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Sample records for neuroblasts dna region

  1. Nerve growth factor enhances DNA synthesis in cultured cerebellar neuroblasts.

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    Confort, C; Charrasse, S; Clos, J

    1991-10-01

    The cerebellar neuroblasts in primary cultures from five-day-old rats bore NGF receptor immunoreactivity, suggesting a potential responsive to NGF. At low plating density, NGF was found to enhance DNA synthesis in these cells in a dose-dependent manner. As these cells synthesize NGF, one possibility to account for the lack of response of neuroblasts plated at high density is that the amount of endogenous trophic agent produced in this culture condition is sufficient to ensure an optimal effect. The results demonstrate that premitotic neuroblasts in the CNS, as well postmitotic neurons, are responsive to NGF. At the early stage of its development, the cerebellum therefore appears to be a very good autocrine model of NGF action.

  2. Microglia from neurogenic and non-neurogenic regions display differential proliferative potential and neuroblast support

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    Gregory Paul Marshall

    2014-07-01

    Full Text Available Microglia isolated from the neurogenic subependymal zone (SEZ and hippocampus (HC are capable of massive in vitro population expansion that is not possible with microglia isolated from non-neurogenic regions. We asked if this regional heterogeneity in microglial proliferative capacity is cell intrinsic, or is conferred by interaction with respective neurogenic or non-neurogenic niches. By combining SEZ and cerebral cortex (CTX primary tissue dissociates to generate heterospatial cultures, we find that exposure to the SEZ environment does not enhance CTX microglia expansion; however, the CTX environment exerts a suppressive effect on SEZ microglia expansion. Furthermore, addition of purified donor SEZ microglia to either CTX- or SEZ-derived cultures suppresses the expansion of host microglia, while the addition of donor CTX microglia enhances the over-all microglia yield. These data suggest that SEZ and CTX microglia possess intrinsic, spatially restricted characteristics that are independent of their in vitro environment, and that they represent unique and functionally distinct populations. Finally, we determined that the repeated supplementation of neurogenic SEZ cultures with expanded SEZ microglia allows for sustained levels of inducible neurogenesis, provided that the ratio of microglia to total cells remains within a fairly narrow range.

  3. Neuroblastic and Schwannian stromal cells of neuroblastoma are derived from a tumoral progenitor cell.

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    Mora, J; Cheung, N K; Juan, G; Illei, P; Cheung, I; Akram, M; Chi, S; Ladanyi, M; Cordon-Cardo, C; Gerald, W L

    2001-09-15

    The coexistence of neuroblastic and Schwannian stromal (SS) cells in differentiating neuroblastoma (NB), and derivation of Schwannian-like cells from neuroblastic clones in vitro, were accepted previously as evidence of a common pluripotent tumor stem line. This paradigm was challenged when SS cells were suggested to be reactive in nature. The advent of microdissection techniques, PCR-based allelic analysis, and in situ fluorescent cytometry made possible the analysis of pure cell populations in fresh surgical specimens, allowing unequivocal determination of clonal origins of various cell subtypes. To overcome the complexity and heterogeneity of three-dimensional tissue structure, we used: (a) Laser-Capture Microdissection to obtain histologically homogeneous cell subtype populations for allelotype analysis at chromosomes 1p36, 11q23, 14q32, and 17q and study of MYCN copy number; (b) multiparametric analysis by Laser-Scanning Cytometry of morphology, DNA content, and immunophenotype of intact cells from touch imprints; and (c) bicolor fluorescence in situ hybridization on touch imprints from manually microdissected neuroblast and stroma-rich areas. Histologically distinct SS and neuroblastic cells isolated by Laser-Capture Microdissection had the same genetic composition in 27 of 28 NB analyzed by allelic imbalance and gene copy number. In all 20 cases studied by Laser-Scanning Cytometry, SS cells identified by morphology and S-100 immunostaining had identical DNA content and GD2-staining pattern as their neuroblastic counterparts. In 7 cases, fluorescence in situ hybridization demonstrated the same chromosomal makeup for SS and neuroblastic cells. These results provide unequivocal evidence that neuroblastic and SS cells in NB are derived from genetically identical neoplastic cells and support the classical paradigm that NB arises from tumoral cells capable of development along multiple lineages.

  4. A model of ischemia-induced neuroblast activation in the adult subventricular zone.

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    Davide Vergni

    Full Text Available We have developed a rat brain organotypic culture model, in which tissue slices contain cortex-subventricular zone-striatum regions, to model neuroblast activity in response to in vitro ischemia. Neuroblast activation has been described in terms of two main parameters, proliferation and migration from the subventricular zone into the injured cortex. We observed distinct phases of neuroblast activation as is known to occur after in vivo ischemia. Thus, immediately after oxygen/glucose deprivation (6-24 hours, neuroblasts reduce their proliferative and migratory activity, whereas, at longer time points after the insult (2 to 5 days, they start to proliferate and migrate into the damaged cortex. Antagonism of ionotropic receptors for extracellular ATP during and after the insult unmasks an early activation of neuroblasts in the subventricular zone, which responded with a rapid and intense migration of neuroblasts into the damaged cortex (within 24 hours. The process is further enhanced by elevating the production of the chemoattractant SDf-1alpha and may also be boosted by blocking the activation of microglia. This organotypic model which we have developed is an excellent in vitro system to study neurogenesis after ischemia and other neurodegenerative diseases. Its application has revealed a SOS response to oxygen/glucose deprivation, which is inhibited by unfavorable conditions due to the ischemic environment. Finally, experimental quantifications have allowed us to elaborate a mathematical model to describe neuroblast activation and to develop a computer simulation which should have promising applications for the screening of drug candidates for novel therapies of ischemia-related pathologies.

  5. The midline protein regulates axon guidance by blocking the reiteration of neuroblast rows within the Drosophila ventral nerve cord.

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    Mary Ann Manavalan

    Full Text Available Guiding axon growth cones towards their targets is a fundamental process that occurs in a developing nervous system. Several major signaling systems are involved in axon-guidance, and disruption of these systems causes axon-guidance defects. However, the specific role of the environment in which axons navigate in regulating axon-guidance has not been examined in detail. In Drosophila, the ventral nerve cord is divided into segments, and half-segments and the precursor neuroblasts are formed in rows and columns in individual half-segments. The row-wise expression of segment-polarity genes within the neuroectoderm provides the initial row-wise identity to neuroblasts. Here, we show that in embryos mutant for the gene midline, which encodes a T-box DNA binding protein, row-2 neuroblasts and their neuroectoderm adopt a row-5 identity. This reiteration of row-5 ultimately creates a non-permissive zone or a barrier, which prevents the extension of interneuronal longitudinal tracts along their normal anterior-posterior path. While we do not know the nature of the barrier, the axon tracts either stall when they reach this region or project across the midline or towards the periphery along this zone. Previously, we had shown that midline ensures ancestry-dependent fate specification in a neuronal lineage. These results provide the molecular basis for the axon guidance defects in midline mutants and the significance of proper specification of the environment to axon-guidance. These results also reveal the importance of segmental polarity in guiding axons from one segment to the next, and a link between establishment of broad segmental identity and axon guidance.

  6. Mcm3 replicative helicase mutation impairs neuroblast proliferation and memory in Drosophila.

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    Blumröder, R; Glunz, A; Dunkelberger, B S; Serway, C N; Berger, C; Mentzel, B; de Belle, J S; Raabe, T

    2016-09-01

    In the developing Drosophila brain, a small number of neural progenitor cells (neuroblasts) generate in a co-ordinated manner a high variety of neuronal cells by integration of temporal, spatial and cell-intrinsic information. In this study, we performed the molecular and phenotypic characterization of a structural brain mutant called small mushroom bodies (smu), which was isolated in a screen for mutants with altered brain structure. Focusing on the mushroom body neuroblast lineages we show that failure of neuroblasts to generate the normal number of mushroom body neurons (Kenyon cells) is the major cause of the smu phenotype. In particular, the premature loss of mushroom body neuroblasts caused a pronounced effect on the number of late-born Kenyon cells. Neuroblasts showed no obvious defects in processes controlling asymmetric cell division, but generated less ganglion mother cells. Cloning of smu uncovered a single amino acid substitution in an evolutionarily conserved protein interaction domain of the Minichromosome maintenance 3 (Mcm3) protein. Mcm3 is part of the multimeric Cdc45/Mcm/GINS (CMG) complex, which functions as a helicase during DNA replication. We propose that at least in the case of mushroom body neuroblasts, timely replication is not only required for continuous proliferation but also for their survival. The absence of Kenyon cells in smu reduced learning and early phases of conditioned olfactory memory. Corresponding to the absence of late-born Kenyon cells projecting to α'/β' and α/β lobes, smu is profoundly defective in later phases of persistent memory. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  7. Postnatal neurogenesis: from neuroblast migration to neuronal integration.

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    Belvindrah, Richard; Lazarini, Françoise; Lledo, Pierre-Marie

    2009-01-01

    Ongoing neurogenesis maintains neuronal replacement in a few regions of the mammalian adult brain. One of these regions, the subventricular zone, generates olfactory bulb interneuron precursors that must migrate through the rostral migratory stream to reach the olfactory bulb circuit. There, they rapidly initiate dendritic growth and establish dendro-dendritic contacts with mitral/tufted cells and potentially other local interneurons. The sequential steps involved in neuroblast maturation during development have been studied extensively over previous years. However, the mechanisms and regulatory factors controlling the recruitment and first steps of synaptic integration of newly-formed neurons in the adult forebrain have only recently started to be elucidated. This review provides an integrated view of our current understanding of fate-choice decision in progenitors, how newborn neurons correctly migrate to specific circuits, how they integrate in olfactory bulb microcircuits, and the function they have to fulfill once they survive. The elucidation of these mechanisms may be crucial to understand the functional role of adult neurogenesis and eventually develop therapeutic strategies aimed at re-routing neuroblasts to altered circuits.

  8. [Clinical picture of neuroblast migratory disorders].

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    Flores-Dinorin, L

    Disturbances of neuroblast migration are prominent among the numerous causes of symptomatic epilepsy and of abnormal neurological development in children. Although their clinical manifestations are generally nonspecific with considerable overlap of symptoms and signs amongst the various disorders, the clinical picture of migratory disorders is continuously being redefined with greater precision and, in some cases, disorders of migration may be grouped into syndromes that are more easily diagnosed during life, in large part because of major advances in recent years in the technology of neuroimaging and in molecular genetics. It is therefore possible to study these patients in greater detail and over longer periods when detected early in life. I have reviewed the clinical manifestations of some of the defined disorders of neuroblast migration: lissencephaly-pachygyria types I and II, pachygyria, schizencephaly, polymicrogyria, special location heterotopia, for example, periventricular heterotopia and subcortical band heterotopia or 'double cortex' syndrome, the latter closely related to isolated lissencephaly type I.

  9. Thyroid hormone promotes transient nerve growth factor synthesis in rat cerebellar neuroblasts.

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    Charrasse, S; Jehan, F; Confort, C; Brachet, P; Clos, J

    1992-01-01

    Primary cultures of cerebellum from 5-day-old rats indicated that proliferating neuroblasts synthesize and release nerve growth factor (NGF). Since NGF promotes DNA synthesis in these cells, our findings demonstrate that the early developing cerebellum is a suitable physiological model for studying the autocrine mitogenic action of NGF. Thyroid deficiency led to a greater reduction in the NGF content of the cerebellum than of the olfactory bulbs or hippocampus. Cerebellar NGF mRNA was also very sensitive to hormone deprivation. Physiological amounts of thyroid hormone stimulated both the mitotic activity and NGF production of cultured cerebellar neuroblasts. A lack of thyroid hormone is known to markedly alter cell formation in the cerebellum where postnatal neurogenesis is highly significant, in contrast to the olfactory bulbs and hippocampus. Taken together, these results suggest that the hormonal control of cell formation in the cerebellum is, at least partly, mediated by the autocrine mitogenic action of NGF. The thyroid hormone could temporally regulate the transient NGF synthesis by cerebellar neuroblasts directly and/or through its ontogenetic action, and hence all the NGF-dependent trophic effects.

  10. Live imaging of Drosophila larval neuroblasts.

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    Lerit, Dorothy A; Plevock, Karen M; Rusan, Nasser M

    2014-07-07

    Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

  11. Sequence analysis of mitochondrial DNA hypervariable region III of ...

    African Journals Online (AJOL)

    The aims of this research were to study mitochondrial DNA hypervariable region III and establish the degree of variation characteristic of a fragment. The mitochondrial DNA (mtDNA) is a small circular genome located within the mitochondria in the cytoplasm of the cell and a smaller 1.2 kb pair fragment, called the control ...

  12. Evidence of a Cell Surface Role for Hsp90 Complex Proteins Mediating Neuroblast Migration in the Subventricular Zone

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    Leo M. Miyakoshi

    2017-05-01

    Full Text Available In most mammalian brains, the subventricular zone (SVZ is a germinative layer that maintains neurogenic activity throughout adulthood. Neuronal precursors arising from this region migrate through the rostral migratory stream (RMS and reach the olfactory bulbs where they differentiate and integrate into the local circuitry. Recently, studies have shown that heat shock proteins have an important role in cancer cell migration and blocking Hsp90 function was shown to hinder cell migration in the developing cerebellum. In this work, we hypothesize that chaperone complexes may have an important function regulating migration of neuronal precursors from the subventricular zone. Proteins from the Hsp90 complex are present in the postnatal SVZ as well as in the RMS. Using an in vitro SVZ explant model, we have demonstrated the expression of Hsp90 and Hop/STI1 by migrating neuroblasts. Treatment with antibodies against Hsp90 and co-chaperone Hop/STI1, as well as Hsp90 and Hsp70 inhibitors hinder neuroblast chain migration. Time-lapse videomicroscopy analysis revealed that cell motility and average migratory speed was decreased after exposure to both antibodies and inhibitors. Antibodies recognizing Hsp90, Hsp70, and Hop/STI1 were found bound to the membranes of cells from primary SVZ cultures and biotinylation assays demonstrated that Hsp70 and Hop/STI1 could be found on the external leaflet of neuroblast membranes. The latter could also be detected in conditioned medium samples obtained from cultivated SVZ cells. Our results suggest that chaperones Hsp90, Hsp70, and co-chaperone Hop/STI1, components of the Hsp90 complex, regulate SVZ neuroblast migration in a concerted manner through an extracellular mechanism.

  13. Prognostic value of partial genetic instability in Neuroblastoma with ? 50% neuroblastic cell content.

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    Piqueras, Marta; Navarro, Samuel; Cañete, Adela; Castel, Victoria; Noguera, Rosa

    2011-01-01

    Abstract Aims. Better understanding of neuroblastoma genetics will improve with genome-wide techniques. However it is not adequated to perform these analyses in samples with less than 60% neuroblastic cell content. We evaluated the utility of FISH on tissue microarrays (TMA) in detecting partial genetic instability (PGI), focussing on samples with ? 50% neuroblastic cells. Methods and results. Alterations of 11q and 17q were detected by FISH on 369 neuroblastic samples included...

  14. Detection of regional DNA methylation using DNA-graphene affinity interactions.

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    Haque, Md Hakimul; Gopalan, Vinod; Yadav, Sharda; Islam, Md Nazmul; Eftekhari, Ehsan; Li, Qin; Carrascosa, Laura G; Nguyen, Nam-Trung; Lam, Alfred K; Shiddiky, Muhammad J A

    2017-01-15

    We report a new method for the detection of regional DNA methylation using base-dependent affinity interaction (i.e., adsorption) of DNA with graphene. Due to the strongest adsorption affinity of guanine bases towards graphene, bisulfite-treated guanine-enriched methylated DNA leads to a larger amount of the adsorbed DNA on the graphene-modified electrodes in comparison to the adenine-enriched unmethylated DNA. The level of the methylation is quantified by monitoring the differential pulse voltammetric current as a function of the adsorbed DNA. The assay is sensitive to distinguish methylated and unmethylated DNA sequences at single CpG resolution by differentiating changes in DNA methylation as low as 5%. Furthermore, this method has been used to detect methylation levels in a collection of DNA samples taken from oesophageal cancer tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. In vitro footprinting of promoter regions within supercoiled plasmid DNA.

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    Sun, Daekyu

    2010-01-01

    Polypurine/polypyrimidine (pPu/pPy) tracts, which exist in the promoter regions of many growth-related genes, have been proposed to be very dynamic in their conformation. In this chapter, we describe a detailed protocol for DNase I and S1 nuclease footprinting experiments with supercoiled plasmid DNA containing the promoter regions to probe whether there are conformational transitions to B-type DNA, melted DNA, and G-quadruplex structures within this tract. This is demonstrated with the proximal promoter region of the human vascular endothelial growth factor (VEGF) gene, which also contains multiple binding sites for Sp1 and Egr-1 transcription factors.

  16. Structure of mitochondrial DNA control region of Argyrosomus ...

    African Journals Online (AJOL)

    The structure of the mitochondrial DNA (mtDNA) control region of Argyrosomus amoyensis was examined in this study. TAS, cTAS, CSB-D to CSB-F and CSB-1 to CSB-3 segments were detected in the species. The results indicated that the structures of these parts were different from that of most fishes. All the mtDNA control ...

  17. Long-term live cell imaging and automated 4D analysis of drosophila neuroblast lineages.

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    Homem, Catarina C F; Reichardt, Ilka; Berger, Christian; Lendl, Thomas; Knoblich, Juergen A

    2013-01-01

    The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.

  18. Long-term live cell imaging and automated 4D analysis of drosophila neuroblast lineages.

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    Catarina C F Homem

    Full Text Available The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.

  19. [Hydrocephaly does not modify neuroblast chain migration in the subventricular zone (SVZ)].

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    González-Pérez, Oscar

    2012-01-01

    Genetic mutations that affect cilia beating have shown that ependymal cilia not only contribute to the movement of cerebrospinal fluid, but also to direct migratory neuroblasts in the subventricular zone. These ciliary disturbances are associated with hydrocephalus. To determine whether hydrocephalus per se alters migration and proliferation of neuroblasts in the subventricular zone, the largest niche of neural stem cells in the adult brain. A vinyl acetate film was surgically inserted into the atrium of the Aqueduct of Sylvius of P60 Balb/C mice. Seven days later, we analyzed the ventricular dilatation, the number of proliferative neural progenitors and migratory neuroblasts, and the organization of neuroblast chains. This model of obstructive hydrocephalus increased the size of the lateral ventricles. No statistically significant differences in cell proliferation (controls 13 ± 2.2 vs. the hydrocephalic group 11 ± 2.9 cells per field) or in the number of neuroblasts (controls 32 ± 3.6 vs. the hydrocephalic group 27 ± 4.8 cells per field). No differences were observed in the migration pattern of neuroblasts. Sub-chronic hydrocephalus did not modify the proliferation of neural precursors or the migration pattern of neuroblasts in the subventricular zone. This suggests that only the CSF flow and the dissolved signaling proteins are the main regulators of the neuronal migration in vivo.

  20. A novel effect of MARCKS phosphorylation by activated PKC: the dephosphorylation of its serine 25 in chick neuroblasts.

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    Andrea Toledo

    Full Text Available MARCKS (Myristoylated Alanine-Rich C Kinase Substrate is a peripheral membrane protein, especially abundant in the nervous system, and functionally related to actin organization and Ca-calmodulin regulation depending on its phosphorylation by PKC. However, MARCKS is susceptible to be phosphorylated by several different kinases and the possible interactions between these phosphorylations have not been fully studied in intact cells. In differentiating neuroblasts, as well as some neurons, there is at least one cell-type specific phosphorylation site: serine 25 (S25 in the chick. We demonstrate here that S25 is included in a highly conserved protein sequence which is a Cdk phosphorylatable region, located far away from the PKC phosphorylation domain. S25 phosphorylation was inhibited by olomoucine and roscovitine in neuroblasts undergoing various states of cell differentiation in vitro. These results, considered in the known context of Cdks activity in neuroblasts, suggest that Cdk5 is the enzyme responsible for this phosphorylation. We find that the phosphorylation by PKC at the effector domain does not occur in the same molecules that are phosphorylated at serine 25. The in situ analysis of the subcellular distribution of these two phosphorylated MARCKS variants revealed that they are also segregated in different protein clusters. In addition, we find that a sustained stimulation of PKC by phorbol-12-myristate-13-acetate (PMA provokes the progressive disappearance of phosphorylation at serine 25. Cells treated with PMA, but in the presence of several Ser/Thr phosphatase (PP1, PP2A and PP2B inhibitors indicated that this dephosphorylation is achieved via a phosphatase 2A (PP2A form. These results provide new evidence regarding the existence of a novel consequence of PKC stimulation upon the phosphorylated state of MARCKS in neural cells, and propose a link between PKC and PP2A activity on MARCKS.

  1. Correlation approach to identify coding regions in DNA sequences

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    Ossadnik, S. M.; Buldyrev, S. V.; Goldberger, A. L.; Havlin, S.; Mantegna, R. N.; Peng, C. K.; Simons, M.; Stanley, H. E.

    1994-01-01

    Recently, it was observed that noncoding regions of DNA sequences possess long-range power-law correlations, whereas coding regions typically display only short-range correlations. We develop an algorithm based on this finding that enables investigators to perform a statistical analysis on long DNA sequences to locate possible coding regions. The algorithm is particularly successful in predicting the location of lengthy coding regions. For example, for the complete genome of yeast chromosome III (315,344 nucleotides), at least 82% of the predictions correspond to putative coding regions; the algorithm correctly identified all coding regions larger than 3000 nucleotides, 92% of coding regions between 2000 and 3000 nucleotides long, and 79% of coding regions between 1000 and 2000 nucleotides. The predictive ability of this new algorithm supports the claim that there is a fundamental difference in the correlation property between coding and noncoding sequences. This algorithm, which is not species-dependent, can be implemented with other techniques for rapidly and accurately locating relatively long coding regions in genomic sequences.

  2. Neurogenesis in the water flea Daphnia magna (Crustacea, Branchiopoda) suggests different mechanisms of neuroblast formation in insects and crustaceans.

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    Ungerer, Petra; Eriksson, Bo Joakim; Stollewerk, Angelika

    2011-09-01

    Within euarthropods, the morphological and molecular mechanisms of early nervous system development have been analysed in insects and several representatives of chelicerates and myriapods, while data on crustaceans are fragmentary. Neural stem cells (neuroblasts) generate the nervous system in insects and in higher crustaceans (malacostracans); in the remaining euarthropod groups, the chelicerates (e.g. spiders) and myriapods (e.g. millipedes), neuroblasts are missing. In the latter taxa, groups of neural precursors segregate from the neuroectoderm and directly differentiate into neurons and glial cells. In all euarthropod groups, achaete-scute homologues are required for neuroblast/neural precursor group formation. In the insects Drosophila melanogaster and Tribolium castaneum achaete-scute homologues are initially expressed in clusters of cells (proneural clusters) in the neuroepithelium but expression becomes restricted to the future neuroblast. Subsequently genes such as snail and prospero are expressed in the neuroblasts which are required for asymmetric division and differentiation. In contrast to insects, malacostracan neuroblasts do not segregate into the embryo but remain in the outer neuroepithelium, similar to vertebrate neural stem cells. It has been suggested that neuroblasts are present in another crustacean group, the branchiopods, and that they also remain in the neuroepithelium. This raises the questions how the molecular mechanisms of neuroblast selection have been modified during crustacean and insect evolution and if the segregation or the maintenance of neuroblasts in the neuroepithelium represents the ancestral state. Here we take advantage of the recently published Daphnia pulex (branchiopod) genome and identify genes in Daphnia magna that are known to be required for the selection and asymmetric division of neuroblasts in the fruit fly D. melanogaster. We unambiguously identify neuroblasts in D. magna by molecular marker gene expression and

  3. Hedgehog signaling acts with the temporal cascade to promote neuroblast cell cycle exit.

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    Phing Chian Chai

    Full Text Available In Drosophila postembryonic neuroblasts, transition in gene expression programs of a cascade of transcription factors (also known as the temporal series acts together with the asymmetric division machinery to generate diverse neurons with distinct identities and regulate the end of neuroblast proliferation. However, the underlying mechanism of how this "temporal series" acts during development remains unclear. Here, we show that Hh signaling in the postembryonic brain is temporally regulated; excess (earlier onset of Hh signaling causes premature neuroblast cell cycle exit and under-proliferation, whereas loss of Hh signaling causes delayed cell cycle exit and excess proliferation. Moreover, the Hh pathway functions downstream of Castor but upstream of Grainyhead, two components of the temporal series, to schedule neuroblast cell cycle exit. Interestingly, hh is likely a target of Castor. Hence, Hh signaling provides a link between the temporal series and the asymmetric division machinery in scheduling the end of neurogenesis.

  4. Revealing large metagenomic regions through long DNA fragment hybridization capture.

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    Gasc, Cyrielle; Peyret, Pierre

    2017-03-14

    High-throughput DNA sequencing technologies have revolutionized genomic analysis, including the de novo assembly of whole genomes from single organisms or metagenomic samples. However, due to the limited capacity of short-read sequence data to assemble complex or low coverage regions, genomes are typically fragmented, leading to draft genomes with numerous underexplored large genomic regions. Revealing these missing sequences is a major goal to resolve concerns in numerous biological studies. To overcome these limitations, we developed an innovative target enrichment method for the reconstruction of large unknown genomic regions. Based on a hybridization capture strategy, this approach enables the enrichment of large genomic regions allowing the reconstruction of tens of kilobase pairs flanking a short, targeted DNA sequence. Applied to a metagenomic soil sample targeting the linA gene, the biomarker of hexachlorocyclohexane (HCH) degradation, our method permitted the enrichment of the gene and its flanking regions leading to the reconstruction of several contigs and complete plasmids exceeding tens of kilobase pairs surrounding linA. Thus, through gene association and genome reconstruction, we identified microbial species involved in HCH degradation which constitute targets to improve biostimulation treatments. This new hybridization capture strategy makes surveying and deconvoluting complex genomic regions possible through large genomic regions enrichment and allows the efficient exploration of metagenomic diversity. Indeed, this approach enables to assign identity and function to microorganisms in natural environments, one of the ultimate goals of microbial ecology.

  5. Mcm3 replicative helicase mutation impairs neuroblast proliferation and memory in Drosophila

    OpenAIRE

    Blumröder, Rebecca; Glunz, Amelie; Dunkelberger, Brian S.; Serway, Christine N.; Berger, Constantin; Mentzel, Benjamin; de Belle, J. Steven; Raabe, Thomas

    2016-01-01

    In the developing Drosophila brain, a small number of neural progenitor cells (neuroblasts) generate in a co-ordinated manner a high variety of neuronal cells by integration of temporal, spatial and cell intrinsic information. In this study, we performed the molecular and phenotypic characterization of a structural brain mutant called small mushroom bodies (smu), which was isolated in a screen for mutants with altered brain structure. Focusing on the mushroom body neuroblast lineages we show ...

  6. Polymorphisms of mitochondrial DNA control region are associated to endometriosis.

    Science.gov (United States)

    Andres, Marina Paula; Cardena, Mari Maki Siria Godoy; Fridman, Cintia; Podgaec, Sergio

    2017-11-09

    Polymorphisms in the control region of mitochondrial DNA (mtDNA) can affect generation of reactive oxygen species and impact in the pathogenesis of endometriosis. This study investigated the association of mtDNA polymorphisms with endometriosis. Patients were divided in two groups: endometriosis (n = 90) and control (n = 92). Inclusion criteria were as follows: women between 18 and 50 years, with histological diagnosis and surgical staging of endometriosis (endometriosis group) or undergoing gynecological surgery for tubal ligation, leiomyoma, or ovarian cysts, with no evidence of endometriosis (control group). DNA extraction was performed from peripheral blood. Sanger sequencing of mtDNA control region was performed, and polymorphisms were determined comparing the sequences obtained with the Cambridge Reference Sequence. The frequency of polymorphisms T16217C (14.4 and 5.4% of endometriosis and control group, respectively; p = 0.049) and G499A (13.3 vs. 4.3%; p = 0.038) was higher in the endometriosis group, while T146C (32.6 vs. 18.9%; p = 0.042) and 573.2C (5.6 vs. 29.3%; p < 0.001) were lower. No difference was observed in haplogroups between groups. mtDNA polymorphisms T16217C and G499A were associated with endometriosis, while T416C and 573.2C were shown to be associated with an absence of disease.

  7. Mechanical forces drive neuroblast morphogenesis and are required for epidermal closure.

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    Wernike, Denise; Chen, Yun; Mastronardi, Karina; Makil, Neetha; Piekny, Alisa

    2016-04-15

    Tissue morphogenesis requires myosin-dependent events such as cell shape changes and migration to be coordinated between cells within a tissue, and/or with cells from other tissues. However, few studies have investigated the simultaneous morphogenesis of multiple tissues in vivo. We found that during Caenorhabditis elegans ventral enclosure, when epidermal cells collectively migrate to cover the ventral surface of the embryo, the underlying neuroblasts (neuronal precursor cells) also undergo morphogenesis. We found that myosin accumulates as foci along the junction-free edges of the ventral epidermal cells to form a ring, whose closure is myosin-dependent. We also observed the accumulation of myosin foci and the adhesion junction proteins E-cadherin and α-catenin in the underlying neuroblasts. Myosin may help to reorganize a subset of neuroblasts into a rosette-like pattern, and decrease their surface area as the overlying epidermal cells constrict. Since myosin is required in the neuroblasts for ventral enclosure, we propose that mechanical forces in the neuroblasts influence constriction of the overlying epidermal cells. In support of this model, disrupting neuroblast cell division or altering their fate influences myosin localization in the overlying epidermal cells. The coordination of myosin-dependent events and forces between cells in different tissues could be a common theme for coordinating morphogenetic events during metazoan development. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. [Heteroplasmy in human mtDNA control region].

    Science.gov (United States)

    Cao, Yang; Wan, Li-Hua; Gu, Lin-Gang; Huang, Ying-Xue; Xiu, Cong-Xian; Hu, Shu-Hui; Mi, Can

    2006-06-01

    To observe the length heteroplasmy and point heteroplasmy in human mtDNA control region. The peripheral blood, buccal cell, and single hair shaft from 50 individuals and 16 family members, related in their maternallineage were analyzed by direct sequencing, and clones from 20 individuals whose mtDNA sequences have a T-C transition at 16189 nt were sequenced. No point heteroplasmy were observed in peripheral blood, buccal cell, single hair shaft from the same individual, neither in maternally related individuals. Length heteroplasmy was observed in those individuals with a homopolymeric tract and the different clones from the same individual has different proportions of length variants, but the hair shafts from the same individual were very similar to the measurements made from blood DNA. No length heteroplasmy was observed between different tissues from the same individual. mtDNA sequences have a characteristic of high consistency and genetic stability, mtDNA sequencing is a suitable tool for forensic applications such as individual identification.

  9. Reduction in subventricular zone-derived olfactory bulb neurogenesis in a rat model of Huntington's disease is accompanied by striatal invasion of neuroblasts.

    Directory of Open Access Journals (Sweden)

    Mahesh Kandasamy

    Full Text Available Huntington's disease (HD is an inherited progressive neurodegenerative disorder caused by an expanded CAG repeat in exon 1 of the huntingtin gene (HTT. The primary neuropathology of HD has been attributed to the preferential degeneration of medium spiny neurons (MSN in the striatum. Reports on striatal neurogenesis have been a subject of debate; nevertheless, it should be considered as an endogenous attempt to repair the brain. The subventricular zone (SVZ might offer a close-by region to supply the degenerated striatum with new cells. Previously, we have demonstrated that R6/2 mice, a widely used preclinical model representing an early onset HD, showed reduced olfactory bulb (OB neurogenesis but induced striatal migration of neuroblasts without affecting the proliferation of neural progenitor cell (NPCs in the SVZ. The present study revisits these findings, using a clinically more relevant transgenic rat model of late onset HD (tgHD rats carrying the human HTT gene with 51 CAG repeats and mimicking many of the neuropathological features of HD seen in patients. We demonstrate that cell proliferation is reduced in the SVZ and OB of tgHD rats compared to WT rats. In the OB of tgHD rats, although cell survival was reduced, the frequency of neuronal differentiation was not altered in the granule cell layer (GCL compared to the WT rats. However, an increased frequency of dopamenergic neuronal differentiation was noticed in the glomerular layer (GLOM of tgHD rats. Besides this, we observed a selective proliferation of neuroblasts in the adjacent striatum of tgHD rats. There was no evidence for neuronal maturation and survival of these striatal neuroblasts. Therefore, the functional role of these invading neuroblasts still needs to be determined, but they might offer an endogenous alternative for stem or neuronal cell transplantation strategies.

  10. DNA barcode regions for differentiating Cattleya walkeriana and C. loddigesii

    Directory of Open Access Journals (Sweden)

    Hernando Rivera-Jiménez

    2017-05-01

    Full Text Available Growers appreciate Cattleya walkeriana and C. loddigesii due to striking shape and rarity. Thus, this study aimed to evaluate the feasibility of DNA barcode regions, namely ITS1, ITS2 and rpoC1, to discriminate between C. walkeriana and C. loddigesii species. DNA barcode regions were successfully amplified using primers designed to amplify plants. We also included sequences from public databases in order to test if these regions were able to discriminate C. walkeriana and C. loddigesii from other Cattleya species. These regions, and their combinations, demonstrated that the ITS1+ITS2 had the highest average interspecific distance (11.1%, followed by rpoC1 (1.06%. For species discrimination, ITS1+ITS2 provided the best results. The combined data set of ITS1+ITS2+rpoC1 also discriminated both species, but did not result in higher rates of discrimination. These results indicate that ITS region is the best option for molecular identification of these two species and from some other species of this genus.

  11. GEAR: genomic enrichment analysis of regional DNA copy number changes.

    Science.gov (United States)

    Kim, Tae-Min; Jung, Yu-Chae; Rhyu, Mun-Gan; Jung, Myeong Ho; Chung, Yeun-Jun

    2008-02-01

    We developed an algorithm named GEAR (genomic enrichment analysis of regional DNA copy number changes) for functional interpretation of genome-wide DNA copy number changes identified by array-based comparative genomic hybridization. GEAR selects two types of chromosomal alterations with potential biological relevance, i.e. recurrent and phenotype-specific alterations. Then it performs functional enrichment analysis using a priori selected functional gene sets to identify primary and clinical genomic signatures. The genomic signatures identified by GEAR represent functionally coordinated genomic changes, which can provide clues on the underlying molecular mechanisms related to the phenotypes of interest. GEAR can help the identification of key molecular functions that are activated or repressed in the tumor genomes leading to the improved understanding on the tumor biology. GEAR software is available with online manual in the website, http://www.systemsbiology.co.kr/GEAR/.

  12. Analysis of human accelerated DNA regions using archaic hominin genomes.

    Directory of Open Access Journals (Sweden)

    Hernán A Burbano

    Full Text Available Several previous comparisons of the human genome with other primate and vertebrate genomes identified genomic regions that are highly conserved in vertebrate evolution but fast-evolving on the human lineage. These human accelerated regions (HARs may be regions of past adaptive evolution in humans. Alternatively, they may be the result of non-adaptive processes, such as biased gene conversion. We captured and sequenced DNA from a collection of previously published HARs using DNA from an Iberian Neandertal. Combining these new data with shotgun sequence from the Neandertal and Denisova draft genomes, we determine at least one archaic hominin allele for 84% of all positions within HARs. We find that 8% of HAR substitutions are not observed in the archaic hominins and are thus recent in the sense that the derived allele had not come to fixation in the common ancestor of modern humans and archaic hominins. Further, we find that recent substitutions in HARs tend to have come to fixation faster than substitutions elsewhere in the genome and that substitutions in HARs tend to cluster in time, consistent with an episodic rather than a clock-like process underlying HAR evolution. Our catalog of sequence changes in HARs will help prioritize them for functional studies of genomic elements potentially responsible for modern human adaptations.

  13. Analysis of Human Accelerated DNA Regions Using Archaic Hominin Genomes

    Science.gov (United States)

    Burbano, Hernán A.; Green, Richard E.; Maricic, Tomislav; Lalueza-Fox, Carles; de la Rasilla, Marco; Rosas, Antonio; Kelso, Janet; Pollard, Katherine S.; Lachmann, Michael; Pääbo, Svante

    2012-01-01

    Several previous comparisons of the human genome with other primate and vertebrate genomes identified genomic regions that are highly conserved in vertebrate evolution but fast-evolving on the human lineage. These human accelerated regions (HARs) may be regions of past adaptive evolution in humans. Alternatively, they may be the result of non-adaptive processes, such as biased gene conversion. We captured and sequenced DNA from a collection of previously published HARs using DNA from an Iberian Neandertal. Combining these new data with shotgun sequence from the Neandertal and Denisova draft genomes, we determine at least one archaic hominin allele for 84% of all positions within HARs. We find that 8% of HAR substitutions are not observed in the archaic hominins and are thus recent in the sense that the derived allele had not come to fixation in the common ancestor of modern humans and archaic hominins. Further, we find that recent substitutions in HARs tend to have come to fixation faster than substitutions elsewhere in the genome and that substitutions in HARs tend to cluster in time, consistent with an episodic rather than a clock-like process underlying HAR evolution. Our catalog of sequence changes in HARs will help prioritize them for functional studies of genomic elements potentially responsible for modern human adaptations. PMID:22412940

  14. Specific requirement of putrescine for the mitogenic action of juvenile hormone on adult insect neuroblasts

    Science.gov (United States)

    Cayre, Myriam; Strambi, Colette; Charpin, Pierre; Augier, Roger; Strambi, Alain

    1997-01-01

    Persistent neurogenesis in an adult insect brain was recently shown to be stimulated by juvenile hormone (JH). This morphogenetic hormone was also shown to act on polyamine biosynthesis. To analyze the possible involvement of polyamines in the neurogenic action of JH, two series of experiments were carried out with adult female crickets, Acheta domesticus: (i) inhibition of the first key enzyme in polyamine biosynthesis, ornithine decarboxylase, with α-difluoromethylornithine (α-DFMO), and examination of the effects of this treatment on the neuroblast proliferation response to JH; and (ii) examination of the effects of putrescine supplementation on the mitotic index of JH-deprived and α-DFMO-treated females. In control females, α-DFMO treatment, as well as JH deprivation, greatly reduced neuroblast proliferation. Putrescine supplementation in α-DFMO-treated insects overcame the effects of α-DFMO, and allowed for detection of putrescine in the neural tissue and stimulation of brain neurogenesis. In JH-deprived females, α-DFMO treatment completely prevented the stimulatory action of JH on neuroblast proliferation and on brain putrescine levels. By contrast, putrescine feeding of JH-deprived animals was able to mimic the stimulatory effect of JH: brain putrescine levels increased and neuroblast proliferation was restored. To our knowledge, this report demonstrates for the first time that in vivo administration of putrescine can mimic the effects of a morphogenetic hormone on adult neuroblast proliferation, and shows the importance of polyamines, especially putrescine, in the transduction of JH message in neural tissue. PMID:9223345

  15. NKCC1 controls GABAergic signaling and neuroblast migration in the postnatal forebrain

    Directory of Open Access Journals (Sweden)

    Murray Kerren

    2011-02-01

    Full Text Available Abstract From an early postnatal period and throughout life there is a continuous production of olfactory bulb (OB interneurons originating from neuronal precursors in the subventricular zone. To reach the OB circuits, immature neuroblasts migrate along the rostral migratory stream (RMS. In the present study, we employed cultured postnatal mouse forebrain slices and used lentiviral vectors to label neuronal precursors with GFP and to manipulate the expression levels of the Na-K-2Cl cotransporter NKCC1. We investigated the role of this Cl- transporter in different stages of postnatal neurogenesis, including neuroblast migration and integration in the OB networks once they have reached the granule cell layer (GCL. We report that NKCC1 activity is necessary for maintaining normal migratory speed. Both pharmacological and genetic manipulations revealed that NKCC1 maintains high [Cl-]i and regulates the resting membrane potential of migratory neuroblasts whilst its functional expression is strongly reduced at the time cells reach the GCL. As in other developing systems, NKCC1 shapes GABAA-dependent signaling in the RMS neuroblasts. Also, we show that NKCC1 controls the migration of neuroblasts in the RMS. The present study indeed indicates that the latter effect results from a novel action of NKCC1 on the resting membrane potential, which is independent of GABAA-dependent signaling. All in all, our findings show that early stages of the postnatal recruitment of OB interneurons rely on precise, orchestrated mechanisms that depend on multiple actions of NKCC1.

  16. Large sequence divergence of mitochondrial DNA genotypes of the control region within populations of the African antelope, kob (Kobus kob)

    DEFF Research Database (Denmark)

    Birungi, J.; Arctander, Peter

    2000-01-01

    conservation genetics, control region, Kobus kob, mitochondrial DNA, population expansion, population structure......conservation genetics, control region, Kobus kob, mitochondrial DNA, population expansion, population structure...

  17. DNA requirements for interaction of the C-terminal region of Ku80 with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs).

    Science.gov (United States)

    Radhakrishnan, Sarvan Kumar; Lees-Miller, Susan P

    2017-09-01

    Non-homologous end joining (NHEJ) is the major pathway for the repair of ionizing radiation induced DNA double strand breaks (DSBs) in human cells. Critical to NHEJ is the DNA-dependent interaction of the Ku70/80 heterodimer with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form the DNA-PK holoenzyme. However, precisely how Ku recruits DNA-PKcs to DSBs ends to enhance its kinase activity has remained enigmatic, with contradictory findings reported in the literature. Here we address the role of the Ku80 C-terminal region (CTR) in the DNA-dependent interaction of Ku70/80 with DNA-PKcs using purified components and defined DNA structures. Our results show that the Ku80 CTR is required for interaction with DNA-PKcs on short segments of blunt ended 25bp dsDNA or 25bp dsDNA with a 15-base poly dA single stranded (ss) DNA extension, but this requirement is less stringent on longer dsDNA molecules (35bp blunt ended dsDNA) or 25bp duplex DNA with either a 15-base poly dT or poly dC ssDNA extension. Moreover, the DNA-PKcs-Ku complex preferentially forms on 25 bp DNA with a poly-pyrimidine ssDNA extension.Our work clarifies the role of the Ku80 CTR and dsDNA ends on the interaction of DNA-PKcs with Ku and provides key information to guide assembly and biology of NHEJ complexes. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Tax impairs DNA replication forks and increases DNA breaks in specific oncogenic genome regions.

    Science.gov (United States)

    Chaib-Mezrag, Hassiba; Lemaçon, Delphine; Fontaine, Hélène; Bellon, Marcia; Bai, Xue Tao; Drac, Marjorie; Coquelle, Arnaud; Nicot, Christophe

    2014-09-04

    Human T-cell leukemia virus type 1 (HTLV-I) is a human retrovirus associated with adult T-cell leukemia (ATL), an aggressive CD4 T-cell proliferative disease with dismal prognosis. The long latency preceding the development of the disease and the low incidence suggests that the virus itself is not sufficient for transformation and that genetic defects are required to create a permissive environment for leukemia. In fact, ATL cells are characterized by profound genetic modifications including structural and numerical chromosome alterations. In this study we used molecular combing techniques to study the effect of the oncoprotein Tax on DNA replication. We found that replication forks have difficulties replicating complex DNA, fork progression is slower, and they pause or stall more frequently in the presence of Tax expression. Our results also show that Tax-associated replication defects are partially compensated by an increase in the firing of back-up origins. Consistent with these effects of Tax on DNA replication, an increase in double strand DNA breaks (DDSB) was seen in Tax expressing cells. Tax-mediated increases in DDSBs were associated with the ability of Tax to activate NF-kB and to stimulate intracellular nitric oxide production. We also demonstrated a reduced expression of human translesion synthesis (TLS) DNA polymerases Pol-H and Pol-K in HTLV-I-transformed T cells and ATL cells. This was associated with an increase in DNA breaks induced by Tax at specific genome regions, such as the c-Myc and the Bcl-2 major breakpoints. Consistent with the notion that the non-homologous end joining (NHEJ) pathway is hyperactive in HTLV-I-transformed cells, we found that inhibition of the NHEJ pathway induces significant killing of HTLV-I transformed cells and patient-derived leukemic ATL cells. Our results suggest that, replication problems increase genetic instability in HTLV-I-transformed cells. As a result, abuse of NHEJ and a defective homologous repair (HR) DNA

  19. Apical constriction is driven by a pulsatile apical myosin network in delaminating Drosophila neuroblasts.

    Science.gov (United States)

    An, Yanru; Xue, Guosheng; Shaobo, Yang; Mingxi, Deng; Zhou, Xiaowei; Yu, Weichuan; Ishibashi, Toyotaka; Zhang, Lei; Yan, Yan

    2017-06-15

    Cell delamination is a conserved morphogenetic process important for the generation of cell diversity and maintenance of tissue homeostasis. Here, we used Drosophila embryonic neuroblasts as a model to study the apical constriction process during cell delamination. We observe dynamic myosin signals both around the cell adherens junctions and underneath the cell apical surface in the neuroectoderm. On the cell apical cortex, the nonjunctional myosin forms flows and pulses, which are termed medial myosin pulses. Quantitative differences in medial myosin pulse intensity and frequency are crucial to distinguish delaminating neuroblasts from their neighbors. Inhibition of medial myosin pulses blocks delamination. The fate of a neuroblast is set apart from that of its neighbors by Notch signaling-mediated lateral inhibition. When we inhibit Notch signaling activity in the embryo, we observe that small clusters of cells undergo apical constriction and display an abnormal apical myosin pattern. Together, these results demonstrate that a contractile actomyosin network across the apical cell surface is organized to drive apical constriction in delaminating neuroblasts. © 2017. Published by The Company of Biologists Ltd.

  20. Pten deletion causes mTorc1-dependent ectopic neuroblast differentiation without causing uniform migration defects.

    Science.gov (United States)

    Zhu, Guo; Chow, Lionel M L; Bayazitov, Ildar T; Tong, Yiai; Gilbertson, Richard J; Zakharenko, Stanislav S; Solecki, David J; Baker, Suzanne J

    2012-09-01

    Neuronal precursors, generated throughout life in the subventricular zone, migrate through the rostral migratory stream to the olfactory bulb where they differentiate into interneurons. We found that the PI3K-Akt-mTorc1 pathway is selectively inactivated in migrating neuroblasts in the subventricular zone and rostral migratory stream, and activated when these cells reach the olfactory bulb. Postnatal deletion of Pten caused aberrant activation of the PI3K-Akt-mTorc1 pathway and an enlarged subventricular zone and rostral migratory stream. This expansion was caused by premature termination of migration and differentiation of neuroblasts and was rescued by inhibition of mTorc1. This phenotype is reminiscent of lamination defects caused by Pten deletion in developing brain that were previously described as defective migration. However, live imaging in acute slices showed that Pten deletion did not cause a uniform defect in the mechanics of directional neuroblast migration. Instead, a subpopulation of Pten-null neuroblasts showed minimal movement and altered morphology associated with differentiation, whereas the remainder showed unimpeded directional migration towards the olfactory bulb. Therefore, migration defects of Pten-null neurons might be secondary to ectopic differentiation.

  1. Iodination as a probe for small regions of disrupted secondary structure in double-stranded DNA

    DEFF Research Database (Denmark)

    Jensen, Kaj Frank; Nes, Ingolf F.; Wells, Robert D.

    1976-01-01

    , if they existed within an otherwise helical DNA fragment 789 base pairs long. Iodination studies were performed on superhelical SV40 DNA and on linear plac DNA. Analysis of the relative amount of iodine in restriction endonuclease fragments of these DNAs revealed the absence of localized single-stranded regions....

  2. Gene Expression Divergence is Coupled to Evolution of DNA Structure in Coding Regions

    Science.gov (United States)

    Dai, Zhiming; Dai, Xianhua

    2011-01-01

    Sequence changes in coding region and regulatory region of the gene itself (cis) determine most of gene expression divergence between closely related species. But gene expression divergence between yeast species is not correlated with evolution of primary nucleotide sequence. This indicates that other factors in cis direct gene expression divergence. Here, we studied the contribution of DNA three-dimensional structural evolution as cis to gene expression divergence. We found that the evolution of DNA structure in coding regions and gene expression divergence are correlated in yeast. Similar result was also observed between Drosophila species. DNA structure is associated with the binding of chromatin remodelers and histone modifiers to DNA sequences in coding regions, which influence RNA polymerase II occupancy that controls gene expression level. We also found that genes with similar DNA structures are involved in the same biological process and function. These results reveal the previously unappreciated roles of DNA structure as cis-effects in gene expression. PMID:22125484

  3. A new database of mitochondrial DNA hypervariable regions I and II sequences from 162 Japanese individuals.

    Science.gov (United States)

    Imaizumi, K; Parsons, T J; Yoshino, M; Holland, M M

    2002-04-01

    A database of mitochondrial DNA (mtDNA) hypervariable region 1 (HV1) and region 2 (HV2) sequences of the mtDNA control region was established from 162 unrelated Japanese individuals. The random match probability and the genetic diversity for this database were 0.96% and 0.997, respectively. Length heteroplasmy in the C-stretch regions located around position 16189 in HVI and 310 in HV2 was observed in 37% and 38% of the samples, respectively. A strategy using internal sequencing primers was devised to obtain confirmed sequences in these length heteroplasmic individuals. This database, combined with other mtDNA sequence databases from the Japanese population, will permit the significance of mtDNA match results to be properly reported in mtDNA typing casework in Japan.

  4. Sequence analysis of mitochondrial DNA hypervariable region III of ...

    African Journals Online (AJOL)

    Aghomotsegin

    2015-07-01

    Jul 1, 2015 ... degradation; third, higher rate of evolution: DNA alterations (mutations) occur in a number of ... The result is that the rate of change, or evolutionary rate, of mitochondrial DNA is about five times greater .... example mass graves in mass disasters, there are newly discovered forensically validated methods ...

  5. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

    NARCIS (Netherlands)

    Schoch, C.L.; Seifert, K.A.; Huhndorf, S.; Robert, V.; Spouge, J.L.; Levesque, C.A.; Chen, W.; Fungal Barcoding Consortium, [No Value

    2012-01-01

    Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it

  6. Rapid turnover of DnaA at replication origin regions contributes to initiation control of DNA replication.

    Science.gov (United States)

    Schenk, Katrin; Hervás, Ana B; Rösch, Thomas C; Eisemann, Marc; Schmitt, Bernhard A; Dahlke, Stephan; Kleine-Borgmann, Luise; Murray, Seán M; Graumann, Peter L

    2017-02-01

    DnaA is a conserved key regulator of replication initiation in bacteria, and is homologous to ORC proteins in archaea and in eukaryotic cells. The ATPase binds to several high affinity binding sites at the origin region and upon an unknown molecular trigger, spreads to several adjacent sites, inducing the formation of a helical super structure leading to initiation of replication. Using FRAP analysis of a functional YFP-DnaA allele in Bacillus subtilis, we show that DnaA is bound to oriC with a half-time of 2.5 seconds. DnaA shows similarly high turnover at the replication machinery, where DnaA is bound to DNA polymerase via YabA. The absence of YabA increases the half time binding of DnaA at oriC, showing that YabA plays a dual role in the regulation of DnaA, as a tether at the replication forks, and as a chaser at origin regions. Likewise, a deletion of soj (encoding a ParA protein) leads to an increase in residence time and to overinitiation, while a mutation in DnaA that leads to lowered initiation frequency, due to a reduced ATPase activity, shows a decreased residence time on binding sites. Finally, our single molecule tracking experiments show that DnaA rapidly moves between chromosomal binding sites, and does not arrest for more than few hundreds of milliseconds. In Escherichia coli, DnaA also shows low residence times in the range of 200 ms and oscillates between spatially opposite chromosome regions in a time frame of one to two seconds, independently of ongoing transcription. Thus, DnaA shows extremely rapid binding turnover on the chromosome including oriC regions in two bacterial species, which is influenced by Soj and YabA proteins in B. subtilis, and is crucial for balanced initiation control, likely preventing fatal premature multimerization and strand opening of DnaA at oriC.

  7. Identification and characterization of neuroblasts in the subventricular zone and rostral migratory stream of the adult human brain

    Science.gov (United States)

    Wang, Congmin; Liu, Fang; Liu, Ying-Ying; Zhao, Cai-Hong; You, Yan; Wang, Lei; Zhang, Jingxiao; Wei, Bin; Ma, Tong; Zhang, Qiangqiang; Zhang, Yue; Chen, Rui; Song, Hongjun; Yang, Zhengang

    2011-01-01

    It is of great interest to identify new neurons in the adult human brain, but the persistence of neurogenesis in the subventricular zone (SVZ) and the existence of the rostral migratory stream (RMS)-like pathway in the adult human forebrain remain highly controversial. In the present study, we have described the general configuration of the RMS in adult monkey, fetal human and adult human brains. We provide evidence that neuroblasts exist continuously in the anterior ventral SVZ and RMS of the adult human brain. The neuroblasts appear singly or in pairs without forming chains; they exhibit migratory morphologies and co-express the immature neuronal markers doublecortin, polysialylated neural cell adhesion molecule and βIII-tubulin. Few of these neuroblasts appear to be actively proliferating in the anterior ventral SVZ but none in the RMS, indicating that neuroblasts distributed along the RMS are most likely derived from the ventral SVZ. Interestingly, no neuroblasts are found in the adult human olfactory bulb. Taken together, our data suggest that the SVZ maintains the ability to produce neuroblasts in the adult human brain. PMID:21577236

  8. Forensic DNA databases in Western Balkan region: retrospectives, perspectives, and initiatives

    Science.gov (United States)

    Marjanović, Damir; Konjhodžić, Rijad; Butorac, Sara Sanela; Drobnič, Katja; Merkaš, Siniša; Lauc, Gordan; Primorac, Damir; Anđelinović, Šimun; Milosavljević, Mladen; Karan, Željko; Vidović, Stojko; Stojković, Oliver; Panić, Bojana; Vučetić Dragović, Anđelka; Kovačević, Sandra; Jakovski, Zlatko; Asplen, Chris; Primorac, Dragan

    2011-01-01

    The European Network of Forensic Science Institutes (ENFSI) recommended the establishment of forensic DNA databases and specific implementation and management legislations for all EU/ENFSI members. Therefore, forensic institutions from Bosnia and Herzegovina, Serbia, Montenegro, and Macedonia launched a wide set of activities to support these recommendations. To assess the current state, a regional expert team completed detailed screening and investigation of the existing forensic DNA data repositories and associated legislation in these countries. The scope also included relevant concurrent projects and a wide spectrum of different activities in relation to forensics DNA use. The state of forensic DNA analysis was also determined in the neighboring Slovenia and Croatia, which already have functional national DNA databases. There is a need for a ‘regional supplement’ to the current documentation and standards pertaining to forensic application of DNA databases, which should include regional-specific preliminary aims and recommendations. PMID:21674821

  9. Morphologic Alteration of Metastatic Neuroblastic Tumor in Bone Marrow after Chemotherapy

    OpenAIRE

    Bae, Go Eun; Suh, Yeon-Lim; Sung, Ki Woong; Kim, Jung-Sun

    2013-01-01

    Background The aim of this study is to evaluate the histologic features of metastatic neuroblastic tumors (NTs) in bone marrow (BM) before and after chemotherapy in comparison with those of primary NTs. Methods A total of 294 biopsies from 48 children diagnosed with NTs with BM metastasis were examined. There were 48 primary neoplasm biopsies, 48 BM biopsies before chemotherapy, 36 primary neoplasm excisional biopsies after chemotherapy, and 162 BM biopsies after chemotherapy. Results Metasta...

  10. Evidence of altered DNA integrity in the brain regions of suicidal victims of Bipolar Depression.

    Science.gov (United States)

    Mustak, Mohammed S; Hegde, Muralidhar L; Dinesh, Athira; Britton, Gabrielle B; Berrocal, Ruben; Subba Rao, K; Shamasundar, N M; Rao, K S J; Sathyanarayana Rao, T S

    2010-07-01

    Deoxyribonucleic acid (DNA) integrity plays a significant role in cell function. There are limited studies with regard to the role of DNA damage in bipolar affective disorder (BP). In the present study, we have assessed DNA integrity, conformation, and stability in the brain region of bipolar depression (BD) patients (n=10) compared to age-matched controls (n=8). Genomic DNA was isolated from 10 postmortem BD patients' brain regions (frontal cortex, Pons, medulla, thalamus, cerebellum, hypothalamus, Parietal, temporal, occipital lobe, and hippocampus) and from the age-matched control subjects. DNA from the frontal cortex, pons, medulla, and thalamus showed significantly higher number of strand breaks in BD (P<0.01) compared to the age-matched controls. However, DNA from the hippocampus region was intact and did not show any strand breaks. The stability studies also indicated that the melting temperature and ethidium bromide binding pattern were altered in the DNA of BD patients' brain regions, except in the hippocampus. The conformation studies showed B-A or secondary B-DNA conformation (instead of the normal B-DNA) in BD patients' brain regions, with the exception of the hippocampus. The levels of redox metals such as Copper (Cu) and Iron (Fe) were significantly elevated in the brain regions of the sufferers of BD, while the Zinc (Zn) level was decreased. In the hippocampus, there was no change in the Fe or Cu levels, whereas, the Zn level was elevated. There was a clear correlation between Cu and Fe levels versus strand breaks in the brain regions of the BD. To date, as far as we are aware, this is a new comprehensive database on stability and conformations of DNA in different brain regions of patients affected with BD. The biological significance of these findings is discussed here.

  11. Combined Scintigraphy and Tumor Marker Analysis Predicts Unfavorable Histopathology of Neuroblastic Tumors with High Accuracy.

    Directory of Open Access Journals (Sweden)

    Wolfgang Peter Fendler

    Full Text Available Our aim was to improve the prediction of unfavorable histopathology (UH in neuroblastic tumors through combined imaging and biochemical parameters.123I-MIBG SPECT and MRI was performed before surgical resection or biopsy in 47 consecutive pediatric patients with neuroblastic tumor. Semi-quantitative tumor-to-liver count-rate ratio (TLCRR, MRI tumor size and margins, urine catecholamine and NSE blood levels of neuron specific enolase (NSE were recorded. Accuracy of single and combined variables for prediction of UH was tested by ROC analysis with Bonferroni correction.34 of 47 patients had UH based on the International Neuroblastoma Pathology Classification (INPC. TLCRR and serum NSE both predicted UH with moderate accuracy. Optimal cut-off for TLCRR was 2.0, resulting in 68% sensitivity and 100% specificity (AUC-ROC 0.86, p < 0.001. Optimal cut-off for NSE was 25.8 ng/ml, resulting in 74% sensitivity and 85% specificity (AUC-ROC 0.81, p = 0.001. Combination of TLCRR/NSE criteria reduced false negative findings from 11/9 to only five, with improved sensitivity and specificity of 85% (AUC-ROC 0.85, p < 0.001.Strong 123I-MIBG uptake and high serum level of NSE were each predictive of UH. Combined analysis of both parameters improved the prediction of UH in patients with neuroblastic tumor. MRI parameters and urine catecholamine levels did not predict UH.

  12. Uncovering the link between malfunctions in Drosophila neuroblast asymmetric cell division and tumorigenesis

    Directory of Open Access Journals (Sweden)

    Kelsom Corey

    2012-11-01

    Full Text Available Abstract Asymmetric cell division is a developmental process utilized by several organisms. On the most basic level, an asymmetric division produces two daughter cells, each possessing a different identity or fate. Drosophila melanogaster progenitor cells, referred to as neuroblasts, undergo asymmetric division to produce a daughter neuroblast and another cell known as a ganglion mother cell (GMC. There are several features of asymmetric division in Drosophila that make it a very complex process, and these aspects will be discussed at length. The cell fate determinants that play a role in specifying daughter cell fate, as well as the mechanisms behind setting up cortical polarity within neuroblasts, have proved to be essential to ensuring that neurogenesis occurs properly. The role that mitotic spindle orientation plays in coordinating asymmetric division, as well as how cell cycle regulators influence asymmetric division machinery, will also be addressed. Most significantly, malfunctions during asymmetric cell division have shown to be causally linked with neoplastic growth and tumor formation. Therefore, it is imperative that the developmental repercussions as a result of asymmetric cell division gone awry be understood.

  13. A fast algorithm for exonic regions prediction in DNA sequences.

    Science.gov (United States)

    Saberkari, Hamidreza; Shamsi, Mousa; Heravi, Hamed; Sedaaghi, Mohammad Hossein

    2013-07-01

    The main purpose of this paper is to introduce a fast method for gene prediction in DNA sequences based on the period-3 property in exons. First, the symbolic DNA sequences were converted to digital signal using the electron ion interaction potential method. Then, to reduce the effect of background noise in the period-3 spectrum, we used the discrete wavelet transform at three levels and applied it on the input digital signal. Finally, the Goertzel algorithm was used to extract period-3 components in the filtered DNA sequence. The proposed algorithm leads to decrease the computational complexity and hence, increases the speed of the process. Detection of small size exons in DNA sequences, exactly, is another advantage of the algorithm. The proposed algorithm ability in exon prediction was compared with several existing methods at the nucleotide level using: (i) specificity - sensitivity values; (ii) receiver operating curves (ROC); and (iii) area under ROC curve. Simulation results confirmed that the proposed method can be used as a promising tool for exon prediction in DNA sequences.

  14. High-Resolution Melting (HRM) of Hypervariable Mitochondrial DNA Regions for Forensic Science.

    Science.gov (United States)

    Dos Santos Rocha, Alípio; de Amorim, Isis Salviano Soares; Simão, Tatiana de Almeida; da Fonseca, Adenilson de Souza; Garrido, Rodrigo Grazinoli; Mencalha, Andre Luiz

    2017-08-23

    Forensic strategies commonly are proceeding by analysis of short tandem repeats (STRs); however, new additional strategies have been proposed for forensic science. Thus, this article standardized the high-resolution melting (HRM) of DNA for forensic analyzes. For HRM, mitochondrial DNA (mtDNA) from eight individuals were extracted from mucosa swabs by DNAzol reagent, samples were amplified by PCR and submitted to HRM analysis to identify differences in hypervariable (HV) regions I and II. To confirm HRM, all PCR products were DNA sequencing. The data suggest that is possible discriminate DNA from different samples by HRM curves. Also, uncommon dual-dissociation was identified in a single PCR product, increasing HRM analyzes by evaluation of melting peaks. Thus, HRM is accurate and useful to screening small differences in HVI and HVII regions from mtDNA and increase the efficiency of laboratory routines based on forensic genetics. © 2017 American Academy of Forensic Sciences.

  15. Adult mouse subventricular zone stem and progenitor cells are sessile and epidermal growth factor receptor negatively regulates neuroblast migration.

    Directory of Open Access Journals (Sweden)

    Yongsoo Kim

    2009-12-01

    Full Text Available The adult subventricular zone (SVZ contains stem and progenitor cells that generate neuroblasts throughout life. Although it is well accepted that SVZ neuroblasts are migratory, recent evidence suggests their progenitor cells may also exhibit motility. Since stem and progenitor cells are proliferative and multipotential, if they were also able to move would have important implications for SVZ neurogenesis and its potential for repair.We studied whether SVZ stem and/or progenitor cells are motile in transgenic GFP+ slices with two photon time lapse microscopy and post hoc immunohistochemistry. We found that stem and progenitor cells; mGFAP-GFP+ cells, bright nestin-GFP+ cells and Mash1+ cells were stationary in the SVZ and rostral migratory stream (RMS. In our search for motile progenitor cells, we uncovered a population of motile betaIII-tubulin+ neuroblasts that expressed low levels of epidermal growth factor receptor (EGFr. This was intriguing since EGFr drives proliferation in the SVZ and affects migration in other systems. Thus we examined the potential role of EGFr in modulating SVZ migration. Interestingly, EGFr(low neuroblasts moved slower and in more tortuous patterns than EGFr-negative neuroblasts. We next questioned whether EGFr stimulation affects SVZ cell migration by imaging Gad65-GFP+ neuroblasts in the presence of transforming growth factor alpha (TGF-alpha, an EGFr-selective agonist. Indeed, acute exposure to TGF-alpha decreased the percentage of motile cells by approximately 40%.In summary, the present study directly shows that SVZ stem and progenitor cells are static, that EGFr is retained on some neuroblasts, and that EGFr stimulation negatively regulates migration. This result suggests an additional role for EGFr signaling in the SVZ.

  16. DNA binding by the Rev1 BRCT region : implications for biological and structural function

    NARCIS (Netherlands)

    Groote, Frederik Hendrik de

    2011-01-01

    Rev1, a protein belonging to the Y-family of polymerases, is a key protein in the process of DNA Translesion Synthesis, and other DNA damage tolerance pathways. Recent studies have shown that its BRCT region is essential for the organisation of TLS events by Rev1. However, the molecular mechanism

  17. The mitochondrial DNA makeup of Romanians: A forensic mtDNA control region database and phylogenetic characterization.

    Science.gov (United States)

    Turchi, Chiara; Stanciu, Florin; Paselli, Giorgia; Buscemi, Loredana; Parson, Walther; Tagliabracci, Adriano

    2016-09-01

    To evaluate the pattern of Romanian population from a mitochondrial perspective and to establish an appropriate mtDNA forensic database, we generated a high-quality mtDNA control region dataset from 407 Romanian subjects belonging to four major historical regions: Moldavia, Transylvania, Wallachia and Dobruja. The entire control region (CR) was analyzed by Sanger-type sequencing assays and the resulting 306 different haplotypes were classified into haplogroups according to the most updated mtDNA phylogeny. The Romanian gene pool is mainly composed of West Eurasian lineages H (31.7%), U (12.8%), J (10.8%), R (10.1%), T (9.1%), N (8.1%), HV (5.4%),K (3.7%), HV0 (4.2%), with exceptions of East Asian haplogroup M (3.4%) and African haplogroup L (0.7%). The pattern of mtDNA variation observed in this study indicates that the mitochondrial DNA pool is geographically homogeneous across Romania and that the haplogroup composition reveals signals of admixture of populations of different origin. The PCA scatterplot supported this scenario, with Romania located in southeastern Europe area, close to Bulgaria and Hungary, and as a borderland with respect to east Mediterranean and other eastern European countries. High haplotype diversity (0.993) and nucleotide diversity indices (0.00838±0.00426), together with low random match probability (0.0087) suggest the usefulness of this control region dataset as a forensic database in routine forensic mtDNA analysis and in the investigation of maternal genetic lineages in the Romanian population. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Association of genetic variations in the mitochondrial DNA control region with presbycusis.

    Science.gov (United States)

    Falah, Masoumeh; Farhadi, Mohammad; Kamrava, Seyed Kamran; Mahmoudian, Saeid; Daneshi, Ahmad; Balali, Maryam; Asghari, Alimohamad; Houshmand, Massoud

    2017-01-01

    The prominent role of mitochondria in the generation of reactive oxygen species, cell death, and energy production contributes to the importance of this organelle in the intracellular mechanism underlying the progression of the common sensory disorder of the elderly, presbycusis. Reduced mitochondrial DNA (mtDNA) gene expression and coding region variation have frequently been reported as being associated with the development of presbycusis. The mtDNA control region regulates gene expression and replication of the genome of this organelle. To comprehensively understand of the role of mitochondria in the progression of presbycusis, we compared variations in the mtDNA control region between subjects with presbycusis and controls. A total of 58 presbycusis patients and 220 control subjects were enrolled in the study after examination by the otolaryngologist and audiology tests. Variations in the mtDNA control region were investigated by polymerase chain reaction and Sanger sequencing. A total of 113 sequence variants were observed in mtDNA, and variants were detected in 100% of patients, with 84% located in hypervariable regions. The frequencies of the variants, 16,223 C>T, 16,311 T>C, 16,249 T>C, and 15,954 A>C, were significantly different between presbycusis and control subjects. The statistically significant difference in the frequencies of four nucleotide variants in the mtDNA control region of presbycusis patients and controls is in agreement with previous experimental evidence and supports the role of mitochondria in the intracellular mechanism underlying presbycusis development. Moreover, these variants have potential as diagnostic markers for individuals at a high risk of developing presbycusis. The data also suggest the possible presence of changes in the mtDNA control region in presbycusis, which could alter regulatory factor binding sites and influence mtDNA gene expression and copy number.

  19. Multiplexed DNA Methylation Analysis of Target Regions Using Microfluidics (Fluidigm).

    Science.gov (United States)

    Adamowicz, Martyna; Maratou, Klio; Aitman, Timothy J

    2018-01-01

    Whole genome shotgun bisulfite sequencing is a method used to generate genome-wide methylation profiles. There are many available protocols to validate the results of this genome-wide method, but they mostly share the limitation of measuring methylation at a small number of CpG positions in small numbers of samples. We developed a multiplexed DNA methylation analysis protocol, which allows for the simultaneous quantitative measurement of cytosine methylation at single nucleotide resolution in 48 PCR amplicons and 48 samples utilizing the microfluidic system established by Fluidigm. Following bisulfite conversion of 500 ng of the target DNA, a PCR reaction is performed using a 48.48 Access Array, which allows parallel amplification of 48 samples by 48 primer pairs. The products of each reaction are labeled with individual, sample specific tags, pooled in a single library and sequenced using the Illumina MiSeq sequencer. The advantages of this system are: speed, small amount of input material, single nucleotide resolution, high coverage of each locus, low cost of simultaneously assaying multiple CpG loci in multiple DNA samples and high reproducibility.

  20. Computer-aided evaluation of neuroblastoma on whole-slide histology images: Classifying grade of neuroblastic differentiation.

    Science.gov (United States)

    Kong, J; Sertel, O; Shimada, H; Boyer, K L; Saltz, J H; Gurcan, M N

    2009-06-01

    Neuroblastoma (NB) is one of the most frequently occurring cancerous tumors in children. The current grading evaluations for patients with this disease require pathologists to identify certain morphological characteristics with microscopic examinations of tumor tissues. Thanks to the advent of modern digital scanners, it is now feasible to scan cross-section tissue specimens and acquire whole-slide digital images. As a result, computerized analysis of these images can generate key quantifiable parameters and assist pathologists with grading evaluations. In this study, image analysis techniques are applied to histological images of haematoxylin and eosin (H&E) stained slides for identifying image regions associated with different pathological components. Texture features derived from segmented components of tissues are extracted and processed by an automated classifier group trained with sample images with different grades of neuroblastic differentiation in a multi-resolution framework. The trained classification system is tested on 33 whole-slide tumor images. The resulting whole-slide classification accuracy produced by the computerized system is 87.88%. Therefore, the developed system is a promising tool to facilitate grading whole-slide images of NB biopsies with high throughput.

  1. Regions of the polytene chromosomes of Drosophila virilis carrying multiple dispersed p Dv 111 DNA sequences

    Energy Technology Data Exchange (ETDEWEB)

    Gubenko, I.S.; Evgen' ev, M.B.

    1986-09-01

    The cloned sequences of p Dv 111 DNA hybridized in situ with more than 170 regions of Drosophila virilis salivary gland chromosomes. Comparative autoradiography of in situ hybridization and the nature of pulse /sup 3/H-thymidine and /sup 3/H-deoxycytidine incorporation into the polytene chromosomes of D. virilis at the puparium formation stage showed that the hybridization sites of p Dv 111 are distributed not only in the heterochromatic regions but also in the euchromatic regions of the chromosomes that are not late replicating. Two distinct bands of hybridization of p Dv 111 /sup 3/H-DNA were observed in the region of the heat shock puff 20CD. The regions of the distal end of chromosome 2, in which breaks appeared during radiation-induced chromosomal rearrangements, hybridized with the p Dv 111 DNA.

  2. Control of Drosophila Type I and Type II central brain neuroblast proliferation by bantam microRNA

    DEFF Research Database (Denmark)

    Weng, Ruifen; Cohen, Stephen M

    2015-01-01

    Post-transcriptional regulation of stem cell self-renewal by microRNAs is emerging as an important mechanism controlling tissue homeostasis. Here, we provide evidence that bantam microRNA controls neuroblast number and proliferation in the Drosophila central brain. Bantam also supports proliferat......Post-transcriptional regulation of stem cell self-renewal by microRNAs is emerging as an important mechanism controlling tissue homeostasis. Here, we provide evidence that bantam microRNA controls neuroblast number and proliferation in the Drosophila central brain. Bantam also supports...... proliferation of transit-amplifying intermediate neural progenitor cells in type II neuroblast lineages. The stem cell factors brat and prospero are identified as bantam targets acting on different aspects of these processes. Thus, bantam appears to act in multiple regulatory steps in the maintenance...

