponding deletion events. Our data implies the existence of an adaptive convergent evolution of these Paramecium deleted DNA segment end sequences. PMID:9171098

  1. Design of an F1 hybrid breeding strategy for ryegrasses based on selection of self-incompatibility locus-specific alleles

    PubMed Central

    Pembleton, Luke W.; Shinozuka, Hiroshi; Wang, Junping; Spangenberg, German C.; Forster, John W.; Cogan, Noel O. I.

    2015-01-01

    Relatively modest levels of genetic gain have been achieved in conventional ryegrass breeding when compared to cereal crops such as maize, current estimates indicating an annual improvement of 0.25–0.6% in dry matter production. This property is partially due to an inability to effectively exploit heterosis through the formation of F1 hybrids. Controlled crossing of ryegrass lines from geographically distant origins has demonstrated the occurrence of heterosis, which can result in increases of dry matter production in the order of 25%. Although capture of hybrid vigor offers obvious advantages for ryegrass cultivar production, to date there have been no effective and commercially suitable methods for obtaining high proportions of F1 hybrid seed. Continued advances in fine-scale genetic and physical mapping of the gametophytic self-incompatibility (SI) loci (S and Z) of ryegrasses are likely in the near future to permit the identification of closely linked genetic markers that define locus-specific haplotypes, allowing prediction of allelic variants and hence compatibility between different plant genotypes. Given the availability of such information, a strategy for efficient generation of ryegrass cultivars with a high proportion of F1 hybrid individuals has been simulated, which is suitable for commercial implementation. Through development of two parental pools with restricted diversity at the SI loci, relative crossing compatibility between pools is increased. Based on simulation of various levels of SI allele diversity restriction, the most effective scheme will generate 83.33% F1 hybrids. Results from the study, including the impact of varying flowering time, are discussed along with a proposed breeding design for commercial application. PMID:26442077

  2. Electromobility Shift Assay Reveals Evidence in Favor of Allele-Specific Binding of RUNX1 to the 5' Hypersensitive Site 4-Locus Control Region.

    PubMed

    Dehghani, Hossein; Ghobakhloo, Sepideh; Neishabury, Maryam

    2016-08-01

    In our previous studies on the Iranian β-thalassemia (β-thal) patients, we identified an association between the severity of the β-thal phenotype and the polymorphic palindromic site at the 5' hypersensitive site 4-locus control region (5'HS4-LCR) of the β-globin gene cluster. Furthermore, a linkage disequilibrium was observed between this region and XmnI-HBG2 in the patient population. Based on this data, it was suggested that the well-recognized phenotype-ameliorating role assigned to positive XmnI could be associated with its linked elements in the LCR. To investigate the functional significance of polymorphisms at the 5'HS4-LCR, we studied its influence on binding of transcription factors. Web-based predictions of transcription factor binding revealed a binding site for runt-related transcription factor 1 (RUNX1), when the allele at the center of the palindrome (TGGGG(A/G)CCCCA) was A but not when it was G. Furthermore, electromobility shift assay (EMSA) presented evidence in support of allele-specific binding of RUNX1 to 5'HS4. Considering that RUNX1 is a well-known regulator of hematopoiesis, these preliminary data suggest the importance of further studies to confirm this interaction and consequently investigate its functional and phenotypical relevance. These studies could help us to understand the molecular mechanism behind the phenotype modifying role of the 5'HS4-LCR polymorphic palindromic region (rs16912979), which has been observed in previous studies. PMID:27492765

  3. Detection of steroid 21-hydroxylase alleles using gene-specific PCR and a multiplexed ligation detection reaction

    SciTech Connect

    Day, D.J.; Barany, F.; Speiser, P.W.

    1995-09-01

    Steroid 21-hydroxylase deficiency is the most common cause of congenital adrenal hyperplasia, an inherited inability to synthesize cortisol that occurs in 1 in 10,000-15,000 births. Affected females are born with ambiguous genitalia, a condition that can be ameliorated by administering dexamethasone to the mother for most of gestation. Prenatal diagnosis is required for accurate treatment of affected females as well as for genetic counseling purposes. Approximately 95% of mutations causing this disorder result from recombinations between the gene encoding the 21-hydroxylase enzyme (CYP21) and a linked, highly homologous pseudogene (CYP21P). Approximately 20% of these mutations are gene deletions, and the remainder are gene conversions that transfer any of nine deleterious mutations from the CYP21P pseudogene to CYP21. We describe a methodology for genetic diagnosis of 21-hydroxylase deficiency that utilizes gene-specific PCR amplification in conjunction with thermostable DNA ligase to discriminate single nucleotide variations in a multiplexed ligation detection assay. The assay has been designed to be used with either fluorescent or radioactive detection of ligation products by electrophoresis on denaturing acrylamide gels and is readily adaptable for use in other disease systems. 30 refs., 5 figs.

  4. PPP1, a plant-specific regulator of transcription controls Arabidopsis development and PIN expression.

    PubMed

    Benjamins, René; Barbez, Elke; Ortbauer, Martina; Terpstra, Inez; Lucyshyn, Doris; Moulinier-Anzola, Jeanette; Khan, Muhammad Asaf; Leitner, Johannes; Malenica, Nenad; Butt, Haroon; Korbei, Barbara; Scheres, Ben; Kleine-Vehn, Jürgen; Luschnig, Christian

    2016-01-01

    Directional transport of auxin is essential for plant development, with PIN auxin transport proteins representing an integral part of the machinery that controls hormone distribution. However, unlike the rapidly emerging framework of molecular determinants regulating PIN protein abundance and subcellular localization, insights into mechanisms controlling PIN transcription are still limited. Here we describe PIN2 PROMOTER BINDING PROTEIN 1 (PPP1), an evolutionary conserved plant-specific DNA binding protein that acts on transcription of PIN genes. Consistent with PPP1 DNA-binding activity, PPP1 reporter proteins are nuclear localized and analysis of PPP1 null alleles and knockdown lines indicated a function as a positive regulator of PIN expression. Furthermore, we show that ppp1 pleiotropic mutant phenotypes are partially reverted by PIN overexpression, and results are presented that underline a role of PPP1-PIN promoter interaction in PIN expression control. Collectively, our findings identify an elementary, thus far unknown, plant-specific DNA-binding protein required for post-embryonic plant development, in general, and correct expression of PIN genes, in particular. PMID:27553690

  5. PPP1, a plant-specific regulator of transcription controls Arabidopsis development and PIN expression

    PubMed Central

    Benjamins, René; Barbez, Elke; Ortbauer, Martina; Terpstra, Inez; Lucyshyn, Doris; Moulinier-Anzola, Jeanette; Khan, Muhammad Asaf; Leitner, Johannes; Malenica, Nenad; Butt, Haroon; Korbei, Barbara; Scheres, Ben; Kleine-Vehn, Jürgen; Luschnig, Christian

    2016-01-01

    Directional transport of auxin is essential for plant development, with PIN auxin transport proteins representing an integral part of the machinery that controls hormone distribution. However, unlike the rapidly emerging framework of molecular determinants regulating PIN protein abundance and subcellular localization, insights into mechanisms controlling PIN transcription are still limited. Here we describe PIN2 PROMOTER BINDING PROTEIN 1 (PPP1), an evolutionary conserved plant-specific DNA binding protein that acts on transcription of PIN genes. Consistent with PPP1 DNA-binding activity, PPP1 reporter proteins are nuclear localized and analysis of PPP1 null alleles and knockdown lines indicated a function as a positive regulator of PIN expression. Furthermore, we show that ppp1 pleiotropic mutant phenotypes are partially reverted by PIN overexpression, and results are presented that underline a role of PPP1-PIN promoter interaction in PIN expression control. Collectively, our findings identify an elementary, thus far unknown, plant-specific DNA-binding protein required for post-embryonic plant development, in general, and correct expression of PIN genes, in particular. PMID:27553690

  6. Segment-specific regulation of epididymal gene expression.

    PubMed

    Sipilä, Petra; Björkgren, Ida

    2016-09-01

    The epididymis is necessary for post-testicular sperm maturation. During their epididymal transit, spermatozoa gain ability for progressive movement and fertilization. The epididymis is composed of several segments that have distinct gene expression profiles that enable the establishment of the changing luminal environment required for sperm maturation. The epididymal gene expression is regulated by endocrine, lumicrine, and paracrine factors in a segment-specific manner. Thus, in addition to its importance for male fertility, the epididymis is a valuable model tissue for studying the regulation of gene expression. This review concentrates on recent advances in understanding the androgen, small RNA, and epigenetically mediated regulation of segment-specific gene expression in the epididymis. PMID:27222594

  7. Laminin Mediates Tissue-specific Gene Expression in Mammary Epithelia

    SciTech Connect

    Streuli, Charles H; Schmidhauser, Christian; Bailey, Nina; Yurchenco, Peter; Skubitz, Amy P. N.; Roskelley, Calvin; Bissell, Mina J

    1995-04-01

    Tissue-specific gene expression in mammary epithelium is dependent on the extracellular matrix as well as hormones. There is good evidence that the basement membrane provides signals for regulating beta-casein expression, and that integrins are involved in this process. Here, we demonstrate that in the presence of lactogenic hormones, laminin can direct expression of the beta-casein gene. Mouse mammary epithelial cells plated on gels of native laminin or laminin-entactin undergo functional differentiation. On tissue culture plastic, mammary cells respond to soluble basement membrane or purified laminin, but not other extracellular matrix components, by synthesizing beta-casein. In mammary cells transfected with chloramphenicol acetyl transferase reporter constructs, laminin activates transcription from the beta-casein promoter through a specific enhancer element. The inductive effect of laminin on casein expression was specifically blocked by the E3 fragment of the carboxy terminal region of the alpha 1 chain of laminin, by antisera raised against the E3 fragment, and by a peptide corresponding to a sequence within this region. Our results demonstrate that laminin can direct tissue-specific gene expression in epithelial cells through its globular domain.

  8. Multiple Sclerosis Risk Allele in CLEC16A Acts as an Expression Quantitative Trait Locus for CLEC16A and SOCS1 in CD4+ T Cells

    PubMed Central

    Gustavsen, Marte W.; Bjølgerud, Anja; Brorson, Ina S.; Celius, Elisabeth G.; Spurkland, Anne; Bos, Steffan D.; Harbo, Hanne F.; Berge, Tone

    2015-01-01

    For multiple sclerosis, genome wide association studies and follow up studies have identified susceptibility single nucleotide polymorphisms located in or near CLEC16A at chromosome 16p13.13, encompassing among others CIITA, DEXI and SOCS1 in addition to CLEC16A. These genetic variants are located in intronic or intergenic regions and display strong linkage disequilibrium with each other, complicating the understanding of their functional contribution and the identification of the direct causal variant(s). Previous studies have shown that multiple sclerosis-associated risk variants in CLEC16A act as expression quantitative trait loci for CLEC16A itself in human pancreatic β-cells, for DEXI and SOCS1 in thymic tissue samples, and for DEXI in monocytes and lymphoblastoid cell lines. Since T cells are major players in multiple sclerosis pathogenesis, we have performed expression analyses of the CIITA-DEXI-CLEC16A-SOCS1 gene cluster in CD4+ and CD8+ T cells isolated from multiple sclerosis patients and healthy controls. We observed a higher expression of SOCS1 and CLEC16A in CD4+ T cells in samples homozygous for the risk allele of CLEC16A rs12927355. Pair-wise linear regression analysis revealed high correlation in gene expression in peripheral T cells of CIITA, DEXI, CLEC16A and SOCS1. Our data imply a possible regulatory role for the multiple sclerosis-associated rs12927355 in CLEC16A. PMID:26203907

  9. Chamber Specific Gene Expression Landscape of the Zebrafish Heart

    PubMed Central

    Singh, Angom Ramcharan; Sivadas, Ambily; Sabharwal, Ankit; Vellarikal, Shamsudheen Karuthedath; Jayarajan, Rijith; Verma, Ankit; Kapoor, Shruti; Joshi, Adita; Scaria, Vinod; Sivasubbu, Sridhar

    2016-01-01

    The organization of structure and function of cardiac chambers in vertebrates is defined by chamber-specific distinct gene expression. This peculiarity and uniqueness of the genetic signatures demonstrates functional resolution attributed to the different chambers of the heart. Altered expression of the cardiac chamber genes can lead to individual chamber related dysfunctions and disease patho-physiologies. Information on transcriptional repertoire of cardiac compartments is important to understand the spectrum of chamber specific anomalies. We have carried out a genome wide transcriptome profiling study of the three cardiac chambers in the zebrafish heart using RNA sequencing. We have captured the gene expression patterns of 13,396 protein coding genes in the three cardiac chambers—atrium, ventricle and bulbus arteriosus. Of these, 7,260 known protein coding genes are highly expressed (≥10 FPKM) in the zebrafish heart. Thus, this study represents nearly an all-inclusive information on the zebrafish cardiac transcriptome. In this study, a total of 96 differentially expressed genes across the three cardiac chambers in zebrafish were identified. The atrium, ventricle and bulbus arteriosus displayed 20, 32 and 44 uniquely expressing genes respectively. We validated the expression of predicted chamber-restricted genes using independent semi-quantitative and qualitative experimental techniques. In addition, we identified 23 putative novel protein coding genes that are specifically restricted to the ventricle and not in the atrium or bulbus arteriosus. In our knowledge, these 23 novel genes have either not been investigated in detail or are sparsely studied. The transcriptome identified in this study includes 68 differentially expressing zebrafish cardiac chamber genes that have a human ortholog. We also carried out spatiotemporal gene expression profiling of the 96 differentially expressed genes throughout the three cardiac chambers in 11 developmental stages and 6

  10. Detection of the BRAF V600E mutation in serous ovarian tumors: a comparative analysis of immunohistochemistry with a mutation-specific monoclonal antibody and allele-specific PCR.

    PubMed

    Bösmüller, Hans; Fischer, Anna; Pham, Deborah L; Fehm, Tanja; Capper, David; von Deimling, Andreas; Bonzheim, Irina; Staebler, Annette; Fend, Falko

    2013-03-01

    Mutations of components of the mitogen-activated protein kinase pathway, mainly BRAF, are common in serous ovarian borderline tumors, whereas high-grade serous ovarian carcinomas rarely show this feature. With the advent of specific kinase inhibitors active against BRAF-mutated cancers, rapid and sensitive detection of the BRAF V600E, by far the most common mutation of this gene, is of great practical relevance. Currently, BRAF mutations are detected by DNA-based techniques. Recently, a monoclonal antibody (VE1) specific for the BRAF V600E protein suitable for archival tissues has been described. In this study, we compared detection of the V600E mutation in serous ovarian tumors by VE1 immunostaining and by allele-specific polymerase chain reaction. All 141 cases of high-grade serous ovarian cancer showed negative or rarely weak, diffuse background VE1 immunostaining, and BRAF wild type was confirmed by molecular analysis in all tested cases. In contrast, 1 (14%) of 7 low-grade serous carcinomas and 22 (71%) of 31 serous borderline tumors revealed moderate to strong VE1 positivity. Immunostaining was clearly evaluable in all cases with sufficient tumor cells, and only rare cases with narrow cytoplasm were difficult to interpret. The V600E mutation was confirmed by allele-specific polymerase chain reaction and sequencing in all VE1-positive cases. Two VE1-positive cases with low epithelial cell content required repeat microdissection to confirm the presence of the mutation. Immunohistochemistry with the VE1 antibody is a specific and sensitive tool for detection of the BRAF V600E mutation in serous ovarian tumors and may provide a practical screening test, especially in tumor samples with low epithelial content. PMID:23089489

  11. Specificity of Facial Expression Labeling Deficits in Childhood Psychopathology

    ERIC Educational Resources Information Center

    Guyer, Amanda E.; McClure, Erin B.; Adler, Abby D.; Brotman, Melissa A.; Rich, Brendan A.; Kimes, Alane S.; Pine, Daniel S.; Ernst, Monique; Leibenluft, Ellen

    2007-01-01

    Background: We examined whether face-emotion labeling deficits are illness-specific or an epiphenomenon of generalized impairment in pediatric psychiatric disorders involving mood and behavioral dysregulation. Method: Two hundred fifty-two youths (7-18 years old) completed child and adult facial expression recognition subtests from the Diagnostic…

  12. Human testis expresses a specific poly(A)-binding protein.

    PubMed

    Féral, C; Guellaën, G; Pawlak, A

    2001-05-01

    In testis mRNA stability and translation initiation are extensively under the control of poly(A)-binding proteins (PABP). Here we have cloned a new human testis-specific PABP (PABP3) of 631 amino acids (70.1 kDa) with 92.5% identical residues to the ubiquitous PABP1. A northern blot of multiple human tissues hybridised with PABP3- and PABP1-specific oligonucleotide probes revealed two PABP3 mRNAs (2.1 and 2.5 kb) detected only in testis, whereas PABP1 mRNA (3.2 kb) was present in all tested tissues. In human adult testis, PABP3 mRNA expression was restricted to round spermatids, whereas PABP1 was expressed in these cells as well as in pachytene spermatocytes. PABP3-specific antibodies identified a protein of 70 kDa in human testis extracts. This protein binds poly(A) with a slightly lower affinity as compared to PABP1. The human PABP3 gene is intronless with a transcription start site 61 nt upstream from the initiation codon. A sequence of 256 bp upstream from the transcription start site drives the promoter activity of PABP3 and its tissue-specific expression. The expression of PABP3 might be a way to bypass PABP1 translational repression and to produce the amount of PABP needed for active mRNA translation in spermatids. PMID:11328870

  13. Disease-specific classification using deconvoluted whole blood gene expression.

    PubMed

    Wang, Li; Oh, William K; Zhu, Jun

    2016-01-01

    Blood-based biomarker assays have an advantage in being minimally invasive. Diagnostic and prognostic models built on peripheral blood gene expression have been reported for various types of disease. However, most of these studies focused on only one disease type, and failed to address whether the identified gene expression signature is disease-specific or more widely applicable across diseases. We conducted a meta-analysis of 46 whole blood gene expression datasets covering a wide range of diseases and physiological conditions. Our analysis uncovered a striking overlap of signature genes shared by multiple diseases, driven by an underlying common pattern of cell component change, specifically an increase in myeloid cells and decrease in lymphocytes. These observations reveal the necessity of building disease-specific classifiers that can distinguish different disease types as well as normal controls, and highlight the importance of cell component change in deriving blood gene expression based models. We developed a new strategy to develop blood-based disease-specific models by leveraging both cell component changes and cell molecular state changes, and demonstrate its superiority using independent datasets. PMID:27596246

  14. Disease-specific classification using deconvoluted whole blood gene expression

    PubMed Central

    Wang, Li; Oh, William K.; Zhu, Jun

    2016-01-01

    Blood-based biomarker assays have an advantage in being minimally invasive. Diagnostic and prognostic models built on peripheral blood gene expression have been reported for various types of disease. However, most of these studies focused on only one disease type, and failed to address whether the identified gene expression signature is disease-specific or more widely applicable across diseases. We conducted a meta-analysis of 46 whole blood gene expression datasets covering a wide range of diseases and physiological conditions. Our analysis uncovered a striking overlap of signature genes shared by multiple diseases, driven by an underlying common pattern of cell component change, specifically an increase in myeloid cells and decrease in lymphocytes. These observations reveal the necessity of building disease-specific classifiers that can distinguish different disease types as well as normal controls, and highlight the importance of cell component change in deriving blood gene expression based models. We developed a new strategy to develop blood-based disease-specific models by leveraging both cell component changes and cell molecular state changes, and demonstrate its superiority using independent datasets. PMID:27596246

  15. Chromosomal fragile site expression in dogs: I. Breed specific differences.

    PubMed

    Stone, D M; Jacky, P B; Hancock, D D; Prieur, D J

    1991-08-01

    Peripheral blood lymphocytes from clinically normal Doberman pinscher and boxer dogs were cultured for folate-sensitive and, in preliminary studies, aphidicolin-inducible fragile site expression. Both autosomal and X chromosomal fragile sites were observed in canine cells cultured under folate/thymidine depletion and in cells cultured in medium containing aphidicolin. Results from the three dogs evaluated for both folate-sensitive and aphidicolin-inducible fragile site expression showed that the frequency of fragile site expression was significantly (P less than 0.05) greater in cells cultured in medium containing aphidicolin than in cells cultured in folate/thymidine-depleted medium. Cells from the boxer dog expressed a high percentage (66.67%) of aphidicolin-inducible fragile sites in contrast to the Doberman pinscher dog in which only 21.10% of the lymphocytes expressed aphidicolin-inducible fragile sites. The frequencies of spontaneous and folate-sensitive fragile site expression did not vary significantly by breed of dog. Age of dog was significantly and positively correlated with frequency of folate-sensitive fragile site expression in dogs of the boxer breed, but not in dogs of the Doberman pinscher breed. The dog X chromosome expressed three folate-sensitive and aphidicolin-inducible fragile sites. The G-band location of these three fragile sites showed homology with three recognized constitutive common fragile sites on the human X chromosome: Xp22, Xq21, and Xq27.2. Two specific autosomal fragile sites were identified, one on the distal end of the long arm of chromosome 1 and one on the distal end of the long arm of chromosome 8. Other autosomal fragile sites were also apparent but could not be assigned reliably to specific chromosomes. PMID:1910263

  16. The environment exerts a greater influence than the transgene on the transcriptome of field-grown wheat expressing the Pm3b allele.

    PubMed

    Quijano, Carolina Diaz; Brunner, Susanne; Keller, Beat; Gruissem, Wilhelm; Sautter, Christof

    2015-02-01

    Wheat provides 20 % of the calories consumed worldwide. Powdery mildew infections of wheat can result in more than 30 % yield loss but it has been demonstrated that wheat overexpressing Pm3b, an allele of the R gene Pm3, has enhanced resistance against powdery mildew under field conditions. A gene expression profile study using GeneChip Wheat Genome Array and Fluidigm 96.96 Dynamic Arrays was performed to obtain insights into the mode of action of Pm3b and to elucidate the molecular basis of pleiotropic effects observed in three out of four independent transgenic events under field conditions. A cluster analysis of the microarray data and a principal component analysis of the Fluidigm 96.96 Dynamic Arrays data showed that transgenic lines and null segregants grouped together. The microarray analysis of samples from fungicide-treated plants revealed that significantly fewer genes were differentially expressed in Pm3b#1 than in Pm3b#2, which had a pleiotropic phenotype in the field, compared to their null segregants. Together, our data provide evidence that the environment influenced gene expression in the Pm3b lines more than the transgene itself. PMID:25095900

  17. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    DOE PAGESBeta

    Ying, Tianlei; Prabakaran, Ponraj; Du, Lanying; Shi, Wei; Feng, Yang; Wang, Yanping; Wang, Lingshu; Li, Wei; Jiang, Shibo; Dimitrov, Dimiter S.; et al

    2015-09-15

    The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (~36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of bindingmore » at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo.« less

  18. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    SciTech Connect

    Ying, Tianlei; Prabakaran, Ponraj; Du, Lanying; Shi, Wei; Feng, Yang; Wang, Yanping; Wang, Lingshu; Li, Wei; Jiang, Shibo; Dimitrov, Dimiter S.; Zhou, Tongqing

    2015-09-15

    The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (~36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of binding at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo.

  19. Detection of disease-specific restriction fragment length polymorphisms in pemphigus vulgaris linked to the DQwl and DQw3 alleles of the HLA-D region

    SciTech Connect

    Szafer, F.; Brautbar, C.; Tzfoni, E.; Frankel, G.; Sherman, L.; Cohen, I.; Hacham-Zadeh, S.; Aberer, W.; Tappeiner, G.; Holubar, K.; Steinman, L.

    1987-09-01

    Pemphigus vulgaris in Israeli Ashkenazi and non-Ashkenazi Jews and in Austrian non-Jewish patients is strongly associated with the DR4 and DRw6 alleles of the HLA-D region class II genes. Restriction fragment length polymorphism analysis was undertaken with DQ..beta.., DQ..cap alpha.., and DR..beta.. cDNA probes. Hybridization with the DQ..beta.. probe identifies Pvu II, BamHI, and EcoRV fragments that absolutely discriminate pemphigus vulgaris patients from healthy DR-, DQ-, and ethnic-matched controls. In contrast the DQ..cap alpha.. and DR..beta.. probes failed to identify disease-specific restriction fragment length polymorphism fragments. These studies indicate that DQw1 and DQw3 polymorphisms carried by pemphigus vulgaris patients may be directly involved in predisposition to the disease or may be tightly linked to the susceptibility gene itself. To our knowledge, this is the first example of an HLA restriction fragment length polymorphism that is highly associated with susceptibility to autoimmune disease.

  20. Rapid KRAS, EGFR, BRAF and PIK3CA Mutation Analysis of Fine Needle Aspirates from Non-Small-Cell Lung Cancer Using Allele-Specific qPCR

    PubMed Central

    Schrumpf, Melanie; Talebian Yazdi, Mehrdad; Ruano, Dina; Forte, Giusi I.; Nederlof, Petra M.; Veselic, Maud; Rabe, Klaus F.; Annema, Jouke T.; Smit, Vincent; Morreau, Hans; van Wezel, Tom

    2011-01-01

    Endobronchial Ultrasound Guided Transbronchial Needle Aspiration (EBUS-TBNA) and Trans-esophageal Ultrasound Scanning with Fine Needle Aspiration (EUS-FNA) are important, novel techniques for the diagnosis and staging of non-small cell lung cancer (NSCLC) that have been incorporated into lung cancer staging guidelines. To guide and optimize treatment decisions, especially for NSCLC patients in stage III and IV, EGFR and KRAS mutation status is often required. The concordance rate of the mutation analysis between these cytological aspirates and histological samples obtained by surgical staging is unknown. Therefore, we studied the extent to which allele-specific quantitative real-time PCR with hydrolysis probes could be reliably performed on EBUS and EUS fine needle aspirates by comparing the results with histological material from the same patient. We analyzed a series of 43 NSCLC patients for whom cytological and histological material was available. We demonstrated that these standard molecular techniques can be accurately applied on fine needle cytological aspirates from NSCLC patients. Importantly, we show that all mutations detected in the histological material of primary tumor were also identified in the cytological samples. We conclude that molecular profiling can be reliably performed on fine needle cytology aspirates from NSCLC patients. PMID:21408138

  1. Junctional and allele-specific residues are critical for MERS-CoV neutralization by an exceptionally potent germline-like antibody

    PubMed Central

    Ying, Tianlei; Prabakaran, Ponraj; Du, Lanying; Shi, Wei; Feng, Yang; Wang, Yanping; Wang, Lingshu; Li, Wei; Jiang, Shibo; Dimitrov, Dimiter S.; Zhou, Tongqing

    2015-01-01

    The MERS-CoV is an emerging virus, which already infected more than 1,300 humans with high (∼36%) mortality. Here, we show that m336, an exceptionally potent human anti-MERS-CoV antibody, is almost germline with only one somatic mutation in the heavy chain. The structure of Fab m336 in complex with the MERS-CoV receptor-binding domain reveals that its IGHV1-69-derived heavy chain provides more than 85% binding surface and that its epitope almost completely overlaps with the receptor-binding site. Analysis of antibodies from 69 healthy humans suggests an important role of the V(D)J recombination-generated junctional and allele-specific residues for achieving high affinity of binding at such low levels of somatic hypermutation. Our results also have important implications for development of vaccine immunogens based on the newly identified m336 epitope as well as for elucidation of mechanisms of neutralization by m336-like antibodies and their elicitation in vivo. PMID:26370782

  2. Apolipoprotein E Epsilon 4 Allele Interacts with Sex and Cognitive Status to Influence All-Cause and Cause-Specific Mortality Among US Older Adults

    PubMed Central

    Beydoun, May A.; Beydoun, Hind A.; Kaufman, Jay S.; An, Yang; Resnick, Susan M.; O'Brien, Richard; Ferrucci, Luigi; Zonderman, Alan B.

    2012-01-01

    Background Apolipoprotein E ε4 (ApoE4 carrier) status, sex and cognitive impairment may interact to affect all-cause and cause-specific mortality risk. Objectives To confirm associations of ApoE4 carrier status, sex and time-dependent cognitive status with mortality risk, and investigate these associations' joint effects in a cohort of community-dwelling US adults. Design & Setting Data from the Baltimore Longitudinal Study of Aging were used. Participants Of n=3,047 (First-visit Age:17–98y, 60.1% men), we selected a sample with complete genetic data and with ≥1 visit at age≥50y (n=1,461). Measurements Time-to-death from all, cardiovascular or non-cardiovascular causes. Results Survival probability was lower for ApoE4 carriers, particularly at oldest ages. Cox proportional hazards model for all-cause mortality yielded a hazard ratio (HR) for ApoE4 carrier vs. non-carriers of 1.31,95%CI:1.02–1.68. This association was also found for cardiovascular mortality. Time-dependent all-cause dementia (HR=1.73, 95%CI:1.33–2.26) and mild cognitive impairment (HR=1.95,95%CI:1.42–2.67) increased all-cause mortality risk, associations also detected for non-cardiovascular mortality. When individuals were free of cognitive impairment, a dose-response relationship with ε4 alleles was found for all-cause mortality (HR=1.40,95%CI:0.94–2.07 for 1 ε4, and HR=2.61; 95%CI:1.12–6.07 for 2 ε4). After Alzheimer's Disease-type (AD) dementia onset, carrying only 1 ε4 allele increased all-cause mortality risk by ~77% compared to non-carriers. ApoE4 carrier status increased all-cause mortality risk in men and interacted with time-dependent AD to increase the risk of this outcome (RERI=2.15; 95% CI:1.22–3.07). Conclusion We found that ApoE4 carrier status increased all-cause and cardiovascular mortality risks, while interacting with sex and time-dependent AD status to affect all-cause mortality. PMID:23581910

  3. Functional Characterization of a Full Length Pregnane X Receptor, Expression in vivo, and Identification of PXR Alleles in Zebrafish (Danio rerio)

    PubMed Central

    Bainy, Afonso C. D.; Kubota, Akira; Goldstone, Jared V.; Lille-Langøy, Roger; Karchner, Sibel I.; Celander, Malin C.; Hahn, Mark E.; Goksøyr, Anders

    2013-01-01

    The pregnane X receptor (PXR) (nuclear receptor NR1I2) is a ligand activated transcription factor, mediating responses to diverse xenobiotic and endogenous chemicals. The properties of PXR in fish are not fully understood. Here we report on cloning and characterization of full-length PXR of zebrafish, Danio rerio, and pxr expression in vivo. Initial efforts gave a cDNA encoding a 430 amino acid protein identified as zebrafish pxr by phylogenetic and synteny analysis. The sequence of the cloned Pxr DNA binding domain was highly conserved, with 74% identity to human PXR, while the ligand-binding domain (LBD) of the cloned sequence was only 44% identical to human PXR-LBD. Sequence variation among clones in the initial effort prompted sequencing of multiple clones from a single fish. There were two prominent variants, one sequence with S183, Y218 and H383 and the other with I183, C218 and N383, which we designate as alleles pxr*1 (nr1i2*1) and pxr*2 (nr1i2*2), respectively. In COS-7 cells co-transfected with a PXR-responsive reporter gene, the full-length Pxr*1 (the more common variant) was activated by known PXR agonists clotrimazole and pregnenolone 16α-carbonitrile but to a lesser extent than the full-length human PXR. Activation of full-length Pxr*1 was only 10% of that with the Pxr*1 LBD. Quantitative real time PCR analysis showed prominent expression of pxr in liver and eye, as well as brain and intestine of adult zebrafish. The pxr was expressed in heart and kidney at levels similar to that in intestine. The expression of pxr in liver was weakly induced by ligands for mammalian PXR or CAR (NR1I3). The results establish a foundation for PXR studies in this vertebrate model. PXR allelic variation and the differences between the full-length PXR and the LBD in reporter assays have implications for assessing the action of PXR ligands in zebrafish. PMID:24121122

  4. MAGEA10 gene expression in non-small cell lung cancer and A549 cells, and the affinity of epitopes with the complex of HLA-A(∗)0201 alleles.

    PubMed

    Wang, Likui; Xu, Yuefang; Luo, Cheng; Sun, Jian; Zhang, Jinlu; Lee, Ming-Wei; Bai, Aiping; Chen, Guanhua; Frenz, Christopher M; Li, Zhengguo; Huang, Wenlin

    2015-09-01

    MAGEA10, a cancer/testis antigens expressed in tumors but not in normal tissues with the exception of testis and placenta, represents an attractive target for cancer immunotherapy. However, suppressive cytoenvironment and requirement of specific HLA-alleles presentation frequently led to immunotherapy failure. In this study MAGEA10 was scarcely expressed in cancer patients, but enhanced by viili polysaccharides, which indicates a possibility of increasing epitopes presentation. Furthermore the correlation of gene expression with methylation, indicated by R(2) value for MAGEA10 that was 3 times higher than the value for other MAGE genes tested, provides an explanation of why MAGEA10 was highly inhibited, this is also seen by Kaplan-Meier analysis because MAGEA10 did not change the patients' lifespan. By using Molecular-Docking method, 3 MAGEA10 peptides were found binding to the groove position of HLA-A(∗)0210 as same as MAGEA4 peptide co-crystallized with HLA-A(∗)0210, which indicates that they could be promising for HLA-A(∗)0201 presentation in immunotherapy. PMID:26058806

  5. Allele-Specific Virulence Attenuation of the Pseudomonas syringae HopZ1a Type III Effector via the Arabidopsis ZAR1 Resistance Protein

    PubMed Central

    Lewis, Jennifer D.; Wu, Ronald

    2010-01-01

    Plant resistance (R) proteins provide a robust surveillance system to defend against potential pathogens. Despite their importance in plant innate immunity, relatively few of the ∼170 R proteins in Arabidopsis have well-characterized resistance specificity. In order to identify the R protein responsible for recognition of the Pseudomonas syringae type III secreted effector (T3SE) HopZ1a, we assembled an Arabidopsis R gene T–DNA Insertion Collection (ARTIC) from publicly available Arabidopsis thaliana insertion lines and screened it for plants lacking HopZ1a-induced immunity. This reverse genetic screen revealed that the Arabidopsis R protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1; At3g50950) is required for recognition of HopZ1a in Arabidopsis. ZAR1 belongs to the coiled-coil (CC) class of nucleotide binding site and leucine-rich repeat (NBS–LRR) containing R proteins; however, the ZAR1 CC domain phylogenetically clusters in a clade distinct from other related Arabidopsis R proteins. ZAR1–mediated immunity is independent of several genes required by other R protein signaling pathways, including NDR1 and RAR1, suggesting that ZAR1 possesses distinct signaling requirements. The closely-related T3SE protein, HopZ1b, is still recognized by zar1 Arabidopsis plants indicating that Arabidopsis has evolved at least two independent R proteins to recognize the HopZ T3SE family. Also, in Arabidopsis zar1 plants HopZ1a promotes P. syringae growth indicative of an ancestral virulence function for this T3SE prior to the evolution of recognition by the host resistance protein ZAR1. Our results demonstrate that the Arabidopsis resistance protein ZAR1 confers allele-specific recognition and virulence attenuation of the Pseudomonas syringae T3SE protein HopZ1a. PMID:20368970

  6. Human rotavirus strains bearing VP4 gene P[6] allele recovered from asymptomatic or symptomatic infections share similar, if not identical, VP4 neutralization specificities.

    PubMed

    Hoshino, Yasutaka; Jones, Ronald W; Ross, Jerri; Santos, Norma; Kapikian, Albert Z

    2003-11-10

    A rotavirus VP4 gene P[6] allele has been documented in a number of countries to be characteristically associated with an endemic predominantly asymptomatic infection in neonates in maternity hospital nurseries. The mechanisms underlying the endemicity and asymptomatic nature of such neonatal infections remain unknown. Rotavirus strains sharing this same P genotype, however, have more recently been recovered from an increasing number of symptomatic diarrheal episodes in infants and young children in various parts of the world. Previously, we have shown that an asymptomatic P[6] rotavirus neonatal infection is not associated with a unique VP7 (G) serotype but may occur in conjunction with various G types. Although amino acid sequence comparisons of the VP4 gene between selected "asymptomatic" and "symptomatic" P[6] rotavirus strains have been reported and yielded information concerning their VP4 genotypes, serotypic comparisons of the outer capsid spike protein VP4 of such viruses have not been studied systematically by two-way cross-neutralizations. We determined the VP4 neutralization specificities of four asymptomatic and four symptomatic P[6] strains: two each of asymptomatic and symptomatic strains by two-way tests, and two each of additional asymptomatic and symptomatic strains by one-way tests. Both asymptomatic and symptomatic P[6] strains were shown to bear similar, if not identical, VP4 neutralization specificities. Thus, P[6] rotavirus strains causing asymptomatic or symptomatic infections did not appear to belong to unique P (VP4) serotypes. In addition, a close VP4 serotypic relationship between human P[6] rotavirus strains and the porcine P[6] rotavirus Gottfried strain was confirmed. PMID:14599785

  7. Quantitative polymerase chain reaction analysis with allele-specific oligonucleotide primers for individual IgH VDJ regions to evaluate tumor burden in myeloma patients.

    PubMed

    Sata, Hiroshi; Shibayama, Hirohiko; Maeda, Ikuhiro; Habuchi, Yoko; Nakatani, Eiji; Fukushima, Kentaro; Fujita, Jiro; Ezoe, Sachiko; Tadokoro, Seiji; Maeda, Tetsuo; Mizuki, Masao; Kosugi, Satoru; Nakagawa, Masashi; Ueda, Shuji; Iida, Masato; Tokumine, Yukihiro; Azenishi, Yasuhiko; Mitsui, Hideki; Oritani, Kenji; Kanakura, Yuzuru

    2015-05-01

    Quantitative polymerase chain reaction (PCR) with patient-specific, allele-specific oligonucleotide (ASO) primers for individual immunoglobulin H VDJ region (ASO-PCR) amplification was performed using several sources of clinical material, including mRNA from peripheral blood cells (PBMNCs), whole bone marrow cells (BMMNCs), and the CD20+ CD38- B-cell population in bone marrow, as well as cell-free DNA from the sera of patients with multiple myeloma (MM). We designed the ASO primers and produced sufficient PCR fragments to evaluate tumor burden in 20 of 30 bone marrow samples at diagnosis. Polymerase chain reaction amplification efficiency depended on primer sequences because the production of ASO-PCR fragments did not correlate with serum M-protein levels. However, the ASO-PCR levels in BMMNCs showed statistically significant correlations with those in PBMNCs and CD20+ CD38- B-cells. The good association between the BMMNC and PBMNC data indicated that PBMNCs could be a suitable source for monitoring minimal residual disease (MRD). In the case of cell-free DNA, ASO-PCR levels showed a unique pattern and remained high even after treatment. Because the sequence information for each ASO-PCR product was identical to the original, the cell-free DNA might also be useful for evaluating MRD. Moreover, the ASO-PCR products were clearly detected in 17 of 22 mRNA samples from CD20+ CD38- populations, suggesting that MM clones might exist in relatively earlier stages of B cells than in plasma cells. Thus, ASO-PCR analysis using various clinical materials is useful for detecting MRD in MM patients as well as for clarifying MM pathogenesis. PMID:25591497

  8. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy

    PubMed Central

    Powell, Sara Kathleen; Rivera-Soto, Ricardo; Gray, Steven James

    2015-01-01

    Over the last five years, the number of clinical trials involving AAV (adeno-associated virus) and lentiviral vectors continue to increase by about 150 trials each year. For continued success, AAV and lentiviral expression cassettes need to be designed to meet each disease's specific needs. This review discusses how viral vector expression cassettes can be engineered with elements to enhance target specificity and increase transgene expression. The key differences relating to target specificity between ubiquitous and tissue-specific promoters are discussed, as well as how endogenous miRNAs and their target sequences have been used to restrict transgene expression. Specifically, relevant studies indicating how cis-acting elements such as introns, WPRE, polyadenylation signals, and the CMV enhancer are highlighted to show their utility for enhancing transgene expression in gene therapy applications. All discussion bears in mind that expression cassettes have space constraints. In conclusion, this review can serve as a menu of vector genome design elements and their cost in terms of space to thoughtfully engineer viral vectors for gene therapy. PMID:25636961

  9. Identification of alleles of carotenoid pathway genes important for zeaxanthin accumulation in potato tubers

    PubMed Central

    Uitdewilligen, Jan G. A. M. L.; Kloosterman, Bjorn A.; Hutten, Ronald C. B.; Visser, Richard G. F.; van Eck, Herman J.

    2010-01-01

    We have investigated the genetics and molecular biology of orange flesh colour in potato (Solanum tuberosum L.). To this end the natural diversity in three genes of the carotenoid pathway was assessed by SNP analyses. Association analysis was performed between SNP haplotypes and flesh colour phenotypes in diploid and tetraploid potato genotypes. We observed that among eleven beta-carotene hydroxylase 2 (Chy2) alleles only one dominant allele has a major effect, changing white into yellow flesh colour. In contrast, none of the lycopene epsilon cyclase (Lcye) alleles seemed to have a large effect on flesh colour. Analysis of zeaxanthin epoxidase (Zep) alleles showed that all (diploid) genotypes with orange tuber flesh were homozygous for one specific Zep allele. This Zep allele showed a reduced level of expression. The complete genomic sequence of the recessive Zep allele, including the promoter, was determined, and compared with the sequence of other Zep alleles. The most striking difference was the presence of a non-LTR retrotransposon sequence in intron 1 of the recessive Zep allele, which was absent in all other Zep alleles investigated. We hypothesise that the presence of this large sequence in intron 1 caused the lower expression level, resulting in reduced Zep activity and accumulation of zeaxanthin. Only genotypes combining presence of the dominant Chy2 allele with homozygosity for the recessive Zep allele produced orange-fleshed tubers that accumulated large amounts of zeaxanthin. Electronic supplementary material The online version of this article (doi:10.1007/s11103-010-9647-y) contains supplementary material, which is available to authorized users. PMID:20490894

  10. Expression and Genetic Activation of Cyclic Di-GMP-Specific Phosphodiesterases in Escherichia coli

    PubMed Central

    Reinders, Alberto; Hee, Chee-Seng; Ozaki, Shogo; Mazur, Adam; Boehm, Alex; Schirmer, Tilman

    2015-01-01

    ABSTRACT Intracellular levels of the bacterial second messenger cyclic di-GMP (c-di-GMP) are controlled by antagonistic activities of diguanylate cyclases and phosphodiesterases. The phosphodiesterase PdeH was identified as a key regulator of motility in Escherichia coli, while deletions of any of the other 12 genes encoding potential phosphodiesterases did not interfere with motility. To analyze the roles of E. coli phosphodiesterases, we demonstrated that most of these proteins are expressed under laboratory conditions. We next isolated suppressor mutations in six phosphodiesterase genes, which reinstate motility in the absence of PdeH by reducing cellular levels of c-di-GMP. Expression of all mutant alleles also led to a reduction of biofilm formation. Thus, all of these proteins are bona fide phosphodiesterases that are capable of interfering with different c-di-GMP-responsive output systems by affecting the global c-di-GMP pool. This argues that E. coli possesses several phosphodiesterases that are inactive under laboratory conditions because they lack appropriate input signals. Finally, one of these phosphodiesterases, PdeL, was studied in more detail. We demonstrated that this protein acts as a transcription factor to control its own expression. Motile suppressor alleles led to a strong increase of PdeL activity and elevated pdeL transcription, suggesting that enzymatic activity and transcriptional control are coupled. In agreement with this, we showed that overall cellular levels of c-di-GMP control pdeL transcription and that this control depends on PdeL itself. We thus propose that PdeL acts both as an enzyme and as a c-di-GMP sensor to couple transcriptional activity to the c-di-GMP status of the cell. IMPORTANCE Most bacteria possess multiple diguanylate cyclases and phosphodiesterases. Genetic studies have proposed that these enzymes show signaling specificity by contributing to distinct cellular processes without much cross talk. Thus, spatial

  11. The expression of one ankyrin pk2 allele of the WO prophage is correlated with the Wolbachia feminizing effect in isopods

    PubMed Central

    2012-01-01

    Background The maternally inherited α-Proteobacteria Wolbachia pipientis is an obligate endosymbiont of nematodes and arthropods, in which they induce a variety of reproductive alterations, including Cytoplasmic Incompatibility (CI) and feminization. The genome of the feminizing wVulC Wolbachia strain harboured by the isopod Armadillidium vulgare has been sequenced and is now at the final assembly step. It contains an unusually high number of ankyrin motif-containing genes, two of which are homologous to the phage-related pk1 and pk2 genes thought to contribute to the CI phenotype in Culex pipiens. These genes encode putative bacterial effectors mediating Wolbachia-host protein-protein interactions via their ankyrin motifs. Results To test whether these Wolbachia homologs are potentially involved in altering terrestrial isopod reproduction, we determined the distribution and expression of both pk1 and pk2 genes in the 3 Wolbachia strains that induce CI and in 5 inducing feminization of their isopod hosts. Aside from the genes being highly conserved, we found a substantial copy number variation among strains, and that is linked to prophage diversity. Transcriptional analyses revealed expression of one pk2 allele (pk2b2) only in the feminizing Wolbachia strains of isopods. Conclusions These results reveal the need to investigate the functions of Wolbachia ankyrin gene products, in particular those of Pk2, and their host targets with respect to host sex manipulation. PMID:22497736

  12. HMG CoA Lyase (HL): Mutation detection and development of a bacterial expression system for screening the activity of mutant alleles from HL-deficient patients

    SciTech Connect

    Robert, M.F.; Ashmarina, L.; Poitier, E.

    1994-09-01

    HL catalyzes the last step of ketogenesis, and autosomal recessive HL deficiency in humans can cause episodes of hypoglycemia and coma. Structurally, HL is a dimer of identical 325-residue peptides which requires a reducing environment to maintain activity. We cloned the human and mouse HL cDNAs and genes and have performed mutation analysis on cells from 30 HL-deficient probands. Using SSCP and also genomic Southern analysis we have identified putative mutations on 53/60 alleles of these patients (88%). To date, we have found 20 mutations: 3 large deletions, 4 termination mutations, 5 frameshift mutations, and 8 missense mutations which we suspect to be pathogenic based on evolutionary conservation and/or our previous studies on purified HL protein. We have also identified 3 polymorphic variants. In order to directly test the activity of the missense mutations, we established a pGEX-based system, using a glutathione S transferase (GST)-HL fusion protein. Expressed wild-type GST-HL was insoluble. We previously located a reactive Cys at the C-terminus of chicken HL which is conserved in human HL. We produced a mutant HL peptide, C323S, which replaced Cys323 with Ser. Purified C323S is soluble and has similar kinetics to wild-type HL. C323S-containing GST-HL is soluble and enzymatically active. We are cloning and expressing the 8 missense mutations.