  3. Postembryonic development of transit amplifying neuroblast lineages in the Drosophila brain

    Directory of Open Access Journals (Sweden)

    Bello Bruno

    2009-12-01

    Full Text Available Abstract Background Specific dorsomedial (DM neuroblast lineages of the Drosophila brain amplify their proliferation through generation of transit amplifying intermediate progenitor cells. Together, these DM neuroblast lineages comprise over 5,000 adult-specific neural cells and thus represent a substantial part of the brain. However, no information is currently available about the structure or function of any of the neural cells in these DM lineages. In this report we use MARCM-based clonal analysis together with immunocytochemical labeling techniques to investigate the type and fate of neural cells generated in the DM lineages. Results Genetic cell lineage-tracing and immunocytochemical marker analysis reveal that DM neuroblasts are multipotent progenitors that produce a set of postembryonic brain glia as well as a large number of adult-specific protocerebral neurons. During larval development the adult-specific neurons of each DM lineage form several spatially separated axonal fascicles, some of which project along larval brain commissural structures that are primordia of midline neuropile. By taking advantage of a specific Gal4 reporter line, the DM-derived neuronal cells can be identified and followed into early pupal stages. During pupal development the neurons of the DM lineages arborize in many parts of the brain and contribute to neuropile substructures of the developing central complex, such as the fan-shaped body, noduli and protocerebral bridge. Conclusions Our findings provide cellular and molecular evidence for the fact that DM neuroblasts are multipotent progenitors; thus, they represent the first identified progenitor cells in the fly brain that have neuroglioblast functions during postembryonic development. Moreover, our results demonstrate that the adult-specific neurons of the DM lineages arborize widely in the brain and also make a major contribution to the developing central complex. These findings suggest that the

  4. Genetic characterization of Phytophthora nicotianae by the analysis of polymorphic regions of the mitochondrial DNA.

    Science.gov (United States)

    A new method based on the analysis of mitochondrial intergenic regions characterized by intraspecific variation in DNA sequences was developed and applied to the study of the plant pathogen Phytophthora nicotianae. Two regions flanked by genes trny and rns and trnw and cox2 were identified by compa...

  5. Efficient DNA barcode regions for classifying Piper species (Piperaceae)

    Science.gov (United States)

    Chaveerach, Arunrat; Tanee, Tawatchai; Sanubol, Arisa; Monkheang, Pansa; Sudmoon, Runglawan

    2016-01-01

    Abstract Piper species are used for spices, in traditional and processed forms of medicines, in cosmetic compounds, in cultural activities and insecticides. Here barcode analysis was performed for identification of plant parts, young plants and modified forms of plants. Thirty-six Piper species were collected and the three barcode regions, matK, rbcL and psbA-trnH spacer, were amplified, sequenced and aligned to determine their genetic distances. For intraspecific genetic distances, the most effective values for the species identification ranged from no difference to very low distance values. However, Piper betle had the highest values at 0.386 for the matK region. This finding may be due to Piper betle being an economic and cultivated species, and thus is supported with growth factors, which may have affected its genetic distance. The interspecific genetic distances that were most effective for identification of different species were from the matK region and ranged from a low of 0.002 in 27 paired species to a high of 0.486. Eight species pairs, Piper kraense and Piper dominantinervium, Piper magnibaccum and Piper kraense, Piper phuwuaense and Piper dominantinervium, Piper phuwuaense and Piper kraense, Piper pilobracteatum and Piper dominantinervium, Piper pilobracteatum and Piper kraense, Piper pilobracteatum and Piper phuwuaense and Piper sylvestre and Piper polysyphonum, that presented a genetic distance of 0.000 and were identified by independently using each of the other two regions. Concisely, these three barcode regions are powerful for further efficient identification of the 36 Piper species. PMID:27829794

  6. Neuron and neuroblast numbers and cytogenesis in the dentate gyrus of aged APP(swe)/PS1(dE9) transgenic mice

    DEFF Research Database (Denmark)

    Olesen, Louise Orum; Sivasaravanaparan, Mithula; Severino, Maurizio

    2017-01-01

    the longitudinal changes in the number of doublecortin-expressing neuroblasts and number of granular neurons in the dentate gyrus of APPswe/PS1dE9 transgenic mice. Furthermore, we investigated the effect of long-term paroxetine treatment on the number of neuroblasts and granular neurons, hippocampal amyloidosis......, and spontaneous alternation behaviour, a measure of spatial working memory, in transgenic mice. We observed no difference in granular neurons between transgenic and wild type mice up till 18 months of age, and no differences with age in wild type mice. The number of neuroblasts and the performance...... in the spontaneous alternation task was reduced in aged transgenic mice. Paroxetine treatment from 9 to 18 months of age reduced hippocampal amyloidosis without affecting the number of neuroblasts or granular neurons. These findings suggest that the amyloidosis affects the differentiation of neuroblasts and spatial...

  7. Multiple initiation sites of DNA replication flanking the origin region of lambda dv genome.

    Science.gov (United States)

    Tsurimoto, T; Matsubara, K

    1984-01-01

    Early replicative intermediates of lambda dv plasmid were prepared by an in vitro replication system in the presence of 2',3'-dideoxycytidine 5'-triphosphate, an inhibitor of DNA chain elongation. Short-chain DNAs produced from regions near the replication origin were purified from the intermediates. A fraction of the DNAs was covalently linked to primer RNA. The transition sites from primer RNA to DNA synthesis were mapped along the nucleotide sequence of the genome, by eliminating the RNA by alkaline hydrolysis and labeling the freshly exposed 5' ends of DNA with 32P. The transition sites were found to be located on both sides of the ori region, which includes four 19-base-pair repeats where one of the lambda specific initiator proteins, O, binds. No transition arose within the ori region. The transition sites are multiple on both sides of the ori region and are clustered in one of the two strands in such a way that DNA syntheses from the two sides converge. The frequency of the "leftward" DNA synthesis is several times higher than that of "rightward" synthesis, reflecting the asymmetric bidirectional replication of lambda dv DNA. Images PMID:6095292

  8. Association of genetic variations in the mitochondrial DNA control region with presbycusis

    Directory of Open Access Journals (Sweden)

    Falah M

    2017-03-01

    Full Text Available Masoumeh Falah,1 Mohammad Farhadi,1 Seyed Kamran Kamrava,1 Saeid Mahmoudian,1 Ahmad Daneshi,1 Maryam Balali,1 Alimohamad Asghari,2 Massoud Houshmand1,3 1ENT and Head & Neck Research Center and Department, Iran University of Medical Sciences, Tehran, Iran; 2Skull Base Research Center, Iran University of Medical Sciences, Tehran, Iran; 3Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Background: The prominent role of mitochondria in the generation of reactive oxygen species, cell death, and energy production contributes to the importance of this organelle in the intracellular mechanism underlying the progression of the common sensory disorder of the elderly, presbycusis. Reduced mitochondrial DNA (mtDNA gene expression and coding region variation have frequently been reported as being associated with the development of presbycusis. The mtDNA control region regulates gene expression and replication of the genome of this organelle. To comprehensively understand of the role of mitochondria in the progression of presbycusis, we compared variations in the mtDNA control region between subjects with presbycusis and controls.Methods: A total of 58 presbycusis patients and 220 control subjects were enrolled in the study after examination by the otolaryngologist and audiology tests. Variations in the mtDNA control region were investigated by polymerase chain reaction and Sanger sequencing.Results: A total of 113 sequence variants were observed in mtDNA, and variants were detected in 100% of patients, with 84% located in hypervariable regions. The frequencies of the variants, 16,223 C>T, 16,311 T>C, 16,249 T>C, and 15,954 A>C, were significantly different between presbycusis and control subjects.Conclusion: The statistically significant difference in the frequencies of four nucleotide variants in the mtDNA control region of presbycusis patients and controls is in agreement with previous experimental

  9. Mitochondrial DNA hypervariable region 1 polymorphism in Singapore Chinese population.

    Science.gov (United States)

    Shee, Cheng-Yap; Chong, Michelle S M; Ng, Irene; Chia, Tet-Fatt

    2005-03-01

    Sequence polymorphisms of hypervariable region 1 were analyzed in 100 unrelated Singaporean Chinese. Ninety-five different haplotypes resulting from 113 variable sites were found between nucleotide positions 16045 and 16364. Single nucleotide polymorphism at nucleotide positions 16223, 16045, 16129, 16362 and 16189 was amongst the five highest frequencies observed in the sequences, whilst the most frequent haplotype was 16045-16223. Based on polymorphic sites observed at HV1, haplogroups A, F1a, M7b1, B5a and D4b were the most commonly observed clusters. The haplotype, nucleotide diversity and the average number of nucleotide differences were found to be 0.999, 0.028 and 9.082, respectively. The cytosine-stretch region located around nucleotide position 16189 was observed in 22% of this population sample. Transitions were found to be more predominant than transversions.

  10. [Polymorphism of hypervariable region in D-loop of mitochondrial DNA].

    Science.gov (United States)

    Takada, Y; Mukaida, M

    1999-06-01

    DNA sequences of PCR products from mitochondrial DNA (mtDNA) of 80 healthy Japanese volunteers (40 pairs, mother and child) were determined by the direct sequencing method for polymorphism. Thirty (15 pairs) of 80 samples analyzed showed a T-to-C transition at position 16189 (T16189C) of the C-stretch region in the hyper-variable region of mtDNA. For seven pairs randomly selected from the 15 T16189C pairs (C-stretch) and a single pair without the transition (non C-stretch), PCR products from the D-loop region were cloned and then sequenced. The repeat number of C in the C-stretch region was found to show heteroplasmy by sequencing multiples clones from each mtDNA. Statistical analyses of the distribution patterns of the repeat number revealed no significant differences between the mother and child in each lineage but significant differences between the lineages. The seven lineages could be then classified into four groups. The result of our data confirmed the existence of heteroplasmic polymorphism in the C-stretch region and the inheritance of the heteroplasmy from mother to child. Therefore, the analysis of heteroplasmy is applicable to individual identification.

  11. [Fluorescence in situ hybridization with DNA probes derived from individual chromosomes and chromosome regions].

    Science.gov (United States)

    Bogomolov, A G; Karamysheva, T V; Rubtsov, N B

    2014-01-01

    A significant part of the eukaryotic genomes consists of repetitive DNA, which can form large clusters or distributed along euchromatic chromosome regions. Repeats located in chromosomal regions make a problem in analysis and identification of the chromosomal material with fluorescence in situ hybridization (FISH). In most cases, the identification of chromosome regions using FISH requires detection of the signal produced with unique sequences. The feasibility, advantages and disadvantages of traditional methods of suppression of repetitive DNA hybridization, methods of repeats-free probe construction and methods of chromosome-specific DNA sequences visualization using image processing of multicolor FISH results are considered in the paper. The efficiency of different techniques for DNA probe generation, different FISH protocols, and image processing of obtained microscopic images depends on the genomic size and structure of analyzing species. This problem was discussed and different approaches were considered for the analysis of the species with very large genome, rare species and species which specimens are too small in size to obtain the amount of genomic and Cot-1 DNA required for suppression of repetitive DNA hybridization.

  12. A non-cell-autonomous role for Ras signaling in C. elegans neuroblast delamination.

    Science.gov (United States)

    Parry, Jean M; Sundaram, Meera V

    2014-11-01

    Receptor tyrosine kinase (RTK) signaling through Ras influences many aspects of normal cell behavior, including epithelial-to-mesenchymal transition, and aberrant signaling promotes both tumorigenesis and metastasis. Although many such effects are cell-autonomous, here we show a non-cell-autonomous role for RTK-Ras signaling in the delamination of a neuroblast from an epithelial organ. The C. elegans renal-like excretory organ is initially composed of three unicellular epithelial tubes, namely the canal, duct and G1 pore cells; however, the G1 cell later delaminates from the excretory system to become a neuroblast and is replaced by the G2 cell. G1 delamination and G2 intercalation involve cytoskeletal remodeling, interconversion of autocellular and intercellular junctions and migration over a luminal extracellular matrix, followed by G1 junction loss. LET-23/EGFR and SOS-1, an exchange factor for Ras, are required for G1 junction loss but not for initial cytoskeletal or junction remodeling. Surprisingly, expression of activated LET-60/Ras in the neighboring duct cell, but not in the G1 or G2 cells, is sufficient to rescue sos-1 delamination defects, revealing that Ras acts non-cell-autonomously to permit G1 delamination. We suggest that, similarly, oncogenic mutations in cells within a tumor might help create a microenvironment that is permissive for other cells to detach and ultimately metastasize. © 2014. Published by The Company of Biologists Ltd.

  13. 'Cross-talk' between Schwannian stroma and neuroblasts promotes neuroblastoma tumor differentiation and inhibits angiogenesis.

    Science.gov (United States)

    Liu, Shuqing; Tian, Yufeng; Chlenski, Alexandre; Yang, Qiwei; Salwen, Helen R; Cohn, Susan L

    2005-10-18

    Neuroblastoma (NB) tumors with abundant Schwannian stroma have a differentiated phenotype, low vascularity, and are associated with a favorable prognosis. These observations have led to the hypothesis that 'cross-talk' between Schwann cells and neuroblasts influences the biology and clinical behavior of NB tumors. In support of this hypothesis, laboratory studies have shown that factors secreted by Schwann cells are capable of promoting NB differentiation, inhibiting angiogenesis, and impairing NB growth. Recently, using a novel NB xenograft model that was designed to directly investigate the affects of infiltrating Schwann cells, we demonstrated that infiltrating mouse Schwann cells can directly impact the phenotype of human NB xenografts in vivo. Taken together, these studies indicate that tumor-stroma interactions are critical in determining the biology of NB tumors. Further research investigating the molecules involved in the 'cross-talk' between Schwann cells and neuroblasts may lead to new treatment strategies that will modify tumor biology and alter the clinically aggressive nature of Schwannian stroma-poor NB tumors.

  14. Fate of neuroblast progeny during postembryonic development of mushroom bodies in the house cricket, Acheta domesticus.

    Science.gov (United States)

    Cayre, M; Malaterre, J; Charpin, P; Strambi, C; Strambi, A

    2000-03-01

    Mushroom bodies represent the main sensory integrative center of the insect brain and probably play a major role in the adaptation of behavioral responses to the environment. Taking into account the continuous neurogenesis of cricket mushroom bodies, we investigated ontogenesis of this brain structure. Using BrdU labeling, we examined the fate of neuroblast progeny during the postembryonic development. Preimaginal Kenyon cells survived throughout larval and imaginal moults and persisted during adulthood. Our results indicate that the location of labelled Kenyon cells in the cortex of the adult cricket mainly depends upon the period when they were produced during development. The present data demonstrate that cricket mushroom bodies grow from the inside out and that, at any developmental stage, the center of the cortex contains the youngest Kenyon cells. This study also allowed us to observe the occurrence of quiescent neuroblasts. Kenyon cell death during postembryonic and adult life seems to be reduced. Although preimaginal Kenyon cells largely contribute to adult mushroom body structure, a permanent remodeling of the mushroom body occurs throughout the whole insect life due to the persistence of neurogenesis in the house cricket. Further studies are needed to understand the functional significance of these findings.

  15. An iternative algorithm for correcting sequencing errors in DNA coding regions

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ying; Mural, R.J.; Uberbacher, E.C.

    1995-12-31

    Insertion and deletion (indel) sequencing errors in DNA coding regions disrupt DNA-to-protein translation frames, and hence make most frame-sensitive coding recognition approaches fail. This paper extends the authors` previous work on indel detection and `correction` algorithms, and presents a more effective algorithm for localizing indels that appear in DNA coding regions and `correcting` the located indels by inserting or deleting DNA bases. The algorithm localizes indels by discovering changes of the preferred translation frames within presumed coding regions, and then `corrects` the indel errors to restore a consistent translation frame within each coding region. An iterative strategy is exploited to repeatedly localize and `correct` indel errors until no more indels can be found. Test results have shown that the algorithm can accurately locate the positions of indels. The technology presented here has proved to be very useful for single pass EST/cDNA or genomic sequences, and is also often beneficial for higher quality sequences from large genomic clones.

  16. The Caenorhabditis elegans NF2/Merlin Molecule NFM-1 Nonautonomously Regulates Neuroblast Migration and Interacts Genetically with the Guidance Cue SLT-1/Slit.

    Science.gov (United States)

    Josephson, Matthew P; Aliani, Rana; Norris, Megan L; Ochs, Matthew E; Gujar, Mahekta; Lundquist, Erik A

    2017-02-01

    During nervous system development, neurons and their progenitors migrate to their final destinations. In Caenorhabditis elegans, the bilateral Q neuroblasts and their descendants migrate long distances in opposite directions, despite being born in the same posterior region. QR on the right migrates anteriorly and generates the AQR neuron positioned near the head, and QL on the left migrates posteriorly, giving rise to the PQR neuron positioned near the tail. In a screen for genes required for AQR and PQR migration, we identified an allele of nfm-1, which encodes a molecule similar to vertebrate NF2/Merlin, an important tumor suppressor in humans. Mutations in NF2 lead to neurofibromatosis type II, characterized by benign tumors of glial tissues. Here we demonstrate that in C. elegans, nfm-1 is required for the ability of Q cells and their descendants to extend protrusions and to migrate, but is not required for direction of migration. Using a combination of mosaic analysis and cell-specific expression, we show that NFM-1 is required nonautonomously, possibly in muscles, to promote Q lineage migrations. We also show a genetic interaction between nfm-1 and the C. elegans Slit homolog slt-1, which encodes a conserved secreted guidance cue. Our results suggest that NFM-1 might be involved in the generation of an extracellular cue that promotes Q neuroblast protrusion and migration that acts with or in parallel to SLT-1 In vertebrates, NF2 and Slit2 interact in axon pathfinding, suggesting a conserved interaction of NF2 and Slit2 in regulating migratory events. Copyright © 2017 by the Genetics Society of America.

  17. Ethanol extract of Oenanthe javanica increases cell proliferation and neuroblast differentiation in the adolescent rat dentate gyrus

    Directory of Open Access Journals (Sweden)

    Bai Hui Chen

    2015-01-01

    Full Text Available Oenanthe javanica is an aquatic perennial herb that belongs to the Oenanthe genus in Apiaceae family, and it displays well-known medicinal properties such as protective effects against glutamate-induced neurotoxicity. However, few studies regarding effects of Oenanthe javanica on neurogenesis in the brain have been reported. In this study, we examined the effects of a normal diet and a diet containing ethanol extract of Oenanthe javanica on cell proliferation and neuroblast differentiation in the subgranular zone of the hippocampal dentate gyrus of adolescent rats using Ki-67 (an endogenous marker for cell proliferation and doublecortin (a marker for neuroblast. Our results showed that Oenanthe javanica extract significantly increased the number of Ki-67-immunoreactive cells and doublecortin-immunoreactive neuroblasts in the subgranular zone of the dentate gyrus in the adolescent rats. In addition, the immunoreactivity of brain-derived neurotrophic factor was significantly increased in the dentate gyrus of the Oenanthe javanica extract-treated group compared with the control group. However, we did not find that vascular endothelial growth factor expression was increased in the Oenanthe javanica extract-treated group compared with the control group. These results indicate that Oenanthe javanica extract improves cell proliferation and neuroblast differentiation by increasing brain-derived neurotrophic factor immunoreactivity in the rat dentate gyrus.

  18. Transcription Restores DNA Repair to Heterochromatin, Determining Regional Mutation Rates in Cancer Genomes

    Directory of Open Access Journals (Sweden)

    Christina L. Zheng

    2014-11-01

    Full Text Available Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs arising in an XPC−/− background. XPC−/− cells lack global genome nucleotide excision repair (GG-NER, thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.

  19. Coding region mitochondrial DNA SNPs: targeting East Asian and Native American haplogroups.

    Science.gov (United States)

    Alvarez-Iglesias, V; Jaime, J C; Carracedo, A; Salas, A

    2007-03-01

    We have developed a single PCR multiplex SNaPshot reaction that consists of 32 coding region SNPs that allows (i) increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and (ii) haplogroup assignments of mtDNA profiles in both human population studies (e.g. anthropological) and medical research. The selected SNPs target the East Asian phylogeny, including its Native American derived branches. We have validated this multiplex assay by genotyping a sample of East Asians (Taiwanese) and Native Americans (Argentineans). In addition to the coding SNP typing, we have sequenced the complete control region for the same samples. The genotyping results (control region plus SNaPshot profiles) are in good agreement with previous human population genetic studies (based on e.g. complete sequencing) and the known mtDNA phylogeny. We observe that the SNaPshot method is reliable, rapid, and cost effective in comparison with other techniques of multiplex SNP genotyping. We discuss the advantages of our SNP genotyping selection with respect to previous attempts, and we highlight the importance of using the known mtDNA phylogeny as a framework for SNP profile interpretation and as a tool to minimize genotyping errors.

  20. Differentially Methylated DNA Regions in Monozygotic Twin Pairs Discordant for Rheumatoid Arthritis

    DEFF Research Database (Denmark)

    Svendsen, Anders J; Gervin, Kristina; Lyle, Robert

    2016-01-01

    OBJECTIVES: In an explorative epigenome-wide association study (EWAS) to search for gene independent, differentially methylated DNA positions and regions (DMRs) associated with rheumatoid arthritis (RA) by studying monozygotic (MZ) twin pairs discordant for RA. METHODS: Genomic DNA was isolated...... from whole blood samples from 28 MZ twin pairs discordant for RA. DNA methylation was measured using the HumanMethylation450 BeadChips. Smoking, anti-cyclic citrullinated peptide antibodies, and immunosuppressive treatment were included as covariates. Pathway analysis was performed using GREAT. RESULTS......: We identified several differentially methylated regions associated with RA, which may represent environmental effects or consequences of the disease and plausible biological pathways pertinent to the pathogenesis of RA....

  1. Heteroplasmy of the human mtDNA control region remains constant during life.

    Science.gov (United States)

    Lagerström-Fermér, M; Olsson, C; Forsgren, L; Syvänen, A C

    2001-05-01

    In a longitudinal, retrospective study, we monitored the level of heteroplasmy at nucleotide position (nt) 309 and nt 16189 of the control region of human mtDNA. As a unique source of DNA, we analyzed multiple cervical-cell samples collected, during 1 or 2 decades, from four women with heteroplasmy at either nt 309 or nt 16189. According to accurate, quantitative analysis by solid-phase minisequencing, the level of heteroplasmy remained stable in the cervical-cell samples from all four women during the time studied. We also analyzed autopsy samples from several different tissues, all containing nt 309 in heteroplasmic form, of one of the women, who was deceased. On the basis of our results, heteroplasmy in the control region of mtDNA seems to be inherited and is not the result of somatic age-related accumulation.

  2. Differences in CART expression and cell cycle behavior discriminate sympathetic neuroblast from chromaffin cell lineages in mouse sympathoadrenal cells.

    Science.gov (United States)

    Chan, Wing Hei; Gonsalvez, David G; Young, Heather M; Southard-Smith, E Michelle; Cane, Kylie N; Anderson, Colin R

    2016-02-01

    Adrenal medullary chromaffin cells and peripheral sympathetic neurons originate from a common sympathoadrenal (SA) progenitor cell. The timing and phenotypic changes that mark this lineage diversification are not fully understood. The present study investigated the expression patterns of phenotypic markers, and cell cycle dynamics, in the adrenal medulla and the neighboring suprarenal ganglion of embryonic mice. The noradrenergic marker, tyrosine hydroxylase (TH), was detected in both presumptive adrenal medulla and sympathetic ganglion cells, but with significantly stronger immunostaining in the former. There was intense cocaine and amphetamine-regulated transcript (CART) peptide immunostaining in most neuroblasts, whereas very few adrenal chromaffin cells showed detectable CART immunostaining. This phenotypic segregation appeared as early as E12.5, before anatomical segregation of the two cell types. Cell cycle dynamics were also examined. Initially, 88% of Sox10 positive (+) neural crest progenitors were proliferating at E10.5. Many SA progenitor cells withdrew from the cell cycle at E11.5 as they started to express TH. Whereas 70% of neuroblasts (TH+/CART+ cells) were back in the cell cycle at E12.5, only around 20% of chromaffin (CART negative) cells were in the cell cycle at E12.5 and subsequent days. Thus, chromaffin cell and neuroblast lineages showed differences in proliferative behavior from their earliest appearance. We conclude that the intensity of TH immunostaining and the expression of CART permit early discrimination of chromaffin cells and sympathetic neuroblasts, and that developing chromaffin cells exhibit significantly lower proliferative activity relative to sympathetic neuroblasts. © 2015 Wiley Periodicals, Inc.

  3. Genetic variability within n-rDNA region of ectomycorrhizal isolates ...

    African Journals Online (AJOL)

    HP

    analysis of the internal transcribed spacers of the nuclear encoded ribosomal RNA (n-RNA) gene region ... taxonomist and time consuming process whereas identification of sporulating ectomycorrhizal fungi in pure culture has been comparatively easy. DNA-based molecular marker ... Morphological features of cultures.

  4. Tandemly repeated sequence in 5'end of mtDNA control region of ...

    African Journals Online (AJOL)

    Extensive length variability was observed in 5' end sequence of the mitochondrial DNA control region of the Japanese Spanish mackerel (Scomberomorus niphonius). This length variability was due to the presence of varying numbers of a 56-bp tandemly repeated sequence and a 46-bp insertion/deletion (indel).

  5. Genetic diversity in the mtDNA control region and population ...

    African Journals Online (AJOL)

    We investigated the genetic structure and phylogeographical patterns of Sardinella zunasi in Northwestern Pacific. The mitochondrial DNA control region was sequenced for 77 individuals of S.zunasi from four localities over most of the species range. A total of 215 polymorphic sites (72 parsimony informative) and 69 ...

  6. Analysis of mtDNA hypervariable region II for increasing the ...

    African Journals Online (AJOL)

    aghomotsegin

    2015-03-11

    Mar 11, 2015 ... Mitochondrial DNA is a useful genetic marker for answering evolutionary questions due to its high copy number, maternal mode of inheritance, and its high rate of evolution. The aims of this research were to study the mitochondria noncoding region by using the sanger sequencing technique and establish ...

  7. Analysis of mtDNA hypervariable region II for increasing the ...

    African Journals Online (AJOL)

    Mitochondrial DNA is a useful genetic marker for answering evolutionary questions due to its high copy number, maternal mode of inheritance, and its high rate of evolution. The aims of this research were to study the mitochondria noncoding region by using the sanger sequencing technique and establish the degree of ...

  8. Bacteriophage Nf DNA region controlling late transcription: structural and functional homology with bacteriophage phi 29.

    Science.gov (United States)

    Nuez, B; Salas, M

    1993-06-25

    The putative region for the control of late transcription of the Bacillus subtilis phage Nf has been identified by DNA sequence homology with the equivalent region of the evolutionary related phage phi 29. A similar arrangement of early and late promoters has been detected in the two phages, suggesting that viral transcription could be regulated in a similar way at late times of the infection. Transcription of late genes requires the presence of a viral early protein, gpF in phage Nf and p4 in phage phi 29, being the latter known to bind to a DNA region located upstream from the phage phi 29 late promoter. We have identified a DNA region located upstream from the putative late promoter of phage Nf that is probably involved in binding protein gpF. Furthermore, we show that the phage phi 29 protein p4 is able to bind to this region and activate transcription from the phage Nf putative late promoter. Sequence alignment has also revealed the existence of significant internal homology between the two early promoters contained in this region of each phage.

  9. Assessment of genetic variability among Indian sheep breeds using mitochondrial DNA cytochrome-b region

    Directory of Open Access Journals (Sweden)

    A. D. Sawaimul

    2014-10-01

    Full Text Available Aim: The present study was conducted to estimate genetic distance, the phylogenetic relationship, and time of divergences using mitochondrial DNA (mtDNA. Materials and Methods: The total 216 unrelated samples were collected from native breeding tract of six Indian sheep breeds. The genomic DNA was isolated and screened for restriction enzyme polymorphisms for cytochrome b (Cyt-b region of mtDNA with seven restriction enzymes. Results: The genetic distance among sheep breeds was ranging between 0.02833 and 0.0946. The phylogenetic analysis revealed that Malpura and Chokla were found closer relationship forming distinct cluster followed by Deccani individual were clustered with Nellore sheep, whereas Nali and Sonadi were distant to each other having separate cluster. Estimated divergence time among Indian sheep breeds were ranging about 1.41-4.73 million years ago (MYA with an average of 3.063±0.27 MYA. It showed that Malpura and Sonadi sheep revealed highest divergence time as 4.73 MYA whereas Malpura and Chokla show the lowest as 1.41 MYA. Conclusion: In conclusion, the restriction fragment length polymorphisms-polymerase chain reaction (RFLP-PCR of the Cyt-b region of mtDNA is suitable and cost effective tool for estimating the genetic variability, phylogenetic relationship, and time of divergence among Indian sheep breeds. These findings will help to formulate proper breeding strategies for conservation and utilization of sheep breeds.

  10. Repetitive sequences in Eurasian lynx (Lynx lynx L.) mitochondrial DNA control region.

    Science.gov (United States)

    Sindičić, Magda; Gomerčić, Tomislav; Galov, Ana; Polanc, Primož; Huber, Duro; Slavica, Alen

    2012-06-01

    Mitochondrial DNA (mtDNA) control region (CR) of numerous species is known to include up to five different repetitive sequences (RS1-RS5) that are found at various locations, involving motifs of different length and extensive length heteroplasmy. Two repetitive sequences (RS2 and RS3) on opposite sides of mtDNA central conserved region have been described in domestic cat (Felis catus) and some other felid species. However, the presence of repetitive sequence RS3 has not been detected in Eurasian lynx (Lynx lynx) yet. We analyzed mtDNA CR of 35 Eurasian lynx (L. lynx L.) samples to characterize repetitive sequences and to compare them with those found in other felid species. We confirmed the presence of 80 base pairs (bp) repetitive sequence (RS2) at the 5' end of the Eurasian lynx mtDNA CR L strand and for the first time we described RS3 repetitive sequence at its 3' end, consisting of an array of tandem repeats five to ten bp long. We found that felid species share similar RS3 repetitive pattern and fundamental repeat motif TACAC.

  11. Localized degradation of foreign DNA strands in cells: Only excising the first nucleotide of 5' region.

    Science.gov (United States)

    Li, Hui; Shen, Wei; Lam, Michael Hon-Wah; Liang, Haojun

    2017-09-15

    Intracellular delivery of foreign DNA probes sharply increases the efficiency of various biodetection protocols. Spherical nucleic acid (SNA) conjugate is a new type of probe that consists of a dense oligonucleotide shell attached typically to a gold nanoparticle core. They are widely used as novel labels for in vitro biodetection and intracellular assay. However, the degradation of foreign DNA still remains a challenge that can cause significant signal leakage (false positive signal). Hence, the site and behavior of intracellular degradation need to be investigated. Herein, we discover a localized degradation behavior that only excises the first nucleotide of 5' terminal from a DNA strand, whereas the residual portion of this strand is unbroken in MCF-7 cell. This novel degradation action totally differs from previous opinion that foreign DNA strand would be digested into tiny fragments or even individual nucleotides in cellular environment. On the basis of these findings, we propose a simple and effective way to avoid degradation-caused false positive that one can bypass the degradable site and choose a secure region to label fluorophore along the DNA stand, when using DNA probes for intracellular biodetection. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Phylogeny of Orchidantha (Lowiaceae) and the Zingiberales Based on Six DNA Regions

    DEFF Research Database (Denmark)

    Johansen, Louise Buchholt

    2005-01-01

    Very little is known about the small, tropical, monogeneric monocotyledon family Lowiaceae within the order Zingiberales. The phylogenetic position of Lowiaceae within Zingiberales is unclear, as are relationships within its single genus Orchidantha, which includes at least 16 species. This paper...... presents a phylogenetic analysis of Zingiberales based on 613 parsimony informative characters of the plastid matK gene, the trnL-trnF region, the rps16 intron, and a conservative part of the nuclear ribosomal ITS region (part of the 18S, 5.8S, and 26S rDNA). The resulting single most parsimonious tree...... indicates that Lowiaceae is sister to all the remaining families of Zingiberales. An analysis of the family (14 species) based on a data set consisting of six plastid and nuclear DNA regions, includes the first use of a cam intron for estimating phylogeny. The results suggest that the nuclear calmodulin...

  13. The Ig heavy chain switch region is a hotspot for insertion of transfected DNA

    Energy Technology Data Exchange (ETDEWEB)

    Baar, J.; Shulman, M.J. [Univ. of Toronto, Ontario (Canada)

    1995-08-15

    The Ig heavy chain switch usually occurs by breaking and rejoining DNA in the switch (S) regions, which consist of tandemly repeated sequences 5{prime} of the constant region exons. Various studies have suggested that S DNA can also recombine with non-S sequences. To measure the frequency of such recombination events, the hybridoma cell line igm692, a deletion mutant that lacks the C{mu}1 and C{mu}2 exons and the 3{prime} end of the S{mu} region, was transfected with a fragment bearing the C{mu}1-2 exons, but no S{mu} DNA. Insertion of this fragment into the residual VDJ-C{mu} intron of igm692 can restore a functional {mu} gene, yielding a transformant that is detected as a plaque-forming cell (PFC). PFCs comprise {approximately}8 x 10{sup -7} of the surviving transfected cells. In 10 of 12 PFCs, the C{mu}1-2 fragment inserted into the 2.5-kb residual S{mu} region, whereas insertion in two cases occurred in the 3.5-kb segment 5{prime} of S{mu}. Using a PCR assay to measure the frequency of insertion of the tranferred fragment elsewhere in the hybridoma genome, we found that {approximately}9% of the surviving tranfected cells had stably acquired the C{mu}1-2 fragment. These results indicate that the S{mu} region is {approximately}100-fold more recombinogenic than the average genomic site, and {approximately}7-fold more recombinogenic than the non-S{mu} segment of the residual VDJ-C{mu}, i.e., the S{mu} region is a hotspot for insertion of transfected DNA.

  14. Mitochondrial DNA variant at HVI region as a candidate of genetic markers of type 2 diabetes

    Science.gov (United States)

    Gumilar, Gun Gun; Purnamasari, Yunita; Setiadi, Rahmat

    2016-02-01

    Mitochondrial DNA (mtDNA) is maternally inherited. mtDNA mutations which can contribute to the excess of maternal inheritance of type 2 diabetes. Due to the high mutation rate, one of the areas in the mtDNA that is often associated with the disease is the hypervariable region I (HVI). Therefore, this study was conducted to determine the genetic variants of human mtDNA HVI that related to the type 2 diabetes in four samples that were taken from four generations in one lineage. Steps being taken include the lyses of hair follicles, amplification of mtDNA HVI fragment using Polymerase Chain Reaction (PCR), detection of PCR products through agarose gel electrophoresis technique, the measurement of the concentration of mtDNA using UV-Vis spectrophotometer, determination of the nucleotide sequence via direct sequencing method and analysis of the sequencing results using SeqMan DNASTAR program. Based on the comparison between nucleotide sequence of samples and revised Cambridge Reference Sequence (rCRS) obtained six same mutations that these are C16147T, T16189C, C16193del, T16127C, A16235G, and A16293C. After comparing the data obtained to the secondary data from Mitomap and NCBI, it were found that two mutations, T16189C and T16217C, become candidates as genetic markers of type 2 diabetes even the mutations were found also in the generations of undiagnosed type 2 diabetes. The results of this study are expected to give contribution to the collection of human mtDNA database of genetic variants that associated to metabolic diseases, so that in the future it can be utilized in various fields, especially in medicine.

  15. Identifications of captive and wild tilapia species existing in Hawaii by mitochondrial DNA control region sequence.

    Directory of Open Access Journals (Sweden)

    Liang Wu

    Full Text Available BACKGROUND: The tilapia family of the Cichlidae includes many fish species, which live in freshwater and saltwater environments. Several species, such as O. niloticus, O. aureus, and O. mossambicus, are excellent for aquaculture because these fish are easily reproduced and readily adapt to diverse environments. Historically, tilapia species, including O. mossambicus, S. melanotheron, and O. aureus, were introduced to Hawaii many decades ago, and the state of Hawaii uses the import permit policy to prevent O. niloticus from coming into the islands. However, hybrids produced from O. niloticus may already be present in the freshwater and marine environments of the islands. The purpose of this study was to identify tilapia species that exist in Hawaii using mitochondrial DNA analysis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we analyzed 382 samples collected from 13 farm (captive and wild tilapia populations in Oahu and the Hawaii Islands. Comparison of intraspecies variation between the mitochondrial DNA control region (mtDNA CR and cytochrome c oxidase I (COI gene from five populations indicated that mtDNA CR had higher nucleotide diversity than COI. A phylogenetic tree of all sampled tilapia was generated using mtDNA CR sequences. The neighbor-joining tree analysis identified seven distinctive tilapia species: O. aureus, O. mossambicus, O. niloticus, S. melanotheron, O. urolepies, T. redalli, and a hybrid of O. massambicus and O. niloticus. Of all the populations examined, 10 populations consisting of O. aureus, O. mossambicus, O. urolepis, and O. niloticus from the farmed sites were relatively pure, whereas three wild populations showed some degree of introgression and hybridization. CONCLUSIONS/SIGNIFICANCE: This DNA-based tilapia species identification is the first report that confirmed tilapia species identities in the wild and captive populations in Hawaii. The DNA sequence comparisons of mtDNA CR appear to be a valid method for

  16. Identification of β Clamp-DNA Interaction Regions That Impair the Ability of E. coli to Tolerate Specific Classes of DNA Damage.

    Science.gov (United States)

    Nanfara, Michael T; Babu, Vignesh M P; Ghazy, Mohamed A; Sutton, Mark D

    The E. coli dnaN-encoded β sliding clamp protein plays a pivotal role in managing the actions on DNA of the 5 bacterial DNA polymerases, proteins involved in mismatch repair, as well as several additional proteins involved in DNA replication. Results of in vitro experiments indicate that the loading of β clamp onto DNA relies on both the DnaX clamp loader complex as well as several discrete sliding clamp-DNA interactions. However, the importance of these DNA interactions to E. coli viability, as well as the ability of the β clamp to support the actions of its numerous partner proteins, have not yet been examined. To determine the contribution of β clamp-DNA interactions to the ability of E. coli to cope with different classes of DNA damage, we used alanine scanning to mutate 22 separate residues mapping to 3 distinct β clamp surfaces known or nearby those known to contact the DNA template, including residues P20-L27 (referred to here as loop I), H148-Y154 (loop II) and 7 different residues lining the central pore of the β clamp through which the DNA template threads. Twenty of these 22 dnaN mutants supported bacterial growth. While none of these 20 conferred sensitivity to hydrogen peroxide or ultra violet light, 12 were sensitized to NFZ, 5 were sensitized to MMS, 8 displayed modestly altered frequencies of DNA damage-induced mutagenesis, and 2 may be impaired for supporting hda function. Taken together, these results demonstrate that discrete β clamp-DNA interaction regions contribute to the ability of E. coli to tolerate specific classes of DNA damage.

  17. Analysis of Canis mitochondrial DNA demonstrates high concordance between the control region and ATPase genes.

    Science.gov (United States)

    Rutledge, Linda Y; Patterson, Brent R; White, Bradley N

    2010-07-16

    Phylogenetic studies of wild Canis species have relied heavily on the mitochondrial DNA control region (mtDNA CR) to infer species relationships and evolutionary lineages. Previous analyses of the CR provided evidence for a North American evolved eastern wolf (C. lycaon), that is more closely related to red wolves (C. rufus) and coyotes (C. latrans) than grey wolves (C. lupus). Eastern wolf origins, however, continue to be questioned. Therefore, we analyzed mtDNA from 89 wolves and coyotes across North America and Eurasia at 347 base pairs (bp) of the CR and 1067 bp that included the ATPase6 and ATPase8 genes. Phylogenies and divergence estimates were used to clarify the evolutionary history of eastern wolves, and regional comparisons of nonsynonomous to synonomous substitutions (dN/dS) at the ATPase6 and ATPase8 genes were used to elucidate the potential role of selection in shaping mtDNA geographic distribution. We found high concordance across analyses between the mtDNA regions studied. Both had a high percentage of variable sites (CR = 14.6%; ATP = 9.7%) and both phylogenies clustered eastern wolf haplotypes monophyletically within a North American evolved lineage apart from coyotes. Divergence estimates suggest the putative red wolf sequence is more closely related to coyotes (DxyCR = 0.01982 +/- 0.00494 SD; DxyATP = 0.00332 +/- 0.00097 SD) than the eastern wolf sequences (DxyCR = 0.03047 +/- 0.00664 SD; DxyATP = 0.00931 +/- 0.00205 SD). Neutrality tests on both genes were indicative of the population expansion of coyotes across eastern North America, and dN/dS ratios suggest a possible role for purifying selection in the evolution of North American lineages. dN/dS ratios were higher in European evolved lineages from northern climates compared to North American evolved lineages from temperate regions, but these differences were not statistically significant. These results demonstrate high concordance between coding and non-coding regions of mtDNA, and provide

  18. Analysis of Canis mitochondrial DNA demonstrates high concordance between the control region and ATPase genes

    Directory of Open Access Journals (Sweden)

    White Bradley N

    2010-07-01

    Full Text Available Abstract Background Phylogenetic studies of wild Canis species have relied heavily on the mitochondrial DNA control region (mtDNA CR to infer species relationships and evolutionary lineages. Previous analyses of the CR provided evidence for a North American evolved eastern wolf (C. lycaon, that is more closely related to red wolves (C. rufus and coyotes (C. latrans than grey wolves (C. lupus. Eastern wolf origins, however, continue to be questioned. Therefore, we analyzed mtDNA from 89 wolves and coyotes across North America and Eurasia at 347 base pairs (bp of the CR and 1067 bp that included the ATPase6 and ATPase8 genes. Phylogenies and divergence estimates were used to clarify the evolutionary history of eastern wolves, and regional comparisons of nonsynonomous to synonomous substitutions (dN/dS at the ATPase6 and ATPase8 genes were used to elucidate the potential role of selection in shaping mtDNA geographic distribution. Results We found high concordance across analyses between the mtDNA regions studied. Both had a high percentage of variable sites (CR = 14.6%; ATP = 9.7% and both phylogenies clustered eastern wolf haplotypes monophyletically within a North American evolved lineage apart from coyotes. Divergence estimates suggest the putative red wolf sequence is more closely related to coyotes (DxyCR = 0.01982 ± 0.00494 SD; DxyATP = 0.00332 ± 0.00097 SD than the eastern wolf sequences (DxyCR = 0.03047 ± 0.00664 SD; DxyATP = 0.00931 ± 0.00205 SD. Neutrality tests on both genes were indicative of the population expansion of coyotes across eastern North America, and dN/dS ratios suggest a possible role for purifying selection in the evolution of North American lineages. dN/dS ratios were higher in European evolved lineages from northern climates compared to North American evolved lineages from temperate regions, but these differences were not statistically significant. Conclusions These results demonstrate high concordance between coding

  19. Homologous recombination-mediated cloning and manipulation of genomic DNA regions using Gateway and recombineering systems.

    Science.gov (United States)

    Rozwadowski, Kevin; Yang, Wen; Kagale, Sateesh

    2008-11-17

    Employing genomic DNA clones to characterise gene attributes has several advantages over the use of cDNA clones, including the presence of native transcription and translation regulatory sequences as well as a representation of the complete repertoire of potential splice variants encoded by the gene. However, working with genomic DNA clones has traditionally been tedious due to their large size relative to cDNA clones and the presence, absence or position of particular restriction enzyme sites that may complicate conventional in vitro cloning procedures. To enable efficient cloning and manipulation of genomic DNA fragments for the purposes of gene expression and reporter-gene studies we have combined aspects of the Gateway system and a bacteriophage-based homologous recombination (i.e. recombineering) system. To apply the method for characterising plant genes we developed novel Gateway and plant transformation vectors that are of small size and incorporate selectable markers which enable efficient identification of recombinant clones. We demonstrate that the genomic coding region of a gene can be directly cloned into a Gateway Entry vector by recombineering enabling its subsequent transfer to Gateway Expression vectors. We also demonstrate how the coding and regulatory regions of a gene can be directly cloned into a plant transformation vector by recombineering. This construct was then rapidly converted into a novel Gateway Expression vector incorporating cognate 5' and 3' regulatory regions by using recombineering to replace the intervening coding region with the Gateway Destination cassette. Such expression vectors can be applied to characterise gene regulatory regions through development of reporter-gene fusions, using the Gateway Entry clones of GUS and GFP described here, or for ectopic expression of a coding region cloned into a Gateway Entry vector. We exemplify the utility of this approach with the Arabidopsis PAP85 gene and demonstrate that the expression

  20. Differential DNA methylation regions in cytokine and transcription factor genomic loci associate with childhood physical aggression.

    Directory of Open Access Journals (Sweden)

    Nadine Provençal

    Full Text Available Animal and human studies suggest that inflammation is associated with behavioral disorders including aggression. We have recently shown that physical aggression of boys during childhood is strongly associated with reduced plasma levels of cytokines IL-1α, IL-4, IL-6, IL-8 and IL-10, later in early adulthood. This study tests the hypothesis that there is an association between differential DNA methylation regions in cytokine genes in T cells and monocytes DNA in adult subjects and a trajectory of physical aggression from childhood to adolescence.We compared the methylation profiles of the entire genomic loci encompassing the IL-1α, IL-6, IL-4, IL-10 and IL-8 and three of their regulatory transcription factors (TF NFkB1, NFAT5 and STAT6 genes in adult males on a chronic physical aggression trajectory (CPA and males with the same background who followed a normal physical aggression trajectory (control group from childhood to adolescence. We used the method of methylated DNA immunoprecipitation with comprehensive cytokine gene loci and TF loci microarray hybridization, statistical analysis and false discovery rate correction. We found differentially methylated regions to associate with CPA in both the cytokine loci as well as in their transcription factors loci analyzed. Some of these differentially methylated regions were located in known regulatory regions whereas others, to our knowledge, were previously unknown as regulatory areas. However, using the ENCODE database, we were able to identify key regulatory elements in many of these regions that indicate that they might be involved in the regulation of cytokine expression.We provide here the first evidence for an association between differential DNA methylation in cytokines and their regulators in T cells and monocytes and male physical aggression.

  1. Non-random expression of ribosomal DNA units in a grasshopper showing high intragenomic variation for the ITS2 region.

    Science.gov (United States)

    Ruiz-Estévez, M; Ruiz-Ruano, F J; Cabrero, J; Bakkali, M; Perfectti, F; López-León, M D; Camacho, J P M

    2015-06-01

    We analyse intragenomic variation of the ITS2 internal transcribed spacer of ribosomal DNA (rDNA) in the grasshopper Eyprepocnemis plorans, by means of tagged PCR 454 amplicon sequencing performed on both genomic DNA (gDNA) and RNA-derived complementary DNA (cDNA), using part of the ITS2 flanking coding regions (5.8S and 28S rDNA) as an internal control for sequencing errors. Six different ITS2 haplotypes (i.e. variants for at least one nucleotide in the complete ITS2 sequence) were found in a single population, one of them (Hap4) being specific to a supernumerary (B) chromosome. The analysis of both gDNA and cDNA from the same individuals provided an estimate of the expression efficiency of the different haplotypes. We found random expression (i.e. about similar recovery in gDNA and cDNA) for three haplotypes (Hap1, Hap2 and Hap5), but significant underexpression for three others (Hap3, Hap4 and Hap6). Hap4 was the most extremely underexpressed and, remarkably, it showed the lowest sequence conservation for the flanking 5.8-28S coding regions in the gDNA reads but the highest conservation (100%) in the cDNA ones, suggesting the preferential expression of mutation-free rDNA units carrying this ITS2 haplotype. These results indicate that the ITS2 region of rDNA is far from complete homogenization in this species, and that the different rDNA units are not expressed at random, with some of them being severely downregulated. © 2015 The Royal Entomological Society.

  2. Detection of genomic variation by selection of a 9 mb DNA region and high throughput sequencing.

    Directory of Open Access Journals (Sweden)

    Sergey I Nikolaev

    Full Text Available Detection of the rare polymorphisms and causative mutations of genetic diseases in a targeted genomic area has become a major goal in order to understand genomic and phenotypic variability. We have interrogated repeat-masked regions of 8.9 Mb on human chromosomes 21 (7.8 Mb and 7 (1.1 Mb from an individual from the International HapMap Project (NA12872. We have optimized a method of genomic selection for high throughput sequencing. Microarray-based selection and sequencing resulted in 260-fold enrichment, with 41% of reads mapping to the target region. 83% of SNPs in the targeted region had at least 4-fold sequence coverage and 54% at least 15-fold. When assaying HapMap SNPs in NA12872, our sequence genotypes are 91.3% concordant in regions with coverage > or = 4-fold, and 97.9% concordant in regions with coverage > or = 15-fold. About 81% of the SNPs recovered with both thresholds are listed in dbSNP. We observed that regions with low sequence coverage occur in close proximity to low-complexity DNA. Validation experiments using Sanger sequencing were performed for 46 SNPs with 15-20 fold coverage, with a confirmation rate of 96%, suggesting that DNA selection provides an accurate and cost-effective method for identifying rare genomic variants.

  3. Direct PCR amplification of the HVSI region in mitochondrial DNA from buccal cell swabs

    Directory of Open Access Journals (Sweden)

    Kovačević-Grujičić Nataša

    2012-01-01

    Full Text Available Amplification of human mitochondrial DNA (mtDNA has been widely used in population genetics, human evolutionary and molecular anthropology studies. mtDNA hypervariable segments I and II (HVSI and HVSII were shown to be a suitable tool in genetic analyses due to the unique properties of mtDNA, such as the lack of recombination, maternal mode of inheritance, rapid evolutionary rate and high population-specific polymorphisms. Here we present a rapid and low-cost method for direct PCR amplification of a 330 bp fragment of HVSI from buccal cell samples. Avoiding the DNA isolation step makes this method appropriate for the analysis of a large number of samples in a short period of time. Since the transportation of samples and fieldwork conditions can affect the quality of samples and subsequent DNA analysis, we tested the effects of long-term storage of buccal cell swabs on the suitability of such samples for direct PCR amplification. We efficiently amplified a 330 bp fragment of HVSI even after the long-term storage of buccal cells at room temperature, +4°C or at -20°C, for up to eight months. All examined PCR products were successfully sequenced, regardless of sample storage time and conditions. Our results suggest that the direct PCR amplification of the HVSI region from buccal cells is a method well suited for large-scale mtDNA population studies.[Acknowledgments. This work was supported by the Ministry of Education and Science of the Republic of Serbia (Grant no. III 47025.

  4. The D1-D2 region of the large subunit ribosomal DNA as barcode for ciliates.

    Science.gov (United States)

    Stoeck, T; Przybos, E; Dunthorn, M

    2014-05-01

    Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our planet and are an essential component for ecosystem function, processes and stability. Accurate identification of these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists. To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria for DNA barcodes defined by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1-D2 region of the ribosomal DNA large subunit (LSU-rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers tested in this study were successful for all tested classes. To test the ability of the D1-D2 region to resolve conspecific and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average conspecific D1-D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic distance analyses, we identified a D1-D2 sequence divergence of barcode for ciliated protists. © 2013 John Wiley & Sons Ltd.

  5. Nuclear DNA content determination in Characiformes fish (Teleostei, Ostariophysi from the Neotropical region

    Directory of Open Access Journals (Sweden)

    Carvalho Margarida Lima

    2002-01-01

    Full Text Available In the present study, nuclear DNA content was analyzed in 53 species of Characiformes fish from the Neotropical region. Diploid number ranged from 2n = 48 in Astyanax fasciatus, Gymnocorymbus ternetzi and Hyphessobrycon griemi to 2n = 102 in Potamorhina squamoralevis, with a modal number of 54 chromosomes. Nuclear DNA content ranged from 1.70 ± 0.04 pg of DNA per diploid nucleus in Acestrorhynchus pantaneiro to 3.94 ± 0.09 pg in Tetragonopterus chalceus. A general analysis showed a mean value of 2.9 pg of DNA per diploid nucleus. Very similar DNA content values were observed in the species of the family Cynodontidae which showed a variation of 3% between the two genera studied. Small variations were observed between populations of Gymnocorymbus ternetzi, Astyanax fasciatus and Moenkhausia sanctaefilomenae (Characidae, Tetragonopterinae. The subfamilies Tetragonopterinae and Acestrorhynchinae (Characidae presented the widest range, about 96%. Even in those families in which diploid number and karyotypic formulae were conserved such as the families Anostomidae, Curimatidae, and Prochilodontidae, episodes leading to losses or gains of genetic material became fixed in their evolutionary history.

  6. Force spectroscopy and fluorescence microscopy of dsDNA-YOYO-1 complexes: implications for the structure of dsDNA in the overstretching region.

    Science.gov (United States)

    Murade, Chandrashekhar U; Subramaniam, Vinod; Otto, Cees; Bennink, Martin L

    2010-06-01

    When individual dsDNA molecules are stretched beyond their B-form contour length, they reveal a structural transition in which the molecule extends 1.7 times its contour length. The nature of this transition is still a subject of debate. In the first model, the DNA helix unwinds and combined with the tilting of the base pairs (which remain intact), results in a stretched form of DNA (also known as S-DNA). In the second model the base pairs break resulting effectively in two single-strands, which is referred to as force-induced melting. Here a combination of optical tweezers force spectroscopy with fluorescence microscopy was used to study the structure of dsDNA in the overstretching regime. When dsDNA was stretched in the presence of 10 nM YOYO-1 an initial increase in total fluorescence intensity of the dye-DNA complex was observed and at an extension where the dsDNA started to overstretch the fluorescence intensity leveled off and ultimately decreased when stretched further into the overstretching region. Simultaneous force spectroscopy and fluorescence polarization microscopy revealed that the orientation of dye molecules did not change significantly in the overstretching region (78.0 degrees +/- 3.2 degrees). These results presented here clearly suggest that, the structure of overstretched dsDNA can be explained accurately by force induced melting.

  7. Comparative analysis of complete mitochondrial DNA control region of four species of Strigiformes.

    Science.gov (United States)

    Xiao, Bing; Ma, Fei; Sun, Yi; Li, Qing-Wei

    2006-11-01

    The sequence of the whole mitochondrial (mt) DNA control region (CR) of four species of Strigiformes was obtained. Length of the CR was 3,290 bp, 2,848 bp, 2,444 bp, and 1,771 bp for Asio flammeus, Asio otus, Athene noctua, and Strix aluco, respectively. Interestingly, the length of the control region was maximum in Asio flammeus among all the avian mtDNA control regions sequenced thus far. In addition, the base composition and organization of mtDNA CR of Asio flammeus were identical to those reported for other birds. On the basis of the differential frequencies of base substitutions, the CR may be divided two variable domains, I and III, and a central conserved domain, II. The 3' end of the CR contained many tandem repeats of varying lengths and repeat numbers. In Asio flammeus, the repeated sequences consisted of a 126 bp sequence that was repeated seven times and a 78 bp sequence that was repeated 14 times. In Asio otus, there were also two repeated sequences, namely a 127 bp sequence that was repeated eight times and a 78 bp sequence that was repeated six times. The control region of Athene noctua contained three sets of repeats: a 89 bp sequence that was repeated three times, a 77 bp sequence that was repeated four times, and a 71 bp sequence that was repeated six times. Strix aluco, however, had only one repeated sequence, a 78 bp sequence that was repeated five times. The results of this study seem to indicate that these tandem repeats may have resulted from slipped-strand mispairing during mtDNA replication. Moreover, there are many conserved motifs within the repeated units. These sequences could form stable stem-loop secondary structures, which suggests that these repeated sequences play an important role in regulating transcription and replication of the mitochondrial genome.

  8. Reconstructing the History of Mesoamerican Populations through the Study of the Mitochondrial DNA Control Region

    OpenAIRE

    Amaya Gorostiza; Víctor Acunha-Alonzo; Lucía Regalado-Liu; Sergio Tirado; Julio Granados; David Sámano; Héctor Rangel-Villalobos; Antonio González-Martín

    2012-01-01

    The study of genetic information can reveal a reconstruction of human population's history. We sequenced the entire mtDNA control region (positions 16.024 to 576 following Cambridge Reference Sequence, CRS) of 605 individuals from seven Mesoamerican indigenous groups and one Aridoamerican from the Greater Southwest previously defined, all of them in present Mexico. Samples were collected directly from the indigenous populations, the application of an individual survey made it possible to remo...

  9. Uncovering the DNA methylation landscape in key regulatory regions within the FADS cluster.

    Science.gov (United States)

    Rahbar, Elaheh; Ainsworth, Hannah C; Howard, Timothy D; Hawkins, Gregory A; Ruczinski, Ingo; Mathias, Rasika; Seeds, Michael C; Sergeant, Susan; Hixson, James E; Herrington, David M; Langefeld, Carl D; Chilton, Floyd H

    2017-01-01

    Genetic variants near and within the fatty acid desaturase (FADS) cluster are associated with polyunsaturated fatty acid (PUFA) biosynthesis, levels of several disease biomarkers and risk of human disease. However, determining the functional mechanisms by which these genetic variants impact PUFA levels remains a challenge. Utilizing an Illumina 450K array, we previously reported strong allele-specific methylation (ASM) associations (p = 2.69×10-29) between a single nucleotide polymorphism (SNP) rs174537 and DNA methylation of CpG sites located in the putative enhancer region between FADS1 and FADS2, in human liver tissue. However, this array only featured 20 CpG sites within this 12kb region. To better understand the methylation landscape within this region, we conducted bisulfite sequencing of the region between FADS1 and FADS2. Liver tissues from 50 male subjects (27 European Americans, 23 African Americans) were obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study, and used to ascertain the genotype at rs174537 and methylation status across the region of interest. Associations between rs174537 genotype and methylation status of 136 CpG sites were determined. Age-adjusted linear regressions were used to assess ASM associations with rs174537 genotype. The majority of CpG sites (117 out of 136, 86%) exhibited high levels of methylation with the greatest variability observed at three key regulatory regions-the promoter regions for FADS1 and FADS2 and a putative enhancer site between the two genes. Eight CpG sites within the putative enhancer region displayed significant (FDR p <0.05) ASM associations with rs174537. These data support the concept that both genetic and epigenetic factors regulate PUFA biosynthesis, and raise fundamental questions as to how genetic variants such as rs174537 impact DNA methylation in distant regulatory regions, and ultimately the capacity of tissues to synthesize PUFAs.

  10. Uncovering the DNA methylation landscape in key regulatory regions within the FADS cluster.

    Directory of Open Access Journals (Sweden)

    Elaheh Rahbar

    Full Text Available Genetic variants near and within the fatty acid desaturase (FADS cluster are associated with polyunsaturated fatty acid (PUFA biosynthesis, levels of several disease biomarkers and risk of human disease. However, determining the functional mechanisms by which these genetic variants impact PUFA levels remains a challenge. Utilizing an Illumina 450K array, we previously reported strong allele-specific methylation (ASM associations (p = 2.69×10-29 between a single nucleotide polymorphism (SNP rs174537 and DNA methylation of CpG sites located in the putative enhancer region between FADS1 and FADS2, in human liver tissue. However, this array only featured 20 CpG sites within this 12kb region. To better understand the methylation landscape within this region, we conducted bisulfite sequencing of the region between FADS1 and FADS2. Liver tissues from 50 male subjects (27 European Americans, 23 African Americans were obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY study, and used to ascertain the genotype at rs174537 and methylation status across the region of interest. Associations between rs174537 genotype and methylation status of 136 CpG sites were determined. Age-adjusted linear regressions were used to assess ASM associations with rs174537 genotype. The majority of CpG sites (117 out of 136, 86% exhibited high levels of methylation with the greatest variability observed at three key regulatory regions-the promoter regions for FADS1 and FADS2 and a putative enhancer site between the two genes. Eight CpG sites within the putative enhancer region displayed significant (FDR p <0.05 ASM associations with rs174537. These data support the concept that both genetic and epigenetic factors regulate PUFA biosynthesis, and raise fundamental questions as to how genetic variants such as rs174537 impact DNA methylation in distant regulatory regions, and ultimately the capacity of tissues to synthesize PUFAs.

  11. Regulation of mitochondrial genome replication by hypoxia: The role of DNA oxidation in D-loop region.

    Science.gov (United States)

    Pastukh, Viktor M; Gorodnya, Olena M; Gillespie, Mark N; Ruchko, Mykhaylo V

    2016-07-01

    Mitochondria of mammalian cells contain multiple copies of mitochondrial (mt) DNA. Although mtDNA copy number can fluctuate dramatically depending on physiological and pathophysiologic conditions, the mechanisms regulating mitochondrial genome replication remain obscure. Hypoxia, like many other physiologic stimuli that promote growth, cell proliferation and mitochondrial biogenesis, uses reactive oxygen species as signaling molecules. Emerging evidence suggests that hypoxia-induced transcription of nuclear genes requires controlled DNA damage and repair in specific sequences in the promoter regions. Whether similar mechanisms are operative in mitochondria is unknown. Here we test the hypothesis that controlled oxidative DNA damage and repair in the D-loop region of the mitochondrial genome are required for mitochondrial DNA replication and transcription in hypoxia. We found that hypoxia had little impact on expression of mitochondrial proteins in pulmonary artery endothelial cells, but elevated mtDNA content. The increase in mtDNA copy number was accompanied by oxidative modifications in the D-loop region of the mitochondrial genome. To investigate the role of this sequence-specific oxidation of mitochondrial genome in mtDNA replication, we overexpressed mitochondria-targeted 8-oxoguanine glycosylase Ogg1 in rat pulmonary artery endothelial cells, enhancing the mtDNA repair capacity of transfected cells. Overexpression of Ogg1 resulted in suppression of hypoxia-induced mtDNA oxidation in the D-loop region and attenuation of hypoxia-induced mtDNA replication. Ogg1 overexpression also reduced binding of mitochondrial transcription factor A (TFAM) to both regulatory and coding regions of the mitochondrial genome without altering total abundance of TFAM in either control or hypoxic cells. These observations suggest that oxidative DNA modifications in the D-loop region during hypoxia are important for increased TFAM binding and ensuing replication of the mitochondrial

  12. Evolution and structural organisation of mitochondrial DNA control region of myiasis-causing flies.

    Science.gov (United States)

    Lessinger, A C; Azeredo-Espin, A M

    2000-03-01

    This study reports the molecular characterization of the mtDNA control region (called the A+T-rich region in insects) of five dipteran species which cause myiasis: Cochliomyia hominivorax Coquerel, Cochliomyia macellaria Fabricius, Chrysomya megacephala Fabricius, Lucilia eximia Wiedemann (Diptera: Calliphoridae) and Dermatobia hominis Linnaeus Jr (Diptera: Oestridae). The control region in these species varies in length from 1000 to 1600 bp. Two structural domains with specific evolutionary patterns were identified. These were (1) conserved sequence blocks containing primary sequence motifs, including dinucleotide pyrimidine-purine series and long T-stretches, located at the 5' end adjacent to the tRNA(Ile) gene and (2) a hypervariable domain at the 3' end characterized by increased nucleotide divergence and size variation. A high frequency of AT transversions at nucleotide substitution level indicated directional mutation pressure. The phylogenetic usefulness of the insect control region is discussed.

  13. Ultrasound-guided core needle biopsy in the diagnosis of neuroblastic tumors in children: a retrospective study on 83 cases.

    Science.gov (United States)

    Zhao, Lihui; Mu, Jie; Du, Ping; Wang, Hailing; Mao, Yiran; Xu, Yong; Xin, Xiaojie; Zang, Fenglin

    2017-03-01

    Ultrasound-guided biopsy technique with the large-core needle has widely been applied in the diagnosis of adult abdominopelvic cavity, thyroid, and neck tumors. There are few reports on ultrasound-guided biopsy using large-core needle in pediatric abdominopelvic cavity tumors. This study was to evaluate the ultrasound features and the diagnostic value of ultrasound-guided core needle biopsy for pediatric neuroblastic tumors. The pediatric patients with neuroblastic tumor that underwent ultrasound examination and ultrasound-guided core needle biopsy from January 2009 to November 2015 were reviewed. A minimum of two cores in each case was obtained. The biopsy results were confirmed by subsequent surgical histopathology. The ultrasound features and the diagnostic accuracy of ultrasound-guided core needle biopsy were evaluated. Eighty-three patients were enrolled into the study. Conventional ultrasound examination showed irregular hypoechoic or mixed echo masses and calcification and liquefied necrosis. The diagnostic accuracy of ultrasound-guided core needle biopsy was 96.4% (80/83). Three cases were misdiagnosed because of inadequate tissue sample. No serious complication, infection, or needle track seeding occurred. Ultrasound-guided core needle biopsy seems to be an accurate, minimally invasive, and safe diagnostic method of pediatric neuroblastic tumor.

  14. The high dosage of earthworm (Eisenia andrei) extract decreases cell proliferation and neuroblast differentiation in the mouse hippocampal dentate gyrus

    Science.gov (United States)

    Yan, Bing Chun; Yoo, Ki-Yeon; Park, Joon Ha; Lee, Choong Hyun; Choi, Jung Hoon

    2011-01-01

    Earthworm extract has shown anticancer characteristics. In the present study, we examined the effect of chronic treatment with a high dose of earthworm (Eisenia andrei) extract (EE) on cell proliferation and neuroblast differentiation in the hippocampal dentate gyrus (DG) of 3-week-old mice using 5-bromo-2'-deoxyuridine (BrdU) and Ki-67 immunohistochemistry for cell proliferation and doublecortin (DCX) immunohistochemistry for neuroblast differentiation, respectively. BrdU-, Ki-67-, and DCX-immunoreactive cells were easily detected in the subgranular zone of the DG in vehicle (saline)-treated mice. However, BrdU-, Ki-67-, and DCX-immunoreactive cells in the 500 mg/kg EE-treated mice decreased distinctively compared to those in the vehicle-treated mice. In addition, brain-derived neurotrophic factor (BDNF) immunoreactivity and its protein level decreased markedly in the DG of the EE-treated group compared to those in the vehicle-treated group. These results indicate that chronic treatment with high dose EE decreased cell proliferation and neuroblast differentiation, and that BDNF immunoreactivity decreased in the DG of EE-treated mice. PMID:22025974

  15. Use of ITS2 region as the universal DNA barcode for plants and animals.

    Science.gov (United States)

    Yao, Hui; Song, Jingyuan; Liu, Chang; Luo, Kun; Han, Jianping; Li, Ying; Pang, Xiaohui; Xu, Hongxi; Zhu, Yingjie; Xiao, Peigen; Chen, Shilin

    2010-10-01

    The internal transcribed spacer 2 (ITS2) region of nuclear ribosomal DNA is regarded as one of the candidate DNA barcodes because it possesses a number of valuable characteristics, such as the availability of conserved regions for designing universal primers, the ease of its amplification, and sufficient variability to distinguish even closely related species. However, a general analysis of its ability to discriminate species in a comprehensive sample set is lacking. In the current study, 50,790 plant and 12,221 animal ITS2 sequences downloaded from GenBank were evaluated according to sequence length, GC content, intra- and inter-specific divergence, and efficiency of identification. The results show that the inter-specific divergence of congeneric species in plants and animals was greater than its corresponding intra-specific variations. The success rates for using the ITS2 region to identify dicotyledons, monocotyledons, gymnosperms, ferns, mosses, and animals were 76.1%, 74.2%, 67.1%, 88.1%, 77.4%, and 91.7% at the species level, respectively. The ITS2 region unveiled a different ability to identify closely related species within different families and genera. The secondary structure of the ITS2 region could provide useful information for species identification and could be considered as a molecular morphological characteristic. As one of the most popular phylogenetic markers for eukaryota, we propose that the ITS2 locus should be used as a universal DNA barcode for identifying plant species and as a complementary locus for CO1 to identify animal species. We have also developed a web application to facilitate ITS2-based cross-kingdom species identification (http://its2-plantidit.dnsalias.org).

  16. Use of ITS2 region as the universal DNA barcode for plants and animals.

    Directory of Open Access Journals (Sweden)

    Hui Yao

    Full Text Available BACKGROUND: The internal transcribed spacer 2 (ITS2 region of nuclear ribosomal DNA is regarded as one of the candidate DNA barcodes because it possesses a number of valuable characteristics, such as the availability of conserved regions for designing universal primers, the ease of its amplification, and sufficient variability to distinguish even closely related species. However, a general analysis of its ability to discriminate species in a comprehensive sample set is lacking. METHODOLOGY/PRINCIPAL FINDINGS: In the current study, 50,790 plant and 12,221 animal ITS2 sequences downloaded from GenBank were evaluated according to sequence length, GC content, intra- and inter-specific divergence, and efficiency of identification. The results show that the inter-specific divergence of congeneric species in plants and animals was greater than its corresponding intra-specific variations. The success rates for using the ITS2 region to identify dicotyledons, monocotyledons, gymnosperms, ferns, mosses, and animals were 76.1%, 74.2%, 67.1%, 88.1%, 77.4%, and 91.7% at the species level, respectively. The ITS2 region unveiled a different ability to identify closely related species within different families and genera. The secondary structure of the ITS2 region could provide useful information for species identification and could be considered as a molecular morphological characteristic. CONCLUSIONS/SIGNIFICANCE: As one of the most popular phylogenetic markers for eukaryota, we propose that the ITS2 locus should be used as a universal DNA barcode for identifying plant species and as a complementary locus for CO1 to identify animal species. We have also developed a web application to facilitate ITS2-based cross-kingdom species identification (http://its2-plantidit.dnsalias.org.

  17. DNA Methylation Analysis of BRD1 Promoter Regions and the Schizophrenia rs138880 Risk Allele.

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    Mads Dyrvig

    Full Text Available The bromodomain containing 1 gene, BRD1 is essential for embryogenesis and CNS development. It encodes a protein that participates in histone modifying complexes and thereby regulates the expression of a large number of genes. Genetic variants in the BRD1 locus show association with schizophrenia and bipolar disorder and risk alleles in the promoter region correlate with reduced BRD1 expression. Insights into the transcriptional regulation of BRD1 and the pathogenic mechanisms associated with BRD1 risk variants, however, remain sparse. By studying transcripts in human HeLa and SH-SY5Y cells we provide evidence for differences in relative expression of BRD1 transcripts with three alternative 5' UTRs (exon 1C, 1B, and 1A. We further show that expression of these transcript variants covaries negatively with DNA methylation proportions in their upstream promoter regions suggesting that promoter usage might be regulated by DNA methylation. In line with findings that the risk allele of the rs138880 SNP in the BRD1 promoter region correlates with reduced BRD1 expression, we find that it is also associated with moderate regional BRD1 promoter hypermethylation in both adipose tissue and blood. Importantly, we demonstrate by inspecting available DNA methylation and expression data that these regions undergo changes in methylation during fetal brain development and that differences in their methylation proportions in fetal compared to postnatal frontal cortex correlate significantly with BRD1 expression. These findings suggest that BRD1 may be dysregulated in both the developing and mature brain of risk allele carriers. Finally, we demonstrate that commonly used mood stabilizers Lithium, Valproate, and Carbamazepine affect the expression of BRD1 in SH-SY5Y cells. Altogether this study indicates a link between genetic risk and epigenetic dysregulation of BRD1 which raises interesting perspectives for targeting the mechanisms pharmacologically.

  18. Correcting sequencing errors in DNA coding regions using a dynamic programming approach

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Y.; Mural, R.J.; Uberbacher, E.C.

    1994-12-01

    This paper presents an algorithm for detecting and ``correcting`` sequencing errors that occur in DNA coding regions. The types of sequencing error addressed include insertions and deletions (indels) of DNA bases. The goal is to provide a capability which makes single-pass or low-redundancy sequence data more informative, reducing the need for high-redundancy sequencing for gene identification and characterization purposes. The algorithm detects sequencing errors by discovering changes in the statistically preferred reading frame within a putative coding region and then inserts a number of ``neutral`` bases at a perceived reading frame transition point to make the putative exon candidate frame consistent. The authors have implemented the algorithm as a front-end subsystem of the GRAIL DNA sequence analysis system to construct a version which is very error tolerant and also intend to use this as a testbed for further development of sequencing error-correction technology. On a test set consisting of 68 Human DNA sequences with 1% randomly generated indels in coding regions, the algorithm detected and corrected 76% of the indels. The average distance between the position of an indel and the predicted one was 9.4 bases. With this subsystem in place, GRAIL correctly predicted 89% of the coding messages with 10% false message on the ``corrected`` sequences, compared to 69% correctly predicted coding messages and 11% falsely predicted messages on the ``corrupted`` sequences using standard GRAIL II method. The method uses a dynamic programming algorithm, and runs in time and space linear to the size of the input sequence.