  13. The KMO allele encoding Arg452 is associated with psychotic features in bipolar disorder type 1, and with increased CSF KYNA level and reduced KMO expression.

    PubMed

    Lavebratt, C; Olsson, S; Backlund, L; Frisén, L; Sellgren, C; Priebe, L; Nikamo, P; Träskman-Bendz, L; Cichon, S; Vawter, M P; Osby, U; Engberg, G; Landén, M; Erhardt, S; Schalling, M

    2014-03-01

    The kynurenine pathway metabolite kynurenic acid (KYNA), modulating glutamatergic and cholinergic neurotransmission, is increased in cerebrospinal fluid (CSF) of patients with schizophrenia or bipolar disorder type 1 with psychotic features. KYNA production is critically dependent on kynurenine 3-monooxygenase (KMO). KMO mRNA levels and activity in prefrontal cortex (PFC) are reduced in schizophrenia. We hypothesized that KMO expression in PFC would be reduced in bipolar disorder with psychotic features and that a functional genetic variant of KMO would associate with this disease, CSF KYNA level and KMO expression. KMO mRNA levels were reduced in PFC of bipolar disorder patients with lifetime psychotic features (P=0.005, n=19) or schizophrenia (P=0.02, n=36) compared with nonpsychotic patients and controls. KMO genetic association to psychotic features in bipolar disorder type 1 was studied in 493 patients and 1044 controls from Sweden. The KMO Arg(452) allele was associated with psychotic features during manic episodes (P=0.003). KMO Arg(452) was studied for association to CSF KYNA levels in an independent sample of 55 Swedish patients, and to KMO expression in 717 lymphoblastoid cell lines and 138 hippocampal biopsies. KMO Arg(452) associated with increased levels of CSF KYNA (P=0.03) and reduced lymphoblastoid and hippocampal KMO expression (P≤0.05). Thus, findings from five independent cohorts suggest that genetic variation in KMO influences the risk for psychotic features in mania of bipolar disorder patients. This provides a possible mechanism for the previous findings of elevated CSF KYNA levels in those bipolar patients with lifetime psychotic features and positive association between KYNA levels and number of manic episodes. PMID:23459468

  14. A risk haplotype of STAT4 for systemic lupus erythematosus is over-expressed, correlates with anti-dsDNA and shows additive effects with two risk alleles of IRF5

    PubMed Central

    Sigurdsson, Snaevar; Nordmark, Gunnel; Garnier, Sophie; Grundberg, Elin; Kwan, Tony; Nilsson, Olof; Eloranta, Maija-Leena; Gunnarsson, Iva; Svenungsson, Elisabet; Sturfelt, Gunnar; Bengtsson, Anders A.; Jönsen, Andreas; Truedsson, Lennart; Rantapää-Dahlqvist, Solbritt; Eriksson, Catharina; Alm, Gunnar; Göring, Harald H.H.; Pastinen, Tomi; Syvänen, Ann-Christine; Rönnblom, Lars

    2008-01-01

    Systemic lupus erythematosus (SLE) is the prototype autoimmune disease where genes regulated by type I interferon (IFN) are over-expressed and contribute to the disease pathogenesis. Because signal transducer and activator of transcription 4 (STAT4) plays a key role in the type I IFN receptor signaling, we performed a candidate gene study of a comprehensive set of single nucleotide polymorphism (SNPs) in STAT4 in Swedish patients with SLE. We found that 10 out of 53 analyzed SNPs in STAT4 were associated with SLE, with the strongest signal of association (P = 7.1 × 10−8) for two perfectly linked SNPs rs10181656 and rs7582694. The risk alleles of these 10 SNPs form a common risk haplotype for SLE (P = 1.7 × 10−5). According to conditional logistic regression analysis the SNP rs10181656 or rs7582694 accounts for all of the observed association signal. By quantitative analysis of the allelic expression of STAT4 we found that the risk allele of STAT4 was over-expressed in primary human cells of mesenchymal origin, but not in B-cells, and that the risk allele of STAT4 was over-expressed (P = 8.4 × 10−5) in cells carrying the risk haplotype for SLE compared with cells with a non-risk haplotype. The risk allele of the SNP rs7582694 in STAT4 correlated to production of anti-dsDNA (double-stranded DNA) antibodies and displayed a multiplicatively increased, 1.82-fold risk of SLE with two independent risk alleles of the IRF5 (interferon regulatory factor 5) gene. PMID:18579578

  15. Expression Cloning of Camelid Nanobodies Specific for Xenopus Embryonic Antigens

    PubMed Central

    Itoh, Keiji; Sokol, Sergei Y.

    2014-01-01

    Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology. PMID:25285446

  16. A Screen for Modifiers of Cilia Phenotypes Reveals Novel MKS Alleles and Uncovers a Specific Genetic Interaction between osm-3 and nphp-4.

    PubMed

    Masyukova, Svetlana V; Landis, Dawn E; Henke, Scott J; Williams, Corey L; Pieczynski, Jay N; Roszczynialski, Kelly N; Covington, Jannese E; Malarkey, Erik B; Yoder, Bradley K

    2016-02-01

    Nephronophthisis (NPHP) is a ciliopathy in which genetic modifiers may underlie the variable penetrance of clinical features. To identify modifiers, a screen was conducted on C. elegans nphp-4(tm925) mutants. Mutations in ten loci exacerbating nphp-4(tm925) ciliary defects were obtained. Four loci have been identified, three of which are established ciliopathy genes mks-1, mks-2, and mks-5. The fourth allele (yhw66) is a missense mutation (S316F) in OSM-3, a kinesin required for cilia distal segment assembly. While osm-3(yhw66) mutants alone have no overt cilia phenotype, nphp-4(tm925);osm-3(yhw66) double mutants lack distal segments and are dye-filling (Dyf) and osmotic avoidance (Osm) defective, similar to osm-3(mn357) null mutants. In osm-3(yhw66) mutants anterograde intraflagellar transport (IFT) velocity is reduced. Furthermore, expression of OSM-3(S316F)::GFP reduced IFT velocities in nphp-4(tm925) mutants, but not in wild type animals. In silico analysis indicates the S316F mutation may affect a phosphorylation site. Putative phospho-null OSM-3(S316F) and phospho-mimetic OSM-3(S316D) proteins accumulate at the cilia base and tip respectively. FRAP analysis indicates that the cilia entry rate of OSM-3(S316F) is slower than OSM-3 and that in the presence of OSM-3(S316F), OSM-3 and OSM-3(S316D) rates decrease. In the presence OSM-3::GFP or OSM-3(S316D)::GFP, OSM-3(S316F)::tdTomato redistributes along the cilium and accumulates in the cilia tip. OSM-3(S316F) and OSM-3(S316D) are functional as they restore cilia distal segment formation in osm-3(mn357) null mutants; however, only OSM-3(S316F) rescues the osm-3(mn357) null Dyf phenotype. Despite rescue of cilia length in osm-3(mn357) null mutants, neither OSM-3(S316F) nor OSM-3(S316D) restores ciliary defects in nphp-4(tm925);osm-3(yhw66) double mutants. Thus, these OSM-3 mutations cause NPHP-4 dependent and independent phenotypes. These data indicate that in addition to regulating cilia protein entry or exit

  17. A Screen for Modifiers of Cilia Phenotypes Reveals Novel MKS Alleles and Uncovers a Specific Genetic Interaction between osm-3 and nphp-4

    PubMed Central

    Williams, Corey L.; Pieczynski, Jay N.; Roszczynialski, Kelly N.; Covington, Jannese E.; Malarkey, Erik B.; Yoder, Bradley K.

    2016-01-01

    Nephronophthisis (NPHP) is a ciliopathy in which genetic modifiers may underlie the variable penetrance of clinical features. To identify modifiers, a screen was conducted on C. elegans nphp-4(tm925) mutants. Mutations in ten loci exacerbating nphp-4(tm925) ciliary defects were obtained. Four loci have been identified, three of which are established ciliopathy genes mks-1, mks-2, and mks-5. The fourth allele (yhw66) is a missense mutation (S316F) in OSM-3, a kinesin required for cilia distal segment assembly. While osm-3(yhw66) mutants alone have no overt cilia phenotype, nphp-4(tm925);osm-3(yhw66) double mutants lack distal segments and are dye-filling (Dyf) and osmotic avoidance (Osm) defective, similar to osm-3(mn357) null mutants. In osm-3(yhw66) mutants anterograde intraflagellar transport (IFT) velocity is reduced. Furthermore, expression of OSM-3(S316F)::GFP reduced IFT velocities in nphp-4(tm925) mutants, but not in wild type animals. In silico analysis indicates the S316F mutation may affect a phosphorylation site. Putative phospho-null OSM-3(S316F) and phospho-mimetic OSM-3(S316D) proteins accumulate at the cilia base and tip respectively. FRAP analysis indicates that the cilia entry rate of OSM-3(S316F) is slower than OSM-3 and that in the presence of OSM-3(S316F), OSM-3 and OSM-3(S316D) rates decrease. In the presence OSM-3::GFP or OSM-3(S316D)::GFP, OSM-3(S316F)::tdTomato redistributes along the cilium and accumulates in the cilia tip. OSM-3(S316F) and OSM-3(S316D) are functional as they restore cilia distal segment formation in osm-3(mn357) null mutants; however, only OSM-3(S316F) rescues the osm-3(mn357) null Dyf phenotype. Despite rescue of cilia length in osm-3(mn357) null mutants, neither OSM-3(S316F) nor OSM-3(S316D) restores ciliary defects in nphp-4(tm925);osm-3(yhw66) double mutants. Thus, these OSM-3 mutations cause NPHP-4 dependent and independent phenotypes. These data indicate that in addition to regulating cilia protein entry or exit

  18. Differential Expression of Specific Dermatan Sulfate Domains in Renal Pathology

    PubMed Central

    Lensen, Joost F. M.; van der Vlag, Johan; Versteeg, Elly M. M.; Wetzels, Jack F. M.; van den Heuvel, Lambert P. W. J.; Berden, Jo H. M.; van Kuppevelt, Toin H.; Rops, Angelique L. W. M. M.

    2015-01-01

    Dermatan sulfate (DS), also known as chondroitin sulfate (CS)-B, is a member of the linear polysaccharides called glycosaminoglycans (GAGs). The expression of CS/DS and DS proteoglycans is increased in several fibrotic renal diseases, including interstitial fibrosis, diabetic nephropathy, mesangial sclerosis and nephrosclerosis. Little, however, is known about structural alterations in DS in renal diseases. The aim of this study was to evaluate the renal expression of two different DS domains in renal transplant rejection and glomerular pathologies. DS expression was evaluated in normal renal tissue and in kidney biopsies obtained from patients with acute interstitial or vascular renal allograft rejection, patients with interstitial fibrosis and tubular atrophy (IF/TA), and from patients with focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy (MGP) or systemic lupus erythematosus (SLE), using our unique specific anti-DS antibodies LKN1 and GD3A12. Expression of the 4/2,4-di-O-sulfated DS domain recognized by antibody LKN1 was decreased in the interstitium of transplant kidneys with IF/TA, which was accompanied by an increased expression of type I collagen, decorin and transforming growth factor beta (TGF-β), while its expression was increased in the interstitium in FSGS, MGP and SLE. Importantly, all patients showed glomerular LKN1 staining in contrast to the controls. Expression of the IdoA-Gal-NAc4SDS domain recognized by GD3A12 was similar in controls and patients. Our data suggest a role for the DS domain recognized by antibody LKN1 in renal diseases with early fibrosis. Further research is required to delineate the exact role of different DS domains in renal fibrosis. PMID:26322947

  19. Gene expression changes during retinal development and rod specification

    PubMed Central

    Carrigan, Matthew; Hokamp, Karsten; Farrar, G. Jane

    2015-01-01

    Purpose Retinitis pigmentosa (RP) typically results from individual mutations in any one of >70 genes that cause rod photoreceptor cells to degenerate prematurely, eventually resulting in blindness. Gene therapies targeting individual RP genes have shown efficacy at clinical trial; however, these therapies require the surviving photoreceptor cells to be viable and functional, and may be economically feasible for only the more commonly mutated genes. An alternative potential treatment strategy, particularly for late stage disease, may involve stem cell transplants into the photoreceptor layer of the retina. Rod progenitors from postnatal mouse retinas can be transplanted and can form photoreceptors in recipient adult retinas; optimal numbers of transplantable cells are obtained from postnatal day 3–5 (P3–5) retinas. These cells can also be expanded in culture; however, this results in the loss of photoreceptor potential. Gene expression differences between postnatal retinas, cultured retinal progenitor cells (RPCs), and rod photoreceptor precursors were investigated to identify gene expression patterns involved in the specification of rod photoreceptors. Methods Microarrays were used to investigate differences in gene expression between cultured RPCs that have lost photoreceptor potential, P1 retinas, and fresh P5 retinas that contain significant numbers of transplantable photoreceptors. Additionally, fluorescence-activated cell sorting (FACS) sorted Rho-eGFP-expressing rod photoreceptor precursors were compared with Rho-eGFP-negative cells from the same P5 retinas. Differential expression was confirmed with quantitative polymerase chain reaction (q-PCR). Results Analysis of the microarray data sets, including the use of t-distributed stochastic neighbor embedding (t-SNE) to identify expression pattern neighbors of key photoreceptor specific genes, resulted in the identification of 636 genes differentially regulated during rod specification. Forty-four of these

  20. A Limousin specific myostatin allele affects longissimus muscle area and fatty acid profiles in a Wagyu-Limousin F2 population.

    PubMed

    Alexander, L J; Kuehn, L A; Smith, T P L; Matukumalli, L K; Mote, B; Koltes, J E; Reecy, J; Geary, T W; Rule, D C; Macneil, M D

    2009-05-01

    A microsatellite-based genome scan of a Wagyu x Limousin F(2) cross population previously demonstrated QTL affecting LM area and fatty acid composition were present in regions near the centromere of BTA2. In this study, we used 70 SNP markers to examine the centromeric 24 megabases (Mb) of BTA2, including the Limousin-specific F94L myostatin allele (AB076403.1; 415C > A) located at approximately 6 Mb on the draft genome sequence of BTA2. A significant effect of the F94L marker was observed (F = 60.17) for LM area, which indicated that myostatin is most likely responsible for the effect. This is consistent with previous reports that the substitution of Leu for Phe at AA 94 of myostatin (caused by the 415C > A transversion) is associated with increased muscle growth. Surprisingly, several fatty acid trait QTL, which affected the amount of unsaturated fats, also mapped to or very near the myostatin marker, including the ratio of C16:1 MUFA to C16:0 saturated fat (F = 16.72), C18:1 to C18:0 (F = 18.88), and total content of MUFA (F = 17.12). In addition, QTL for extent of marbling (F = 14.73) approached significance (P = 0.05), and CLA concentration (F = 9.22) was marginally significant (P = 0.18). We also observed associations of SNP located at 16.3 Mb with KPH (F = 15.00) and for the amount of SFA (F = 12.01). These results provide insight into genetic differences between the Wagyu and Limousin breeds and may lead to a better tasting and healthier product for consumers through improved selection for lipid content of beef. PMID:19213716

  1. Brief communication: Evolution of a specific O allele (O1vG542A) supports unique ancestry of Native Americans.

    PubMed

    Villanea, Fernando A; Bolnick, Deborah A; Monroe, Cara; Worl, Rosita; Cambra, Rosemary; Leventhal, Alan; Kemp, Brian M

    2013-08-01

    In this study, we explore the geographic and temporal distribution of a unique variant of the O blood group allele called O1v(G542A) , which has been shown to be shared among Native Americans but is rare in other populations. O1v(G542A) was previously reported in Native American populations in Mesoamerica and South America, and has been proposed as an ancestry informative marker. We investigated whether this allele is also found in the Tlingit and Haida, two contemporary indigenous populations from Alaska, and a pre-Columbian population from California. If O1v(G542A) is present in Na-Dene speakers (i.e., Tlingits), it would indicate that Na-Dene speaking groups share close ancestry with other Native American groups and support a Beringian origin of the allele, consistent with the Beringian Incubation Model. If O1v(G542A) is found in pre-Columbian populations, it would further support a Beringian origin of the allele, rather than a more recent introduction of the allele into the Americas via gene flow from one or more populations which have admixed with Native Americans over the past five centuries. We identified this allele in one Na-Dene population at a frequency of 0.11, and one ancient California population at a frequency of 0.20. Our results support a Beringian origin of O1v(G542A) , which is distributed today among all Native American groups that have been genotyped in appreciable numbers at this locus. This result is consistent with the hypothesis that Na-Dene and other Native American populations primarily derive their ancestry from a single source population. PMID:23868176

  2. The expression pattern of small nucleolar and small Cajal body-specific RNAs characterizes distinct molecular subtypes of multiple myeloma

    PubMed Central

    Ronchetti, D; Todoerti, K; Tuana, G; Agnelli, L; Mosca, L; Lionetti, M; Fabris, S; Colapietro, P; Miozzo, M; Ferrarini, M; Tassone, P; Neri, A

    2012-01-01

    Small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs) are non-coding RNAs involved in the maturation of other RNA molecules and generally located in the introns of host genes. It is now emerging that altered sno/scaRNAs expression may have a pathological role in cancer. This study elucidates the patterns of sno/scaRNAs expression in multiple myeloma (MM) by profiling purified malignant plasma cells from 55 MMs, 8 secondary plasma cell leukemias (sPCLs) and 4 normal controls. Overall, a global sno/scaRNAs downregulation was found in MMs and, even more, in sPCLs compared with normal plasma cells. Whereas SCARNA22 resulted the only sno/scaRNA characterizing the translocation/cyclin D4 (TC4) MM, TC2 group displayed a distinct sno/scaRNA signature overexpressing members of SNORD115 and SNORD116 families located in a region finely regulated by an imprinting center at 15q11, which, however, resulted overall hypomethylated in MMs independently of the SNORD115 and SNORD116 expression levels. Finally, integrative analyses with available gene expression and genome-wide data revealed the occurrence of significant sno/scaRNAs/host genes co-expression and the putative influence of allelic imbalances on specific snoRNAs expression. Our data extend the current view of sno/scaRNAs deregulation in cancer and add novel information to the bio-molecular complexity of plasma cell dyscrasias. PMID:23178508

  3. Cell-type specific expression of a dominant negative PKA mutation in mice.

    PubMed

    Willis, Brandon S; Niswender, Colleen M; Su, Thomas; Amieux, Paul S; McKnight, G Stanley

    2011-01-01

    We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology. PMID:21533282

  4. Modulated Expression of Specific tRNAs Drives Gene Expression and Cancer Progression.

    PubMed

    Goodarzi, Hani; Nguyen, Hoang C B; Zhang, Steven; Dill, Brian D; Molina, Henrik; Tavazoie, Sohail F

    2016-06-01

    Transfer RNAs (tRNAs) are primarily viewed as static contributors to gene expression. By developing a high-throughput tRNA profiling method, we find that specific tRNAs are upregulated in human breast cancer cells as they gain metastatic activity. Through loss-of-function, gain-of-function, and clinical-association studies, we implicate tRNAGluUUC and tRNAArgCCG as promoters of breast cancer metastasis. Upregulation of these tRNAs enhances stability and ribosome occupancy of transcripts enriched for their cognate codons. Specifically, tRNAGluUUC promotes metastatic progression by directly enhancing EXOSC2 expression and enhancing GRIPAP1-constituting an "inducible" pathway driven by a tRNA. The cellular proteomic shift toward a pro-metastatic state mirrors global tRNA shifts, allowing for cell-state and cell-type transgene expression optimization through codon content quantification. TRNA modulation represents a mechanism by which cells achieve altered expression of specific transcripts and proteins. TRNAs are thus dynamic regulators of gene expression and the tRNA codon landscape can causally and specifically impact disease progression. PMID:27259150

  5. Rosa26 Locus Supports Tissue-Specific Promoter Driving Transgene Expression Specifically in Pig

    PubMed Central

    Ma, Jing; Huang, Tianqing; Jiang, Dandan; Xie, Bingteng; Wu, Meiling; Wang, Jiaqiang; Song, Yuran; Wang, Ying; He, Yilong; Sun, Jialu; Hu, Kui; Guo, Runfa; Wang, Liu; Zhou, Qi; Mu, Yanshuang; Liu, Zhonghua

    2014-01-01

    Genetically modified pigs have become a popular model system in fundamental research, agricultural and biomedical applications. However, random integration often result in unstable expression of transgene and unpredictable phenotypes. The Rosa26 locus has been widely used to produce genetic modified animals with high and consistent expressing of transgene in mouse, human and rat, as it can be targeted efficiently and is not subject to gene-silencing effects. Recently, the first case of reporter gene targeting pigs in porcine Rosa26 (pRosa26) locus was reported. In the study, full sequence of pRosa26 locus was further characterized, and the pRosa26 promoter (pR26) was cloned and we evidenced that the new porcine endogenous promoter is suitable for driving transgene expression in a high and stable manner by avoiding DNA methylation. Furthermore, elongation factor 1a promoter (EF1a) -driven GFP reporter and Myostatin promoter (MyoP)-driven Follistatin (Fst) were successfully targeted into the pRosa26 locusby traditional homologous recombination (HR) strategy. EF1a showed high activity and hypomethylation at the locus. And, muscle-specific promoter MyoP was activated strictly in muscle of the pRosa26 targeted pigs, indicating Rosa26 locus supports tissue-specific promoter driving transgene expression in its own manner. The study provided further demonstration on biomedical and agricultural applications of porcine Rosa26 promoter and locus. PMID:25232950

  6. Extensive Allelic Diversity of MHC Class I in Wild Mallard Ducks.

    PubMed

    Fleming-Canepa, Ximena; Jensen, Shawna M; Mesa, Christine M; Diaz-Satizabal, Laura; Roth, Alexa J; Parks-Dely, Julie A; Moon, Debra A; Wong, Janet P; Evseev, Danyel; Gossen, Desolie A; Tetrault, David G; Magor, Katharine E

    2016-08-01

    MHC class I is critically involved in defense against viruses, and diversity from polygeny and polymorphism contributes to the breadth of the immune response and health of the population. In this article, we examine MHC class I diversity in wild mallard ducks, the natural host and reservoir of influenza A viruses. We previously showed domestic ducks predominantly use UAA, one of five MHC class I genes, but whether biased expression is also true for wild mallards is unknown. Using RT-PCR from blood, we examined expressed MHC class I alleles from 38 wild mallards (Anas platyrhynchos) and identified 61 unique alleles, typically 1 or 2 expressed alleles in each individual. To determine whether expressed alleles correspond to UAA adjacent to TAP2 as in domestic ducks, we cloned and sequenced genomic UAA-TAP2 fragments from all mallards, which matched transcripts recovered and allowed us to assign most alleles as UAA Allelic differences are primarily located in α1 and α2 domains in the residues known to interact with peptide in mammalian MHC class I, suggesting the diversity is functional. Most UAA alleles have unique residues in the cleft predicting distinct specificity; however, six alleles have an unusual conserved cleft with two cysteine residues. Residues that influence peptide-loading properties and tapasin involvement in chicken are fixed in duck alleles and suggest tapasin independence. Biased expression of one MHC class I gene may make viral escape within an individual easy, but high diversity in the population places continual pressure on the virus in the reservoir species. PMID:27342841

  7. MYD88 L265P in Waldenström macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction

    PubMed Central

    Xu, Lian; Hunter, Zachary R.; Yang, Guang; Zhou, Yangsheng; Cao, Yang; Liu, Xia; Morra, Enrica; Trojani, Alessandra; Greco, Antonino; Arcaini, Luca; Varettoni, Maria; Brown, Jennifer R.; Tai, Yu-Tzu; Anderson, Kenneth C.; Munshi, Nikhil C.; Patterson, Christopher J.; Manning, Robert J.; Tripsas, Christina K.; Lindeman, Neal I.