  19. Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin

    Directory of Open Access Journals (Sweden)

    Mayra Eduardoff

    2017-09-01

    Full Text Available The analysis of mitochondrial DNA (mtDNA has proven useful in forensic genetics and ancient DNA (aDNA studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR is commonly sequenced using established Sanger-type Sequencing (STS protocols involving fragment sizes down to approximately 150 base pairs (bp. Recent developments include Massively Parallel Sequencing (MPS of (multiplex PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less, and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples, and tested challenging forensic samples (n = 2 as well as compromised solid tissue samples (n = 15 up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS

  20. Optimized mtDNA Control Region Primer Extension Capture Analysis for Forensically Relevant Samples and Highly Compromised mtDNA of Different Age and Origin

    Science.gov (United States)

    Eduardoff, Mayra; Xavier, Catarina; Strobl, Christina; Casas-Vargas, Andrea; Parson, Walther

    2017-01-01

    The analysis of mitochondrial DNA (mtDNA) has proven useful in forensic genetics and ancient DNA (aDNA) studies, where specimens are often highly compromised and DNA quality and quantity are low. In forensic genetics, the mtDNA control region (CR) is commonly sequenced using established Sanger-type Sequencing (STS) protocols involving fragment sizes down to approximately 150 base pairs (bp). Recent developments include Massively Parallel Sequencing (MPS) of (multiplex) PCR-generated libraries using the same amplicon sizes. Molecular genetic studies on archaeological remains that harbor more degraded aDNA have pioneered alternative approaches to target mtDNA, such as capture hybridization and primer extension capture (PEC) methods followed by MPS. These assays target smaller mtDNA fragment sizes (down to 50 bp or less), and have proven to be substantially more successful in obtaining useful mtDNA sequences from these samples compared to electrophoretic methods. Here, we present the modification and optimization of a PEC method, earlier developed for sequencing the Neanderthal mitochondrial genome, with forensic applications in mind. Our approach was designed for a more sensitive enrichment of the mtDNA CR in a single tube assay and short laboratory turnaround times, thus complying with forensic practices. We characterized the method using sheared, high quantity mtDNA (six samples), and tested challenging forensic samples (n = 2) as well as compromised solid tissue samples (n = 15) up to 8 kyrs of age. The PEC MPS method produced reliable and plausible mtDNA haplotypes that were useful in the forensic context. It yielded plausible data in samples that did not provide results with STS and other MPS techniques. We addressed the issue of contamination by including four generations of negative controls, and discuss the results in the forensic context. We finally offer perspectives for future research to enable the validation and accreditation of the PEC MPS method for

  1. The presence of chimeric DNA consisting of 5' regions of the hemoglobin and nucleosome assembly protein-1 genes in Paramecium caudatum macronuclear genomic DNA.

    Science.gov (United States)

    Nishiyama, Norihito; Mikami, Kazuyuki; Ochiai, Takehiko; Yamauchi, Kiyoshi

    2009-04-01

    We detected an unexpected small-sized DNA fragment during polymerase chain reaction (PCR) analysis of the heterogeneity of a macronuclear intergenic region of Paramecium caudatum. Southern blotting of total genomic DNA with the PCR product as a probe indicated that the small-sized DNA fragment constituted part of the macronuclear genome. Sequencing revealed that the PCR product was a chimeric DNA structure that may be generated by tail-to-tail fusion of the 5' region of the hemoglobin (hb) gene to most of the nucleosome assembly protein-1 (nap-1) gene. Short tandem repeats consisting of tetra- and tri-nucleotides exist at the putative cleavage sites in the hb and nap-1 genes, respectively. This feature differs from those found at the boundaries of TA-internal eliminated sequences in the P. aurelia complex and at transposable elements in other species. This suggests that the chimeric DNA is generated by a novel mechanism. Although the chimeric DNA contains the hb and nap-1 promoters, transcripts corresponding to the chimeric DNA were not detected by reverse transcription (RT)-PCR analysis during vegetative cell growth. Possible roles of chimeric DNA are discussed.

  2. BioCode: two biologically compatible Algorithms for embedding data in non-coding and coding regions of DNA.

    Science.gov (United States)

    Haughton, David; Balado, Félix

    2013-04-09

    In recent times, the application of deoxyribonucleic acid (DNA) has diversified with the emergence of fields such as DNA computing and DNA data embedding. DNA data embedding, also known as DNA watermarking or DNA steganography, aims to develop robust algorithms for encoding non-genetic information in DNA. Inherently DNA is a digital medium whereby the nucleotide bases act as digital symbols, a fact which underpins all bioinformatics techniques, and which also makes trivial information encoding using DNA straightforward. However, the situation is more complex in methods which aim at embedding information in the genomes of living organisms. DNA is susceptible to mutations, which act as a noisy channel from the point of view of information encoded using DNA. This means that the DNA data embedding field is closely related to digital communications. Moreover it is a particularly unique digital communications area, because important biological constraints must be observed by all methods. Many DNA data embedding algorithms have been presented to date, all of which operate in one of two regions: non-coding DNA (ncDNA) or protein-coding DNA (pcDNA). This paper proposes two novel DNA data embedding algorithms jointly called BioCode, which operate in ncDNA and pcDNA, respectively, and which comply fully with stricter biological restrictions. Existing methods comply with some elementary biological constraints, such as preserving protein translation in pcDNA. However there exist further biological restrictions which no DNA data embedding methods to date account for. Observing these constraints is key to increasing the biocompatibility and in turn, the robustness of information encoded in DNA. The algorithms encode information in near optimal ways from a coding point of view, as we demonstrate by means of theoretical and empirical (in silico) analyses. Also, they are shown to encode information in a robust way, such that mutations have isolated effects. Furthermore, the

  3. FBIS: A regional DNA barcode archival & analysis system for Indian fishes.

    Science.gov (United States)

    Nagpure, Naresh Sahebrao; Rashid, Iliyas; Pathak, Ajey Kumar; Singh, Mahender; Singh, Shri Prakash; Sarkar, Uttam Kumar

    2012-01-01

    DNA barcode is a new tool for taxon recognition and classification of biological organisms based on sequence of a fragment of mitochondrial gene, cytochrome c oxidase I (COI). In view of the growing importance of the fish DNA barcoding for species identification, molecular taxonomy and fish diversity conservation, we developed a Fish Barcode Information System (FBIS) for Indian fishes, which will serve as a regional DNA barcode archival and analysis system. The database presently contains 2334 sequence records of COI gene for 472 aquatic species belonging to 39 orders and 136 families, collected from available published data sources. Additionally, it contains information on phenotype, distribution and IUCN Red List status of fishes. The web version of FBIS was designed using MySQL, Perl and PHP under Linux operating platform to (a) store and manage the acquisition (b) analyze and explore DNA barcode records (c) identify species and estimate genetic divergence. FBIS has also been integrated with appropriate tools for retrieving and viewing information about the database statistics and taxonomy. It is expected that FBIS would be useful as a potent information system in fish molecular taxonomy, phylogeny and genomics. The database is available for free at http://mail.nbfgr.res.in/fbis/

  4. Assessing the value of DNA barcodes and other priority gene regions for molecular phylogenetics of Lepidoptera.

    Directory of Open Access Journals (Sweden)

    John James Wilson

    Full Text Available BACKGROUND: Despite apparently abundant amounts of observable variation and species diversity, the order Lepidoptera exhibits a morphological homogeneity that has provided only a limited number of taxonomic characters and led to widespread use of nucleotides for inferring relationships. This study aims to characterize and develop methods to quantify the value of priority gene regions designated for Lepidoptera molecular systematics. In particular, I assess how the DNA barcode segment of the mitochondrial COI gene performs across a broad temporal range given its number one position of priority, most sequenced status, and the conflicting opinions on its phylogenetic performance. METHODOLOGY/PRINCIPAL FINDINGS: Gene regions commonly sequenced for lepidoptera phylogenetics were scored using multiple measures across three categories: practicality, which includes universality of primers and sequence quality; phylogenetic utility; and phylogenetic signal. I found that alternative measures within a category often appeared correlated, but high scores in one category did not necessarily translate into high scores in another. The DNA barcode was easier to sequence than other genes, and had high scores for utility but low signal above the genus level. CONCLUSIONS/SIGNIFICANCE: Given limited financial resources and time constraints, careful selection of gene regions for molecular phylogenetics is crucial to avoid wasted effort producing partially informative data. This study introduces an approach to assessing the value of gene regions prior to the initiation of new studies and presents empirical results to help guide future selections.

  5. Phylogenetic patterns in the genus Manihot (Euphorbiaceae) inferred from analyses of nuclear and chloroplast DNA regions.

    Science.gov (United States)

    Chacón, Juliana; Madriñán, Santiago; Debouck, Daniel; Rodriguez, Fausto; Tohme, Joe

    2008-10-01

    From a phylogenetic perspective, the genus Manihot can be considered as an orphan group of plants, and the scientific knowledge acquired has been mainly related to cassava, one of the most important crops in poor tropical countries. The goal of the majority of evolutionary studies in the genus has been to decipher the domestication process and identify the closest relatives of cassava. Few investigations have focused on wild Manihot species, and the phylogeny of the genus is still unclear. In this study the DNA sequence variation from two chloroplast regions, the nuclear DNA gene G3pdh and two nuclear sequences derived from the 3'-end of two cassava ESTs, were used in order to infer the phylogenetic relationships among a subset of wild Manihot species, including two species from Cnidoscolus as out-groups. Maximum parsimony and Bayesian analyses were conducted for each data set and for a combined matrix due to the low variation of each region when analyzed independently. A penalized likelihood analysis of the chloroplast region trnL-trnF, calibrated with various age estimates for genera in the Euphorbiaceae extracted from the literature was used to determine the ages of origin and diversification of the genus. The two Mesoamerican species sampled form a well-defined clade. The South American species can be grouped into clades of varying size, but the relationships amongst them cannot be established with the data available. The age of the crown node of Manihot was estimated at 6.6 million years ago. Manihot esculenta varieties do not form a monophyletic group that is consistent with the possibility of multiple introgressions of genes from other wild species. The low levels of variation observed in the DNA regions sampled suggest a recent and explosive diversification of the genus, which is confirmed by our age estimates.

  6. Repair of DNA damage induced by anthanthrene, a polycyclic aromatic hydrocarbon (PAH) without bay or fjord regions

    DEFF Research Database (Denmark)

    Madsen, Claus Desler; Johannessen, Christian; Rasmussen, Lene Juel

    2009-01-01

    is known about the repair of DNA damage resulting from metabolites from PAHs without bay and fjord regions. We have investigated the six-ringed PAH anthanthrene (dibenzo[def,mno]chrysene), which does not posses bay or fjord motifs. We analyzed the repair profile of human cell extracts and of cell cultures...... in response to DNA damage induced by cytochrome P450-activated anthanthrene. In cell extracts, functional nucleotide excision repair (NER) and mismatch repair (MMR) activities were necessary to trigger a response to anthanthrene metabolite-induced DNA damage. In cell cultures, NER was responsible...... proposed for metabolic activation of PAHs involves the cytochrome P450 enzymes. The DNA damaging potential of cytochrome P450-activated PAHs is generally associated with their bay and fjord regions, and the DNA repair response of PAHs containing such regions has been thoroughly studied. However, little...

  7. Assembly of pyrene-modified DNA/RNA duplexes incorporating a G-rich single strand region.

    Science.gov (United States)

    Seio, Kohji; Tokugawa, Munefumi; Tsunoda, Hirosuke; Ohkubo, Akihiro; Arisaka, Fumio; Sekine, Mitsuo

    2013-12-15

    The structural properties of a DNA/RNA duplex having a pyrene residue at the 5' end of DNA and a G-rich single strand region at the 3' end of RNA were studied in detail. Fluorescence and ultracentrifugation analyses indicated the formation of a complex containing four DNA/RNA duplexes, which required a pyrene residue, G-rich sequence, RNA-type backbone, and high salt concentration. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. High Sequence Variations in Mitochondrial DNA Control Region among Worldwide Populations of Flathead Mullet Mugil cephalus

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    Brian Wade Jamandre

    2014-01-01

    Full Text Available The sequence and structure of the complete mtDNA control region (CR of M. cephalus from African, Pacific, and Atlantic populations are presented in this study to assess its usefulness in phylogeographic studies of this species. The mtDNA CR sequence variations among M. cephalus populations largely exceeded intraspecific polymorphisms that are generally observed in other vertebrates. The length of CR sequence varied among M. cephalus populations due to the presence of indels and variable number of tandem repeats at the 3′ hypervariable domain. The high evolutionary rate of the CR in this species probably originated from these mutations. However, no excessive homoplasic mutations were noticed. Finally, the star shaped tree inferred from the CR polymorphism stresses a rapid radiation worldwide, in this species. The CR still appears as a good marker for phylogeographic investigations and additional worldwide samples are warranted to further investigate the genetic structure and evolution in M. cephalus.

  9. The effectiveness of three regions in mitochondrial genome for aphid DNA barcoding: a case in Lachininae.

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    Rui Chen

    Full Text Available BACKGROUND: The mitochondrial gene COI has been widely used by taxonomists as a standard DNA barcode sequence for the identification of many animal species. However, the COI region is of limited use for identifying certain species and is not efficiently amplified by PCR in all animal taxa. To evaluate the utility of COI as a DNA barcode and to identify other barcode genes, we chose the aphid subfamily Lachninae (Hemiptera: Aphididae as the focus of our study. We compared the results obtained using COI with two other mitochondrial genes, COII and Cytb. In addition, we propose a new method to improve the efficiency of species identification using DNA barcoding. METHODOLOGY/PRINCIPAL FINDINGS: Three mitochondrial genes (COI, COII and Cytb were sequenced and were used in the identification of over 80 species of Lachninae. The COI and COII genes demonstrated a greater PCR amplification efficiency than Cytb. Species identification using COII sequences had a higher frequency of success (96.9% in "best match" and 90.8% in "best close match" and yielded lower intra- and higher interspecific genetic divergence values than the other two markers. The use of "tag barcodes" is a new approach that involves attaching a species-specific tag to the standard DNA barcode. With this method, the "barcoding overlap" can be nearly eliminated. As a result, we were able to increase the identification success rate from 83.9% to 95.2% by using COI and the "best close match" technique. CONCLUSIONS/SIGNIFICANCE: A COII-based identification system should be more effective in identifying lachnine species than COI or Cytb. However, the Cytb gene is an effective marker for the study of aphid population genetics due to its high sequence diversity. Furthermore, the use of "tag barcodes" can improve the accuracy of DNA barcoding identification by reducing or removing the overlap between intra- and inter-specific genetic divergence values.

  10. Enhancement of Dopaminergic Differentiation in Proliferating Midbrain Neuroblasts by Sonic Hedgehog and Ascorbic Acid

    Science.gov (United States)

    Volpicelli, Floriana; Consales, Claudia; Caiazzo, Massimiliano; Colucci-D'Amato, Luca; Perrone-Capano, Carla; di Porzio, Umberto

    2004-01-01

    We analyzed the molecular mechanisms involved in the acquisition and maturation of dopaminergic (DA) neurons generated in vitro from rat ventral mesencephalon (MES) cells in the presence of mitogens or specific signaling molecules. The addition of basic fibroblast growth factor (bFGF) to MES cells in serum-free medium stimulates the proliferation of neuroblasts but delays DA differentiation. Recombinant Sonic hedgehog (SHH) protein increases up to three fold the number of tyrosine hydroxylase (TH)-positive cells and their differentiation, an effect abolished by anti-SHH antibodies. The expanded cultures are rich in nestin-positive neurons, glial cells are rare, all TH+ neurons are DA, and all DA and GABAergic markers analyzed are expressed. Adding ascorbic acid to bFGF/SHH-treated cultures resulted in a further five- to seven-fold enhancement of viable DA neurons. This experimental system also provides a powerful tool to generate DA neurons from single embryos. Our strategy provides an enriched source of MES DA neurons that are useful for analyzing molecular mechanisms controlling their function and for experimental regenerative approaches in DA dysfunction. PMID:15303305

  11. Notch maintains Drosophila type II neuroblasts by suppressing expression of the Fez transcription factor Earmuff.

    Science.gov (United States)

    Li, Xiaosu; Xie, Yonggang; Zhu, Sijun

    2016-07-15

    Notch signaling is crucial for maintaining neural stem cell (NSC) self-renewal and heterogeneity; however, the underlying mechanism is not well understood. In Drosophila, loss of Notch prematurely terminates the self-renewal of larval type II neuroblasts (NBs, the Drosophila NSCs) and transforms type II NBs into type I NBs. Here, we demonstrate that Notch maintains type II NBs by suppressing the activation of earmuff (erm) by Pointed P1 (PntP1). We show that loss of Notch or components of its canonical pathway leads to PntP1-dependent ectopic Erm expression in type II NBs. Knockdown of Erm significantly rescues the loss-of-Notch phenotypes, and misexpression of Erm phenocopies the loss of Notch. Ectopically expressed Erm promotes the transformation of type II NBs into type I NBs by inhibiting PntP1 function and expression in type II NBs. Our work not only elucidates a key mechanism of Notch-mediated maintenance of type II NB self-renewal and identity, but also reveals a novel function of Erm. © 2016. Published by The Company of Biologists Ltd.

  12. Antipsychotic drugs alter neuronal development including ALM neuroblast migration and PLM axonal outgrowth in Caenorhabditis elegans.

    Science.gov (United States)

    Donohoe, Dallas R; Weeks, Kathrine; Aamodt, Eric J; Dwyer, Donard S

    2008-01-01

    Antipsychotic drugs are increasingly being prescribed for children and adolescents, and are used in pregnant women without a clear demonstration of safety in these populations. Global effects of these drugs on neurodevelopment (e.g., decreased brain size) have been reported in rats, but detailed knowledge about neuronal effects and mechanisms of action are lacking. Here we report on the evaluation of a comprehensive panel of antipsychotic drugs in a model organism (Caenorhabditis elegans) that is widely used to study neuronal development. Specifically, we examined the effects of the drugs on neuronal migration and axonal outgrowth in mechanosensory neurons visualized with green fluorescent protein expressed from the mec-3 promoter. Clozapine, fluphenazine, and haloperidol produced deficits in the development and migration of ALM neurons and axonal outgrowth in PLM neurons. The defects included failure of neuroblasts to migrate to the proper location, and excessive growth of axons past their normal termination point, together with abnormal morphological features of the processes. Although the antipsychotic drugs are potent antagonists of dopamine and serotonin receptors, the neurodevelopmental deficits were not rescued by co-incubation with serotonin or the dopaminergic agonist, quinpirole. Other antipsychotic drugs, risperidone, aripiprazole, quetiapine, trifluoperazine and olanzapine, also produced modest, but detectable, effects on neuronal development. This is the first report that antipsychotic drugs interfere with neuronal migration and axonal outgrowth in a developing nervous system.

  13. Myosin II promotes the anisotropic loss of the apical domain during Drosophila neuroblast ingression.

    Science.gov (United States)

    Simões, Sérgio; Oh, Youjin; Wang, Michael F Z; Fernandez-Gonzalez, Rodrigo; Tepass, Ulrich

    2017-05-01

    Epithelial-mesenchymal transitions play key roles in development and cancer and entail the loss of epithelial polarity and cell adhesion. In this study, we use quantitative live imaging of ingressing neuroblasts (NBs) in Drosophila melanogaster embryos to assess apical domain loss and junctional disassembly. Ingression is independent of the Snail family of transcriptional repressors and down-regulation of Drosophila E-cadherin (DEcad) transcription. Instead, the posttranscriptionally regulated decrease in DEcad coincides with the reduction of cell contact length and depends on tension anisotropy between NBs and their neighbors. A major driver of apical constriction and junctional disassembly are periodic pulses of junctional and medial myosin II that result in progressively stronger cortical contractions during ingression. Effective contractions require the molecular coupling between myosin and junctions and apical relaxation of neighboring cells. Moreover, planar polarization of myosin leads to the loss of anterior-posterior junctions before the loss of dorsal-ventral junctions. We conclude that planar-polarized dynamic actomyosin networks drive apical constriction and the anisotropic loss of cell contacts during NB ingression. © 2017 Simões et al.

  14. Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi

    Science.gov (United States)

    Daniel L. Lindner; Tor Carlsen; Henrik Nilsson; Marie Davey; Trond Schumacher; Havard. Kauserud

    2013-01-01

    The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular...

  15. Indices of methylation in sperm DNA from fertile men differ between distinct geographical regions.

    Science.gov (United States)

    Consales, C; Leter, G; Bonde, J P E; Toft, G; Eleuteri, P; Moccia, T; Budillon, A; Jönsson, B A G; Giwercman, A; Pedersen, H S; Ludwicki, J K; Zviezdai, V; Heederik, D; Spanò, M

    2014-09-01

    Which are the main determinants, if any, of sperm DNA methylation levels? Geographical region resulted associated with the sperm methylation status assessed on genome-wide repetitive sequences. DNA methylation level, assessed on repetitive sequences from peripheral blood lymphocyte, can vary with age, gender, alcohol consumption and white blood cell counts. A cross-sectional study. Individual data were collected from 269 young healthy men of proven fertility living in three geographical regions: Inuits from Greenland, Caucasians from Warsaw (Poland) and Kharkiv (Ukraine). Semen samples were collected between May 2002 and February 2004 and aliquots were immediately frozen. We estimated sperm DNA global methylation level (DGML) in two ways. First DNA methylation in repetitive DNA sequences (LINE-1, Satα and Alu) was quantified by PCR pyrosequencing after bisulfite conversion and second by flow cytometry (FCM) using fluorescently labeled monoclonal antibodies anti-5-methylcytosine. We analyzed whether personal characteristics and habits, body mass index, semen quality parameters, sperm chromatin integrity, biomarkers of accessory gland function and the plasma concentration of reproductive hormones were associated with sperm DNA methylation levels in men. Associations were evaluated by analysis of variance and linear regression analyses. The geographical location emerged as the main determinant when using the methylation level in repetitive sequences. FCM DGML results were not associated with those from repetitive sequence analysis. No other consistent associations between methylation markers and the assessed variables were identified across countries. The methods used are only surrogates of the actual sperm methylome and the methylation levels at individual specific loci were not explored. Sperm DGML is relatively independent from semen quality parameters and is a new candidate biomarker for epidemiological studies of the impact of environmental contaminants on male

  16. A genetic association study of DNA methylation levels in the DRD4 gene region finds associations with nearby SNPs.

    Science.gov (United States)

    Docherty, Sophia J; Davis, Oliver Sp; Haworth, Claire Ma; Plomin, Robert; D'Souza, Ursula; Mill, Jonathan

    2012-06-12

    Dopamine receptor D4(DRD4) polymorphisms have been associated with a number of psychiatric disorders, but little is known about the mechanism of these associations. DNA methylation is linked to the regulation of gene expression and plays a vital role in normal cellular function, with abnormal DNA methylation patterns implicated in a range of disorders. Recent evidence suggests DNA methylation can be influenced by cis-acting DNA sequence variation, that is, DNA sequence variation located nearby on the same chromosome. To investigate the potential influence of cis-acting genetic elements within DRD4, we analysed DRD4 promoter DNA methylation levels in the transformed lymphoblastoid cell-line DNA of 89 individuals (from 30 family-trios). Five SNPs located +/- 10kb of the promoter region were interrogated for associations with DNA methylation levels. Four significant SNP associations were found with DNA methylation (rs3758653, rs752306, rs11246228 and rs936465). The associations of rs3758653 and rs936465 with DNA methylation were tested and nominally replicated (p-value associations. DRD4 has been implicated in several psychiatric disease phenotypes and our results shed light upon the possible mode of action of SNP associations in this region.

  17. 16S-23S rDNA internal transcribed spacer regions in four Proteus species.

    Science.gov (United States)

    Cao, Boyang; Wang, Min; Liu, Lei; Zhou, Zhemin; Wen, Shaoping; Rozalski, Antoni; Wang, Lei

    2009-04-01

    Proteus is a Gram-negative, facultative anaerobic bacterium. In this study, 813 Proteus 16S-23S rDNA internal transcribed spacer (ITS) sequences were determined from 46 Proteus strains, including 388 ITS from 22 P. mirabilis strains, 211 ITS from 12 P. vulgaris strains, 169 ITS from 10 P. penneri strains, and 45 ITS from 2 P. myxofaciens strains. The Proteus strains carry mainly two types of ITS, ITS(Glu) (containing tRNA(Glu (UUC)) gene) and ITS(Ile+Ala) (containing tRNA(Ile (GAU)) and tRNA(Ala (UGC)) gene), and are in the forms of 28 variants with 25 genomic origins. The ITS sequences are a mosaic-like structure consisting of three conservative regions and two variable regions. The nucleotide identity of ITS subtypes in strains of the same species ranges from 96.2% to 100%. The divergence of Proteus ITS divergence was most likely due to intraspecies recombinations or horizontal transfers of sequence blocks. The phylogenetic relationship deduced from the second variable region of ITS sequences of the three facultative human pathogenic species P. mirabilis, P. vulgaris and P. penneri is similar with that based on 16S rDNA sequences, but has higher resolution to differentiate closely related P. vulgaris and P. penneri. This study is the first comprehensive study of ITS in four Proteus species and laid solid foundation for the development of high-throughput technology for quick and accurate identification of the important foodborne and nosocomial pathogens.

  18. DNA Methylation of Regulatory Regions of Imprinted Genes at Birth and Its Relation to Infant Temperament

    Directory of Open Access Journals (Sweden)

    Bernard F. Fuemmeler

    2016-01-01

    Full Text Available BACKGROUND DNA methylation of the differentially methylated regions (DMRs of imprinted genes is relevant to neurodevelopment. METHODS DNA methylation status of the DMRs of nine imprinted genes in umbilical cord blood leukocytes was analyzed in relation to infant behaviors and temperament (n = 158. RESULTS MEG3 DMR levels were positively associated with internalizing ( β = 0.15, P = 0.044 and surgency ( β = 0.19, P = 0.018 behaviors, after adjusting for birth weight, gender, gestational age at birth, maternal age at delivery, race/ethnicity, education level, smoking status, parity, and a history of anxiety or depression. Higher methylation levels at the intergenic MEG3-IG methylation regions were associated with surgency ( β = 0.28, P = 0.0003 and PEG3 was positively related to externalizing ( β = 0.20, P = 0.01 and negative affectivity ( β = 0.18, P = 0.02. CONCLUSION While the small sample size limits inference, these pilot data support gene-specific associations between epigenetic differences in regulatory regions of imprinted domains at birth and later infant temperament.

  19. Genetic structure of Florida green turtle rookeries as indicated by mitochondrial DNA control region sequences

    Science.gov (United States)

    Shamblin, Brian M.; Bagley, Dean A.; Ehrhart, Llewellyn M.; Desjardin, Nicole A.; Martin, R. Erik; Hart, Kristen M.; Naro-Maciel, Eugenia; Rusenko, Kirt; Stiner, John C.; Sobel, Debra; Johnson, Chris; Wilmers, Thomas; Wright, Laura J.; Nairn, Campbell J.

    2014-01-01

    Green turtle (Chelonia mydas) nesting has increased dramatically in Florida over the past two decades, ranking the Florida nesting aggregation among the largest in the Greater Caribbean region. Individual beaches that comprise several hundred kilometers of Florida’s east coast and Keys support tens to thousands of nests annually. These beaches encompass natural to highly developed habitats, and the degree of demographic partitioning among rookeries was previously unresolved. We characterized the genetic structure of ten Florida rookeries from Cape Canaveral to the Dry Tortugas through analysis of 817 base pair mitochondrial DNA (mtDNA) control region sequences from 485 nesting turtles. Two common haplotypes, CM-A1.1 and CM-A3.1, accounted for 87 % of samples, and the haplotype frequencies were strongly partitioned by latitude along Florida’s Atlantic coast. Most genetic structure occurred between rookeries on either side of an apparent genetic break in the vicinity of the St. Lucie Inlet that separates Hutchinson Island and Jupiter Island, representing the finest scale at which mtDNA structure has been documented in marine turtle rookeries. Florida and Caribbean scale analyses of population structure support recognition of at least two management units: central eastern Florida and southern Florida. More thorough sampling and deeper sequencing are necessary to better characterize connectivity among Florida green turtle rookeries as well as between the Florida nesting aggregation and others in the Greater Caribbean region.

  20. Investigation of length heteroplasmy in mitochondrial DNA control region by massively parallel sequencing.

    Science.gov (United States)

    Lin, Chun-Yen; Tsai, Li-Chin; Hsieh, Hsing-Mei; Huang, Chia-Hung; Yu, Yu-Jen; Tseng, Bill; Linacre, Adrian; Lee, James Chun-I

    2017-09-01

    Accurate sequencing of the control region of the mitochondrial genome is notoriously difficult due to the presence of polycytosine bases, termed C-tracts. The precise number of bases that constitute a C-tract and the bases beyond the poly cytosines may not be accurately defined when analyzing Sanger sequencing data separated by capillary electrophoresis. Massively parallel sequencing has the potential to resolve such poor definition and provides the opportunity to discover variants due to length heteroplasmy. In this study, the control region of mitochondrial genomes from 20 samples was sequenced using both standard Sanger methods with separation by capillary electrophoresis and also using massively parallel DNA sequencing technology. After comparison of the two sets of generated sequence, with the exception of the C-tracts where length heteroplasmy was observed, all sequences were concordant. Sequences of three segments 16184-16193, 303-315 and 568-573 with C-tracts in HVI, II and III can be clearly defined from the massively parallel sequencing data using the program SEQ Mapper. Multiple sequence variants were observed in the length of C-tracts longer than 7 bases. Our report illustrates the accurate designation of all the length variants leading to heteroplasmy in the control region of the mitochondrial genome that can be determined by SEQ Mapper based on data generated by massively parallel DNA sequencing. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Combination of native and denaturing PAGE for the detection of protein binding regions in long fragments of genomic DNA

    Directory of Open Access Journals (Sweden)

    Metsis Madis

    2008-06-01

    Full Text Available Abstract Background In a traditional electrophoresis mobility shift assay (EMSA a 32P-labeled double-stranded DNA oligonucleotide or a restriction fragment bound to a protein is separated from the unbound DNA by polyacrylamide gel electrophoresis (PAGE in nondenaturing conditions. An extension of this method uses the large population of fragments derived from long genomic regions (approximately 600 kb for the identification of fragments containing protein binding regions. With this method, genomic DNA is fragmented by restriction enzymes, fragments are amplified by PCR, radiolabeled, incubated with nuclear proteins and the resulting DNA-protein complexes are separated by two-dimensional PAGE. Shifted DNA fragments containing protein binding sites are identified by using additional procedures, i. e. gel elution, PCR amplification, cloning and sequencing. Although the method allows simultaneous analysis of a large population of fragments, it is relatively laborious and can be used to detect only high affinity protein binding sites. Here we propose an alternative and straightforward strategy which is based on a combination of native and denaturing PAGE. This strategy allows the identification of DNA fragments containing low as well as high affinity protein binding regions, derived from genomic DNA ( Results We have combined an EMSA-based selection step with subsequent denaturing PAGE for the localization of protein binding regions in long (up to10 kb fragments of genomic DNA. Our strategy consists of the following steps: digestion of genomic DNA with a 4-cutter restriction enzyme (AluI, BsuRI, TruI, etc, separation of low and high molecular weight fractions of resultant DNA fragments, 32P-labeling with Klenow polymerase, traditional EMSA, gel elution and identification of the shifted bands (or smear by denaturing PAGE. The identification of DNA fragments containing protein binding sites is carried out by running the gel-eluted fragments alongside

  2. Conserved DNA motifs in the type II-A CRISPR leader region.

    Science.gov (United States)

    Van Orden, Mason J; Klein, Peter; Babu, Kesavan; Najar, Fares Z; Rajan, Rakhi

    2017-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3' end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3' leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3' leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci.

  3. Fingerprinting of cell lines by directed amplification of minisatellite-region DNA (DAMD

    Directory of Open Access Journals (Sweden)

    Silva L.M.

    2001-01-01

    Full Text Available The development of in vitro propagation of cells has been an extraordinary technical advance for several biological studies. The correct identification of the cell line used, however, is crucial, as a mistaken identity or the presence of another contaminating cell may lead to invalid and/or erroneous conclusions. We report here the application of a DNA fingerprinting procedure (directed amplification of minisatellite-region DNA, developed by Heath et al. [Nucleic Acids Research (1993 21: 5782-5785], to the characterization of cell lines. Genomic DNA of cells in culture was extracted and amplified by PCR in the presence of VNTR core sequences, and the amplicons were separated by agarose gel electrophoresis. After image capture with a digital camera, the banding profiles obtained were analyzed using a software (AnaGel specially developed for the storage and analysis of electrophoretic fingerprints. The fingerprints are useful for construction of a data base for identification of cell lines by comparison to reference profiles as well as comparison of similar lines from different sources and periodic follow-up of cells in culture.

  4. Comparing the Potential for Identification of Lactobacillus spp. of 16S rDNA Variable Regions

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    Diego Mauricio Riaño-Pachón

    2013-07-01

    Full Text Available 0 0 1 248 1368 Universidad de los Andes 11 3 1613 14.0 Normal 0 21 false false false ES-TRAD JA X-NONE 16s rDNA is used for bacterial identification because its variation rate between species allows differentiation. The gene for this ribosomal subunit has 9 variable regions and some of them give more information than others. We were interested in evaluating the potential for species identification of each region and their combinations. We extracted the V1 to V8 regions of 16s rDNA from different strains and species of Lactobacillus and analyzed them using STAP (ss-RNA Taxonomy Assigning Pipeline and RDP (Ribosomal Database Project multiclassifier packages. Phylogenetic trees obtained by maximum likelihood analyses were compared. Classification results show that many regions give the correct genus classification using RDP and STAP, however they are not enough to classify up to the level of species. V5V6 region presents the highest quantity of informative fragments but also present the highest rate of false negatives. V1V3 region presents the highest rate of true positives (species using STAP and the region V5V8 in RDP (genus.The phylogenetic result shows that the reference topology could be obtained using different combination of regions as V1V3 and V1V8.The experimental validation was done using commercial strains from a probiotic tampon. Sequencing analysis show that the V1V3 region gives the same information and result as the complete 16s rDNA; the three isolated strains correspond to the strains indicated in the product. We conclude that the V1V3 region is the minimum required region to classify Lactobacillus spp. in the correct way and this region is useful in metagenomics to analyze probiotics samples  COMPARACIÓN ENTRE EL POTENCIAL DE LAS REGIONES VARIABLES DEL 16S rDNA PARA LA IDENTIFICACIÓN DE Lactobacillus spp (LactobacilliaceaeEl 16s rDNA es utilizado para la identificación bacteriana dada su tasa de variaci

  5. Quantitative analysis of DNA methylation in the promoter region of the methylguanine-O(6) -DNA-methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis.

    Science.gov (United States)

    Goedecke, Simon; Mühlisch, Jörg; Hempel, Georg; Frühwald, Michael C; Wünsch, Bernhard

    2015-12-01

    Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine-O(6) -DNA-Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non-methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non-linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Methyl thiophanate as a DNA minor groove binder produces MT-Cu(II)-DNA ternary complex preferably with AT rich region for initiation of DNA damage.

    Science.gov (United States)

    Saquib, Quaiser; Al-Khedhairy, Abdulaziz A; Alarifi, Saud A; Dutta, Sansa; Dasgupta, Swagata; Musarrat, Javed

    2010-07-01

    Interaction of a genotoxic fungicide methyl thiophanate (MT) has been studied in vitro with calf thymus DNA. Fluorescence quenching data revealed the binding constant (K(a)=3.23 x 10(4)M(-1)) and binding capacity (n=1.1) of MT with ctDNA. Ligand displacement studies using specific probes suggested the MT binding at DNA minor groove. The docking analysis further substantiated MT interaction with at least three AT base pairs within the DNA groove. A discernable change in E(0)' value with decreased peak currents in cyclic voltammogram, and peak shifts in CD spectra reflected the formation of MT-ctDNA and MT-ctDNA-Cu(II) complexes. The results elucidate the significance of specific MT-DNA interactions as an initiating event in MT-induced DNA damage. (c) 2010 Elsevier B.V. All rights reserved.

  7. The next generation of target capture technologies - large DNA fragment enrichment and sequencing determines regional genomic variation of high complexity.

    Science.gov (United States)

    Dapprich, Johannes; Ferriola, Deborah; Mackiewicz, Kate; Clark, Peter M; Rappaport, Eric; D'Arcy, Monica; Sasson, Ariella; Gai, Xiaowu; Schug, Jonathan; Kaestner, Klaus H; Monos, Dimitri

    2016-07-09

    The ability to capture and sequence large contiguous DNA fragments represents a significant advancement towards the comprehensive characterization of complex genomic regions. While emerging sequencing platforms are capable of producing several kilobases-long reads, the fragment sizes generated by current DNA target enrichment technologies remain a limiting factor, producing DNA fragments generally shorter than 1 kbp. The DNA enrichment methodology described herein, Region-Specific Extraction (RSE), produces DNA segments in excess of 20 kbp in length. Coupling this enrichment method to appropriate sequencing platforms will significantly enhance the ability to generate complete and accurate sequence characterization of any genomic region without the need for reference-based assembly. RSE is a long-range DNA target capture methodology that relies on the specific hybridization of short (20-25 base) oligonucleotide primers to selected sequence motifs within the DNA target region. These capture primers are then enzymatically extended on the 3'-end, incorporating biotinylated nucleotides into the DNA. Streptavidin-coated beads are subsequently used to pull-down the original, long DNA template molecules via the newly synthesized, biotinylated DNA that is bound to them. We demonstrate the accuracy, simplicity and utility of the RSE method by capturing and sequencing a 4 Mbp stretch of the major histocompatibility complex (MHC). Our results show an average depth of coverage of 164X for the entire MHC. This depth of coverage contributes significantly to a 99.94 % total coverage of the targeted region and to an accuracy that is over 99.99 %. RSE represents a cost-effective target enrichment method capable of producing sequencing templates in excess of 20 kbp in length. The utility of our method has been proven to generate superior coverage across the MHC as compared to other commercially available methodologies, with the added advantage of producing longer sequencing

  8. Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme.

    Directory of Open Access Journals (Sweden)

    Anurag Kumar Sinha

    2017-10-01

    Full Text Available Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant.

  9. Sequencing the hypervariable regions of human mitochondrial DNA using massively parallel sequencing: Enhanced data acquisition for DNA samples encountered in forensic testing.

    Science.gov (United States)

    Davis, Carey; Peters, Dixie; Warshauer, David; King, Jonathan; Budowle, Bruce

    2015-03-01

    Mitochondrial DNA testing is a useful tool in the analysis of forensic biological evidence. In cases where nuclear DNA is damaged or limited in quantity, the higher copy number of mitochondrial genomes available in a sample can provide information about the source of a sample. Currently, Sanger-type sequencing (STS) is the primary method to develop mitochondrial DNA profiles. This method is laborious and time consuming. Massively parallel sequencing (MPS) can increase the amount of information obtained from mitochondrial DNA samples while improving turnaround time by decreasing the numbers of manipulations and more so by exploiting high throughput analyses to obtain interpretable results. In this study 18 buccal swabs, three different tissue samples from five individuals, and four bones samples from casework were sequenced at hypervariable regions I and II using STS and MPS. Sample enrichment for STS and MPS was PCR-based. Library preparation for MPS was performed using Nextera® XT DNA Sample Preparation Kit and sequencing was performed on the MiSeq™ (Illumina, Inc.). MPS yielded full concordance of base calls with STS results, and the newer methodology was able to resolve length heteroplasmy in homopolymeric regions. This study demonstrates short amplicon MPS of mitochondrial DNA is feasible, can provide information not possible with STS, and lays the groundwork for development of a whole genome sequencing strategy for degraded samples. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. In situ genomic DNA extraction for PCR analysis of regions of interest in four plant species and one filamentous fungi

    Directory of Open Access Journals (Sweden)

    Luis E. Rojas

    2014-07-01

    Full Text Available The extraction methods of genomic DNA are usually laborious and hazardous to human health and the environment by the use of organic solvents (chloroform and phenol. In this work a protocol for in situ extraction of genomic DNA by alkaline lysis is validated. It was used in order to amplify regions of DNA in four species of plants and fungi by polymerase chain reaction (PCR. From plant material of Saccharum officinarum L., Carica papaya L. and Digitalis purpurea L. it was possible to extend different regions of the genome through PCR. Furthermore, it was possible to amplify a fragment of avr-4 gene DNA purified from lyophilized mycelium of Mycosphaerella fijiensis. Additionally, it was possible to amplify the region ap24 transgene inserted into the genome of banana cv. `Grande naine' (Musa AAA. Key words: alkaline lysis, Carica papaya L., Digitalis purpurea L., Musa, Saccharum officinarum L.

  11. Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome.

    Science.gov (United States)

    Erliandri, Indri; Fu, Haiqing; Nakano, Megumi; Kim, Jung-Hyun; Miga, Karen H; Liskovykh, Mikhail; Earnshaw, William C; Masumoto, Hiroshi; Kouprina, Natalay; Aladjem, Mirit I; Larionov, Vladimir

    2014-10-01

    In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  12. Mitotic effects of monochromatic ultraviolet radiation at 225, 265, and 280 nm on eleven stages of the cell cycle of the grasshopper neuroblast in culture. I. Overall retardation from the stage irradiated to nuclear membrane breakdown

    Energy Technology Data Exchange (ETDEWEB)

    Carlson, J.G.

    1976-10-01

    Neuroblasts of Chortophaga viridifasciata (DeGeer) in culture were exposed to different doses of 225, 265, or 280 nm ultraviolet radiations at 11 different stages and substages of the mitotic cycle and individually selected cells were timed to breakdown of the nuclear membrane. Comparisons of the effectiveness of different wavelengths on the different stages were based on the dose that reduced the cell progression rate to 67 percent of normal (D/sub 67/) and the slope of the regression line, i.e., the control to treated time (C/T) ratio change/erg/mm/sup 2/ at the D/sub 67/ level. Cells of the prereplication period (metaphase + anaphase + early telophase) and the S phase (middle and late telophase + interphase + very early prophase) are equally sensitive to uv and contrast sharply with the much lower sensitivity of those in the postreplication period (early and middle prophase). This can best be interpreted if chromosomal DNA is the chromophore for uv-induced mitotic retardation. Cells in the prereplication period at exposure show no wavelength effect. In the S phase all stages except middle telophase and all stages combined are significantly more sensitive to 265 and 280 nm than to 225 nm. Of the postreplication stages, early prophase is retarded significantly more by 280 than by 225 or 265 nm. The C/T ratio/erg/mm/sup 2/ is greater after exposure to 265 nm at all prereplication and replication stages, but exhibits no consistent wavelength pattern during the postreplication period. Evidence based on the orientation of the neuroblast with respect to the uv-source suggests that the chromophore for mitotic retardation does not reside within the centrosome and related structures, but may be present, at least partly, in the nucleolus.

  13. In search of coding and non-coding regions of DNA sequences based on balanced estimation of diffusion entropy.

    Science.gov (United States)

    Zhang, Jin; Zhang, Wenqing; Yang, Huijie

    2016-01-01

    Identification of coding regions in DNA sequences remains challenging. Various methods have been proposed, but these are limited by species-dependence and the need for adequate training sets. The elements in DNA coding regions are known to be distributed in a quasi-random way, while those in non-coding regions have typical similar structures. For short sequences, these statistical characteristics cannot be extracted correctly and cannot even be detected. This paper introduces a new way to solve the problem: balanced estimation of diffusion entropy (BEDE).

  14. [Variation of human mitochondrial DNA: distribution of hot spots in hypervariable segment I of the major noncoding region].

    Science.gov (United States)

    Maliarchuk, B A; Derenko, M V

    2001-07-01

    Mitochondrial DNA (mtDNA) samples belonging to fifteen phylogenetically related mtDNA types specific to the populations of Europe (H, V, J, T, U, K, I, W, and X) and Northern Asia (A, C, D, G, Y, and Z) were typed for sequence variation in hypervariable segment I (HVSI). The approach used allowed to distinguish several hypervariable sites at nucleotide positions 16093, 16129, 16189, 16311, and 16362. Identical mutations at these sites were found in 10-11 out of 15 mtDNA groups examined. Positions 16126, 16172, 16192, 16256, 16261, 16291, 16293, and 16298 appeared to be less variable, since parallel mutations at these sites were found in 6-8 European and Asian mtDNA groups. The examples of the effects of mutations in hypervariable positions at the major noncoding mtDNA region on the frequency of reverse mutations in other mtDNA regions are presented. It was shown that such effects of nucleotide context on the mutation rate could be observed in phylogenetic mtDNA networks such as cyclic structures like rhombs and cubes. Analogous structures in the networks could be seen also in the case of the appearance of recombinant mtDNA types resulted from homologous recombination between mtDNA molecules in heteroplasmic mixture. The problem of the effect of polynucleotide context on the intensity of mtDNA mutagenesis is discussed. Recombination processes along with site-directed mutagenesis caused by action of genetic factors (of nuclear genome) and/or of the environment are considered as possible mechanisms of mitochondrial genome evolution.

  15. Analysis of a DNA polymorphic region in the gtfB and gtfC genes of Streptococcus mutans.

    Science.gov (United States)

    Chia, J S; Lin, S W; Hsu, T Y; Chen, J Y; Kwan, H W; Yang, C S

    1993-04-01

    We have previously demonstrated the existence of DNA polymorphisms at the 5' coding regions of the gtfB and gtfC genes specifying the streptococcal glucosyltransferases (J.S. Chia, T.Y. Hsu, L.J. Teng, J.Y. Chen, L.J. Hahn, and C.S. Yang, Infect. Immun. 59:1656-1660, 1991). DNA sequence analysis by polymerase chain reaction and direct sequencing revealed that while several nucleotide changes were identified, accounting for the polymorphisms, the amino acids which they code for remain unchanged. The polymorphic region is located in a highly conserved amino terminus of the glucosyltransferases. A peptide of 19 amino acids from this region reversed the inhibiting activity of an antiserum raised against the proteins coded for by the gtfB and gtfC genes. The results suggest that the polymorphic region, varying in DNA but not in amino acid sequences, might specify some biological function.

  16. [Screening of the gene mutation in D-loop region of mitochondrial DNA in oral squamous cell carcinoma].

    Science.gov (United States)

    Sun, Yang; Yuan, Rong-tao; Chen, Wan-tao; Bu, Ling-xue; Jia, Mu-yun

    2013-05-01

    To investigate the gene mutation in D-loop region of mitochondrial DNA (mtDNA) in oral squamous cell carcinoma (OSCC) tissue and to explore the role of the gene mutation in D-loop region in the OSCC tumorigenesis. mtDNA was obtained from cancer, paracancerous and normal mucosa tissues of thirty patients with OSCC. The D-loop regions of mtDNA were amplified with PCR, sequencing and then analyzed by Chromas software and BLAST to identify the mutation site. Mutation in the D-loop region was found in eight cases, with the mutation rate of 27%. There were nine mutations totally, including one point mutation, two base deletions, three insertion mutations, three heterozygous mutations. In these mutations, base deletions were different from each other and heterozygous mutations had no same mutation form, while the three insertion mutations were same, the insertion of base C. One case had T/A heterozygous mutation and base C insertion at the same time. There were mutations in mtDNA D-loop in OSCC, but the relationship between occurrence of OSCC and mutation of mtDNA needs further study.

  17. Conserved DNA motifs in the type II-A CRISPR leader region

    Science.gov (United States)

    Babu, Kesavan; Najar, Fares Z.

    2017-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3′ end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3′ leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3′ leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci. PMID:28392985

  18. Conserved DNA motifs in the type II-A CRISPR leader region

    Directory of Open Access Journals (Sweden)

    Mason J. Van Orden

    2017-04-01

    Full Text Available The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3′ end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3′ leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710 and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3′ leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci.

  19. Polymorphic sequence in the ND3 region of Java endemic Ploceidae birds mitochondrial DNA

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    R. SUSANTI

    2011-04-01

    Full Text Available Susanti R (2011 Polymorphic sequence in the ND3 region of Java endemic Ploceidae birds mitochondrial DNA. Biodiversitas 12: 70-75. As part of biodiversity, Ploceidae bird family must be kept away from extinction and degradation of gene-diversity. This research was aimed to analyze ND3 gene from mitochondrial DNA of Java Island endemic of Ploceidae bird. Each species of Ploceidae birds family was identified based on their morphological character, then the blood sample was taken from the birds nail vein. DNA was isolated from blood using Dixit method. Fragment of ND3 gene was amplified using PCR method with specific primer pairs and sequenced using dideoxy termination method with ABI automatic sequencer. Multiple alignment of ND3 nucleotide sequences were analyzed using ClustalW of MEGA-3.1 program. Estimation of genetic distance and phylogenetic tree construction were analyzed with Neighbor-Joining method and calculation of distance matrix with Kimura 2 –parameter. The result of Java Island endemic of Ploceidae bird family exploration showed that Erythrura hyperythra and Lonchura ferruginosa can not be found anymore in nature, but the Lonchura malacca that are not actually Java island endemic was also found. Nucleotide sequence of mitochondrial ND3 gene of Ploceidae bird family showed a quite high polymorphism, with 122 substitutions from 334 nucleotides analyzed. Phylogenetic tree of nucleotide sequence of Ploceidae bird family formed 2 clusters. One cluster consisted of the Ploceus hypoxanthus, Ploceus philippinus, Ploceus manyar and Passer montanus, and the others species were included in the second cluster. ND3 gene sequence data from this Ploceidae family need to be analyzed further to see possible relationship with a particular phenotype.

  20. Diversity of prophage DNA regions of Streptococcus agalactiae clonal lineages from adults and neonates with invasive infectious disease.

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    Mazen Salloum

    Full Text Available The phylogenetic position and prophage DNA content of the genomes of 142 S. agalactiae (group-B streptococcus, GBS isolates responsible for bacteremia and meningitis in adults and neonates were studied and compared. The distribution of the invasive isolates between the various serotypes, sequence types (STs and clonal complexes (CCs differed significantly between adult and neonatal isolates. Use of the neighbor-net algorithm with the PHI test revealed evidence for recombination in the population studied (PHI, P = 2.01 × 10(-6, and the recombination-mutation ratio (R/M was 6:7. Nevertheless, the estimated R/M ratio differed between CCs. Analysis of the prophage DNA regions of the genomes of the isolates assigned 90% of the isolates to five major prophage DNA groups: A to E. The mean number of prophage DNA fragments amplified per isolate varied from 2.6 for the isolates of prophage DNA group E to 4.0 for the isolates of prophage DNA group C. The isolates from adults and neonates with invasive diseases were distributed differently between the various prophage DNA groups (P < 0.00001. Group C prophage DNA fragments were found in 52% of adult invasive isolates, whereas 74% of neonatal invasive isolates had prophage DNA fragments of groups A and B. Differences in prophage DNA content were also found between serotypes, STs and CCs (P < 0.00001. All the ST-1 and CC1 isolates, mostly of serotype V, belonged to the prophage DNA group C, whereas 84% of the ST-17 and CC17 isolates, all of serotype III, belonged to prophage DNA groups A and B. These data indicate that the transduction mechanisms, i.e., gene transfer from one bacterium to another by a bacteriophage, underlying genetic recombination in S. agalactiae species, are specific to each intraspecies lineage and population of strains responsible for invasive diseases in adults and neonates.

  1. Sequence analysis of mtDNA COI barcode region revealed three haplotypes within Culex pipiens assemblage.

    Science.gov (United States)

    Koosha, Mona; Oshaghi, Mohammad Ali; Sedaghat, Mohammad Mehdi; Vatandoost, Hassan; Azari-Hamidian, Shahyad; Abai, Mohammad Reza; Hanafi-Bojd, Ahmad Ali; Mohtarami, Fatemeh

    2017-10-01

    Members of the Culex (Culex) pipiens assemblage are known vectors of deadly encephalitides, periodic filariasis, and West Nile virus throughout the world. However, members of this assemblage are morphologically indistinguishable or hard to distinguish and play distinct roles in transmission of the diseases. The current study aimed to provide further evidence on utility of the two most popular nuclear (ITS2-rDNA) and mitochondrial (COI barcode region) genetic markers to identify members of the assemblage. Culex pipiens assemblage specimens from different climate zones of Iran were collected and identified to species level based on morphological characteristics. Nucleotide sequences of the loci for the specimens plus available data in the GenBank were analyzed to find species specific genetic structures useful for diagnosis purposes. ITS2 region was highly divergent within species or populations suggesting lack of consistency as a reliable molecular marker. In contrast, sequence analysis of 710 bp of COI gene revealed three fixed haplotypes named here "C, T, H" within the assemblage which can be distinguished by HaeIII and AluI enzymes. There were a correlation between the haplotypes and the world climate regions, where the haplotypes H/T and C are present mainly in temperate and tropical regions of the world, respectively. In the New world, Australia, and Japan only haplotype H is found. In conjunction between tropical and temperate regions such Iran, China, and Turkey, a mix of C/H or C/H/T are present. Although, the haplotypes are not strictly species-specific, however, Cx. quinquefasciatus was mainly of haplotype C. Due to the lack of mating barrier and questionable taxonomic situation of the complex members, the mentioned haplotypes in combination with other morphological and molecular characters might be used to address the genetic structure of the studied populations. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Hybridization study of developmental plastid gene expression in mustard (Sinapsis alba L.) with cloned probes for most plastid DNA regions.

    Science.gov (United States)

    Link, G

    1984-07-01

    An approach to assess the extent of developmental gene expression of various regions of plastid (pt)DNA in mustard (Sinapis alba L.) is described. It involves cloning of most ptDNA regions. The cloned regions then serve as hybridization probes to detect and assess the abundance of complementary RNA sequences represented in total plastid RNA. By comparison of the hybridization pattern observed with plastid RNA from either dark-grown or light-grown plants it was found that many ptDNA regions are constitutively expressed, while several 'inducible' regions account for much higher transcript levels in the chloroplast than in the etioplast stage. The reverse situation, i.e. 'repressed' regions which would account for higher transcript levels in the etioplast, was not observed. The hybridization results obtained with RNA from 'intermediatetype' plastids suggest that transient gene expression is a common feature during light-induced chloroplast development. The time-course of gene expression differs for various ptDNA regions.

  3. Evolutionary analysis of DNA-protein-coding regions based on a genetic code cube metric.

    Science.gov (United States)

    Sanchez, Robersy

    2014-01-01

    The right estimation of the evolutionary distance between DNA or protein sequences is the cornerstone of the current phylogenetic analysis based on distance methods. Herein, it is demonstrated that the Manhattan distance (dw), weighted by the evolutionary importance of the nucleotide bases in the codon, is a naturally derived metric in the standard genetic code cube inserted into the three-dimensional Euclidean space. Based on the application of distance dw, a novel evolutionary model is proposed. This model includes insertion/deletion mutations that are very important for cancer studies, but usually discarded in classical evolutionary models. In this study, the new evolutionary model was applied to the phylogenetic analysis of the DNA protein-coding regions of 13 mammal mitochondrial genomes and of four cancer genetic- susceptibility genes (ATM, BRCA1, BRCA2 and p53) from nine mammals. The opossum (a marsupial) was used as an out-group species for both sets of sequences. The new evolutionary model yielded the correct topology, while the current models failed to separate the evolutionarily distant species of mouse and opossum.

  4. Intraspecific polymorphism of rDNA among five Nosema bombycis isolates from different geographic regions in China.

    Science.gov (United States)

    Liu, Handeng; Pan, Guoqing; Luo, Bo; Li, Tian; Yang, Qiong; Vossbrinck, Charles R; Debrunner-Vossbrinck, Bettina A; Zhou, Zeyang

    2013-05-01

    The microsporidian Nosema bombycis is the causative agent of pébrine, a highly infectious disease of the silkworm Bombyx mori. Three regions of the multicopy rDNA gene were examined in order to investigate the relationships among five Nosema isolates from various regions of China. Ribosomal DNA alleles are present on each of the 18 chromosomes of N. bombycis and show a high degree of variation. In this study the small subunit (SSU) rDNA, internal transcribed spacer (ITS) and intergenic spacer (IGS) regions for up to 10 different rDNA copies from each N. bombycis isolate were cloned and sequenced. As expected we see greater polymorphism in the ITS region (88 variable sites in 179 nucleotides) and IGS (200 variable sites in 279 nucleotides) than in the SSU rDNA (24 variable sites in 1232 nucleotides). Phylogenetic analysis shows greater differences between alleles within an isolate than between the same alleles from different isolates. The data reveal two very different groups, one from the Sichuan province and the other with a broad distribution including four provinces in southeast China and Japan. The Sichuan isolate does not have any rDNA alleles with sequences identical to those in the other isolates, implying that it is a separate, non-intermixing, population or perhaps a separate species from the other isolates. In light of the polymorphic nature of the rDNA alleles in N. bombycis and their presence on every chromosome, the rDNA gene may be useful for understanding the movement and ultimately the source of pébrine infections. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Naturally occurring V region connected antibodies inhibit anti-dsDNA antibody reactivity with dsDNA.

    Science.gov (United States)

    Srdic-Rajic, Tatjana; Jurisic, Vladimir; Andrejevic, Sladjana; Bonaci-Nikolic, Branka; Bowker, Timothy; Concas, Daniela; Metlas, Radmila

    2012-01-01

    The production of autoantibodies against a vast array of self antigens, most notably double stranded (ds) DNA, characterized systemic lupus erythematosus (SLE). The purpose of this work is to study specific Ig fractions isolated from normal human serum (NHS) and their effect on the binding of anti-double-stranded deoxyribonucleic acid (dsDNA) antibodies (Abs) to dsDNA. A fraction named immunoglobulin G (IgG)-reactive IgG was purified from total NHS IgG by absorption onto (CNBr)-activated Sepharose 4B linked to intact IgG molecules (IgG-Sepharose column). IgG-reactive IgG was co-incubated with systemic lupus erythematosus (SLE) patient's serum and binding of the anti-dsDNA Abs to dsDNA was measured by enzyme-linked immunosorbent assay (ELISA). Co-incubation of SLE patient's serum with IgG-reactive IgG resulted in a dose-dependent reduction in binding of anti-dsDNA Abs to dsDNA. A reduction greater than 70% was observed at a concentration of 300 μg of IgG-reactive IgG per mL of a 400-fold diluted SLE patient's serum whereas total NHS IgG, at the same concentration, resulted in a 10% reduction in binding. The purification process used to isolate IgG-reactive IgG was based on interactions between intact Ig rather than on interactions between F(ab')(2) portions. IgG(2) is the predominant immunoglobulin (Ig) subclass in IgG-reactive IgG. Thus, IgG(2) might have an important role in the connectivity characteristics of NHS IgG. The capacity of IgG-reactive IgG to inhibit anti-DNA Ab binding to dsDNA may have potential application in the treatment of SLE. This targeted biological approach may provide an alternative strategy to immunosuppressants. Copyright © 2011 Elsevier GmbH. All rights reserved.

  6. Bio-CAP: a versatile and highly sensitive technique to purify and characterise regions of non-methylated DNA

    Science.gov (United States)

    Blackledge, Neil P.; Long, Hannah K.; Zhou, Jin C.; Kriaucionis, Skirmantas; Patient, Roger; Klose, Robert J.

    2012-01-01

    Across vertebrate genomes methylation of cytosine residues within the context of CpG dinucleotides is a pervasive epigenetic mark that can impact gene expression and has been implicated in various developmental and disease-associated processes. Several biochemical approaches exist to profile DNA methylation, but recently an alternative approach based on profiling non-methylated CpGs was developed. This technique, called CxxC affinity purification (CAP), uses a ZF-CxxC (CxxC) domain to specifically capture DNA containing clusters of non-methylated CpGs. Here we describe a new CAP approach, called biotinylated CAP (Bio-CAP), which eliminates the requirement for specialized equipment while dramatically improving and simplifying the CxxC-based DNA affinity purification. Importantly, this approach isolates non-methylated DNA in a manner that is directly proportional to the density of non-methylated CpGs, and discriminates non-methylated CpGs from both methylated and hydroxymethylated CpGs. Unlike conventional CAP, Bio-CAP can be applied to nanogram quantities of genomic DNA and in a magnetic format is amenable to efficient parallel processing of samples. Furthermore, Bio-CAP can be applied to genome-wide profiling of non-methylated DNA with relatively small amounts of input material. Therefore, Bio-CAP is a simple and streamlined approach for characterizing regions of the non-methylated DNA, whether at specific target regions or genome wide. PMID:22156374

  7. Genetic characterization of the Aceh cattle using phenotypic, mitochondrial DNA of D-loop region and microsatellite DNA analyses.

    Science.gov (United States)

    Abdullah, M A N; Martojo, H; Noor, R R; Solihin, D D

    2012-01-01

    The present study reports the phenotypic variation of body weight and body size, the genetic variation of D-loop of mtDNA and microsatellite DNA allele in Aceh cattle in Indonesia within the frame of the design of a conservation programme for this indigenous species. Aceh cattle differ from Bali, Madura, Java-Ongole and Pesisir cattle, but its ancestry relates it closest to Pesisir, thus adding more information to its entry from the Indian sub-continent. © 2012 Blackwell Verlag GmbH.

  8. DNA Barcoding: Amplification and sequence analysis of rbcl and matK genome regions in three divergent plant species

    Directory of Open Access Journals (Sweden)

    Javed Iqbal Wattoo

    2016-11-01

    Full Text Available Background: DNA barcoding is a novel method of species identification based on nucleotide diversity of conserved sequences. The establishment and refining of plant DNA barcoding systems is more challenging due to high genetic diversity among different species. Therefore, targeting the conserved nuclear transcribed regions would be more reliable for plant scientists to reveal genetic diversity, species discrimination and phylogeny. Methods: In this study, we amplified and sequenced the chloroplast DNA regions (matk+rbcl of Solanum nigrum, Euphorbia helioscopia and Dalbergia sissoo to study the functional annotation, homology modeling and sequence analysis to allow a more efficient utilization of these sequences among different plant species. These three species represent three families; Solanaceae, Euphorbiaceae and Fabaceae respectively. Biological sequence homology and divergence of amplified sequences was studied using Basic Local Alignment Tool (BLAST. Results: Both primers (matk+rbcl showed good amplification in three species. The sequenced regions reveled conserved genome information for future identification of different medicinal plants belonging to these species. The amplified conserved barcodes revealed different levels of biological homology after sequence analysis. The results clearly showed that the use of these conserved DNA sequences as barcode primers would be an accurate way for species identification and discrimination. Conclusion: The amplification and sequencing of conserved genome regions identified a novel sequence of matK in native species of Solanum nigrum. The findings of the study would be applicable in medicinal industry to establish DNA based identification of different medicinal plant species to monitor adulteration.

  9. Association of the RENT complex with nontranscribed and coding regions of rDNA and a regional requirement for the replication fork block protein Fob1 in rDNA silencing

    Science.gov (United States)

    Huang, Julie; Moazed, Danesh

    2003-01-01

    Silencing within the yeast rDNA repeats inhibits hyperrecombination, represses transcription from foreign promoters, and extends replicative life span. rDNA silencing is mediated by a Sir2-containing complex called RENT (regulator of nucleolar silencing and telophase exit). We show that the Net1 (also called Cfi1) and Sir2 subunits of RENT localize primarily to two distinct regions within rDNA: in one of the nontranscribed spacers (NTS1) and around the Pol I promoter, extending into the 35S rRNA coding region. Binding to NTS1 overlaps the recombination hotspot and replication fork barrier elements, which have been shown previously to require the Fob1 protein for their activities. In cells lacking Fob1, silencing and the association of RENT subunits are abolished specifically at NTS1, while silencing and association at the Pol I promoter region are unaffected or increased. We find that Net1 and Sir2 are physically associated with Fob1 and subunits of RNA polymerase I. Together with the localization data, these results suggest the existence of two distinct modes for the recruitment of the RENT complex to rDNA and reveal a role for Fob1 in rDNA silencing and in the recruitment of the RENT complex. Furthermore, the Fob1-dependent associations of Net1 and Sir2 with the recombination hotspot region strongly suggest that Sir2 acts directly at this region to carry out its inhibitory effect on rDNA recombination and accelerated aging. PMID:12923057

  10. The marker choice: Unexpected resolving power of an unexplored CO1 region for layered DNA barcoding approaches.

    Science.gov (United States)

    Rach, Jessica; Bergmann, Tjard; Paknia, Omid; DeSalle, Rob; Schierwater, Bernd; Hadrys, Heike

    2017-01-01

    The potential of DNA barcoding approaches to identify single species and characterize species compositions strongly depends on the marker choice. The prominent "Folmer region", a 648 basepair fragment at the 5' end of the mitochondrial CO1 gene, has been traditionally applied as a universal DNA barcoding region for metazoans. In order to find a suitable marker for biomonitoring odonates (dragonflies and damselflies), we here explore a new region of the CO1 gene (CO1B) for DNA barcoding in 51 populations of 23 dragonfly and damselfly species. We compare the "Folmer region", the mitochondrial ND1 gene (NADH dehydrogenase 1) and the new CO1 region with regard to (i) speed and reproducibility of sequence generation, (ii) levels of homoplasy and (iii) numbers of diagnostic characters for discriminating closely related sister taxa and populations. The performances of the gene regions regarding these criteria were quite different. Both, the amplification of CO1B and ND1 was highly reproducible and CO1B showed the highest potential for discriminating sister taxa at different taxonomic levels. In contrast, the amplification of the "Folmer region" using the universal primers was difficult and the third codon positions of this fragment have experienced nucleotide substitution saturation. Most important, exploring this new barcode region of the CO1 gene identified a higher discriminating power between closely related sister taxa. Together with the design of layered barcode approaches adapted to the specific taxonomic "environment", this new marker will further enhance the discrimination power at the species level.

  11. Control region mutations and the 'common deletion' are frequent in the mitochondrial DNA of patients with esophageal squamous cell carcinoma

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    Tang Ze-Zong

    2004-07-01

    Full Text Available Abstract Background North central China has some of the highest rates of esophageal squamous cell carcinoma in the world with cumulative mortality surpassing 20%. Mitochondrial DNA (mtDNA accumulates more mutations than nuclear DNA and because of its high abundance has been proposed as a early detection device for subjects with cancer at various sites. We wished to examine the prevalence of mtDNA mutation and polymorphism in subjects from this high risk area of China. Methods We used DNA samples isolated from tumors, adjacent normal esophageal tissue, and blood from 21 esophageal squamous cell carcinoma cases and DNA isolated from blood from 23 healthy persons. We completely sequenced the control region (D-Loop from each of these samples and used a PCR assay to assess the presence of the 4977 bp common deletion. Results Direct DNA sequencing revealed that 7/21 (33%, 95% CI = 17–55% tumor samples had mutations in the control region, with clustering evident in the hyper-variable segment 1 (HSV1 and the homopolymeric stretch surrounding position 309. The number of mutations per subject ranged from 1 to 16 and there were a number of instances of heteroplasmy. We detected the 4977 bp 'common deletion' in 92% of the tumor and adjacent normal esophageal tissue samples examined, whereas no evidence of the common deletion was found in corresponding peripheral blood samples. Conclusions Control region mutations were insufficiently common to warrant attempts to develop mtDNA mutation screening as a clinical test for ESCC. The common deletion was highly prevalent in the esophageal tissue of cancer cases but absent from peripheral blood. The potential utility of the common deletion in an early detection system will be pursued in further studies.

  12. Tetrahelical structural family adopted by AGCGA-rich regulatory DNA regions

    Science.gov (United States)

    Kocman, Vojč; Plavec, Janez

    2017-05-01

    Here we describe AGCGA-quadruplexes, an unexpected addition to the well-known tetrahelical families, G-quadruplexes and i-motifs, that have been a focus of intense research due to their potential biological impact in G- and C-rich DNA regions, respectively. High-resolution structures determined by solution-state nuclear magnetic resonance (NMR) spectroscopy demonstrate that AGCGA-quadruplexes comprise four 5'-AGCGA-3' tracts and are stabilized by G-A and G-C base pairs forming GAGA- and GCGC-quartets, respectively. Residues in the core of the structure are connected with edge-type loops. Sequences of alternating 5'-AGCGA-3' and 5'-GGG-3' repeats could be expected to form G-quadruplexes, but are shown herein to form AGCGA-quadruplexes instead. Unique structural features of AGCGA-quadruplexes together with lower sensitivity to cation and pH variation imply their potential biological relevance in regulatory regions of genes responsible for basic cellular processes that are related to neurological disorders, cancer and abnormalities in bone and cartilage development.

  13. Molecular phylogenetic analysis of Indonesia Solanaceae based on DNA sequences of internal transcribed spacer region

    Science.gov (United States)

    Hidayat, Topik; Priyandoko, Didik; Islami, Dina Karina; Wardiny, Putri Yunitha

    2016-02-01

    Solanaceae is one of largest family in Angiosperm group with highly diverse in morphological character. In Indonesia, this group of plant is very popular due to its usefulness as food, ornamental and medicinal plants. However, investigation on phylogenetic relationship among the member of this family in Indonesia remains less attention. The purpose of this study was to evaluate the phylogenetics relationship of the family especially distributed in Indonesia. DNA sequences of Internal Transcribed Spacer (ITS) region of 19 species of Solanaceae and three species of outgroup, which belongs to family Convolvulaceae, Apocynaceae, and Plantaginaceae, were isolated, amplified, and sequenced. Phylogenetic tree analysis based on parsimony method was conducted with using data derived from the ITS-1, 5.8S, and ITS-2, separately, and the combination of all. Results indicated that the phylogenetic tree derived from the combined data established better pattern of relationship than separate data. Thus, three major groups were revealed. Group 1 consists of tribe Datureae, Cestreae, and Petunieae, whereas group 2 is member of tribe Physaleae. Group 3 belongs to tribe Solaneae. The use of the ITS region as a molecular markers, in general, support the global Solanaceae relationship that has been previously reported.

  14. The role of a static bend in the DNA of the aroF regulatory region of Escherichia coli.

    Science.gov (United States)

    Cobbett, C; Dickson, B; Farmer, L

    1989-01-30

    DNA fragments containing the regulatory regions of two genes repressed by the tyrR gene product exhibit retarded electrophoretic mobility in polyacrylamide gels indicating the presence of static bends in the DNA. In the aroF gene this bend has been localized to a region containing two TyrR protein-binding sites. A point mutation between these two sites reduced the degree of bending observed but did not reduce the level of repression, indicating the static bend may not be an important component of the repression mechanism.

  15. Phylogeny and Biogeography of the Genus Ainsliaea (Asteraceae) in the Sino-Japanese Region based on Nuclear rDNA and Plastid DNA Sequence Data

    Science.gov (United States)

    Mitsui, Yuki; Chen, Shao-Tien; Zhou, Zhe-Kun; Peng, Ching-I.; Deng, Yun-Fei; Setoguchi, Hiroaki

    2008-01-01

    Background and Aims The flora of the Sino-Japanese plant region of eastern Asia is distinctively rich compared with other floristic regions in the world. However, knowledge of its floristic evolution is fairly limited. The genus Ainsliaea is endemic to and distributed throughout the Sino-Japanese region. Its interspecific phylogenetic relationships have not been resolved. The aim is to provide insight into floristic evolution in eastern Asia on the basis of a molecular phylogenetic analysis of Ainsliaea species. Methods Cladistic analyses of the sequences of two nuclear (ITS, ETS) and one plastid (ndhF) regions were carried out individually and using the combined data from the three markers. Key Results Phylogenetic analyses of three DNA regions confirmed that Ainsliaea is composed of three major clades that correspond to species distributions. Evolution of the three lineages was estimated to have occurred around 1·1 MYA during the early Pleistocene. Conclusions The results suggest that Ainsliaea species evolved allopatrically and that the descendants were isolated in the eastern (between SE China and Japan, through Taiwan and the Ryukyu Islands) and western (Yunnan Province and its surrounding areas, including the Himalayas, the temperate region of Southeast Asia, and Sichuan Province) sides of the Sino-Japanese region. The results suggest that two distinct lineages of Ainsliaea have independently evolved in environmentally heterogeneous regions within the Sino-Japanese region. These regions have maintained rich and original floras due to their diverse climates and topographies. PMID:17981878

  16. Reconstructing the history of Mesoamerican populations through the study of the mitochondrial DNA control region.

    Science.gov (United States)

    Gorostiza, Amaya; Acunha-Alonzo, Víctor; Regalado-Liu, Lucía; Tirado, Sergio; Granados, Julio; Sámano, David; Rangel-Villalobos, Héctor; González-Martín, Antonio

    2012-01-01

    The study of genetic information can reveal a reconstruction of human population's history. We sequenced the entire mtDNA control region (positions 16.024 to 576 following Cambridge Reference Sequence, CRS) of 605 individuals from seven Mesoamerican indigenous groups and one Aridoamerican from the Greater Southwest previously defined, all of them in present Mexico. Samples were collected directly from the indigenous populations, the application of an individual survey made it possible to remove related or with other origins samples. Diversity indices and demographic estimates were calculated. Also AMOVAs were calculated according to different criteria. An MDS plot, based on FST distances, was also built. We carried out the construction of individual networks for the four Amerindian haplogroups detected. Finally, barrier software was applied to detect genetic boundaries among populations. The results suggest: a common origin of the indigenous groups; a small degree of European admixture; and inter-ethnic gene flow. The process of Mesoamerica's human settlement took place quickly influenced by the region's orography, which development of genetic and cultural differences facilitated. We find the existence of genetic structure is related to the region's geography, rather than to cultural parameters, such as language. The human population gradually became fragmented, though they remained relatively isolated, and differentiated due to small population sizes and different survival strategies. Genetic differences were detected between Aridoamerica and Mesoamerica, which can be subdivided into "East", "Center", "West" and "Southeast". The fragmentation process occurred mainly during the Mesoamerican Pre-Classic period, with the Otomí being one of the oldest groups. With an increased number of populations studied adding previously published data, there is no change in the conclusions, although significant genetic heterogeneity can be detected in Pima and Huichol groups

  17. Reconstructing the history of Mesoamerican populations through the study of the mitochondrial DNA control region.

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    Amaya Gorostiza

    Full Text Available The study of genetic information can reveal a reconstruction of human population's history. We sequenced the entire mtDNA control region (positions 16.024 to 576 following Cambridge Reference Sequence, CRS of 605 individuals from seven Mesoamerican indigenous groups and one Aridoamerican from the Greater Southwest previously defined, all of them in present Mexico. Samples were collected directly from the indigenous populations, the application of an individual survey made it possible to remove related or with other origins samples. Diversity indices and demographic estimates were calculated. Also AMOVAs were calculated according to different criteria. An MDS plot, based on FST distances, was also built. We carried out the construction of individual networks for the four Amerindian haplogroups detected. Finally, barrier software was applied to detect genetic boundaries among populations. The results suggest: a common origin of the indigenous groups; a small degree of European admixture; and inter-ethnic gene flow. The process of Mesoamerica's human settlement took place quickly influenced by the region's orography, which development of genetic and cultural differences facilitated. We find the existence of genetic structure is related to the region's geography, rather than to cultural parameters, such as language. The human population gradually became fragmented, though they remained relatively isolated, and differentiated due to small population sizes and different survival strategies. Genetic differences were detected between Aridoamerica and Mesoamerica, which can be subdivided into "East", "Center", "West" and "Southeast". The fragmentation process occurred mainly during the Mesoamerican Pre-Classic period, with the Otomí being one of the oldest groups. With an increased number of populations studied adding previously published data, there is no change in the conclusions, although significant genetic heterogeneity can be detected in Pima and

  18. DNA Barcoding Identification of Kadsurae Caulis and Spatholobi Caulis Based on Internal Transcribed Spacer 2 Region and Secondary Structure Prediction.

    Science.gov (United States)

    Yu, Xiaoxue; Xie, Zhiyong; Wu, Junwei; Tao, Junfei; Xu, Xinjun

    2016-05-01

    Kadsurae Caulis and Spatholobi Caulis have very similar Chinese names. Their commodities were hard to distinguish because their stems were very alike after dried and processed. These two herbal drugs were often mixed in clinical use. Authenticity assurance is crucial for quality control of herbal drugs. Therefore, it is essential to establish a method for identifying the two herbs. In this paper, we used the DNA barcoding technology, based on the internal transcribed spacer 2 (ITS2) regions, to differentiate Kadsurae Caulis and Spatholobi Caulis. The ITS2 of these two herbs were very different. They were successfully differentiated using the DNA barcoding technique. DNA barcoding was a promising and reliable tool for the identification of medicinal plants. It can be a powerful complementary method for traditional authentication. The internal transcribed spacer 2 (ITS2) regions between Kadsurae Caulis and Spatholobi Caulis varied considerably, totally 139 variable sitesSample 1 was not Kadsurae Caulis as it labeled, but it should be Spatholobi Caulis in fact based on ITS2 regionThe secondary structure can also separate Kadsurae Caulis and Spatholobi Caulis effectivelyDNA barcoding provided an accurate and strong prove to identify these two herbs. Abbreviations used: CTAB: hexadecyltrimethylammonium bromide, DNA: deoxyribonucleic acid, ITS2:internal transcribed spacer 2, PCR: polymerase chain reaction.

  19. Genetic relationships of grizzly bears (Ursus arctos) in the Prudhoe Bay region of Alaska: inference from microsatellite DNA, mitochondrial DNA, and field observations.

    Science.gov (United States)

    Cronin, M; Shideler, R; Hechtel, J; Strobeck, C; Paetkau, D

    1999-01-01

    Grizzly bears are abundant in the region of the Prudhoe Bay oil fields in northern Alaska. We used field observations and molecular genetic data to identify parent-offspring and sibling relationships among bears in this region. We determined genotypes at 14 microsatellite DNA loci and the cytochrome b gene of mitochondrial DNA (mtDNA) for 36 bears. We identified 17 possible mother-offspring pairs and 8 possible father-offspring pairs. This includes verification of the relationships of 14 mother-offspring pairs identified from field observations. Three additional mother-offspring pairs and all eight father-offspring pairs were determined from genetic and age data. Relatedness coefficients based on numbers of shared alleles between individuals were as expected: approximately 0.50 for parent-offspring and sibling pairs and approximately 0.75 for a father-offspring pair resulting from a father-daughter mating. The level of genetic variation (mean number of alleles per locus = 6.6, mean heterozygosity = 70%) and allele frequencies in grizzly bears in the Prudhoe Bay region are similar to those in other parts of the species' range.

  20. Accelerated construction of a regional DNA-barcode reference library: Caddisflies (Trichoptera) in the Great Smoky Mountains National Park

    Science.gov (United States)

    Zhou, X.; Robinson, J.L.; Geraci, C.J.; Parker, C.R.; Flint, O.S.; Etnier, D.A.; Ruiter, D.; DeWalt, R.E.; Jacobus, L.M.; Hebert, P.D.N.

    2011-01-01

    Deoxyribonucleic acid (DNA) barcoding is an effective tool for species identification and lifestage association in a wide range of animal taxa. We developed a strategy for rapid construction of a regional DNA-barcode reference library and used the caddisflies (Trichoptera) of the Great Smoky Mountains National Park (GSMNP) as a model. Nearly 1000 cytochrome c oxidase subunit I (COI) sequences, representing 209 caddisfly species previously recorded from GSMNP, were obtained from the global Trichoptera Barcode of Life campaign. Most of these sequences were collected from outside the GSMNP area. Another 645 COI sequences, representing 80 species, were obtained from specimens collected in a 3-d bioblitz (short-term, intense sampling program) in GSMNP. The joint collections provided barcode coverage for 212 species, 91% of the GSMNP fauna. Inclusion of samples from other localities greatly expedited construction of the regional DNA-barcode reference library. This strategy increased intraspecific divergence and decreased average distances to nearest neighboring species, but the DNA-barcode library was able to differentiate 93% of the GSMNP Trichoptera species examined. Global barcoding projects will aid construction of regional DNA-barcode libraries, but local surveys make crucial contributions to progress by contributing rare or endemic species and full-length barcodes generated from high-quality DNA. DNA taxonomy is not a goal of our present work, but the investigation of COI divergence patterns in caddisflies is providing new insights into broader biodiversity patterns in this group and has directed attention to various issues, ranging from the need to re-evaluate species taxonomy with integrated morphological and molecular evidence to the necessity of an appropriate interpretation of barcode analyses and its implications in understanding species diversity (in contrast to a simple claim for barcoding failure).

  1. Haplotypes and variable position detection in the mitochondrial DNA coding region encompassing nucleotide positions 10,716-11,184.

    Science.gov (United States)

    Hameed, Imad Hadi; Abdulzahra, Ameer Ibrahim; Jebor, Mohammed Abdullah; Kqueen, Cheah Yoke; Ommer, Aamera Jaber

    2015-08-01

    This study evaluates the mitochondrial noncoding regions by using the Sanger sequencing method for application in Forensic Science. FTA® Technology (FTA™ paper DNA extraction) was utilized to extract DNA. Portion of coding region encompassing positions from (10,716 to 11,184) amplified in accordance with the Anderson reference sequence. PCR products purified by EZ-10 spin column were then sequenced and detected using the ABI 3730 × L DNA Analyzer. A new polymorphic positions 10,750 and 10,790 that are described may be suitable sources in future for identification purpose. The data obtained can be used to identify variable nucleotide positions characterized by frequent occurrence, most promising for identification variants.

  2. The neuroblast and angioblast chemotaxic factor SDF-1 (CXCL12 expression is briefly up regulated by reactive astrocytes in brain following neonatal hypoxic-ischemic injury

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    Walker Aisha L

    2005-10-01

    Full Text Available Abstract Background Stromal cell-derived factor 1 (SDF-1 or CXCL12 is chemotaxic for CXCR4 expressing bone marrow-derived cells. It functions in brain embryonic development and in response to ischemic injury in helping guide neuroblast migration and vasculogenesis. In experimental adult stroke models SDF-1 is expressed perivascularly in the injured region up to 30 days after the injury, suggesting it could be a therapeutic target for tissue repair strategies. We hypothesized that SDF-1 would be expressed in similar temporal and spatial patterns following hypoxic-ischemic (HI injury in neonatal brain. Results Twenty-five 7-day-old C57BL/J mice underwent HI injury. SDF-1 expression was up regulated up to 7 days after the injury but not at the later time points. The chief sites of SDF-1 up regulation were astrocytes, their foot processes along blood vessels and endothelial cells. Conclusion The localization of SDF-1 along blood vessels in the HI injury zone suggests that these perivascular areas are where chemotaxic signaling for cellular recruitment originates and that reactive astrocytes are major mediators of this process. The associated endothelium is likely to be the site for vascular attachment and diapedesis of CXCR4 receptor expressing cells to enter the injured tissue. Here we show that, relative to adults, neonates have a significantly smaller window of opportunity for SDF-1 based vascular chemotaxic recruitment of bone marrow-derived cells. Therefore, without modification, following neonatal HI injury there is only a narrow period of time for endogenous SDF-1 mediated chemotaxis and recruitment of reparative cells, including exogenously administered stem/progenitor cells.

  3. Identification of Candida species by PCR and restriction fragment length polymorphism analysis of intergenic spacer regions of ribosomal DNA.

    OpenAIRE

    Williams, D. W.; Wilson, M. J.; Lewis, M A; Potts, A J

    1995-01-01

    The PCR was used to amplify a targeted region of the ribosomal DNA from 84 Candida isolates. Unique product sizes were obtained for Candida guilliermondii, Candida (Torulopsis) glabrata, and Candida pseudotropicalis. Isolates of Candida albicans, Candida tropicalis, Candida stellatoidea, Candida parapsilosis, and Candida krusei could be identified following restriction digestion of the PCR products.

  4. Genomic Mapping of Human DNA provides Evidence of Difference in Stretch between AT and GC rich regions

    Science.gov (United States)

    Reifenberger, Jeffrey; Dorfman, Kevin; Cao, Han

    Human DNA is a not a polymer consisting of a uniform distribution of all 4 nucleic acids, but rather contains regions of high AT and high GC content. When confined, these regions could have different stretch due to the extra hydrogen bond present in the GC basepair. To measure this potential difference, human genomic DNA was nicked with NtBspQI, labeled with a cy3 like fluorophore at the nick site, stained with YOYO, loaded into a device containing an array of nanochannels, and imaged. Over 473,000 individual molecules of DNA, corresponding to roughly 30x coverage of a human genome, were collected and aligned to the human reference. Based on the known AT/GC content between aligned pairs of labels, the stretch was measured for regions of similar size but different AT/GC content. We found that regions of high GC content were consistently more stretched than regions of high AT content between pairs of labels varying in size between 2.5 kbp and 500 kbp. We measured that for every 1% increase in GC content there was roughly a 0.06% increase in stretch. While this effect is small, it is important to take into account differences in stretch between AT and GC rich regions to improve the sensitivity of detection of structural variations from genomic variations. NIH Grant: R01-HG006851.

  5. 5' coding region of the follicular epithelium yolk polypeptide 2 cDNA in the moth, Plodia interpunctella, contains an extended coding region.

    Science.gov (United States)

    Shirk, P D; Perera, O P

    1998-01-01

    The 5' region of YP2 cDNA, a follicular epithelium yolk protein subunit in the moth, Plodia interpunctella, shows that the polypeptide contains an extended internal coding region. Partial cDNA clones for YP2 were isolated from a pharate adult female ovarian cDNA expression library in Lambda Zap II by screening with antigen selected YP2 antiserum. The 5' sequence of the YP2 transcript was determined by 5' RACE PCR of ovarian mRNA using YP2 sequence-specific nested primers. The combined cDNA and 5' RACE sequencing showed the YP2 transcript to be 1971 bp in length up to the poly(A) tail with a single open reading frame for a predicted polypeptide of 616 amino acids. Northern analysis showed a single YP2 transcript to be present in ovarian RNA that was approximately 2 kb in length. The predicted amino acid sequence for YP2 from P. interpunctella is most closely related to egg specific protein (ESP) from Bombyx mori and the partial YP2 sequence from Galleria mellonella. YP2 from P. interpunctella also is similar to vertebrate lipases and contains a conserved lipid binding region. However, the 5' coding region of YP2 from P. interpunctella contains an in-frame insert of approximately 438 bp that had replaced an approximately 270-bp region as compared with ESP from B. mori and YP2 of G. mellonella. This suggests that the insert occurred by a recombinational event internal to the YP2 structural gene of P. interpunctella.

  6. Genetic architecture of trout from Albania as revealed by mtDNA control region variation

    Science.gov (United States)

    2009-01-01

    To determine the genetic architecture of trout in Albania, 87 individuals were collected from 19 riverine and lacustrine sites in Albania, FYROM and Greece. All individuals were analyzed for sequence variation in the mtDNA control region. Among fourteen haplotypes detected, four previously unpublished haplotypes, bearing a close relationship to haplotypes of the Adriatic and marmoratus lineages of Salmo trutta, were revealed. Ten previously described haplotypes, characteristic of S. ohridanus, S. letnica and the Adriatic and Mediterranean lineages of S. trutta, were also detected. Haplotypes detected in this study were placed in a well supported branch of S. ohridanus, and a cluster of Mediterranean – Adriatic – marmoratus haplotypes, which were further delimited into three subdivisions of Mediterranean, marmoratus, and a previously non-described formation of four Adriatic haplotypes (Balkan cluster). Haplotypes of the Balkan cluster and the other Adriatic haplotypes, do not represent a contiguous haplotype lineage and appear not to be closely related, indicating independent arrivals into the Adriatic drainage and suggesting successive colonization events. Despite the presence of marmoratus haplotypes in Albania, no marbled phenotype was found, confirming previously reported findings that there is no association between this phenotype and marmoratus haplotypes. PMID:19284692

  7. Control Region-mtDNA heterogeneity of Kalimantan false gharial (Tomistoma schiegelii population: a preliminary study

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    Hellen Hellen

    2003-06-01

    Full Text Available The preliminary genetic study on Kalimantan false gharial from the wild was reported. Eleven tail scutes were collected fromeleven individuals that originally consisting of two individuals from Kapuas River, one individual from Sentarum Lake, Jelai River,Mapam River, Perian Lake, and Lamandau River, two individuals from Barito River and three individuals from Mahakam River. PCRamplifying and sequencing 451 nucleotides in average that can be aligned at the same length of control region mitochondrial DNA.Among 11 individuals found eight polymorphic sites that consisted four haplotypes (A, B, C, and D respectively, which is haplotypeA is dominant. Based on phylogenetic tree that constructed by Tamura-Nei parameter, false gharial population in Kalimantan can bedivided into two population groups; there were Central-Eastern Kalimantan population group and Western Kalimantan populationgroup. Based on the hypothesis of landmasses separating between central-eastern Kalimantan and western Kalimantan that known asSchwaner Mountains, the genetic distance D = 1.53% was expected to be equal to 20 million years.

  8. Length heteroplasmy in the first hypervariable segment of the human mtDNA control region.

    Science.gov (United States)

    Bendall, K E; Sykes, B C

    1995-08-01

    The first hypervariable segment of the human mtDNA control region contains a homopolymeric tract of cytosines between nt 16184 and 16193, interrupted at position 16189 by a thymine, according to the Cambridge reference sequence. A variant commonly found in population screening is a T-to-C transition at nt 16189, resulting in an uninterrupted homopolymeric tract. Direct sequencing of individuals with this variant produces a characteristic blurred sequence in nucleotides beyond the tract. Sequencing clones from these individuals revealed that this is caused by high levels of length heteroplasmy in the homopolymeric tract and low levels of length heteroplasmy in the four adenines following the tract. We have developed a rapid method involving densitometry of sequencing gels to quantify the relative proportions of different length variants present in an individual. We have used this to study the proportions of length variants in individuals from three twin pairs and two maternal lineages. While unrelated individuals usually have different proportions of length variants, all maternally related individuals studied have the same proportions, even if they are only distantly related. It is not obvious how identical heteroplasmic profiles are maintained in maternally related individuals, but some possible mechanisms are suggested.

  9. Quantitative analysis of DNA methylation at all human imprinted regions reveals preservation of epigenetic stability in adult somatic tissue

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    Woodfine Kathryn

    2011-01-01

    Full Text Available Abstract Background Genes subject to genomic imprinting are mono-allelically expressed in a parent-of-origin dependent manner. Each imprinted locus has at least one differentially methylated region (DMR which has allele specific DNA methylation and contributes to imprinted gene expression. Once DMRs are established, they are potentially able to withstand normal genome reprogramming events that occur during cell differentiation and germ-line DMRs are stably maintained throughout development. These DMRs, in addition to being either maternally or paternally methylated, have differences in whether methylation was acquired in the germ-line or post fertilization and are present in a variety of genomic locations with different Cytosine-phosphate guanine (CpG densities and CTCF binding capacities. We therefore examined the stability of maintenance of DNA methylation imprints and determined the normal baseline DNA methylation levels in several adult tissues for all imprinted genes. In order to do this, we first developed and validated 50 highly specific, quantitative DNA methylation pyrosequencing assays for the known DMRs associated with human imprinted genes. Results Remarkable stability of the DNA methylation imprint was observed in all germ-line DMRs and paternally methylated somatic DMRs (which maintained average methylation levels of between 35% - 65% in all somatic tissues, independent of gene expression. Maternally methylated somatic DMRs were found to have more variation with tissue specific methylation patterns. Most DMRs, however, showed some intra-individual variability for DNA methylation levels in peripheral blood, suggesting that more than one DMR needs to be examined in order to get an overall impression of the epigenetic stability in a tissue. The plasticity of DNA methylation at imprinted genes was examined in a panel of normal and cancer cell lines. All cell lines showed changes in DNA methylation, especially at the paternal germ

  10. Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing.

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    Nilesh V Khade

    Full Text Available Yeast Rad52 (yRad52 has two important functions at homologous DNA recombination (HR; annealing complementary single-strand DNA (ssDNA molecules and recruiting Rad51 recombinase onto ssDNA (recombination mediator activity. Its human homolog (hRAD52 has a lesser role in HR, and apparently lacks mediator activity. Here we show that yRad52 can load human Rad51 (hRAD51 onto ssDNA complexed with yeast RPA in vitro. This is biochemically equivalent to mediator activity because it depends on the C-terminal Rad51-binding region of yRad52 and on functional Rad52-RPA interaction. It has been reported that the N-terminal two thirds of both yRad52 and hRAD52 is essential for binding to and annealing ssDNA. Although a second DNA binding region has been found in the C-terminal region of yRad52, its role in ssDNA annealing is not clear. In this paper, we also show that the C-terminal region of yRad52, but not of hRAD52, is involved in ssDNA annealing. This suggests that the second DNA binding site is required for the efficient ssDNA annealing by yRad52. We propose an updated model of Rad52-mediated ssDNA annealing.

  11. Species identification of medicinal pteridophytes by a DNA barcode marker, the chloroplast psbA-trnH intergenic region.

    Science.gov (United States)

    Ma, Xin-Ye; Xie, Cai-Xiang; Liu, Chang; Song, Jing-Yuan; Yao, Hui; Luo, Kun; Zhu, Ying-Jie; Gao, Ting; Pang, Xiao-Hui; Qian, Jun; Chen, Shi-Lin

    2010-01-01

    Medicinal pteridophytes are an important group used in traditional Chinese medicine; however, there is no simple and universal way to differentiate various species of this group by morphological traits. A novel technology termed "DNA barcoding" could discriminate species by a standard DNA sequence with universal primers and sufficient variation. To determine whether DNA barcoding would be effective for differentiating pteridophyte species, we first analyzed five DNA sequence markers (psbA-trnH intergenic region, rbcL, rpoB, rpoC1, and matK) using six chloroplast genomic sequences from GeneBank and found psbA-trnH intergenic region the best candidate for availability of universal primers. Next, we amplified the psbA-trnH region from 79 samples of medicinal pteridophyte plants. These samples represented 51 species from 24 families, including all the authentic pteridophyte species listed in the Chinese pharmacopoeia (2005 version) and some commonly used adulterants. We found that the sequence of the psbA-trnH intergenic region can be determined with both high polymerase chain reaction (PCR) amplification efficiency (94.1%) and high direct sequencing success rate (81.3%). Combined with GeneBank data (54 species cross 12 pteridophyte families), species discriminative power analysis showed that 90.2% of species could be separated/identified successfully by the TaxonGap method in conjunction with the Basic Local Alignment Search Tool 1 (BLAST1) method. The TaxonGap method results further showed that, for 37 out of 39 separable species with at least two samples each, between-species variation was higher than the relevant within-species variation. Thus, the psbA-trnH intergenic region is a suitable DNA marker for species identification in medicinal pteridophytes.

  12. Graphene Functionalized Scaffolds Reduce the Inflammatory Response and Supports Endogenous Neuroblast Migration when Implanted in the Adult Brain.

    Science.gov (United States)

    Zhou, Kun; Motamed, Sepideh; Thouas, George A; Bernard, Claude C; Li, Dan; Parkington, Helena C; Coleman, Harold A; Finkelstein, David I; Forsythe, John S

    2016-01-01

    Electroactive materials have been investigated as next-generation neuronal tissue engineering scaffolds to enhance neuronal regeneration and functional recovery after brain injury. Graphene, an emerging neuronal scaffold material with charge transfer properties, has shown promising results for neuronal cell survival and differentiation in vitro. In this in vivo work, electrospun microfiber scaffolds coated with self-assembled colloidal graphene, were implanted into the striatum or into the subventricular zone of adult rats. Microglia and astrocyte activation levels were suppressed with graphene functionalization. In addition, self-assembled graphene implants prevented glial scarring in the brain 7 weeks following implantation. Astrocyte guidance within the scaffold and redirection of neuroblasts from the subventricular zone along the implants was also demonstrated. These findings provide new functional evidence for the potential use of graphene scaffolds as a therapeutic platform to support central nervous system regeneration.

  13. Graphene Functionalized Scaffolds Reduce the Inflammatory Response and Supports Endogenous Neuroblast Migration when Implanted in the Adult Brain.

    Directory of Open Access Journals (Sweden)

    Kun Zhou

    Full Text Available Electroactive materials have been investigated as next-generation neuronal tissue engineering scaffolds to enhance neuronal regeneration and functional recovery after brain injury. Graphene, an emerging neuronal scaffold material with charge transfer properties, has shown promising results for neuronal cell survival and differentiation in vitro. In this in vivo work, electrospun microfiber scaffolds coated with self-assembled colloidal graphene, were implanted into the striatum or into the subventricular zone of adult rats. Microglia and astrocyte activation levels were suppressed with graphene functionalization. In addition, self-assembled graphene implants prevented glial scarring in the brain 7 weeks following implantation. Astrocyte guidance within the scaffold and redirection of neuroblasts from the subventricular zone along the implants was also demonstrated. These findings provide new functional evidence for the potential use of graphene scaffolds as a therapeutic platform to support central nervous system regeneration.

  14. Differentially methylated DNA regions in monozygotic twin pairs discordant for rheumatoid arthritis.An epigenome - wide study.

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    Anders Jørgen Svendsen

    2016-11-01

    Full Text Available Objectives: In an explorative epigenome-wide association study (EWAS to search for gene independent, differentially methylated DNA positions (DMPs and regions (DMRs associated with rheumatoid arthritis (RA by studying monozygotic (MZ twin pairs discordant for rheumatoid arthritis.Methods: Genomic DNA was isolated from whole blood samples from 28 monozygotic (MZ twin pairs discordant for RA. DNA methylation was measured using the HumanMethylation450 BeadChips. Smoking, anti-cyclic citrullinated peptide antibodies and immunosuppressive treatment were included as covariates. Pathway analysis was performed using GREAT.Results: Smoking was significantly associated with hypomethylation of a DMR overlapping the promoter region of the RNF5 and the AGPAT1, which are implicated in inflammation and autoimmunity, whereas DMARD treatment induced hypermethylation of the same region. Additionally, the promotor region of both S100A6 and EFCAB4B were hypomethylated and both genes have previously been associated with RA. We replicated several candidate genes identified in a previous EWAS in treatment naïve RA singletons. Gene set analysis indicated the involvement of immunologic signatures and cancer-related pathways in RA.Conclusion: We identified several differentially methylated regions associated with RA which may represent environmental effects or consequences of the disease and plausible biological pathways pertinent to the pathogenesis of rheumatoid arthritis

  15. SDN-1/Syndecan Acts in Parallel to the Transmembrane Molecule MIG-13 to Promote Anterior Neuroblast Migration.

    Science.gov (United States)

    Sundararajan, Lakshmi; Norris, Megan L; Lundquist, Erik A

    2015-05-28

    The Q neuroblasts in Caenorhabditis elegans display left-right asymmetry in their migration, with QR and descendants on the right migrating anteriorly, and QL and descendants on the left migrating posteriorly. Initial QR and QL migration is controlled by the transmembrane receptors UNC-40/DCC, PTP-3/LAR, and the Fat-like cadherin CDH-4. After initial migration, QL responds to an EGL-20/Wnt signal that drives continued posterior migration by activating MAB-5/Hox activity in QL but not QR. QR expresses the transmembrane protein MIG-13, which is repressed by MAB-5 in QL and which drives anterior migration of QR descendants. A screen for new Q descendant AQR and PQR migration mutations identified mig-13 as well as hse-5, the gene encoding the glucuronyl C5-epimerase enzyme, which catalyzes epimerization of glucuronic acid to iduronic acid in the heparan sulfate side chains of heparan sulfate proteoglycans (HSPGs). Of five C. elegans HSPGs, we found that only SDN-1/Syndecan affected Q migrations. sdn-1 mutants showed QR descendant AQR anterior migration defects, and weaker QL descendant PQR migration defects. hse-5 affected initial Q migration, whereas sdn-1 did not. sdn-1 and hse-5 acted redundantly in AQR and PQR migration, but not initial Q migration, suggesting the involvement of other HSPGs in Q migration. Cell-specific expression studies indicated that SDN-1 can act in QR to promote anterior migration. Genetic interactions between sdn-1, mig-13, and mab-5 suggest that MIG-13 and SDN-1 act in parallel to promote anterior AQR migration and that SDN-1 also controls posterior migration. Together, our results indicate previously unappreciated complexity in the role of multiple signaling pathways and inherent left-right asymmetry in the control of Q neuroblast descendant migration. Copyright © 2015 Sundararajan et al.

  16. EGL-20/Wnt and MAB-5/Hox Act Sequentially to Inhibit Anterior Migration of Neuroblasts in C. elegans.

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    Matthew P Josephson

    Full Text Available Directed neuroblast and neuronal migration is important in the proper development of nervous systems. In C. elegans the bilateral Q neuroblasts QR (on the right and QL (on the left undergo an identical pattern of cell division and differentiation but migrate in opposite directions (QR and descendants anteriorly and QL and descendants posteriorly. EGL-20/Wnt, via canonical Wnt signaling, drives the expression of MAB-5/Hox in QL but not QR. MAB-5 acts as a determinant of posterior migration, and mab-5 and egl-20 mutants display anterior QL descendant migrations. Here we analyze the behaviors of QR and QL descendants as they begin their anterior and posterior migrations, and the effects of EGL-20 and MAB-5 on these behaviors. The anterior and posterior daughters of QR (QR.a/p after the first division immediately polarize and begin anterior migration, whereas QL.a/p remain rounded and non-migratory. After ~1 hour, QL.a migrates posteriorly over QL.p. We find that in egl-20/Wnt, bar-1/β-catenin, and mab-5/Hox mutants, QL.a/p polarize and migrate anteriorly, indicating that these molecules normally inhibit anterior migration of QL.a/p. In egl-20/Wnt mutants, QL.a/p immediately polarize and begin migration, whereas in bar-1/β-catenin and mab-5/Hox, the cells transiently retain a rounded, non-migratory morphology before anterior migration. Thus, EGL-20/Wnt mediates an acute inhibition of anterior migration independently of BAR-1/β-catenin and MAB-5/Hox, and a later, possible transcriptional response mediated by BAR-1/β-catenin and MAB-5/Hox. In addition to inhibiting anterior migration, MAB-5/Hox also cell-autonomously promotes posterior migration of QL.a (and QR.a in a mab-5 gain-of-function.

  17. HIF2A and IGF2 Expression Correlates in Human Neuroblastoma Cells and Normal Immature Sympathetic Neuroblasts

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    Sofie Mohlin

    2013-03-01

    Full Text Available During normal sympathetic nervous system (SNS development, cells of the ganglionic lineage can malignantly transform and develop into the childhood tumor neuroblastoma. Hypoxia-inducible transcription factors (HIFs mediate cellular responses during normal development and are central in the adaptation to oxygen shortage. HIFs are also implicated in the progression of several cancer forms, and high HIF-2α expression correlates with disseminated disease and poor outcome in neuroblastoma. During normal SNS development, HIF2A is transiently expressed in neuroblasts and chromaffin cells. SNS cells can, during development, be distinguished by distinct gene expression patterns, and insulin-like growth factor 2 (IGF2 is a marker of sympathetic chromaffin cells, whereas sympathetic neuroblasts lack IGF2 expression. Despite the neuronal derivation of neuroblastomas, we show that neuroblastoma cell lines and specimens express IGF2 and that expression of HIF2A and IGF2 correlates, with the strongest correlation in high-stage tumors. In neuroblastoma, both IGF2 and HIF2A are hypoxia-driven and knocking down IGF2 at hypoxia resulted in downregulated HIF2A levels. HIF-2α and IGF2 were strongly expressed in subsets of immature neuroblastoma cells, suggesting that these two genes could be co-expressed also at early stages of SNS development. We show that IGF2 is indeed expressed in sympathetic chain ganglia at embryonic week 6.5, a developmental stage when HIF-2α is present. These findings provide a rationale for the unexpected IGF2 expression in neuroblastomas and might suggest that IGF2 and HIF2A positive neuroblastoma cells are arrested at an embryonic differentiation stage corresponding to the stage when sympathetic chain ganglia begins to coalesce.

  18. Diagnostic value of diffusion-weighted MRI for tumor characterization, differentiation and monitoring in pediatric patients with neuroblastic tumors

    Energy Technology Data Exchange (ETDEWEB)

    Neubauer, Henning [Univ. Hospital Ulm (Germany). Dept. of Diagnostic and Interventional Radiology; Univ. Hospital Wuerzburg (Germany). Dept. of Diagnostic and Interventional Radiology; Li, Mengxia [Univ. Hospital Wuerzburg (Germany). Dept. of Radiation Oncology; Mueller, Verena Rabea [Univ. Hospital Wuerzburg (Germany). Dept. of Paediatrics; Pabst, Thomas [Univ. Hospital Wuerzburg (Germany). Dept. of Diagnostic and Interventional Radiology; Beer, Meinrad [Univ. Hospital Ulm (Germany). Dept. of Diagnostic and Interventional Radiology

    2017-07-15

    We explored the diagnostic value of diffusion-weighted MRI (DWI) for tumor characterization, differentiation and therapy monitoring in pediatric patients with extracranial neuroblastic tumors. All 29 patients (14 girls, median age: 3 years) with neuroblastoma (NB, n = 19), ganglioneuroblastoma (GNB, n = 4) and ganglioneuroma (GN, n = 6) who had had at least one in-house DWI examination since 2005 were identified and retrospectively analyzed. Two independent blinded readers measured ADC values (unit: 10-3 mm{sup 2}/s) and signal intensity ratios (SIRs) of the primary tumor and, if applicable, of the tumor after chemotherapy, metastases and tumor relapse. The pre-treatment ADC was 0.90 ± 0.23 in NB/GNB and 1.70 ± 0.36 in GN without overlap between the two entities for both readers, 0.67 ± 0.14 in metastases and 0.72 ± 0.18 in tumor relapse. With chemotherapy, mean ADC increased to 1.54 ± 0.33 in NB/GNB and to 1.23 ± 0.27 in metastases (p < 0.05). The median SIRs of various tumor lesions vs. liver, vs. muscle tissue and vs. adjacent tissue were significantly higher on DWI (range: 2.4 -9.9) than on ce-T1w (range: 1.0 - 1.8, all p < 0.05). The coefficient of variation (CV) was ≤ 8.0% for ADC and ≤ 16.4% for signal intensity data. Based on mean ADC, DWI distinguishes between NB/GNB and GN with high certainty and provides plausible quantitative data on tumor response to therapy. Lesion conspicuity, as measured by SIR, is superior on DWI, compared to ce-T1w. DWI as a noninvasive, radiation-free and widely available imaging technique should be an integral part of MR imaging for neuroblastic tumors and should undergo prospective evaluation in multicenter studies.

  19. DNA methylation patterns of behavior-related gene promoter regions dissect the gray wolf from domestic dog breeds.

    Science.gov (United States)

    Banlaki, Zsofia; Cimarelli, Giulia; Viranyi, Zsofia; Kubinyi, Eniko; Sasvari-Szekely, Maria; Ronai, Zsolt

    2017-06-01

    A growing body of evidence highlights the relationship between epigenetics, especially DNA methylation, and population divergence as well as speciation. However, little is known about how general the phenomenon of epigenetics-wise separation of different populations is, or whether population assignment is, possible based on solely epigenetic marks. In the present study, we compared DNA methylation profiles between four different canine populations: three domestic dog breeds and their ancestor the gray wolf. Altogether, 79 CpG sites constituting the 65 so-called CpG units located in the promoter regions of genes affecting behavioral and temperamental traits (COMT, HTR1A, MAOA, OXTR, SLC6A4, TPH1, WFS1)-regions putatively targeted during domestication and breed selection. Methylation status of buccal cells was assessed using EpiTYPER technology. Significant inter-population methylation differences were found in 52.3% of all CpG units investigated. DNA methylation profile-based hierarchical cluster analysis indicated an unambiguous segregation of wolf from domestic dog. In addition, one of the three dog breeds (Golden Retriever) investigated also formed a separate, autonomous group. The findings support that population segregation is interrelated with shifts in DNA methylation patterns, at least in putative selection target regions, and also imply that epigenetic profiles could provide a sufficient basis for population assignment of individuals.

  20. Minding the gap: Frequency of indels in mtDNA control region sequence data and influence on population genetic analyses

    Science.gov (United States)

    Pearce, J.M.

    2006-01-01

    Insertions and deletions (indels) result in sequences of various lengths when homologous gene regions are compared among individuals or species. Although indels are typically phylogenetically informative, occurrence and incorporation of these characters as gaps in intraspecific population genetic data sets are rarely discussed. Moreover, the impact of gaps on estimates of fixation indices, such as FST, has not been reviewed. Here, I summarize the occurrence and population genetic signal of indels among 60 published studies that involved alignments of multiple sequences from the mitochondrial DNA (mtDNA) control region of vertebrate taxa. Among 30 studies observing indels, an average of 12% of both variable and parsimony-informative sites were composed of these sites. There was no consistent trend between levels of population differentiation and the number of gap characters in a data block. Across all studies, the average influence on estimates of ??ST was small, explaining only an additional 1.8% of among population variance (range 0.0-8.0%). Studies most likely to observe an increase in ??ST with the inclusion of gap characters were those with control region DNA appears small, dependent upon total number of variable sites in the data block, and related to species-specific characteristics and the spatial distribution of mtDNA lineages that contain indels. ?? 2006 Blackwell Publishing Ltd.

  1. Extent and divergence of heteroplasmy of the DNA barcoding region in Anapodisma miramae (Orthoptera: Acrididae).

    Science.gov (United States)

    Kang, Ah Rang; Kim, Min Jee; Park, In Ah; Kim, Kee Young; Kim, Iksoo

    2016-09-01

    A partial sequence of the mitochondrial cytochrome oxidase subunit I (COI) gene is widely used as a molecular marker for species identification in animals, also termed a DNA barcode. However, the presence of more than one sequence type in a single individual, also known as heteroplasmy, is one of the shortcomings of barcode identification. In this study, we examined the extent and divergence of COI heteroplasmy, including nuclear-encoded mitochondrial pseudogenes (NUMTs), at the genomic-DNA level from 13 insect species including orthopteran Anapodisma miramae, and a long fragment of mitochondrial DNA and cDNA from A. miramae as templates. When multiple numbers of clones originated from genomic DNA were sequenced, heteroplasmy was prevalent in all species and NUMTs were observed in five species. Long fragment DNA (∼13.5 kb) also is a source of heteroplasmic amplification, but the divergent haplotypes and NUMTs obtained from genomic DNA were not detected in A. miramae. On the other hand, cDNA was relatively heteroplasmy-free. Consistently, one dominant haplotype was always obtained from the genomic DNA-origin clones in all species and also from the long fragment- and cDNA-origin clones in the two tested individuals of A. miramae. Furthermore, the dominant haplotype was identical in sequence, regardless of the DNA source in A. miramae. Thus, one possible solution to avoid the barcoding problem in relationship to heteroplasmy could be the acquisition of multiple numbers of barcoding sequences to determine a dominant haplotype that can be assigned as barcoding sequence for a given species.

  2. Frequent non-reciprocal exchange in microsatellite-containing-DNA-regions of vertebrates

    DEFF Research Database (Denmark)

    Ziegler, J.O.; Wälther, M.; Linzer, T.R.

    2009-01-01

    Microsatellites are DNA-fragments containing short repetitive motifs with 2-10 bp. They are highly variable in most species and distributed throughout the whole genome. It is broadly accepted that their high degree of variability is closely associated with mispairing of DNA-strands during the rep...

  3. Single genetic stock of kawakawa Euthynnus affinis (Cantor, 1849) along the Indian coast inferred from sequence analyses of mitochondrial DNA D-loop region

    Digital Repository Service at National Institute of Oceanography (India)

    GirishKumar; Kunal, S.P.; Menezes, M.R.; Meena, R.

    , genetic variation was assessed using sequence analyses of Mitochondrial DNA (mtDNA) D-loop region. A 500 bp segment of D-loop region was sequenced in 400 samples collected from eight localities (Veraval (VE), Ratnagiri (RA), Kochi (KO), Kavaratti (KA...

  4. Intragenomic variation in the ITS rDNA region obscures phylogenetic relationships and inflates estimates of operational taxonomic units in genus Laetiporus

    Science.gov (United States)

    Daniel L. Lindner; Mark T. Banik

    2011-01-01

    Regions of rDNA are commonly used to infer phylogenetic relationships among fungal species and as DNA barcodes for identification. These regions occur in large tandem arrays, and concerted evolution is believed to reduce intragenomic variation among copies within these arrays, although some variation still might exist. Phylogenetic studies typically use consensus...

  5. Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity.

    Science.gov (United States)

    Wyszomirski, Karol H; Curth, Ute; Alves, Jürgen; Mackeldanz, Petra; Möncke-Buchner, Elisabeth; Schutkowski, Mike; Krüger, Detlev H; Reuter, Monika

    2012-04-01

    For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod(2)Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.

  6. Reconstructing the History of Mesoamerican Populations through the Study of the Mitochondrial DNA Control Region

    Science.gov (United States)

    Gorostiza, Amaya; Acunha-Alonzo, Víctor; Regalado-Liu, Lucía; Tirado, Sergio; Granados, Julio; Sámano, David; Rangel-Villalobos, Héctor; González-Martín, Antonio

    2012-01-01

    The study of genetic information can reveal a reconstruction of human population’s history. We sequenced the entire mtDNA control region (positions 16.024 to 576 following Cambridge Reference Sequence, CRS) of 605 individuals from seven Mesoamerican indigenous groups and one Aridoamerican from the Greater Southwest previously defined, all of them in present Mexico. Samples were collected directly from the indigenous populations, the application of an individual survey made it possible to remove related or with other origins samples. Diversity indices and demographic estimates were calculated. Also AMOVAs were calculated according to different criteria. An MDS plot, based on FST distances, was also built. We carried out the construction of individual networks for the four Amerindian haplogroups detected. Finally, barrier software was applied to detect genetic boundaries among populations. The results suggest: a common origin of the indigenous groups; a small degree of European admixture; and inter-ethnic gene flow. The process of Mesoamerica’s human settlement took place quickly influenced by the region’s orography, which development of genetic and cultural differences facilitated. We find the existence of genetic structure is related to the region’s geography, rather than to cultural parameters, such as language. The human population gradually became fragmented, though they remained relatively isolated, and differentiated due to small population sizes and different survival strategies. Genetic differences were detected between Aridoamerica and Mesoamerica, which can be subdivided into “East”, “Center”, “West” and “Southeast”. The fragmentation process occurred mainly during the Mesoamerican Pre-Classic period, with the Otomí being one of the oldest groups. With an increased number of populations studied adding previously published data, there is no change in the conclusions, although significant genetic heterogeneity can be detected in Pima

  7. Inter-individual variation in DNA methylation is largely restricted to tissue-specific differentially methylated regions in maize.

    Science.gov (United States)

    Lauria, Massimiliano; Echegoyen-Nava, Rodrigo Antonio; Rodríguez-Ríos, Dalia; Zaina, Silvio; Lund, Gertrud

    2017-02-23

    Variation in DNA methylation across distinct genetic populations, or in response to specific biotic or abiotic stimuli, has typically been studied in leaf DNA from pooled individuals using either reduced representation bisulfite sequencing, whole genome bisulfite sequencing (WGBS) or methylation sensitive amplified polymorphism (MSAP). The latter represents a useful alterative when sample size is large, or when analysing methylation changes in genomes that have yet to be sequenced. In this study we compared variation in methylation across ten individual leaf and endosperm samples from maize hybrid and inbred lines using MSAP. We also addressed the methodological implications of analysing methylation variation using pooled versus individual DNA samples, in addition to the validity of MSAP compared to WGBS. Finally, we analysed a subset of variable and non-variable fragments with respect to genomic location, vicinity to repetitive elements and expression patterns across leaf and endosperm tissues. On average, 30% of individuals showed inter-individual methylation variation, mostly of leaf and endosperm-specific differentially methylated DNA regions. With the exception of low frequency demethylation events, the bulk of inter-individual methylation variation (84 and 80% in leaf and endosperm, respectively) was effectively captured in DNA from pooled individuals. Furthermore, available genome-wide methylation data largely confirmed MSAP leaf methylation profiles. Most variable methylation that mapped within genes was associated with CG methylation, and many of such genes showed tissue-specific expression profiles. Finally, we found that the hAT DNA transposon was the most common class II transposable element found in close proximity to variable DNA regions. The relevance of our results with respect to future studies of methylation variation is the following: firstly, the finding that inter-individual methylation variation is largely restricted to tissue

  8. Methylation of the DCLK1 promoter region in circulating free DNA and its prognostic value in lung cancer patients.

    Science.gov (United States)

    Powrózek, T; Krawczyk, P; Nicoś, M; Kuźnar-Kamińska, B; Batura-Gabryel, H; Milanowski, J

    2016-04-01

    The possibility of detection of suppressor genes methylation in circulating free DNA (cf-DNA) of cancer patients and the lack of methylation in healthy individuals makes this epigenetic alternation an ideal diagnostic marker of neoplastic processes. Moreover, hypermethylation in several genes promoter was described as a biomarker of lung cancer. Methylation in the gene encoding doublecortin-like kinase 1 (DCLK1) is observed in patients with colorectal cancer and cholangiocarcinoma. However, there are no studies concerning DCLK1 methylation in lung cancer patients. The aims of the study was to evaluate the frequency of DCLK1 promoter methylation in cf-DNA of lung cancer patients and of healthy persons as well as the usefulness of this test for predicting the lung cancer course. DCLK1 methylation status was evaluated in DNA isolated from peripheral blood plasma from 65 lung cancer patients and 95 healthy individuals. After DNA bisulfitation, DCLK1 methylation was determined using the qMSP-PCR technique. Moreover, the presence of DCLK1 methylation was correlated with the overall survival (OS) probability of lung cancer patients. DCLK1 promoter methylation was detected in 32 lung cancer patients (49.2 %) and 8 healthy individuals (8.4 %). The methylation of the region before transcription start site (TSS) and the region after TSS of DCLK1 gene was detected in 28 and 11 patients, respectively. In seven cases (10.8 %), the DCLK1 promoter methylation in both regions was reported simultaneously. The methylation was observed slightly frequently in patients with small cell lung cancer (75 % of SCLC patients). The median overall survival of patients with DCLK1 promoter methylation was lower than that of patients without DCLK1 gene modification (p = methylation may be useful in both early diagnosis and prediction of the course of lung cancer.

  9. Nuclear DNA content in 20 species of Siluriformes (Teleostei: Ostariophysi from the Neotropical region

    Directory of Open Access Journals (Sweden)

    Paulo César Fenerich

    2004-01-01

    Full Text Available In the present study, 20 species of Siluriformes fish were analyzed in order to determine their nuclear DNA content and compare these data with their diploid number. In addition, the extension and importance of the changes that occurred during the process of diversification in the group of Neotropical freshwater catfish were investigated. The only species studied of the family Doradidae, Rhinodoras d'orbignyi (2n = 58, presented 3.46 ± 0.13 pg of DNA. Among the species of the family Heptapteridae, the values of nuclear DNA content and the diploid numbers ranged from 1.13 ± 0.09 pg of DNA in Pimelodella sp. (2n = 46 to 2.38 ± 0.07 pg of DNA in Imparfinis mirini (2n = 58. The family Loricariidae showed the widest variation in diploid number and nuclear DNA content values, ranging from 2n = 52 and 3.96 ± 0.22 pg of DNA in Liposarcus anisitsi to 2n = 76 and 4.90 ± 0.12 pg of DNA in Hypostomus sp. 4. In this group, two local samples of Pimelodus maculatus (Pimelodidae were analyzed, and both exhibited 2n = 56, but different nuclear DNA content values (2.68 ± 0.22 pg and 2.82 ± 0.20 pg, respectively. Among the Pseudopimelodidae species analyzed, Pseudopimelodus mangurus (2n = 54 showed 2.23 ± 0.15 pg and Microglanis cottoides (2n = 54 exhibited 2.50 ± 0.18 pg of DNA. Two species of Trichomycterus (Trichomycteridae also presented the same diploid number, 2n = 54 chromosomes, but, while the species from the Quinta stream presented a DNA content of 2.62 ± 0.19 pg, in the sample from the Capivara river this value was 2.30 ± 0.23 pg. In the analyzed species, the results showed that the changes in DNA content were frequently not followed by changes in the diploid number. This fact permits to suggest that, in addition to structural chromosome rearrangements, other mechanisms, including deletions, duplications and polyploidy, could be involved in the process of species differentiation in the representatives of the fish order Siluriformes.

  10. A Composite Method Based on Formal Grammar and DNA Structural Features in Detecting Human Polymerase II Promoter Region

    Science.gov (United States)

    Datta, Sutapa; Mukhopadhyay, Subhasis

    2013-01-01

    An important step in understanding gene regulation is to identify the promoter regions where the transcription factor binding takes place. Predicting a promoter region de novo has been a theoretical goal for many researchers for a long time. There exists a number of in silico methods to predict the promoter region de novo but most of these methods are still suffering from various shortcomings, a major one being the selection of appropriate features of promoter region distinguishing them from non-promoters. In this communication, we have proposed a new composite method that predicts promoter sequences based on the interrelationship between structural profiles of DNA and primary sequence elements of the promoter regions. We have shown that a Context Free Grammar (CFG) can formalize the relationships between different primary sequence features and by utilizing the CFG, we demonstrate that an efficient parser can be constructed for extracting these relationships from DNA sequences to distinguish the true promoter sequences from non-promoter sequences. Along with CFG, we have extracted the structural features of the promoter region to improve upon the efficiency of our prediction system. Extensive experiments performed on different datasets reveals that our method is effective in predicting promoter sequences on a genome-wide scale and performs satisfactorily as compared to other promoter prediction techniques. PMID:23437045

  11. A composite method based on formal grammar and DNA structural features in detecting human polymerase II promoter region.

    Directory of Open Access Journals (Sweden)

    Sutapa Datta

    Full Text Available An important step in understanding gene regulation is to identify the promoter regions where the transcription factor binding takes place. Predicting a promoter region de novo has been a theoretical goal for many researchers for a long time. There exists a number of in silico methods to predict the promoter region de novo but most of these methods are still suffering from various shortcomings, a major one being the selection of appropriate features of promoter region distinguishing them from non-promoters. In this communication, we have proposed a new composite method that predicts promoter sequences based on the interrelationship between structural profiles of DNA and primary sequence elements of the promoter regions. We have shown that a Context Free Grammar (CFG can formalize the relationships between different primary sequence features and by utilizing the CFG, we demonstrate that an efficient parser can be constructed for extracting these relationships from DNA sequences to distinguish the true promoter sequences from non-promoter sequences. Along with CFG, we have extracted the structural features of the promoter region to improve upon the efficiency of our prediction system. Extensive experiments performed on different datasets reveals that our method is effective in predicting promoter sequences on a genome-wide scale and performs satisfactorily as compared to other promoter prediction techniques.

  12. [Database establishment of the whole rDNA ITS region of Dendrobium species of "fengdou" and authentication by analysis of their sequences].

    Science.gov (United States)

    Ding, Xiao-yu; Wang, Zheng-tao; Xu, Hong; Xu, Luo-san; Zhou, Kai-ya

    2002-07-01

    To establish the whole rDNA ITS region sequence database of various Dendrobium species of "Fengdou" and to authenticate exactly the inspected species of "Fengdou". The rDNA ITS regions of various Dendrobium species of "Fengdou" were amplified and sequenced. The database of their rDNA ITS regions was established in order to authenticate the inspected species by means of the softwares of CLUSTRAL and MEGA which were used to analyze the rDNA ITS region. A database of the rDNA ITS sequences of 21 species of Dendrobium has been established. The notable and stable differences of the interspecies of the rDNA ITS regions have been demonstrated. The numbers of transitions and transversions among 21 species are 11-122. The variable sites are 341 while the informative sites are 195. The ITS sequence differences between the outgroup species (Pholidota yunnanensis) and species of "Fengdou" are obvious. The numbers of transitions and transversions are 131-161. The population differences of the rDNA ITS region of various species of "Fengdou" are very small (0-6). On the basis of the database of various Dendrobium species of "Fengdou" and two genetics software, the botanical origin of the inspected species of "Fengdou" has been authenticated successfully by sequencing the rDNA ITS regions.

  13. Pharmacoepigenetics of the role of DNA methylation in μ-opioid receptor expression in different human brain regions.

    Science.gov (United States)

    Knothe, Claudia; Oertel, Bruno G; Ultsch, Alfred; Kettner, Mattias; Schmidt, Peter Harald; Wunder, Cora; Toennes, Stefan W; Geisslinger, Gerd; Lötsch, Jörn

    2016-12-01

    Exposure to opioids has been associated with epigenetic effects. Studies in rodents suggested a role of varying degrees of DNA methylation in the differential regulation of μ-opioid receptor expression across the brain. In a translational investigation, using tissue acquired postmortem from 21 brain regions of former opiate addicts, representing a human cohort with chronic opioid exposure, μ-opioid receptor expression was analyzed at the level of DNA methylation, mRNA and protein. While high or low μ-opioid receptor expression significantly correlated with local OPRM1 mRNA levels, there was no corresponding association with OPRM1 methylation status. Additional experiments in human cell lines showed that changes in DNA methylation associated with changes in μ-opioid expression were an order of magnitude greater than differences in brain. Hence, different degrees of DNA methylation associated with chronic opioid exposure are unlikely to exert a major role in the region-specificity of μ-opioid receptor expression in the human brain.

  14. Simple species identification of Trichinella isolates by amplification and sequencing of the 5S ribosomal DNA intergenic spacer region.

    Science.gov (United States)

    De Bruyne, Aymeric; Yera, Hélène; Le Guerhier, Franck; Boireau, Pascal; Dupouy-Camet, Jean

    2005-09-05

    We developed a PCR-based assay using a single primer pair to amplify the 5S ribosomal DNA intergenic spacer region to identify Trichinella isolates. In our method, amplified products are directly sequenced on both strands and compared to GenBank sequences. Using this method, we were able to identify Trichinella spiralis, T. britovi and T. nativa. This method permits rapid species identification of Trichinella isolates; however, further evaluation is required before recommending this approach for routine use.

  15. Identification of differentially methylated BRCA1 and CRISP2 DNA regions as blood surrogate markers for cardiovascular disease

    OpenAIRE

    Istas, Geoffrey; Declerck, Ken; Pudenz, Maria; Szic, Katarzyna Szarc vel; Lendinez-Tortajada, Veronica; Leon-Latre, Montserrat; Heyninck,Karen; Haegeman, Guy; Casasnovas, Jose A.; Tellez-Plaza, Maria; Gerhauser, Clarissa; Heiss, Christian; Rodriguez-Mateos, Ana; Berghe, Wim Vanden

    2017-01-01

    Genome-wide Illumina InfiniumMethylation 450?K DNA methylation analysis was performed on blood samples from clinical atherosclerosis patients (n?=?8) and healthy donors (n?=?8) in the LVAD study (NCT02174133, NCT01799005). Multiple differentially methylated regions (DMR) could be identified in atherosclerosis patients, related to epigenetic control of cell adhesion, chemotaxis, cytoskeletal reorganisations, cell proliferation, cell death, estrogen receptor pathways and phagocytic immune respo...

  16. Comparison of DNA variants in the PRNP and NF1 regions between bovine spongiform encephalopathy and control cattle.

    Science.gov (United States)

    Geldermann, H; He, H; Bobal, P; Bartenschlager, H; Preuss, S

    2006-10-01

    DNA from 252 bovine spongiform encephalopathy (BSE) cattle and 376 non-diseased control cattle were genotyped for nine loci in the prion protein (PRNP) gene region, three loci in the neurofibromin 1 (NF1) region and four control loci on different chromosomes. The allele and genotype frequencies of the control loci were similar in BSE and control cattle. In the analysed 7.4 Mb PRNP region, the largest differences between BSE and control cattle were found for the loci REG2, R16 and R18, which are located between +300 and +5600 bp, spanning PRNP introns 1 to 2. Carriers of the REG2 genotype 128/128 were younger at BSE diagnosis than those with the other genotypes (128/140 or 140/140). The predominant haplotype REG2 128 bp-R18 173 bp occurred more frequently (P < 0.001), and the second-most frequent haplotype (REG2 140 bp-R18 175 bp) occurred less frequently (P < 0.05) in BSE than in control cattle. The largest frequency differences between BSE and control groups were observed in the Brown Swiss breed. Across all breeds, most of the same alleles and haplotypes of the PRNP region were associated with BSE. In the 23-cM NF1 region, associations with BSE incidence were found for the RM222 allele and for the DIK4009 genotype frequencies. Cattle carrying RM222 genotypes with the 127- or 129-bp alleles were about half a year older at BSE incidence than those with other genotypes. Across the breeds, different alleles and genotypes of the NF1 region were associated with BSE. The informative DNA markers were used to localize the genetic disposition to BSE and may be useful for the identification of the causative DNA variants.

  17. Remarkably low mtDNA control-region diversity and shallow population structure in Pacific cod Gadus macrocephalus.

    Science.gov (United States)

    Liu, M; Lu, Z C; Gao, T X; Yanagimoto, T; Sakurai, Y

    2010-10-01

    To investigate the genetic diversity and describe the population structure in Gadus macrocephalus, a 452 base pair (bp) fragment of the mitochondrial DNA control region was analysed in 259 individuals. The results showed remarkably low nucleotide diversity and a lack of genealogical structure. Small but significant genetic differentiations, however, were detected among north-western Pacific populations, but no large-scale regional differences were detected. These results indicate that populations of G. macrocephalus in the north-western Pacific are genetically subdivided and represent evolutionary lineages that should be managed individually. © 2010 The Authors. Journal compilation © 2010 The Fisheries Society of the British Isles.

  18. Targeted Ablation and Reorganization of the Principal Preplate Neurons and Their Neuroblasts Identified by Golli Promoter Transgene Expression in the Neocortex of Mice

    Directory of Open Access Journals (Sweden)

    Yuan-Yun Xie

    2009-10-01

    Full Text Available The present study delineates the cellular responses of dorsal pallium to targeted genetic ablation of the principal preplate neurons of the neocortex. Ganciclovir treatment during prenatal development (E11-E13; where E is embryonic day of mice selectively killed cells with shared S-phase vulnerability and targeted expression of a GPT [golli promoter transgene, linked to HSV-TK (herpes simplex virus-thymidine kinase, τ-eGFP (τ-enhanced green fluorescent protein and lacZ (lacZ galactosidase reporters] localized in preplate neurons. Morphogenetic fates of attacked neurons and neuroblasts, and their successors, were assessed by multiple labelling in time-series comparisons between ablated (HSV-TK+/0 and control (HSV-TK0/0 littermates. During ablation generation, neocortical growth was suppressed, and compensatory reorganization of non-GPT ventricular zone progenitors of dorsal pallium produced replacements for killed GPT neuroblasts. Replacement and surviving GPT neuroblasts then produced replacements for killed GPT neurons. Near-normal restoration of their complement delayed the settlement of GPT neurons into the reconstituted preplate, which curtailed the outgrowth of pioneer corticofugal axons. Based on this evidence, we conclude that specific cell killing in ablated mice can eliminate a major fraction of GPT neurons, with insignificant bystander killing. Also, replacement GPT neurons in ablated mice originate exclusively by proliferation from intermediate progenitor GPT neuroblasts, whose complement is maintained by non-GPT progenitors for inductive regulation of the total complement of GPT neurons. Finally, GPT neurons in both normal and ablated mice meet all morphogenetic criteria, including the ‘outside-in’ vertical gradient of settlement, presently used to identify principal preplate neurons. In ablated mice, delayed organization of these neurons desynchronizes and isolates developing neocortex from the rest of the brain, and

  19. High fructose consumption induces DNA methylation at PPARα and CPT1A promoter regions in the rat liver

    Energy Technology Data Exchange (ETDEWEB)

    Ohashi, Koji [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Munetsuna, Eiji [Department of Biochemistry, Fujita Health University School of Medicine, Toyoake (Japan); Yamada, Hiroya, E-mail: hyamada@fujita-hu.ac.jp [Department of Hygiene, Fujita Health University School of Medicine, Toyoake (Japan); Ando, Yoshitaka [Department of Joint Research Laboratory of Clinical Medicine, Fujita Health University Hospital, Toyoake (Japan); Yamazaki, Mirai; Taromaru, Nao; Nagura, Ayuri; Ishikawa, Hiroaki [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Suzuki, Koji [Department of Public Health, Fujita Health University School of Health Sciences, Toyoake (Japan); Teradaira, Ryoji [Department of Clinical Biochemistry, Fujita Health University School of Health Sciences, Toyoake (Japan); Hashimoto, Shuji [Department of Hygiene, Fujita Health University School of Medicine, Toyoake (Japan)

    2015-12-04

    DNA methylation status is affected by environmental factors, including nutrition. Fructose consumption is considered a risk factor for the conditions that make up metabolic syndrome such as dyslipidemia. However, the pathogenetic mechanism by which fructose consumption leads to metabolic syndrome is unclear. Based on observations that epigenetic modifications are closely related to induction of metabolic syndrome, we hypothesized that fructose-induced metabolic syndrome is caused by epigenetic alterations. Male SD rats were designated to receive water or 20% fructose solution for 14 weeks. mRNA levels for peroxisome proliferator-activated receptor alpha (PPARα) and carnitine palmitoyltransferase 1A (CPT1A) was analyzed using Real-time PCR. Restriction digestion and real-time PCR (qAMP) was used for the analysis of DNA methylation status. Hepatic lipid accumulation was also observed by fructose intake. Fructose feeding also significantly decreased mRNA levels for PPARα and CPT1A. qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status, and pathogenesis of metabolic syndrome induced by fructose relates to DNA methylation status. - Highlights: • No general consensus has been reached regarding the molecular mechanisms of the pathogenesis of fructose-induced diseases. • Significant increase in hepatic total methylation level was observed after fructose-supplemented feeding. • Fructose feeding significantly decreased mRNA levels for PPARα and CPT1A. • qAMP analysis demonstrated the hypermethylation of promoter regions of PPARα and CTP1A genes. • Fructose-mediated attenuated gene expression may be mediated by alterations of DNA methylation status in rat liver.

  20. [Analysis of genetic diversity of Russian regional populations based on common STR markers used in DNA identification].

    Science.gov (United States)

    Pesik, V Yu; Fedunin, A A; Agdzhoyan, A T; Utevska, O M; Chukhraeva, M I; Evseeva, I V; Churnosov, M I; Lependina, I N; Bogunov, Yu V; Bogunova, A A; Ignashkin, M A; Yankovsky, N K; Balanovska, E V; Orekhov, V A; Balanovsky, O P

    2014-06-01

    We conducted the first genetic analysis of a wide a range of rural Russian populations in European Russia with a panel of common DNA markers commonly used in criminalistics genetic identification. We examined a total of 647 samples from indigenous ethnic Russian populations in Arkhangelsk, Belgorod, Voronezh, Kursk, Rostov, Ryazan, and Orel regions. We employed a multiplex genotyping kit, COrDIS Plus, to genotype Short Tandem Repeat (STR) loci, which included the genetic marker panel officially recommended for DNA identification in the Russian Federation, the United States, and the European Union. In the course of our study, we created a database of allelic frequencies, examined the distribution of alleles and genotypes in seven rural Russian populations, and defined the genetic relationships between these populations. We found that, although multidimensional analysis indicated a difference between the Northern gene pool and the rest of the Russian European populations, a pairwise comparison using 19 STR markers among all populations did not reveal significant differences. This is in concordance with previous studies, which examined up to 12 STR markers of urban Russian populations. Therefore, the database of allelic frequencies created in this study can be applied for forensic examinations and DNA identification among the ethnic Russian population over European Russia. We also noted a decrease in the levels of heterozygosity in the northern Russian population compared to ethnic populations in southern and central Russia, which is consistent with trends identified previously using classical gene markers and analysis of mitochondrial DNA.

  1. Structure of the DNA-binding and RNA-polymerase-binding region of transcription antitermination factor λQ.

    Science.gov (United States)

    Vorobiev, Sergey M; Gensler, Yocheved; Vahedian-Movahed, Hanif; Seetharaman, Jayaraman; Su, Min; Huang, Janet Y; Xiao, Rong; Kornhaber, Gregory; Montelione, Gaetano T; Tong, Liang; Ebright, Richard H; Nickels, Bryce E

    2014-03-04

    The bacteriophage λ Q protein is a transcription antitermination factor that controls expression of the phage late genes as a stable component of the transcription elongation complex. To join the elongation complex, λQ binds a specific DNA sequence element and interacts with RNA polymerase that is paused during early elongation. λQ binds to the paused early-elongation complex through interactions between λQ and two regions of RNA polymerase: region 4 of the σ(70) subunit and the flap region of the β subunit. We present the 2.1 Å resolution crystal structure of a portion of λQ containing determinants for interaction with DNA, interaction with region 4 of σ(70), and interaction with the β flap. The structure provides a framework for interpreting prior genetic and biochemical analysis and sets the stage for future structural studies to elucidate the mechanism by which λQ alters the functional properties of the transcription elongation complex. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. DNA variants within the 5'-flanking region of milk-protein-encoding genes II. The β-lactoglobulin-encoding gene.

    Science.gov (United States)

    Wagner, V A; Schild, T A; Geldermann, H

    1994-09-01

    For the detection of polymorphisms within the 5'-flanking region of the β-lactoglobulin (-LG) -encoding gene a nucleotide sequence containing 795 bp of the promoter and 59 bp of exon I was cloned and sequenced. After comparing the sequence from the DNA of 11 diverse cows (different breeds and milk-protein yields), 14 singlebp substitutions were identified within the 5'-flanking region and two in the 5'-untranslated region (5'-UTR) of exon I. Some of the variants are located in potential binding sites for trans-acting factors or in the 5'-UTR. A PCR-based RFLP analysis was performed, and the genotypes of an additional 60 cows were identified at five variable 5'-flanking sites. The results reveal three frequent combinations between the A and B alleles of the protein-coding region and the novel 5'-flanking DNA variants. This finding may explain the differences of the protein-variant-dependent β-LG synthesis (A>B) observed in vivo. A sequence comparison of the bovine and ovine promoters reveals an homology of 92.8% and shows a higher degree of conservation between positions -600 and -300.

  3. Potential non-B DNA regions in the human genome are associated with higher rates of nucleotide mutation and expression variation.

    Science.gov (United States)

    Du, Xiangjun; Gertz, E Michael; Wojtowicz, Damian; Zhabinskaya, Dina; Levens, David; Benham, Craig J; Schäffer, Alejandro A; Przytycka, Teresa M

    2014-11-10

    While individual non-B DNA structures have been shown to impact gene expression, their broad regulatory role remains elusive. We utilized genomic variants and expression quantitative trait loci (eQTL) data to analyze genome-wide variation propensities of potential non-B DNA regions and their relation to gene expression. Independent of genomic location, these regions were enriched in nucleotide variants. Our results are consistent with previously observed mutagenic properties of these regions and counter a previous study concluding that G-quadruplex regions have a reduced frequency of variants. While such mutagenicity might undermine functionality of these elements, we identified in potential non-B DNA regions a signature of negative selection. Yet, we found a depletion of eQTL-associated variants in potential non-B DNA regions, opposite to what might be expected from their proposed regulatory role. However, we also observed that genes downstream of potential non-B DNA regions showed higher expression variation between individuals. This coupling between mutagenicity and tolerance for expression variability of downstream genes may be a result of evolutionary adaptation, which allows reconciling mutagenicity of non-B DNA structures with their location in functionally important regions and their potential regulatory role. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  4. Length variations in the COII-tRNA(Lys) intergenic region of mitochondrial DNA in Indonesian populations.

    Science.gov (United States)

    Handoko, H Y; Lum, J K; Gustiani; Rismalia; Kartapradja, H; Sofro, A S; Marzuki, S

    2001-04-01

    The prevalence of a 9-base-pair (bp) deletion between the mitochondrial cytochrome oxidase II (MTCOX*2) and lysine tRNA (MTTK) genes (region V) has been used to estimate the genetic relationships among Asian and Pacific populations. Many East Asian and Pacific Island populations have been examined previously, but the mitochondrial DNA (mtDNA) diversity of the intervening Indonesian archipelago has not previously been systematically examined. The 17,500 islands of Indonesia currently contain nearly 213 million people and extensive cultural, linguistic, and, presumably, genetic diversity. This study of 1091 individuals representing 15 ethnic groups is the most extensive mtDNA survey to date of the Indonesian archipelago. Six distinct length polymorphisms in region V were observed within these 15 populations. The 9-bp deletion was found in every population examined at frequencies comparable to those of previously examined East Asian populations and substantially lower than those in most Pacific Island populations. Despite the inclusion of Austronesian-speaking populations and a Papuan-speaking population, there was no statistically significant heterogeneity in the frequency of the 9-bp deletion among the 15 populations (p = 0.09). These data indicate that substantial gene flow occurred among the populations at some time in the past. Our observations of no significant correlations between genetic and geographic distances (r = -0.04, p = 0.53) coupled with the extensive cultural and linguistic differences currently within the archipelago suggest that little gene flow among neighboring populations has occurred recently.

  5. Identification of differentially methylated BRCA1 and CRISP2 DNA regions as blood surrogate markers for cardiovascular disease.

    Science.gov (United States)

    Istas, Geoffrey; Declerck, Ken; Pudenz, Maria; Szic, Katarzyna Szarc Vel; Lendinez-Tortajada, Veronica; Leon-Latre, Montserrat; Heyninck, Karen; Haegeman, Guy; Casasnovas, Jose A; Tellez-Plaza, Maria; Gerhauser, Clarissa; Heiss, Christian; Rodriguez-Mateos, Ana; Berghe, Wim Vanden

    2017-07-11

    Genome-wide Illumina InfiniumMethylation 450 K DNA methylation analysis was performed on blood samples from clinical atherosclerosis patients (n = 8) and healthy donors (n = 8) in the LVAD study (NCT02174133, NCT01799005). Multiple differentially methylated regions (DMR) could be identified in atherosclerosis patients, related to epigenetic control of cell adhesion, chemotaxis, cytoskeletal reorganisations, cell proliferation, cell death, estrogen receptor pathways and phagocytic immune responses. Furthermore, a subset of 34 DMRs related to impaired oxidative stress, DNA repair, and inflammatory pathways could be replicated in an independent cohort study of donor-matched healthy and atherosclerotic human aorta tissue (n = 15) and human carotid plaque samples (n = 19). Upon integrated network analysis, BRCA1 and CRISP2 DMRs were identified as most central disease-associated DNA methylation biomarkers. Differentially methylated BRCA1 and CRISP2 regions were verified by MassARRAY Epityper and pyrosequencing assays and could be further replicated in blood, aorta tissue and carotid plaque material of atherosclerosis patients. Moreover, methylation changes at BRCA1 and CRISP2 specific CpG sites were consistently associated with subclinical atherosclerosis measures (coronary calcium score and carotid intima media thickness) in an independent sample cohort of middle-aged men with subclinical cardiovascular disease in the Aragon Workers' Health Study (n = 24). Altogether, BRCA1 and CRISP2 DMRs hold promise as novel blood surrogate markers for early risk stratification and CVD prevention.

  6. Photoabsorption study of Bacillus megaterium, DNA and Related Biological Materials in the Phosphorus K-edge Region

    Science.gov (United States)

    Frigo, Sean P.; McNulty,Ian; Richmond, Robert C.; Ehret, Charles F.

    2003-01-01

    We have measured the x-ray transmission spectra of several biologically related samples in the phosphorus K-edge absorption region. These include red phosphorus, hydrated sodium phosphate (Na3PO4 12 H2O), deoxyribonucleic acid (DNA), adenosinetriphosphate (ATP), diolylphosphatidyl choline (DOPC), and Bacillus megaterium spores. Red phosphorus essentially displays an edge-jump. All other spectra are similar in form and energy position, where each is dominated by a narrower, more intense first peak and a broader but less intense second peak. The corresponding K-edge absorption thresholds are shifted towards higher energy relative to that for red phosphorus, as expected for increasing degrees of phosphorus oxidation. The B.meguterium spectrum has aspects common to both the phosphate and DNA spectra and is therefore interpreted as a composite of spectra arising from DNA/RNA and phosphates within the spore. The B. megaterium spore spectrum provides needed information for resonant radiation damage studies in the phosphorus K-edge absorption region by identifying candidate photoexcitations. In addition, the absorption spectra will be useful in macromolecular crystallography studies employing anomalous dispersion effects at the phosphorus K-edge.

  7. Photoreactivity of the linker region of two consecutive G-quadruplexes formed by human telomeric DNA.

    Science.gov (United States)

    Li, Yue; Sugiyama, Hiroshi

    2015-05-25

    We report the application of a photoreaction method for probing two consecutive G-quadruplexes formed by human telomeric DNA. This method can discriminate the loop structure located between two consecutive G-quadruplexes formed by eight TTAGGG repeats in K(+) and Na(+) solutions.

  8. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

    Czech Academy of Sciences Publication Activity Database

    Schoch, C.L.; Seifert, K.A.; Huhndorf, S.; Robert, V.; Spouge, J.L.; Levesque, C.A.; Chen, W.; Bolchacova, E.; Voigt, K.; Crous, P.W.; Miller, A.N.; Wingfield, M.J.; Aime, M.C.; An, K.D.; Bai, F.Y.; Barreto, R.W.; Bergeron, M.J.; Blackwell, M.; Boekhout, T.; Bogale, M.; Boonyuen, N.; Burgaz, A.R.; Buyck, B.; Cai, L.; Cai, Q.; Cardinali, G.; Chaverri, P.; Coppins, B.J.; Crespo, A.; Cubas, P.; Cummings, C.; Damm, U.; de Beer, Z.W.; de Hoog, G.S.; Del-Prado, R.; Dentinger, B.; Dieguez-Uribeondo, J.; Divakar, P.K.; Douglas, B.; Duenas, M.; Duong, T.A.; Eberhardt, U.; Edwards, J.E.; Elshahed, M.S.; Fliegerová, Kateřina; Furtado, M.; Garcia, M.A.; Ge, Z.W.; Griffith, G.W.; Griffiths, K.; Groenewald, J.Z.; Groenewald, M.; Grube, M.; Gryzenhout, M.; Guo, L.D.; Hagen, F.; Hambleton, S.; Hamelin, R.C.; Hansen, K.; Harrold, P.; Heller, G.; Herrera, C.; Hirayama, K.; Hirooka, Y.; Ho, H.M.; Hoffmann, K.; Hofstetter, V.; Hognabba, F.; Hollingsworth, P.M.; Hong, S.B.; Hosaka, K.; Houbraken, J.; Hughes, K.; Huhtinen, S.; Hyde, K.D.; James, T.; Johnson, E.M.; Johnson, J.E.; Johnston, P.R.; Jones, E.B.; Kelly, L.J.; Kirk, P.M.; Knapp, D.G.; Koljalg, U.; Kovacs, G.M.; Kurtzman, C.P.; Landvik, S.; Leavitt, S.D.; Liggenstoffer, A.S.; Liimatainen, K.; Lombard, L.; Luangsa-Ard, J.J.; Lumbsch, H.T.; Maganti, H.; Maharachchikumbura, S.S.; Martin, M.P.; May, T.W.; McTaggart, A.R.; Methven, A.S.; Meyer, W.; Moncalvo, J.M.; Mongkolsamrit, S.; Nagy, L.G.; Nilsson, R.H.; Niskanen, T.; Nyilasi, I.; Okada, G.; Okane, I.; Olariaga, I.; Otte, J.; Papp, T.; Park, D.; Petkovits, T.; Pino-Bodas, R.; Quaedvlieg, W.; Raja, H.A.; Redecker, D.; Rintoul, T.; Ruibal, C.; Sarmiento-Ramirez, J.M.; Schmitt, I.; Schussler, A.; Shearer, C.; Sotome, K.; Stefani, F.O.; Stenroos, S.; Stielow, B.; Stockinger, H.; Suetrong, S.; Suh, S.O.; Sung, G.H.; Suzuki, M.; Tanaka, K.; Tedersoo, L.; Telleria, M.T.; Tretter, E.; Untereiner, W.A.; Urbina, H.; Vagvolgyi, C.; Vialle, A.; Vu, T.D.; Walther, G.; Wang, Q.M.; Wang, Y.; Weir, B.S.; Weiss, M.; White, M.M.; Xu, J.; Yahr, R.; Yang, Z.L.; Yurkov, A.; Zamora, J.C.; Zhang, N.; Zhuang, W.Y.; Schindel, D.