    2013-01-01

    By whole-genome and/or Sanger sequencing, we recently identified a somatic mutation (MYD88 L265P) that stimulates nuclear factor κB activity and is present in >90% of Waldenström macroglobulinemia (WM) patients. MYD88 L265P was absent in 90% of immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) patients. We therefore developed conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays for more sensitive detection and quantification of MYD88 L265P. Using either assay, MYD88 L265P was detected in 97 of 104 (93%) WM and 13 of 24 (54%) IgM MGUS patients and was either absent or rarely expressed in samples from splenic marginal zone lymphoma (2/20; 10%), CLL (1/26; 4%), multiple myeloma (including IgM cases, 0/14), and immunoglobulin G MGUS (0/9) patients as well as healthy donors (0/40; P < 1.5 × 10−5 for WM vs other cohorts). Real-time AS-PCR identified IgM MGUS patients progressing to WM and showed a high rate of concordance between MYD88 L265P ΔCT and BM disease involvement (r = 0.89, P = .008) in WM patients undergoing treatment. These studies identify MYD88 L265P as a widely present mutation in WM and IgM MGUS patients using highly sensitive and specific AS-PCR assays with potential use in diagnostic discrimination and/or response assessment. The finding of this mutation in many IgM MGUS patients suggests that MYD88 L265P may be an early oncogenic event in WM pathogenesis. PMID:23321251

  8. Isolation, identification and expression of specific human CD133 antibodies.

    PubMed

    Xia, Jing; Zhang, Ying; Qian, Jun; Zhu, Xiaojun; Zhang, Yafen; Zhang, Jianqiong; Zhao, Gang

    2013-01-01

    CD133, a 120 KDa glycoprotein is a transmembrane glycoprotein which has been recently used as a cancer stem cell (CSCs) marker in a variety of carcinomas. CD133(+) cells possess strong tumorigenicity, responsible for tumor initiation and maintenance. Therefore, the goal of our study was to develop a novel CD133 humanized antibody as a promising target for cancer therapy. CD133 purified proteins were used for panning the naive human-semi-synthetic Tomlinson I + J phagemid library. The second extracellular domain (loop1) and the third extracellular domain (loop2) of CD133 were expressed in E. coli. In this study, we adopted a novel five-round selection strategy based on moderate stringent selection during the first rounds. This unique strategy was aimed at avoiding the loss of rare phages with high affinity to target proteins. After the five rounds of specific panning, six phage-antibody clones which specifically recognized recombinant human CD133 protein were obtained. The desirable phage clone named CD133-scFv-1 was cloned into the expression vector, then induced and purified. We show that CD133-scFv-1 and commercial murine antibody 293C3 could compete with each other in the indirect competitive immunoassay. Our work may lay the groundwork for future studies involving biological functions and applications of the CD133 humanized antibody. PMID:24271022

  9. A hierarchy of ECM-mediated signalling tissue-specific gene expression regulates tissue-specific gene expression

    SciTech Connect

    Roskelley, Calvin D; Srebrow, Anabella; Bissell, Mina J

    1995-10-07

    A dynamic and reciprocal flow of information between cells and the extracellular matrix contributes significantly to the regulation of form and function in developing systems. Signals generated by the extracellular matrix do not act in isolation. Instead, they are processed within the context of global signalling hierarchies whose constituent inputs and outputs are constantly modulated by all the factors present in the cell's surrounding microenvironment. This is particularly evident in the mammary gland, where the construction and subsequent destruction of such a hierarchy regulates changes in tissue-specific gene expression, morphogenesis and apoptosis during each developmental cycle of pregnancy, lactation and involution.

  10. Clinical expression of facioscapulohumeral muscular dystrophy in carriers of 1–3 D4Z4 reduced alleles: experience of the FSHD Italian National Registry

    PubMed Central

    Nikolic, Ana; Ricci, Giulia; Sera, Francesco; Bucci, Elisabetta; Govi, Monica; Mele, Fabiano; Rossi, Marta; Ruggiero, Lucia; Vercelli, Liliana; Ravaglia, Sabrina; Brisca, Giacomo; Fiorillo, Chiara; Villa, Luisa; Maggi, Lorenzo; Cao, Michelangelo; D'Amico, Maria Chiara; Siciliano, Gabriele; Antonini, Giovanni; Santoro, Lucio; Mongini, Tiziana; Moggio, Maurizio; Morandi, Lucia; Pegoraro, Elena; Angelini, Corrado; Di Muzio, Antonio; Rodolico, Carmelo; Tomelleri, Giuliano; Grazia D'Angelo, Maria; Bruno, Claudio; Berardinelli, Angela; Tupler, Rossella

    2016-01-01

    Objectives Facioscapulohumeral muscular dystrophy type 1 (FSHD1) has been genetically linked to reduced numbers (≤8) of D4Z4 repeats at 4q35. Particularly severe FSHD cases, characterised by an infantile onset and presence of additional extra-muscular features, have been associated with the shortest D4Z4 reduced alleles with 1–3 repeats (1–3 DRA). We searched for signs of perinatal onset and evaluated disease outcome through the systematic collection of clinical and anamnestic records of de novo and familial index cases and their relatives, carrying 1–3 DRA. Setting Italy. Participants 66 index cases and 33 relatives carrying 1–3 DRA. Outcomes The clinical examination was performed using the standardised FSHD evaluation form with validated inter-rater reliability. To investigate the earliest signs of disease, we designed the Infantile Anamnestic Questionnaire (IAQ). Comparison of age at onset was performed using the non-parametric Wilcoxon rank-sum or Kruskal-Wallis test. Comparison of the FSHD score was performed using a general linear model and Wald test. Kaplan-Meier survival analysis was used to estimate the age-specific cumulative motor impairment risk. Results No patients had perinatal onset. Among index cases, 36 (54.5%) showed the first signs by 10 years of age. The large majority of patients with early disease onset (26 out of 36, 72.2%) were de novo; whereas the majority of patients with disease onset after 10 years of age were familial (16, 53.3%). Comparison of the disease severity outcome between index cases with age at onset before and over 10 years of age, failed to detect statistical significance (Wald test p value=0.064). Of 61 index cases, only 17 (27.9%) presented extra-muscular conditions. Relatives carrying 1–3 DRA showed a large clinical variability ranging from healthy subjects, to patients with severe motor impairment. Conclusions The size of the D4Z4 allele is not always predictive of severe clinical outcome. The high

  11. LINC00507 Is Specifically Expressed in the Primate Cortex and Has Age-Dependent Expression Patterns.

    PubMed

    Mills, James D; Ward, Melanie; Chen, Bei Jun; Iyer, Anand M; Aronica, Eleonora; Janitz, Michael

    2016-08-01

    Over the past decade, there has been an increase in the appreciation of the role of non-coding RNA in the development of organism phenotype. It is possible to divide the non-coding elements of the transcriptome into three categories: short non-coding RNAs, circular RNAs and long non-coding RNAs. Long non-coding RNAs are those transcripts that are greater than 200 nts in length and lack any significant open reading frames that produce proteins greater then 100 amino acids. Long intervening non-coding RNAs (lincRNAs) are a subclass of long non-coding RNAs. In contrast to protein coding RNAs, lincRNAs are expressed in a more tissue- and species-specific manner. In particular, many lincRNAs are only conserved amongst higher primates. This coupled with the propensity of many lincRNAs to be expressed in the brain, suggests that they are in fact one of the major drivers of organism complexity. We analysed 39 lincRNAs that are expressed in the frontal cortex and identified LINC00507 as being expressed in a cortex-specific manner in non-human primates and humans. The expression patterns of LINC00507 appear to be age-dependent, suggesting it may be involved in brain development of higher primates. Moreover, the analysis of LINC00507 potential to bind ribosomes revealed that this previously identified non-coding transcript may harbour a micropeptide. PMID:27059230

  12. Isolation and characterization of new alleles of the cyclin-dependent kinase gene CDC28 with cyclin-specific functional and biochemical defects.

    PubMed

    Levine, K; Oehlen, L J; Cross, F R

    1998-01-01

    The G1 cyclin Cln2 negatively regulates the mating-factor pathway. In a genetic screen to identify factors required for this regulation, we identified an allele of CDC28 (cdc28-csr1) that blocked this function of Cln2. Cln2 immunoprecipitated from cdc28-csr1 cells was completely defective in histone H1 kinase activity, due to defects in Cdc28 binding and activation by Cln2. In contrast, Clb2-associated H1 kinase and Cdc28 binding was normal in immunoprecipitates from these cells. cdc28-csr1 was significantly deficient in other aspects of genetic interaction with Cln2. The cdc28-csr1 mutation was determined to be Q188P, in the T loop distal to most of the probable Cdk-cyclin interaction regions. We performed random mutagenesis of CDC28 to identify additional alleles incapable of causing CLN2-dependent mating-factor resistance but capable of complementing cdc28 temperature-sensitive and null alleles. Two such mutants had highly defective Cln2-associated kinase, but, surprisingly, two other mutants had levels of Cln2-associated kinase near to wild-type levels. We performed a complementary screen for CDC28 mutants that could cause efficient Cln2-dependent mating-factor resistance but not complement a cdc28 null allele. Most such mutants were found to alter residues essential for kinase activity; the proteins had little or no associated kinase activity in bulk or in association with Cln2. Several of these mutants also functioned in another assay for CLN2-dependent function not involving the mating-factor pathway, complementing the temperature sensitivity of a cln1 cln3 cdc28-csr1 strain. These results could indicate that Cln2-Cdc28 kinase activity is not directly relevant to some CLN2-mediated functions. Mutants of this sort should be useful in differentiating the function of Cdc28 complexed with different cyclin regulatory subunits. PMID:9418876

  13. Allele-specific germ cell epimutation in the spacer promoter of the 45S ribosomal RNA gene after Cr(III) exposure

    SciTech Connect

    Shiao, Y.-H. . E-mail: shiao@mail.ncifrcf.gov; Crawford, Erik B.; Anderson, Lucy M.; Patel, Pritesh; Ko, Kinarm

    2005-06-15

    Paternal exposure of mice to Cr(III) causes increased tumor risk in offspring; an epigenetic mechanism has been hypothesized. Representational difference analysis of gene methylation in sperm revealed hypomethylation in the 45S ribosomal RNA (rRNA) gene after Cr(III) exposure, compared with controls. The most striking effects were seen in the rRNA spacer promoter, a region in the intergenic region of rRNA gene clusters that can influence transcription. Methylation of the rRNA spacer promoter has not been studied heretofore. Sperm DNAs from Cr(III)-treated and control mice were modified by the bisulfite method followed by PCR amplification of the spacer promoter, including 27 CpG sites. Cloning and dideoxy sequencing identified sequence variants (T or G at base -2214) in the spacer promoter. The T allele had less DNA methylation than the G allele in control mice (17 of 17 clones vs. 42 of 72 clones, P = 0.0004). In spite of diversity of sperm DNA methylation patterns, the DNA clones from Cr(III)-exposed mice had fewer methylated CpG sites, by an average of 19% (P < 0.0001). This difference was limited to the G allele. The pyrosequencing technique was applied to quantify the percentage of methylation directly from amplified PCR products. Strikingly, for nine CpG sites including the spacer promoter core region, hypomethylation was highly significant in the Cr(III)-treated group (paired T test, P < 0.0001). Thus, one allele of the 45S rRNA spacer promoter is hypomethylated in sperm germ cells after Cr(III) exposure. This epimutation may lead to increase of tumor risk in the offspring.

  14. Generation of mice with a conditional Lbh null allele.

    PubMed

    Lindley, Linsey E; Briegel, Karoline J

    2013-07-01

    Limb bud and heart (LBH) is a developmentally expressed, tissue-specific transcription cofactor in vertebrates that acts in the WNT signaling pathway, a genetic program critical for embryogenesis and adult tissue homeostasis. Aberrant gain-of-function of LBH is implicated in both human congenital disease and cancer. The normal physiological function of LBH has remained elusive owing to a lack of genetic loss-of-function models. Here, we have generated mice with a conditional null allele of Lbh by flanking exon 2 with loxP sites (Lbh(flox)). Homozygous Lbh(flox) and Lbh(loxP) mice, in which the Neo cassette was removed through FLPe-mediated recombination, were viable and fertile, indicating that these conditional Lbh alleles are fully functional. Lbh(loxP) mice were then crossed with a Rosa26-Cre line, resulting in ubiquitous deletion of exon 2 and abolishment of LBH protein expression. Mice homozygous for the Lbh null allele (Lbh(Δ)(2)) displayed normal embryonic development and postnatal growth with morphologies indistinguishable from wild-type littermates. However, mammary gland development, which occurs primarily after birth, was perturbed. Thus, the conditional Lbh allele will be a valuable tool to uncover the currently unknown tissue-specific roles of LBH in postnatal development and disease. PMID:23495064

  15. Expression and Trans-Specific Polymorphism of Self-Incompatibility RNases in Coffea (Rubiaceae)

    PubMed Central

    Nowak, Michael D.; Davis, Aaron P.; Anthony, François; Yoder, Anne D.

    2011-01-01

    Self-incompatibility (SI) is widespread in the angiosperms, but identifying the biochemical components of SI mechanisms has proven to be difficult in most lineages. Coffea (coffee; Rubiaceae) is a genus of old-world tropical understory trees in which the vast majority of diploid species utilize a mechanism of gametophytic self-incompatibility (GSI). The S-RNase GSI system was one of the first SI mechanisms to be biochemically characterized, and likely represents the ancestral Eudicot condition as evidenced by its functional characterization in both asterid (Solanaceae, Plantaginaceae) and rosid (Rosaceae) lineages. The S-RNase GSI mechanism employs the activity of class III RNase T2 proteins to terminate the growth of “self” pollen tubes. Here, we investigate the mechanism of Coffea GSI and specifically examine the potential for homology to S-RNase GSI by sequencing class III RNase T2 genes in populations of 14 African and Madagascan Coffea species and the closely related self-compatible species Psilanthus ebracteolatus. Phylogenetic analyses of these sequences aligned to a diverse sample of plant RNase T2 genes show that the Coffea genome contains at least three class III RNase T2 genes. Patterns of tissue-specific gene expression identify one of these RNase T2 genes as the putative Coffea S-RNase gene. We show that populations of SI Coffea are remarkably polymorphic for putative S-RNase alleles, and exhibit a persistent pattern of trans-specific polymorphism characteristic of all S-RNase genes previously isolated from GSI Eudicot lineages. We thus conclude that Coffea GSI is most likely homologous to the classic Eudicot S-RNase system, which was retained since the divergence of the Rubiaceae lineage from an ancient SI Eudicot ancestor, nearly 90 million years ago. PMID:21731641

  16. Elementary school age children's comprehension of specific idiomatic expressions.

    PubMed

    Brinton, B; Fujiki, M; Mackey, T A

    1985-08-01

    This study explored the ability of elementary school age children to comprehend six idiomatic expressions. Eighty linguistically normal children, 20 from each of four different grade levels (kindergarten, second grade, fourth grade, and sixth grade) participated as subjects. All of the children completed a task designed to probe comprehension of specific idioms. A short story was presented, after which the subjects were required to identify events in the story, which were described using idiomatic phrases. When examined as a group, comprehension of the idioms studied improved with increasing age. However, when examined individually, performance was found to be highly variable from idiom to idiom. These results are discussed with regard to clinical implications in the assessment and management of language-disordered children. PMID:4019816

  17. Mouse strain specific gene expression differences for illumina microarray expression profiling in embryos

    PubMed Central

    2012-01-01

    Background In the field of mouse genetics the advent of technologies like microarray based expression profiling dramatically increased data availability and sensitivity, yet these advanced methods are often vulnerable to the unavoidable heterogeneity of in vivo material and might therefore reflect differentially expressed genes between mouse strains of no relevance to a targeted experiment. The aim of this study was not to elaborate on the usefulness of microarray analysis in general, but to expand our knowledge regarding this potential “background noise” for the widely used Illumina microarray platform surpassing existing data which focused primarily on the adult sensory and nervous system, by analyzing patterns of gene expression at different embryonic stages using wild type strains and modern transgenic models of often non-isogenic backgrounds. Results Wild type embryos of 11 mouse strains commonly used in transgenic and molecular genetic studies at three developmental time points were subjected to Illumina microarray expression profiling in a strain-by-strain comparison. Our data robustly reflects known gene expression patterns during mid-gestation development. Decreasing diversity of the input tissue and/or increasing strain diversity raised the sensitivity of the array towards the genetic background. Consistent strain sensitivity of some probes was attributed to genetic polymorphisms or probe design related artifacts. Conclusion Our study provides an extensive reference list of gene expression profiling background noise of value to anyone in the field of developmental biology and transgenic research performing microarray expression profiling with the widely used Illumina microarray platform. Probes identified as strain specific background noise further allow for microarray expression profiling on its own to be a valuable tool for establishing genealogies of mouse inbred strains. PMID:22583621

  18. Isolated 3-Methylcrotonyl-CoA Carboxylase Deficiency: Evidence for an Allele-Specific Dominant Negative Effect and Responsiveness to Biotin Therapy

    PubMed Central

    Baumgartner, Matthias R.; Dantas, M. Fernanda; Suormala, Terttu; Almashanu, Shlomo; Giunta, Cecilia; Friebel, Dolores; Gebhardt, Boris; Fowler, Brian; Hoffmann, Georg F.; Baumgartner, E. Regula; Valle, David

    2004-01-01

    Deficiency of 3-methylcrotonyl-CoA carboxylase (MCC) results in elevated excretion of 3-methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA). MCC is a heteromeric mitochondrial enzyme comprising biotin-containing α subunits and smaller β subunits, encoded by MCCA and MCCB, respectively. Mutations in these genes cause isolated MCC deficiency, an autosomal recessive disorder with a variable phenotype that ranges from severe neonatal to asymptomatic adult forms. No reported patients have responded to biotin therapy. Here, we describe two patients with a biochemical and, in one case, clinical phenotype of MCC deficiency, both of whom were responsive to biotin. The first patient presented at 3 months with seizures and progressive psychomotor retardation. Metabolic investigation at 2 years revealed elevated excretion of 3-MCG and 3-HIVA, suggesting MCC deficiency. High-dose biotin therapy was associated with a dramatic reduction in seizures, normalization of the electroencephalogram, and correction of the organic aciduria, within 4 weeks. MCC activity in fibroblasts was 25% of normal levels. The second patient, a newborn detected by tandem-mass-spectrometry newborn screening, displayed the same biochemical phenotype and remained asymptomatic with biotin up to the age of 18 months. In both patients, sequence analysis of the complete open reading frames of MCCA and MCCB revealed heterozygosity for MCCA-R385S and for the known polymorphic variant MCCA-P464H but revealed no other coding alterations. MCCA-R385S is unusual, in that it has a normal amount of MCCα protein but confers no MCC activity. We show that MCCA-R385S, but not other MCCA missense alleles, reduces the MCC activity of cotransfected MCCA–wild-type allele. Our results suggest that MCCA-R385S is a dominant negative allele and is biotin responsive in vivo. PMID:15359379

  19. Bisphenol A interferes with thyroid specific gene expression.

    PubMed

    Gentilcore, Daniela; Porreca, Immacolata; Rizzo, Francesca; Ganbaatar, Erdentuya; Carchia, Emanuele; Mallardo, Massimo; De Felice, Mario; Ambrosino, Concetta

    2013-02-01

    Bisphenol A (BPA) is an endocrine-disrupting chemical that leads to low-dose human exposure due to its ability to leach from chemically derived products, as polycarbonate plastics and epoxy resin. In addition to its known xeno-endocrine action, BPA exerts a wide range of metabolic effects. Despite the documented BPA exposure outcomes on synthesis of thyroid hormones, there are not any data available on its actions on the thyroid follicular cells, site of synthesis of the thyroid hormones. Recently, it has been shown that several environmental pollutants, as BPA, can exert a thyroid disrupting activity. In this study, we employed in vitro and in vivo (zebrafish) models to examine the effects of BPA in regulating the expression of genes involved in the thyroid hormone synthesis and of their transcriptional regulators at BPA doses as low as 10(-9)M, a dose that is environmentally pertinent and far below the one detected in infants plasma. In both systems we could detect an altered expression of the genes involved in thyroid hormones synthesis and of thyroid specific transcriptional factors in BPA dose and time dependent manner. Our results suggest that BPA exerts a direct effect on thyroid follicular cell. We show that these cells can "sense" very low amount of BPA. Thus they, potentially, represent an ideal in vitro system to develop assays to detect BPA and other pollutants with thyroid disrupting activity at level far below the ones considered to be environmental relevant. Moreover, this report may provide new insight into the mode of BPA-induced deregulation of physiological processes as well as on the extensively debated molecular pathways underlying its biological activities. PMID:23238275

  20. Germ line activation of the Tie2 and SMMHC promoters causes noncell-specific deletion of floxed alleles Address for reprint requests and other correspondence: C. D. Sigmund, Depts. of Internal Medicine and Physiology & Biophysics, 3181B Medical Education and Biomedical Research Facility, Roy J. and Lucille A. Carver Collage of Medicine, Univ. of Iowa, Iowa City, IA 52242 (e-mail: curt-sigmund@uiowa.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

    PubMed Central

    de Lange, Willem J.; Halabi, Carmen M.; Beyer, Andreas M.; Sigmund, Curt D.

    2008-01-01

    Tissue-specific knockouts generated through Cre-loxP recombination have become an important tool to manipulate the mouse genome. Normally, two successive rounds of breeding are performed to generate mice carrying two floxed target-gene alleles and a transgene expressing Cre-recombinase tissue-specifically. We show herein that two promoters commonly used to generate endothelium-specific (Tie2) and smooth muscle-specific [smooth muscle myosin heavy chain (Smmhc)] knockout mice exhibit activity in the female and male germ lines, respectively. This can result in the inheritance of a null allele in the second generation that is not tissue specific. Careful experimental design is required therefore to ensure that tissue-specific knockouts are indeed tissue specific and that appropriate controls are used to compare strains. PMID:18612081

  1. Identification of a New Class of Negative Regulators Affecting Sporulation-Specific Gene Expression in Yeast

    PubMed Central

    Benni, M. L.; Neigeborn, L.