    2012-01-01

    Roč. 109, č. 16 (2012), s. 6241-6246 ISSN 0027-8424 Institutional research plan: CEZ:AV0Z50450515 Keywords : DNA barcoding * fungal biodiversity Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 9.737, year: 2012

  9. Microsatellite DNA suggests regional structure in the fusiform rust fungus Cronartium quercuum f. sp fusiforme

    Science.gov (United States)

    T.L Kubisiak; J.H. Roberds; P.C. Spaine; R.L. Doudrick

    2004-01-01

    This paper reports results obtained from microsatellite DNA analysis of genetic structure for populations of the native fungus Cronartium quercuum f. sp fusiforme infecting loblolly pine (Pinus taeda L.) over much of this host's natural range. Mostly all fusiform rust galls formed under field conditions are...

  10. A collaborative EDNAP exercise on SNaPshot™-based mtDNA control region typing

    DEFF Research Database (Denmark)

    Weiler, NEC; Baca, K; Ballard, D

    2017-01-01

    A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (m...

  11. Early Holocenic and Historic mtDNA African Signatures in the Iberian Peninsula: The Andalusian Region as a Paradigm.

    Directory of Open Access Journals (Sweden)

    Candela L Hernández

    Full Text Available Determining the timing, identity and direction of migrations in the Mediterranean Basin, the role of "migratory routes" in and among regions of Africa, Europe and Asia, and the effects of sex-specific behaviors of population movements have important implications for our understanding of the present human genetic diversity. A crucial component of the Mediterranean world is its westernmost region. Clear features of transcontinental ancient contacts between North African and Iberian populations surrounding the maritime region of Gibraltar Strait have been identified from archeological data. The attempt to discern origin and dates of migration between close geographically related regions has been a challenge in the field of uniparental-based population genetics. Mitochondrial DNA (mtDNA studies have been focused on surveying the H1, H3 and V lineages when trying to ascertain north-south migrations, and U6 and L in the opposite direction, assuming that those lineages are good proxies for the ancestry of each side of the Mediterranean. To this end, in the present work we have screened entire mtDNA sequences belonging to U6, M1 and L haplogroups in Andalusians--from Huelva and Granada provinces--and Moroccan Berbers. We present here pioneer data and interpretations on the role of NW Africa and the Iberian Peninsula regarding the time of origin, number of founders and expansion directions of these specific markers. The estimated entrance of the North African U6 lineages into Iberia at 10 ky correlates well with other L African clades, indicating that U6 and some L lineages moved together from Africa to Iberia in the Early Holocene. Still, founder analysis highlights that the high sharing of lineages between North Africa and Iberia results from a complex process continued through time, impairing simplistic interpretations. In particular, our work supports the existence of an ancient, frequently denied, bridge connecting the Maghreb and Andalusia.

  12. Comparability of multiple data types from the Bering Strait region: cranial and dental metrics and nonmetrics, mtDNA, and Y-chromosome DNA.

    Science.gov (United States)

    Herrera, Brianne; Hanihara, Tsunehiko; Godde, Kanya

    2014-07-01

    Different data types have previously been shown to have the same microevolutionary patterns in worldwide data sets. However, peopling of the New World studies have shown a difference in migration paths and timings using multiple types of data, spurring research to understand why this is the case. This study was designed to test the degree of similarity in evolutionary patterns by using cranial and dental metric and nonmetric data, along with Y-chromosome DNA and mtDNA. The populations used included Inuits from Alaska, Canada, Siberia, Greenland, and the Aleutian Islands. For comparability, the populations used for the cranial and molecular data were from similar geographic regions or had a shared population history. Distance, R and kinship matrices were generated for use in running Mantel tests, PROTEST analyses, and Procrustes analyses. A clear patterning was seen, with the craniometric data being most highly correlated to the mtDNA data and the cranial nonmetric data being most highly correlated with the Y-chromosome data, while the phenotypic data were also linked. This patterning is suggestive of a possible male or female inheritance, or the correlated data types are affected by the same or similar evolutionary forces. The results of this study indicate cranial traits have some degree of heritability. Moreover, combining data types leads to a richer knowledge of biological affinity. This understanding is important for bioarchaeological contexts, in particular, peopling of the New World studies where focusing on reconciling the results from comparing multiple data types is necessary to move forward. © 2014 Wiley Periodicals, Inc.

  13. Ethanol-induced transcriptional activation of programmed cell death 4 (Pdcd4 is mediated by GSK-3β signaling in rat cortical neuroblasts.

    Directory of Open Access Journals (Sweden)

    Amanjot Kaur Riar

    Full Text Available Ingestion of ethanol (ETOH during pregnancy induces grave abnormalities in developing fetal brain. We have previously reported that ETOH induces programmed cell death 4 (PDCD4, a critical regulator of cell growth, in cultured fetal cerebral cortical neurons (PCNs and in the cerebral cortex in vivo and affect protein synthesis as observed in Fetal Alcohol Spectrum Disorder (FASD. However, the mechanism which activates PDCD4 in neuronal systems is unclear and understanding this regulation may provide a counteractive strategy to correct the protein synthesis associated developmental changes seen in FASD. The present study investigates the molecular mechanism by which ethanol regulates PDCD4 in cortical neuroblasts, the immediate precursor of neurons. ETOH treatment significantly increased PDCD4 protein and transcript expression in spontaneously immortalized rat brain neuroblasts. Since PDCD4 is regulated at both the post-translational and post-transcriptional level, we assessed ETOH's effect on PDCD4 protein and mRNA stability. Chase experiments demonstrated that ETOH does not significantly impact either PDCD4 protein or mRNA stabilization. PDCD4 promoter-reporter assays confirmed that PDCD4 is transcriptionally regulated by ETOH in neuroblasts. Given a critical role of glycogen synthase kinase 3β (GSK-3β signaling in regulating protein synthesis and neurotoxic mechanisms, we investigated the involvement of GSK-3β and showed that multifunctional GSK-3β was significantly activated in response to ETOH in neuroblasts. In addition, we found that ETOH-induced activation of PDCD4 was inhibited by pharmacologic blockade of GSK-3β using inhibitors, lithium chloride (LiCl and SB-216763 or siRNA mediated silencing of GSK-3β. These results suggest that ethanol transcriptionally upregulates PDCD4 by enhancing GSK-3β signaling in cortical neuroblasts. Further, we demonstrate that canonical Wnt-3a/GSK-3β signaling is involved in regulating PDCD4 protein

  14. Human DNA repair genes possess potential G-quadruplex sequences in their promoters and 5`-untranslated regions.

    Science.gov (United States)

    Fleming, Aaron M; Zhu, Judy; Ding, Yun; Visser, Joshua A; Zhu, Julia; Burrows, Cynthia J

    2018-01-10

    The cellular response to oxidative stress includes transcriptional changes, particularly for genes involved in DNA repair. Recently, our laboratory demonstrated that oxidation of 2`-deoxyguanosine (G) to 8-oxo-7,8-dihydro-2`-deoxyguanosine (OG) in G-rich potential G-quadruplex sequences (PQSs) in gene promoters impacts the level of gene expression up or down depending on the position of the PQS in the promoter. In the present report, bioinformatic analysis found that the 390 human DNA repair genes in the genome ontology initiative harbor 2,936 PQSs in their promoters and 5`-untranslated regions (5`-UTRs). The average density of PQSs in human DNA repair genes was found to be nearly twofold greater than the average density of PQSs in all coding and non-coding human genes (7.5 vs. 4.3 per gene). The distribution of the PQSs in the DNA repair genes on the non-transcribed (coding) vs. transcribed strands reflects that of PQSs in all human genes. Next, literature data were interrogated to select 30 PQSs to catalog their ability to adopt G-quadruplex (G4) folds in vitro using five different experimental tests. The G4 characterization experiments concluded that 26 of the 30 sequences could adopt G4 topologies in solution. Last, four PQSs were synthesized into the promoter of a luciferase plasmid and co-transfected with the G4-specific ligands pyridostatin, Phen-DC3, or BRACO-19 in human cells to determine whether the PQSs could adopt G4 folds. The cell studies identified changes in luciferase expression when the G4 ligands were present, and the magnitude of the expression changes dependent on the PQS and the coding vs. template strand on which the sequence resided. Our studies demonstrate PQSs exist at a high density in human DNA repair gene promoters and a subset of the identified sequences fold in vitro and in vivo.

  15. Differential DNA methylation of microRNAs within promoters, intergenic and intragenic regions of type 2 diabetic, pre-diabetic and non-diabetic individuals.

    Science.gov (United States)

    Pheiffer, Carmen; Erasmus, Rajiv T; Kengne, Andre P; Matsha, Tandi E

    2016-04-01

    Accumulating evidence supports the role of epigenetic modifications, and in particular DNA methylation and non-coding RNAs in the pathophysiology of type 2 diabetes. Alterations in methylation patterns within promoter regions are linked with aberrant transcription and pathological gene expression; however the role of methylation within non-promoter regions is not yet fully elucidated. We performed whole genome methylated DNA immunoprecipitation sequencing (MeDIP-Seq) in peripheral-blood-derived DNA from age-gender-body mass index (BMI)-ethnicity matched type 2 diabetic, pre-diabetic and non-diabetic individuals. The density of methylation normalized to the average length of the promoter, intergenic and intragenic regions and to CpG count was 3.17, 9.80 and 0.09 for the promoter, intergenic and intragenic regions, respectively. Methylation within these regions varied according to glucose tolerance status and was associated with hypermethylation rather than hypomethylation. MicroRNA-DNA methylation peaks accounted for 4.8% of the total number of peaks detected. Differential DNA methylation of these microRNA peaks was observed during dysglycemia, with the promoter, intergenic and intragenic regions accounting for 2%, 95% and 3% respectively, of the differentially methylated microRNA peaks. Genome-wide DNA methylation varied according to glucose tolerance. Methylation within non-promoter regions accounted for the majority of differentially methylated peaks identified, thus highlighting the importance of DNA methylation within these non-promoter regions in the pathogenesis of type 2 diabetes. This study suggests that DNA methylation within intergenic regions is a mechanism regulating microRNAs, another increasingly important epigenetic factor, during type 2 diabetes. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  16. Brain region-specific expression of MeCP2 isoforms correlates with DNA methylation within Mecp2 regulatory elements.

    Directory of Open Access Journals (Sweden)

    Carl O Olson

    Full Text Available MeCP2 is a critical epigenetic regulator in brain and its abnormal expression or compromised function leads to a spectrum of neurological disorders including Rett Syndrome and autism. Altered expression of the two MeCP2 isoforms, MeCP2E1 and MeCP2E2 has been implicated in neurological complications. However, expression, regulation and functions of the two isoforms are largely uncharacterized. Previously, we showed the role of MeCP2E1 in neuronal maturation and reported MeCP2E1 as the major protein isoform in the adult mouse brain, embryonic neurons and astrocytes. Recently, we showed that DNA methylation at the regulatory elements (REs within the Mecp2 promoter and intron 1 impact the expression of Mecp2 isoforms in differentiating neural stem cells. This current study is aimed for a comparative analysis of temporal, regional and cell type-specific expression of MeCP2 isoforms in the developing and adult mouse brain. MeCP2E2 displayed a later expression onset than MeCP2E1 during mouse brain development. In the adult female and male brain hippocampus, both MeCP2 isoforms were detected in neurons, astrocytes and oligodendrocytes. Furthermore, MeCP2E1 expression was relatively uniform in different brain regions (olfactory bulb, striatum, cortex, hippocampus, thalamus, brainstem and cerebellum, whereas MeCP2E2 showed differential enrichment in these brain regions. Both MeCP2 isoforms showed relatively similar distribution in these brain regions, except for cerebellum. Lastly, a preferential correlation was observed between DNA methylation at specific CpG dinucleotides within the REs and Mecp2 isoform-specific expression in these brain regions. Taken together, we show that MeCP2 isoforms display differential expression patterns during brain development and in adult mouse brain regions. DNA methylation patterns at the Mecp2 REs may impact this differential expression of Mecp2/MeCP2 isoforms in brain regions. Our results significantly contribute

  17. Brain Region-Specific Expression of MeCP2 Isoforms Correlates with DNA Methylation within Mecp2 Regulatory Elements

    Science.gov (United States)

    Liyanage, Vichithra R. B.; Rastegar, Mojgan

    2014-01-01

    MeCP2 is a critical epigenetic regulator in brain and its abnormal expression or compromised function leads to a spectrum of neurological disorders including Rett Syndrome and autism. Altered expression of the two MeCP2 isoforms, MeCP2E1 and MeCP2E2 has been implicated in neurological complications. However, expression, regulation and functions of the two isoforms are largely uncharacterized. Previously, we showed the role of MeCP2E1 in neuronal maturation and reported MeCP2E1 as the major protein isoform in the adult mouse brain, embryonic neurons and astrocytes. Recently, we showed that DNA methylation at the regulatory elements (REs) within the Mecp2 promoter and intron 1 impact the expression of Mecp2 isoforms in differentiating neural stem cells. This current study is aimed for a comparative analysis of temporal, regional and cell type-specific expression of MeCP2 isoforms in the developing and adult mouse brain. MeCP2E2 displayed a later expression onset than MeCP2E1 during mouse brain development. In the adult female and male brain hippocampus, both MeCP2 isoforms were detected in neurons, astrocytes and oligodendrocytes. Furthermore, MeCP2E1 expression was relatively uniform in different brain regions (olfactory bulb, striatum, cortex, hippocampus, thalamus, brainstem and cerebellum), whereas MeCP2E2 showed differential enrichment in these brain regions. Both MeCP2 isoforms showed relatively similar distribution in these brain regions, except for cerebellum. Lastly, a preferential correlation was observed between DNA methylation at specific CpG dinucleotides within the REs and Mecp2 isoform-specific expression in these brain regions. Taken together, we show that MeCP2 isoforms display differential expression patterns during brain development and in adult mouse brain regions. DNA methylation patterns at the Mecp2 REs may impact this differential expression of Mecp2/MeCP2 isoforms in brain regions. Our results significantly contribute towards characterizing

  18. Temporal fluctuation in North East Baltic Sea region cattle population revealed by mitochondrial and Y-chromosomal DNA analyses.

    Directory of Open Access Journals (Sweden)

    Marianna Niemi

    Full Text Available Ancient DNA analysis offers a way to detect changes in populations over time. To date, most studies of ancient cattle have focused on their domestication in prehistory, while only a limited number of studies have analysed later periods. Conversely, the genetic structure of modern cattle populations is well known given the undertaking of several molecular and population genetic studies.Bones and teeth from ancient cattle populations from the North-East Baltic Sea region dated to the Prehistoric (Late Bronze and Iron Age, 5 samples, Medieval (14, and Post-Medieval (26 periods were investigated by sequencing 667 base pairs (bp from the mitochondrial DNA (mtDNA and 155 bp of intron 19 in the Y-chromosomal UTY gene. Comparison of maternal (mtDNA haplotypes genetic diversity in ancient cattle (45 samples with modern cattle populations in Europe and Asia (2094 samples revealed 30 ancient mtDNA haplotypes, 24 of which were shared with modern breeds, while 6 were unique to the ancient samples. Of seven Y-chromosomal sequences determined from ancient samples, six were Y2 and one Y1 haplotype. Combined data including Swedish samples from the same periods (64 samples was compared with the occurrence of Y-chromosomal haplotypes in modern cattle (1614 samples.The diversity of haplogroups was highest in the Prehistoric samples, where many haplotypes were unique. The Medieval and Post-Medieval samples also show a high diversity with new haplotypes. Some of these haplotypes have become frequent in modern breeds in the Nordic Countries and North-Western Russia while other haplotypes have remained in only a few local breeds or seem to have been lost. A temporal shift in Y-chromosomal haplotypes from Y2 to Y1 was detected that corresponds with the appearance of new mtDNA haplotypes in the Medieval and Post-Medieval period. This suggests a replacement of the Prehistoric mtDNA and Y chromosomal haplotypes by new types of cattle.

  19. Genomic Clustering of differential DNA methylated regions (epimutations) associated with the epigenetic transgenerational inheritance of disease and phenotypic variation.

    Science.gov (United States)

    Haque, M Muksitul; Nilsson, Eric E; Holder, Lawrence B; Skinner, Michael K

    2016-06-01

    A variety of environmental factors have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation in numerous species. Exposure to environmental factors such as toxicants can promote epigenetic changes (epimutations) involving alterations in DNA methylation to produce specific differential DNA methylation regions (DMRs). The germline (e.g. sperm) transmission of epimutations is associated with epigenetic transgenerational inheritance phenomena. The current study was designed to determine the genomic locations of environmentally induced transgenerational DMRs and assess their potential clustering. The exposure specific DMRs (epimutations) from a number of different studies were used. The clustering approach identified areas of the genome that have statistically significant over represented numbers of epimutations. The location of DMR clusters was compared to the gene clusters of differentially expressed genes found in tissues and cells associated with the transgenerational inheritance of disease. Such gene clusters, termed epigenetic control regions (ECRs), have been previously suggested to regulate gene expression in regions spanning up to 2-5 million bases. DMR clusters were often found to associate with inherent gene clusters within the genome. The current study used a number of epigenetic datasets from previous studies to identify novel DMR clusters across the genome. Observations suggest these clustered DMR within an ECR may be susceptible to epigenetic reprogramming and dramatically influence genome activity.

  20. Molecular Identification of Dendrobium Species (Orchidaceae Based on the DNA Barcode ITS2 Region and Its Application for Phylogenetic Study

    Directory of Open Access Journals (Sweden)

    Shangguo Feng

    2015-09-01

    Full Text Available The over-collection and habitat destruction of natural Dendrobium populations for their commercial medicinal value has led to these plants being under severe threat of extinction. In addition, many Dendrobium plants are similarly shaped and easily confused during the absence of flowering stages. In the present study, we examined the application of the ITS2 region in barcoding and phylogenetic analyses of Dendrobium species (Orchidaceae. For barcoding, ITS2 regions of 43 samples in Dendrobium were amplified. In combination with sequences from GenBank, the sequences were aligned using Clustal W and genetic distances were computed using MEGA V5.1. The success rate of PCR amplification and sequencing was 100%. There was a significant divergence between the inter- and intra-specific genetic distances of ITS2 regions, while the presence of a barcoding gap was obvious. Based on the BLAST1, nearest distance and TaxonGAP methods, our results showed that the ITS2 regions could successfully identify the species of most Dendrobium samples examined; Second, we used ITS2 as a DNA marker to infer phylogenetic relationships of 64 Dendrobium species. The results showed that cluster analysis using the ITS2 region mainly supported the relationship between the species of Dendrobium established by traditional morphological methods and many previous molecular analyses. To sum up, the ITS2 region can not only be used as an efficient barcode to identify Dendrobium species, but also has the potential to contribute to the phylogenetic analysis of the genus Dendrobium.

  1. Molecular Identification of Dendrobium Species (Orchidaceae) Based on the DNA Barcode ITS2 Region and Its Application for Phylogenetic Study.

    Science.gov (United States)

    Feng, Shangguo; Jiang, Yan; Wang, Shang; Jiang, Mengying; Chen, Zhe; Ying, Qicai; Wang, Huizhong

    2015-09-11

    The over-collection and habitat destruction of natural Dendrobium populations for their commercial medicinal value has led to these plants being under severe threat of extinction. In addition, many Dendrobium plants are similarly shaped and easily confused during the absence of flowering stages. In the present study, we examined the application of the ITS2 region in barcoding and phylogenetic analyses of Dendrobium species (Orchidaceae). For barcoding, ITS2 regions of 43 samples in Dendrobium were amplified. In combination with sequences from GenBank, the sequences were aligned using Clustal W and genetic distances were computed using MEGA V5.1. The success rate of PCR amplification and sequencing was 100%. There was a significant divergence between the inter- and intra-specific genetic distances of ITS2 regions, while the presence of a barcoding gap was obvious. Based on the BLAST1, nearest distance and TaxonGAP methods, our results showed that the ITS2 regions could successfully identify the species of most Dendrobium samples examined; Second, we used ITS2 as a DNA marker to infer phylogenetic relationships of 64 Dendrobium species. The results showed that cluster analysis using the ITS2 region mainly supported the relationship between the species of Dendrobium established by traditional morphological methods and many previous molecular analyses. To sum up, the ITS2 region can not only be used as an efficient barcode to identify Dendrobium species, but also has the potential to contribute to the phylogenetic analysis of the genus Dendrobium.

  2. Detection of human papillomavirus DNA in pterygia from different geographical regions

    Science.gov (United States)

    Piras, F; Moore, P S; Ugalde, J; Perra, M T; Scarpa, A; Sirigu, P

    2003-01-01

    Background/aims: The aetiology and pathogenesis of pterygia remain unclear and the involvement of human papillomavirus (HPV) is controversial. 41 pterygia from two geographic locations were evaluated for the presence of HPV DNA. Methods: 41 pterygium biopsies (17 from Italy and 24 from Ecuador) were analysed using the L1C1 and PU-1ML primer sets by polymerase chain reaction (PCR) and DNA sequence analysis. Results: 22 of the 41 pterygia (54%) were positive for HPV, including all 17 Italian cases and 5/24 (21%) Ecuadorean cases. DNA sequencing of the 22 positive cases showed that 11 were HPV type 52, four were type 54, five were candHPV90, and two of unknown genotype. Conclusions: The major differences in the frequency of HPV in geographically distant populations might suggest a possible explanation for the vast differences in the reported detection rates. Three subtypes of HPV were found in this sample of pterygia. None the less, these results suggest that HPV may have a pathogenic role in pterygium. PMID:12812887

  3. Age-dependent guanine oxidation in DNA of different brain regions of Wistar rats and prematurely aging OXYS rats.

    Science.gov (United States)

    Sattarova, Evgeniya A; Sinitsyna, Olga I; Vasyunina, Elena A; Duzhak, Alexander B; Kolosova, Nataliya G; Zharkov, Dmitry O; Nevinsky, Georgy A

    2013-06-01

    Oxidative damage to the cell, including the formation of 8-oxoG, has been regarded as a significant factor in carcinogenesis and aging. An inbred prematurely aging rat strain (OXYS) is characterized by high sensitivity to oxidative stress, lipid peroxidation, protein oxidation, DNA rearrangements, and pathological conditions paralleling several human degenerative diseases including learning and memory deterioration. We have used monoclonal antibodies against a common pre-mutagenic base lesion 8-oxoguanine (8-oxoG) and 8-oxoguanine DNA glycosylase (OGG1) in combination with indirect immunofluorescence microscopy and image analysis to follow the relative amounts and distribution of 8-oxoG and OGG1 in various cells of different brain regions from OXYS and control Wistar rats. It was shown that 8-oxoG increased with age in mature neurons, nestin- and glial fibrillary acidic protein (GFAP)-positive cells of hippocampus and frontal cortex in both strains of rats, with OXYS rats always displaying statistically significantly higher levels of oxidative DNA damage than Wistar rats. The relative content of 8-oxoG and OGG1 in nestin- and GFAP-positive cells was higher than in mature neurons in both Wistar and OXYS rats. However, there was no significant interstrain difference in the content of OGG1 for all types of cells and brain regions analyzed, and no difference in the relative content of 8-oxoG between different brain regions. Oxidation of guanine may play an important role in the development of age-associated decrease in memory and learning capability of OXYS rats. The findings are important for validation of the OXYS rat strain as a model of mammalian aging. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Genome-wide mapping of imprinted differentially methylated regions by DNA methylation profiling of human placentas from triploidies

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    Yuen Ryan KC

    2011-07-01

    Full Text Available Abstract Background Genomic imprinting is an important epigenetic process involved in regulating placental and foetal growth. Imprinted genes are typically associated with differentially methylated regions (DMRs whereby one of the two alleles is DNA methylated depending on the parent of origin. Identifying imprinted DMRs in humans is complicated by species- and tissue-specific differences in imprinting status and the presence of multiple regulatory regions associated with a particular gene, only some of which may be imprinted. In this study, we have taken advantage of the unbalanced parental genomic constitutions in triploidies to further characterize human DMRs associated with known imprinted genes and identify novel imprinted DMRs. Results By comparing the promoter methylation status of over 14,000 genes in human placentas from ten diandries (extra paternal haploid set and ten digynies (extra maternal haploid set and using 6 complete hydatidiform moles (paternal origin and ten chromosomally normal placentas for comparison, we identified 62 genes with apparently imprinted DMRs (false discovery rate FAM50B, as well as novel imprinted DMRs associated with known imprinted genes (for example, CDKN1C and RASGRF1 can be identified by using this approach. Furthermore, we have demonstrated how comparison of DNA methylation for known imprinted genes (for example, GNAS and CDKN1C between placentas of different gestations and other somatic tissues (brain, kidney, muscle and blood provides a detailed analysis of specific CpG sites associated with tissue-specific imprinting and gestational age-specific methylation. Conclusions DNA methylation profiling of triploidies in different tissues and developmental ages can be a powerful and effective way to map and characterize imprinted regions in the genome.

  5. Specific Variants in the MLH1 Gene Region May Drive DNA Methylation, Loss of Protein Expression, and MSI-H Colorectal Cancer

    OpenAIRE

    Mrkonjic, Miralem; Roslin, Nicole M.; Greenwood, Celia M.; Raptis, Stavroula; Pollett, Aaron; Laird, Peter W.; Pethe, Vaijayanti V.; Chiang, Theodore; Daftary, Darshana; Dicks, Elizabeth; Thibodeau, Stephen N.; Gallinger, Steven; Parfrey, Patrick S.; Younghusband, H. Banfield; Potter, John D.

    2010-01-01

    Background We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734) and microsatellite unstable (MSI-H) colorectal cancers (CRCs) in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and...

  6. Regions of common inter-individual DNA methylation differences in human monocytes: genetic basis and potential function.

    Science.gov (United States)

    Schröder, Christopher; Leitão, Elsa; Wallner, Stefan; Schmitz, Gerd; Klein-Hitpass, Ludger; Sinha, Anupam; Jöckel, Karl-Heinz; Heilmann-Heimbach, Stefanie; Hoffmann, Per; Nöthen, Markus M; Steffens, Michael; Ebert, Peter; Rahmann, Sven; Horsthemke, Bernhard

    2017-07-26

    There is increasing evidence for inter-individual methylation differences at CpG dinucleotides in the human genome, but the regional extent and function of these differences have not yet been studied in detail. For identifying regions of common methylation differences, we used whole genome bisulfite sequencing data of monocytes from five donors and a novel bioinformatic strategy. We identified 157 differentially methylated regions (DMRs) with four or more CpGs, almost none of which has been described before. The DMRs fall into different chromatin states, where methylation is inversely correlated with active, but not repressive histone marks. However, methylation is not correlated with the expression of associated genes. High-resolution single nucleotide polymorphism (SNP) genotyping of the five donors revealed evidence for a role of cis-acting genetic variation in establishing methylation patterns. To validate this finding in a larger cohort, we performed genome-wide association studies (GWAS) using SNP genotypes and 450k array methylation data from blood samples of 1128 individuals. Only 30/157 (19%) DMRs include at least one 450k CpG, which shows that these arrays miss a large proportion of DNA methylation variation. In most cases, the GWAS peak overlapped the CpG position, and these regions are enriched for CREB group, NF-1, Sp100 and CTCF binding motifs. In two cases, there was tentative evidence for a trans-effect by KRAB zinc finger proteins. Allele-specific DNA methylation occurs in discrete chromosomal regions and is driven by genetic variation in cis and trans, but in general has little effect on gene expression.

  7. Temporal transcription of the lactococcal temperate phage TP901-1 and DNA sequence of the early promoter region

    DEFF Research Database (Denmark)

    Madsen, Hans Peter Lynge; Hammer, Karin

    1998-01-01

    , of which at least two (the integrase gene and putative repressor) are needed for lysogeny, and the divergent and longer transcriptional unit from PL, presumably encoding functions required for the lytic life cycle. ORFs with homology to proteins involved in DNA replication were identified on the latter......Transcriptional analysis by Northern blotting identified clusters of early, middle and late transcribed regions of the temperate lactococcal bacteriophage TP901-1 during one-step growth experiments. The latent period was found to be 65 min and the burst size 40 +/- 10. The eight early transcripts...

  8. Z-DNA-forming sites identified by ChIP-Seq are associated with actively transcribed regions in the human genome.

    Science.gov (United States)

    Shin, So-I; Ham, Seokjin; Park, Jihwan; Seo, Seong Hye; Lim, Chae Hyun; Jeon, Hyeongrin; Huh, Jounghyun; Roh, Tae-Young

    2016-07-03

    Z-DNA, a left-handed double helical DNA is structurally different from the most abundant B-DNA. Z-DNA has been known to play a significant role in transcription and genome stability but the biological meaning and positions of Z-DNA-forming sites (ZFSs) in the human genome has not been fully explored. To obtain genome-wide map of ZFSs, Zaa with two Z-DNA-binding domains was used for ChIP-Seq analysis. A total of 391 ZFSs were found and their functions were examined in vivo A large portion of ZFSs was enriched in the promoter regions and contain sequences with high potential to form Z-DNA. Genes containing ZFSs were occupied by RNA polymerase II at the promoters and showed high levels of expression. Moreover, ZFSs were significantly related to active histone marks such as H3K4me3 and H3K9ac. The association of Z-DNA with active transcription was confirmed by the reporter assay system. Overall, our results suggest that Z-DNA formation depends on chromatin structure as well as sequence composition, and is associated with active transcription in human cells. The global information about ZFSs positioning will provide a useful resource for further understanding of DNA structure-dependent transcriptional regulation. © The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

  9. Newly Characterized Region of CP190 Associates with Microtubules and Mediates Proper Spindle Morphology in Drosophila Stem Cells.

    Directory of Open Access Journals (Sweden)

    Karen M Plevock

    Full Text Available CP190 is a large, multi-domain protein, first identified as a centrosome protein with oscillatory localization over the course of the cell cycle. During interphase it has a well-established role within the nucleus as a chromatin insulator. Upon nuclear envelope breakdown, there is a striking redistribution of CP190 to centrosomes and the mitotic spindle, in addition to the population at chromosomes. Here, we investigate CP190 in detail by performing domain analysis in cultured Drosophila S2 cells combined with protein structure determination by X-ray crystallography, in vitro biochemical characterization, and in vivo fixed and live imaging of cp190 mutant flies. Our analysis of CP190 identifies a novel N-terminal centrosome and microtubule (MT targeting region, sufficient for spindle localization. This region consists of a highly conserved BTB domain and a linker region that serves as the MT binding domain. We present the 2.5 Å resolution structure of the CP190 N-terminal 126 amino acids, which adopts a canonical BTB domain fold and exists as a stable dimer in solution. The ability of the linker region to robustly localize to MTs requires BTB domain-mediated dimerization. Deletion of the linker region using CRISPR significantly alters spindle morphology and leads to DNA segregation errors in the developing Drosophila brain neuroblasts. Collectively, we highlight a multivalent MT-binding architecture in CP190, which confers distinct subcellular cytoskeletal localization and function during mitosis.

  10. Utility of mtDNA-COI Barcode Region for Phylogenetic Relationship and Diagnosis of Five Common Pest Cockroaches

    Directory of Open Access Journals (Sweden)

    Saedeh Sadat Hashemi-Aghdam

    2017-06-01

    Full Text Available Background: Cockroaches are of vital importance medically and hygienically as they can disperse human patho­genic agents and are especially responsible for food contamination and spreading of food borne pathogens. In this study, part of mtDNA-COI gene of five common pest cockroaches was tested for diagnostic and phylogenetic pur­poses.Methods: We have described barcode region of mtDNA-COI gene of five cockroach species: Blattella germanica, Blatta orientalis, Periplaneta americana, Shelfordella lateralis, and Supella longipalpa, along with the development of a PCR-RFLP method for rapid detection and differentiation of these health pest species.Results: The PCR generates a single 710 bp-sized amplicon in all cockroach specimens, followed by direct se­quencing. AluI predicted from the sequencing data provided different RFLP profiles among five species. There was a significant intra-species variation within the American cockroach populations, but no genetic variation within other species. Accordingly, phylogenetic analysis demonstrates common monophyly for cockroach families in agreement with conventional taxonomy. However S. longipalpa (Ectobiidae diverged as an early ancestor of other cockroaches and was not associated with other Ectobiidae.Conclusion: The PCR-RFLP protocol might be useful when the conventional taxonomic methods are not able to identify specimens, particularly when only small body parts of specimens are available or they are in a decaying condition. mtDNA-COI gene shows potentially useful for studying phylogenetic relationships of Blattodea order.

  11. Utility of mtDNA-COI Barcode Region for Phylogenetic Relationship and Diagnosis of Five Common Pest Cockroaches.

    Science.gov (United States)

    Hashemi-Aghdam, Saedeh Sadat; Rafie, Golnaz; Akbari, Sanaz; Oshaghi, Mohammad Ali

    2017-06-01

    Cockroaches are of vital importance medically and hygienically as they can disperse human pathogenic agents and are especially responsible for food contamination and spreading of food borne pathogens. In this study, part of mtDNA-COI gene of five common pest cockroaches was tested for diagnostic and phylogenetic purposes. We have described barcode region of mtDNA-COI gene of five cockroach species: Blattella germanica, Blatta orientalis, Periplaneta americana, Shelfordella lateralis, and Supella longipalpa, along with the development of a PCR-RFLP method for rapid detection and differentiation of these health pest species. The PCR generates a single 710 bp-sized amplicon in all cockroach specimens, followed by direct sequencing. AluI predicted from the sequencing data provided different RFLP profiles among five species. There was a significant intra-species variation within the American cockroach populations, but no genetic variation within other species. Accordingly, phylogenetic analysis demonstrates common monophyly for cockroach families in agreement with conventional taxonomy. However S. longipalpa (Ectobiidae) diverged as an early ancestor of other cockroaches and was not associated with other Ectobiidae. The PCR-RFLP protocol might be useful when the conventional taxonomic methods are not able to identify specimens, particularly when only small body parts of specimens are available or they are in a decaying condition. mtDNA-COI gene shows potentially useful for studying phylogenetic relationships of Blattodea order.

  12. Force spectroscopy and fluorescence microscopy of dsDNA–YOYO-1 complexes: implications for the structure of dsDNA in the overstretching region

    Science.gov (United States)

    Murade, Chandrashekhar U.; Subramaniam, Vinod; Otto, Cees; Bennink, Martin L.

    2010-01-01

    When individual dsDNA molecules are stretched beyond their B-form contour length, they reveal a structural transition in which the molecule extends 1.7 times its contour length. The nature of this transition is still a subject of debate. In the first model, the DNA helix unwinds and combined with the tilting of the base pairs (which remain intact), results in a stretched form of DNA (also known as S-DNA). In the second model the base pairs break resulting effectively in two single-strands, which is referred to as force-induced melting. Here a combination of optical tweezers force spectroscopy with fluorescence microscopy was used to study the structure of dsDNA in the overstretching regime. When dsDNA was stretched in the presence of 10 nM YOYO-1 an initial increase in total fluorescence intensity of the dye–DNA complex was observed and at an extension where the dsDNA started to overstretch the fluorescence intensity leveled off and ultimately decreased when stretched further into the overstretching region. Simultaneous force spectroscopy and fluorescence polarization microscopy revealed that the orientation of dye molecules did not change significantly in the overstretching region (78.0°± 3.2°). These results presented here clearly suggest that, the structure of overstretched dsDNA can be explained accurately by force induced melting. PMID:20129944

  13. [Polymorphism of mitochondrial genome noncoding regions in the three Kazakh populations inhabited different areas of Kazakhstan and in the samples of DNA from ancient people of Kazakhstan Altai].

    Science.gov (United States)

    Aĭtkhozhina, N A; Dzisiuk, N V; Liudvikova, E K

    2004-01-01

    Polymorphism of major noncoding region of mitochondrial DNA (mtDNA D-loop, 528 bp in length) from the three modem kazakh populations and from DNA samples of ancient people inhabited modern Kazakhstani Altai were studied. PCR and RFLP analysis of 13 sites of restriction--BamHI, EcoRV, Sau3AI (1 restriction site), KpnI (2 sites), HaeIII (3 sites), RsaI (5 restriction sites), were carried out. The distribution of each site frequencies was determined. Nucleotide diversity (h) and genetic distance between different kazakh population and other populations of world were estimated. The same RFLP analysis of the mitochondrial DNA control region was carried out for the paleogenomic samples. It was shown that two samples of ancient mitochondrial DNA were monomorphous throughout all analyzed restriction sites.

  14. Characterization of human control region sequences of the African American SWGDAM forensic mtDNA data set.

    Science.gov (United States)

    Allard, Marc W; Polanskey, Deborah; Miller, Kevin; Wilson, Mark R; Monson, Keith L; Budowle, Bruce

    2005-03-10

    The scientific working group on DNA analysis Methods (SWGDAM) mitochondrial DNA (mtDNA) population data set is used to infer the relative rarity of control region mtDNA profiles obtained from evidence samples and of profiles used for identification of missing persons. In this study, the African American haplogroup patterns in the SWGDAM data were analyzed in a phylogenetic context to determine relevant single nucleotide polymorphisms (SNPs) and to describe haplogroup distributions for Africans observed in these data sets. Over 200 SNPs (n=217) were observed in the African American data set (n=1148). These SNPs ranged from having 1-39 changes in the phylogenetic tree, with sites 152 and 16519 being the most variable. On average there were 5.8 changes for a character on the tree. The most variable sites (with 19 or more changes each) observed included 16093, 16129, 16189, 16311, 16362, 16519, 146, 150, 152, 189, and 195. These rapidly changing sites are consistent with other published analyses. Only 34 SNPs are needed to identify all clusters containing 10 or more individuals in the African American data set. The results show that the African American SWGDAM mtDNA data set contains variation consistent with that described in continental African populations. Thirteen of the 18 haplogroups previously observed in African populations were observed and include: L1a, L1b, L1c, L2a, L2b, L2c, L3b, L3d, L3e1, L3e2, L3e3, L3e4 and L3f. Haplogroup L2a is the most commonly observed cluster (18.8%) in the African American data set. The next most common haplogroups in the African American data set include the clusters L1c (11.0%), L1b (9.1%), L3e2 (9.0%) and L3b (8.1%). Approximately 8% of the haplogroups observed within African Americans were common in European Caucasians or East Asians; these were H (n=32), J (n=4), K (n=5), T (n=2), U5 (n=6), U6 (n=9 also known from North Africa), A (n=12), B (n=7), C (n=4), and M (n=16), respectively. The European Caucasian and East Asian

  15. Hyper-variable regions in 18S rDNA of Strongyloides spp. as markers for species-specific diagnosis.

    Science.gov (United States)

    Hasegawa, Hideo; Hayashida, Shotaro; Ikeda, Yatsukaho; Sato, Hiroshi

    2009-03-01

    Four hyper-variable regions (HVR-I to -IV) found in 18S ribosomal DNA sequences were compared among 34 isolates of 15 species of the genus Strongyloides to evaluate their diagnostic value. HVR-I to -III were short, and plural species exhibit the same nucleotide arrangement. Meanwhile, HVR-IV had 23 to 39 nucleotides, showing species-specific arrangements, except Strongyloides ransomi and Strongyloides venezuelensis, which had the same nucleotide sequence in HVR-IV but were readily distinguished by the difference in HVR-I and -III. Isolates of Strongyloides stercoralis from humans of USA, Japan, and Philippines, chimpanzees, and dogs had an identical sequence in this region. Meanwhile, intraspecific polymorphism in HVR-IV nucleotide arrangement was observed among isolates of Strongyloides fuelleborni and Strongyloides callosciureus, presumably reflecting process of geographical dispersal and adaptation to the hosts.

  16. Hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA

    Energy Technology Data Exchange (ETDEWEB)

    Hood, E.E.; Helmer, G.L.; Fraley, R.T.; Chilton, M.D.

    1986-12-01

    A binary-vectory strategy was used to study the hypervirulence of Agrobacterium tumefaciens A281, an L,L-succinamopine strain. Strain A281 is hypervirulent on several solanaceous plants. Plasmids were constructed (pCS65 and pCS277) carrying either the transferred DNA (T-DNA) or the remainder of the tumor-inducing (Ti) plasmid (pEHA101) from this strain and tested each of these constructs were tested in trans with complementary each of regions from heterologous Ti plasmids. Hypervirulence on tobacco could be reconstructed in a bipartite strain with the L,L-succinamopine T-DNA and the vir region on separate plasmids. pEHA101 was able to complement octopine T-DNA to hypervirulence on tobacco and tomato plants. Nopaline T-DNA was complemented better on tomato plants by pEHA101 than it was by its own nopaline vir region, but not to hypervirulence. L,L-Succinamopine T-DNA could not be complemented to hypervirulence on tobacco and tomato plants with either heterologous vir region. From these results the authors suggest that the hypervirulence of strain A281 is due to non-T-DNA sequences on the Ti plasmid.

  17. Fine organization of genomic regions tagged to the 5S rDNA locus of the bread wheat 5B chromosome.

    Science.gov (United States)

    Sergeeva, Ekaterina M; Shcherban, Andrey B; Adonina, Irina G; Nesterov, Michail A; Beletsky, Alexey V; Rakitin, Andrey L; Mardanov, Andrey V; Ravin, Nikolai V; Salina, Elena A

    2017-11-14

    The multigene family encoding the 5S rRNA, one of the most important structurally-functional part of the large ribosomal subunit, is an obligate component of all eukaryotic genomes. 5S rDNA has long been a favored target for cytological and phylogenetic studies due to the inherent peculiarities of its structural organization, such as the tandem arrays of repetitive units and their high interspecific divergence. The complex polyploid nature of the genome of bread wheat, Triticum aestivum, and the technically difficult task of sequencing clusters of tandem repeats mean that the detailed organization of extended genomic regions containing 5S rRNA genes remains unclear. This is despite the recent progress made in wheat genomic sequencing. Using pyrosequencing of BAC clones, in this work we studied the organization of two distinct 5S rDNA-tagged regions of the 5BS chromosome of bread wheat. Three BAC-clones containing 5S rDNA were identified in the 5BS chromosome-specific BAC-library of Triticum aestivum. Using the results of pyrosequencing and assembling, we obtained six 5S rDNA- containing contigs with a total length of 140,417 bp, and two sets (pools) of individual 5S rDNA sequences belonging to separate, but closely located genomic regions on the 5BS chromosome. Both regions are characterized by the presence of approximately 70-80 copies of 5S rDNA, however, they are completely different in their structural organization. The first region contained highly diverged short-type 5S rDNA units that were disrupted by multiple insertions of transposable elements. The second region contained the more conserved long-type 5S rDNA, organized as a single tandem array. FISH using probes specific to both 5S rDNA unit types showed differences in the distribution and intensity of signals on the chromosomes of polyploid wheat species and their diploid progenitors. A detailed structural organization of two closely located 5S rDNA-tagged genomic regions on the 5BS chromosome of bread

  18. The Application of DNA Barcodes for the Identification of Marine Crustaceans from the North Sea and Adjacent Regions.

    Directory of Open Access Journals (Sweden)

    Michael J Raupach

    Full Text Available During the last years DNA barcoding has become a popular method of choice for molecular specimen identification. Here we present a comprehensive DNA barcode library of various crustacean taxa found in the North Sea, one of the most extensively studied marine regions of the world. Our data set includes 1,332 barcodes covering 205 species, including taxa of the Amphipoda, Copepoda, Decapoda, Isopoda, Thecostraca, and others. This dataset represents the most extensive DNA barcode library of the Crustacea in terms of species number to date. By using the Barcode of Life Data Systems (BOLD, unique BINs were identified for 198 (96.6% of the analyzed species. Six species were characterized by two BINs (2.9%, and three BINs were found for the amphipod species Gammarus salinus Spooner, 1947 (0.4%. Intraspecific distances with values higher than 2.2% were revealed for 13 species (6.3%. Exceptionally high distances of up to 14.87% between two distinct but monophyletic clusters were found for the parasitic copepod Caligus elongatus Nordmann, 1832, supporting the results of previous studies that indicated the existence of an overlooked sea louse species. In contrast to these high distances, haplotype-sharing was observed for two decapod spider crab species, Macropodia parva Van Noort & Adema, 1985 and Macropodia rostrata (Linnaeus, 1761, underlining the need for a taxonomic revision of both species. Summarizing the results, our study confirms the application of DNA barcodes as highly effective identification system for the analyzed marine crustaceans of the North Sea and represents an important milestone for modern biodiversity assessment studies using barcode sequences.

  19. Extensive Pyrosequencing Reveals Frequent Intra-Genomic Variations of Internal Transcribed Spacer Regions of Nuclear Ribosomal DNA

    Science.gov (United States)

    Li, Dezhu; Sun, Yongzhen; Niu, Yunyun; Chen, Zhiduan; Luo, Hongmei; Pang, Xiaohui; Sun, Zhiying; Liu, Chang; Lv, Aiping; Deng, Youping; Larson-Rabin, Zachary; Wilkinson, Mike; Chen, Shilin

    2012-01-01

    Background Internal transcribed spacer of nuclear ribosomal DNA (nrDNA) is already one of the most popular phylogenetic and DNA barcoding markers. However, the existence of its multiple copies has complicated such usage and a detailed characterization of intra-genomic variations is critical to address such concerns. Methodology/Principal Findings In this study, we used sequence-tagged pyrosequencing and genome-wide analyses to characterize intra-genomic variations of internal transcribed spacer 2 (ITS2) regions from 178 plant species. We discovered that mutation of ITS2 is frequent, with a mean of 35 variants per species. And on average, three of the most abundant variants make up 91% of all ITS2 copies. Moreover, we found different congeneric species share identical variants in 13 genera. Interestingly, different species across different genera also share identical variants. In particular, one minor variant of ITS2 in Eleutherococcus giraldii was found identical to the ITS2 major variant of Panax ginseng, both from Araliaceae family. In addition, DNA barcoding gap analysis showed that the intra-genomic distances were markedly smaller than those of the intra-specific or inter-specific variants. When each of 5543 variants were examined for its species discrimination efficiency, a 97% success rate was obtained at the species level. Conclusions Identification of identical ITS2 variants across intra-generic or inter-generic species revealed complex species evolutionary history, possibly, horizontal gene transfer and ancestral hybridization. Although intra-genomic multiple variants are frequently found within each genome, the usage of the major variants alone is sufficient for phylogeny construction and species determination in most cases. Furthermore, the inclusion of minor variants further improves the resolution of species identification. PMID:22952830

  20. Hydrolysis of RNA/DNA hybrids containing nonpolar pyrimidine isosteres defines regions essential for HIV type 1 polypurine tract selection.

    Science.gov (United States)

    Rausch, Jason W; Qu, Jin; Yi-Brunozzi, Hye Young; Kool, Eric T; Le Grice, Stuart F J

    2003-09-30

    Both x-ray crystallography and chemical footprinting indicate that bases of the HIV type 1 (HIV-1) polypurine tract (PPT)-containing RNA/DNA hybrid deviate from standard Watson-Crick base pairing. However, the contribution of these structural anomalies to the accuracy of plus-strand primer selection by HIV-1 reverse transcriptase is not immediately clear. To address this issue, DNA templates harboring single and pairwise non-hydrogen-bonding isosteres of cytosine (2-fluoro-4-methylbenzene deoxyribonucleoside) and thymine (2,4-difluoro-5-methylbenzene deoxyribonucleoside) were synthesized and hybridized to PPT-containing RNA primers as a means of locally removing hydrogen bonding and destabilizing paired structure. Cleavage of these hybrids was examined with p66/p51 HIV-1 reverse transcriptase and a mutant carrying an alteration in the p66 RNase H primer shown to specifically impair PPT processing. Analog insertion within the PPT (rG):(dC) and central (rA):(dT) tracts repositioned the RNase H domain such that the RNA/DNA hybrid was cleaved 3-4 bp from the site of insertion, a distance corresponding closely to the spatial separation between the catalytic center and RNase H primer grip. However, PPT processing was significantly impaired when the junction between these tracts was substituted. Substitutions within the upstream (rA):(dT) tract, where maximum distortion had previously been observed, destroyed PPT processing. Collectively, our scanning mutagenesis approach implicates multiple regions of the PPT in the accuracy with which it is excised from (+) U3 RNA and DNA, and also provides evidence for close cooperation between the RNase H primer grip and catalytic center in achieving this cleavage.

  1. Regional Variation in mtDNA of the Lesser Prairie-Chicken

    Science.gov (United States)

    Hagen, Christian A.; Pitman, James C.; Sandercock, Brett K.; Wolfe, Don H.; Robel, Robel J.; Applegate, Roger D.; Oyler-McCance, Sara J.

    2010-01-01

    Cumulative loss of habitat and long-term decline in the populations of the Lesser Prairie-Chicken (Tympanuchus pallidicinctus) have led to concerns for the species' viability throughout its range in the southern Great Plains. For more efficient conservation past and present distributions of genetic variation need to be understood. We examined the distribution of mitochondrial DNA (mtDNA) variation in the Lesser Prairie-Chicken across Kansas, Colorado, Oklahoma, and New Mexico. Throughout the range we found little genetic differentiation except for the population in New Mexico, which was significantly different from most other publications. We did, however, find significant isolation by distance at the rangewide scale (r=0.698). We found no relationship between haplotype phylogeny and geography, and our analyses provide evidence for a post-glacial population expansion within the species that is consistent with the idea that speciation within Tympanuchus is recent. Conservation actions that increase the likelihood of genetically viable populations in the future should be evaluated for implementation.

  2. Retrotransposons are specified as DNA replication origins in the gene-poor regions of Arabidopsis heterochromatin.

    Science.gov (United States)

    Vergara, Zaida; Sequeira-Mendes, Joana; Morata, Jordi; Peiró, Ramón; Hénaff, Elizabeth; Costas, Celina; Casacuberta, Josep M; Gutierrez, Crisanto

    2017-08-21

    Genomic stability depends on faithful genome replication. This is achieved by the concerted activity of thousands of DNA replication origins (ORIs) scattered throughout the genome. The DNA and chromatin features determining ORI specification are not presently known. We have generated a high-resolution genome-wide map of 3230 ORIs in cultured Arabidopsis thaliana cells. Here, we focused on defining the features associated with ORIs in heterochromatin. In pericentromeric gene-poor domains ORIs associate almost exclusively with the retrotransposon class of transposable elements (TEs), in particular of the Gypsy family. ORI activity in retrotransposons occurs independently of TE expression and while maintaining high levels of H3K9me2 and H3K27me1, typical marks of repressed heterochromatin. ORI-TEs largely colocalize with chromatin signatures defining GC-rich heterochromatin. Importantly, TEs with active ORIs contain a local GC content higher than the TEs lacking them. Our results lead us to conclude that ORI colocalization with retrotransposons is determined by their transposition mechanism based on transcription, and a specific chromatin landscape. Our detailed analysis of ORIs responsible for heterochromatin replication has implications on the mechanisms of ORI specification in other multicellular organisms in which retrotransposons are major components of heterochromatin and of the entire genome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Discrimination of juvenile yellowfin (Thunnus albacares and bigeye (T. obesus Tunas using mitochondrial DNA control region and liver morphology.

    Directory of Open Access Journals (Sweden)

    Ivane R Pedrosa-Gerasmio

    Full Text Available Yellowfin tuna, Thunnus albacares (Bonnaterre, 1788 and bigeye tuna, Thunnus obesus (Lowe, 1839 are two of the most economically important tuna species in the world. However, identification of their juveniles, especially at sizes less than 40 cm, is very difficult, often leading to misidentification and miscalculation of their catch estimates. Here, we applied the mitochondrial DNA control region D-loop, a recently validated genetic marker used for identifying tuna species (Genus Thunnus, to discriminate juvenile tunas caught by purse seine and ringnet sets around fish aggregating devices (FADs off the Southern Iloilo Peninsula in Central Philippines. We checked individual identifications using the Neighbor-Joining Method and compared results with morphometric analyses and the liver phenotype. We tested 48 specimens ranging from 13 to 31 cm fork length. Morpho-meristic analyses suggested that 12 specimens (25% were bigeye tuna and 36 specimens (75% were yellowfin tuna. In contrast, the genetic and liver analyses both showed that 5 specimens (10% were bigeye tuna and 43 (90% yellowfin tuna. This suggests that misidentification can occur even with highly stringent morpho-meristic characters and that the mtDNA control region and liver phenotype are excellent markers to discriminate juveniles of yellowfin and bigeye tunas.

  4. High-quality mtDNA control region sequences from 680 individuals sampled across the Netherlands to establish a national forensic mtDNA reference database

    NARCIS (Netherlands)

    L.C. Chaitanya (Lakshmi); M. van Oven (Mannis); S. Brauer (Silke); B. Zimmermann (Bettina); G. Huber (Gabriela); C. Xavier (Catarina); W. Parson (Walther); P. de Knijff (Peter); M.H. Kayser (Manfred)

    2016-01-01

    textabstractThe use of mitochondrial DNA (mtDNA) for maternal lineage identification often marks the last resort when investigating forensic and missing-person cases involving highly degraded biological materials. As with all comparative DNA testing, a match between evidence and reference sample

  5. Sequence Analysis of the Ribosomal DNA Intergenic Spacer 1 Regions of Trichosporon Species

    Science.gov (United States)

    Sugita, Takashi; Nakajima, Masamitsu; Ikeda, Reiko; Matsushima, Toshiharu; Shinoda, Takako

    2002-01-01

    We determined the sequence of the intergenic spacer (IGS) 1 region, which is located between the 26S and 5S rRNA genes, in 25 species of the genus Trichosporon. IGS 1 sequences varied in length from 195 to 719 bp. Comparative sequence analysis suggested that the divergence of IGS 1 sequences has been greater than that of the internal transcribed spacer regions. We also identified five genotypes of T. asahii, which is a major causative agent of deep-seated trichosporonosis, based on the IGS 1 sequences of 43 strains. Most of the isolates that originated in Japan were of genotype 1, whereas the American isolates were of genotype 3 or 5. Our results suggest that analysis of IGS regions provides a powerful method to distinguish between phylogenetically closely related species and that a geographic substructure may exist among T. asahii clinical isolates. PMID:11980969

  6. Species diversity of planktonic gastropods (Pteropoda and Heteropoda) from six ocean regions based on DNA barcode analysis

    Science.gov (United States)

    Jennings, Robert M.; Bucklin, Ann; Ossenbrügger, Holger; Hopcroft, Russell R.

    2010-12-01

    Pteropods and heteropods are two distinct groups of holoplanktonic gastropods whose species and genetic diversity remain poorly understood, despite their ubiquity in the world's oceans. Some species apparently attain near cosmopolitan distributions, implying long-distance dispersal or cryptic species assemblages. We present the first multi-regional and species-rich molecular dataset of holoplanktonic gastropods, comprising DNA barcodes from the mitochondrial cytochrome c oxidase I subunit gene (COI) from 115 individuals of 41 species sampled from six ocean regions across the globe. Molecular analysis and assessment of barcoding utility supported the validity of several morphological subspecies and forms (e.g. of Creseis virgula and Limacina helicina), while others were not supported (e.g. Cavolinia uncinata). Significant genetic variation was observed among conspecific specimens collected in different geographic regions for some species, particularly in euthecosomatous pteropods. Several species of euthecosomes showed no evidence of genetic separation among distant ocean regions. Overall, we suggest some taxonomic revision of the holoplanktonic gastropods will be required, pending a more complete molecular inventory of these groups.

  7. DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region.

    OpenAIRE

    Hediger, M A; Johnson, D F; Nierlich, D P; Zabin, I

    1985-01-01

    The lac operon of Escherichia coli spans approximately 5300 base pairs and includes the lacZ, lacY, and lacA genes in addition to the operator, promoter, and transcription termination regions. We report here the sequence of the lacA gene and the region distal to it, confirming the sequence of thiogalactoside transacetylase and completing the sequence of the lac operon. The lacA gene is characterized by use of rare codons, suggesting an origin from a plasmid, transposon, or virus gene. UUG is ...

  8. Promoter activity associated with the intergenic regions of banana bunchy top virus DNA-1 to -6 in transgenic tobacco and banana cells.

    Science.gov (United States)

    Dugdale, B; Beetham, P R; Becker, D K; Harding, R M; Dale, J L

    1998-10-01

    Promoter regions associated with each of the six ssDNA components of banana bunchy top virus (BBTV) have been characterized. DNA segments incorporating the intergenic regions of BBTV DNA-1 to -6 were isolated and fused to the uidA (beta-glucuronidase) reporter gene to assess promoter activity. In tobacco cell suspensions, the BBTV DNA-2 and -6 promoters generated levels of GUS expression 2-fold greater and similar to the 800 bp CaMV 35S promoter, respectively. Deletion analysis of the BBTV DNA-6 promoter suggested all the necessary promoter elements required for strong expression were located within 239 nucleotides upstream of the translational start codon. In transgenic tobacco plants, the BBTV-derived promoters generally provided a weak, tissue-specific GUS expression pattern restricted to phloem-associated cells. However, in callus derived from tobacco leaf tissue, GUS expression directed by the BBTV DNA-6 promoter was strong and, in some lines, comparable to the CaMV 35S promoter. Detectable promoter activity associated with the BBTV promoters in banana embryogenic cells was only observed using a sensitive green fluorescent protein (GFP) reporter. Promoters derived from BBTV DNA-4 and -5 generated the highest levels of transient activity, which were greater than that of the maize ubi-1 promoter. In transgenic banana plants, the activity of the BBTV DNA-6 promoter was restricted to the phloem of leaves and roots, stomata and root meristems.

  9. Genetic variability within n-rDNA region of ectomycorrhizal isolates ...

    African Journals Online (AJOL)

    The region was first amplified by polymerase chain reaction with specific primers and then cleaved with different restriction enzymes. The degree of polymorphism, although extensive, proved inadequate for proper identification of most of the isolates. Depending on the restriction enzymes used, the genera or species could ...

  10. Indices of methylation in sperm DNA from fertile men differ between distinct geographical regions

    DEFF Research Database (Denmark)

    Consales, C; Leter, G; Bonde, J P E

    2014-01-01

    sequences from peripheral blood lymphocyte, can vary with age, gender, alcohol consumption and white blood cell counts. STUDY DESIGN, SIZE, DURATION: A cross-sectional study. Individual data were collected from 269 young healthy men of proven fertility living in three geographical regions: Inuits from...

  11. Interaction of a nodule specific, trans-acting factor with distinct DNA elements in the soybean leghaemoglobin Ibc(3) 5' upstream region

    DEFF Research Database (Denmark)

    Jensen, Erik Østergaard; Marcker, Kjeld A; Schell, J

    1988-01-01

    Nuclear extracts from soybean nodules, leaves and roots were used to investigate protein-DNA interactions in the 5' upstream (promoter) region of the soybean leghaemoglobin lbc(3) gene. Two distinct regions were identified which strongly bind a nodule specific factor. A Bal31 deletion analysis......, but with different affinities. Elements 1 and 2 share a common motif, although their AT-rich DNA sequences differ. Element 2 is highly conserved at an analogous position in other soybean lb gene 5' upstream regions. Udgivelsesdato: 1988-May...

  12. Striatal astrocytes produce neuroblasts in an excitotoxic model of Huntington's disease.

    Science.gov (United States)

    Nato, Giulia; Caramello, Alessia; Trova, Sara; Avataneo, Valeria; Rolando, Chiara; Taylor, Verdon; Buffo, Annalisa; Peretto, Paolo; Luzzati, Federico

    2015-03-01

    In the adult brain, subsets of astrocytic cells residing in well-defined neurogenic niches constitutively generate neurons throughout life. Brain lesions can stimulate neurogenesis in otherwise non-neurogenic regions, but whether local astrocytic cells generate neurons in these conditions is unresolved. Here, through genetic and viral lineage tracing in mice, we demonstrate that striatal astrocytes become neurogenic following an acute excitotoxic lesion. Similar to astrocytes of adult germinal niches, these activated parenchymal progenitors express nestin and generate neurons through the formation of transit amplifying progenitors. These results shed new light on the neurogenic potential of the adult brain parenchyma. © 2015. Published by The Company of Biologists Ltd.

  13. 5-Bromo-2’-deoxyuridine induces visible morphological alteration in the DNA puffs of the anterior salivary gland region of Bradysia hygida (Diptera, Sciaridae

    Directory of Open Access Journals (Sweden)

    J.C. de Almeida

    2010-12-01

    Full Text Available 5-Bromo-2’-deoxyuridine (BrdUrd has long been known to interfere with cell differentiation. We found that treatment ofBradysia hygida larvae with BrdUrd during DNA puff anlage formation in the polytene chromosomes of the salivary gland S1 region noticeably affects anlage morphology. However, it does not affect subsequent metamorphosis to the adult stage. The chromatin of the chromosomal sites that would normally form DNA puffs remains very compact and DNA puff expansion does not occur with administration of 4 to 8 mM BrdUrd. Injection of BrdUrd at different ages provoked a gradient of compaction of the DNA puff chromatin, leading to the formation of very small to almost normal puffs. By immunodetection, we show that the analogue is preferentially incorporated into the DNA puff anlages. When BrdUrd is injected in a mixture with thymidine, it is not incorporated into the DNA, and normal DNA puffs form. Therefore, incorporation of this analogue into the amplified DNA seems to be the cause of this extreme compaction. Autoradiographic experiments and silver grains counting showed that this treatment decreases the efficiency of RNA synthesis at DNA puff anlages.

  14. GMDH-GA Hybrid Model Extracting Exon Region from DNA Sequences

    OpenAIRE

    Ohta, Kouji; Yoshihara, Ikuo; Yamamori, Kunihito; Yasunaga, Moritoshi

    2004-01-01

    Abstract ###A model building method based on Group Method of Data Handling (GMDH) optimized by ###GA is developed for extracting exon regions. GMDH, that is originally a method to construct ###higher order polynomial model, is extended to constructing higher order logical model. ###The model built by proposed method is compared with Genetic Programming (GP)-based ###model as to the extraction rate of best, worst and average. The proposed method is superior to GP ###as to extraction rate of al...

  15. Comparison of DNA walking methods for isolation of transgene-flanking regions in GM potato.

    Science.gov (United States)

    Cullen, Danny; Harwood, Wendy; Smedley, Mark; Davies, Howard; Taylor, Mark

    2011-09-01

    An important aspect in the safety assessment of transgenic plants is the exact location of transgene insertion sites within the host genome. However, robust standard operating procedures are not currently available. Using potato as a test species, different methodologies for the determination of insertion sites using a range of published protocols and commercially available kits were assessed in transgenic lines of varying degrees of complexity, from low copy number to complex re-transformed and co-transformed lines. Three commercial kits, APAgene™ GOLD Genome Walking Kit (BIO S&T), DNA Walking SpeedUp™ Kit II (Seegene), and Universal Vectorette™ System (Sigma) were compared with an adaptor-mediated PCR technique. Overall, the APAgene™ kit was used with a high success rate with low copy number potato lines, and also more complex co- and re-transformed lines, and adhering to key confirmation steps it was possible to obtain flanking sequence ranging in size from 300 to 2,500 bp and eliminate PCR artefacts from the analysis.

  16. DNA sequence of the lactose operon: the lacA gene and the transcriptional termination region.

    Science.gov (United States)

    Hediger, M A; Johnson, D F; Nierlich, D P; Zabin, I

    1985-10-01

    The lac operon of Escherichia coli spans approximately 5300 base pairs and includes the lacZ, lacY, and lacA genes in addition to the operator, promoter, and transcription termination regions. We report here the sequence of the lacA gene and the region distal to it, confirming the sequence of thiogalactoside transacetylase and completing the sequence of the lac operon. The lacA gene is characterized by use of rare codons, suggesting an origin from a plasmid, transposon, or virus gene. UUG is the translation initiation codon. A preliminary examination of 3' end of the lac messenger in the region distal to the lacA gene indicates several endpoints. A predominant one is located at the 3' end of a G + C-rich hairpin structure, which may be involved in termination of transcription or in post-transcriptional processing. An open reading frame of 702 base pairs is present on the complementary strand downstream from lacA.

  17. Identification and characterization of the regions involved in the nuclear translocation of the heterodimeric leishmanial DNA topoisomerase IB.

    Directory of Open Access Journals (Sweden)

    Christopher F Prada

    Full Text Available Leishmania donovani, the causative organism for visceral leishmaniasis, contains a unique heterodimeric DNA-topoisomerase IB (LdTopIB. LdTopIB is a heterodimer made up of a large subunit and a small subunit that must interact with each other to build an active enzyme able to solve the topological tensions on the DNA. As LdTopIB is located within the nucleus, one or more nuclear localization signals (NLS should exist to ensure its nuclear translocation. In this report three novel NLS have been identified through a sequential deletion study of the genes encoding of both subunits fused to that encoding the green fluorescent protein (GFP. NLS1 is a highly basic sequence of 43 amino acids in the C-terminal extension of the large protomer. We found two well-defined sequences in the small protomer: NLS2 is a 10-amino acid motif located in the N-terminal extension of the protein; NLS3 consists of a complex region of 28 amino acids placed in the vicinity of the catalytic Tyr-222 included at the conserved SKINY signature within the C-terminal. Furthermore, by means of yeast cell viability assays, conducted with several LdTopIB chimeras lacking any of the NLS motives, we have revealed that both subunits are transported independently to the nucleus. There was no evidence of LdTopIB accumulation in mitochondria or association to the kinetoplast DNA network. The results rule out the former hypothesis, which attributes nucleocytoplasmic transport of LdTopIB entirely to the large subunit. The LdTopIB is localized to the nucleus only.

  18. Structural features of DNA are conserved in the promoter region of orthologous genes across different strains of Helicobacter pylori.

    Science.gov (United States)

    Kumar, Aditya; Manivelan, Vasumathi; Bansal, Manju

    2016-09-01

    Promoter regions play a key role in the process of transcription initiation and gene expression, hence promoter identification is an inherent component of the genome annotation process. Identification and characterization of promoters in fully sequenced genomes is a challenging and complex task. An analysis of sequence-dependent DNA structural properties in the promoter region of orthologous and non-orthologous genes can help in characterizing promoters and also provide insights into transcription initiation. Various structural properties, such as duplex stability, protein-induced bendability and intrinsic curvature of promoter sequences have been calculated and compared for 10 different strains of Helicobacter pylori genomes, and it is found that promoter regions in orthologous and non-orthologous genes show distinct trends for these properties, with orthologous genes showing sharper low-stability peak, lower bendability and higher curvature. The average GC content of orthologous genes is higher than that of non-orthologous genes, and relative stability-based promoter annotation tool PromPredict performs better for orthologous genes than non-orthologous genes. The characteristic sequence-dependent structural properties of promoters show significant differences between orthologous and non-orthologous genes. Interestingly, these structural properties of promoters are conserved, but the genes themselves vary in their evolutionary selection rate. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  19. Processing by MRE11 is involved in the sensitivity of subtelomeric regions to DNA double-strand breaks.

    Science.gov (United States)

    Muraki, Keiko; Han, Limei; Miller, Douglas; Murnane, John P

    2015-09-18

    The caps on the ends of chromosomes, called telomeres, keep the ends of chromosomes from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion. However, subtelomeric regions are sensitive to DSBs, which in normal cells is responsible for ionizing radiation-induced cell senescence and protection against oncogene-induced replication stress, but promotes chromosome instability in cancer cells that lack cell cycle checkpoints. We have previously reported that I-SceI endonuclease-induced DSBs near telomeres in a human cancer cell line are much more likely to generate large deletions and gross chromosome rearrangements (GCRs) than interstitial DSBs, but found no difference in the frequency of I-SceI-induced small deletions at interstitial and subtelomeric DSBs. We now show that inhibition of MRE11 3'-5' exonuclease activity with Mirin reduces the frequency of large deletions and GCRs at both interstitial and subtelomeric DSBs, but has little effect on the frequency of small deletions. We conclude that large deletions and GCRs are due to excessive processing of DSBs, while most small deletions occur during classical nonhomologous end joining (C-NHEJ). The sensitivity of subtelomeric regions to DSBs is therefore because they are prone to undergo excessive processing, and not because of a deficiency in C-NHEJ in subtelomeric regions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  20. cDNA sequence, genomic organization, and evolutionary conservation of a novel gene from the WAGR region

    Energy Technology Data Exchange (ETDEWEB)

    Schwartz, F.; Eisenman, R.; Knoll, J.; Bruns, G. [Children`s Hospital and Department of Pediatrics, Boston, MA (United States)

    1995-09-20

    A new gene (239FB) with predominant and differential expression in fetal brain has recently been isolated from a chromosome 11p13-p14 boundary area near FSHB. The corresponding mRNA has an open reading frame of 294 amino acids, a 3` untranslated region of 1247 nucleotides, and a highly GC-rich 5` untranslated region. The coding and 3` UT sequence is specified by 6 exons within nearly 87 kb of isolated genomic locus. The 5` end region of the transcript maps adjacent to the only genomically defined CpG island in a chromosomal subregion that may be associated with part of the mental retardation of some WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome patients. In addition to nucleotide and amino acid similarity to an EST from a normalized infant brain cDNA library, the predicted protein has extensive similarity to Caenorhbditis elegans polypeptides of, as yet, unknown function. The 239FB locus is, therefore, likely part of a family of genes with two members expressed in human brain. The extensive conservation of the predicted protein suggests a fundamental function of the gene product and will enable evaluation of the role of the 239FB gene in neurogenesis in model organisms. 48 refs., 4 figs., 1 tab.

  1. Phylogenetic analysis of encapsulated and non-encapsulated Trichinella species by studying the 5S rDNA tandemly repeated intergenic region.

    Science.gov (United States)

    van der Giessen, J W B; Fonville, M; Briels, I; Pozio, E

    2005-09-05

    The identification of sequence regions in the genomes of pathogens which can be useful to distinguish among species and genotypes, is of great importance for epidemiological, molecular, and phylogenetic studies. The 5S ribosomal DNA intergenic spacer region has been identified as a good target to distinguish among eight Trichinella species and genotypes. The recent discovery of two non-encapsulated species in this genus, Trichinella papuae and Trichinella zimbabwensis, which can infect both mammals and reptiles, has suggested analyzing their 5S rDNA. Amplification of the tandem repeats of the 5S rDNA intergenic region of encapsulated species of Trichinella shows a 751bp fragment, whereas the three non-encapsulated species show a fragment of 800bp with T. pseudospiralis showing an additional fragment of 522bp. Although the size of the 800bp PCR fragments of T. papuae and T. zimbabwensis are similar to that of T. pseudospiralis, there are differences in the 5S rDNA intergenic regions among the three non-encapsulated species. Phylogenetic analysis of the 5S rDNA intergenic regions shows a clustering together of the three non-encapsulated Trichinella species that is well separated from the encapsulated ones. In addition, a single PCR-based method allows distinguishing non-encapsulated and encapsulated species.

  2. The comparison of rDNA spacer regions of Nosema ceranae isolates from different hosts and locations.

    Science.gov (United States)

    Huang, Wei-Fone; Bocquet, Michel; Lee, Ker-Chang; Sung, I-Hsin; Jiang, Jing-Hao; Chen, Yue-Wen; Wang, Chung-Hsiung

    2008-01-01

    Nosema ceranae is a common microsporidian pathogen, one of two Nosema species that cause "nosema disease" in honeybees, Apis cerana and Apis mellifera. Samples of N. ceranae rDNA from isolates collected in different locations were sequenced and one 5S rRNA was found to be upstream of SSUrRNA. The rDNA arrangement, 5'-5S rRNA-IGS-SSUrRNA-ITS-LSUrRNA-3', was found in all isolates. In order to better understand the distribution relationship between N. ceranae isolates from A. cerana and A. mellifera, their rRNA spacer regions were also sequenced for analysis. Results showed that there are no significant differences between the IGS sequences of the isolates and no difference in the ITS sequence with the exception of one transition found in an isolate from Martinique. These isolates showed consistency in the IGS phylogenic analysis suggesting that no transmission barrier exists between A. mellifera and A. cerana and there is no difference between isolates from geography separated areas.

  3. Identification and verification of hybridoma-derived monoclonal antibody variable region sequences using recombinant DNA technology and mass spectrometry.