    1997-01-01

    We characterized two yeast loci, MDS3 and PMD1, that negatively regulate sporulation. Initiation of sporulation is mediated by the meiotic activator IME1, which relies on MCK1 for maximal expression. We isolated the MDS3-1 allele (encoding a truncated form of Mds3p) as a suppressor that restores IME1 expression in mck1 mutants. mds3 null mutations confer similar suppression phenotypes as MDS3-1, indicating that Mds3p is a negative regulator of sporulation and the MDS3-1 allele confers a dominant-negative phenotype. PMD1 is predicted to encode a protein sharing significant similarity with Mds3p. mds3 pmd1 double mutants are better suppressors of mck1 than is either single mutant, indicating that Mds3p and Pmd1p function synergistically. Northern blot analysis revealed that suppression is due to increased IME1 transcript accumulation. The roles of Mds3p and Pmd1p are not restricted to the MCK1 pathway because mds3 pmd1 mutations also suppress IME1 expression defects associated with MCK1-independent sporulation mutants. Furthermore, mds3 pmd1 mutants express significant levels of IME1 even in vegetative cells and this unscheduled expression results in premature sporulation. These phenotypes and interactions with RAS2-Val19 suggest that unscheduled derepression of IME1 is probably due to a defect in recognition of nutritional status. PMID:9383076

  2. Erythropoietin gene expression: developmental-stage specificity, cell-type specificity, and hypoxia inducibility.

    PubMed

    Suzuki, Norio

    2015-01-01

    Erythrocytes play an essential role in the delivery of oxygen from the lung to every organ; a decrease in erythrocytes (anemia) causes hypoxic stress and tissue damage. To maintain oxygen homeostasis in adult mammals, when the kidney senses hypoxia, it secretes an erythroid growth factor, erythropoietin (Epo), which stimulates erythropoiesis in the bone marrow. Recently, studies using genetically modified mice have shown that the in vivo expression profile of the Epo gene changes dramatically during development. The first Epo-producing cells emerge in the neural crest and neuroepithelium of mid-stage embryos and support primitive erythropoiesis in the yolk sac. Subsequently, Epo from the hepatocytes stimulates erythropoiesis in the fetal liver of later stage embryos in a paracrine manner. In fact, erythroid lineage cells comprise the largest cell population in the fetal liver, and hepatocytes are distributed among the erythroid cell clusters. Adult erythropoiesis in the bone marrow requires Epo that is secreted by renal Epo-producing cells (REP cells). REP cells are widely distributed in the renal cortex and outer medulla. Hypoxia-inducible Epo production both in hepatocytes and REP cells is controlled at the gene transcription level that is mainly mediated by the hypoxia-inducible transcription factor (HIF) pathway. These mouse studies further provide insights into the molecular mechanisms of the cell-type specific, hypoxia-inducible expression of the Epo gene, which involves multiple sets of cis- and trans-regulatory elements. PMID:25786542

  3. Development of an allele-specific PCR assay for simultaneous sero-typing of avian pathogenic Escherichia coli predominant O1, O2, O18 and O78 strains.

    PubMed

    Wang, Shaohui; Meng, Qingmei; Dai, Jianjun; Han, Xiangan; Han, Yue; Ding, Chan; Liu, Haiwen; Yu, Shengqing

    2014-01-01

    Systemic infections by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. E. coli strains belonging to serotypes O1, O2, O18 and O78 are preferentially associated with avian colibacillosis. The rfb gene cluster controlling O antigen synthesis is usually various among different E. coli serotypes. In present study, the rfb gene clusters of E. coli serotypes O1, O2, O18 and O78 were characterized and compared. Based on the serotype-specific genes in rfb gene cluster, an allele-specific polymerase chain reaction (PCR) assay was developed. This PCR assay was highly specific and reliable for sero-typing of APEC O1, O2, O18 and O78 strains. The sensitivity of the assay was determined as 10 pg DNA or 10 colony forming units (CFUs) bacteria for serotypes O2 and O18 strains, and 500 pg DNA or 1,000 CFUs bacteria for serotypes O1 and O78 strains. Using this PCR system, APEC isolates and the infected tissue samples were categorized successfully. Furthermore, it was able to differentiate the serotypes for the samples with multi-agglutination in the traditional serum agglutination assay. Therefore, the allele-specific PCR is more simple, rapid and accurate assay for APEC diagnosis, epidemiologic study and vaccine development. PMID:24805368

  4. Gene trapping uncovers sex-specific mechanisms for upstream stimulatory factors 1 and 2 in angiotensinogen expression.

    PubMed

    Park, Sungmi; Liu, Xuebo; Davis, Deborah R; Sigmund, Curt D

    2012-06-01

    A single-nucleotide polymorphism (C/A) located within an E-box at the -20 position of the human angiotensinogen (AGT) promoter may regulate transcriptional activation through differential recruitment of the transcription factors upstream stimulatory factor (USF) 1 and 2. To study the contribution of USF1 on AGT gene expression, mice carrying a (-20C) human AGT (hAGT) transgene were bred with mice harboring a USF1 gene trap allele designed to knock down USF1 expression. USF1 mRNA was reduced relative to controls in liver (9 ± 1%), perigenital adipose (16 ± 3%), kidney (17 ± 1%), and brain (34 ± 2%) in double-transgenic mice. This decrease was confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation analyses revealed a decrease in USF1, with retention of USF2 binding at the hAGT promoter in the liver of male mice. hAGT expression was reduced in the liver and other tissues of female but not male mice. The decrease in endogenous AGT expression was insufficient to alter systolic blood pressure at baseline but caused reduced systolic blood pressure in female USF1 gene trap mice fed a high-fat diet. Treatment of USF1 knockdown males with intravenous adenoviral short hairpin RNA targeting USF2 resulted in reduced expression of USF1, USF2, and hAGT protein. Our data from chromatin immunoprecipitation assays suggests that this decrease in hAGT is attributed to decreased USF2 binding to the hAGT promoter. In conclusion, both USF1 and USF2 are essential for AGT transcriptional regulation, and distinct sex-specific and tissue-specific mechanisms are involved in the activities of these transcription factors in vivo. PMID:22547438

  5. Topologically associated domains enriched for lineage-specific genes reveal expression-dependent nuclear topologies during myogenesis

    PubMed Central

    Neems, Daniel S.; Garza-Gongora, Arturo G.; Smith, Erica D.; Kosak, Steven T.

    2016-01-01

    The linear distribution of genes across chromosomes and the spatial localization of genes within the nucleus are related to their transcriptional regulation. The mechanistic consequences of linear gene order, and how it may relate to the functional output of genome organization, remain to be fully resolved, however. Here we tested the relationship between linear and 3D organization of gene regulation during myogenesis. Our analysis has identified a subset of topologically associated domains (TADs) that are significantly enriched for muscle-specific genes. These lineage-enriched TADs demonstrate an expression-dependent pattern of nuclear organization that influences the positioning of adjacent nonenriched TADs. Therefore, lineage-enriched TADs inform cell-specific genome organization during myogenesis. The reduction of allelic spatial distance of one of these domains, which contains Myogenin, correlates with reduced transcriptional variability, identifying a potential role for lineage-specific nuclear topology. Using a fusion-based strategy to decouple mitosis and myotube formation, we demonstrate that the cell-specific topology of syncytial nuclei is dependent on cell division. We propose that the effects of linear and spatial organization of gene loci on gene regulation are linked through TAD architecture, and that mitosis is critical for establishing nuclear topologies during cellular differentiation. PMID:26957603

  6. Transcription Coactivators p300 and CBP Are Necessary for Photoreceptor-Specific Chromatin Organization and Gene Expression

    PubMed Central

    Hennig, Anne K.; Peng, Guang-Hua; Chen, Shiming

    2013-01-01

    Rod and cone photoreceptor neurons in the mammalian retina possess specialized cellular architecture and functional features for converting light to a neuronal signal. Establishing and maintaining these characteristics requires appropriate expression of a specific set of genes, which is tightly regulated by a network of photoreceptor transcription factors centered on the cone-rod homeobox protein CRX. CRX recruits transcription coactivators p300 and CBP to acetylate promoter-bound histones and activate transcription of target genes. To further elucidate the role of these two coactivators, we conditionally knocked out Ep300 and/or CrebBP in differentiating rods or cones, using opsin-driven Cre recombinase. Knockout of either factor alone exerted minimal effects, but loss of both factors severely disrupted target cell morphology and function: the unique nuclear chromatin organization seen in mouse rods was reversed, accompanied by redistribution of nuclear territories associated with repressive and active histone marks. Transcription of many genes including CRX targets was severely impaired, correlating with reduced histone H3/H4 acetylation (the products of p300/CBP) on target gene promoters. Interestingly, the presence of a single wild-type allele of either coactivator prevented many of these defects, with Ep300 more effective than Cbp. These results suggest that p300 and CBP play essential roles in maintaining photoreceptor-specific structure, function and gene expression. PMID:23922782

  7. Imprinting control regions (ICRs) are marked by mono-allelic bivalent chromatin when transcriptionally inactive

    PubMed Central

    Maupetit-Méhouas, Stéphanie; Montibus, Bertille; Nury, David; Tayama, Chiharu; Wassef, Michel; Kota, Satya K.; Fogli, Anne; Cerqueira Campos, Fabiana; Hata, Kenichiro; Feil, Robert; Margueron, Raphael; Nakabayashi, Kazuhiko; Court, Franck; Arnaud, Philippe

    2016-01-01

    Parental allele-specific expression of imprinted genes is mediated by imprinting control regions (ICRs) that are constitutively marked by DNA methylation imprints on the maternal or paternal allele. Mono-allelic DNA methylation is strictly required for the process of imprinting and has to be faithfully maintained during the entire life-span. While the regulation of DNA methylation itself is well understood, the mechanisms whereby the opposite allele remains unmethylated are unclear. Here, we show that in the mouse, at maternally methylated ICRs, the paternal allele, which is constitutively associated with H3K4me2/3, is marked by default by H3K27me3 when these ICRs are transcriptionally inactive, leading to the formation of a bivalent chromatin signature. Our data suggest that at ICRs, chromatin bivalency has a protective role by ensuring that DNA on the paternal allele remains unmethylated and protected against spurious and unscheduled gene expression. Moreover, they provide the proof of concept that, beside pluripotent cells, chromatin bivalency is the default state of transcriptionally inactive CpG island promoters, regardless of the developmental stage, thereby contributing to protect cell identity. PMID:26400168

  8. Computational deconvolution of genome wide expression data from Parkinson's and Huntington's disease brain tissues using population-specific expression analysis

    PubMed Central

    Capurro, Alberto; Bodea, Liviu-Gabriel; Schaefer, Patrick; Luthi-Carter, Ruth; Perreau, Victoria M.

    2015-01-01

    The characterization of molecular changes in diseased tissues gives insight into pathophysiological mechanisms and is important for therapeutic development. Genome-wide gene expression analysis has proven valuable for identifying biological processes in neurodegenerative diseases using post mortem human brain tissue and numerous datasets are publically available. However, many studies utilize heterogeneous tissue samples consisting of multiple cell types, all of which contribute to global gene expression values, confounding biological interpretation of the data. In particular, changes in numbers of neuronal and glial cells occurring in neurodegeneration confound transcriptomic analyses, particularly in human brain tissues where sample availability and controls are limited. To identify cell specific gene expression changes in neurodegenerative disease, we have applied our recently published computational deconvolution method, population specific expression analysis (PSEA). PSEA estimates cell-type-specific expression values using reference expression measures, which in the case of brain tissue comprises mRNAs with cell-type-specific expression in neurons, astrocytes, oligodendrocytes and microglia. As an exercise in PSEA implementation and hypothesis development regarding neurodegenerative diseases, we applied PSEA to Parkinson's and Huntington's disease (PD, HD) datasets. Genes identified as differentially expressed in substantia nigra pars compacta neurons by PSEA were validated using external laser capture microdissection data. Network analysis and Annotation Clustering (DAVID) identified molecular processes implicated by differential gene expression in specific cell types. The results of these analyses provided new insights into the implementation of PSEA in brain tissues and additional refinement of molecular signatures in human HD and PD. PMID:25620908

  9. The A Allele of the rs1990760 Polymorphism in the IFIH1 Gene Is Associated with Protection for Arterial Hypertension in Type 1 Diabetic Patients and with Expression of This Gene in Human Mononuclear Cells

    PubMed Central

    Bouças, Ana P.; Brondani, Letícia A.; Souza, Bianca M.; Lemos, Natália E.; de Oliveira, Fernanda S.; Canani, Luis H.; Crispim, Daisy

    2013-01-01

    Background The rs1990760 polymorphism of interferon induced with helicase C domain 1 (IFIH1) has been associated with type 1 diabetes mellitus (T1DM). Here, we investigated whether this polymorphism is associated with T1DM or its clinical characteristics in a Brazilian population, and if IFIH1 gene expression in mononuclear cells from T1DM patients differs according to the genotypes of this polymorphism. A meta-analysis was also conducted to evaluate if the rs1990760 polymorphism is associated with T1DM. Methods Frequencies of the rs1990760 polymorphism were analyzed in 527 T1DM patients and in 517 healthy subjects. IFIH1 gene expressions according to genotypes were measured in a sub-sample of 26 T1DM patients by quantitative real-time PCR. Results Our data show the association of the A allele with risk to T1DM under a dominant model of inheritance [odds ratio (OR) = 1.421, P = 0.037], adjusting for ethnicity. The meta-analysis revealed significant association between the rs199760A allele and risk for T1DM for all analyzed inheritance models. Surprisingly, T1DM patients carrying the A allele showed lower levels of systolic (P = 0.001) and diastolic (P = 1×10−10) blood pressures as compared to G/G carriers. Furthermore, the A/A genotype seems to be associated with protection to arterial hypertension (AH) after adjustment for covariates (OR = 0.339, P = 0.019). IFIH1 gene expression in mononuclear cells from 26 T1DM patients did not differ among genotypes (P = 0.274). Nevertheless, IFIH1 gene expression was increased in mononuclear cells from T1DM patients with AH as compared with T1DM patients without AH [6.7 (1.7–2.0) vs. 1.8 (1.3–7.1) arbitrary units; P = 0.036]. The association with blood pressures and AH was not observed in patients with type 2 diabetes mellitus. Conclusions Our results indicate that the rs1990760 polymorphism is associated with T1DM. Interestingly, the rs1990760 A allele seems to be associated with

  10. Inducible loss of one Apc allele in Lrig1-expressing progenitor cells results in multiple distal colonic tumors with features of familial adenomatous polyposis

    PubMed Central

    Powell, Anne E.; Vlacich, Gregory; Zhao, Zhen-Yang; McKinley, Eliot T.; Washington, M. Kay; Manning, H. Charles

    2014-01-01

    Individuals with familial adenomatous polyposis (FAP) harbor a germline mutation in adenomatous polyposis coli (APC). The major clinical manifestation is development of multiple colonic tumors at a young age due to stochastic loss of the remaining APC allele. Extracolonic features, including periampullary tumors, gastric abnormalities, and congenital hypertrophy of the retinal pigment epithelium, may occur. The objective of this study was to develop a mouse model that simulates these features of FAP. We combined our Lrig1-CreERT2/+ mice with Apcfl/+ mice, eliminated one copy of Apc in leucine-rich repeats and immunoglobulin-like domains protein 1 (Lrig1)-positive (Lrig1+) progenitor cells with tamoxifen injection, and monitored tumor formation in the colon by colonoscopy and PET. Initial loss of one Apc allele in Lrig1+ cells results in a predictable pattern of preneoplastic changes, culminating in multiple distal colonic tumors within 50 days of induction, as well as the extracolonic manifestations of FAP mentioned above. We show that tumor formation can be monitored by noninvasive PET imaging. This inducible stem cell-driven model recapitulates features of FAP and offers a tractable platform on which therapeutic interventions can be monitored over time by colonoscopy and noninvasive imaging. PMID:24833705

  11. Allele-Independent Turnover of Human Leukocyte Antigen (HLA) Class Ia Molecules.

    PubMed

    Prevosto, Claudia; Usmani, M Farooq; McDonald, Sarah; Gumienny, Aleksandra M; Key, Tim; Goodman, Reyna S; Gaston, J S Hill; Deery, Michael J; Busch, Robert

    2016-01-01

    Major histocompatibility complex class I (MHCI) glycoproteins present cytosolic peptides to CD8+ T cells and regulate NK cell activity. Their heavy chains (HC) are expressed from up to three MHC gene loci (human leukocyte antigen [HLA]-A, -B, and -C in humans), whose extensive polymorphism maps predominantly to the antigen-binding groove, diversifying the bound peptide repertoire. Codominant expression of MHCI alleles is thus functionally critical, but how it is regulated is not fully understood. Here, we have examined the effect of polymorphism on the turnover rates of MHCI molecules in cell lines with functional MHCI peptide loading pathways and in monocyte-derived dendritic cells (MoDCs). Proteins were labeled biosynthetically with heavy water (2H2O), folded MHCI molecules immunoprecipitated, and tryptic digests analysed by mass spectrometry. MHCI-derived peptides were assigned to specific alleles and isotypes, and turnover rates quantified by 2H incorporation, after correcting for cell growth. MHCI turnover half-lives ranged from undetectable to a few hours, depending on cell type, activation state, donor, and MHCI isotype. However, in all settings, the turnover half-lives of alleles of the same isotype were similar. Thus, MHCI protein turnover rates appear to be allele-independent in normal human cells. We propose that this is an important feature enabling the normal function and codominant expression of MHCI alleles. PMID:27529174

  12. Allele-Independent Turnover of Human Leukocyte Antigen (HLA) Class Ia Molecules

    PubMed Central

    Prevosto, Claudia; Usmani, M. Farooq; McDonald, Sarah; Gumienny, Aleksandra M.; Key, Tim; Goodman, Reyna S.; Gaston, J. S. Hill; Deery, Michael J.; Busch, Robert

    2016-01-01

    Major histocompatibility complex class I (MHCI) glycoproteins present cytosolic peptides to CD8+ T cells and regulate NK cell activity. Their heavy chains (HC) are expressed from up to three MHC gene loci (human leukocyte antigen [HLA]-A, -B, and -C in humans), whose extensive polymorphism maps predominantly to the antigen-binding groove, diversifying the bound peptide repertoire. Codominant expression of MHCI alleles is thus functionally critical, but how it is regulated is not fully understood. Here, we have examined the effect of polymorphism on the turnover rates of MHCI molecules in cell lines with functional MHCI peptide loading pathways and in monocyte-derived dendritic cells (MoDCs). Proteins were labeled biosynthetically with heavy water (2H2O), folded MHCI molecules immunoprecipitated, and tryptic digests analysed by mass spectrometry. MHCI-derived peptides were assigned to specific alleles and isotypes, and turnover rates quantified by 2H incorporation, after correcting for cell growth. MHCI turnover half-lives ranged from undetectable to a few hours, depending on cell type, activation state, donor, and MHCI isotype. However, in all settings, the turnover half-lives of alleles of the same isotype were similar. Thus, MHCI protein turnover rates appear to be allele-independent in normal human cells. We propose that this is an important feature enabling the normal function and codominant expression of MHCI alleles. PMID:27529174

  13. Cell Surface Expression Level Variation between Two Common Human Leukocyte Antigen Alleles, HLA-A2 and HLA-B8, Is Dependent on the Structure of the C Terminal Part of the Alpha 2 and the Alpha 3 Domains

    PubMed Central

    Dellgren, Christoffer; Nehlin, Jan O.; Barington, Torben

    2015-01-01

    Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses. Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of-B7 (P = 0.002). Transfection experiments with full-length HLA-A2 and -B8 encoding plasmids confirmed this (54,031 molecules per cell vs. 2,466, respectively, P = 0.001) independently of transcript levels suggesting a post-transcriptional regulation. Using chimeric constructs we found that the cytoplasmic tail and the transmembrane region had no impact on the differential cell surface expression. In contrast, ~65% of the difference could be mapped to the six C-terminal amino acids of the alpha 2 domain and the alpha 3 domain (amino acids 176–284), i.e. amino acids not previously shown to be of importance for differential expression levels of HLA class I molecules. We suggest that the differential cell surface expression of two common HLA-A and–B alleles is regulated by a post-translational mechanism that may involve hitherto unrecognized molecules. PMID:26258424

  14. Caste-Specific and Sex-Specific Expression of Chemoreceptor Genes in a Termite

    PubMed Central

    Mikheyev, Alexander; Tin, Mandy M. Y.; Watanabe, Yutaka; Matsuura, Kenji

    2016-01-01

    The sophisticated colony organization of eusocial insects is primarily maintained through the utilization of pheromones. The regulation of these complex social interactions requires intricate chemoreception systems. The recent publication of the genome of Zootermopsis nevadensis opened a new avenue to study molecular basis of termite caste systems. Although there has been a growing interest in the termite chemoreception system that regulates their sophisticated caste system, the relationship between division of labor and expression of chemoreceptor genes remains to be explored. Using high-throughput mRNA sequencing (RNA-seq), we found several chemoreceptors that are differentially expressed among castes and between sexes in a subterranean termite Reticulitermes speratus. In total, 53 chemoreception-related genes were annotated, including 22 odorant receptors, 7 gustatory receptors, 12 ionotropic receptors, 9 odorant-binding proteins, and 3 chemosensory proteins. Most of the chemoreception-related genes had caste-related and sex-related expression patterns; in particular, some chemoreception genes showed king-biased or queen-biased expression patterns. Moreover, more than half of the genes showed significant age-dependent differences in their expression in female and/or male reproductives. These results reveal a strong relationship between the evolution of the division of labor and the regulation of chemoreceptor gene expression, thereby demonstrating the chemical communication and underlining chemoreception mechanism in social insects. PMID:26760975

  15. Caste-Specific and Sex-Specific Expression of Chemoreceptor Genes in a Termite.

    PubMed

    Mitaka, Yuki; Kobayashi, Kazuya; Mikheyev, Alexander; Tin, Mandy M Y; Watanabe, Yutaka; Matsuura, Kenji

    2016-01-01

    The sophisticated colony organization of eusocial insects is primarily maintained through the utilization of pheromones. The regulation of these complex social interactions requires intricate chemoreception systems. The recent publication of the genome of Zootermopsis nevadensis opened a new avenue to study molecular basis of termite caste systems. Although there has been a growing interest in the termite chemoreception system that regulates their sophisticated caste system, the relationship between division of labor and expression of chemoreceptor genes remains to be explored. Using high-throughput mRNA sequencing (RNA-seq), we found several chemoreceptors that are differentially expressed among castes and between sexes in a subterranean termite Reticulitermes speratus. In total, 53 chemoreception-related genes were annotated, including 22 odorant receptors, 7 gustatory receptors, 12 ionotropic receptors, 9 odorant-binding proteins, and 3 chemosensory proteins. Most of the chemoreception-related genes had caste-related and sex-related expression patterns; in particular, some chemoreception genes showed king-biased or queen-biased expression patterns. Moreover, more than half of the genes showed significant age-dependent differences in their expression in female and/or male reproductives. These results reveal a strong relationship between the evolution of the division of labor and the regulation of chemoreceptor gene expression, thereby demonstrating the chemical communication and underlining chemoreception mechanism in social insects. PMID:26760975

  16. Identification of Enterococcus faecalis antigens specifically expressed in vivo

    PubMed Central

    Shet, Uttom K.; Park, Sang-Won; Lim, Hyun-Pil; Yun, Kwi-Dug; Kang, Seong Soo; Kim, Se Eun

    2015-01-01

    Objectives Molecular mechanism of the pathogenicity of Enterococcus faecalis (E. faecalis), a suspected endodontic pathogen, has not yet been adequately elucidated due to limited information on its virulence factors. Here we report the identification of in vivo expressed antigens of E. faecalis by using a novel immunoscreening technique called change-mediated antigen technology (CMAT) and an experimental animal model of endodontic infection. Materials and Methods Among 4,500 E. coli recombinant clones screened, 19 positive clones reacted reproducibly with hyperimmune sera obtained from rabbits immunized with E. faecalis cells isolated from an experimental endodontic infection. DNA sequences from 16 of these in vivo-induced (IVI) genes were determined. Results Identified protein antigens of E. faecalis included enzymes involved in housekeeping functions, copper resistance protein, putative outer membrane proteins, and proteins of unknown function. Conclusions In vivo expressed antigens of E. faecalis could be identified by using a novel immune-screening technique CMAT and an experimental animal model of endodontic infection. Detailed analysis of these IVI genes will lead to a better understanding of the molecular mechanisms involved in the endodontic infection of E. faecalis. PMID:26587417

  17. Improved allele-specific PCR assays for detection of clarithromycin and fluoroquinolone resistant of Helicobacter pylori in gastric biopsies: identification of N87I mutation in GyrA.