    Science.gov (United States)

    Babrak, Lmar; McGarvey, Jeffery A; Stanker, Larry H; Hnasko, Robert

    2017-10-01

    Antibody engineering requires the identification of antigen binding domains or variable regions (VR) unique to each antibody. It is the VR that define the unique antigen binding properties and proper sequence identification is essential for functional evaluation and performance of recombinant antibodies (rAb). This determination can be achieved by sequence analysis of immunoglobulin (Ig) transcripts obtained from a monoclonal antibody (MAb) producing hybridoma and subsequent expression of a rAb. However the polyploidy nature of a hybridoma cell often results in the added expression of aberrant immunoglobulin-like transcripts or even production of anomalous antibodies which can confound production of rAb. An incorrect VR sequence will result in a non-functional rAb and de novo assembly of Ig primary structure without a sequence map is challenging. To address these problems, we have developed a methodology which combines: 1) selective PCR amplification of VR from both the heavy and light chain IgG from hybridoma, 2) molecular cloning and DNA sequence analysis and 3) tandem mass spectrometry (MS/MS) on enzyme digests obtained from the purified IgG. Peptide analysis proceeds by evaluating coverage of the predicted primary protein sequence provided by the initial DNA maps for the VR. This methodology serves to both identify and verify the primary structure of the MAb VR for production as rAb. Published by Elsevier Ltd.

  4. Phylogeny of the basal angiosperm genus Pseuduvaria (Annonaceae) inferred from five chloroplast DNA regions, with interpretation of morphological character evolution.

    Science.gov (United States)

    Su, Yvonne C F; Smith, Gavin J D; Saunders, Richard M K

    2008-07-01

    Phylogenetic relationships within the magnoliid basal angiosperm genus Pseuduvaria (Annonaceae) are investigated using chloroplast DNA sequences from five regions: psbA-trnH spacer, trnL-F, matK, rbcL, and atpB-rbcL spacer. Over 4000 nucleotides from 51 species (of the total 53) were sequenced. The five cpDNA datasets were analyzed separately and in combination using maximum parsimony (MP), maximum likelihood (ML), and Bayesian methods. The phylogenetic trees constructed using all three phylogenetic methods, based on the combined data, strongly support the monophyly of Pseuduvaria following the inclusion of Craibella phuyensis. The trees generated using MP were less well resolved, but relationships are similar to those obtained using the other methods. ML and Bayesian analyses recovered trees with short branch lengths, showing five main clades. This study highlights the evolutionary changes in seven selected morphological characters (floral sex, stamen and carpel numbers, inner petal color, presence of inner petal glands, flowering peduncle length, and monocarp size). Although floral unisexuality is ancestral within the genus, several evolutionary lineages reveal reversal to bisexuality. Other phylogenetic transitions include the evolution of sapromyophily, and fruit-bat frugivory and seed dispersal, thus allowing a wide range of adaptations for species survival.

  5. Phylogenetic relationships in Solanaceae and related species based on cpDNA sequence from plastid trnE-trnT region

    Directory of Open Access Journals (Sweden)

    Danila Montewka Melotto-Passarin

    2008-01-01

    Full Text Available Intergenic spacers of chloroplast DNA (cpDNA are very useful in phylogenetic and population genetic studiesof plant species, to study their potential integration in phylogenetic analysis. The non-coding trnE-trnT intergenic spacer ofcpDNA was analyzed to assess the nucleotide sequence polymorphism of 16 Solanaceae species and to estimate its ability tocontribute to the resolution of phylogenetic studies of this group. Multiple alignments of DNA sequences of trnE-trnT intergenicspacer made the identification of nucleotide variability in this region possible and the phylogeny was estimated by maximumparsimony and rooted with Convolvulaceae Ipomoea batatas, the most closely related family. Besides, this intergenic spacerwas tested for the phylogenetic ability to differentiate taxonomic levels. For this purpose, species from four other families wereanalyzed and compared with Solanaceae species. Results confirmed polymorphism in the trnE-trnT region at different taxonomiclevels.

  6. DNA Sequence Variants in the Five Prime Untranslated Region of the Cyclooxygenase-2 Gene Are Commonly Found in Healthy Dogs and Gray Wolves.

    Science.gov (United States)

    Safra, Noa; Hayward, Louisa J; Aguilar, Miriam; Sacks, Benjamin N; Westropp, Jodi L; Mohr, F Charles; Mellersh, Cathryn S; Bannasch, Danika L

    2015-01-01

    The aim of this study was to investigate the frequency of regional DNA variants upstream to the translation initiation site of the canine Cyclooxygenase-2 (Cox-2) gene in healthy dogs. Cox-2 plays a role in various disease conditions such as acute and chronic inflammation, osteoarthritis and malignancy. A role for Cox-2 DNA variants in genetic predisposition to canine renal dysplasia has been proposed and dog breeders have been encouraged to select against these DNA variants. We sequenced 272-422 bases in 152 dogs unaffected by renal dysplasia and found 19 different haplotypes including 11 genetic variants which had not been described previously. We genotyped 7 gray wolves to ascertain the wildtype variant and found that the wolves we analyzed had predominantly the second most common DNA variant found in dogs. Our results demonstrate an elevated level of regional polymorphism that appears to be a feature of healthy domesticated dogs.

  7. DNA Sequence Variants in the Five Prime Untranslated Region of the Cyclooxygenase-2 Gene Are Commonly Found in Healthy Dogs and Gray Wolves.

    Directory of Open Access Journals (Sweden)

    Noa Safra

    Full Text Available The aim of this study was to investigate the frequency of regional DNA variants upstream to the translation initiation site of the canine Cyclooxygenase-2 (Cox-2 gene in healthy dogs. Cox-2 plays a role in various disease conditions such as acute and chronic inflammation, osteoarthritis and malignancy. A role for Cox-2 DNA variants in genetic predisposition to canine renal dysplasia has been proposed and dog breeders have been encouraged to select against these DNA variants. We sequenced 272-422 bases in 152 dogs unaffected by renal dysplasia and found 19 different haplotypes including 11 genetic variants which had not been described previously. We genotyped 7 gray wolves to ascertain the wildtype variant and found that the wolves we analyzed had predominantly the second most common DNA variant found in dogs. Our results demonstrate an elevated level of regional polymorphism that appears to be a feature of healthy domesticated dogs.

  8. Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Pedro Carnieli Junior

    Full Text Available Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.

  9. Genetic variation in wild and hatchery population of Catla catla (Hamilton, 1822) analyzed through mtDNA cytochrome b region.

    Science.gov (United States)

    Behera, Bijay Kumar; Kunal, Swaraj Priyaranjan; Baisvar, Vishwamitra Singh; Meena, Dharmendra Kumar; Panda, Debarata; Pakrashi, Sudip; Paria, Prasenjit; Das, Pronob; Debnath, Dipesh; Parida, Pranaya Kumar; Das, Basanta Kumar; Jena, Joykrushna

    2018-01-01

    Catla (Catla catla) is a one of the most harvested Indian major carps and is widely cultured fish species in Indian subcontinent. In the present study, genetic variability between hatchery and wild stocks of Catla was surveyed using sequence data of mitochondrial DNA of partial 307 bp of cytochrome b region. A total of 174 Catla individuals were examined from three different river basins and hatcheries. Significant genetic heterogeneity was observed for the sequence data (FST = 0.308, p ≤ 0.001). However, analysis of molecular variance (AMOVA) resulted in insignificant genetic differentiation among the samples of three rivers and culture zones (FCT = -0.10, p = 0.44). The result suggested a significant genetic variation within different riverine system, low genetic differentiation among samples from river basins and a lack of genetic variation in hatchery populations.

  10. Molecular phylogeny of Acer monspessulanum L. subspecies from Iran inferred using the ITS region of nuclear ribosomal DNA

    Directory of Open Access Journals (Sweden)

    HANIF KHADEMI

    2016-04-01

    Full Text Available Abstract. Khademi H, Mehregan I, Assadi M, Nejadsatari T, Zarre S. 2015. Molecular phylogeny of Acer monspessulanum L. subspecies from Iran inferred using the ITS region of nuclear ribosomal DNA. Biodiversitas 17: 16-23. This study was carried out on the Acer monspessulanum complex growing wild in Iran. Internal transcribed spacer (ITS sequences for 75 samples representing five different subspecies of Acer monspessulanum were analyzed. Beside this, 86 previously published ITS sequences from GenBank were used to test the monophyly of the complex worldwide. Phylogenetic analyses were conducted using Bayesian inference and maximum parsimony. The results indicate that most samples of A. monspessulanum species from Iran were part of a monophyletic clade with 8 samples of A. ibericum from Georgia, A. hyrcanum from Iran and one of A. sempervirens from Greece (PP= 1; BS= 79%. Our results indicate that use of morphological characteristics coupled with molecular data will be most effective.

  11. 45S rDNA regions are chromosome fragile sites expressed as gaps in vitro on metaphase chromosomes of root-tip meristematic cells in Lolium spp.

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    Jing Huang

    Full Text Available BACKGROUND: In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported. METHODS AND RESULTS: During the course of cytological mapping of rDNA on ryegrass chromosomes, we found that the number of chromosomes plus chromosome fragments was often more than the expected 14 in most cells for Lolium perenne L. cv. Player by close cytological examination using a routine chromosome preparation procedure. Further fluorescent in situ hybridization (FISH using 45S rDNA as a probe indicated that the root-tip cells having more than a 14-chromosome plus chromosome fragment count were a result of chromosome breakage or gap formation in vitro (referred to as chromosome lesions at 45S rDNA sites, and 86% of the cells exhibited chromosome breaks or gaps and all occurred at the sites of 45S rDNA in Lolium perenne L. cv. Player, as well as in L. multiflorum Lam. cv. Top One. Chromatin depletion or decondensation occurred at various locations within the 45S rDNA regions, suggesting heterogeneity of lesions of 45S rDNA sites with respect to their position within the rDNA region. CONCLUSIONS: The chromosome lesions observed in this study are very similar cytologically to that of fragile sites observed in human chromosomes, and thus we conclude that the high frequency of chromosome lesions in vitro in Lolium species is the result of the expression of 45S rDNA fragile sites. Possible causes for the spontaneous expression of fragile sites and their potential biological significance are discussed.

  12. Mitochondrial DNA regionalism and historical demography in the extant populations of Chirocephalus kerkyrensis (Branchiopoda: Anostraca.

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    Valerio Ketmaier

    Full Text Available BACKGROUND: Mediterranean temporary water bodies are important reservoirs of biodiversity and host a unique assemblage of diapausing aquatic invertebrates. These environments are currently vanishing because of increasing human pressure. Chirocephalus kerkyrensis is a fairy shrimp typical of temporary water bodies in Mediterranean plain forests and has undergone a substantial decline in number of populations in recent years due to habitat loss. We assessed patterns of genetic connectivity and phylogeographic history in the seven extant populations of the species from Albania, Corfu Is. (Greece, Southern and Central Italy. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed sequence variation at two mitochondrial DNA genes (Cytochrome Oxidase I and 16s rRNA in all the known populations of C. kerkyrensis. We used multiple phylogenetic, phylogeographic and coalescence-based approaches to assess connectivity and historical demography across the whole distribution range of the species. C. kerkyrensis is genetically subdivided into three main mitochondrial lineages; two of them are geographically localized (Corfu Is. and Central Italy and one encompasses a wide geographic area (Albania and Southern Italy. Most of the detected genetic variation (≈81% is apportioned among the aforementioned lineages. CONCLUSIONS/SIGNIFICANCE: Multiple analyses of mismatch distributions consistently supported both past demographic and spatial expansions with the former predating the latter; demographic expansions were consistently placed during interglacial warm phases of the Pleistocene while spatial expansions were restricted to cold periods. Coalescence methods revealed a scenario of past isolation with low levels of gene flow in line with what is already known for other co-distributed fairy shrimps and suggest drift as the prevailing force in promoting local divergence. We recommend that these evolutionary trajectories should be taken in proper consideration in any effort

  13. Regionalized pathology correlates with augmentation of mtDNA copy numbers in a patient with myoclonic epilepsy with ragged-red fibers (MERRF-syndrome.

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    Anja Brinckmann

    Full Text Available Human patients with myoclonic epilepsy with ragged-red fibers (MERRF suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G. In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV, transcription factors (NRF1 and TFAM, and VDAC1 (Porin as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89-100%. However, mtDNA copy numbers were increased 3-7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs. Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that "futile" stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome.

  14. 45S rDNA Regions Are Chromosome Fragile Sites Expressed as Gaps In Vitro on Metaphase Chromosomes of Root-Tip Meristematic Cells in Lolium spp

    OpenAIRE

    Jing Huang; Lu Ma; Fei Yang; Shui-zhang Fei; Lijia Li

    2008-01-01

    BACKGROUND: In humans, chromosome fragile sites are regions that are especially prone to forming non-staining gaps, constrictions or breaks in one or both of the chromatids on metaphase chromosomes either spontaneously or following partial inhibition of DNA synthesis and have been well identified. So far, no plant chromosome fragile sites similar to those in human chromosomes have been reported. METHODS AND RESULTS: During the course of cytological mapping of rDNA on ryegrass chromosomes, we ...

  15. coMET: visualisation of regional epigenome-wide association scan results and DNA co-methylation patterns.

    Science.gov (United States)

    Martin, Tiphaine C; Yet, Idil; Tsai, Pei-Chien; Bell, Jordana T

    2015-04-28

    Epigenome-wide association scans (EWAS) are an increasingly powerful and widely-used approach to assess the role of epigenetic variation in human complex traits. However, this rapidly emerging field lacks dedicated visualisation tools that can display features specific to epigenetic datasets. We developed coMET, an R package and online tool for visualisation of EWAS results in a genomic region of interest. coMET generates a regional plot of epigenetic-phenotype association results and the estimated DNA methylation correlation between CpG sites (co-methylation), with further options to visualise genomic annotations based on ENCODE data, gene tracks, reference CpG-sites, and user-defined features. The tool can be used to display phenotype association signals and correlation patterns of microarray or sequencing-based DNA methylation data, such as Illumina Infinium 450k, WGBS, or MeDIP-seq, as well as other types of genomic data, such as gene expression profiles. The software is available as a user-friendly online tool from http://epigen.kcl.ac.uk/comet and as an R Bioconductor package. Source code, examples, and full documentation are also available from GitHub. Our new software allows visualisation of EWAS results with functional genomic annotations and with estimation of co-methylation patterns. coMET is available to a wide audience as an online tool and R package, and can be a valuable resource to interpret results in the fast growing field of epigenetics. The software is designed for epigenetic data, but can also be applied to genomic and functional genomic datasets in any species.

  16. Origins and dispersal of the mitochondrial DNA region V 9 bp deletion and insertion in Nigeria and the Ivory Coast

    Energy Technology Data Exchange (ETDEWEB)

    Merriwether, D.A.; Huston, S.L.; Bunker, C.A. [Univ. of Pittsburgh, PA (United States)] [and others

    1994-09-01

    An intergenic region V Mitochondrial DNA (mtDNA) 9 bp deletion located between the genes for tRNA{sup LYS} and cytochrome oxidase II was discovered in a small percentage of Nigerian and Ivory Coast natives. Previously this deletion has been described as Asian-specific and has been reported throughout the New World, Asia, S.E. Asia, and the Pacific Islands at frequencies ranging from 0% to 100%. In the New World and the Pacific Islands, the deletion is almost always accompanied by an Hae III restriction site gain at nt 16517. All 9 occurrences of the deletion observed in Africa (from four different populations) co-occur with the Hae III 16517 site gain, indicating that the African deletion probably shares a common origin with the deletion described as {open_quotes}Asian-specific{close_quotes}. The deletion was found in Benin and Sokoto, Nigeria in 2/54 Edo Bini, 1/2 Edo Ishan, 3/99 Hausa, 0/18 Fulani, and 0/16 other Nigerians. The deletion was also detected in 3/115 Ivory Coast natives from Abidjan. A 9 bp insertion (triplication) was observed in 1/115 Ivory Coast natives. The triplicated individual also possessed the Hae III 16517 site gain. The fragment containing the African deletion was sequenced and found to be identical in sequence to the Asian deletion region. D-loop sequence of nts 15975 to 00048 revealed that 2 of the 3 Ivory Coast deleted individuals and 1 of the 6 Nigerians deleted (Hausa) had a T-C transition at nt position 16189 which is common in New World-deleted individuals. These results raise the possibility that the occurrence of this deletion predates the separation of Asian and African populations from a common ancestral populations, or that the deletion has occurred more than once in human evolution. Either explanation requires that caution be exercised when using the 9 bp deletion as a population marker.

  17. Genotyping human ancient mtDNA control and coding region polymorphisms with a multiplexed Single-Base-Extension assay: the singular maternal history of the Tyrolean Iceman

    Directory of Open Access Journals (Sweden)

    Egarter-Vigl Eduard

    2009-06-01

    Full Text Available Abstract Background Progress in the field of human ancient DNA studies has been severely restricted due to the myriad sources of potential contamination, and because of the pronounced difficulty in identifying authentic results. Improving the robustness of human aDNA results is a necessary pre-requisite to vigorously testing hypotheses about human evolution in Europe, including possible admixture with Neanderthals. This study approaches the problem of distinguishing between authentic and contaminating sequences from common European mtDNA haplogroups by applying a multiplexed Single-Base-Extension assay, containing both control and coding region sites, to DNA extracted from the Tyrolean Iceman. Results The multiplex assay developed for this study was able to confirm that the Iceman's mtDNA belongs to a new European mtDNA clade with a very limited distribution amongst modern data sets. Controlled contamination experiments show that the correct results are returned by the multiplex assay even in the presence of substantial amounts of exogenous DNA. The overall level of discrimination achieved by targeting both control and coding region polymorphisms in a single reaction provides a methodology capable of dealing with most cases of homoplasy prevalent in European haplogroups. Conclusion The new genotyping results for the Iceman confirm the extreme fallibility of human aDNA studies in general, even when authenticated by independent replication. The sensitivity and accuracy of the multiplex Single-Base-Extension methodology forms part of an emerging suite of alternative techniques for the accurate retrieval of ancient DNA sequences from both anatomically modern humans and Neanderthals. The contamination of laboratories remains a pressing concern in aDNA studies, both in the pre and post-PCR environments, and the adoption of a forensic style assessment of a priori risks would significantly improve the credibility of results.

  18. Genotyping human ancient mtDNA control and coding region polymorphisms with a multiplexed Single-Base-Extension assay: the singular maternal history of the Tyrolean Iceman.

    Science.gov (United States)

    Endicott, Phillip; Sanchez, Juan J; Pichler, Irene; Brotherton, Paul; Brooks, Jerome; Egarter-Vigl, Eduard; Cooper, Alan; Pramstaller, Peter

    2009-06-19

    Progress in the field of human ancient DNA studies has been severely restricted due to the myriad sources of potential contamination, and because of the pronounced difficulty in identifying authentic results. Improving the robustness of human aDNA results is a necessary pre-requisite to vigorously testing hypotheses about human evolution in Europe, including possible admixture with Neanderthals. This study approaches the problem of distinguishing between authentic and contaminating sequences from common European mtDNA haplogroups by applying a multiplexed Single-Base-Extension assay, containing both control and coding region sites, to DNA extracted from the Tyrolean Iceman. The multiplex assay developed for this study was able to confirm that the Iceman's mtDNA belongs to a new European mtDNA clade with a very limited distribution amongst modern data sets. Controlled contamination experiments show that the correct results are returned by the multiplex assay even in the presence of substantial amounts of exogenous DNA. The overall level of discrimination achieved by targeting both control and coding region polymorphisms in a single reaction provides a methodology capable of dealing with most cases of homoplasy prevalent in European haplogroups. The new genotyping results for the Iceman confirm the extreme fallibility of human aDNA studies in general, even when authenticated by independent replication. The sensitivity and accuracy of the multiplex Single-Base-Extension methodology forms part of an emerging suite of alternative techniques for the accurate retrieval of ancient DNA sequences from both anatomically modern humans and Neanderthals. The contamination of laboratories remains a pressing concern in aDNA studies, both in the pre and post-PCR environments, and the adoption of a forensic style assessment of a priori risks would significantly improve the credibility of results.

  19. DNA methylation of the IGF2/H19 imprinting control region and adiposity distribution in young adults

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    Huang Rae-Chi

    2012-11-01

    Full Text Available Abstract Background The insulin-like growth factor 2 (IGF2 and H19 imprinted genes control growth and body composition. Adverse in-utero environments have been associated with obesity-related diseases and linked with altered DNA methylation at the IGF2/H19 locus. Postnatally, methylation at the IGF2/H19 imprinting control region (ICR has been linked with cerebellum weight. We aimed to investigate whether decreased IGF2/H19 ICR methylation is associated with decreased birth and childhood anthropometry and increased contemporaneous adiposity. DNA methylation in peripheral blood (n = 315 at 17 years old was measured at 12 cytosine-phosphate-guanine sites (CpGs, analysed as Sequenom MassARRAY EpiTYPER units within the IGF2/H19 ICR. Birth size, childhood head circumference (HC at six time-points and anthropometry at age 17 years were measured. DNA methylation was investigated for its association with anthropometry using linear regression. Results The principal component of IGF2/H19 ICR DNA methylation (representing mean methylation across all CpG units positively correlated with skin fold thickness (at four CpG units (P-values between 0.04 to 0.001 and subcutaneous adiposity (P = 0.023 at age 17, but not with weight, height, BMI, waist circumference or visceral adiposity. IGF2/H19 methylation did not associate with birth weight, length or HC, but CpG unit 13 to 14 methylation was negatively associated with HC between 1 and 10 years. β-coefficients of four out of five remaining CpG units also estimated lower methylation with increasing childhood HC. Conclusions As greater IGF2/H19 methylation was associated with greater subcutaneous fat measures, but not overall, visceral or central adiposity, we hypothesize that obesogenic pressures in youth result in excess fat being preferentially stored in peripheral fat depots via the IGF2/H19 domain. Secondly, as IGF2/H19 methylation was not associated with birth size but negatively with early childhood HC, we

  20. Japanese Wolves are Genetically Divided into Two Groups Based on an 8-Nucleotide Insertion/Deletion within the mtDNA Control Region.

    Science.gov (United States)

    Ishiguro, Naotaka; Inoshima, Yasuo; Yanai, Tokuma; Sasaki, Motoki; Matsui, Akira; Kikuchi, Hiroki; Maruyama, Masashi; Hongo, Hitomi; Vostretsov, Yuri E; Gasilin, Viatcheslav; Kosintsev, Pavel A; Quanjia, Chen; Chunxue, Wang

    2016-02-01

    The mitochondrial DNA (mtDNA) control region (198- to 598-bp) of four ancient Canis specimens (two Canis mandibles, a cranium, and a first phalanx) was examined, and each specimen was genetically identified as Japanese wolf. Two unique nucleotide substitutions, the 78-C insertion and the 482-G deletion, both of which are specific for Japanese wolf, were observed in each sample. Based on the mtDNA sequences analyzed, these four specimens and 10 additional Japanese wolf samples could be classified into two groups- Group A (10 samples) and Group B (4 samples)-which contain or lack an 8-bp insertion/deletion (indel), respectively. Interestingly, three dogs (Akita-b, Kishu 25, and S-husky 102) that each contained Japanese wolf-specific features were also classified into Group A or B based on the 8-bp indel. To determine the origin or ancestor of the Japanese wolf, mtDNA control regions of ancient continental Canis specimens were examined; 84 specimens were from Russia, and 29 were from China. However, none of these 113 specimens contained Japanese wolf-specific sequences. Moreover, none of 426 Japanese modern hunting dogs examined contained these Japanese wolf-specific mtDNA sequences. The mtDNA control region sequences of Groups A and B appeared to be unique to grey wolf and dog populations.

  1. Increased levels of oxidative DNA damage in pesticide sprayers in Thessaly Region (Greece). Implications of pesticide exposure.

    Science.gov (United States)

    Koureas, Michalis; Tsezou, Aspasia; Tsakalof, Andreas; Orfanidou, Timoklia; Hadjichristodoulou, Christos

    2014-10-15

    The widespread use of pesticides substances nowadays largely guarantees the protection of crops and people from undesired pests. However, exposure to pesticides was related to a variety of human health effects. The present study was conducted in the region of Thessaly which is characterized by intensive agricultural activities and wide use of pesticides. The study aimed at estimating the oxidative damage to DNA in different subpopulations in Thessaly region (Greece) and investigating its correlation with exposure to pesticides and other potential risk factors. In total, the study involved 80 pesticide sprayers, 85 rural residents and 121 individuals, inhabitants of the city of Larissa. Demographic characteristics, habits, medical history and exposure history of the participants to pesticides were recorded by personal interviews. Blood and urine samples were collected from all participants. For the measurement of exposure to organophosphorus insecticides, dialkylphosphate (DAP) metabolites were quantified in urine, by gas chromatography-mass spectrometry. Genomic DNA was extracted from peripheral blood samples and the oxidation by-product 8-hydroxydeoxyguanosine (8-OHdG) was determined by Enzyme Immuno-Assay. Urinary metabolite concentrations were not associated with 8-OHdG levels but it was found that pesticide sprayers had significantly higher levels of 8-OHdG (p=0.007) in comparison to the control group. Last season's exposure to insecticides and fungicides, expressed as total area treated multiplied by the number of applications, showed a statistically significant association with the risk of having high 8-OHdG levels [RR: 2.19 (95%CI:1.09-4.38) and RR: 2.32 (95% CI:1.16-4.64) respectively]. Additionally, from the subgroups of pesticides examined, seasonal exposure to neonicotinoid insecticides [RR: 2.22 (95% CI:1.07-4.63)] and glufosinate ammonium [RR: 3.26 (95% CI:1.38-7.69)] was found to have the greater impact on 8-OHdG levels. This study produced findings

  2. Structure of the DNA-bound BRCA1 C-terminal region from human replication factor C p140 and model of the protein-DNA complex

    NARCIS (Netherlands)

    Kobayashi, M.; AB, E.; Bonvin, A.M.J.J.|info:eu-repo/dai/nl/113691238; Siegal, G.

    2010-01-01

    BRCA1 C-terminal domain (BRCT)-containing proteins are found widely throughout the animal and bacteria kingdoms where they are exclusively involved in cell cycle regulation and DNA metabolism. Whereas most BRCT domains are involved in protein-protein interactions, a small subset has bona fide DNA

  3. Multicolor chromosome banding (MCB) with YAC/BAC-based probes and region-specific microdissection DNA libraries.

    Science.gov (United States)

    Liehr, T; Weise, A; Heller, A; Starke, H; Mrasek, K; Kuechler, A; Weier, H-U G; Claussen, U

    2002-01-01

    Multicolor chromosome banding (MCB) allows the delineation of chromosomal regions with a resolution of a few megabasepairs, i.e., slightly below the size of most visible chromosome bands. Based on the hybridization of overlapping region-specific probe libraries, chromosomal subregions are hybridized with probes that fluoresce in distinct wavelength intervals, so they can be assigned predefined pseudo-colors during the digital imaging and visualization process. The present study demonstrates how MCB patterns can be produced by region-specific microdissection derived (mcd) libraries as well as collections of yeast or bacterial artificial chromosomes (YACs and BACs, respectively). We compared the efficiency of an mcd library based approach with the hybridization of collections of locus-specific probes (LSP) for fluorescent banding of three rather differently sized human chromosomes, i.e., chromosomes 2, 13, and 22. The LSP sets were comprised of 107 probes specific for chromosome 2, 82 probes for chromosome 13, and 31 probes for chromosome 22. The results demonstrated a more homogeneous coverage of chromosomes and thus, more desirable banding patterns using the microdissection library-based MCB. This may be related to the observation that chromosomes are difficult to cover completely with YAC and/or BAC clones as single-color fluorescence in situ hybridization (FISH) experiments showed. Mcd libraries, on the other hand, provide high complexity probes that work well as region-specific paints, but do not readily allow positioning of breakpoints on genetic or physical maps as required for the positional cloning of genes. Thus, combinations of mcd libraries and locus-specific large insert DNA probes appear to be the most efficient tools for high-resolution cytogenetic analyses. Copyright 2002 S. Karger AG, Basel

  4. Scalloped a member of the Hippo tumor suppressor pathway controls mushroom body size in Drosophila brain by non-canonical regulation of neuroblast proliferation.

    Science.gov (United States)

    Rohith, Basavanahalli Nanjundaiah; Shyamala, Baragur Venkatanarayanasetty

    2017-12-15

    Cell proliferation, growth and survival are three different basic processes which converge at determining a fundamental property -the size of an organism. Scalloped (Sd) is the first characterised transcriptional partner to Yorkie (Yki), the downstream effector of the Hippo pathway which is a highly potential and evolutionarily conserved regulator of organ size. Here we have studied the hypomorphic effect of sd on the development of Mushroom Bodies (MBs) in Drosophila brain. We show that, sd non-function results in an increase in the size of MBs. We demonstrate that, sd regulation on MB size operates through multiple routes. Sd expressed in the differentiated MB neurons, imposes non-cell autonomous repression on the proliferation of MB precursor cells, and Sd expression in the MB neuroblasts (NB) cell autonomously represses mushroom body neuroblast (MBNB) proliferation. Further Sd in Kenyon cells (KCs) imparts a cell autonomous restriction on their growth. Our findings are distinctive because, while the classical sd loss of function phenotypes in eye, wing and lymph gland are reported as loss of tissue or reduced organ size, the present study shows that, Sd inactivation in the developing MB, promotes precursor cell proliferation and results in an increase in the organ size. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Effects of total saponins of Panax notoginseng on immature neuroblasts in the adult olfactory bulb following global cerebral ischemia/reperfusion

    Directory of Open Access Journals (Sweden)

    Xu He

    2015-01-01

    Full Text Available The main active components extracted from Panax notoginseng are total saponins. They have been shown to inhibit platelet aggregation, increase cerebral blood flow, improve neurological behavior, decrease infarct volume and promote proliferation and differentiation of neural stem cells in the hippocampus and lateral ventricles. However, there is a lack of studies on whether total saponins of Panax notoginseng have potential benefits on immature neuroblasts in the olfactory bulb following ischemia and reperfusion. This study established a rat model of global cerebral ischemia and reperfusion using four-vessel occlusion. Rats were administered total saponins of Panax notoginseng at 75 mg/kg intraperitoneally 30 minutes after ischemia then once a day, for either 7 or 14 days. Total saponins of Panax notoginseng enhanced the number of doublecortin (DCX + neural progenitor cells and increased co-localization of DCX with neuronal nuclei and phosphorylated cAMP response element-binding/DCX + neural progenitor cells in the olfactory bulb at 7 and 14 days post ischemia. These findings indicate that following global brain ischemia/reperfusion, total saponins of Panax notoginseng promote differentiation of DCX + cells expressing immature neuroblasts in the olfactory bulb and the underlying mechanism is related to the activation of the signaling pathway of cyclic adenosine monophosphate response element binding protein.

  6. A novel sandwich hybridization method for selecting cDNAs from large genomic regions: Identification of cDNAs from the cloned genomic DNA spanning the XLRP locus

    Energy Technology Data Exchange (ETDEWEB)

    Yan, D.; McHenry, C.; Fujita, R. [Univ. of Michigan, Ann Arbor, MI (United States)] [and others

    1994-09-01

    We have developed an efficient hybridization-based cDNA-selection method. A sandwich of three species - single-stranded cDNA, tagged RNA derived from genomic DNA, and biotinylated RNA complementary to the tag - allows specific retention of hybrids on an avidin-matrix. Previously, using model experiments, we demonstrated highly specific and efficient selection of a retinal gene, NRL, from complex mixtures of cDNA clones, using a sub-library from a 5 kb NRL genomic clone. We have now applied this selection strategy to isolate cDNAs from human adult retina and fetal eye libraries, with the {open_quotes}genomic RNA{close_quotes} derived from two YAC clones (OTC-C and 55B) spanning the region of X-linked retinitis pigmentosa (XLRP) locus RP3 at Xp21.1. Effectiveness of the selection-method was monitored by enrichment of TCTEX-1L gene that maps within the 55B YAC. Of the 15 selected cDNA clones that hybridized to the 55B YAC DNA, five appear to the map to specific cosmid clones derived from the 55B YAC. Inserts in these selected cDNA clones range from 0.5 to 2.3 kb in size. Additional clones are now being isolated and characterized. This procedure should be independent of the size or complexity of genomic DNA being used for selection, allow for the isolation of full-length cDNAs, and may have wider application.

  7. Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor.

    Science.gov (United States)

    Rintala, Anniina; Pietilä, Sami; Munukka, Eveliina; Eerola, Erkki; Pursiheimo, Juha-Pekka; Laiho, Asta; Pekkala, Satu; Huovinen, Pentti

    2017-04-01

    Next-generation sequencing (NGS) is currently the method of choice for analyzing gut microbiota composition. As gut microbiota composition is a potential future target for clinical diagnostics, it is of utmost importance to enhance and optimize the NGS analysis procedures. Here, we have analyzed the impact of DNA extraction and selected 16S rDNA primers on the gut microbiota NGS results. Bacterial DNA from frozen stool specimens was extracted with 5 commercially available DNA extraction kits. Special attention was paid to the semiautomated DNA extraction methods that could expedite the analysis procedure, thus being especially suitable for clinical settings. The microbial composition was analyzed with 2 distinct protocols: 1 targeting the V3-V4 and the other targeting the V4-V5 area of the bacterial 16S rRNA gene. The overall effect of DNA extraction on the gut microbiota 16S rDNA profile was relatively small, whereas the 16S rRNA gene target region had an immense impact on the results. Furthermore, semiautomated DNA extraction methods clearly appeared suitable for NGS procedures, proposing that application of these methods could importantly reduce hands-on time and human errors without compromising the validity of results.

  8. Sensitivity to the photoperiod and potential migratory features of neuroblasts in the adult sheep hypothalamus.

    Science.gov (United States)

    Batailler, Martine; Derouet, Laura; Butruille, Lucile; Migaud, Martine

    2016-07-01

    Adult neurogenesis, a process that consists in the generation of new neurons from adult neural stem cells, represents a remarkable illustration of the brain structural plasticity abilities. The hypothalamus, a brain region that plays a key role in the neuroendocrine regulations including reproduction, metabolism or food intake, houses neural stem cells located within a hypothalamic neurogenic niche. In adult sheep, a seasonal mammalian species, previous recent studies have revealed photoperiod-dependent changes in the hypothalamic cell proliferation rate. In addition, doublecortin (DCX), a microtubule-associated protein expressed in immature migrating neurons, is highly present in the vicinity of the hypothalamic neurogenic niche. With the aim to further explore the mechanism underlying adult sheep hypothalamic neurogenesis, we first show that new neuron production is also seasonally regulated since the density of DCX-positive cells changes according to the photoperiodic conditions at various time points of the year. We then demonstrate that cyclin-dependant kinase-5 (Cdk5) and p35, two proteins involved in DCX phosphorylation and known to be critically involved in migration processes, are co-expressed with DCX in young hypothalamic neurons and are capable of in vivo interaction. Finally, to examine the migratory potential of these adult-born neurons, we reveal the rostro-caudal extent of DCX labeling on hypothalamic sagittal planes. DCX-positive cells are found in the most rostral nuclei of the hypothalamus, including the preoptic area many of which co-expressed estrogen receptor-α. Thus, beyond the confirmation of the high level of neuron production during short photoperiod in sheep, our results bring new and compelling elements in support of the existence of a hypothalamic migratory path that is responsive to seasonal stimuli.

  9. Regionalized GC content of template DNA as a predictor of PCR success.

    Science.gov (United States)

    Benita, Yair; Oosting, Ronald S; Lok, Martin C; Wise, Michael J; Humphery-Smith, Ian

    2003-08-15

    A set of 1438 human exons was subjected to nested PCR. The initial success rate using a standard PCR protocol required for ligation-independent cloning was 83.4%. Logistic regression analysis was conducted on 27 primer- and template-related characteristics, of which most could be ignored apart from those related to the GC content of the template. Overall GC content of the template was a good predictor for PCR success; however, specificity and sensitivity values for predicted outcome were improved to 84.3 and 94.8%, respectively, when regionalized GC content was employed. This represented a significant improvement in predictability with respect to GC content alone (P sliding window of 21 bp across the target sequence. Fine-tuning of PCR conditions is not practicable for all target sequences whenever a large number of genes of different lengths and GC content are to be amplified in parallel, particularly if total open reading frame or domain coverage is essential for recombinant protein synthesis. Thus, the present method is proposed as a means of grouping subsets of genes possessing potentially difficult target sequences so that PCR conditions can be optimized separately in order to obtain improved outcomes.

  10. Mitochondrial DNA reveals regional and interregional importance of the central Mediterranean African shelf for loggerhead sea turtles (Caretta caretta

    Directory of Open Access Journals (Sweden)

    Paolo Casale

    2008-09-01

    Full Text Available The wide north African continental shelf in the central Mediterranean is known to be one of the few important areas in the basin for loggerhead turtles in the neritic stage. In order to assess the origin of these turtles, sequences of the mtDNA control region were obtained from 70 turtles caught by bottom trawlers in the area, and compared with known sequences from turtles from Mediterranean and Atlantic nesting sites. Five haplotypes were identified (Haplotype diversity = 0.262; nucleotide diversity = 5.4×10-3. Specific haplotypes indicate contributions from distant rookeries such as Turkey and the Atlantic, which shows that Atlantic turtles entering the Mediterranean while in the oceanic phase use at least one Mediterranean continental shelf as a neritic foraging ground. A new haplotype and another one previously found only in foraging areas, highlight the genetic information gaps for nesting sites, which undermine powerful mixed stock analyses. Despite these limitations, the results reveal the regional importance of the study area as a neritic foraging ground for turtles that are probably from most of the Mediterranean nesting aggregates. Therefore, reducing turtle mortality resulting from the high fishing effort in the area should be regarded as key for Mediterranean turtle conservation and is also possibly important for Atlantic populations.

  11. [Discrimination of psychoactive fungi (commonly called "magic mushrooms") based on the DNA sequence of the internal transcribed spacer region].

    Science.gov (United States)

    Maruyama, Takuro; Shirota, Osamu; Kawahara, Nobuo; Yokoyama, Kazumasa; Makino, Yukiko; Goda, Yukihiro

    2003-02-01

    'Magic mushrooms' (MMs) are psychoactive fungi containing the hallucinogenic compounds, psilocin (1) and psilocybin (2). Since June 6, 2002, these fungi have been regulated by the Narcotics and Psychotropics Control Law in Japan. Because there are many kinds of MMs and they are sold even as dry powders in local markets, it is very difficult to identify the original species of the MMs by morphological observation. Therefore, we investigated the internal transcribed spacer (ITS) region in the ribosomal RNA gene of MMs obtained in Japanese markets to classify them by a genetic approach. Based on the size and nucleotide sequence of the ITS region amplified by PCR, tested MMs were classified into 6 groups. Furthermore, a comparison of the DNA sequences of the MMs with those of authentic samples or with those found in the databases (GenBank, EMBL and DDBJ) made it possible to identify the species of tested MMs. Analysis by LC revealed that psilocin (1) was contained at the highest level in Panaeolus cyanescens among the MMs, but was absent in the Amanita species.

  12. Phylogenetic relations of humans and African apes from DNA sequences in the Psi eta-globin region

    Energy Technology Data Exchange (ETDEWEB)

    Miyamoto, M.M.; Slightom, J.L.; Goodman, M.

    1987-10-16

    Sequences from the upstream and downstream flanking DNA regions of the Psi eta-globin locus in Pan troglodytes (common chimpanzee), Gorilla gorilla (gorilla), and Pongo pygmaeus (orangutan, the closest living relative to Homo, Pan, and Gorilla) provided further data for evaluating the phylogenetic relations of humans and African apes. These newly sequenced orthologs (an additional 4.9 kilobase pairs (kbp) for each species) were combined with published Psi eta-gene sequences and then compared to the same orthologous stretch (a continuous 7.1-kbp region) available for humans. Phylogenetic analysis of these nucleotide sequences by the parsimony method indicated (i) that human and chimpanzee are more closely related to each other than either is to gorilla and (ii) that the slowdown in the rate of sequence evolution evident in higher primates is especially pronounced in humans. These results indicate that features unique to African apes (but not to humans) are primitive and that even local molecular clocks should be applied with caution.

  13. PCR-mediated Detection of Xanthomonas oryzae pv. oryzae by Amplification of the 16S-23S rDNA Spacer Region Sequence

    OpenAIRE

    Naoto, Adachi; Takashi, OKU; Ishikawa Agriculture Research Center; Hiroshima Prefectural University, School of Bioresources

    2000-01-01

    A detection method specific for Xanthomonas oryzae pv. oryzae, the pathogen responsible for bacterial blight of rice, was based on the polymerase chain reaction (PCR) and designed by amplifying the 16S-23S rDNA apacer region from this bacterium. The nucleotide sequence of the spacer region between the 16S and 23S rDNA, consisting of approximately 580-bp, from X. oryzae pv. oryzae, X. campestris pv. alfalfae, X. campestris pv. campestris, X. campestris pv. cannabis, X. campestris pv. citri, X....

  14. Lack of Structural Variation but Extensive Length Polymorphisms and Heteroplasmic Length Variations in the Mitochondrial DNA Control Region of Highly Inbred Crested Ibis, Nipponia nippon.

    Directory of Open Access Journals (Sweden)

    Xue-Lian He

    Full Text Available The animal mitochondrial DNA (mtDNA length polymorphism and heteroplasmy are accepted to be universal. Here we report the lack of structural variation but the presence of length polymorphism as well as heteroplasmy in mtDNA control region of an endangered avian species - the Crested Ibis (Nipponia nippon. The complete control region was directly sequenced while the distribution pattern and inheritance of the length variations were examined using both direct sequencing and genotyping of the PCR fragments from captive birds with pedigrees, wild birds and a historical specimen. Our results demonstrated that there was no structural variation in the control region, however, different numbers of short tandem repeats with an identical motif of CA3CA2CA3 at the 3'-end of the control region determined the length polymorphisms among and heteroplasmy within individual birds. There were one to three predominant fragments in every bird; nevertheless multiple minor fragments coexist in all birds. These extremely high polymorphisms were suggested to have derived from the 'replication slippage' of a perfect microsatellite evolution following the step-wise mutational model. The patterns of heteroplasmy were found to be shifted between generations and among siblings but rather stable between blood and feather samples. This study provides the first evidence of a very extensive mtDNA length polymorphism and heteroplasmy in the highly inbred Crested Ibis which carries an mtDNA genome lack of structural genetic diversity. The analysis of pedigreed samples also sheds light on the transmission of mtDNA length heteroplasmy in birds following the genetic bottleneck theory. Further research focusing on the generation and transmission of particular mtDNA heteroplasmy patterns in single germ line of Crested Ibis is encouraged by this study.

  15. Genetic analysis of mitochondrial DNA control region variations in four tribes of Khyber Pakhtunkhwa, Pakistan.

    Science.gov (United States)

    Bhatti, Shahzad; Aslamkhan, M; Abbas, Sana; Attimonelli, Marcella; Aydin, Hikmet Hakan; de Souza, Erica Martinha Silva

    2017-09-01

    Due to its geo strategic position at the crossroad of Asia, Pakistan has gained crucial importance of playing its pivotal role in subsequent human migratory events, both prehistoric and historic. This human movement became possible through an ancient overland network of trails called "The Silk Route" linking Asia Minor, Middle East China, Central Asia and Southeast Asia. This study was conducted to analyze complete mitochondrial control region samples of 100 individuals of four major Pashtun tribes namely, Bangash, Khattak, Mahsuds and Orakzai in the province of Khyber Pakhtunkhwa, Pakistan. All Pashtun tribes revealed high genetic diversity which is comparable to the other Central Asian, Southeast Asian and European populations. The configuration of genetic variation and heterogeneity further unveiled through Multidimensional Scaling, Principal Component Analysis and phylogenetic analysis. The results revealed that Pashtun are the composite mosaic of West Eurasian ancestry of numerous geographic origin. They received substantial gene flow during different invasive movements and have a high element of the Western provenance. The most common haplogroups reported in this study are: South Asian haplogroups M (28%) and R (8%); whereas, West Asians haplogroups are present, albeit in high frequencies (67%) and widespread over all; HV (15%), U (17%), H (9%), J (8%), K (8%), W (4%), N (3%) and T (3%). Moreover, we linked the unexplored genetic connection between Ashkenazi Jews and Pashtun. The presence of specific haplotypes J1b (4%) and K1a1b1a (5%) pointed to a genetic connection of Jewish conglomeration in Khattak tribe. This was a result of an ancient genetic influx in the early Neolithic period that led to the formation of a diverse genetic substratum in present day Pashtun.

  16. Euchromatin islands in large heterochromatin domains are enriched for CTCF binding and differentially DNA-methylated regions

    Directory of Open Access Journals (Sweden)

    Wen Bo

    2012-10-01

    Full Text Available Abstract Background The organization of higher order chromatin is an emerging epigenetic mechanism for understanding development and disease. We and others have previously observed dynamic changes during differentiation and oncogenesis in large heterochromatin domains such as Large Organized Chromatin K (lysine modifications (LOCKs, of histone H3 lysine-9 dimethylation (H3K9me2 or other repressive histone posttranslational modifications. The microstructure of these regions has not previously been explored. Results We analyzed the genome-wide distribution of H3K9me2 in two human pluripotent stem cell lines and three differentiated cells lines. We identified > 2,500 small regions with very low H3K9me2 signals in the body of LOCKs, which were termed as euchromatin islands (EIs. EIs are 6.5-fold enriched for DNase I Hypersensitive Sites and 8-fold enriched for the binding of CTCF, the major organizer of higher-order chromatin. Furthermore, EIs are 2–6 fold enriched for differentially DNA-methylated regions associated with tissue types (T-DMRs, reprogramming (R-DMRs and cancer (C-DMRs. Gene ontology (GO analysis suggests that EI-associated genes are functionally related to organ system development, cell adhesion and cell differentiation. Conclusions We identify the existence of EIs as a finer layer of epigenomic architecture within large heterochromatin domains. Their enrichment for CTCF sites and DNAse hypersensitive sites, as well as association with DMRs, suggest that EIs play an important role in normal epigenomic architecture and its disruption in disease.

  17. Transglutaminase-catalyzed incorporation of polyamines masks the DNA-binding region of the transcription factor Relish.

    Science.gov (United States)

    Maki, Kouki; Shibata, Toshio; Kawabata, Shun-Ichiro

    2017-04-14

    In Drosophila, the final immune deficiency (IMD) pathway-dependent signal is transmitted through proteolytic conversion of the nuclear factor-κB (NF-κB)-like transcription factor Relish to the active N-terminal fragment Relish-N. Relish-N is then translocated from the cytosol into the nucleus for the expression of IMD-controlled genes. We previously demonstrated that transglutaminase (TG) suppresses the IMD pathway by polymerizing Relish-N to inhibit its nuclear translocation. Conversely, we also demonstrated that orally ingested synthetic amines, such as monodansylcadaverine (DCA) and biotin-labeled pentylamine, are TG-dependently incorporated into Relish-N, causing the nuclear translocation of modified Relish-N in gut epithelial cells. It remains unclear, however, whether polyamine-containing Relish-N retains transcriptional activity. Here, we used mass spectrometry analysis of a recombinant Relish-N modified with DCA by TG activity after proteolytic digestion and show that the DCA-modified Gln residues are located in the DNA-binding region of Relish-N. TG-catalyzed DCA incorporation inhibited binding of Relish-N to the Rel-responsive element in the NF-κB-binding DNA sequence. Subcellular fractionation of TG-expressing Drosophila S2 cells indicated that TG was localized in both the cytosol and nucleus. Of note, natural polyamines, including spermidine and spermine, competitively inhibited TG-dependent DCA incorporation into Relish-N. Moreover, in vivo experiments demonstrated that Relish-N was modified by spermine and that this modification reduced transcription of IMD pathway-controlled cecropin A1 and diptericin genes. These findings suggest that intracellular TG regulates Relish-N-mediated transcriptional activity by incorporating polyamines into Relish-N and via protein-protein cross-linking. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Genetic diversity in captive and wild Matschie's tree kangaroo (Dendrolagus matschiei) from Huon Peninsula, Papua New Guinea, based on mtDNA control region sequences.

    Science.gov (United States)

    McGreevy, Thomas J; Dabek, Lisa; Gomez-Chiarri, Marta; Husband, Thomas P

    2009-05-01

    The Association of Zoos and Aquariums (AZA) Matschie's tree kangaroo (Dendrolagus matschiei) population is at a critical point for assessing long-term viability. This population, established from 19 genetically uncharacterized D. matschiei, has endured a founder effect because only four individuals contributed the majority of offspring. The highly variable mitochondrial DNA (mtDNA) control region was sequenced for five of the female-founders by examining extant representatives of their maternal lineage and compared with wild (n = 13) and captive (n = 18) D. matschiei from Papua New Guinea (PNG). AZA female-founder D. matschiei control region haplotype diversity was low, compared with captive D. matschiei held in PNG. AZA D. matschiei have only two control region haplotypes because four out of five AZA female-founder D. matschiei had an identical sequence. Both AZA haplotypes were identified among the 17 wild and captive D. matschiei haplotypes from PNG. Genomic DNA extracted from wild D. matschiei fecal samples was a reliable source of mtDNA that could be used for a larger scale study. We recommend a nuclear DNA genetic analysis to more fully characterize AZA D. matschiei genetic diversity and to assist their Species Survival Plan((R)). An improved understanding of D. matschiei genetics will contribute substantially to the conservation of these unique animals both in captivity and the wild.

  19. Characterization of cytosine methylated regions and 5-cytosine DNA methyltransferase (Ehmeth) in the protozoan parasite Entamoeba histolytica

    Science.gov (United States)

    Fisher, Ohad; Siman-Tov, Rama; Ankri, Serge

    2004-01-01

    The DNA methylation status of the protozoan parasite Entamoeba histolytica was heretofore unknown. In the present study, we developed a new technique, based on the affinity of methylated DNA to 5-methylcytosine antibodies, to identify methylated DNA in this parasite. Ribosomal DNA and ribosomal DNA circles were isolated by this method and we confirmed the validity of our approach by sodium bisulfite sequencing. We also report the identification and the characterization of a gene, Ehmeth, encoding a DNA methyltransferase strongly homologous to the human DNA methyltransferase 2 (Dnmt2). Immunofluorescence microscopy using an antibody raised against a recombinant Ehmeth showed that Ehmeth is concentrated in the nuclei of trophozoites. The recombinant Ehmeth has a weak but significant methyltransferase activity when E.histolytica genomic DNA is used as substrate. 5-Azacytidine (5-AzaC), an inhibitor of DNA methyltransferase, was used to study in vivo the role of DNA methylation in E.histolytica. Genomic DNA of trophozoites grown with 5-AzaC (23 µM) was undermethylated and the ability of 5-AzaC-treated trophozoites to kill mammalian cells or to cause liver abscess in hamsters was strongly impaired. PMID:14715927

  20. The nucleotide sequence of the variable region in Trypanosoma brucei completes the sequence analysis of the maxicircle component of mitochondrial kinetoplast DNA

    NARCIS (Netherlands)

    Sloof, P.; de Haan, A.; Eier, W.; van Iersel, M.; Boel, E.; van Steeg, H.; Benne, R.

    1992-01-01

    The nucleotide sequence of two non-contiguous DNA fragments of 4.0 and 2.2 kb, respectively, of the kinetoplast maxicircle of Trypanosoma brucei brucei EATRO strain 427 has been determined, completing the sequence analysis of the so-called variable region (see also de Vries et al., 1988, Mol.

  1. Development and evaluation of specific PCR primers targeting the ribosomal DNA-internal transcribed spacer (ITS) region of peritrich ciliates in environmental samples

    Science.gov (United States)

    Su, Lei; Zhang, Qianqian; Gong, Jun

    2017-07-01

    Peritrich ciliates are highly diverse and can be important bacterial grazers in aquatic ecosystems. Morphological identifications of peritrich species and assemblages in the environment are time-consuming and expertise-demanding. In this study, two peritrich-specific PCR primers were newly designed to amplify a fragment including the internal transcribed spacer (ITS) region of ribosomal rDNA from environmental samples. The primers showed high specificity in silico, and in tests with peritrich isolates and environmental DNA. Application of these primers in clone library construction and sequencing yielded exclusively sequences of peritrichs for water and sediment samples. We also found the ITS1, ITS2, ITS, D1 region of 28S rDNA, and ITS+D1 region co-varied with, and generally more variable than, the V9 region of 18S rDNA in peritrichs. The newly designed specific primers thus provide additional tools to study the molecular diversity, community composition, and phylogeography of these ecologically important protists in different systems.

  2. Phylogeny of Cercis based on DNA sequences of nuclear ITS and four plastid regions: implications for transatlantic historical biogeography.

    Science.gov (United States)

    Fritsch, Peter W; Cruz, Boni C

    2012-03-01

    The disjunct genus Cercis has been used to test models of Northern Hemisphere historical biogeography. Previous phylogenetic estimates employing DNA sequences of the ITS region and (in one study) those of ndhF recovered a well supported clade of North American and western Eurasian species that was nested within a paraphyletic group of Chinese species. Resolution and clade support within the tree were otherwise low and the monophyly of Cercis canadensis was uncertain. Here we conduct a phylogenetic analysis of Cercis with a higher number of regions (ITS, ndhF, rpoB-trnC, trnT-trnD, and trnS-trnG) and samples than in previous studies. Results corroborate the initial divergence between the Chinese species Cercis chingii and the rest of the genus. Support is newly found both for a clade of the two North American species as sister to the western Eurasian species, and for the monophyly of C. canadensis. As in a previous study, divergence between North American and western Eurasian Cercis was estimated as mid-Miocene (ca. 13 million years ago), and the ancestor in which this divergence occurred was inferred to be xerophytic. Contrary to previous studies, however, our data infer strictly east-to-west vicariance. The timing of the transatlantic divergence in Cercis is too recent to be explained by a postulated continuous belt of semi-arid vegetation between North America and Europe in the Paleogene, suggesting instead the presence of a Miocene North Atlantic corridor for semi-arid plants. In the absence of strong evidence from other sources, the possibility that Cercis has been able to quickly adapt from mesophytic antecedents to semi-arid conditions whenever the latter have arisen in the Northern Hemisphere can be considered a plausible alternative, although parsimony optimization renders this scenario two steps longer. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Primary analysis of DNA polymorphisms in the TRIM region (MHC subregion) of the Japanese quail, Coturnix japonica.

    Science.gov (United States)

    Suzuki, Shingo; Hosomichi, Kazuyoshi; Yokoyama, Kana; Tsuda, Kaoru; Hara, Hiromi; Yoshida, Yutaka; Fujiwara, Akira; Mizutani, Makoto; Shiina, Takashi; Kono, Tomohiro; Hanzawa, Kei

    2013-01-01

    Based on sequences of two cosmid clones from Japanese quail (Coturnix japonica, Coja), we confirmed that the syntenic cluster, GNB2L1∼BTN1∼BTN2, is located in the quail TRIM subregion of the quail major histocompatibility complex (MHC Coja) region. These cosmids also included four CjBG loci and one CjLEC locus; therefore, the quail TRIM subregion was thought to be adjacent to the BG/LEC subregion. We then identified three polymorphic markers - CjHEP21, CjTRIM39.2 and CjBTN2 - in the TRIM subregion that may be useful for the functional analysis of the MHC-Coja region. We examined MHC-Coja sequences from 321 individual quails sampled from 11 inbred strains, and we found eight alleles for each of the three genes - CjHEP21, CjTRIM39.2 and CjBTN2. These polymorphisms represent the first avian DNA markers in the TRIM subregion. Additionally, we discovered a quail-specific VNTR (variable number of long tandem repeats, 133-137 bp) in intron 7 of CjBTN2. We identified 25 haplotypes in the sample of 321 quail; these haplotypes comprised combinations of all 24 alleles of the three polymorphic genes. We suggest that there are two recombination hotspots, one between each pair of adjacent loci. All strains, except AMRP, contained multiple haplotypes; the AMRP strain contained a single, apparently fixed haplotype. © 2012 Japanese Society of Animal Science.

  4. Contribution of the C-terminal tri-lysine regions of human immunodeficiency virus type 1 integrase for efficient reverse transcription and viral DNA nuclear import

    Directory of Open Access Journals (Sweden)

    Fowke Keith R

    2005-10-01

    Full Text Available Abstract Background In addition to mediating the integration process, HIV-1 integrase (IN has also been implicated in different steps during viral life cycle including reverse transcription and viral DNA nuclear import. Although the karyophilic property of HIV-1 IN has been well demonstrated using a variety of experimental approaches, the definition of domain(s and/or motif(s within the protein that mediate viral DNA nuclear import and its mechanism are still disputed and controversial. In this study, we performed mutagenic analyses to investigate the contribution of different regions in the C-terminal domain of HIV-1 IN to protein nuclear localization as well as their effects on virus infection. Results Our analysis showed that replacing lysine residues in two highly conserved tri-lysine regions, which are located within previously described Region C (235WKGPAKLLWKGEGAVV and sequence Q (211KELQKQITK in the C-terminal domain of HIV-1 IN, impaired protein nuclear accumulation, while mutations for RK263,4 had no significant effect. Analysis of their effects on viral infection in a VSV-G pseudotyped RT/IN trans-complemented HIV-1 single cycle replication system revealed that all three C-terminal mutant viruses (KK215,9AA, KK240,4AE and RK263,4AA exhibited more severe defect of induction of β-Gal positive cells and luciferase activity than an IN class 1 mutant D64E in HeLa-CD4-CCR5-β-Gal cells, and in dividing as well as non-dividing C8166 T cells, suggesting that some viral defects are occurring prior to viral integration. Furthermore, by analyzing viral DNA synthesis and the nucleus-associated viral DNA level, the results clearly showed that, although all three C-terminal mutants inhibited viral reverse transcription to different extents, the KK240,4AE mutant exhibited most profound effect on this step, whereas KK215,9AA significantly impaired viral DNA nuclear import. In addition, our analysis could not detect viral DNA integration in each C

  5. Geographic structure and demographic history of Iranian brown bear (Ursus arctos based on mtDNA control region sequences

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Ashrafzadeh

    2015-12-01

    Full Text Available In recent years, the brown bear's range has declined and its populations in some areas have faced extinction. Therefore, to have a comprehensive picture of genetic diversity and geographic structure of populations is essential for effective conservation strategies. In this research, we sequenced a 271bp segment of mtDNA control region of seven Iranian brown bears, where a total dataset of 467 sequences (brown and polar bears were used in analyses. Overall, 113 different haplotypes and 77 polymorphic sites were identified within the segment. Based on phylogenetic analyses, Iranian brown bears were not nested in any other clades. The low values of Nm (range=0.014-0.187 and high values of Fst (range=0.728-0.972 among Iranian bears and others revealed a genetically significant differentiation. We aren't found any significant signal of demographic reduction in Iranian bears. The time to the most recent common ancestor of Iranian brown bears (Northern Iran was found to be around 19000 BP.

  6. Molecular Identification of Isolated Fungi from Unopened Containers of Greek Yogurt by DNA Sequencing of Internal Transcribed Spacer Region

    Directory of Open Access Journals (Sweden)

    Irshad M. Sulaiman

    2014-06-01

    Full Text Available In our previous study, we described the development of an internal transcribed spacer (ITS1 sequencing method, and used this protocol in species-identification of isolated fungi collected from the manufacturing areas of a compounding company known to have caused the multistate fungal meningitis outbreak in the United States. In this follow-up study, we have analyzed the unopened vials of Greek yogurt from the recalled batch to determine the possible cause of microbial contamination in the product. A total of 15 unopened vials of Greek yogurt belonging to the recalled batch were examined for the detection of fungi in these samples known to cause foodborne illness following conventional microbiological protocols. Fungi were isolated from all of the 15 Greek yogurt samples analyzed. The isolated fungi were genetically typed by DNA sequencing of PCR-amplified ITS1 region of rRNA gene. Analysis of data confirmed all of the isolated fungal isolates from the Greek yogurt to be Rhizomucor variabilis. The generated ITS1 sequences matched 100% with the published sequences available in GenBank. In addition, these yogurt samples were also tested for the presence of five types of bacteria (Salmonella, Listeria, Staphylococcus, Bacillus and Escherichia coli causing foodborne disease in humans, and found negative for all of them.

  7. Robust Phylogeny of Tetrastigma (Vitaceae Based on Ten Plastid DNA Regions: Implications for Infrageneric Classification and Seed Character Evolution

    Directory of Open Access Journals (Sweden)

    Li-Min Lu

    2017-04-01

    Full Text Available Tetrastigma (Miq. Planch. is one of the most species-rich genera of the economically and agronomically important grape family Vitaceae. It includes ca. 95 species widely distributed in the tropics and subtropics of Asia and Australia. Species of Tetrastigma exhibit great diversity in both vegetative and reproductive characters. Here we inferred a well-supported phylogeny of Tetrastigma based on ten chloroplast DNA regions with an expanded taxon sampling of 72 species and two varieties. Our molecular results support six major clades within Tetrastigma and the relationships among these clades were well-resolved. We also documented seed morphology of 44 species covering the six major clades of the genus. Ancestral states of eight characters (seed shape, seed surface rumination pattern, chalaza length/width ratio, chalaza position, ventral infold position, ventral infold divergence, ventral infold depth in cross section, and endosperm shape were reconstructed in Mesquite and R with four models. Character optimizations suggest that all character states have evolved multiple times except that the irregular-shaped surface rumination has derived only once in Tetrastigma. We evaluated the taxonomic importance of seed morphology and identified potential morphological evidence to support each major clade. Our comprehensive analyses of Tetrastigma shed insights into the infrageneric classification of this morphologically diverse and ecologically important genus in tropical and subtropical Asia.

  8. Identification of Rhizoctonia solani isolates from sugar beet roots by analyzing the ITS region of ribosomal DNA

    Directory of Open Access Journals (Sweden)

    Stojšin Vera B.

    2007-01-01

    Full Text Available Rhizoctonia solani (Kühn is one of the most important sugar beet pathogens Rhizoctonia solani anastomosis groups (AGs 2-2 and 4 are proven to be the most common pathogenic strains on sugar beet. AG 2-2 (intraspecific groups IIIB and IV can cause root and crown rot while damping-off of seedlings is most frequently attributed to AG 4. Four isolates of R. solani from sugar beet roots showing characteristic crown and root rot symptoms, collected from different localities in Vojvodina Province, were chosen and compared to the well-characterized R. solani isolate R9, AG 2-2 IV, from the USA. All Vojvodinian isolates showed medium level of pathogenicity and were able to cause crown and root rot symptoms on inoculated sugar beet roots. Based on anastomosis reaction, isolates from Vojvodina did not belong to the AG 2-2 group. Sequencing of the ITS (internal transcribed spacer region of ribosomal DNA was performed on the Vojvodinian isolates from R9 in order to determine their relatedness. Sequence analysis showed that these isolates were different than R9 and were closely related (99-100% sequence homology to anastomosis group 4, subgroup HG II.

  9. Assessing genetic diversity of wild and hatchery samples of the Chinese sucker (Myxocyprinus asiaticus) by the mitochondrial DNA control region.

    Science.gov (United States)

    Wu, Jiayun; Wu, Bo; Hou, Feixia; Chen, Yongbai; Li, Chong; Song, Zhaobin

    2016-01-01

    To restore the natural populations of Chinese sucker (Myxocyprinus asiaticus), a hatchery release program has been underway for nearly 10 years. Using DNA sequences of the mitochondrial control region, we assessed the genetic diversity and genetic structure among samples collected from three sites of the wild population as well as from three hatcheries. The haplotype diversity of the wild samples (h = 0.899-0.975) was significantly higher than that of the hatchery ones (h = 0.296-0.666), but the nucleotide diversity was almost identical between them (π = 0.0170-0.0280). Relatively high gene flow was detected between the hatchery and wild samples. Analysis of effective population size indicated that M. asiaticus living in the Yangtze River has been expanding following a bottleneck in the recent past. Our results suggest the hatchery release programs for M. asiaticus have not reduced the genetic diversity, but have influenced the genetic structure of the species in the upper Yangtze River.

  10. Purine analog substitution of the HIV-1 polypurine tract primer defines regions controlling initiation of plus-strand DNA synthesis.

    Science.gov (United States)

    Rausch, Jason W; Le Grice, Stuart F J

    2007-01-01

    Despite extensive study, the mechanism by which retroviral reverse transciptases (RTs) specifically utilize polypurine tract (PPT) RNA for initiation of plus-strand DNA synthesis remains unclear. Three sequence motifs within or adjacent to the purine-rich elements are highly conserved, namely, a rU:dA tract region immediately 5' to the PPT, an rA:dT-rich sequence constituting the upstream portion of the PPT and a downstream rG:dC tract. Using an in vitro HIV-1 model system, we determined that the former two elements define the 5' terminus of the (+)-strand primer, whereas the rG:dC tract serves as the primary determinant of initiation specificity. Subsequent analysis demonstrated that G-->A or A-->G substitution at PPT positions -2, -4 and +1 (relative to the scissile phosphate) substantially reduces (+)-strand priming. We explored this observation further using PPT substrates substituted with a variety of nucleoside analogs [inosine (I), purine riboside (PR), 2-aminopurine (2-AP), 2,6-diaminopurine (2,6-DAP), isoguanine (iG)], or one of the naturally occurring bases at these positions. Our results demonstrate that for PPT positions -2 or +1, substituting position 2 of the purine was an important determinant of cleavage specificity. In addition, cleavage specificity was greatly affected by substituting -4G with an analog containing a 6-NH2 moiety.

  11. Novel Bacteriocinogenic Lactobacillus plantarum Strains and Their Differentiation by Sequence Analysis of 16S rDNA, 16S-23S and 23S-5S Intergenic Spacer Regions and Randomly Amplified Polymorphic DNA Analysis

    Directory of Open Access Journals (Sweden)

    Morteza Shojaei Moghadam

    2010-01-01

    Full Text Available Six strains of bacteriocinogenic Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 and RI11 isolated from Malaysian foods were investigated for their structural bacteriocin genes. A new combination of plantaricin EF and plantaricin W bacteriocin structural genes was successfully amplified from all studied strains, suggesting that they were novel bacteriocin-producing L. plantarum strains. A four-base pair variable region was detected in the short 16S-23S intergenic spacer regions of the studied strains by a comparative analysis with 17 L. plantarum strains deposited in the GenBank, implying they were new genotypes. The studied L. plantarum strains were subsequently differentiated into four groups on the basis of the detected four-base pair variable region of the short 16S-23S intergenic spacer region. Further analysis of the DNA sequence of 23S-5S intergenic spacer region revealed only one type of 23S-5S intergenic spacer region present in the studied strains, indicating it was highly conserved among the studied L. plantarum strains. Three randomly amplified polymorphic DNA experiments using three different combinations of arbitrary primers successfully differentiated the studied L. plantarum strains from each other, confirming they were different strains. In conclusion, the studied L. plantarum strains were shown to be novel bacteriocin producers and high level of strain discrimination could be achieved with a combination of randomly amplified polymorphic DNA analysis and the analysis of the variable region of short 16S-23S intergenic spacer region present in L. plantarum strains.

  12. Section-level relationships of North American Agalinis (Orobanchaceae based on DNA sequence analysis of three chloroplast gene regions

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    Neel Maile C

    2004-06-01

    Full Text Available Abstract Background The North American Agalinis are representatives of a taxonomically difficult group that has been subject to extensive taxonomic revision from species level through higher sub-generic designations (e.g., subsections and sections. Previous presentations of relationships have been ambiguous and have not conformed to modern phylogenetic standards (e.g., were not presented as phylogenetic trees. Agalinis contains a large number of putatively rare taxa that have some degree of taxonomic uncertainty. We used DNA sequence data from three chloroplast genes to examine phylogenetic relationships among sections within the genus Agalinis Raf. (=Gerardia, and between Agalinis and closely related genera within Orobanchaceae. Results Maximum likelihood analysis of sequences data from rbcL, ndhF, and matK gene regions (total aligned length 7323 bp yielded a phylogenetic tree with high bootstrap values for most branches. Likelihood ratio tests showed that all but a few branch lengths were significantly greater than zero, and an additional likelihood ratio test rejected the molecular clock hypothesis. Comparisons of substitution rates between gene regions based on linear models of pairwise distance estimates between taxa show both ndhF and matK evolve more rapidly than rbcL, although the there is substantial rate heterogeneity within gene regions due in part to rate differences among codon positions. Conclusions Phylogenetic analysis supports the monophyly of Agalinis, including species formerly in Tomanthera, and this group is sister to a group formed by the genera Aureolaria, Brachystigma, Dasistoma, and Seymeria. Many of the previously described sections within Agalinis are polyphyletic, although many of the subsections appear to form natural groups. The analysis reveals a single evolutionary event leading to a reduction in chromosome number from n = 14 to n = 13 based on the sister group relationship of section Erectae and section Purpureae

  13. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

    Science.gov (United States)

    Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R.; Guerrero, Justin A.; Shen, Tian; Casali, Paolo

    2017-01-01

    Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S–S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52+/+ counterparts, which display normal CSR, Rad52−/− B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S–S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S–S recombination, as emphasized by the significantly greater CSR reduction in Rad52−/− versus Rad52+/+ B cells on Ku86 knockdown. PMID:28176781

  14. Somatic point mutations in mtDNA control region are influenced by genetic background and associated with healthy aging: a GEHA study

    DEFF Research Database (Denmark)

    Rose, Giuseppina; Romeo, Giuseppe; Dato, Serena

    2010-01-01

    Tissue specific somatic mutations occurring in the mtDNA control region have been proposed to provide a survival advantage. Data on twins and on relatives of long-lived subjects suggested that the occurrence/accumulation of these mutations may be genetically influenced. To further investigate....... We found a significant correlation of the mtDNA control region heteroplasmy between sibs, confirming a genetic influence on this phenomenon. Furthermore, many subjects showed heteroplasmy due to mutations different from the C150T transition. In these cases heteroplasmy was correlated within sibpairs...... in Finnish and northern Italian samples, but not in southern Italians. This suggested that the genetic contribution to control region mutations may be population specific. Finally, we observed a possible correlation between heteroplasmy and Hand Grip strength, one of the best markers of physical performance...

  15. The Use and Effectiveness of Triple Multiplex System for Coding Region Single Nucleotide Polymorphism in Mitochondrial DNA Typing of Archaeologically Obtained Human Skeletons from Premodern Joseon Tombs of Korea

    Directory of Open Access Journals (Sweden)

    Chang Seok Oh

    2015-01-01

    Full Text Available Previous study showed that East Asian mtDNA haplogroups, especially those of Koreans, could be successfully assigned by the coupled use of analyses on coding region SNP markers and control region mutation motifs. In this study, we tried to see if the same triple multiplex analysis for coding regions SNPs could be also applicable to ancient samples from East Asia as the complementation for sequence analysis of mtDNA control region. By the study on Joseon skeleton samples, we know that mtDNA haplogroup determined by coding region SNP markers successfully falls within the same haplogroup that sequence analysis on control region can assign. Considering that ancient samples in previous studies make no small number of errors in control region mtDNA sequencing, coding region SNP analysis can be used as good complimentary to the conventional haplogroup determination, especially of archaeological human bone samples buried underground over long periods.

  16. DNA methylation in an enhancer region of the FADS cluster is associated with FADS activity in human liver.

    Science.gov (United States)

    Howard, Timothy D; Mathias, Rasika A; Seeds, Michael C; Herrington, David M; Hixson, James E; Shimmin, Lawrence C; Hawkins, Greg A; Sellers, Matthew; Ainsworth, Hannah C; Sergeant, Susan; Miller, Leslie R; Chilton, Floyd H

    2014-01-01

    Levels of omega-6 (n-6) and omega-3 (n-3), long chain polyunsaturated fatty acids (LcPUFAs) such as arachidonic acid (AA; 20:4, n-6), eicosapentaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3) impact a wide range of biological activities, including immune signaling, inflammation, and brain development and function. Two desaturase steps (Δ6, encoded by FADS2 and Δ5, encoded by FADS1) are rate limiting in the conversion of dietary essential 18 carbon PUFAs (18C-PUFAs) such as LA (18:2, n-6) to AA and α-linolenic acid (ALA, 18:3, n-3) to EPA and DHA. GWAS and candidate gene studies have consistently identified genetic variants within FADS1 and FADS2 as determinants of desaturase efficiencies and levels of LcPUFAs in circulating, cellular and breast milk lipids. Importantly, these same variants are documented determinants of important cardiovascular disease risk factors (total, LDL, and HDL cholesterol, triglycerides, CRP and proinflammatory eicosanoids). FADS1 and FADS2 lie head-to-head (5' to 5') in a cluster configuration on chromosome 11 (11q12.2). There is considerable linkage disequilibrium (LD) in this region, where multiple SNPs display association with LcPUFA levels. For instance, rs174537, located ∼ 15 kb downstream of FADS1, is associated with both FADS1 desaturase activity and with circulating AA levels (p-value for AA levels = 5.95 × 10(-46)) in humans. To determine if DNA methylation variation impacts FADS activities, we performed genome-wide allele-specific methylation (ASM) with rs174537 in 144 human liver samples. This approach identified highly significant ASM with CpG sites between FADS1 and FADS2 in a putative enhancer signature region, leading to the hypothesis that the phenotypic associations of rs174537 are likely due to methylation differences. In support of this hypothesis, methylation levels of the most significant probe were strongly associated with FADS1 and, to a lesser degree, FADS2 activities.