    PubMed

    Trespalacios, Alba A; Rimbara, Emiko; Otero, William; Reddy, Rita; Graham, David Y

    2015-04-01

    Molecular testing can rapidly detect Helicobacter pylori susceptibility using gastric biopsies. Allele-specific polymerase chain reaction (ASP-PCR) was used to identify H. pylori 23S rRNA and gyrA mutation using gastric biopsies from Colombian patients and confirmed by PCR and sequencing of the 23S rRNA and gyrA genes. The sensitivity and specificity of ASP-PCR were compared with susceptibilities measured by agar dilution. Samples included gastric biopsies from 107 biopsies with H. pylori infections and 20 H. pylori negative. The sensitivity and specificity of ASP-PCR for the 23S rRNA gene were both 100%. The sensitivity and specificity of ASP-PCR for the gyrA gene, published in 2007 by Nishizawa et al., were 52% and 92.7%, respectively; the lower sensitivity was due to the presence of mutation N87I in our samples, which were not detected by the test. In this study, we designed new primers to detect the mutation N87I in GyrA. The ASP-PCR was performed with the original primers plus the new primers. The molecular test with the new primers improved the sensitivity to 100%. In conclusion, ASP-PCR provides a specific and rapid means of predicting resistance to clarithromycin and levofloxacin in gastric biopsies. PMID:25600075

  18. PrimerSNP: a web tool for whole-genome selection of allele-specific and common primers of phylogenetically-related bacterial genomic sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The increasing number of genomic sequences of bacteria makes it possible to select unique SNPs of a particular strain/species at the whole genome level and thus design specific primers based on the SNPs. The high similarity of genomic sequences among phylogenetically-related bacteria requires the id...

  19. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase.

    PubMed

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana; Gritsenko, Natalia; Rask, Lene; Mainbakh, Yuli; Zilberstein, Yael; Yagil, Ezra; Kolot, Mikhail

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system. PMID:27117628

  20. Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase

    PubMed Central

    Elias, Amer; Spector, Itay; Sogolovsky-Bard, Ilana; Gritsenko, Natalia; Rask, Lene; Mainbakh, Yuli; Zilberstein, Yael; Yagil, Ezra; Kolot, Mikhail

    2016-01-01

    Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system. PMID:27117628

  1. The Sarcoglycan complex is expressed in the cerebrovascular system and is specifically regulated by astroglial Cx30 channels.

    PubMed

    Boulay, Anne-Cécile; Saubaméa, Bruno; Cisternino, Salvatore; Mignon, Virginie; Mazeraud, Aurélien; Jourdren, Laurent; Blugeon, Corinne; Cohen-Salmon, Martine

    2015-01-01

    Astrocytes, the most prominent glial cell type in the brain, send specialized processes called endfeet, around blood vessels and express a large molecular repertoire regulating the cerebrovascular system physiology. One of the most striking properties of astrocyte endfeet is their enrichment in gap junction proteins Connexin 43 and 30 (Cx43 and Cx30) allowing in particular for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. In this study, we addressed the specific role of Cx30 at the gliovascular interface. Using an inactivation mouse model for Cx30 (Cx30(Δ/Δ); Δ means deleted allele) we showed that absence of Cx30 does not affect blood-brain barrier (BBB) organization and permeability. However, it results in the cerebrovascular fraction, in a strong upregulation of Sgcg encoding γ-Sarcoglycan (γ-SG), a member of the Dystrophin-associated protein complex (DAPC) connecting cytoskeleton and the extracellular matrix. The same molecular event occurs in Cx30(T5M/T5M) mutated mice, where Cx30 channels are closed, demonstrating that Sgcg regulation relied on Cx30 channel functions. We further characterized the expression of other Sarcoglycan complex (SGC) molecules in the cerebrovascular system and showed the presence of α-, β-, δ-, γ-, ε- and ζ- SG, as well as Sarcospan. Their expression was however not modified in Cx30(Δ/Δ). These results suggest that a full SGC might be present in the cerebrovascular system, and that expression of one of its member, γ-SG, depends on Cx30 channels. As described in skeletal muscles, the SGC may contribute to membrane stabilization and signal transduction in the cerebrovascular system, which may therefore be regulated by Cx30 channel-mediated functions. PMID:25698924

  2. The Sarcoglycan complex is expressed in the cerebrovascular system and is specifically regulated by astroglial Cx30 channels

    PubMed Central

    Boulay, Anne-Cécile; Saubaméa, Bruno; Cisternino, Salvatore; Mignon, Virginie; Mazeraud, Aurélien; Jourdren, Laurent; Blugeon, Corinne; Cohen-Salmon, Martine

    2015-01-01

    Astrocytes, the most prominent glial cell type in the brain, send specialized processes called endfeet, around blood vessels and express a large molecular repertoire regulating the cerebrovascular system physiology. One of the most striking properties of astrocyte endfeet is their enrichment in gap junction proteins Connexin 43 and 30 (Cx43 and Cx30) allowing in particular for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. In this study, we addressed the specific role of Cx30 at the gliovascular interface. Using an inactivation mouse model for Cx30 (Cx30Δ/Δ; Δ means deleted allele) we showed that absence of Cx30 does not affect blood-brain barrier (BBB) organization and permeability. However, it results in the cerebrovascular fraction, in a strong upregulation of Sgcg encoding γ-Sarcoglycan (γ-SG), a member of the Dystrophin-associated protein complex (DAPC) connecting cytoskeleton and the extracellular matrix. The same molecular event occurs in Cx30T5M/T5M mutated mice, where Cx30 channels are closed, demonstrating that Sgcg regulation relied on Cx30 channel functions. We further characterized the expression of other Sarcoglycan complex (SGC) molecules in the cerebrovascular system and showed the presence of α-, β-, δ-, γ-, ε- and ζ- SG, as well as Sarcospan. Their expression was however not modified in Cx30Δ/Δ. These results suggest that a full SGC might be present in the cerebrovascular system, and that expression of one of its member, γ-SG, depends on Cx30 channels. As described in skeletal muscles, the SGC may contribute to membrane stabilization and signal transduction in the cerebrovascular system, which may therefore be regulated by Cx30 channel-mediated functions. PMID:25698924

  3. Tissue Specificity and Sex-Specific Regulatory Variation Permit the Evolution of Sex-Biased Gene Expression.

    PubMed

    Dean, Rebecca; Mank, Judith E

    2016-09-01

    Genetic correlations between males and females are often thought to constrain the evolution of sexual dimorphism. However, sexually dimorphic traits and the underlying sexually dimorphic gene expression patterns are often rapidly evolving. We explore this apparent paradox by measuring the genetic correlation in gene expression between males and females (Cmf) across broad evolutionary timescales, using two RNA-sequencing data sets spanning multiple populations and multiple species. We find that unbiased genes have higher Cmf than sex-biased genes, consistent with intersexual genetic correlations constraining the evolution of sexual dimorphism. However, we found that highly sex-biased genes (both male and female biased) also had higher tissue specificity, and unbiased genes had greater expression breadth, suggesting that pleiotropy may constrain the breakdown of intersexual genetic correlations. Finally, we show that genes with high Cmf showed some degree of sex-specific changes in gene expression in males and females. Together, our results suggest that genetic correlations between males and females may be less important in constraining the evolution of sex-biased gene expression than pleiotropy. Sex-specific regulatory variation and tissue specificity may resolve the paradox of widespread sex bias within a largely shared genome. PMID:27501094

  4. Tissue-specific and differentiation-specific expression of a human K14 keratin gene in transgenic mice

    SciTech Connect

    Vassar, R.; Rosenberg, M.; Tyner, A.; Fuchs, E. ); Ross, S. )

    1989-03-01

    A construct containing {approx}2,500 base pairs (bp) of 5{prime} upstream and {approx}700 bp of 3{prime} downstream sequence was used to drive the expression of an intronless human K14 gene in vitro and in vivo. To track the expression of the gene, a small sequence encoding the antigenic portion of neuropeptide substance P was inserted in frame 5{prime} to the TGA translation stop codon of the gene. Surprisingly, this gene was expressed promiscuously in a wide variety of cultured cells transiently transfected with the construct. In contrast, when introduced into the germ line of transgenic mice, the construct was expressed in a fashion analogous to the endogenous K14 gene--namely, in the basal layer of stratified squamous epithelia. The results suggest that some regulatory mechanism is overridden as a consequence of transient transfection but that sequences that can control proper K14 expression are present in the construct. The appropriate tissue-specific and differentiation-specific expression of K14{center dot}P in transgenic mice is an important first step in characterizing a promoter that could be employed to drive the foreign expression of drug-related genes in the epidermis of skin grafts.

  5. Novel alleles of the transforming growth factor β-1 regulatory region and exon 1.

    PubMed

    Arrieta-Bolaños, E; Madrigal, J A; Shaw, B E

    2015-06-01

    Transforming growth factor β-1, encoded by the TGFB1 gene, is a cytokine that plays a central role in many physiologic and pathogenic processes with pleiotropic effects. Regulatory activity for this gene has been shown for 3.0 kb between positions -2665 and +423 from its translational start site. At least 17 TGFB1 regulatory region and exon 1 alleles have been defined on the basis of 18 polymorphic sites. Polymorphisms in TGFB1's regulatory region have been associated with differential levels of expression of this cytokine and to genetic risk in cancer and transplantation. In this report, we present 19 novel TGFB1 regulatory region and exon 1 alleles: p018-p036. Amplification of TGFB1's regulatory region was performed with an in-house protocol, and novel alleles were defined by either allele-specific amplification and/or molecular cloning of the amplicons, followed by sequencing in isolation. Three of these novel alleles (p018, p019, and p020) are shown to be formed by novel combinations of the aforementioned known polymorphic positions. Another 16 novel alleles are shown to carry additional known and unknown single-nucleotide polymorphisms. Polymorphism in TGFB1's regulatory region could have an impact on important processes, including embryogenesis, hematopoiesis, carcinogenesis, angiogenesis, fibrosis, immune responses, and transplantation, making its characterization necessary. PMID:25808355

  6. Ubiquitously expressed genes participate in cell-specific functions via alternative promoter usage.

    PubMed

    Feng, Guihai; Tong, Man; Xia, Baolong; Luo, Guan-Zheng; Wang, Meng; Xie, Dongfang; Wan, Haifeng; Zhang, Ying; Zhou, Qi; Wang, Xiu-Jie

    2016-09-01

    How do different cell types acquire their specific identities and functions is a fundamental question of biology. Previously significant efforts have been devoted to search for cell-type-specifically expressed genes, especially transcription factors, yet how do ubiquitously expressed genes participate in the formation or maintenance of cell-type-specific features remains largely unknown. Here, we have identified 110 mouse embryonic stem cell (mESC) specifically expressed transcripts with cell-stage-specific alternative transcription start sites (SATS isoforms) from 104 ubiquitously expressed genes, majority of which have active epigenetic modification- or stem cell-related functions. These SATS isoforms are specifically expressed in mESCs, and tend to be transcriptionally regulated by key pluripotency factors through direct promoter binding. Knocking down the SATS isoforms of Nmnat2 or Usp7 leads to differentiation-related phenotype in mESCs. These results demonstrate that cell-type-specific transcription factors are capable to produce cell-type-specific transcripts with alternative transcription start sites from ubiquitously expressed genes, which confer ubiquitously expressed genes novel functions involved in the establishment or maintenance of cell-type-specific features. PMID:27466324

  7. Tissue specific expression of avian vitellogenin gene is correlated with DNA hypomethylation and in vivo specific protein-DNA interactions.

    PubMed

    Jost, J P; Saluz, H P; McEwan, I; Feavers, I M; Hughes, M; Reiber, S; Liang, H M; Vaccaro, M

    1990-01-30

    The avian vitellogenin gene is expressed only in the liver of egg-laying hens. It can, however, be activated in immature chicks or roosters by oestradiol. Parallel to the onset of transcription, there is a demethylation of specific mCpGs in the promoter region and in the oestrogen response element (ERE). The methylation pattern in the promoter region is hormone and expression specific, whereas in the ERE it is only hormone and not organ specific. The demethylation occurring in the promoter region is correlated with the appearance of DNase I hypersensitivity sites and changes in the specific protein-DNA interactions. In vivo genomic footprinting of the ERE with varying concentrations of dimethylsulphate revealed, upon gene activation, only minor changes in the protein-DNA interaction. We present evidence that there is another protein that binds with high affinity to the ERE, besides the oestrogen receptor. PMID:1968660

  8. 454 next generation-sequencing outperforms allele-specific PCR, Sanger sequencing, and pyrosequencing for routine KRAS mutation analysis of formalin-fixed, paraffin-embedded samples

    PubMed Central

    Altimari, Annalisa; de Biase, Dario; De Maglio, Giovanna; Gruppioni, Elisa; Capizzi, Elisa; Degiovanni, Alessio; D’Errico, Antonia; Pession, Annalisa; Pizzolitto, Stefano; Fiorentino, Michelangelo; Tallini, Giovanni

    2013-01-01

    Detection of KRAS mutations in archival pathology samples is critical for therapeutic appropriateness of anti-EGFR monoclonal antibodies in colorectal cancer. We compared the sensitivity, specificity, and accuracy of Sanger sequencing, ARMS-Scorpion (TheraScreen®) real-time polymerase chain reaction (PCR), pyrosequencing, chip array hybridization, and 454 next-generation sequencing to assess KRAS codon 12 and 13 mutations in 60 nonconsecutive selected cases of colorectal cancer. Twenty of the 60 cases were detected as wild-type KRAS by all methods with 100% specificity. Among the 40 mutated cases, 13 were discrepant with at least one method. The sensitivity was 85%, 90%, 93%, and 92%, and the accuracy was 90%, 93%, 95%, and 95% for Sanger sequencing, TheraScreen real-time PCR, pyrosequencing, and chip array hybridization, respectively. The main limitation of Sanger sequencing was its low analytical sensitivity, whereas TheraScreen real-time PCR, pyrosequencing, and chip array hybridization showed higher sensitivity but suffered from the limitations of predesigned assays. Concordance between the methods was k = 0.79 for Sanger sequencing and k > 0.85 for the other techniques. Tumor cell enrichment correlated significantly with the abundance of KRAS-mutated deoxyribonucleic acid (DNA), evaluated as ΔCt for TheraScreen real-time PCR (P = 0.03), percentage of mutation for pyrosequencing (P = 0.001), ratio for chip array hybridization (P = 0.003), and percentage of mutation for 454 next-generation sequencing (P = 0.004). Also, 454 next-generation sequencing showed the best cross correlation for quantification of mutation abundance compared with all the other methods (P < 0.001). Our comparison showed the superiority of next-generation sequencing over the other techniques in terms of sensitivity and specificity. Next-generation sequencing will replace Sanger sequencing as the reference technique for diagnostic detection of KRAS mutation in archival tumor tissues. PMID

  9. Cloning and expression of human neuron-specific enolase cDNA in Escherichia coli.

    PubMed

    Pavlov, K A; Gurina, O I; Antonova, O M; Semenova, A V; Chekhonin, V P

    2011-12-01

    cDNA fragment encoding neuron-specific enolase was amplified from the cDNA library of human brain. Then the fragment was cloned for expression in E. coli using the vector pET28-a. High level of neuron-specific enolase expression was confirmed by SDS-PAAG electrophoresis and immunochemical identity by immunoblot analysis. The constructed producer strain is the cheapest source of neuron-specific enolase suitable for the use in diagnostic applications. PMID:22808461

  10. Association between suicide attempt and a tri-allelic functional polymorphism in serotonin transporter gene promoter in Chinese patients with schizophrenia.

    PubMed

    Hung, Chi-Fa; Lung, For-Wey; Chen, Chien-Hsiun; O'Nions, Elizabeth; Hung, Tai-Hsin; Chong, Mian-Yoon; Wu, Ching-Kuan; Wen, Jung-Kwang; Lin, Pao-Yen

    2011-10-31

    Mounting evidence supports the association between a polymorphism in the serotonin transporter gene promoter region (5-HTTLPR) and suicidal behaviour. Recently, a novel variant of the 5-HTTLPR L allele was identified. The previously unknown L(G) allele produced similar levels of gene expression to the S allele and might have been misclassified as a "high-expression" allele in previous association studies. In this study, we aimed to compare the genotype distribution of the tri-allelic 5-HTTLPR polymorphism in 168 Chinese patients with schizophrenia, including 60 suicide attempters and 108 non-suicide attempters. In our analysis, which used the L(A) dominant model, it was found that the L(A) allele carriers were significantly more likely to have attempted suicide (p=0.035). Further analysis showed this association existed only in male patients (p=0.012). A similar association between the L(A) allele and violent suicide attempt was also found (p=0.028). In addition, logistic regression confirmed our findings that male L(A) allele carriers were at a higher risk of suicide, although the lack of a significant association in females may reflect insufficient power due to small sample size. However, no association was found when we examined the traditional bi-allelic 5-HTTLPR. These findings differ from those reported in Caucasian subjects, where no associations have been reported. Different genetic backgrounds may give rise to different allelic distribution, causing differential effects on the expression of endophenotypes of suicide behaviours. Although the potential influence of multiple comparisons might weaken our findings, our study provides preliminary evidence for a potentially gender-specific role of a "high-expression" 5-HTTLPR polymorphism in susceptibility to suicide in Chinese patients with schizophrenia. PMID:21964390

  11. Allelic Imbalance in Regulation of ANRIL through Chromatin Interaction at 9p21 Endometriosis Risk Locus.

    PubMed

    Nakaoka, Hirofumi; Gurumurthy, Aishwarya; Hayano, Takahide; Ahmadloo, Somayeh; Omer, Waleed H; Yoshihara, Kosuke; Yamamoto, Akihito; Kurose, Keisuke; Enomoto, Takayuki; Akira, Shigeo; Hosomichi, Kazuyoshi; Inoue, Ituro

    2016-04-01

    Genome-wide association studies (GWASs) have discovered numerous single nucleotide polymorphisms (SNPs) associated with human complex disorders. However, functional characterization of the disease-associated SNPs remains a formidable challenge. Here we explored regulatory mechanism of a SNP on chromosome 9p21 associated with endometriosis by leveraging "allele-specific" functional genomic approaches. By re-sequencing 1.29 Mb of 9p21 region and scrutinizing DNase-seq data from the ENCODE project, we prioritized rs17761446 as a candidate functional variant that was in perfect linkage disequilibrium with the original GWAS SNP (rs10965235) and located on DNase I hypersensitive site. Chromosome conformation capture followed by high-throughput sequencing revealed that the protective G allele of rs17761446 exerted stronger chromatin interaction with ANRIL promoter. We demonstrated that the protective allele exhibited preferential binding affinities to TCF7L2 and EP300 by bioinformatics and chromatin immunoprecipitation (ChIP) analyses. ChIP assays for histone H3 lysine 27 acetylation and RNA polymerase II reinforced the enhancer activity of the SNP site. The allele specific expression analysis for eutopic endometrial tissues and endometrial carcinoma cell lines showed that rs17761446 was a cis-regulatory variant where G allele was associated with increased ANRIL expression. Our work illuminates the allelic imbalances in a series of transcriptional regulation from factor binding to gene expression mediated by chromatin interaction underlie the molecular mechanism of 9p21 endometriosis risk locus. Functional genomics on common disease will unlock functional aspect of genotype-phenotype correlations in the post-GWAS stage. PMID:27055116

  12. A comparative approach to understanding tissue-specific expression of uncoupling protein 1 expression in adipose tissue.

    PubMed

    Shore, Andrew; Emes, Richard D; Wessely, Frank; Kemp, Paul; Cillo, Clemente; D'Armiento, Maria; Hoggard, Nigel; Lomax, Michael A

    2012-01-01

    The thermoregulatory function of brown adipose tissue (BAT) is due to the tissue-specific expression of uncoupling protein 1 (UCP1) which is thought to have evolved in early mammals. We report that a CpG island close to the UCP1 transcription start site is highly conserved in all 29 vertebrates examined apart from the mouse and xenopus. Using methylation sensitive restriction digest and bisulfite mapping we show that the CpG island in both the bovine and human is largely un-methylated and is not related to differences in UCP1 expression between white and BAT. Tissue-specific expression of UCP1 has been proposed to be regulated by a conserved 5' distal enhancer which has been reported to be absent in marsupials. We demonstrate that the enhancer, is also absent in five eutherians as well as marsupials, monotremes, amphibians, and fish, is present in pigs despite UCP1 having become a pseudogene, and that absence of the enhancer element does not relate to BAT-specific UCP1 expression. We identify an additional putative 5' regulatory unit which is conserved in 14 eutherian species but absent in other eutherians and vertebrates, but again unrelated to UCP1 expression. We conclude that despite clear evidence of conservation of regulatory elements in the UCP1 5' untranslated region, this does not appear to be related to species or tissues-specific expression of UCP1. PMID:23293654

  13. Validation of a Multiplex Allele-Specific Polymerase Chain Reaction Assay for Detection of KRAS Gene Mutations in Formalin-Fixed, Paraffin-Embedded Tissues from Colorectal Cancer Patients

    PubMed Central

    Seekhuntod, Sirirat; Thavarungkul, Paninee; Chaichanawongsaroj, Nuntaree

    2016-01-01

    Background Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations. Methods We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D). We performed MAS-PCR assay analysis for KRAS on DNA isolated from 270 formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues. Sequences of all 270 samples were determined by pyrosequencing. Seven known point-mutation DNA samples diluted with wild-type DNA were assayed to determine the limitation of detection and reproducibility of the MAS-PCR assay. Results Overall, the results of MAS-PCR assay were in good concordance with pyrosequencing, and only seven discordant samples were found. The MAS-PCR assay reproducibly detected 1 to 2% mutant alleles. The most common mutations were G13D in codon 13 (49.17%), G12D (25.83%) and G12V (12.50%) in codon 12. Conclusion The MAS-PCR assay provides a rapid, cost-effective, and reliable diagnostic tool for accurate detection of KRAS mutations in routine FFPE colorectal cancer tissues. PMID:26812617

  14. Highly specific expression of luciferase gene in lungs of naive nude mice directed by prostate-specific antigen promoter

    SciTech Connect

    Li Hongwei; Li Jinzhong; Helm, Gregory A.; Pan Dongfeng . E-mail: Dongfeng_pan@yahoo.com

    2005-09-09

    PSA promoter has been demonstrated the utility for tissue-specific toxic gene therapy in prostate cancer models. Characterization of foreign gene overexpression in normal animals elicited by PSA promoter should help evaluate therapy safety. Here we constructed an adenovirus vector (AdPSA-Luc), containing firefly luciferase gene under the control of the 5837 bp long prostate-specific antigen promoter. A charge coupled device video camera was used to non-invasively image expression of firefly luciferase in nude mice on days 3, 7, 11 after injection of 2 x 10{sup 9} PFU of AdPSA-Luc virus via tail vein. The result showed highly specific expression of the luciferase gene in lungs of mice from day 7. The finding indicates the potential limitations of the suicide gene therapy of prostate cancer based on selectivity of PSA promoter. By contrary, it has encouraging implications for further development of vectors via PSA promoter to enable gene therapy for pulmonary diseases.

  15. Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase.

    PubMed

    Yaoi, Katsuro; Mitsuishi, Yasushi

    2002-12-13

    A novel oligoxyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH), with a molecular mass of 97 kDa and a pI of 6.1, was isolated from the fungus Geotrichum sp. M128. Analysis of substrate specificity using various xyloglucan oligosaccharide structures revealed that OXG-RCBH had exoglucanase activity. It recognized the reducing end of oligoxyloglucan and released two glucosyl residue segments from the main chain. The full-length cDNA encoding OXG-RCBH was cloned and sequenced, and it had a 2436-bp open reading frame encoding an 812amino acid protein. The deduced protein showed approximately 35% identity to members of glycoside hydrolase family 74. The cDNA encoding OXG-RCBH was then expressed in Escherichia coli. Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant OXG-RCBH was obtained. PMID:12374797

  16. Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168.

    PubMed

    Anjum, Awais; Brathwaite, Kelly J; Aidley, Jack; Connerton, Phillippa L; Cummings, Nicola J; Parkhill, Julian; Connerton, Ian; Bayliss, Christopher D

    2016-06-01

    Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5'CCCGA and 5'CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits. PMID:26786317

  17. Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168

    PubMed Central

    Anjum, Awais; Brathwaite, Kelly J.; Aidley, Jack; Connerton, Phillippa L.; Cummings, Nicola J.; Parkhill, Julian; Connerton, Ian; Bayliss, Christopher D.

    2016-01-01

    Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5′CCCGA and 5′CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits. PMID:26786317

  18. Proliferation and Foxp3 Expression in Virus-Specific Memory CD8+ T Lymphocytes

    PubMed Central

    Hoji, Aki; Coro, Alfonso; Ng, Hwee L.; Jamieson, Beth D.

    2008-01-01

    Abstract Foxp3 plays a critical role in development of CD4+ regulatory T lymphocytes (Tregs). It was originally proposed as a specific marker for Tregs, but recent studies have shown that Foxp3 can be expressed in proliferating CD8+ and CD4+ T lymphocytes. We further investigated the association between Foxp3 expression and proliferation of peripheral blood CD4+ and CD8+ T lymphocytes and focused on virus-specific memory CD8+ T lymphocytes. We found that resting peripheral blood bulk and cytomegalovirus- or HIV-1-specific CD8+ T lymphocytes do not normally express Foxp3. However, stimulation in vitro triggered these cells to express Foxp3 as well as CD25, and the addition of interleukin-2 possibly enhanced the expression of Foxp3. These data demonstrate that proliferation itself is sufficient to induce the Treg-like phenotype. Given that others have demonstrated Treg functional activity in such “induced Tregs,” these results suggest that virus-specific CD8+ T lymphocytes have the capacity to acquire regulatory functions. Although the implications of Foxp3 expression in virus-specific CD8+ T lymphocytes in the immunologic control of persistent HIV-1 viremia remain to be determined, our results are consistent with Foxp3 expression playing an essential role in regulation of cell proliferation and functional outcomes for HIV-1-specific CD8+ T lymphocytes. PMID:18620494

  19. Genome-wide screen of genes imprinted in sorghum endosperm, and the roles of allelic differential cytosine methylation.

    PubMed

    Zhang, Meishan; Li, Ning; He, Wenan; Zhang, Huakun; Yang, Wei; Liu, Bao

    2016-02-01

    Imprinting is an epigenetic phenomenon referring to allele-biased expression of certain genes depending on their parent of origin. Accumulated evidence suggests that, while imprinting is a conserved mechanism across kingdoms, the identities of the imprinted genes are largely species-specific. Using deep RNA sequencing of endosperm 14 days after pollination in sorghum, 5683 genes (29.27% of the total 19 418 expressed genes) were found to harbor diagnostic single nucleotide polymorphisms between two parental lines. The analysis of parent-of-origin expression patterns in the endosperm of a pair of reciprocal F1 hybrids between the two sorghum lines led to identification of 101 genes with ≥ fivefold allelic expression difference in both hybrids, including 85 maternal expressed genes (MEGs) and 16 paternal expressed genes (PEGs). Thirty of these genes were previously identified as imprinted in endosperm of maize (Zea mays), rice (Oryza sativa) or Arabidopsis, while the remaining 71 genes are sorghum-specific imprinted genes relative to these three plant species. Allele-biased expression of virtually all of the 14 tested imprinted genes (nine MEGs and five PEGs) was validated by pyrosequencing using independent sources of RNA from various developmental stages and dissected parts of endosperm. Forty-six imprinted genes (30 MEGs and 16 PEGs) were assayed by quantitative RT-PCR, and the majority of them showed endosperm-specific or preferential expression relative to embryo and other tissues. DNA methylation analysis of the 5' upstream region and gene body for seven imprinted genes indicated that, while three of the four PEGs were associated with hypomethylation of maternal alleles, no MEG was associated with allele-differential methylation. PMID:26718755

  20. Construction of a BALB/c congenic mouse, C.C3H-Lpsd, that expresses the Lpsd allele: analysis of chromosome 4 markers surrounding the Lps gene.

    PubMed Central

    Vogel, S N; Wax, J S; Perera, P Y; Padlan, C; Potter, M; Mock, B A

    1994-01-01

    Development of a congenic BALB/c mouse strain that contains a segment of chromosome 4 including the Lpsd allele of the lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ strain is presented. On the basis of LPS-induced spleen cell mitogenesis, macrophage tumor necrosis factor secretion, and tyrosine phosphorylation in vitro and lethality in galactosamine-sensitized mice in vivo, the C.C3H-Lpsd strain provides a model of LPS hyporesponsiveness that is comparable to that of the parental C3H/HeJ strain. Analysis of markers in this region indicates that length of the donor fragment is approximately 5.5 centimorgans. Thus, the C.C3H-Lpsd strain provides an important genetic tool for analysis of markers in this region and for examining functional effects of Lpsd expression on the BALB/c background. Images PMID:7927709

  1. Cardiac-specific activation of Cre expression at late fetal development

    SciTech Connect

    Opherk, Jan P.; Yampolsky, Peter; Hardt, Stefan E.; Schoels, Wolfgang; Katus, Hugo A.; Koenen, Michael . E-mail: koenen@mpimf-heidelberg.mpg.de; Zehelein, Joerg

    2007-07-27

    In a first step towards dissecting molecular mechanisms that contribute to the development of cardiac diseases, we have generated transgenic mice that express a Cre-GFP fusion protein under the transcriptional control of a 4.3 kb murine cardiac Troponin I gene (cTnI) promoter. Cre-GFP expression, similar in three transgenic lines, is described in one line. In mouse embryos, transgenic for the Cre-GFP and ROSA lacZ reporter allele, first Cre-mediated recombination appeared at 16.5 dpc selectively at the heart. Like the endogenous cTnI gene, transgenic Cre expression showed a slow rise through fetal development that increased neonatally. Bitransgenic hearts, stained at 30 days of age, showed intense signals in ventricular and atrial myocytes while no recombination occurred in other tissues. The delayed onset of Cre activity in cTnI-Cre mice could provide a useful genetic tool to evaluate the function of loxP targeted cardiac genes without interference of recombination during early heart development.

  2. Murine somatic cell nuclear transfer using reprogrammed donor cells expressing male germ cell-specific genes.

    PubMed

    Kang, Hoin; Park, Jong Im; Roh, Sangho

    2016-01-01

    In vivo-matured mouse oocytes were enucleated, and a single murine embryonic fibroblast (control or reprogrammed by introducing extracts from murine testis tissue, which showed expression of male germ cell-specific genes) was injected into the cytoplasm of the oocytes. The rate of blastocyst development and expression levels of Oct-4, Eomes and Cdx-2 were not significantly different in both experimental groups. However, the expression levels of Nanog, Sox9 and Glut-1 were significantly increased when reprogrammed cells were used as donor nuclei. Increased expression of Nanog can be supportive of complete reprogramming of somatic cell nuclear transfer murine embryos. The present study suggested that donor cells expressing male germ cell-specific genes can be reconstructed and can develop into embryos with normal high expression of developmentally essential genes. PMID:26369430

  3. Computational dissection of tissue contamination for identification of colon cancer-specific expression profiles.

    PubMed

    Türeci, Ozlem; Ding, Jiayi; Hilton, Holly; Bian, Hongjin; Ohkawa, Hitomi; Braxenthaler, Michael; Seitz, Gerhard; Raddrizzani, Laura; Friess, Helmut; Buchler, Markus; Sahin, Ugur; Hammer, Juergen

    2003-03-01

    Microarray profiles of bulk tumor tissues reflect gene expression corresponding to malignant cells as well as to many different types of contaminating normal cells. In this report, we assess the feasibility of querying baseline multitissue transcriptome databases to dissect disease-specific genes. Using colon cancer as a model tumor, we show that the application of Boolean operators (AND, OR, BUTNOT) for database searches leads to genes with expression patterns of interest. The BUTNOT operator for example allows the assignment of "expression signatures" to normal tissue specimens. These expression signatures were then used to computationally identify contaminating cells within conventionally dissected tissue specimens. The combination of several logic operators together with an expression database based on multiple human tissue specimens can resolve the problem of tissue contamination, revealing novel cancer-specific gene expression. Several markers, previously not known to be colon cancer associated, are provided. PMID:12631577

  4. Autoimmune diabetes-prone NOD mice express the Lyt2{sup a} (Lyt2.1) and Lyt3{sup a} (Lyt3.1) alleles of CD8

    SciTech Connect

    Johnson-Tardieu, J.M.; Cornelius, J.G.; Ye, X.

    1996-06-01

    Predisposition to Type I insulin-dependent diabetes (IDD) has a strong underlying genetic basis involving class II major histocompatibility complex (MHC) genes as well as several non-MHC genetic systems. In the non-obese diabetic (NOD) mouse, a model for human IDD, genes associated with the appearance of immune cell infiltrates in the pancreatic islets (insulitis) and/or overt IDD have been mapped to chromosomes 1, 3, 6, 11, and 17. A recent report has suggested that CD8+ lymphocytes of the NOD mouse might be deficient in the expression of the CD8{Beta} molecule, a protein encoded by a gene on chromosome 6. The CD8{Beta} molecule is a T-cell surface marker, the lack of which could affect selection in the thymus, possibly permitting auto-reactive T-cell clones to populate the peripheral lymphoid tissues. For this reason, we examined the expression of the CD8 molecule by lymphocytes in the NOD mouse. Results indicate that the NOD mouse is not deficient in its transcription of detectable mRNA encoding either the CD8{alpha} or {Beta} subunits. However, the NOD mouse expresses the Lyt2{sup a} and Lyt3{sup a} alleles, suggesting that a portion of chromosome 6 centromeric to the diabetes-susceptibility genetic region is derived from an ancestry common to AKR and, like AKR, the CD8{alpha} and CD8{Beta}3.1 (but not CD8{Beta}3.2) subunits are detected on the cell surface of T lymphocytes of the NOD mouse. Interestingly, though, the CD8{Beta}3.1 molecule may not be expressed in the NOD mouse to the same extent as it is expressed in the AKR/J mouse, suggesting the possibility that the NOD mouse possesses a defect somewhere between transcription and cell surface expression of the CD8{Beta} molecule. 36 refs., 5 figs.

  5. The Expressive Elaboration of Imaginative Narratives by Children with Specific Language Impairment

    ERIC Educational Resources Information Center

    Ukrainetz, Teresa A.; Gillam, Ronald B.

    2009-01-01

    Purpose: This study investigated the expressive elaboration of narratives from children with specific language impairment (SLI). Method: Forty-eight 6- and 8-year-old children with SLI were compared with forty-eight 6- and 8-year-old typical language (TL) children. Two imaginative narratives were scored for 14 elements of expressive elaboration in…

  6. Microarray analysis of female- and larval-specific gene expression in the horn fly, Haematobia irritans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dominant conditional lethal gene systems are being investigated as population control technologies against agricultural insect pests. One of the critical components of these systems is a highly expressed female-specific gene promoter which can be used to drive expression of a lethality-inducing gene...

  7. Allelic Imbalance in Regulation of ANRIL through Chromatin Interaction at 9p21 Endometriosis Risk Locus

    PubMed Central

    Nakaoka, Hirofumi; Gurumurthy, Aishwarya; Hayano, Takahide; Ahmadloo, Somayeh; Omer, Waleed H; Yoshihara, Kosuke; Yamamoto, Akihito; Kurose, Keisuke; Enomoto, Takayuki; Akira, Shigeo; Hosomichi, Kazuyoshi; Inoue, Ituro

    2016-01-01

    Genome-wide association studies (GWASs) have discovered numerous single nucleotide polymorphisms (SNPs) associated with human complex disorders. However, functional characterization of the disease-associated SNPs remains a formidable challenge. Here we explored regulatory mechanism of a SNP on chromosome 9p21 associated with endometriosis by leveraging “allele-specific” functional genomic approaches. By re-sequencing 1.29 Mb of 9p21 region and scrutinizing DNase-seq data from the ENCODE project, we prioritized rs17761446 as a candidate functional variant that was in perfect linkage disequilibrium with the original GWAS SNP (rs10965235) and located on DNase I hypersensitive site. Chromosome conformation capture followed by high-throughput sequencing revealed that the protective G allele of rs17761446 exerted stronger chromatin interaction with ANRIL promoter. We demonstrated that the protective allele exhibited preferential binding affinities to TCF7L2 and EP300 by bioinformatics and chromatin immunoprecipitation (ChIP) analyses. ChIP assays for histone H3 lysine 27 acetylation and RNA polymerase II reinforced the enhancer activity of the SNP site. The allele specific expression analysis for eutopic endometrial tissues and endometrial carcinoma cell lines showed that rs17761446 was a cis-regulatory variant where G allele was associated with increased ANRIL expression. Our work illuminates the allelic imbalances in a series of transcriptional regulation from factor binding to gene expression mediated by chromatin interaction underlie the molecular mechanism of 9p21 endometriosis risk locus. Functional genomics on common disease will unlock functional aspect of genotype-phenotype correlations in the post-GWAS stage. PMID:27055116

  8. Cloning, expression, and regulation of tissue-specific genes in Drosophila

    SciTech Connect

    Korochkin, L.I.

    1995-08-01

    The family of esterase genes was studied in various Drosophilia species. These genes are classified as tissue-specific and housekeeping ones. The expression of tissue-specific esterases in the male reproductive system of Drosophilia species from the virilis and melanogaster groups was thoroughly examined. Modifier genes controlling activity level, time of synthesis, and distribution in cells of the tissue-specific esterase isozyme from the ejaculatory bulb were revealed. The structural gene coding of this enzyme was isolated, cloned, and sequenced. This gene was shown to be similar in different Drosophilia species; the transcriptional level of tissue specificity of this gene was determined. The possibility of transformating the tissue-specific gene into a housekeeping one was demonstrated. In different Drosophilia species, this gene can be expressed in different parts of the reproductive system. In transgenic males carrying the gene of another species, the foreign gene is expressed as in the donor. 68 refs., 11 figs.

  9. Getting down to specifics: profiling gene expression and protein-DNA interactions in a cell type-specific manner

    PubMed Central

    McClure, Colin D.; Southall, Tony D.

    2015-01-01

    The majority of multicellular organisms are comprised of an extraordinary range of cell types, with different properties and gene expression profiles. Understanding what makes each cell type unique, and how their individual characteristics are attributed, are key questions for both developmental and neurobiologists alike. The brain is an excellent example of the cellular diversity expressed in the majority of eukaryotes. The mouse brain comprises of approximately 75 million neurons varying in morphology, electrophysiology, and preferences for synaptic partners. A powerful process in beginning to pick apart the mechanisms that specify individual characteristics of the cell, as well as their fate, is to profile gene expression patterns, chromatin states, and transcriptional networks in a cell type-specific manner, i.e. only profiling the cells of interest in a particular tissue. Depending on the organism, the questions being investigated, and the material available, certain cell type-specific profiling methods are more suitable than others. This chapter reviews the approaches presently available for selecting and isolating specific cell types and evaluates their key features. PMID:26410031

  10. Systemic Inflammation Affects Human Osteocyte-Specific Protein and Cytokine Expression.

    PubMed

    Pathak, Janak L; Bakker, Astrid D; Luyten, Frank P; Verschueren, Patrick; Lems, Willem F; Klein-Nulend, Jenneke; Bravenboer, Nathalie

    2016-06-01

    Bone remodeling can be disturbed in active rheumatoid arthritis (RA), possibly as a result of elevated levels of circulating inflammatory cytokines. Osteocyte-specific proteins and cytokines play a vital role in bone remodeling by orchestrating bone formation and/or bone resorption. Therefore, we aimed to investigate the effect of RA-serum or inflammatory cytokines on expression of human osteocyte-specific proteins and cytokines. Human trabecular bone chips were cultured with RA-serum or inflammatory cytokines for 7-days. Live-dead staining was performed to assess cell viability. Gene expression of osteocyte-specific proteins and cytokines was analyzed by qPCR. Immuno-staining was performed for osteocyte-specific markers. Approximately 60 % of the osteocytes on the bone chips were alive at day-7. Cells in or on the bone chips did express the gene for osteocyte markers SOST, FGF23, DMP1, and MEPE, and the cytokines IL-1β, IL-6, and TNFα at day 0 and 7. Active RA-serum treatment enhanced IL-1β, TNFα, SOST, and DKK1 gene expression. IL-1β treatment enhanced IL-1β, TNFα, IL-6, IL-8, FGF23, and SOST gene expression. TNFα treatment enhanced IL-1β, TNFα, IL-6, IL-8, and FGF23 gene expression. IL-8 treatment enhanced TNFα, IL-8, and FGF23 gene expression. A combination of IL-1β, IL-6, and TNFα treatment synergistically upregulated IL-1β, IL-6, and IL-8 gene expression, as well as enhanced TNFα, OPG, SOST, and FGF23, and inhibited DKK1 gene expression. In conclusion, gene expression of human osteocyte-specific proteins and cytokines was affected by RA-serum, and exogenous recombinant cytokines treatment suggesting that osteocytes could provide a new target to prevent systemic inflammation-induced bone loss in RA. PMID:26887974

  11. Fucci2a: A bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice

    PubMed Central

    Mort, Richard Lester; Ford, Matthew Jonathan; Sakaue-Sawano, Asako; Lindstrom, Nils Olof; Casadio, Angela; Douglas, Adam Thomas; Keighren, Margaret Anne; Hohenstein, Peter; Miyawaki, Atsushi; Jackson, Ian James

    2014-01-01

    Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes—there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development. PMID:25486356

  12. Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice.

    PubMed

    Mort, Richard Lester; Ford, Matthew Jonathan; Sakaue-Sawano, Asako; Lindstrom, Nils Olof; Casadio, Angela; Douglas, Adam Thomas; Keighren, Margaret Anne; Hohenstein, Peter; Miyawaki, Atsushi; Jackson, Ian James

    2014-01-01

    Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes-there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development. PMID:25486356

  13. The VpreB1 enhancer drives developmental stage-specific gene expression in vivo.

    PubMed

    Licence, Steve; Persson, Christine; Mundt, Cornelia; Mårtensson, Inga-Lill

    2003-04-01

    In adult mice, the VpreB genes are expressed in bone marrow progenitor (pro-) and precursor (pre-) B cells. As part of the pre-B cell receptor, the proteins are crucial for the proliferation of these cells and consequently normal B lymphocyte development. Using cell lines, we identified a lineage- and developmental-stage-specific VpreB1 enhancer. Here, we analyze its specificity in vivo by generating transgenic mice in which expression of a reporter gene (human CD122) is regulated by the VpreB1 enhancer in the context of its own promoter. All transgenic lines expressed the reporter gene in the bone marrow in a copy number-independent manner, whereas expression levels were integration site-dependent. While the enhancer is not tissue specific, within the B cell lineage the expression pattern of human CD122 mimicked that of endogenous VpreB1. Thus, low levels were detected in pro-B cells, high levels in pre-BI and slightly lower levels in pre-BII cells; no expression was detected in immature/mature B cells. Furthermore, when in vitro cultured transgenic pre-B cells differentiated into immature B cells there was concomitant down-regulation of human CD122 and endogenous VpreB1. Thus the VpreB1 enhancer is sufficient to ensure developmental stage-specific expression of a reporter gene in B lymphocytes in vivo. PMID:12672078

  14. Context Specific and Differential Gene Co-expression Networks via Bayesian Biclustering.

    PubMed

    Gao, Chuan; McDowell, Ian C; Zhao, Shiwen; Brown, Christopher D; Engelhardt, Barbara E

    2016-07-01

    Identifying latent structure in high-dimensional genomic data is essential for exploring biological processes. Here, we consider recovering gene co-expression networks from gene expression data, where each network encodes relationships between genes that are co-regulated by shared biological mechanisms. To do this, we develop a Bayesian statistical model for biclustering to infer subsets of co-regulated genes that covary in all of the samples or in only a subset of the samples. Our biclustering method, BicMix, allows overcomplete representations of the data, computational tractability, and joint modeling of unknown confounders and biological signals. Compared with related biclustering methods, BicMix recovers latent structure with higher precision across diverse simulation scenarios as compared to state-of-the-art biclustering methods. Further, we develop a principled method to recover context specific gene co-expression networks from the estimated sparse biclustering matrices. We apply BicMix to breast cancer gene expression data and to gene expression data from a cardiovascular study cohort, and we recover gene co-expression networks that are differential across ER+ and ER- samples and across male and female samples. We apply BicMix to the Genotype-Tissue Expression (GTEx) pilot data, and we find tissue specific gene networks. We validate these findings by using our tissue specific networks to identify trans-eQTLs specific to one of four primary tissues. PMID:27467526

  15. Context Specific and Differential Gene Co-expression Networks via Bayesian Biclustering

    PubMed Central

    McDowell, Ian C.; Zhao, Shiwen; Brown, Christopher D.; Engelhardt, Barbara E.