  17. DNA methylation in an enhancer region of the FADS cluster is associated with FADS activity in human liver.

    Directory of Open Access Journals (Sweden)

    Timothy D Howard

    Full Text Available Levels of omega-6 (n-6 and omega-3 (n-3, long chain polyunsaturated fatty acids (LcPUFAs such as arachidonic acid (AA; 20:4, n-6, eicosapentaenoic acid (EPA; 20:5, n-3 and docosahexaenoic acid (DHA; 22:6, n-3 impact a wide range of biological activities, including immune signaling, inflammation, and brain development and function. Two desaturase steps (Δ6, encoded by FADS2 and Δ5, encoded by FADS1 are rate limiting in the conversion of dietary essential 18 carbon PUFAs (18C-PUFAs such as LA (18:2, n-6 to AA and α-linolenic acid (ALA, 18:3, n-3 to EPA and DHA. GWAS and candidate gene studies have consistently identified genetic variants within FADS1 and FADS2 as determinants of desaturase efficiencies and levels of LcPUFAs in circulating, cellular and breast milk lipids. Importantly, these same variants are documented determinants of important cardiovascular disease risk factors (total, LDL, and HDL cholesterol, triglycerides, CRP and proinflammatory eicosanoids. FADS1 and FADS2 lie head-to-head (5' to 5' in a cluster configuration on chromosome 11 (11q12.2. There is considerable linkage disequilibrium (LD in this region, where multiple SNPs display association with LcPUFA levels. For instance, rs174537, located ∼ 15 kb downstream of FADS1, is associated with both FADS1 desaturase activity and with circulating AA levels (p-value for AA levels = 5.95 × 10(-46 in humans. To determine if DNA methylation variation impacts FADS activities, we performed genome-wide allele-specific methylation (ASM with rs174537 in 144 human liver samples. This approach identified highly significant ASM with CpG sites between FADS1 and FADS2 in a putative enhancer signature region, leading to the hypothesis that the phenotypic associations of rs174537 are likely due to methylation differences. In support of this hypothesis, methylation levels of the most significant probe were strongly associated with FADS1 and, to a lesser degree, FADS2 activities.

  18. Characterization of a DNA sequence family in the Prader-Willi/Angelman syndrome chromosome region in 15q11-q13

    Energy Technology Data Exchange (ETDEWEB)

    Dittrich, B.; Knoblauch, H.; Buiting, K.; Horsthemke, B. (Universitaetsklinikum Essen (Germany))

    1993-04-01

    IR4-3R (D15S11) is an anonymous DNA sequence from human chromosome 15. Using YAC cloning and restriction enzyme analysis, the authors have found that IR4-3R detects five related DNA sequences, which are spread over 700 kb within the Prader-Willi/Angelman syndrome chromosome region in 15q11-q 13. The RsaI and StyI polymorphisms, which were described previously, are associated with the most proximal copy of IR4-3R and are in strong linkage disequilibrium. IR4-3R represents the third DNA sequence family that has been identified in 15q11-q13. 14 refs., 2 figs., 1 tab.

  19. Novel sequencing strategy for repetitive DNA in a Drosophila BAC clone reveals that the centromeric region of the Y chromosome evolved from a telomere.

    Science.gov (United States)

    Méndez-Lago, María; Wild, Jadwiga; Whitehead, Siobhan L; Tracey, Alan; de Pablos, Beatriz; Rogers, Jane; Szybalski, Waclaw; Villasante, Alfredo

    2009-04-01

    The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.

  20. Mitochondrial DNA variation within and among regional populations of longtail macaques (Macaca fascicularis) in relation to other species of the fascicularis group of macaques.

    Science.gov (United States)

    Smith, David Glenn; McDonough, John W; George, Debra A

    2007-02-01

    An 835 base pair (bp) fragment of mitochondrial DNA (mtDNA) was sequenced to characterize genetic variation within and among 1,053 samples comprising five regional populations each of longtail macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta), and one sample each of Japanese (M. fuscata) and Taiwanese (M. cyclopis) macaques. The mtDNA haplotypes of longtail macaques clustered in two large highly structured clades (Fas1 and Fas2) of a neighbor-joining tree that were reciprocally monophyletic with respect to those representing rhesus macaques, Japanese macaques, and Taiwanese macaques. Both clades exhibited haplotypes of Indonesian and Malaysian longtail macaques widely dispersed throughout them; however, longtail macaques from Indochina, Philippines, and Mauritius each clustered in a separate well-defined clade together with one or a few Malaysian and/or Indonesian longtail macaques, suggesting origins on the Sunda shelf. Longtail macaques from Malaysia and Indonesia were far more genetically diverse, and those from Mauritius were far less diverse than any other population studied. Nucleotide diversity between mtDNA sequences of longtail macaques from different geographic regions is, in some cases, greater than that between Indian and Chinese rhesus macaques. Approximately equal amounts of genetic diversity are due to differences among animals in the same regional population, different regional populations, and different species. A greater proportion of genetic variance was explained by interspecies differences when Japanese and Taiwanese macaques were regarded as regional populations of rhesus macaques than when they were treated as separate species. Rhesus macaques from China were more closely related to both Taiwanese and Japanese macaques than to their own conspecifics from India.

  1. Fungal community analysis in the deep-sea sediments of the Pacific Ocean assessed by comparison of ITS, 18S and 28S ribosomal DNA regions

    Science.gov (United States)

    Xu, Wei; Luo, Zhu-Hua; Guo, Shuangshuang; Pang, Ka-Lai

    2016-03-01

    We investigated the diversity of fungal communities in 6 different deep-sea sediment samples of the Pacific Ocean based on three different types of clone libraries, including internal transcribed spacer (ITS), 18S rDNA, and 28S rDNA regions. A total of 1978 clones were generated from 18 environmental clone libraries, resulting in 140 fungal operational taxonomic units (OTUs), including 18 OTUs from ITS, 44 OTUs from 18S rDNA, and 78 OTUs from 28S rDNA gene primer sets. The majority of the recovered sequences belonged to diverse phylotypes of the Ascomycota and Basidiomycota. Additionally, our study revealed a total of 46 novel fungal phylotypes, which showed low similarities (<97%) with available fungal sequences in the GenBank, including a novel Zygomycete lineage, suggesting possible new fungal taxa occurring in the deep-sea sediments. The results suggested that 28S rDNA is an efficient target gene to describe fungal community in deep-sea environment.

  2. A DNA fragment from Xq21 replaces a deleted region containing the entire FVIII gene in a severe hemophilia A patient

    Energy Technology Data Exchange (ETDEWEB)

    Murru, S.; Casula, L.; Moi, P. [Insituto di Clinica e Biologia dell` Eta Evolutiva, Cagliari (Italy)] [and others

    1994-09-15

    In this paper the authors report the molecular characterization of a large deletion that removes the entire Factor VIII gene in a severe hemophilia A patient. Accurate DNA analysis of the breakpoint region revealed that a large DNA fragment replaced the 300-kb one, which was removed by the deletion. Pulsed-field gel electrophoresis analysis revealed that the size of the inserted fragment is about 550 kb. In situ hybridization demonstrated that part of the inserted region normally maps to Xq21 and to the tip of the short arm of the Y chromosome (Yp). In this patient this locus is present both in Xq21 and in Xq28, in addition to the Yp, being thus duplicated in the X chromosome. Sequence analysis of the 3` breakpoint suggested that an illegitimate recombination is probably the cause of this complex rearrangement. 52 refs., 7 figs.

  3. Phylogenetic relationships of intraspecific forms of the house mouse Mus musculus: Analysis of variability of the control region (D-loop) of mitochondrial DNA.

    Science.gov (United States)

    Maltsev, A N; Stakheev, V V; Bogdanov, A S; Fomina, E S; Kotenkova, E V

    2015-11-01

    Analysis of the control region of mitochondrial DNA (mtDNA) or D-loop of 96 house mice (Mus musculus) from Russia, Moldova, Armenia, Azerbaijan, Kazakhstan, and Turkmenistan has been used to reconstruct the phylogenetic relationships and phylogeographic patterns of intraspecific forms. New data on the phylogenetic structure of the house mouse are presented. Three phylogroups can be reliably distinguished in the eastern part of the M. musculus species range, the first one mainly comprising the haplotypes of mice from Transcaucasia (Armenia); the second one, the haplotypes of mice from Kazakhstan; and the third one, the haplotypes of mice from Siberia and some other regions. The morphological subspecies M. m. wagneri and M. m. gansuensis have proved to be genetically heterogeneous and did not form discrete phylogroups in the phylogenetic tree.

  4. Reconfiguration of nucleosome-depleted regions at distal regulatory elements accompanies DNA methylation of enhancers and insulators in cancer

    National Research Council Canada - National Science Library

    Taberlay, Phillippa C; Statham, Aaron L; Kelly, Theresa K; Clark, Susan J; Jones, Peter A

    2014-01-01

    .... Here, we evaluate the scope of global epigenetic alterations at enhancers and insulator elements in prostate and breast cancer cells using simultaneous genome-wide mapping of DNA methylation and nucleosome occupancy (NOMe-seq...

  5. Genetic Characteristic of Indonesian Local Ducks Based on Single Nucleotide Polymorphism (SNP) Analysis in D-loop Region Mitochondria DNA

    OpenAIRE

    Purwantini, Dattadewi; Ismoyowati, Ismoyowati

    2014-01-01

    . Penelitian ini bertujuan untuk mengetahui karakteristik genetik dan polimorfisme itik lokal Indonesia yaitu itik Magelang, Tegal, Mojosari, Bali dan Alabio berdasarkan analisis Single Nucleotide Polymorphism (SNP) daerah D-loop mtDNA. Tujuan jangka panjangnya adalah menetapkan marker atau penanda genetik berdasarkan SNP daerah D-loop mtDNA spesifik yang dapat membedakan itik-itik lokal yang ada di Indonesia. Selanjutnya digunakan sebagai alat bantu seleksi untuk konservasi, pembibitan da...

  6. Morphology And Genetic Diversity Of Mitochondrial Dna D-loop Region Using Pcr-rflp Analysis In Magelang Duck And Other Native Duck

    OpenAIRE

    D. Purwantini; T. Yuwanta; T. Hartatik; Ismoyowati, I

    2013-01-01

    The aim of this study was to investigate the different of plumage colors on morphological diversityof Magelang duck and genetic diversity using PCR-RFLP mtDNA D-loop region analysis of Magelangduck and four others native duck population (Tegal, Mojosari, Bali and Alabio duck) in Indonesia. Bloodsample was taken from 50 Magelang ducks and 20 of each native ducks. Morphological characteristicsof body measurement, production ability and egg quality of Magelang duck were analyzed usingCompletely ...

  7. [DNA of some regions of constitutive heterochromatin is demethylated and decondensed in MRC5 and A431 cells].

    Science.gov (United States)

    Vaĭsertreĭger, I S R; Podgornaia, O I; Enukashvili, N I

    2007-01-01

    It is believed that satellite DNA is compact and transcriptionally inert during interphase. We determined localization, range of compactization and methylation state of the centromeric and pericentromeric satellite DNA using the method of fluorescence hybridization in situ (FISH) combined with the antibody immunostaining against the methylated DNA. We investigated the tissue cells (the cells of placenta and lymphocytes), primary (MRC5 fibroblasts) and malignant (A431) cell cultures. Centromeric satellite DNA was condensed and stained with antibodies against 5-methylcytosine in all the cases. Pericentromeric satellite 3 of the chromosome 1 was condensed in lymphocytes, placenta cells and young culture of fibroblasts. The unwrapping of satellite 3 of the chromosome 1 has been observed in the senescent MRC5 fibroblasts and in the malignant cell line A431. The compact areas of pericentromeric satellites were stained with antibodies against the methylated DNA, white the decondensed areas were'nt stained. Thus, we observed pericentromeric satellite 3 decondensation in senescent fibroblasts culture MRC5 and in cell line A431. The decondensation was accompanied by the partial demethylation of the satellite DNA, which is believed to belong to constitutive heterochromatin.

  8. Procerain B, a cysteine protease from Calotropis procera, requires N-terminus pro-region for activity: cDNA cloning and expression with pro-sequence.

    Science.gov (United States)

    Nandana, Vidhyadhar; Singh, Sushant; Singh, Abhay Narayan; Dubey, Vikash Kumar

    2014-11-01

    We have previously reported isolation and characterization of a novel plant cysteine protease, Procerain B, from the latex of Calotropis procera. Our initial attempts for active recombinant Procerain B in Escherichiacoli expression system was not successful. The reason for inactive enzyme production was attributed to the absence of 5' pro-region in the Procerain B cDNA that may be involved in proper folding and production of mature active protein. The current manuscript reports the cloning of full length Procerain B for the production of the active protein. The complete cDNA sequence of Procerain B with pro-region sequence was obtained by using RNA ligase mediated rapid amplification of 5' cDNA ends (RLM-RACE). The N-terminus pro-sequence region consists of 127 amino acids and characterized as the member of inhibitory I29 family. Further the three dimensional structure of full length Procerain B was modelled by homology modelling using X-ray crystal structure of procaricain (PDB ID: 1PCI). N-terminus pro-sequence of full length Procerain B runs along the active site cleft. Full length Procerain B was expressed in prokaryotic system and activated in vitro at pH 4.0. This is the first study reporting the production of active recombinant cysteine protease from C.procera. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. MORPHOLOGY AND GENETIC DIVERSITY OF MITOCHONDRIAL DNA D-LOOP REGION USING PCR-RFLP ANALYSIS IN MAGELANG DUCK AND OTHER NATIVE DUCK

    Directory of Open Access Journals (Sweden)

    D. Purwantini

    2014-10-01

    Full Text Available The aim of this study was to investigate the different of plumage colors on morphological diversityof Magelang duck and genetic diversity using PCR-RFLP mtDNA D-loop region analysis of Magelangduck and four others native duck population (Tegal, Mojosari, Bali and Alabio duck in Indonesia. Bloodsample was taken from 50 Magelang ducks and 20 of each native ducks. Morphological characteristicsof body measurement, production ability and egg quality of Magelang duck were analyzed usingCompletely Randomized Design with 11 plumage colors as treatments. PCR technique was administeredto amplify fragments in mtDNA D-loop region and PCR products were digested with endonucleaserestriction enzyme AluI and HaeIII. The result showed that morphology diversity of Magelang duck wasstatistically affected by different plumage colors. PCR-RFLP analysis using AluI and HaeIII restrictionenzyme resulted in six combinations of restriction fragment pattern shown in six haplotypes (A, B, C, D,E and F. Haplotype difference showed genetic diversity in the population of Magelang duck and theother native ducks. In conclusion, the different plumage colors affected morphology diversity ofMagelang duck. Genetic diversity of Indonesian native duck population could be identified by usingPCR-RFLP analysis on mtDNA D-loop region.

  10. MORPHOLOGY AND GENETIC DIVERSITY OF MITOCHONDRIAL DNA D-LOOP REGION USING PCR-RFLP ANALYSIS IN MAGELANG DUCK AND OTHER NATIVE DUCK

    Directory of Open Access Journals (Sweden)

    D. Purwantini

    2013-03-01

    Full Text Available The aim of this study was to investigate the different of plumage colors on morphological diversity of Magelang duck and genetic diversity using PCR-RFLP mtDNA D-loop region analysis of Magelang duck and four others native duck population (Tegal, Mojosari, Bali and Alabio duck in Indonesia. Blood sample was taken from 50 Magelang ducks and 20 of each native ducks. Morphological characteristics of body measurement, production ability and egg quality of Magelang duck were analyzed using Completely Randomized Design with 11 plumage colors as treatments. PCR technique was administered to amplify fragments in mtDNA D-loop region and PCR products were digested with endonuclease restriction enzyme AluI and HaeIII. The result showed that morphology diversity of Magelang duck was statistically affected by different plumage colors. PCR-RFLP analysis using AluI and HaeIII restriction enzyme resulted in six combinations of restriction fragment pattern shown in six haplotypes (A, B, C, D, E and F. Haplotype difference showed genetic diversity in the population of Magelang duck and the other native ducks. In conclusion, the different plumage colors affected morphology diversity of Magelang duck. Genetic diversity of Indonesian native duck population could be identified by using PCR-RFLP analysis on mtDNA D-loop region.

  11. Phylogenetic relationships of Scomberomorus commerson using sequence analysis of the mtDNA D-loop region in the Persian Gulf, Oman Sea and Arabian Sea

    Directory of Open Access Journals (Sweden)

    Ana Mansourkiaei

    2016-04-01

    Full Text Available Abstract Narrow-barred Spanish mackerel, Scomberomorus commerson, is an epipelagic and migratory species of family Scombridae which have a significant role in terms of ecology and fishery. 100 samples were collected from the Persian Gulf, Oman Sea and Arabian Sea. Part of their dorsal fins was snipped and transferred to micro-tubes containing ethanol; then, DNAs were extracted and HRM-Real Time PCR was performed to designate representative specimens for sequencing. Phylogenetic relationships of S. commerson from Persian Gulf, Oman Sea and Arabian Sea were investigated using sequence data of mitochondrial DNA D-loop region. None clustered Neighbor Joining tree indicated the proximity amid S. commerson in four sites. As numbers demonstrated in sequence analyses of mitochondrial DNA D-Loop region a sublimely high degree of genetic similarity among S. commerson from the Persian Gulf and Oman Sea were perceived, thereafter, having one stock structure of S. commerson in four regions were proved, and this approximation can be merely justified by their migration process along the coasts of Oman Sea and Persian Gulf. Therefore, the assessment of distribution patterns of 20 haplotypes in the constructed phylogenetic tree using mtDNA D-Loop sequences ascertained that no significant clustering according to the sampling sites was concluded.

  12. ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients.

    Science.gov (United States)

    Martínez-Galán, Joaquina; Torres-Torres, Blanca; Núñez, María Isabel; López-Peñalver, Jesús; Del Moral, Rosario; Ruiz De Almodóvar, José Mariano; Menjón, Salomón; Concha, Angel; Chamorro, Clara; Ríos, Sandra; Delgado, Juan Ramón

    2014-02-04

    Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5' CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient's resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment.

  13. Concordant genetic distinctness of the phylogroup of the Siberian chipmunk from the Korean peninsula (Tamias sibiricus barberi), reexamined with nuclear DNA c-myc gene exon 2 and mtDNA control region sequences.

    Science.gov (United States)

    Koh, Hung Sun; Zhang, Minghai; Bayarlkhagva, Damdingiin; Ham, Eui Jeong; Kim, Jin Seong; Jang, Kyung Hee; Park, Nam Jeong

    2010-08-01

    We reexamined Tamias sibiricus barberi from Korea by sequencing c-myc exon 2 and the mtDNA control region. In the c-myc exon, the monogenic T. s. barberi differed from the monogenic T. s. orientalis (nucleotide distance 0.48%; 3 variable sites at 168, 306, and 552), whereas T. s. orientalis was identical to T. s. sibiricus. In the control region, T. s. barberi differed from T. s. orientalis (distance 6.84%) and T. s. sibiricus (9.35%). We considered the concordant, extensive gaps between the phylogroup of T. s. barberi and other subspecies of T. sibiricus in the c-myc gene, control region, and cytochrome b gene to be evidence of a lack of intergradation through North Korea from T. s. barberi to T. s. orientalis. Our results, showing the genetic and morphological distinctness of T. s. barberi, support that this phylogroup is a distinct species.

  14. DNA methylation patterns in human tissues of uniparental origin using a zinc-Finger gene (ZNF127) from the Angelman/Prader-Willi region

    Energy Technology Data Exchange (ETDEWEB)

    Mowery-Rushton, P.A.; Surti, U.; Locker, J. [Univ. of Pittsburgh, PA (United States)] [and others

    1996-01-11

    In order to further our understanding of the epigenetic modification of DNA and its role in imprinting, we examined DNA methylation patterns of human tissues of uniparental origin. We used complete hydatidiform moles (CHM), which are totally androgenetic conceptions, to examine the paternal methylation pattern in the absence of a maternal contribution and we used ovarian teratomas to represent the maternal counterpart. We carried out an analysis of DNA methylation of a gene which has been shown to contain sites which are differentially methylated in a parent-specific fashion. The gene, ZNF127, is located on chromosome 15q11-q13 in the region associated with Prader-Willi and Angelman syndromes. The parent-of-origin DNA methylation has been postulated to reflect the presence of an imprint and recent studies have confirmed that ZNF127 is differentially expressed only from the paternal chromosome. We identified a unique pattern of hyper- and hypomethylated sites in androgenetic conceptions which was nearly identical to the paternal pattern found in sperm. This may represent the paternal germ-line methylation imprint. We also studied partial hydatidiform moles, non-molar triploid conceptions, normal chorionic villi, and somatic tissue. These all demonstrated a modified DNA methylation pattern characteristic of normal chorionic villi with only limited findings of the imprint. Our results suggest that human androgenetic conceptions may provide an excellent model to analyze epigenetic DNA modifications, such as methylation, in imprinted genes. The paternal allele-specific methylation imprint will also be useful clinically to confirm the androgenetic nature of suspected molar conceptions in which parental blood samples may not be available. 55 refs., 3 figs.

  15. Control region sequences for East Asian individuals in the Scientific Working Group on DNA Analysis Methods forensic mtDNA data set.

    Science.gov (United States)

    Allard, Marc W; Wilson, Mark R; Monson, Keith L; Budowle, Bruce

    2004-03-01

    The Scientific Working Group on DNA Analysis Methods (SWGDAM) mitochondrial DNA (mtDNA) population data set is used to infer the relative rarity of mtDNA profiles obtained from evidence samples and of profiles used to identify missing persons. In this study, the East Asian haplogroup patterns in the SWGDAM data sets were analyzed in a phylogenetic context to determine relevant single nucleotide polymorphisms (SNPs) and to describe haplogroup distributions for Asians (n = 753; with a breakdown of individuals from China n = 356, Korea n = 182, Japan n = 163, and Thailand n = 52). We focus on the patterns observed in the SWGDAM Chinese data set and refer to interesting differences in the smaller subgroup data sets for the other East Asian populations (Japanese, Korean, and Thai). A total of 218 SNPs were observed in the data set, including 37 observed positions not previously reported. In the largest of the East Asian SWGDAM data sets (Chinese), these SNPs ranged from having 1 to 29 changes in the phylogenetic tree, with site 16519 being the most variable. On average there were 4.5 changes for a character on the tree. The most variable sites (with 14 or more changes each listed from fastest to slowest) observed were 16519 (L = 29), 16311 (L = 27), 152 (L = 24), 146 (L = 21), 16172 (L = 17), 16189 (L = 17), 195 (L = 16), 16362 (L = 15), 16093 (L = 14), 16129 (L = 14) and 150 (L = 14). These rapidly changing sites are consistent with other published analyses. Only 28 SNPs are needed to identify all clusters containing 1% (n = 7) or more individuals in the East Asian data set. All 36 haplogroups previously observed in East Asian populations were also seen in the SWGDAM data sets and include: A, B, B4, B4a, B4b, B5a, B5b, C, D, D4, D4a, D4b, D5, D5a, F, F1, F1a, F1b, F1c, F2a, G2, G2a, M, M7a1, M7b, M7b1, M7b2, M7c, M8a, M9, M10, N9a, R, R9a, Y, and Z. Haplogroups A, B4a, D4, and F1a were the most commonly observed clusters in the Chinese data set (the largest of the data

  16. Global MYCN transcription factor binding analysis in neuroblastoma reveals association with distinct E-box motifs and regions of DNA hypermethylation.

    LENUS (Irish Health Repository)

    Murphy, Derek M

    2009-01-01

    BACKGROUND: Neuroblastoma, a cancer derived from precursor cells of the sympathetic nervous system, is a major cause of childhood cancer related deaths. The single most important prognostic indicator of poor clinical outcome in this disease is genomic amplification of MYCN, a member of a family of oncogenic transcription factors. METHODOLOGY: We applied MYCN chromatin immunoprecipitation to microarrays (ChIP-chip) using MYCN amplified\\/non-amplified cell lines as well as a conditional knockdown cell line to determine the distribution of MYCN binding sites within all annotated promoter regions. CONCLUSION: Assessment of E-box usage within consistently positive MYCN binding sites revealed a predominance for the CATGTG motif (p<0.0016), with significant enrichment of additional motifs CATTTG, CATCTG, CAACTG in the MYCN amplified state. For cell lines over-expressing MYCN, gene ontology analysis revealed enrichment for the binding of MYCN at promoter regions of numerous molecular functional groups including DNA helicases and mRNA transcriptional regulation. In order to evaluate MYCN binding with respect to other genomic features, we determined the methylation status of all annotated CpG islands and promoter sequences using methylated DNA immunoprecipitation (MeDIP). The integration of MYCN ChIP-chip and MeDIP data revealed a highly significant positive correlation between MYCN binding and DNA hypermethylation. This association was also detected in regions of hemizygous loss, indicating that the observed association occurs on the same homologue. In summary, these findings suggest that MYCN binding occurs more commonly at CATGTG as opposed to the classic CACGTG E-box motif, and that disease associated over expression of MYCN leads to aberrant binding to additional weaker affinity E-box motifs in neuroblastoma. The co-localization of MYCN binding and DNA hypermethylation further supports the dual role of MYCN, namely that of a classical transcription factor affecting the

  17. Effects of curcumin (Curcuma longa) on learning and spatial memory as well as cell proliferation and neuroblast differentiation in adult and aged mice by upregulating brain-derived neurotrophic factor and CREB signaling.

    Science.gov (United States)

    Nam, Sung Min; Choi, Jung Hoon; Yoo, Dae Young; Kim, Woosuk; Jung, Hyo Young; Kim, Jong Whi; Yoo, Miyoung; Lee, Sanghee; Kim, Chul Jung; Yoon, Yeo Sung; Hwang, In Koo

    2014-06-01

    Aging is a progressive process, and it may lead to the initiation of neurological diseases. In this study, we investigated the effects of wild Indian Curcuma longa using a Morris water maze paradigm on learning and spatial memory in adult and D-galactose-induced aged mice. In addition, the effects on cell proliferation and neuroblast differentiation were assessed by immunohistochemistry for Ki67 and doublecortin (DCX) respectively. The aging model in mice was induced through the subcutaneous administration of D-galactose (100 mg/kg) for 10 weeks. C. longa (300 mg/kg) or its vehicle (physiological saline) was administered orally to adult and D-galactose-treated mice for the last three weeks before sacrifice. The administration of C. longa significantly shortened the escape latency in both adult and D-galactose-induced aged mice and significantly ameliorated D-galactose-induced reduction of cell proliferation and neuroblast differentiation in the subgranular zone of hippocampal dentate gyrus. In addition, the administration of C. longa significantly increased the levels of phosphorylated CREB and brain-derived neurotrophic factor in the subgranular zone of dentate gyrus. These results indicate that C. longa mitigates D-galactose-induced cognitive impairment, associated with decreased cell proliferation and neuroblast differentiation, by activating CREB signaling in the hippocampal dentate gyrus.

  18. Additive or synergistic effects of aluminum on the reduction of neural stem cells, cell proliferation, and neuroblast differentiation in the dentate gyrus of high-fat diet-fed mice.

    Science.gov (United States)

    Nam, Sung Min; Kim, Jong Whi; Yoo, Dae Young; Kim, Woosuk; Jung, Hyo Young; Hwang, In Koo; Seong, Je Kyung; Yoon, Yeo Sung

    2014-01-01

    Aluminum is the most plentiful metal on the Earth's crust, and its usage in cooking utensils, cosmetics, drinking containers, food additives, pharmaceutical products, and building materials provides many opportunities for potential aluminum consumption. However, its toxicity is low and harmful effects only develop with large-scale deposition of aluminum. In this study, we investigated the effects of subchronic exposure to aluminum (40 mg/kg/day) on neural stem cells, cell proliferation, neuroblast differentiation, and mature neurons in the dentate gyrus of the hippocampus. These experiments were performed in both high-fat diet and low-fat diet-fed C57BL/6J mice via immunohistochemistry using the relevant marker for each cell type, including nestin, Ki67, doublecortin, and NeuN. Subchronic exposure to aluminum in both low-fat and high-fat diet-fed mice reduced neural stem cells, cell proliferation, and neuroblast differentiation without any changes in mature neurons. Furthermore, this reduction effect was exacerbated in high-fat diet-fed mice. These results suggest that aluminum accelerates the reduction of neural stem cells, cell proliferation, and neuroblast differentiation additively or synergistically in high-fat diet-fed mice without any harmful changes in mature neurons.

  19. Non-detection of human herpesvirus 8 (HHV-8) DNA in HHV-8-seropositive blood donors from three Brazilian regions.

    Science.gov (United States)

    Levi, José Eduardo; Nascimento, Maria Claudia; Sumita, Laura Masami; de Souza, Vanda Akico Ueda Fick; Freire, Wilton S; Mayaud, Philippe; Pannuti, Claudio S

    2011-01-01

    Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the etiologic agent of all forms of Kaposi's sarcoma, primary effusion lymphoma and the plasmablastic cell variant of multicentric Castleman disease. In endemic areas of sub-Saharan Africa, blood transfusions have been associated with a substantial risk of HHV-8 transmission. By contrast, several studies among healthy blood donors from North America have failed to detect HHV-8 DNA in samples of seropositive individuals. In this study, using a real-time PCR assay, we investigated the presence of HHV-8 DNA in whole-blood samples of 803 HHV-8 blood donors from three Brazilian states (São Paulo, Amazon, Bahia) who tested positive for HHV-8 antibodies, in a previous multicenter study. HHV-8 DNA was not detected in any sample. Our findings do not support the introduction of routine HHV-8 screening among healthy blood donors in Brazil. (WC = 140).

  20. The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function

    Science.gov (United States)

    Nitharwal, Ram Gopal; Paul, Subhankar; Dar, Ashraf; Choudhury, Nirupam Roy; Soni, Rajesh K; Prusty, Dhaneswar; Sinha, Sukrat; Kashav, Tara; Mukhopadhyay, Gauranga; Chaudhuri, Tapan Kumar; Gourinath, Samudrala; Dhar, Suman Kumar

    2007-01-01

    Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function. PMID:17430964

  1. [Genetic diversity of the locus COI-COII of mitochondrial DNA in honeybee populations (Apis mellifera L.) from the Tomsk region].

    Science.gov (United States)

    Ostroverkhova, N V; Konusova, O L; Kucher, A N; Kireeva, T N; Vorotov, A A; Belykh, E A

    2015-01-01

    An assessment of the genetic diversity of the COI-COII mtDNA locus in honeybee populations from the Tomsk region was conducted. Three variants of the COI-COII mtDNA locus were registered: PQQ, PQQQ (typical for Middle Russian race), and Q (typical for southern breeds). It was established that 64% of bee colonies of the maternal line originate from the Middle Russian honeybee race, 28% of bee colonies originate from southern species, and 8% are mixed bee colonies. The southern parts of the region show a higher genetic diversity of honey bees as compared to northern regions, which are dominated by bee colonies (96%) and apiaries (73%) that are homogeneous for the genetic variant of locus COI-COII. The Tomsk region has no large areas with bee colonies maternally originating from the Middle Russian breed; only a few apiaries (both in the northern and southern areas) were revealed in which all bees originated from the Middle Russian breed.

  2. DNA barcoding of marine crustaceans from the Estuary and Gulf of St Lawrence: a regional-scale approach.

    Science.gov (United States)

    Radulovici, Adriana E; Sainte-Marie, Bernard; Dufresne, France

    2009-05-01

    Marine crustaceans are known as a group with a high level of morphological and ecological diversity but are difficult to identify by traditional approaches and usually require the help of highly trained taxonomists. A faster identification method, DNA barcoding, was found to be an effective tool for species identification in many metazoan groups including some crustaceans. Here we expand the DNA barcode database with a case study involving 80 malacostracan species from the Estuary and Gulf of St Lawrence. DNA sequences for 460 specimens grouped into clusters corresponding to known morphological species in 95% of cases. Genetic distances between species were on average 25 times higher than within species. Intraspecific divergence was high (3.78-13.6%) in specimens belonging to four morphological species, suggesting the occurrence of cryptic species. Moreover, we detected the presence of an invasive amphipod species in the St Lawrence Estuary. This study reconfirms the usefulness of DNA barcoding for the identification of marine crustaceans. © 2009 Blackwell Publishing Ltd.

  3. Non-oncogenic T-region mutants of Agrobacterium tumefaciens do transfer T-DNA into plant cells

    NARCIS (Netherlands)

    Hille, Jacques; Wullems, George; Schilperoort, Rob

    1983-01-01

    A new procedure for site-directed mutagenesis has been applied to the shooting and rooting loci of T-DNA of an octopine Ti-plasmid of Agrobacterium tumefaciens. Mutants have been obtained which induced tumours that either developed shoots or produced more roots than normally observed. Double

  4. Comparative analyses of coding and noncoding DNA regions indicate that Acropora (Anthozoa: Scleractina) possesses a similar evolutionary tempo of nuclear vs. mitochondrial genomes as in plants.

    Science.gov (United States)

    Chen, I-Ping; Tang, Chung-Yu; Chiou, Chih-Yung; Hsu, Jia-Ho; Wei, Nuwei Vivian; Wallace, Carden C; Muir, Paul; Wu, Henry; Chen, Chaolun Allen

    2009-01-01

    Evidence suggests that the mitochondrial (mt)DNA of anthozoans is evolving at a slower tempo than their nuclear DNA; however, parallel surveys of nuclear and mitochondrial variations and calibrated rates of both synonymous and nonsynonymous substitutions across taxa are needed in order to support this scenario. We examined species of the scleractinian coral genus Acropora, including previously unstudied species, for molecular variations in protein-coding genes and noncoding regions of both nuclear and mt genomes. DNA sequences of a calmodulin (CaM)-encoding gene region containing three exons, two introns and a 411-bp mt intergenic spacer (IGS) spanning the cytochrome b (cytb) and NADH 2 genes, were obtained from 49 Acropora species. The molecular evolutionary rates of coding and noncoding regions in nuclear and mt genomes were compared in conjunction with published data, including mt cytochrome b, the control region, and nuclear Pax-C introns. Direct sequencing of the mtIGS revealed an average interspecific variation comparable to that seen in published data for mt cytb. The average interspecific variation of the nuclear genome was two to five times greater than that of the mt genome. Based on the calibration of the closure of Panama Isthmus (3.0 mya) and closure of the Tethy Seaway (12 mya), synonymous substitution rates ranged from 0.367% to 1.467% Ma(-1) for nuclear CaM, which is about 4.8 times faster than those of mt cytb (0.076-0.303% Ma(-1)). This is similar to the findings in plant genomes that the nuclear genome is evolving at least five times faster than those of mitochondrial counterparts.

  5. DNA methylation aberrations rather than polymorphisms of FZD3 gene increase the risk of spina bifida in a high-risk region for neural tube defects.

    Science.gov (United States)

    Shangguan, Shaofang; Wang, Li; Chang, Shaoyan; Lu, Xiaoling; Wang, Zhen; Wu, Lihua; Wang, Jianhua; Wang, Xiuwei; Guan, Zhen; Bao, Yihua; Zhao, Huizhi; Zou, Jizhen; Niu, Bo; Zhang, Ting

    2015-01-01

    Animal models of neural tube defects (NTDs) have indicated roles for the Fzd3 gene and the planar cell polarity signaling pathway in convergent extension. We investigated the involvement of FZD3 in genetic and epigenetic mechanisms associated with human NTDs, especially spina bifida. We explored the effects of variants spanning the FZD3 gene in NTDs and examined the role of aberrant methylation of the FZD3 promoter on gene expression in brain tissue in spina bifida. Six FZD3 single nucleotide polymorphisms were genotyped using a MassARRAY system in tissue from 165 NTD fetuses and 152 controls. DNA methylation aberrations in the FZD3 promoter region were detected using a MassARRAY EpiTYPER (17 CpG units from -500 to -2400 bp from the transcription start site) in brain tissue from 77 spina bifida and 74 control fetuses. None of the six single nucleotide polymorphisms evaluated were significantly associated with spina bifida, but the mean methylation level was significantly higher in spina bifida samples (13.70%) compared with control samples (10.91%) (p = 0.001). In terms of specific sites, DNA methylation levels were significantly higher in the spina bifida samples at 14 of the 17 CpG units, which mostly included in R2 region. FZD3 mRNA expression was negatively correlated with methylation of the FZD3 promoter region, especially the R2 region (R = 0.970; p = 0.001) in HeLa cells. The results of this study suggest that DNA methylation plays an important role in FZD3 gene expression regulation and may be associated with an increased risk of spina bifida. © 2014 Wiley Periodicals, Inc.

  6. A two-locus global DNA barcode for land plants: the coding rbcL gene complements the non-coding trnH-psbA spacer region.

    Science.gov (United States)

    Kress, W John; Erickson, David L

    2007-06-06

    A useful DNA barcode requires sufficient sequence variation to distinguish between species and ease of application across a broad range of taxa. Discovery of a DNA barcode for land plants has been limited by intrinsically lower rates of sequence evolution in plant genomes than that observed in animals. This low rate has complicated the trade-off in finding a locus that is universal and readily sequenced and has sufficiently high sequence divergence at the species-level. Here, a global plant DNA barcode system is evaluated by comparing universal application and degree of sequence divergence for nine putative barcode loci, including coding and non-coding regions, singly and in pairs across a phylogenetically diverse set of 48 genera (two species per genus). No single locus could discriminate among species in a pair in more than 79% of genera, whereas discrimination increased to nearly 88% when the non-coding trnH-psbA spacer was paired with one of three coding loci, including rbcL. In silico trials were conducted in which DNA sequences from GenBank were used to further evaluate the discriminatory power of a subset of these loci. These trials supported the earlier observation that trnH-psbA coupled with rbcL can correctly identify and discriminate among related species. A combination of the non-coding trnH-psbA spacer region and a portion of the coding rbcL gene is recommended as a two-locus global land plant barcode that provides the necessary universality and species discrimination.

  7. Single nucleotide polymorphisms in the D-loop region of mitochondrial DNA is associated with the kidney survival time in chronic kidney disease patients.

    Science.gov (United States)

    Xu, Jinsheng; Guo, Zhanjun; Bai, Yaling; Zhang, Junxia; Cui, Liwen; Zhang, Huiran; Zhang, Shenglei; Ai, Xiaolu

    2015-02-01

    The mitochondrial displacement loop (D-loop) is known to accumulate mutations and SNPs at a higher frequency than other regions of mitochondrial DNA (mtDNA). We had identified chronic kidney disease (CKD) risk-associated SNPs in the D-loop of CKD patients previously. In this study, we investigated the association of SNPs in the D-loop of mtDNA with the kidney survival of CKD. The D-loop region of mtDNA was sequenced for 119 CKD patients from the inpatient of the Fourth Hospital of Hebei Medical University. The Kaplan-Meier method was used to identify disease outcome-associated SNPs in the D-loop of CKD patients. The Cox proportional hazards model was used to identify risk factors for the kidney survival of CKD. In the present study, we identified 20 SNPs with a frequency higher than 5% and assessed the relationship of these SNPs with kidney survival time in CKD patients, a SNP of 146 was identified by log-rank test for statistically significant prediction of the kidney survival time. In an overall multivariate analysis, allele 146 was identified as an independent predictor of kidney survival time in CKD patients. The survival time of kidney in the CKD patients with 146C was significantly shorter than that of kidney in CKD patients with 146T (relative risk, 2.336; 95% CI, 1.319-3.923; p = 0.001). SNPs in the D-loop can predict the kidney survival of CKD patients. Analysis of genetic polymorphisms in the mitochondrial D-loop can help to identify CKD patient subgroup at high risk of a poor disease outcome.

  8. Mitochondrial DNA control region sequence variation suggests an independent origin of an {open_quotes}Asian-specific{close_quotes} 9-bp deletion in Africans

    Energy Technology Data Exchange (ETDEWEB)

    Soodyall, H.; Redd, A.; Vigilant [Pennsylvania State Univ., Univeristy Park, PA (United States)] [and others

    1994-09-01

    The intergenic noncoding region between the cytochrome oxidase II and lysyl tRNA genes of human mitochondrial DNA (mtDNA) is associated with two tandemly arranged copies of a 9-bp sequence. A deletion of one of these repeats has been found at varying frequencies in populations of Asian descent, and is commonly referred to as an {open_quotes}Asian-specific{close_quotes} marker. We report here that the 9-bp deletion is also found at a frequency of 10.2% (66/649) in some indigenous African populations, with frequencies of 28.6% (20/70) in Pygmies, 26.6% (12/45) in Malawians and 15.4% (31/199) in southeastern Bantu-speaking populations. The deletion was not found in 123 Khoisan individuals nor in 209 western Bantu-speaking individuals, with the exception of 3 individuals from one group that was admixed with Pygmies. Sequence analysis of the two hypervariable segments of the mtDNA control region reveals that the types associated with the African 9-bp deletion are different from those found in Asian-derived populations with the deletion. Phylogenetic analysis separates the {open_quotes}African{close_quotes} and {open_quotes}Asian{close_quotes} 9-bp deletion types into two different clusters which are statistically supported. Mismatch distributions based on the number of differences between pairs of mtDNA types are consistent with this separation. These findings strongly support the view that the 9-bp deletion originated independently in Africa and in Asia.

  9. The historical demography and genetic variation of the endangered Cycas multipinnata (Cycadaceae in the red river region, examined by chloroplast DNA sequences and microsatellite markers.

    Directory of Open Access Journals (Sweden)

    Yi-Qing Gong

    Full Text Available Cycas multipinnata C.J. Chen & S.Y. Yang is a cycad endemic to the Red River drainage region that occurs under evergreen forest on steep limestone slopes in Southwest China and northern Vietnam. It is listed as endangered due to habitat loss and over-collecting for the ornamental plant trade, and only several populations remain. In this study, we assess the genetic variation, population structure, and phylogeography of C. multipinnata populations to help develop strategies for the conservation of the species. 60 individuals from six populations were used for chloroplast DNA (cpDNA sequencing and 100 individuals from five populations were genotyped using 17 nuclear microsatellites. High genetic differentiation among populations was detected, suggesting that pollen or seed dispersal was restricted within populations. Two main genetic clusters were observed in both the cpDNA and microsatellite loci, corresponding to Yunnan China and northern Vietnam. These clusters indicated low levels of gene flow between the regions since their divergence in the late Pleistocene, which was inferred from both Bayesian and coalescent analysis. In addition, the result of a Bayesian skyline plot based on cpDNA portrayed a long history of constant population size followed by a decline in the last 50,000 years of C. multipinnata that was perhaps affected by the Quaternary glaciations, a finding that was also supported by the Garza-Williamson index calculated from the microsatellite data. The genetic consequences produced by climatic oscillations and anthropogenic disturbances are considered key pressures on C. multipinnata. To establish a conservation management plan, each population of C. multipinnata should be recognized as a Management Unit (MU. In situ and ex situ actions, such as controlling overexploitation and creating a germplasm bank with high genetic diversity, should be urgently implemented to preserve this species.

  10. A study of variation in rDNA ITS regions shows that two haplotypes coexist within a single aphid genome.

    Science.gov (United States)

    Fenton, B; Malloch, G; Germa, F

    1998-06-01

    We report variation in the rDNA internal transcribed spacers (ITSs) of aphid species, the first for these insects. Variation at 6 sites within ITS1 sequences of the green peach aphid, Myzus persicae, identified two haplotypes coexisting within the same individuals, indicating that molecular drive has not homogenised different copies of rDNA. During this study, we found that PCR can cause a precise 58-bp loss in the amplified copies of an ITS haplotype (type 1). This occurs in all detectable copies under routine PCR conditions, at different annealing temperatures and with Pfu and Taq polymerases. In addition, "hot-start" PCR exclusively copied a different, rare haplotype (type 2). These observations have important considerations for using PCR, as large deletions in PCR products may not reflect real deletions in the genome, and changes in PCR conditions may be needed to copy cryptic haplotypes.

  11. Somatic diversification in the heavy chain variable region genes expressed by human autoantibodies bearing a lupus-associated nephritogenic anti-DNA idiotype

    Energy Technology Data Exchange (ETDEWEB)

    Demaison, C.; Chastagner, P.; Theze, J.; Zouali, M. (Institut Pasteur, Paris (France))

    1994-01-18

    Monoclonal anti-DNA antibodies bearing a lupus nephritis-associated idiotype were derived from five patients with systemic lupus erythematosus (SLE). Genes encoding their heavy (H)-chain variable (V[sub H]) regions were cloned and sequenced. When compared with their closest V[sub h] germ-line gene relatives, these sequences exhibit a number of silent (S) and replacement (R) substitutions. The ratios of R/S mutations were much higher in the complementarity-determining regions (CDRs) of the antibodies than in the framework regions. Molecular amplification of genomic V[sub H] genes and Southern hybridization with somatic CDR2-specific oligonucleotide probes showed that the configuration of the V[sub H] genes corresponding to V[sub H] sequences in the nephritogenic antibodies is not present in the patient's own germ-line DNA, implying that the B-cell clones underwent somatic mutation in vivo. These findings, together with the characteristics of the diversity and junctional gene elements utilized to form the antibody, indicate that these autoantibodies have been driven through somatic selection processes reminiscent of those that govern antibody responses triggered by exogenous stimuli.

  12. Identification and Individualization of Lophophora using DNA Analysis of the trnL/trnF Region and rbcL Gene.

    Science.gov (United States)

    Ng, Adrienne E; Sandoval, Ernesto; Murphy, Terence M

    2016-01-01

    Lophophora williamsii (peyote) is a small, spineless, greenish-blue cactus found in Mexico and the southwestern United States. Ingestion of the cactus can result in hallucinations due to its content of mescaline. In the United States, L. williamsii is classified as a Schedule I controlled substance. In this study, we use DNA analysis of the chloroplast trnL/trnF region and chloroplast rbcL gene to identify the individuals of Lophophora. Using the rbcL gene, Lophophora specimens could be distinguished from outgroups, but species within the genus could not be distinguished. The trnL/trnF region split the Lophophora genus into several groups based on the length and substructure of an AT-rich segment of the sequence. Our results indicate that the genetic variability at the trnL/trnF locus is greater than previously recognized. Although DNA structures at the trnL/trnF region and rbcL gene do not align with the classification of Lophophora species, they can be used to aid in forensic analysis. © 2015 American Academy of Forensic Sciences.

  13. Genetic Variability of Service Tree (Sorbus domestica L. in the Hungarian Middle Mountains – Based on cpDNA Analysis in Two Regions

    Directory of Open Access Journals (Sweden)

    NYÁRI, László

    2010-01-01

    Full Text Available A genetic inventory was conducted at maternally inherited chloroplast DNA(cpDNA gene loci of 196 adult service trees (S. domestica. The sampled trees representautochthonous collectives/populations originating from 2 distant regions, from contrastinghabitats, a forested area (eastern part of the Dunazug Mountains and cultured habitats (ZemplénMountains, respectively.Strong intrapopulation variation was observed; percentages of molecular variance were: betweenregions 27%, among populations/regions 6%, within populations 67%. Considering all samples, themajor part of total diversity (ht = 0.752 was contributed by intrapopulation diversity (hs = 0.583.Species diversity was represented differently in individual populations. E.g. the populationKácsárd contains only one haplotype: the doubtless sign of local human cultivation. The populationBuda Hills has an average differentiation considering the whole sampled material but the highest whenevaluating the region north from Budapest separately. That points to the dispersion after anintroduction event, probably parallel to adaptive radiation under selection influence.In the study genetically polymorphic populations containing unique haplotypes weredetected, providing important information for forest management, gene conservation andnature protection activities. The described work is part of ex situ gene conservation projects ofthe species in Hungary.

  14. Salamander Hox clusters contain repetitive DNA and expanded non-coding regions: a typical Hox structure for non-mammalian tetrapod vertebrates?

    Science.gov (United States)

    Voss, Stephen Randal; Putta, Srikrishna; Walker, John A; Smith, Jeramiah J; Maki, Nobuyasu; Tsonis, Panagiotis A

    2013-04-05

    Hox genes encode transcription factors that regulate embryonic and post-embryonic developmental processes. The expression of Hox genes is regulated in part by the tight, spatial arrangement of conserved coding and non-coding sequences. The potential for evolutionary changes in Hox cluster structure is thought to be low among vertebrates; however, recent studies of a few non-mammalian taxa suggest greater variation than originally thought. Using next generation sequencing of large genomic fragments (>100 kb) from the red spotted newt (Notophthalamus viridescens), we found that the arrangement of Hox cluster genes was conserved relative to orthologous regions from other vertebrates, but the length of introns and intergenic regions varied. In particular, the distance between hoxd13 and hoxd11 is longer in newt than orthologous regions from vertebrate species with expanded Hox clusters and is predicted to exceed the length of the entire HoxD clusters (hoxd13-hoxd4) of humans, mice, and frogs. Many repetitive DNA sequences were identified for newt Hox clusters, including an enrichment of DNA transposon-like sequences relative to non-coding genomic fragments. Our results suggest that Hox cluster expansion and transposon accumulation are common features of non-mammalian tetrapod vertebrates.

  15. Evaluation of variation in control region sequences for Hispanic individuals in the SWGDAM mtDNA data set.

    Science.gov (United States)

    Allard, Marc W; Polanskey, Deborah; Wilson, Mark R; Monson, Keith L; Budowle, Bruce

    2006-05-01

    The Scientific Working Group on DNA Analysis Methods (SWGDAM) Hispanic data set was analyzed to determine the diversity, phylogeny, and relevant single nucleotide polymorphisms (SNPs) that describe haplogroup patterns for Hispanic Americans (N=686), and to assess the degree of admixture regarding mitochondrial DNA (mtDNA). The largest component of admixture based on mtDNA analysis derives from the four major haplogroups previously observed in Native American ancestry, including A (29.3%), B (15.7%), C (20.6%), and D (4.8%). European (17.8%) and African (11.8%) haplogroups also were observed within this data set. Hispanic SWGDAM samples from the southwest, compared with other SWGDAM Hispanic samples, were observed to have a greater percent of Native American haplogroups present (79.9%), and fewer African American haplogroups (4.5%). A total of 234 SNPs were observed in the data set, including 36 newly reported variable positions. These SWGDAM Hispanic data set SNPs ranged from having 1 to 31 changes (Length=L) on the phylogenetic tree, with site 16519 being the most variable. On average, there were 3.9 character changes for each variable position on the tree. The most variable sites (with 13 or more changes each listed from fastest to slowest) observed were 16519 (L=31), 16189 (L=23), 152 (L=23), 16311 (L=19), 146 (L=17), 195 (L=17), 16093 (L=15), 16362 (L=14), 16129 (L=13), 150 (L=13), and 153 (L=13). These sites are consistent with other reports on highly variable positions. A total of 27 SNPs were chosen to identify all clusters containing 1% (N=7) or more individuals in the SWGDAM Hispanic data set. The descriptive analyses revealed that the SWGDAM Hispanic data set is similar to published Native American and Hispanic data sets.

  16. Non-Detection of Human Herpesvirus 8 (HHV-8) DNA in HHV-8-Seropositive Blood Donors from Three Brazilian Regions

    Science.gov (United States)

    Levi, José Eduardo; Nascimento, Maria Claudia; Sumita, Laura Masami; de Souza, Vanda Akico Ueda Fick; Freire, Wilton S.; Mayaud, Philippe; Pannuti, Claudio S.

    2011-01-01

    Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the etiologic agent of all forms of Kaposi's sarcoma, primary effusion lymphoma and the plasmablastic cell variant of multicentric Castleman disease. In endemic areas of sub-Saharan Africa, blood transfusions have been associated with a substantial risk of HHV-8 transmission. By contrast, several studies among healthy blood donors from North America have failed to detect HHV-8 DNA in samples of seropositive individuals. In this study, using a real-time PCR assay, we investigated the presence of HHV-8 DNA in whole-blood samples of 803 HHV-8 blood donors from three Brazilian states (São Paulo, Amazon, Bahia) who tested positive for HHV-8 antibodies, in a previous multicenter study. HHV-8 DNA was not detected in any sample. Our findings do not support the introduction of routine HHV-8 screening among healthy blood donors in Brazil. (WC = 140). PMID:21858163

  17. Rapid isolation of microsatellite DNAs and identification of polymorphic mitochondrial DNA regions in the fish rotan (Perccottus glenii) invading European Russia

    Science.gov (United States)

    King, Timothy L.; Eackles, Michael S.; Reshetnikov, Andrey N.

    2015-01-01

    Human-mediated translocations and subsequent large-scale colonization by the invasive fish rotan (Perccottus glenii Dybowski, 1877; Perciformes, Odontobutidae), also known as Amur or Chinese sleeper, has resulted in dramatic transformations of small lentic ecosystems. However, no detailed genetic information exists on population structure, levels of effective movement, or relatedness among geographic populations of P. glenii within the European part of the range. We used massively parallel genomic DNA shotgun sequencing on the semiconductor-based Ion Torrent Personal Genome Machine (PGM) sequencing platform to identify nuclear microsatellite and mitochondrial DNA sequences in P. glenii from European Russia. Here we describe the characterization of nine nuclear microsatellite loci, ascertain levels of allelic diversity, heterozygosity, and demographic status of P. glenii collected from Ilev, Russia, one of several initial introduction points in European Russia. In addition, we mapped sequence reads to the complete P. glenii mitochondrial DNA sequence to identify polymorphic regions. Nuclear microsatellite markers developed for P. glenii yielded sufficient genetic diversity to: (1) produce unique multilocus genotypes; (2) elucidate structure among geographic populations; and (3) provide unique perspectives for analysis of population sizes and historical demographics. Among 4.9 million filtered P. glenii Ion Torrent PGM sequence reads, 11,304 mapped to the mitochondrial genome (NC_020350). This resulted in 100 % coverage of this genome to a mean coverage depth of 102X. A total of 130 variable sites were observed between the publicly available genome from China and the studied composite mitochondrial genome. Among these, 82 were diagnostic and monomorphic between the mitochondrial genomes and distributed among 15 genome regions. The polymorphic sites (N = 48) were distributed among 11 mitochondrial genome regions. Our results also indicate that sequence reads generated

  18. Specific Variants in the MLH1 Gene Region May Drive DNA Methylation, Loss of Protein Expression, and MSI-H Colorectal Cancer

    Science.gov (United States)

    Mrkonjic, Miralem; Roslin, Nicole M.; Greenwood, Celia M.; Raptis, Stavroula; Pollett, Aaron; Laird, Peter W.; Pethe, Vaijayanti V.; Chiang, Theodore; Daftary, Darshana; Dicks, Elizabeth; Thibodeau, Stephen N.; Gallinger, Steven; Parfrey, Patrick S.; Younghusband, H. Banfield; Potter, John D.; Hudson, Thomas J.; McLaughlin, John R.; Green, Roger C.; Zanke, Brent W.; Newcomb, Polly A.; Paterson, Andrew D.; Bapat, Bharati

    2010-01-01

    Background We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734) and microsatellite unstable (MSI-H) colorectal cancers (CRCs) in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability. Methodology/Principal Findings We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls) and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls) and from Seattle (591 cases, 629 controls). Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promoter-methylation status and MLH1 immunohisotchemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value  = 0.72 vs. 2.3×10−4 when the SNP was examined alone). Conclusions/Significance The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both. PMID:20967208

  19. Specific variants in the MLH1 gene region may drive DNA methylation, loss of protein expression, and MSI-H colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Miralem Mrkonjic

    Full Text Available We previously identified an association between a mismatch repair gene, MLH1, promoter SNP (rs1800734 and microsatellite unstable (MSI-H colorectal cancers (CRCs in two samples. The current study expanded on this finding as we explored the genetic basis of DNA methylation in this region of chromosome 3. We hypothesized that specific polymorphisms in the MLH1 gene region predispose it to DNA methylation, resulting in the loss of MLH1 gene expression, mismatch-repair function, and consequently to genome-wide microsatellite instability.We first tested our hypothesis in one sample from Ontario (901 cases, 1,097 controls and replicated major findings in two additional samples from Newfoundland and Labrador (479 cases, 336 controls and from Seattle (591 cases, 629 controls. Logistic regression was used to test for association between SNPs in the region of MLH1 and CRC, MSI-H CRC, MLH1 gene expression in CRC, and DNA methylation in CRC. The association between rs1800734 and MSI-H CRCs, previously reported in Ontario and Newfoundland, was replicated in the Seattle sample. Two additional SNPs, in strong linkage disequilibrium with rs1800734, showed strong associations with MLH1 promoter methylation, loss of MLH1 protein, and MSI-H CRC in all three samples. The logistic regression model of MSI-H CRC that included MLH1-promoter-methylation status and MLH1 immunohistochemistry status fit most parsimoniously in all three samples combined. When rs1800734 was added to this model, its effect was not statistically significant (P-value  = 0.72 vs. 2.3×10(-4 when the SNP was examined alone.The observed association of rs1800734 with MSI-H CRC occurs through its effect on the MLH1 promoter methylation, MLH1 IHC deficiency, or both.

  20. Study of the genetic origin of the Mexican creole donkey (Equus asinus) by means of the analysis of the D-loop region of mitochondrial DNA.

    Science.gov (United States)

    Lopez Lopez, C; Alonso, R; de Aluja, A S

    2005-11-01

    The aim of this work was to analyse the genetic origin of the Mexican Creole donkey, as well as its genetic diversity, by comparison with Spanish and African donkey populations by means of the D-loop region of mitochondrial DNA. To this end, the genomic DNA of 68 Mexican Creole donkeys from eight geographical regions in six States of the Republic of Mexico and from a Sicilian donkey was obtained. By the polymerase chain-reaction technique (PCR) a fragment of 541 bp was amplified, corresponding to the most informative region of the mitochondrial DNA, the D-loop. The fragments were subsequently sequenced. The analysed sequences revealed 10 new Mexican haplotypes that were different from those of the Spanish and African breeds with which they were compared, showing high levels of genetic diversity. Analysis of the phylogenetic relationships in the different Creole varieties showed a tendency of origin towards Spanish breeds, mainly the Andaluza, Zamorano-Leonesa and Majorera from the Canary Islands; these in turn showed an African origin, seven Mexican haplotypes and three haplotypes similar to those analysed by Aranguren and colleagues (2004) of Spanish and African breeds being obtained. This work allows us to reach the preliminary conclusion that the origin of Mexican Creole donkey populations in the different states of the Republic of Mexico is clearly of Iberian origin, the Spanish donkey breed Andaluza being the main one contributing to the populations of the Mexican Creole donkeys, followed by the Spanish breeds Zamorano-Leonesa and Majorera from the Canary Islands, and that the populations possess high levels of genetic diversity.

  1. Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex.

    Science.gov (United States)

    Koumbaris, George; Kypri, Elena; Tsangaras, Kyriakos; Achilleos, Achilleas; Mina, Petros; Neofytou, Maria; Velissariou, Voula; Christopoulou, Georgia; Kallikas, Ioannis; González-Liñán, Alicia; Benusiene, Egle; Latos-Bielenska, Anna; Marek, Pietryga; Santana, Alfredo; Nagy, Nikoletta; Széll, Márta; Laudanski, Piotr; Papageorgiou, Elisavet A; Ioannides, Marios; Patsalis, Philippos C

    2016-06-01

    There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%-100%) cases of trisomy 21, 16/16 (95% CI, 79.4%-100%) cases of trisomy 18, 5/5 (95% CI, 47.8%-100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%-100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%-100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT. © 2016 American Association for Clinical Chemistry.

  2. Identification of sequence polymorphism in the D-Loop region of mitochondrial DNA as a risk factor for hepatocellular carcinoma with distinct etiology

    Directory of Open Access Journals (Sweden)

    Zhang Ruixing

    2010-09-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC is frequently preceded by hepatitis virus infection or alcohol abuse. Genetic backgrounds may increase susceptibility to HCC from these exposures. Methods Mitochondrial DNA (mtDNA of peripheral blood, tumor, and/or adjacent non-tumor tissue from 49 hepatitis B virus-related and 11 alcohol-related HCC patients, and from 38 controls without HCC were examined for single nucleotide polymorphisms (SNPs and mutations in the D-Loop region. Results Single nucleotide polymorphisms (SNPs in the D-loop region of mt DNA were examined in HCC patients. Individual SNPs, namely the 16266C/T, 16293A/G, 16299A/G, 16303G/A, 242C/T, 368A/G, and 462C/T minor alleles, were associated with increased risk for alcohol- HCC, and the 523A/del was associated with increased risks of both HCC types. The mitochondrial haplotypes under the M haplogroup with a defining 489C polymorphism were detected in 27 (55.1% of HBV-HCCand 8 (72.7% of alcohol- HCC patients, and in 15 (39.5% of controls. Frequencies of the 489T/152T, 489T/523A, and 489T/525C haplotypes were significantly reduced in HBV-HCC patients compared with controls. In contrast, the haplotypes of 489C with 152T, 249A, 309C, 523Del, or 525Del associated significantly with increase of alcohol-HCC risk. Mutations in the D-Loop region were detected in 5 adjacent non-tumor tissues and increased in cancer stage (21 of 49 HBV-HCC and 4 of 11 alcohol- HCC, p Conclusions In sum, mitochondrial haplotypes may differentially predispose patients to HBV-HCC and alcohol-HCC. Mutations of the mitochondrial D-Loop sequence may relate to HCC development.

  3. Genetic discrimination for three gynogenetic clones of silver carp Hypophthalmichthys molitrix, based on restriction endonuclease analysis of Nd5-Nd6 region of mitochondrial DNA

    Science.gov (United States)

    Zhou, Jianfeng; Ye, Yuzhen; Wu, Qingjiang

    2005-03-01

    Three artificial gynogenetic clones of silver carp were produced for the analysis of restriction enzyme digestion patterns of ND5-ND6 region from mtDNA of the clones. It is revealed that all intraclonal individuals shared completely the same digestion patterns but among interclonal individuals did not. The three clones were mixed and cultured in a pond together for two years, and restriction endonuclease digestion patterns of ND5 ND6 were used as genetic markers to assess the growth performance of each clone.

  4. The domain structure of Helicobacter pylori DnaB helicase: the N-terminal domain can be dispensable for helicase activity whereas the extreme C-terminal region is essential for its function

    OpenAIRE

    Nitharwal, Ram Gopal; Paul, Subhankar; Dar, Ashraf; Choudhury, Nirupam Roy; Soni, Rajesh K; Prusty, Dhaneswar; Sinha, Sukrat; Kashav, Tara; Mukhopadhyay, Gauranga; Chaudhuri, Tapan Kumar; Gourinath, Samudrala; Dhar, Suman Kumar

    2007-01-01

    Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase...

  5. Regional occurrence, high frequency but low diversity of mitochondrial DNA haplogroup d1 suggests a recent dog-wolf hybridization in Scandinavia

    Science.gov (United States)

    Klütsch, C F C; Seppälä, E H; Fall, T; Uhlén, M; Hedhammar, Å; Lohi, H; Savolainen, P

    2011-01-01

    The domestic dog mitochondrial DNA (mtDNA)-gene pool consists of a homogenous mix of haplogroups shared among all populations worldwide, indicating that the dog originated at a single time and place. However, one small haplogroup, subclade d1, found among North Scandinavian/Finnish spitz breeds at frequencies above 30%, has a clearly separate origin. We studied the genetic and geographical diversity for this phylogenetic group to investigate where and when it originated and whether through independent domestication of wolf or dog-wolf crossbreeding. We analysed 582 bp of the mtDNA control region for 514 dogs of breeds earlier shown to harbour d1 and possibly related northern spitz breeds. Subclade d1 occurred almost exclusively among Swedish/Finnish Sami reindeer-herding spitzes and some Swedish/Norwegian hunting spitzes, at a frequency of mostly 60–100%. Genetic diversity was low, with only four haplotypes: a central, most frequent, one surrounded by two haplotypes differing by an indel and one differing by a substitution. The substitution was found in a single lineage, as a heteroplasmic mix with the central haplotype. The data indicate that subclade d1 originated in northern Scandinavia, at most 480–3000 years ago and through dog-wolf crossbreeding rather than a separate domestication event. The high frequency of d1 suggests that the dog-wolf hybrid phenotype had a selective advantage. PMID:20497152

  6. Regional occurrence, high frequency but low diversity of mitochondrial DNA haplogroup d1 suggests a recent dog-wolf hybridization in Scandinavia.

    Science.gov (United States)

    Klütsch, C F C; Seppälä, E H; Fall, T; Uhlén, M; Hedhammar, A; Lohi, H; Savolainen, P

    2011-02-01

    The domestic dog mitochondrial DNA (mtDNA)-gene pool consists of a homogenous mix of haplogroups shared among all populations worldwide, indicating that the dog originated at a single time and place. However, one small haplogroup, subclade d1, found among North Scandinavian/Finnish spitz breeds at frequencies above 30%, has a clearly separate origin. We studied the genetic and geographical diversity for this phylogenetic group to investigate where and when it originated and whether through independent domestication of wolf or dog-wolf crossbreeding. We analysed 582 bp of the mtDNA control region for 514 dogs of breeds earlier shown to harbour d1 and possibly related northern spitz breeds. Subclade d1 occurred almost exclusively among Swedish/Finnish Sami reindeer-herding spitzes and some Swedish/Norwegian hunting spitzes, at a frequency of mostly 60-100%. Genetic diversity was low, with only four haplotypes: a central, most frequent, one surrounded by two haplotypes differing by an indel and one differing by a substitution. The substitution was found in a single lineage, as a heteroplasmic mix with the central haplotype. The data indicate that subclade d1 originated in northern Scandinavia, at most 480-3000 years ago and through dog-wolf crossbreeding rather than a separate domestication event. The high frequency of d1 suggests that the dog-wolf hybrid phenotype had a selective advantage. © 2010 The Authors, Animal Genetics © 2010 Stichting International Foundation for Animal Genetics.

  7. DNA methylation of specific CpG sites in the promoter region regulates the transcription of the mouse oxytocin receptor.

    Directory of Open Access Journals (Sweden)

    Shimrat Mamrut

    Full Text Available Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter.

  8. Variation in whole DNA methylation in red maple (Acer rubrum) populations from a mining region: association with metal contamination and cation exchange capacity (CEC) in podzolic soils.

    Science.gov (United States)

    Kalubi, K N; Mehes-Smith, M; Spiers, G; Omri, A

    2017-04-01

    Although a number of publications have provided convincing evidence that abiotic stresses such as drought and high salinity are involved in DNA methylation reports on the effects of metal contamination, pH, and cation exchange on DNA modifications are limited. The main objective of the present study is to determine the relationship between metal contamination and Cation exchange capacity (CEC) on whole DNA modifications. Metal analysis confirms that nickel and copper are the main contaminants in sampled sites within the Greater Sudbury Region (Ontario, Canada) and liming has increased soil pH significantly even after 30 years following dolomitic limestone applications. The estimated CEC values varied significantly among sites, ranging between 1.8 and 10.5 cmol(+) kg -1 , with a strong relationship being observed between CEC and pH (r = 0.96**). Cation exchange capacity, significantly lower in highly metal contaminated sites compared to both reference and less contaminated sites, was higher in the higher organic matter limed compared to unlimed sites. There was a significant variation in the level of cytosine methylation among the metal-contaminated sites. Significant and strong negative correlations between [5mdC]/[dG] and bioavailable nickel (r = -0.71**) or copper (r = -0.72**) contents were observed. The analysis of genomic DNA for adenine methylation in this study showed a very low level of [6N-mdA]/dT] in Acer rubrum plants analyzed ranging from 0 to 0.08%. Significant and very strong positive correlation was observed between [6N-mdA]/dT] and soil bioavailable nickel (r = 0.78**) and copper (r = 0.88**) content. This suggests that the increased bioavailable metal levels associated with contamination by nickel and copper particulates are associated with cytosine and adenine methylation.

  9. Specific identification of Western Atlantic Ocean scombrids using mitochondrial DNA cytochrome c oxidase subunit I (COI) gene region sequences

    National Research Council Canada - National Science Library

    Paine, Melissa A; McDowell, Jan R; Graves, John E

    2007-01-01

    .... The mitochondrial cytochrome c oxidase subunit I (COI) gene region was evaluated as a molecular marker for the specific identification of the 17 members of the family Scombridae common to the western Atlantic Ocean...

  10. Induction of DNA Demethylation Depending on Two Sets of Sox2 and Adjacent Oct3/4 Binding Sites (Sox-Oct Motifs) within the Mouse H19/Insulin-like Growth Factor 2 (Igf2) Imprinted Control Region

    Science.gov (United States)

    Hori, Naohiro; Yamane, Mariko; Kouno, Kaori; Sato, Kenzo

    2012-01-01

    DNA demethylation is used to establish and maintain an unmethylated state. The molecular mechanisms to induce DNA demethylation at a particular genomic locus remain unclear. The mouse H19/insulin-like growth factor 2 (Igf2) imprinted control region (ICR) is a methylation state-sensitive insulator that regulates transcriptional activation of both genes. The unmethylated state of the ICR established in female germ cells is maintained during development, resisting the wave of genome-wide de novo methylation. We previously demonstrated that a DNA fragment (fragment b) derived from this ICR-induced DNA demethylation when it was transfected into undifferentiated mouse embryonal carcinoma cell lines. Moreover, two octamer motifs within fragment b were necessary to induce this DNA demethylation. Here, we demonstrated that both octamer motifs and their flanking sequences constitute Sox-Oct motifs (SO1 and SO2) and that the SO1 region, which requires at least four additional elements, including the SO2 region, contributes significantly to the induction of high-frequency DNA demethylation as a Sox-Oct motif. Moreover, RNAi-mediated inhibition of Oct3/4 expression in P19 cells resulted in a reduced DNA demethylation frequency of fragment b but not of the adenine phosphoribosyltransferase gene CpG island. The Sox motif of SO1 could function as a sensor for a hypermethylated state of the ICR to repress demethylation activity. These results indicate that Sox-Oct motifs in the ICR determine the cell type, DNA region, and allele specificity of DNA demethylation. We propose a link between the mechanisms for maintenance of the unmethylated state of the H19/Igf2 ICR and the undifferentiated cell-specific induction of DNA demethylation. PMID:23115243

  11. Detection of Coxiella burnetii DNA in Peridomestic and Wild Animals and Ticks in an Endemic Region (Canary Islands, Spain).

    Science.gov (United States)

    Bolaños-Rivero, Margarita; Carranza-Rodríguez, Cristina; Rodríguez, Noe F; Gutiérrez, Carlos; Pérez-Arellano, José-Luis

    2017-09-01

    Coxiella burnetii, the etiological agent of human Q fever, can infect mammals, birds, and arthropods. The Canary Islands (Spain) are considered an endemic territory, with a high prevalence in both humans and livestock. Nonetheless, there is no epidemiological information about the wild and peridomestic cycles of C. burnetii. Tissue samples from rodents on farms (100) and wild rabbits (129) were collected and assessed by PCR to detect C. burnetii DNA. In parallel, ticks were also collected from vegetation (1169), livestock (335), domestic dogs (169), and wild animals (65). Globally, eight rodents (8%) and two rabbits (1.5%) were found to be positive, with the spleen being the most affected organ. Tick species identified were Hyalomma lusitanicum, Rhipicephalus turanicus, Rhipicephalus sanguineus, and Rhipicephalus pusillus. Hyalomma lusitanicum (80%) was the main species identified in vegetation, livestock, and wild animals, whereas Rhipicephalus sanguineus was the most prevalent in domestic dogs. Overall, C. burnetii DNA was detected in 6.1% of the processed ticks, distributed between those removed from livestock (11.3%), domestic dogs (6.9%), and from wild animals (6%). Ticks from vegetation were all negative. Results suggest that, in the Canary Islands, C. burnetii develops in a peridomestic rather than a wild cycle.

  12. Investigating the prehistory of Tungusic peoples of Siberia and the Amur-Ussuri region with complete mtDNA genome sequences and Y-chromosomal markers.

    Directory of Open Access Journals (Sweden)

    Ana T Duggan

    Full Text Available Evenks and Evens, Tungusic-speaking reindeer herders and hunter-gatherers, are spread over a wide area of northern Asia, whereas their linguistic relatives the Udegey, sedentary fishermen and hunter-gatherers, are settled to the south of the lower Amur River. The prehistory and relationships of these Tungusic peoples are as yet poorly investigated, especially with respect to their interactions with neighbouring populations. In this study, we analyse over 500 complete mtDNA genome sequences from nine different Evenk and even subgroups as well as their geographic neighbours from Siberia and their linguistic relatives the Udegey from the Amur-Ussuri region in order to investigate the prehistory of the Tungusic populations. These data are supplemented with analyses of Y-chromosomal haplogroups and STR haplotypes in the Evenks, Evens, and neighbouring Siberian populations. We demonstrate that whereas the North Tungusic Evenks and Evens show evidence of shared ancestry both in the maternal and in the paternal line, this signal has been attenuated by genetic drift and differential gene flow with neighbouring populations, with isolation by distance further shaping the maternal genepool of the Evens. The Udegey, in contrast, appear quite divergent from their linguistic relatives in the maternal line, with a mtDNA haplogroup composition characteristic of populations of the Amur-Ussuri region. Nevertheless, they show affinities with the Evenks, indicating that they might be the result of admixture between local Amur-Ussuri populations and Tungusic populations from the north.