    2016-01-01

    Identifying latent structure in high-dimensional genomic data is essential for exploring biological processes. Here, we consider recovering gene co-expression networks from gene expression data, where each network encodes relationships between genes that are co-regulated by shared biological mechanisms. To do this, we develop a Bayesian statistical model for biclustering to infer subsets of co-regulated genes that covary in all of the samples or in only a subset of the samples. Our biclustering method, BicMix, allows overcomplete representations of the data, computational tractability, and joint modeling of unknown confounders and biological signals. Compared with related biclustering methods, BicMix recovers latent structure with higher precision across diverse simulation scenarios as compared to state-of-the-art biclustering methods. Further, we develop a principled method to recover context specific gene co-expression networks from the estimated sparse biclustering matrices. We apply BicMix to breast cancer gene expression data and to gene expression data from a cardiovascular study cohort, and we recover gene co-expression networks that are differential across ER+ and ER- samples and across male and female samples. We apply BicMix to the Genotype-Tissue Expression (GTEx) pilot data, and we find tissue specific gene networks. We validate these findings by using our tissue specific networks to identify trans-eQTLs specific to one of four primary tissues. PMID:27467526

  16. Transcriptional Profiling Identifies Location-Specific and Breed-Specific Differentially Expressed Genes in Embryonic Myogenesis in Anas Platyrhynchos

    PubMed Central

    Zhang, Rong-Ping; Liu, He-He; Liu, Jun-Ying; Hu, Ji-Wei; Yan, Xi-Ping; Wang, Ding-Min-Cheng; Li, Liang; Wang, Ji-Wen

    2015-01-01

    Skeletal muscle growth and development are highly orchestrated processes involving significant changes in gene expressions. Differences in the location-specific and breed-specific genes and pathways involved have important implications for meat productions and meat quality. Here, RNA-Seq was performed to identify differences in the muscle deposition between two muscle locations and two duck breeds for functional genomics studies. To achieve those goals, skeletal muscle samples were collected from the leg muscle (LM) and the pectoral muscle (PM) of two genetically different duck breeds, Heiwu duck (H) and Peking duck (P), at embryonic 15 days. Functional genomics studies were performed in two experiments: Experiment 1 directly compared the location-specific genes between PM and LM, and Experiment 2 compared the two breeds (H and P) at the same developmental stage (embryonic 15 days). Almost 13 million clean reads were generated using Illumina technology (Novogene, Beijing, China) on each library, and more than 70% of the reads mapped to the Peking duck (Anas platyrhynchos) genome. A total of 168 genes were differentially expressed between the two locations analyzed in Experiment 1, whereas only 8 genes were differentially expressed when comparing the same location between two breeds in Experiment 2. Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes pathways (KEGG) were used to functionally annotate DEGs (differentially expression genes). The DEGs identified in Experiment 1 were mainly involved in focal adhesion, the PI3K-Akt signaling pathway and ECM-receptor interaction pathways (corrected P-value<0.05). In Experiment 2, the DEGs were associated with only the ribosome signaling pathway (corrected P-value<0.05). In addition, quantitative real-time PCR was used to confirm 15 of the differentially expressed genes originally detected by RNA-Seq. A comparative transcript analysis of the leg and pectoral muscles of two duck breeds not only improves our

  17. Specific gene expression patterns of 108 schizophrenia-associated loci in cortex.

    PubMed

    Ohi, Kazutaka; Shimada, Takamitsu; Nitta, Yusuke; Kihara, Hiroaki; Okubo, Hiroaki; Uehara, Takashi; Kawasaki, Yasuhiro

    2016-07-01

    The latest genome-wide association study of schizophrenia identified 108 distinct genomic loci that contribute to schizophrenia. Brain development and function depend on the precise regulation of gene expression. The expression of many genes is differentially regulated across brain regions and developmental time points. We investigated the specific gene expression patterns arising from the 108 schizophrenia-associated loci using multiple publicly available databases and multiple regional brain datasets from developing and adult post-mortem human brains. The temporal-spatial expression analysis revealed that the genes in these loci were intensively enriched in the cortex during several developmental stages. These cortex-specific genes were particularly expressed in the fetal brain and adult neocortex. PMID:27061659

  18. Sharing and Specificity of Co-expression Networks across 35 Human Tissues

    PubMed Central

    Pierson, Emma; Koller, Daphne; Battle, Alexis; Mostafavi, Sara

    2015-01-01

    To understand the regulation of tissue-specific gene expression, the GTEx Consortium generated RNA-seq expression data for more than thirty distinct human tissues. This data provides an opportunity for deriving shared and tissue specific gene regulatory networks on the basis of co-expression between genes. However, a small number of samples are available for a majority of the tissues, and therefore statistical inference of networks in this setting is highly underpowered. To address this problem, we infer tissue-specific gene co-expression networks for 35 tissues in the GTEx dataset using a novel algorithm, GNAT, that uses a hierarchy of tissues to share data between related tissues. We show that this transfer learning approach increases the accuracy with which networks are learned. Analysis of these networks reveals that tissue-specific transcription factors are hubs that preferentially connect to genes with tissue specific functions. Additionally, we observe that genes with tissue-specific functions lie at the peripheries of our networks. We identify numerous modules enriched for Gene Ontology functions, and show that modules conserved across tissues are especially likely to have functions common to all tissues, while modules that are upregulated in a particular tissue are often instrumental to tissue-specific function. Finally, we provide a web tool, available at mostafavilab.stat.ubc.ca/GNAT, which allows exploration of gene function and regulation in a tissue-specific manner. PMID:25970446

  19. Human platelet antigen 1 (HPA 1) genotyping with 5' nuclease assay and sequence-specific primers reveals a single nucleotide deletion in intron 2 of the HPA 1a allele of platelet glycoprotein IIIa.

    PubMed

    Kjaer, Killie Mette; Jaegtvik, Sissel; Husebekk, Anne; Skogen, Bjorn

    2002-05-01

    We have established a 5' nuclease assay (5' NA) for human platelet antigen (HPA) 1a/b allelic discrimination. The assay is based on the simultaneous amplification and detection of the two targets in a one-tube system. The results are read optically, immediately after termination of the polymerase chain reaction (PCR), and no post-PCR processing is necessary. This genotyping procedure is less time-consuming and cheaper than our conventional sequence-specific primer PCR (SSP-PCR), which is run as a two-tube test, with verification of the results after electrophoresis in agarose gel. The reduction of analytical steps, simplification of the procedure and potential for automation were important advantages for our choice of system. This test system is more suitable for large-scale testing and fits better for our screening programme for HPA 1bb determination. DNA from 1093 individuals were tested in parallel with the SSP-PCR and the 5' NA. One thousand and ninety-one samples gave identical results in SSP-PCR and 5' NA. Upon repeated testing, two samples consistently came out as HPA 1bb in SSP-PCR and HPA 1ab in 5' NA. DNA sequencing revealed a defect located in an intronic area that corresponds to the consensus primer used for the SSP-PCR HPA 1a typing. PMID:11972525

  20. Expression of BCR-ABL1 oncogene relative to ABL1 gene changes overtime in chronic myeloid leukemia

    SciTech Connect

    Gupta, Manu; Milani, Lili; Hermansson, Monica; Simonsson, Bengt; Markevaern, Berit; Syvaenen, Ann Christine; Barbany, Gisela

    2008-02-15

    Using a quantitative single nucleotide polymorphism (SNP) assay we have investigated the changes in the expression of the BCR-ABL1 oncogene relative to the wild-type ABL1 and BCR alleles in cells from chronic myeloid leukemia (CML) patients not responding to therapy. The results show a progressive increase in the BCR-ABL1 oncogene expression at the expense of decreased expression of the ABL1 allele, not involved in the fusion. No relative changes in the expression of the two BCR alleles were found. These results demonstrate that allele-specific changes in gene expression, with selective, progressive silencing of the wild-type ABL1 allele in favor of the oncogenic BCR-ABL1 allele occur in CML patients with therapy-resistant disease.

  1. The Evolutionary Panorama of Organ-Specifically Expressed or Repressed Orthologous Genes in Nine Vertebrate Species

    PubMed Central

    Shen, Libing; Liu, Gangbiao; Zou, Yangyun; Zhou, Zhan; Su, Zhixi; Gu, Xun

    2015-01-01

    RNA sequencing (RNA-Seq) technology provides the detailed transcriptomic information for a biological sample. Using the RNA-Seq data of six organs from nine vertebrate species, we identified a number of organ-specifically expressed or repressed orthologous genes whose expression patterns are mostly conserved across nine species. Our analyses show the following results: (i) About 80% of these genes have a chordate or more ancient origin and more than half of them are the legacy of one or multiple rounds of large-scale gene duplication events. (ii) Their evolutionary rates are shaped by the organ in which they are expressed or repressed, e.g. the genes specially expressed in testis and liver generally evolve more than twice as fast as the ones specially expressed in brain and cerebellum. The organ-specific transcription factors were discriminated from these genes. The ChIP-seq data from the ENCODE project also revealed the transcription-related factors that might be involved in regulating human organ-specifically expressed or repressed genes. Some of them are shared by all six human organs. The comparison of ENCODE data with mouse/chicken ChIP-seq data proposes that organ-specifically expressed or repressed orthologous genes are regulated in various combinatorial fashions in different species, although their expression features are conserved among these species. We found that the duplication events in some gene families might help explain the quick organ/tissue divergence in vertebrate lineage. The phylogenetic analysis of testis-specifically expressed genes suggests that some of them are prone to develop new functions for other organs/tissues. PMID:25679776

  2. In silico analysis of stomach lineage specific gene set expression pattern in gastric cancer

    SciTech Connect

    Pandi, Narayanan Sathiya Suganya, Sivagurunathan; Rajendran, Suriliyandi

    2013-10-04

    Highlights: •Identified stomach lineage specific gene set (SLSGS) was found to be under expressed in gastric tumors. •Elevated expression of SLSGS in gastric tumor is a molecular predictor of metabolic type gastric cancer. •In silico pathway scanning identified estrogen-α signaling is a putative regulator of SLSGS in gastric cancer. •Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. -- Abstract: Stomach lineage specific gene products act as a protective barrier in the normal stomach and their expression maintains the normal physiological processes, cellular integrity and morphology of the gastric wall. However, the regulation of stomach lineage specific genes in gastric cancer (GC) is far less clear. In the present study, we sought to investigate the role and regulation of stomach lineage specific gene set (SLSGS) in GC. SLSGS was identified by comparing the mRNA expression profiles of normal stomach tissue with other organ tissue. The obtained SLSGS was found to be under expressed in gastric tumors. Functional annotation analysis revealed that the SLSGS was enriched for digestive function and gastric epithelial maintenance. Employing a single sample prediction method across GC mRNA expression profiles identified the under expression of SLSGS in proliferative type and invasive type gastric tumors compared to the metabolic type gastric tumors. Integrative pathway activation prediction analysis revealed a close association between estrogen-α signaling and SLSGS expression pattern in GC. Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. In conclusion, our results highlight that estrogen mediated regulation of SLSGS in gastric tumor is a molecular predictor of metabolic type GC and prognostic factor in GC.

  3. Tissue-specific transcript annotation and expression profiling with complementary next-generation sequencing technologies

    PubMed Central

    Hestand, Matthew S.; Klingenhoff, Andreas; Scherf, Matthias; Ariyurek, Yavuz; Ramos, Yolande; van Workum, Wilbert; Suzuki, Makoto; Werner, Thomas; van Ommen, Gert-Jan B.; den Dunnen, Johan T.; Harbers, Matthias; 't Hoen, Peter A.C.

    2010-01-01

    Next-generation sequencing is excellently suited to evaluate the abundance of mRNAs to study gene expression. Here we compare two alternative technologies, cap analysis of gene expression (CAGE) and serial analysis of gene expression (SAGE), for the same RNA samples. Along with quantifying gene expression levels, CAGE can be used to identify tissue-specific transcription start sites, while SAGE monitors 3′-end usage. We used both methods to get more insight into the transcriptional control of myogenesis, studying differential gene expression in differentiated and proliferating C2C12 myoblast cells with statistical evaluation of reproducibility and differential gene expression. Both CAGE and SAGE provided highly reproducible data (Pearson's correlations >0.92 among biological triplicates). With both methods we found around 10 000 genes expressed at levels 2 transcripts per million (0.3 copies per cell), with an overlap of 86%. We identified 4304 and 3846 genes differentially expressed between proliferating and differentiated C2C12 cells by CAGE and SAGE, respectively, with an overlap of 2144. We identified 196 novel regulatory regions with preferential use in proliferating or differentiated cells. Next-generation sequencing of CAGE and SAGE libraries provides consistent expression levels and can enrich current genome annotations with tissue-specific promoters and alternative 3′-UTR usage. PMID:20615900

  4. A conserved apomixis-specific polymorphism is correlated with exclusive exonuclease expression in premeiotic ovules of apomictic boechera species.

    PubMed

    Corral, José M; Vogel, Heiko; Aliyu, Olawale M; Hensel, Götz; Thiel, Thomas; Kumlehn, Jochen; Sharbel, Timothy F

    2013-12-01

    Apomixis (asexual seed production) is characterized by meiotically unreduced egg cell production (apomeiosis) followed by its parthenogenetic development into offspring that are genetic clones of the mother plant. Fertilization (i.e. pseudogamy) of the central cell is important for the production of a functional endosperm with a balanced 2:1 maternal:paternal genome ratio. Here, we present the APOLLO (for apomixis-linked locus) gene, an Aspartate Glutamate Aspartate Aspartate histidine exonuclease whose transcripts are down-regulated in sexual ovules entering meiosis while being up-regulated in apomeiotic ovules at the same stage of development in plants of the genus Boechera. APOLLO has both "apoalleles," which are characterized by a set of linked apomixis-specific polymorphisms, and "sexalleles." All apomictic Boechera spp. accessions proved to be heterozygous for the APOLLO gene (having at least one apoallele and one sexallele), while all sexual genotypes were homozygous for sexalleles. Apoalleles contained a 20-nucleotide polymorphism present in the 5' untranslated region that contains specific transcription factor-binding sites for ARABIDOPSIS THALIANA HOMEOBOX PROTEIN5, LIM1 (for LINEAGE ABNORMAL11, INSULIN1, MECHANOSENSORY PROTEIN3), SORLIP1AT (for SEQUENCES OVERREPRESENTED IN LIGHT-INDUCED PROMOTERS IN ARABIDOPSIS THALIANA1), SORLIP2AT, and POLYA SIGNAL1. In the same region, sexalleles contain transcription factor-binding sites for DNA BINDING WITH ONE FINGER2, DNA BINDING WITH ONE FINGER3, and PROLAMIN BOX-BINDING FACTOR. Our results suggest that the expression of a single deregulated allele could induce the cascade of events leading to asexual female gamete formation in an apomictic plant. PMID:24163323

  5. Identification and characterization of promoters specifically and strongly expressed in maize embryos.

    PubMed

    Liu, Xiaoqing; Tian, Jian; Zhou, Xiaojin; Chen, Rumei; Wang, Lei; Zhang, Chunyi; Zhao, Jun; Fan, Yunliu

    2014-12-01

    The use of maize seeds as bioreactors has several advantages for the production of recombinant proteins in plant biotechnology, but available embryo-specific and strong promoters are limited. Here, we describe a genome-scale microarray-based approach to identify embryo-specifically and strongly expressed genes and their promoters in maize. We identified 28 embryo-preferred and abundantly expressed genes based on our microarray data. These embryo-preferred genes were further analysed using the UniGene database and by quantitative reverse transcriptase-PCR leading to the identification of seven genes (Zm.2098, Zm.13387, Zm.66589, Zm.85502, Zm.68129, Zm.3896 and Zm.2941) as embryo-specific genes with higher expression levels relative to maize globulin-1. The putative promoters of five embryo-specific genes (Zm.13387, Zm.66589, Zm.85502, Zm.3896 and Zm.2941) were isolated and all exhibited strong promoter activities when transiently expressed in maize embryos of 20 DAP. The embryo specificity and expression levels of the promoters of four genes (Zm.13387, Zm.85502, Zm.3896 and Zm.2941) were further examined in transgenic maize plants, revealing that they are strong promoters in embryos of all four developmental stages tested compared with reference globulin-1 promoter. Moreover, Zm.2941 and Zm.3896 promoters are stringently embryo-specific promoters, while Zm.85502 promoter is basically embryo specific yet wounding inducible in non-seed tissues, and Zm.13387 promoter is developmentally expressed in both embryo and aleurone with wounding-induced activity in non-seed tissues. Our study provides novel embryo-specific and strong promoters that are suitable for production of high-level recombinant proteins in maize embryos. PMID:25052028

  6. Histone Modifications at Human Enhancers Reflect Global Cell Type-Specific Gene Expression

    PubMed Central

    Heintzman, Nathaniel D.; Hon, Gary C.; Hawkins, R. David; Kheradpour, Pouya; Stark, Alexander; Harp, Lindsey F.; Ye, Zhen; Lee, Leonard K.; Stuart, Rhona K.; Ching, Christina W.; Ching, Keith A.; Antosiewicz, Jessica E.; Liu, Hui; Zhang, Xinmin; Green, Roland D.; Stewart, Ron; Thomson, James A.; Crawford, Gregory E.; Kellis, Manolis; Ren, Bing

    2010-01-01

    The human body is composed of diverse cell types with distinct functions. While it is known that lineage specification depends on cell specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene1–3, the relative roles of these regulatory elements in this process is not clear. We have previously developed a chromatin immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers, and insulators in the human genome4–6. Here, we use the same approach to identify these elements in multiple cell types and investigated their roles in cell type-specific gene expression. We observed that chromatin state at promoters and CTCF-binding at insulators are largely invariant across diverse cell types. By contrast, enhancers are marked with highly cell type-specific histone modification patterns, strongly correlate to cell type-specific gene expression programs on a global scale, and are functionally active in a cell type-specific manner. Our results defined over 55,000 potential transcriptional enhancers in the human genome, significantly expanding the current catalog of human enhancers and highlighting the role of these elements in cell type-specific gene expression. PMID:19295514

  7. Gene expression profiles of hepatic cell-type specific marker genes in progression of liver fibrosis

    PubMed Central

    Takahara, Yoshiyuki; Takahashi, Mitsuo; Wagatsuma, Hiroki; Yokoya, Fumihiko; Zhang, Qing-Wei; Yamaguchi, Mutsuyo; Aburatani, Hiroyuki; Kawada, Norifumi

    2006-01-01

    AIM: To determine the gene expression profile data for the whole liver during development of dimethylni-trosamine (DMN)-induced hepatic fibrosis. METHODS: Marker genes were identified for different types of hepatic cells, including hepatic stellate cells (HSCs), Kupffer cells (including other inflammatory cells), and hepatocytes, using independent temporal DNA microarray data obtained from isolated hepatic cells. RESULTS: The cell-type analysis of gene expression gave several key results and led to formation of three hypotheses: (1) changes in the expression of HSC-specific marker genes during fibrosis were similar to gene expression data in in vitro cultured HSCs, suggesting a major role of the self-activating characteristics of HSCs in formation of fibrosis; (2) expression of mast cell-specific marker genes reached a peak during liver fibrosis, suggesting a possible role of mast cells in formation of fibrosis; and (3) abnormal expression of hepatocyte-specific marker genes was found across several metabolic pathways during fibrosis, including sulfur-containing amino acid metabolism, fatty acid metabolism, and drug metabolism, suggesting a mechanistic relationship between these abnormalities and symptoms of liver fibrosis. CONCLUSION: Analysis of marker genes for specific hepatic cell types can identify the key aspects of fibrogenesis. Sequential activation of inflammatory cells and the self-supporting properties of HSCs play an important role in development of fibrosis. PMID:17072980

  8. Lamina-specific alterations in cortical GABA(A) receptor subunit expression in schizophrenia.

    PubMed

    Beneyto, Monica; Abbott, Andrew; Hashimoto, Takanori; Lewis, David A

    2011-05-01

    Dysfunction of the dorsolateral prefrontal cortex (DLPFC) in schizophrenia is associated with lamina-specific alterations in particular subpopulations of interneurons. In pyramidal cells, postsynaptic γ-aminobutyric acid (GABA(A)) receptors containing different α subunits are inserted preferentially in distinct subcellular locations targeted by inputs from specific interneuron subpopulations. We used in situ hybridization to quantify the laminar expression of α1, α2, α3, and α5 subunit, and of β1-3 subunit, mRNAs in the DLFPC of schizophrenia, and matched normal comparison subjects. In subjects with schizophrenia, mean GABA(A) α1 mRNA expression was 17% lower in layers 3 and 4, α2 expression was 14% higher in layer 2, α5 expression was 15% lower in layer 4, and α3 expression did not differ relative to comparison subjects. The mRNA expression of β2, which preferentially assembles with α1 subunits, was also 20% lower in layers 3 and 4, whereas β1 and β3 mRNA levels were not altered in schizophrenia. These expression differences were not attributable to medication effects or other potential confounds. These findings suggest that GABA neurotransmission in the DLPFC is altered at the postsynaptic level in a receptor subunit- and layer-specific manner in subjects with schizophrenia and support the hypothesis that GABA neurotransmission in this illness is predominantly impaired in certain cortical microcircuits. PMID:20843900

  9. Detection of BRAF Mutations Using a Fully Automated Platform and Comparison with High Resolution Melting, Real-Time Allele Specific Amplification, Immunohistochemistry and Next Generation Sequencing Assays, for Patients with Metastatic Melanoma

    PubMed Central

    Harlé, Alexandre; Salleron, Julia; Franczak, Claire; Dubois, Cindy; Filhine-Tressarieu, Pierre; Leroux, Agnès; Merlin, Jean-Louis

    2016-01-01

    Background Metastatic melanoma is a severe disease with one of the highest mortality rate in skin diseases. Overall survival has significantly improved with immunotherapy and targeted therapies. Kinase inhibitors targeting BRAF V600 showed promising results. BRAF genotyping is mandatory for the prescription of anti-BRAF therapies. Methods Fifty-nine formalin-fixed paraffin-embedded melanoma samples were assessed using High-Resolution-Melting (HRM) PCR, Real-time allele-specific amplification (RT-ASA) PCR, Next generation sequencing (NGS), immunohistochemistry (IHC) and the fully-automated molecular diagnostics platform IdyllaTM. Sensitivity, specificity, positive predictive value and negative predictive value were calculated using NGS as the reference standard to compare the different assays. Results BRAF mutations were found in 28(47.5%), 29(49.2%), 31(52.5%), 29(49.2%) and 27(45.8%) samples with HRM, RT-ASA, NGS, IdyllaTM and IHC respectively. Twenty-six (81.2%) samples were found bearing a c.1799T>A (p.Val600Glu) mutation, three (9.4%) with a c.1798_1799delinsAA (p.Val600Lys) mutation and one with c.1789_1790delinsTC (p.Leu597Ser) mutation. Two samples were found bearing complex mutations. Conclusions HRM appears the less sensitive assay for the detection of BRAF V600 mutations. The RT-ASA, IdyllaTM and IHC assays are suitable for routine molecular diagnostics aiming at the prescription of anti-BRAF therapies. IdyllaTM assay is fully-automated and requires less than 2 minutes for samples preparation and is the fastest of the tested assays. PMID:27111917

  10. Molecular characterization of antigen B2 subunit in two genotypes of Echinococcus granulosus from Indian bubaline isolates, its stage specific expression and serological evaluation.

    PubMed

    Pan, D; Bera, A K; Bandyopadhyay, S; Das, S; Rana, T; Das, S K; Bandyopadhyay, S; Manna, B; Bhattacharya, D

    2011-03-01

    Echinococcus granulosus is a parasitic helminth which affects both man and animals. During infection with larval stage of the organism secretory and membrane-bound (S/M) proteins play a meaningful role for evasion of immune system. Antigen B (AgB) is one of them. Present investigation has defined sequence diversity of AgB2 subunit of cattle and buffalo isolates of the organism. A total of 55 isolates were screened by polymerase chain reaction based single stranded conformation polymorphism (PCR-SSCP). Subsequently, six conformers could be detected. Based on predicted amino acid sequences of 90 amino acid residues, three clusters could be deduced. Sequence information of two buffalo isolates was homo