  13. Phylogenetic analysis of widely cultivated Ganoderma in China based on the mitochondrial V4-V6 region of SSU rDNA.

    Science.gov (United States)

    Zhou, X W; Su, K Q; Zhang, Y M

    2015-02-02

    Ganoderma mushroom is one of the most prescribed traditional medicines and has been used for centuries, particularly in China, Japan, Korea, and other Asian countries. In this study, different strains of Ganoderma spp and the genetic relationships of the closely related strains were identified and investigated based on the V4-V6 region of mitochondrial small subunit ribosomal DNA of the Ganoderma species. The sizes of the mitochondrial ribosomal DNA regions from different Ganoderma species showed 2 types of sequences, 2.0 or 0.5 kb. A phylogenetic tree was constructed, which revealed a high level of genetic diversity in Ganoderma species. Ganoderma lucidum G05 and G. eupense G09 strains were clustered into a G. resinaceum group. Ganoderma spp G29 and G22 strains were clustered into a G. lucidum group. However, Ganoderma spp G19, G20, and G21 strains were clustered into a single group, the G. lucidum AF214475, G. sinense, G. strum G17, G. strum G36, and G. sinense G10 strains contained an intron and were clustered into other groups.

  14. Organization of the TC and TE cT-DNA regions in Nicotiana otophora and functional analysis of three diverged TE-6b genes.

    Science.gov (United States)

    Chen, Ke; de Borne, François Dorlhac; Sierro, Nicolas; Ivanov, Nikolai V; Alouia, Malek; Koechler, Sandrine; Otten, Léon

    2018-02-03

    Nicotiana otophora contains Agrobacterium-derived T-DNA sequences introduced by horizontal gene transfer (Chen et al., 2014). Sixty-nine contigs were assembled into four different cT-DNAs, totalling 83 kb. TC and TE result from two successive transformation events, each followed by duplication, yielding two TC and two TE inserts. TC is also found in other Nicotiana species, whereas TE is unique for N. otophora. Both cT-DNA regions are partially duplicated inverted repeats. Analysis of the cT-DNA divergence patterns allowed reconstruction of the evolution of the TC and TE regions. TC and TE carry ten intact open reading frames. Three of these are TE-6b genes, derived from a single 6b gene carried by the Agrobacterium strain which inserted TE in the N. otophora ancestor. 6b genes have so far only been found on A. tumefaciens or A. vitis T-DNAs and strongly modify plant growth (Chen and Otten, 2016). The TE-6b genes were expressed in N. tabacum under the constitutive 2x35S promoter. TE-1-6b-R and TE-2-6b led to shorter plants, dark-green leaves, a strong increase in leaf vein development, and modified petiole wings. TE-1-6b-L expression led to a similar phenotype, but in addition, leaves show outgrowths at the margins, flowers were modified and plants became viviparous, i. e. embryos germinated in the capsules at an early stage of their development. Embryos could be rescued by culture in vitro. The TE-6b phenotypes are very different from the earlier described 6b phenotypes and could provide new insight into the mode of action of the 6b genes. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  15. Evidence on How a Conserved Glycine in the Hinge Region of HapR Regulates Its DNA Binding Ability: LESSONS FROM A NATURAL VARIANT.

    Energy Technology Data Exchange (ETDEWEB)

    M Dongre; N Singh; C Dureja; N Peddada; A Solanki; F Ashish; S Raychaudhuri

    2011-12-31

    HapR has been recognized as a quorum-sensing master regulator in Vibrio cholerae. Because it controls a plethora of disparate cellular events, the absence of a functional HapR affects the physiology of V. cholerae to a great extent. In the current study, we pursued an understanding of an observation of a natural protease-deficient non-O1, non-O139 variant V. cholerae strain V2. Intriguingly, a nonfunctional HapR (henceforth designated as HapRV2) harboring a substitution of glycine to aspartate at position 39 of the N-terminal hinge region has been identified. An in vitro gel shift assay clearly suggested the inability of HapRV2 to interact with various cognate promoters. Reinstatement of glycine at position 39 restores DNA binding ability of HapRV2 (HapRV2G), thereby rescuing the protease-negative phenotype of this strain. The elution profile of HapRV2 and HapRV2G proteins in size-exclusion chromatography and their circular dichroism spectra did not reflect any significant differences to explain the functional discrepancies between the two proteins. To gain insight into the structure-function relationship of these two proteins, we acquired small/wide angle x-ray scattering data from samples of the native and G39D mutant. Although Guinier analysis and indirect Fourier transformation of scattering indicated only a slight difference in the shape parameters, structure reconstruction using dummy amino acids concluded that although HapR adopts a 'Y' shape similar to its crystal structure, the G39D mutation in hinge drastically altered the DNA binding domains by bringing them in close proximity. This altered spatial orientation of the helix-turn-helix domains in this natural variant provides the first structural evidence on the functional role of the hinge region in quorum sensing-related DNA-binding regulatory proteins of Vibrio spp.

  16. The 172-kb genomic DNA region of the O. rufipogon yld1.1 locus: comparative sequence analysis with O. sativa ssp. japonica and O. sativa ssp. indica.

    Science.gov (United States)

    Song, Beng-Kah; Hein, Ingo; Druka, Arnis; Waugh, Robbie; Marshall, David; Nadarajah, Kalaivani; Yap, Soon-Joo; Ratnam, Wickneswari

    2009-02-01

    Common wild rice (Oryza rufipogon) plays an important role by contributing to modern rice breeding. In this paper, we report the sequence and analysis of a 172-kb genomic DNA region of wild rice around the RM5 locus, which is associated with the yield QTL yld1.1. Comparative sequence analysis between orthologous RM5 regions from Oryza sativa ssp. japonica, O. sativa ssp. indica and O. rufipogon revealed a high level of conserved synteny in the content, homology, structure, orientation, and physical distance of all 14 predicted genes. Twelve of the putative genes were supported by matches to proteins with known function, whereas two were predicted by homology to rice and other plant expressed sequence tags or complementary DNAs. The remarkably high level of conservation found in coding, intronic and intergenic regions may indicate high evolutionary selection on the RM5 region. Although our analysis has not defined which gene(s) determine the yld1.1 phenotype, allelic variation and the insertion of transposable elements, among other nucleotide changes, represent potential variation responsible for the yield QTL. However, as suggested previously, two putative receptor-like protein kinase genes remain the key suspects for yld1.1.

  17. Polymorphism and genomic structure of the A+T-rich region of mitochondrial DNA in the oriental mole cricket, Gryllotalpa Orientalis (Orthoptera: Gryllotalpidae).

    Science.gov (United States)

    Kim, Iksoo; Cha, So Young; Kim, Mi Ae; Lee, Young Shin; Lee, Kwang Sik; Choi, Yong Soo; Hwang, Jae Sam; Jin, Byung Rae; Han, Yeon Soo

    2007-08-01

    The complete A+T-rich region of mitochondrial DNA has been cloned and sequenced from 48 individuals of Gryllotalpa orientalis (Orthoptera: Gryllotalpidae), collected from five Korean localities. Thirty-six haplotypes acquired from 48 individuals were found to range in size from 917 to 925 bp, with a sequence divergence from 0.1% to 14.3% and an A+T content from 74.5 to 76.3%. Phylogeographic analysis of the G. orientalis haplotypes showed the presence of two clearly differentiated mitochondrial clades, separated by 13.4% of a minimum uncorrected sequence divergence, which suggests the presence of an unknown, similar Gryllotalpa species or a once isolated G. orientalis population. Structural analysis in search of the conserved structural elements previously described in the caeliferan Orthoptera and Diptera revealed that the G. orientalis A+T-rich region harbored a stretch of the [TA(A)]n sequence, which has been suggested to be involved in the control of transcription or replication. In contrast to the abundance of the sequence stretches containing the stem-and-loop structure in the G. orientalis A+T-rich region, the 3' flanking sequence "G(A)nT," which is well conserved in a variety of organisms, including the caeliferan Orthoptera and Diptera, was not conserved in the A+T-rich region of G. orientalis.

  18. Nucleotide sequence of the promoter-distal region of the tra operon of plasmid R100, including traI (DNA helicase I) and traD genes.

    Science.gov (United States)

    Yoshioka, Y; Fujita, Y; Ohtsubo, E

    1990-07-05

    The nucleotide sequence of the promoter-distal region of the tra operon of R100 was determined. There are five open reading frames in the region between traT and finO, and their protein products were identified. Nucleotide sequences of plasmid F corresponding to the junction regions among the open reading frames seen in R100 were also determined. Comparison of these nucleotide sequences revealed strong homology in the regions containing traD, traI and an open reading frame (named orfD). The TraD protein (83,899 Da) contains three hydrophobic regions, of which two are located near the amino-terminal region. This protein also contains a possible ATP-binding consensus sequence at the amino-terminal region and a characteristic repeated peptide sequence (Gln-Gln-Pro)10 at the carboxy-terminal region. The TraI protein (191,679 Da) contains the sequence motif conserved in an ATP-dependent DNA helicase superfamily in its carboxy-terminal region. The protein product of orfD, which is probably a new tra gene (named traX), contains 65% hydrophobic amino acids, especially rich in alanine and leucine. There exist non-homologous regions between R100 and F that could be represented as four I-D (insertion or deletion) loops in heteroduplex molecules. Assignment of each loop to the strand of R100 or F was , however, found to be the reverse from that previously assumed. The three I-D loops that were located between traT and traD, between traD and traI, and between traI and finO had no terminal inverted repeat sequences nor had they any homology with known insertion sequences, while the fourth was IS3, located within the finO gene of F. The sequences in the I-D loops, except IS3, may also code for proteins that are, however, likely to be nonessential for transfer of plasmids.

  19. Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen.

    Science.gov (United States)

    Bauer, Asli; Podola, Lilli; Mann, Philipp; Missanga, Marco; Haule, Antelmo; Sudi, Lwitiho; Nilsson, Charlotta; Kaluwa, Bahati; Lueer, Cornelia; Mwakatima, Maria; Munseri, Patricia J; Maboko, Leonard; Robb, Merlin L; Tovanabutra, Sodsai; Kijak, Gustavo; Marovich, Mary; McCormack, Sheena; Joseph, Sarah; Lyamuya, Eligius; Wahren, Britta; Sandström, Eric; Biberfeld, Gunnel; Hoelscher, Michael; Bakari, Muhammad; Kroidl, Arne; Geldmacher, Christof

    2017-09-15

    Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gagp37 and two vaccinations with MVA-CMDR encoding subtype A Gagp55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ+) Gag-specific T-cell responses were dominated by CD4+ T cells (P viruses. While including multiple variants for a given immunogen in prime-boost vaccination strategies is one approach that aims to improve coverage for global virus variants, the immunologic consequences of this strategy have been poorly defined so far. It is unclear whether inclusion of multiple variants in prime-boost vaccination strategies improves recognition of variant viruses by T cells and by which mechanisms this would be achieved, either by improved cross-recognition of multiple variants for a given antigenic region or through preferential targeting of antigenic regions more conserved between prime and boost. Engineering vaccines to induce adaptive immune responses that preferentially target conserved antigenic regions of viral vulnerability might facilitate better immune control after preventive and therapeutic vaccination for HIV and for other variable viruses. Copyright © 2017 American Society for Microbiology.

  20. Ancient paralogy in the cpDNA trnL-F region in Annonaceae: implications for plant molecular systematics

    NARCIS (Netherlands)

    Pirie, M.D.; Vargas, M.P.B.; Botermans, M.; Bakker, F.T.; Chatrou, L.W.

    2007-01-01

    The plastid trnL-F region has proved useful in molecular phylogenetic studies addressing diverse evolutionary questions from biogeographic history to character evolution in a broad range of plant groups. An important assumption for phylogenetic reconstruction is that data used in combined analyses

  1. BENZO[A]PYRENE AND ITS K-REGION DIOL INDUCE DNA DAMAGE IN C3H10T1/2C18 CELLS AS MEASURED BY THE ALKALINE SINGLE CELL GEL (COMET) ASSAY

    Science.gov (United States)

    160. Benzo[a]pyrene and its K-region diol induce DNA damage in C3HlOTl/2Cl8 cells as measured by the alkaline single cell gel (Comet) assay In a continuing series of studies on the genotoxicity ofK-region dihydrodiols of polycyclic aromatic hydrocarbons, we have repo...

  2. The 5S rDNA gene family in mollusks: characterization of transcriptional regulatory regions, prediction of secondary structures, and long-term evolution, with special attention to Mytilidae mussels.

    Science.gov (United States)

    Vizoso, Miguel; Vierna, Joaquín; González-Tizón, Ana M; Martínez-Lage, Andrés

    2011-01-01

    Several reports on the characterization of 5S ribosomal DNA (5S rDNA) in various animal groups have been published to date, but there is a lack of studies analyzing this gene family in a much broader context. Here, we have studied 5S rDNA variation in several molluskan species, including bivalves, gastropods, and cephalopods. The degree of conservation of transcriptional regulatory regions was analyzed in these lineages, revealing a conserved TATA-like box in the upstream region. The evolution of the 120 bp coding region (5S) was also studied, suggesting the occurrence of paralogue groups in razor clams, clams, and cockles. In addition, 5S rDNA sequences from 11 species and 7 genus of Mytilidae Rafinesque, 1815 mussels were sampled and studied in detail. Four different 5S rDNA types, based on the nontranscribed spacer region were identified. The phylogenetic analyses performed within each type showed a between-species gene clustering pattern, suggesting ancestral polymorphism. Moreover, some putative pseudogenized 5S copies were also identified. Our report, together with previous studies that found high degree of intragenomic divergence in bivalve species, suggests that birth-and-death evolution may be the main force driving the evolution of 5S rDNA in these animals, even at the genus level.

  3. X-ray crystal structure of the N-terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition: N-Terminal Region of M-MuLV Integrase

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Rongjin [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Aiyer, Sriram [Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Cote, Marie L. [Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, Piscataway New Jersey 08854; Xiao, Rong [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Jiang, Mei [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Acton, Thomas B. [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Roth, Monica J. [Department of Pharmacology, Robert Wood Johnson Medical School, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Montelione, Gaetano T. [Center for Advanced Biotechnology and Medicine, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway New Jersey 08854; Department of Biochemistry, Robert Wood Johnson Medical School, UMDNJ, Piscataway New Jersey 08854

    2017-02-03

    The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N-terminal domain (NTD), the catalytic core domain, and the C-terminal domain. The NTD includes an HHCC zinc finger-like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M-MuLV) IN N-terminal region (NTR) constructs that both include an N-terminal extension domain (NED, residues 1–44) and an HHCC zinc-finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR1–105) and 8–105 (NTR8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR1–105 packs as a dimer and NTR8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti-parallel β-strands and an α-helix, similar to the NED of prototype foamy virus (PFV) IN. These three β-strands form an extended β-sheet with another β-strand in the HHCC Zn2+ binding domain, which is a unique structural feature for the M-MuLV IN. The HHCC Zn2+ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M-MuLV. Proteins 2017; 85:647–656.

  4. Seladin-1 is a fundamental mediator of the neuroprotective effects of estrogen in human neuroblast long-term cell cultures.

    Science.gov (United States)

    Luciani, Paola; Deledda, Cristiana; Rosati, Fabiana; Benvenuti, Susanna; Cellai, Ilaria; Dichiara, Francesca; Morello, Matteo; Vannelli, Gabriella Barbara; Danza, Giovanna; Serio, Mario; Peri, Alessandro

    2008-09-01

    Estrogen exerts neuroprotective effects and reduces beta-amyloid accumulation in models of Alzheimer's disease (AD). A few years ago, a new neuroprotective gene, i.e. seladin-1 (for selective AD indicator-1), was identified and found to be down-regulated in AD vulnerable brain regions. Seladin-1 inhibits the activation of caspase-3, a key modulator of apoptosis. In addition, it has been demonstrated that the seladin-1 gene encodes 3beta-hydroxysterol Delta24-reductase, which catalyzes the synthesis of cholesterol from desmosterol. We have demonstrated previously that in fetal neuroepithelial cells, 17beta-estradiol (17betaE2), raloxifene, and tamoxifen exert neuroprotective effects and increase the expression of seladin-1. The aim of the present study was to elucidate whether seladin-1 is directly involved in estrogen-mediated neuroprotection. Using the small interfering RNA methodology, significantly reduced levels of seladin-1 mRNA and protein were obtained in fetal neuroepithelial cells. Seladin-1 silencing determined the loss of the protective effect of 17betaE2 against beta-amyloid and oxidative stress toxicity and caspase-3 activation. A computer-assisted analysis revealed the presence of half-palindromic estrogen responsive elements upstream from the coding region of the seladin-1 gene. A 1490-bp region was cloned in a luciferase reporter vector, which was transiently cotransfected with the estrogen receptor alpha in Chinese hamster ovarian cells. The exposure to 17betaE2, raloxifene, tamoxifen, and the soy isoflavones genistein and zearalenone increased luciferase activity, thus suggesting a functional role for the half-estrogen responsive elements of the seladin-1 gene. Our data provide for the first time a direct demonstration that seladin-1 may be considered a fundamental mediator of the neuroprotective effects of estrogen.

  5. Loss of genetic variability in a hatchery strain of Senegalese sole (Solea senegalensis revealed by sequence data of the mitochondrial DNA control region and microsatellite markers

    Directory of Open Access Journals (Sweden)

    Pablo Sánchez

    2012-06-01

    Full Text Available Comparisons of the levels of genetic variation within and between a hatchery F1 (FAR, n=116 of Senegalese sole, Solea senegalensis, and its wild donor population (ATL, n = 26, both native to the SW Atlantic coast of the Iberian peninsula, as well as between the wild donor population and a wild western Mediterranean sample (MED, n=18, were carried out by characterizing 412 base pairs of the nucleotide sequence of the mitochondrial DNA control region I, and six polymorphic microsatellite loci. FAR showed a substantial loss of genetic variability (haplotypic diversity, h=0.49±0.066; nucleotide diversity, π=0.006±0.004; private allelic richness, pAg=0.28 to its donor population ATL (h=0.69±0.114; π=0.009±0.006; pAg=1.21. Pairwise FST values of microsatellite data were highly significant (P < 0.0001 between FAR and ATL (0.053 and FAR and MED (0.055. The comparison of wild samples revealed higher values of genetic variability in MED than in ATL, but only with mtDNA CR-I sequence data (h=0.948±0.033; π=0.030±0.016. However, pairwise ΦST and FST values between ATL and MED were highly significant (P < 0.0001 with mtDNA CR-I (0.228 and with microsatellite data (0.095, respectively. While loss of genetic variability in FAR could be associated with the sampling error when the broodstock was established, the results of parental and sibship inference suggest that most of these losses can be attributed to a high variance in reproductive success among members of the broodstock, particularly among females.

  6. Chronic Fatigue Syndrome and DNA Hypomethylation of the Glucocorticoid Receptor Gene Promoter 1F Region: Associations With HPA Axis Hypofunction and Childhood Trauma.

    Science.gov (United States)

    Vangeel, Elise; Van Den Eede, Filip; Hompes, Titia; Izzi, Benedetta; Del Favero, Jurgen; Moorkens, Greta; Lambrechts, Diether; Freson, Kathleen; Claes, Stephan

    2015-10-01

    Chronic fatigue syndrome (CFS) has been associated with hypothalamic-pituitary-adrenal axis hypofunction and enhanced glucocorticoid receptor (GR) sensitivity. In addition, childhood trauma is considered a major risk factor for the syndrome. This study examines DNA methylation of the GR gene (NR3C1) in CFS and associations with childhood sexual and physical trauma. Quantification of DNA methylation within the 1F promoter region of NR3C1 was performed in 76 female patients (46 with no/mild and 30 with moderate/severe childhood trauma) and 19 healthy controls by using Sequenom EpiTYPER. Further, we examined the association of NR3C1-1F promoter methylation with the outcomes of the low-dose (0.5 mg) dexamethasone/corticotropin-releasing factor test in a subset of the study population. Mann-Whitney U tests and Spearman correlations were used for statistical analyses. Overall NR3C1-1F DNA methylation was lower in patients with CFS than in controls. After cytosine guanine dinucleotide (CpG)-specific analysis, CpG_1.5 remained significant after Bonferroni correction (adjusted p = .0014). Within the CFS group, overall methylation (ρ = 0.477, p = .016) and selective CpG units (CpG_1.5: ρ = 0.538, p = .007; CpG_12.13: ρ = 0.448, p = .025) were positively correlated with salivary cortisol after dexamethasone administration. There was no significant difference in NR3C1-1F methylation between traumatized and nontraumatized patients. We found evidence of NR3C1 promoter hypomethylation in female patients with CFS and the functional relevance of these differences was consistent with the hypothalamic-pituitary-adrenalaxis hypofunction hypothesis (GR hypersuppression). However, we found no evidence of an additional effect of childhood trauma on CFS via alterations in NR3C1 methylation.

  7. Generational comparisons (F1 versus F3) of vinclozolin induced epigenetic transgenerational inheritance of sperm differential DNA methylation regions (epimutations) using MeDIP-Seq.

    Science.gov (United States)

    Beck, Daniel; Sadler-Riggleman, Ingrid; Skinner, Michael K

    2017-07-01

    Environmentally induced epigenetic transgenerational inheritance of disease and phenotypic variation has been shown to involve DNA methylation alterations in the germline (e.g. sperm). These differential DNA methylation regions (DMRs) are termed epimutations and in part transmit the transgenerational phenotypes. The agricultural fungicide vinclozolin exposure of a gestating female rat has previously been shown to promote transgenerational disease and epimutations in F3 generation (great-grand-offspring) animals. The current study was designed to investigate the actions of direct fetal exposure on the F1 generation rat sperm DMRs compared to the F3 transgenerational sperm DMRs. A protocol involving methylated DNA immunoprecipitation (MeDIP) followed by next-generation sequencing (Seq) was used in the current study. Bioinformatics analysis of the MeDIP-Seq data was developed and several different variations in the bioinformatic analysis were evaluated. Observations indicate needs to be considered. Interestingly, the F1 generation DMRs were found to be fewer in number and for the most part distinct from the F3 generation epimutations. Observations suggest the direct exposure induced F1 generation sperm DMRs appear to promote in subsequent generations alterations in the germ cell developmental programming that leads to the distinct epimutations in the F3 generation. This may help explain the differences in disease and phenotypes between the direct exposure F1 generation and transgenerational F3 generation. Observations demonstrate a distinction between the direct exposure versus transgenerational epigenetic programming induced by environmental exposures and provide insights into the molecular mechanisms involved in the epigenetic transgenerational inheritance phenomenon.

  8. Genome-wide Anaplasma phagocytophilum AnkA-DNA interactions are enriched in intergenic regions and gene promoters and correlate with infection-induced differential gene expression.

    Directory of Open Access Journals (Sweden)

    J Stephen Dumler

    2016-09-01

    Full Text Available Anaplasma phagocytophilum, an obligate intracellular prokaryote, infects neutrophils and alters cardinal functions via reprogrammed transcription. Large contiguous regions of neutrophil chromosomes are differentially expressed during infection. Secreted A. phagocytophilum effector AnkA transits into the neutrophil or granulocyte nucleus to complex with DNA in heterochromatin across all chromosomes. AnkA binds to gene promoters to dampen cis-transcription and also has features of matrix attachment region (MAR-binding proteins that regulate three-dimensional chromatin architecture and coordinate transcriptional programs encoded in topologically-associated chromatin domains. We hypothesize that identification of additional AnkA binding sites will better delineate how A. phagocytophilum infection results in reprogramming of the neutrophil genome. Using AnkA-binding ChIP-seq, we showed that AnkA binds broadly throughout all chromosomes in a reproducible pattern, especially at: i intergenic regions predicted to be matrix attachment regions (MARs; ii within predicted lamina-associated domains; and iii at promoters ≤3,000 bp upstream of transcriptional start sites. These findings provide genome-wide support for AnkA as a regulator of cis-gene transcription. Moreover, the dominant mark of AnkA in distal intergenic regions known to be AT-enriched, coupled with frequent enrichment in the nuclear lamina, provides strong support for its role as a MAR-binding protein and genome re-organizer. AnkA must be considered a prime candidate to promote neutrophil reprogramming and subsequent functional changes that belie improved microbial fitness and pathogenicity.

  9. Double differential cross sections for proton induced electron emission from molecular analogues of DNA constituents for energies in the Bragg peak region

    Science.gov (United States)

    Rudek, Benedikt; Bennett, Daniel; Bug, Marion U.; Wang, Mingjie; Baek, Woon Yong; Buhr, Ticia; Hilgers, Gerhard; Champion, Christophe; Rabus, Hans

    2016-09-01

    For track structure simulations in the Bragg peak region, measured electron emission cross sections of DNA constituents are required as input for developing parameterized model functions representing the scattering probabilities. In the present work, double differential cross sections were measured for the electron emission from vapor-phase pyrimidine, tetrahydrofuran, and trimethyl phosphate that are structural analogues to the base, the sugar, and the phosphate residue of the DNA, respectively. The range of proton energies was from 75 keV to 135 keV, the angles ranged from 15° to 135°, and the electron energies were measured from 10 eV to 200 eV. Single differential and total electron emission cross sections are derived by integration over angle and electron energy and compared to the semi-empirical Hansen-Kocbach-Stolterfoht (HKS) model and a quantum mechanical calculation employing the first Born approximation with corrected boundary conditions (CB1). The CB1 provides the best prediction of double and single differential cross section, while total cross sections can be fitted with semi-empirical models. The cross sections of the three samples are proportional to their total number of valence electrons.

  10. [Polymorphism of the mtDNA control region in wild reindeer Rangifer tarandus (Mammalia: Artiodactyla) from the European part of Russia].

    Science.gov (United States)

    Baranova, A I; Kholodova, M V; Davydov, A V; Rozhkov, Iu I

    2012-09-01

    Genetic diversity ofwild reindeer (Rangifer tarandus) inhabiting the European part of Russia, including Komi Republic, Arkhangelsk oblast, Murmansk oblast, and the Republic of Karelia was characterized using sequence polymorphism of the mtDNA control region. Despite of currently low population number of wild reindeer, they were characterized by a high level of genetic diversity (pi = 0.018; H= 0.872 to 0.914). Phylogenetic analysis showed close relationships between European reindeer and wild reindeer of Siberia. In reindeer from Murmansk oblast a haplotype in common with the wild reindeer form Southwestern Norway was described. The reindeer sample examined contained no haplotypes earlier described for the reindeer of Central Norway. It is suggested that in recent past wild reindeer from the European north of Russia formed one population with the reindeer from the north of the Asian part of Eurasia.

  11. DNA barcoding of Gobiid fishes (Perciformes: Gobiidae) from eastern and northeastern India with new record of a Gobionellinae species for the region.

    Science.gov (United States)

    Laskar, Boni Amin; Kumar, Vikas; Kundu, Shantanu; Tyagi, Kaomud; Singha, Devkant; Chakraborty, Rajasree; Chatterjee, Sumantika; Saha, Soumitra

    2017-07-01

    The study attempted identification of Gobiid fishes from freshwaters in the east and northeast India on a collection of 20 specimens. The DNA barcode data delineated the collected samples into three species clades in the neighbor-joining tree. The results confirmed the identification of five sample sequences belonging to the subfamily Gobionellinae due to cohesive cladding with Awaous congeners. This is a new subfamily record for the northeastern region. Another 15 sample sequences showed conspecific cladding with Glossogobius giuris in the database. Among the 15 sample sequences, 14 sequences cladded with G. giuris sequences of Indian specimens while one sample sequence cladded with G. giuris sequences of South African specimens. This indicated the presence of either a hidden species or a previously synonymized species in the G. giuris complex.

  12. Green turtles (Chelonia mydas foraging at Arvoredo Island in Southern Brazil: genetic characterization and mixed stock analysis through mtDNA control region haplotypes

    Directory of Open Access Journals (Sweden)

    Maíra Carneiro Proietti

    2009-01-01

    Full Text Available We analyzed mtDNA control region sequences of green turtles (Chelonia mydas from Arvoredo Island, a foraging ground in southern Brazil, and identified eight haplotypes. Of these, CM-A8 (64% and CM-A5 (22% were dominant, the remainder presenting low frequencies ( 0.05. Mixed Stock Analysis, incorporating eleven Atlantic and one Mediterranean rookery as possible sources of individuals, indicated Ascension and Aves islands as the main contributing stocks to the Arvoredo aggregation (68.01% and 22.96%, respectively. These results demonstrate the extensive relationships between Arvoredo Island and other Atlantic foraging and breeding areas. Such an understanding provides a framework for establishing adequate management and conservation strategies for this endangered species.

  13. Green turtles (Chelonia mydas) foraging at Arvoredo Island in Southern Brazil: Genetic characterization and mixed stock analysis through mtDNA control region haplotypes

    Science.gov (United States)

    2009-01-01

    We analyzed mtDNA control region sequences of green turtles (Chelonia mydas) from Arvoredo Island, a foraging ground in southern Brazil, and identified eight haplotypes. Of these, CM-A8 (64%) and CM-A5 (22%) were dominant, the remainder presenting low frequencies (Rocas/Noronha, in Brazil (p > 0.05). Mixed Stock Analysis, incorporating eleven Atlantic and one Mediterranean rookery as possible sources of individuals, indicated Ascension and Aves islands as the main contributing stocks to the Arvoredo aggregation (68.01% and 22.96%, respectively). These results demonstrate the extensive relationships between Arvoredo Island and other Atlantic foraging and breeding areas. Such an understanding provides a framework for establishing adequate management and conservation strategies for this endangered species. PMID:21637527

  14. DNA microsatellite region for a reliable quantification of soft wheat adulteration in durum wheat-based foodstuffs by real-time PCR.

    Science.gov (United States)

    Sonnante, Gabriella; Montemurro, Cinzia; Morgese, Anita; Sabetta, Wilma; Blanco, Antonio; Pasqualone, Antonella

    2009-11-11

    Italian industrial pasta and durum wheat typical breads must be prepared using exclusively durum wheat semolina. Previously, a microsatellite sequence specific of the wheat D-genome had been chosen for traceability of soft wheat in semolina and bread samples, using qualitative and quantitative Sybr green-based real-time experiments. In this work, we describe an improved method based on the same soft wheat genomic region by means of a quantitative real-time PCR using a dual-labeled probe. Standard curves based on dilutions of 100% soft wheat flour, pasta, or bread were constructed. Durum wheat semolina, pasta, and bread samples were prepared with increasing amounts of soft wheat to verify the accuracy of the method. Results show that reliable quantifications were obtained especially for the samples containing a lower amount of soft wheat DNA, fulfilling the need to verify labeling of pasta and typical durum wheat breads.

  15. Placentas from pregnancies conceived by IVF/ICSI have a reduced DNA methylation level at the H19 and MEST differentially methylated regions.

    Science.gov (United States)

    Nelissen, Ewka C M; Dumoulin, John C M; Daunay, Antoine; Evers, Johannes L H; Tost, Jörg; van Montfoort, Aafke P A

    2013-04-01

    Does IVF/ICSI have an effect on the epigenetic regulation of the human placenta? We found a reduced DNA methylation level at the H19 and MEST differentially methylated regions (DMRs), and an increased RNA expression of H19 in placentas from pregnancies conceived by IVF/ICSI when compared with placentas from spontaneous conception. Changes in fetal environment are associated with adverse health outcomes. The placenta is pivotal for intrauterine environment. Animal studies show that epigenetic regulation plays an important role in these environment-induced phenotypic effects. Also, the preimplantation embryo environment affects birthweight as well as the risk of chronic adult diseases. Epigenetic processes are sensitive to the environment, especially during the period around conception. Placental tissue was collected from 35 spontaneously conceived pregnancies and 35 IVF/ICSI (5 IVF, 30 ICSI) derived pregnancies. We quantitatively analysed the DNA methylation patterns of a number of consecutive CpGs in the core regions of DMRs and other regulatory regions of imprinted genes, since these are involved in placental and fetal growth and development. By using pyrosequencing, the DNA methylation at seven germline-derived primary DMRs was analysed quantitatively. Five of these are maternally methylated (MEST isoform α and β, PEG3, KCNQ1OT1 and SNRPN) and two are paternally methylated [H19 DMR and the intergenic region between DLK1 and MEG3 (IG-DMR)]. The post-fertilization-derived secondary DMRs, IGF2 (DMR0 and 2) and IG-DMR (CG7, also called MEG3 DMR), and the MEG3 promoter region were examined as well. In case of differential methylation between the two groups, the effect on gene expression was assessed by quantitative real-time PCR. Both the promoter region of MEST isoform α and β and the 6th CTCF binding site within the H19 DMR were significantly hypomethylated in the IVF/ICSI group. The phenomenon was consistently observed over all CpG sites analysed and not

  16. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  17. DNA Replication Origins in Immunoglobulin Switch Regions Regulate Class Switch Recombination in an R-Loop-Dependent Manner.

    Science.gov (United States)

    Wiedemann, Eva-Maria; Peycheva, Mihaela; Pavri, Rushad

    2016-12-13

    Class switch recombination (CSR) at the immunoglobulin heavy chain (IgH) locus generates antibody isotypes. CSR depends on double-strand breaks (DSBs) induced by activation-induced cytidine deaminase (AID). Although DSB formation and repair machineries are active in G1 phase, efficient CSR is dependent on cell proliferation and S phase entry; however, the underlying mechanisms are obscure. Here, we show that efficient CSR requires the replicative helicase, the Mcm complex. Mcm proteins are enriched at IgH switch regions during CSR, leading to assembly of facultative replication origins that require Mcm helicase function for productive CSR. Assembly of CSR-associated origins is facilitated by R loops and promotes the physical proximity (synapsis) of recombining switch regions, which is reduced by R loop inhibition or Mcm complex depletion. Thus, R loops contribute to replication origin specification that promotes DSB resolution in CSR. This suggests a mechanism for the dependence of CSR on S phase and cell division. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  18. Genetic relationships among some subspecies of the Peregrine Falcon (Falco peregrinus L.), inferred from mitochondrial DNA control-region sequences

    Science.gov (United States)

    White, Clayton M.; Sonsthagen, Sarah A.; Sage, George K.; Anderson, Clifford; Talbot, Sandra L.

    2013-01-01

    The ability to successfully colonize and persist in diverse environments likely requires broad morphological and behavioral plasticity and adaptability, and this may partly explain why the Peregrine Falcon (Falco peregrinus) exhibits a large range of morphological characteristics across their global distribution. Regional and local differences within Peregrine Falcons were sufficiently variable that ∼75 subspecies have been described; many were subsumed, and currently 19 are generally recognized. We used sequence information from the control region of the mitochondrial genome to test for concordance between genetic structure and representatives of 12 current subspecies and from two areas where subspecies distributions overlap. Haplotypes were broadly shared among subspecies, and all geographic locales shared a widely distributed common haplotype (FalconCR2). Haplotypes were distributed in a star-like phylogeny, consistent with rapid expansion of a recently derived species, with observed genetic patterns congruent with incomplete lineage sorting and/or differential rates of evolution on morphology and neutral genetic characters. Hierarchical analyses of molecular variance did not uncover genetic partitioning at the continental level, despite strong population-level structure (FST = 0.228). Similar analyses found weak partitioning, albeit significant, among subspecies (FCT = 0.138). All reconstructions placed the hierofalcons' (Gyrfalcon [F. rusticolus] and Saker Falcon [F. cherrug]) haplotypes in a well-supported clade either basal or unresolved with respect to the Peregrine Falcon. In addition, haplotypes representing Taita Falcon (F. fasciinucha) were placed within the Peregrine Falcon clade.

  19. Syntenic homology of human unique DNA sequences within chromossome regions 5q31, 10q22, 13q32-33 and 19q13.1 in the great apes

    Directory of Open Access Journals (Sweden)

    Vallente-Samonte Rhea U.

    2000-01-01

    Full Text Available Homologies between chromosome banding patterns and DNA sequences in the great apes and humans suggest an apparent common origin for these two lineages. The availability of DNA probes for specific regions of human chromosomes (5q31, 10q22, 13q32-33 and 19q13.1 led us to cross-hybridize these to chimpanzee (Pan troglodytes, PTR, gorilla (Gorilla gorilla, GGO and orangutan (Pongo pygmaeus, PPY chromosomes in a search for equivalent regions in the great apes. Positive hybridization signals to the chromosome 5q31-specific DNA probe were observed at HSA 5q31, PTR 4q31, GGO 4q31 and PPY 4q31, while fluorescent signals using the chromosome 10q22-specific DNA probe were noted at HSA 10q22, PTR 8q22, GGO 8q22 and PPY 7q22. The chromosome arms showing hybridization signals to the Quint-EssentialTM 13-specific DNA probe were identified as HSA 13q32-33, PTR 14q32-33, GGO 14q32-33 and PPY 14q32-33, while those presenting hybridization signals to the chromosome 19q13.1-specific DNA probe were identified as HSA 19q13.1, PTR 20q13, GGO 20q13 and PPY 20q13. All four probes presumably hybridized to homologous chromosomal locations in the apes, which suggests a homology of certain unique DNA sequences among hominoid species.

  20. COMPARACIÓN ENTRE EL POTENCIAL DE LAS REGIONES VARIABLES DEL 16S rDNA PARA LA IDENTIFICACIÓN DE Lactobacillus spp. (LACTOBACILLIACEAE

    Directory of Open Access Journals (Sweden)

    LAURA N. GONZÁLEZ GARCÍA

    2013-01-01

    Full Text Available El 16s rDNA es utilizado para la identificación bacteriana dada su tasa de variación entre especies. Algunas de las regiones variables de la subunidad ribosomal son más informativas que otras por lo cual en este estudio se evalúa el potencial de identificación aportado por cada región y combinaciones entre ellas. Se extrajeron las regiones variables V1 a la V8 del 16s rDNA de diferentes cepas y especies de Lactobacillus y se analizaron mediante los paquetes de STAP (ss-RNA Taxonomy Assigning Pipeline y RDP (Ribosomal Database Project multiclassifier. Adicionalmente se evaluaron árboles filogenéticos de máxima verosimilitud. Nuestros resultados muestran que la mayoría de regiones variables logran dar una correcta clasificación hasta género, sin embargo no son suficientes para clasificar hasta especie usando STAP. La región que presenta el mayor número de amplímeros es V5V6, sin embargo es la que presenta la mayor cantidad de falsos negativos. La que presenta el mayor número de verdaderos positivos es V1V3 (especie para STAP y V5V8(género para RDP. Las filogenias evaluadas mostraron que la topología de referencia se puede obtener con diferentes combinaciones de regiones variables e.g., V1V3 y V1V8. El estudio experimental de las cepas contenidas en un tampón comercial mostró que el amplicón V1V8 y el V1V3 dan una misma clasificación correcta. Proponemos la región V1V3 como la región mínima para clasificación correcta de Lactobacillus spp.. En conclusión, la región mínima para clasificar especies del género Lactobacillus es la V1V3, la cual es útil para estudios meta- genómicos de muestras de probióticos.

  1. Phylogenetic Analysis of a 'Jewel Orchid' Genus Goodyera (Orchidaceae) Based on DNA Sequence Data from Nuclear and Plastid Regions.

    Science.gov (United States)

    Hu, Chao; Tian, Huaizhen; Li, Hongqing; Hu, Aiqun; Xing, Fuwu; Bhattacharjee, Avishek; Hsu, Tianchuan; Kumar, Pankaj; Chung, Shihwen

    2016-01-01

    A molecular phylogeny of Asiatic species of Goodyera (Orchidaceae, Cranichideae, Goodyerinae) based on the nuclear ribosomal internal transcribed spacer (ITS) region and two chloroplast loci (matK and trnL-F) was presented. Thirty-five species represented by 132 samples of Goodyera were analyzed, along with other 27 genera/48 species, using Pterostylis longifolia and Chloraea gaudichaudii as outgroups. Bayesian inference, maximum parsimony and maximum likelihood methods were used to reveal the intrageneric relationships of Goodyera and its intergeneric relationships to related genera. The results indicate that: 1) Goodyera is not monophyletic; 2) Goodyera could be divided into four sections, viz., Goodyera, Otosepalum, Reticulum and a new section; 3) sect. Reticulum can be further divided into two subsections, viz., Reticulum and Foliosum, whereas sect. Goodyera can in turn be divided into subsections Goodyera and a new subsection.

  2. High antiviral effect of TiO2·PL–DNA nanocomposites targeted to conservative regions of (−RNA and (+RNA of influenza A virus in cell culture

    Directory of Open Access Journals (Sweden)

    Asya S. Levina

    2016-08-01

    Full Text Available Background: The development of new antiviral drugs based on nucleic acids is under scrutiny. An important problem in this aspect is to find the most vulnerable conservative regions in the viral genome as targets for the action of these agents. Another challenge is the development of an efficient system for their delivery into cells. To solve this problem, we proposed a TiO2·PL–DNA nanocomposite consisting of titanium dioxide nanoparticles and polylysine (PL-containing oligonucleotides.Results: The TiO2·PL–DNA nanocomposites bearing the DNA fragments targeted to different conservative regions of (−RNA and (+RNA of segment 5 of influenza A virus (IAV were studied for their antiviral activity in MDCK cells infected with the H1N1, H5N1, and H3N2 virus subtypes. Within the negative strand of each of the studied strains, the efficiency of DNA fragments increased in the direction of its 3’-end. Thus, the DNA fragment aimed at the 3’-noncoding region of (−RNA was the most efficient and inhibited the reproduction of different IAV subtypes by 3–4 orders of magnitude. Although to a lesser extent, the DNA fragments targeted at the AUG region of (+RNA and the corresponding region of (−RNA were also active. For all studied viral subtypes, the nanocomposites bearing the DNA fragments targeted to (−RNA appeared to be more efficient than those containing fragments aimed at the corresponding (+RNA regions.Conclusion: The proposed TiO2·PL–DNA nanocomposites can be successfully used for highly efficient and site-specific inhibition of influenza A virus of different subtypes. Some patterns of localization of the most vulnerable regions in IAV segment 5 for the action of DNA-based drugs were found. The (−RNA strand of IAV segment 5 appeared to be more sensitive as compared to (+RNA.

  3. DNA barcoding and regional diversity of understudied Micropeplinae (Coleoptera: Staphylinidae) in Southwest China: phylogenetic implications and a new Micropeplus from Mount Emei.

    Science.gov (United States)

    Grebennikov, Vasily V; Smetana, Aleš

    2015-02-18

    Extensive litter sampling at eight forested localities in Yunnan and Sichuan detected 381 specimens of Micropeplinae rove beetles. DNA barcoding data from 85 representative specimens were analysed to delimit species and infer their relationships. Statistical methods were implemented to assess regional species diversity of understudied Micropeplinae. The total number of sampled Micropeplinae species varied between 14 and 17, depending on a splitting versus lumping approach for allopatric populations. A single Micropeplinae species was sampled in six of eight studied localities, three species were found on Mount Gongga, while ten species were discovered on hyperdiverse Mount Emei in Sichuan. All Micropeplinae specimens from our samples belong either to the genus Cerapeplus, or to three other inclusive groups temporarily retained inside Micropeplus sensu lato. Each of the three groups potentially represents a separate genus: tesserula group, sculptus group and Micropeplus sensu stricto. A new species Micropeplus jason sp. n. from Mount Emei in Sichuan is described. Numerous illustrations introduce regional fauna and clarify the discussed morphological characters.

  4. Molecular characterization of the Indian poultry nodular tapeworm, Raillietina echinobothrida (Cestoda: Cyclophyllidea: Davaineidae) based on rDNA internal transcribed spacer 2 region.

    Science.gov (United States)

    Ramnath; Jyrwa, D B; Dutta, A K; Das, B; Tandon, V

    2014-03-01

    The nodular tapeworm, Raillietina echinobothrida is a well studied avian gastrointestinal parasite of family Davaineidae (Cestoda: Cyclophyllidea). It is reported to be the largest in size and second most prevalent species infecting chicken in north-east India. In the present study, morphometrical methods coupled with the molecular analysis of the second internal transcribed spacer (ITS2) region of ribosomal DNA were employed for precise identification of the parasite. The annotated ITS2 region was found to be 446 bp long and further utilized to elucidate the phylogenetic relationships and its species-interrelationships at the molecular level. In phylogenetic analysis similar topology was observed among the trees obtained by distance-based neighbor-joining as well as character-based maximum parsimony tree building methods. The query sequence R. echinobothrida is well aligned and placed within the Davaineidae group, with all Raillietina species well separated from the other cyclophyllidean (taeniid and hymenolepid) cestodes, while Diphyllobothrium latum (Pseudophyllidea: Diphyllobothriidae) was rooted as an out-group. Sequence similarities indeed confirmed our hypothesis that Raillietina spp. are neighboring the position with other studied species of order Cyclophyllidea against the out-group order Pseudophyllidea. The present study strengthens the potential of ITS2 as a reliable marker for phylogenetic reconstructions.

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

    Energy Technology Data Exchange (ETDEWEB)

    Onodera, Osamu; Oyake, Mutsuo; Takano, Hiroki [Niigata Univ. (Japan)] [and others

    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.

  6. Origin and genetic diversity of Egyptian native chickens based on complete sequence of mitochondrial DNA D-loop region.

    Science.gov (United States)

    Osman, Sayed A-M; Yonezawa, Takahiro; Nishibori, Masahide

    2016-06-01

    Domestic chickens (Gallus gallus) play a significant role, ranging from food and entertainment to religion and ornamentation. However, the details on their domestication process are still controversial, especially the origin and evolution of African chickens. Egypt is thought to be important place for this event because of its geographic location as well as its long history of civilization. However, the genetic component and structure of Egyptian native chicken (ENC) have not been studied so far. The aim of this study is to clarify the origin and evolution of African chickens through assessing the genetic diversities and structure of five ENC breeds using the mitochondrial D-loop sequences. Our results suggest there is genetic differentiation between the pure native breeds and the improved native breeds. The latter breeds were established by the hybridization of the pure native and the exotic breeds. The pure native breeds were estimated to be established about 800 years ago. Subsequently, we extensively analyzed the D-loop sequences from the ENC as well as the globally collected chickens (2,010 individuals in total). Our phylogenetic tree among the regional populations shows African chickens can be separated to two distinct clades. The first clade consists of North African (Egypt), Central African (Sudan and Cameroon), European, and West (and Central) Asian chickens. The second clade consists of East African (Kenya, Malawi, and Zimbabwe) and Pacific chickens. It suggests the dual origins of African native chickens. The first group was probably originated from South Asia, and then migrated to West Asia, and finally arrived to Africa thorough Egypt. The second group migrated from Pacific to East Africa via Indian Ocean probably by Austronesian people. This dual origin hypothesis as well as estimated divergence times in this study is harmonious with the archaeological and historical evidences. Our migration analysis suggests there is limited gene flow within African

  7. Regions of IkappaBalpha that are critical for its inhibition of NF-kappaB.DNA interaction fold upon binding to NF-kappaB.

    Science.gov (United States)

    Truhlar, Stephanie M E; Torpey, Justin W; Komives, Elizabeth A

    2006-12-12

    Nuclear factor kappaB (NF-kappaB) transcription factors regulate genes responsible for critical cellular processes. IkappaBalpha, -beta, and -epsilon bind to NF-kappaBs and inhibit their transcriptional activity. The NF-kappaB-binding domains of IkappaBs contain six ankyrin repeats (ARs), which adopt a beta-hairpin/alpha-helix/loop/alpha-helix/loop architecture. IkappaBalpha appears compactly folded in the IkappaBalpha.NF-kappaB crystal structure, but biophysical studies suggested that IkappaBalpha might be flexible even when bound to NF-kappaB. Amide H/(2)H exchange in free IkappaBalpha suggests that ARs 2-4 are compact, but ARs 1, 5, and 6 are conformationally flexible. Amide H/(2)H exchange is one of few techniques able to experimentally identify regions that fold upon binding. Comparison of amide H/(2)H exchange in free and NF-kappaB-bound IkappaBalpha reveals that the beta-hairpins in ARs 5 and 6 fold upon binding to NF-kappaB, but AR 1 remains highly solvent accessible. These regions are implicated in various aspects of NF-kappaB regulation, such as controlling degradation of IkappaBalpha, enabling high-affinity interaction with different NF-kappaB dimers, and preventing NF-kappaB from binding to its target DNA. Thus, IkappaBalpha conformational flexibility and regions of IkappaBalpha folding upon binding to NF-kappaB are important attributes for its regulation of NF-kappaB transcriptional activity.

  8. The substructure of three repetitive DNA regions of Schistosoma haematobium group species as a potential marker for species recognition and interbreeding detection.

    Science.gov (United States)

    Abbasi, Ibrahim; Webster, Bonnie L; King, Charles H; Rollinson, David; Hamburger, Joseph

    2017-08-01

    Schistosoma haematobium is the causative agent of human urogenital schistosomiasis affecting ~112 million people in Africa and the Middle East. The parasite is transmitted by snails of the genus Bulinus, which also transmit other closely related human and animal schistosomes. The accurate discrimination of S. haematobium from species infecting animals will aid effective control and elimination programs. Previously we have shown the utility of different repetitive nuclear DNA sequences (DraI, sh73bp, and sh77bp) for the identification of S. haematobium-group species and inter-repeat sequences for discriminating S. haematobium from S. bovis. In this current study we clarify the structural arrangement and association between the three repetitive sequences (DraI, sh73bp, and sh77bp) in both S. haematobium and S. bovis, with a unique repeat linker being found in S. haematobium (Sh64bp repeat linker) and in S. bovis (Sb30bp repeat linker). Sequence data showed that the 3'-end of the repeat linker was connected to the DraI repetitive sequence array, and at the 5'-end of the repeat linker sh73bp and sh77bp were arranged in an alternating manner. Species-specific oligonucleotides were designed targeting the species-specific repeat linkers and used in a reverse line blot (RLB) hybridization assay enabling differentiation between S. haematobium and S. bovis. The assay was used to discriminate natural infections in wild caught Bulinus globosus. This research enabled the characterisation of species-specific DNA regions that enabled the design of species-specific oligonucleotides that can be used to rapidly differentiate between S. haematobium and S. bovis and also have the potential to aid the detection of natural hybridization between these two species.

  9. Mitochondrial cytochrome c oxidase subunit 1 gene and nuclear rDNA regions of Enterobius vermicularis parasitic in captive chimpanzees with special reference to its relationship with pinworms in humans.

    Science.gov (United States)

    Nakano, Tadao; Okamoto, Munehiro; Ikeda, Yatsukaho; Hasegawa, Hideo

    2006-12-01

    Sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) gene, nuclear internal transcribed spacer 2 (ITS2) region of ribosomal DNA (rDNA), and 5S rDNA of Enterobius vermicularis from captive chimpanzees in five zoos/institutions in Japan were analyzed and compared with those of pinworm eggs from humans in Japan. Three major types of variants appearing in both CO1 and ITS2 sequences, but showing no apparent connection, were observed among materials collected from the chimpanzees. Each one of them was also observed in pinworms in humans. Sequences of 5S rDNA were identical in the materials from chimpanzees and humans. Phylogenetic analysis of CO1 gene revealed three clusters with high bootstrap value, suggesting considerable divergence, presumably correlated with human evolution, has occurred in the human pinworms. The synonymy of E. gregorii with E. vermicularis is supported by the molecular evidence.

  10. Intragenomic sequence variation at the ITS1 - ITS2 region and at the 18S and 28S nuclear ribosomal DNA genes of the New Zealand mud snail, Potamopyrgus antipodarum (Hydrobiidae: mollusca)

    Science.gov (United States)

    Hoy, Marshal S.; Rodriguez, Rusty J.

    2013-01-01

    Molecular genetic analysis was conducted on two populations of the invasive non-native New Zealand mud snail (Potamopyrgus antipodarum), one from a freshwater ecosystem in Devil's Lake (Oregon, USA) and the other from an ecosystem of higher salinity in the Columbia River estuary (Hammond Harbor, Oregon, USA). To elucidate potential genetic differences between the two populations, three segments of nuclear ribosomal DNA (rDNA), the ITS1-ITS2 regions and the 18S and 28S rDNA genes were cloned and sequenced. Variant sequences within each individual were found in all three rDNA segments. Folding models were utilized for secondary structure analysis and results indicated that there were many sequences which contained structure-altering polymorphisms, which suggests they could be nonfunctional pseudogenes. In addition, analysis of molecular variance (AMOVA) was used for hierarchical analysis of genetic variance to estimate variation within and among populations and within individuals. AMOVA revealed significant variation in the ITS region between the populations and among clones within individuals, while in the 5.8S rDNA significant variation was revealed among individuals within the two populations. High levels of intragenomic variation were found in the ITS regions, which are known to be highly variable in many organisms. More interestingly, intragenomic variation was also found in the 18S and 28S rDNA, which has rarely been observed in animals and is so far unreported in Mollusca. We postulate that in these P. antipodarum populations the effects of concerted evolution are diminished due to the fact that not all of the rDNA genes in their polyploid genome should be essential for sustaining cellular function. This could lead to a lessening of selection pressures, allowing mutations to accumulate in some copies, changing them into variant sequences.                   

  11. Combinatorial H3K9acS10ph Histone Modification in IgH Locus S Regions Targets 14-3-3 Adaptors and AID to Specify Antibody Class-Switch DNA Recombination

    Directory of Open Access Journals (Sweden)

    Guideng Li

    2013-11-01

    Full Text Available Class-switch DNA recombination (CSR is central to the antibody response, in that it changes the immunoglobulin heavy chain (IgH constant region, thereby diversifying biological effector functions of antibodies. The activation-induced cytidine deaminase (AID-centered CSR machinery excises and rejoins DNA between an upstream (donor and a downstream (acceptor S region, which precede the respective constant region DNA. AID is stabilized on S regions by 14-3-3 adaptors. These adaptors display a high affinity for 5′-AGCT-3′ repeats, which recur in all S regions. However, how 14-3-3, AID, and the CSR machinery target exclusively the donor and acceptor S regions is poorly understood. Here, we show that histone methyltransferases and acetyltransferases are induced by CD40 or Toll-like receptor signaling and catalyze H3K4me3 and H3K9ac/K14ac histone modifications, which are enriched in S regions but do not specify the S region targets of CSR. By contrast, the combinatorial H3K9acS10ph modification specifically marks the S regions set to recombine and directly recruits 14-3-3 adaptors for AID stabilization there. Inhibition of the enzymatic activity of GCN5 and PCAF histone acetyltransferases reduces H3K9acS10ph in S regions, 14-3-3 and AID stabilization, and CSR. Thus, H3K9acS10ph is a histone code that is “written” specifically in S regions and is “read” by 14-3-3 adaptors to target AID for CSR as an important biological outcome.

  12. HPV16 DNA status is a strong prognosticator of loco-regional control after postoperative radiochemotherapy of locally advanced oropharyngeal carcinoma: results from a multicentre explorative study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG).

    Science.gov (United States)

    Lohaus, Fabian; Linge, Annett; Tinhofer, Inge; Budach, Volker; Gkika, Eleni; Stuschke, Martin; Balermpas, Panagiotis; Rödel, Claus; Avlar, Melanie; Grosu, Anca-Ligia; Abdollahi, Amir; Debus, Jürgen; Bayer, Christine; Belka, Claus; Pigorsch, Steffi; Combs, Stephanie E; Mönnich, David; Zips, Daniel; von Neubeck, Cläre; Baretton, Gustavo B; Löck, Steffen; Thames, Howard D; Krause, Mechthild; Baumann, Michael

    2014-12-01

    To investigate the impact of HPV status in patients with locally advanced head and neck squamous cell carcinoma (HNSCC), who received surgery and cisplatin-based postoperative radiochemotherapy. For 221 patients with locally advanced squamous cell carcinoma of the hypopharynx, oropharynx or oral cavity treated at the 8 partner sites of the German Cancer Consortium, the impact of HPV DNA, p16 overexpression and p53 expression on outcome were retrospectively analysed. The primary endpoint was loco-regional tumour control; secondary endpoints were distant metastases and overall survival. In the total patient population, univariate analyses revealed a significant impact of HPV16 DNA positivity, p16 overexpression, p53 positivity and tumour site on loco-regional tumour control. Multivariate analysis stratified for tumour site showed that positive HPV 16 DNA status correlated with loco-regional tumour control in patients with oropharyngeal carcinoma (p=0.02) but not in the oral cavity carcinoma group. Multivariate evaluation of the secondary endpoints in the total population revealed a significant association of HPV16 DNA positivity with overall survival (ploco-regional tumour control after postoperative cisplatin-based radiochemotherapy of locally advanced oropharyngeal carcinoma and is now being explored in a prospective validation trial. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  13. A novel type of unstable homogeneously staining region with a head-to-tail arrangement: spontaneous decay and reintegration of DNA elements into a plethora of new chromosomal sites.

    Science.gov (United States)

    Koehler, U; Abken, H; Grummt, F; Wienberg, J; Weidle, U H

    1995-01-01

    After transfection of amplification-promoting DNA elements into mammalian cells, homogeneously staining regions (HSRs) are formed by high copy numbers of transfected DNA arranged in head-to-tail polymers. Here, we wanted to evaluate the stability of this type of HSR during prolonged cultivation of transfected cells in selective medium. Thymidine kinase-deficient mouse L cells were transfected with pAPR4tk DNA harboring the amplification-promoting element 4 (APR4) linked to the gene for thymidine kinase (TK) or, alternatively, transfected with a DNA construct (pARP4t-PA) carrying, in addition, the expression cassette for human tissue-type plasminogen activator (t-PA). After transfection, one or two HSRs per cell were formed that disintegrated spontaneously after 25-40 wk of continuous cultivation in the presence of selective HAT (hypoxanthine-aminopterin-thymidine) medium. Unexpectedly, plasmid DNA reinserted into a plethora of new chromosomal sites, as revealed by in situ hybridization and Southern blot analysis. Coincidently, secretion of t-PA decreased to 10-20% of its original level. After transfection of pAPR4tk DNA lacking the t-PA expression cassette, HSR decay and reintegration of plasmid constructs into multiple chromosomal sites were also observed, whereas the ptk vector without an amplification-promoting DNA element did not form an HSR after transfection. We conclude that, in contrast to the pattern of known structures with head-to-tail arrangements, the HSR formed by amplification-promoting DNA elements represents a novel type of HSR that disintegrates by transposition into a plethora of new chromosomal integration sites. This process is mediated by the amplification-promoting DNA element itself and can be observed even when selective pressure is maintained.

  14. Sequence variants of the CRH 5'-flanking region: effects on DNA-protein interactions studied by EMSA in PC12 cells.

    Science.gov (United States)

    Wagner, Uta; Wahle, Matthias; Malysheva, Olga; Wagner, Ulf; Häntzschel, Holm; Baerwald, Christoph

    2006-06-01

    Recently, studies in adult rheumatoid arthritis patients have shown an association with four single-nucleotide polymorphisms (SNPs) in the 3.7-kb regulatory region of human corticotropin-releasing hormone (hCRH) gene located at positions -3531, -3371, -2353, and -684 bp. Three of these novel polymorphisms are in absolute linkage disequilibrium, resulting in three combined alleles, named A1B1, A2B1, and A2B2. To study whether the described polymorphic nucleotide sequences in the 5' region of the hCRH gene interfere with binding of nuclear proteins, an electric mobility shift assay (EMSA) was performed. At position -2353 bp, a specific DNA protein complex was detected for the wild-type sequence only, possibly interfering with a binding site for the activating transcription factor 6 (ATF6). In contrast, no difference could be detected for the other SNPs. However, at position -684, a quantitative difference in protein binding due to cAMP incubation could be observed. To further investigate whether these SNPs in the CRH promoter are associated with an altered regulation of the CRH gene, we performed a luciferase reporter gene assay with transiently transfected rat pheochromocytoma cells PC12. Incubation with 8-Br-cAMP alone or in combination with cytokines enhanced significantly the promoter activity in PC12 cells. The promoter haplotypes studied exhibited a differential capacity to modulate CRH gene expression. In all our experiments, haplotype A1B1 showed the most pronounced influence on promoter activity. Taken together, our results demonstrate a differential binding capacity of nuclear proteins of the promoter polymorphisms resulting in a different gene regulation. Most probably the SNP at position -2,353 plays a major role in mediating these differences.

  15. Environmental stress affects DNA methylation of a CpG rich promoter region of serotonin transporter gene in a nurse cohort.

    Science.gov (United States)

    Alasaari, Jukka S; Lagus, Markus; Ollila, Hanna M; Toivola, Auli; Kivimäki, Mika; Vahtera, Jussi; Kronholm, Erkki; Härmä, Mikko; Puttonen, Sampsa; Paunio, Tiina

    2012-01-01

    Shift-working nurses are exposed to a stressful work environment, which puts them at an increased risk for burnout and depression. We explored the effect of environmental stress on serotonin transporter gene (SLC6A4) promoter methylation among nurses from high and low work stress environments. Using bisulfite sequencing, we investigated the methylation status of five CpG residues of a CpG-rich region in the promoter of SLC6A4 by comparing female shift working nurses from a high work stress environment (n = 24) to low work stress environment (n = 25). We also analyzed the association of 5-HTTLPR polymorphism at 5' end of SLC6A4. Work stress was assessed by the Karasek's Model and possible signs of burnout or depression were measured by the Maslach Burnout Index General Survey and Beck Depression Index. Methylation levels were assessed by bisulfite sequencing of DNA extracted from peripheral blood leucocytes. Restriction enzyme treatment followed by standard PCR was used to identify 5-HTTLPR genotypes. We found that nurses in the high stress environment had significantly lower promoter methylation levels at all five CpG residues compared to nurses in the low stress environment (pburnout was not significantly associated to methylation levels. However, when mutually adjusted for both, burnout and work stress were significant contributors (p = 0.038 and pdepressed mood in long-term stress.

  16. Persistent heteroplasmy of a mutation in the human mtDNA control region: hypermutation as an apparent consequence of simple-repeat expansion/contraction.

    Science.gov (United States)

    Howell, N; Smejkal, C B

    2000-05-01

    In the genealogical and phylogenetic analyses that are reported here, we obtained evidence for an unusual pattern of mutation/reversion in the human mitochondrial genome. The cumulative results indicate that, when there is a T-->C polymorphism at nt 16189 and a C-->T substitution at nt 16192, there is an extremely high rate of reversion (hypermutation) at the latter site. The apparent reversion rate is sufficiently high that there is persistent heteroplasmy at nt 16192 in maternal lineages and at the phylogenetic level, a situation that is similar to that observed for the rapid expansion/contraction of simple repeats within the control region. This is the first specific instance in which the mutation frequency at one site in the D-loop is markedly influenced by the local sequence "context." The 16189 T-->C polymorphism lengthens a (C:G)n simple repeat, which then undergoes expansion and contraction, probably through replication slippage. This proclivity toward expansion/contraction is more pronounced when there is a C residue, rather than a T, at nt 16192. The high T-->C reversion frequency at nt 16192 apparently is the result of polymerase misincorporation or slippage during replication, the same mechanism that also causes the expansion/contraction of this simple-repeat sequence. In addition to the first analysis of this mitochondrial hypermutation process, these results also yield mechanistic insights into the expansion/contraction of simple-repeat sequences in mtDNA.

  17. Chloroplast DNA analysis of Tunisian cork oak populations (Quercus suber L.): sequence variations and molecular evolution of the trnL (UAA)-trnF (GAA) region.

    Science.gov (United States)

    Abdessamad, A; Baraket, G; Sakka, H; Ammari, Y; Ksontini, M; Hannachi, A Salhi

    2016-10-24

    Sequences of the trnL-trnF spacer and combined trnL-trnF region in chloroplast DNA of cork oak (Quercus suber L.) were analyzed to detect polymorphisms and to elucidate molecular evolution and demographic history. The aligned sequences varied in length and nucleotide composition. The overall ratio of transition/transversion (ti/tv) of 0.724 for the intergenic spacer and 0.258 for the pooled sequences were estimated, and indicated that transversions are more frequent than transitions. The molecular evolution and demographic history of Q. suber were investigated. Neutrality tests (Tajima's D and Fu and Li) ruled out the null hypothesis of a strictly neutral model, and Fu's Fs and Ramos-Onsins and Rozas' R2 confirmed the recent expansion of cork oak trees, validating its persistency in North Africa since the last glaciation during the Quaternary. The observed uni-modal mismatch distribution and the Harpending's raggedness index confirmed the demographic history model for cork oak. A phylogenetic dendrogram showed that the distribution of Q. suber trees occurs independently of geographical origin, the relief of the population site, and the bioclimatic stages. The molecular history and cytoplasmic diversity suggest that in situ and ex situ conservation strategies can be recommended for preserving landscape value and facing predictable future climatic changes.

  18. Phylogenetic relationships in Solanaceae and related species based on cpDNA sequence from plastid trnE-trnT region

    OpenAIRE

    Danila Montewka Melotto-Passarin; Irving Joseph Berger; Keini Dressano; Valentina de Fátima De Martin; Giancarlo Conde Xavier Oliveira; Ralph Bock; Helaine Carrer

    2008-01-01

    Intergenic spacers of chloroplast DNA (cpDNA) are very useful in phylogenetic and population genetic studies of plant species, to study their potential integration in phylogenetic analysis. The non-coding trnE-trnT intergenic spacer of cpDNA was analyzed to assess the nucleotide sequence polymorphism of 16 Solanaceae species and to estimate its ability to contribute to the resolution of phylogenetic studies of this group. Multiple alignments of DNA sequences of trnE-trnT intergenic spacer mad...

  19. Using occupancy modelling to compare environmental DNA to traditional field methods for regional-scale monitoring of an endangered aquatic species.

    Science.gov (United States)

    Schmelzle, Molly C; Kinziger, Andrew P

    2016-07-01

    Environmental DNA (eDNA) monitoring approaches promise to greatly improve detection of rare, endangered and invasive species in comparison with traditional field approaches. Herein, eDNA approaches and traditional seining methods were applied at 29 research locations to compare method-specific estimates of detection and occupancy probabilities for endangered tidewater goby (Eucyclogobius newberryi). At each location, multiple paired seine hauls and water samples for eDNA analysis were taken, ranging from two to 23 samples per site, depending upon habitat size. Analysis using a multimethod occupancy modelling framework indicated that the probability of detection using eDNA was nearly double (0.74) the rate of detection for seining (0.39). The higher detection rates afforded by eDNA allowed determination of tidewater goby occupancy at two locations where they have not been previously detected and at one location considered to be locally extirpated. Additionally, eDNA concentration was positively related to tidewater goby catch per unit effort, suggesting eDNA could potentially be used as a proxy for local tidewater goby abundance. Compared to traditional field sampling, eDNA provided improved occupancy parameter estimates and can be applied to increase management efficiency across a broad spatial range and within a diversity of habitats. © 2015 John Wiley & Sons Ltd.

  20. The C-terminal region of Rad52 is essential for Rad52 nuclear and nucleolar localization, and accumulation at DNA damage sites immediately after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Koike, Manabu, E-mail: m_koike@nirs.go.jp [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Yutoku, Yasutomo [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Graduate School of Science, Chiba University, Yayoicho, Inage-ku, Chiba 263-8522 (Japan); Koike, Aki [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

    2013-05-31

    Highlights: •Rad52 might play a key role in the repair of DSB immediately after irradiation. •EYFP-Rad52 accumulates rapidly at DSB sites and colocalizes with Ku80. •Accumulation of Rad52 at DSB sites is independent of the core NHEJ factors. •Localization and recruitment of Rad52 to DSB sites are dependent on the Rad52 CTR. •Basic amino acids in Rad52 CTR are highly conserved among vertebrate species. -- Abstract: Rad52 plays essential roles in homologous recombination (HR) and repair of DNA double-strand breaks (DSBs) in Saccharomyces cerevisiae. However, in vertebrates, knockouts of the Rad52 gene show no hypersensitivity to agents that induce DSBs. Rad52 localizes in the nucleus and forms foci at a late stage following irradiation. Ku70 and Ku80, which play an essential role in nonhomologous DNA-end-joining (NHEJ), are essential for the accumulation of other core NHEJ factors, e.g., XRCC4, and a HR-related factor, e.g., BRCA1. Here, we show that the subcellular localization of EYFP-Rad52(1–418) changes dynamically during the cell cycle. In addition, EYFP-Rad52(1–418) accumulates rapidly at microirradiated sites and colocalizes with the DSB sensor protein Ku80. Moreover, the accumulation of EYFP-Rad52(1–418) at DSB sites is independent of the core NHEJ factors, i.e., Ku80 and XRCC4. Furthermore, we observed that EYFP-Rad52(1–418) localizes in nucleoli in CHO-K1 cells and XRCC4-deficient cells, but not in Ku80-deficient cells. We also found that Rad52 nuclear localization, nucleolar localization, and accumulation at DSB sites are dependent on eight amino acids (411–418) at the end of the C-terminal region of Rad52 (Rad52 CTR). Furthermore, basic amino acids on Rad52 CTR are highly conserved among mammalian, avian, and fish homologues, suggesting that Rad52 CTR is important for the regulation and function of Rad52 in vertebrates. These findings also suggest that the mechanism underlying the regulation of subcellular localization of Rad52 is

  1. Long-term follow up of human T-cell responses to conserved HIV-1 regions elicited by DNA/simian adenovirus/MVA vaccine regimens.

    Directory of Open Access Journals (Sweden)

    Nathifa Moyo

    Full Text Available Durability of vaccine-elicited immune responses is one of the key determinants for vaccine success. Our aim is to develop a vaccination strategy against the human immunodeficiency virus type 1 (HIV-1, which induces protective and durable CD8+ T-cell responses. The central theorem of our approach is to focus T cells on highly conserved regions of the HIV-1 proteome and this is achieved through the use of the first-generation conserved vaccine immunogen HIVconsv. This immunogen vectored by plasmid DNA, simian adenovirus and poxvirus MVA was tested in healthy, HIV-1-negative adults in UK and induced high magnitudes of HIVconsv-specific plurifunctional CD8+ T cells capable of in vitro HIV-1 inhibition. Here, we assessed the durability of these responses.Vaccine recipients in trial HIV-CORE 002 were invited to provide a blood sample at 1 and 2 years after vaccination. Their PBMCs were tested in IFN-γ ELISPOT, 25-analyte Luminex, CFSE proliferation and intracellular cytokine staining assays, the last enhanced by HLA-peptide dextramer analysis.12/12 (1 year and 8/8 (2 years returning subjects had median (range of 990 (150-2495 and 763 (70-1745 IFN-γ SFU/106 PBMC specific for HIVconsv, respectively, and recognized 5 (1-6 out of 6 peptide pools at 2 years. Over one-half of the HIVconsv-specific cells expressed at least 3 functions IFN-γ, TNF-α and CD107a, and were capable of proliferation. Among dextramer-reactive cells, naïve, transitional, effector and terminally differentiated memory subsets were similarly represented.First generation HIVconsv vaccine induced human T cells, which were plurifunctional and persisted for at least 2 years.ClinicalTrials.gov NCT01151319.

  2. Molecular phylogenetics of the species-rich angiosperm genus Goniothalamus (Annonaceae) inferred from nine chloroplast DNA regions: Synapomorphies and putative correlated evolutionary changes in fruit and seed morphology.

    Science.gov (United States)

    Tang, Chin Cheung; Thomas, Daniel C; Saunders, Richard M K

    2015-11-01

    A phylogenetic study of the genus Goniothalamus (Annonaceae) is presented using maximum parsimony, maximum likelihood and Bayesian approaches, with 65 species sampled (48.5% of the genus) based on sequences of nine chloroplast DNA regions (11,214 aligned positions). The resultant phylogeny clearly indicates that Goniothalamus is monophyletic. Preliminary research initially focused on identifying synapomorphies and estimating the phylogenetic signal of selected morphological characters based on parsimony and likelihood ancestral character state reconstructions. This prescreening of characters enabled 40 to be selected for further study, and of these 15 are shown here to demonstrate significant phylogenetic signal and to provide clear synapomorphies for several infrageneric clades. Although floral structure in Goniothalamus is comparatively uniform, suggesting a common basic pattern of pollination ecology, fruit and seed morphology in the genus is very diverse and is presumably associated with different patterns of frugivory. The present study assesses correlations amongst fruit and seed characters which are putatively of functional importance with regard to frugivory and dispersal. One-way phylogenetic ANOVA indicates significant phylogenetically independent correlation between the following fruit and seed characters: fruits borne on older branches and/or on the main trunk have larger monocarps than fruits borne on young branches; and monocarps that contain seeds with a hairy testa are larger than those with glabrous seeds. We discuss fruit morphologies and potential explanations for the inferred correlations, and suggest that they may be the result of adaptation to different frugivores (birds, larger non-volant animal and primate seed dispersers, respectively). Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Characterization of muntjac DNA

    Energy Technology Data Exchange (ETDEWEB)

    Davis, R.C.

    1981-05-27

    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.

  4. Deciphering the Combinatorial DNA-binding Code of the CCAAT-binding Complex and the Iron-regulatory Basic Region Leucine Zipper (bZIP) Transcription Factor HapX*

    Science.gov (United States)

    Hortschansky, Peter; Ando, Eriko; Tuppatsch, Katja; Arikawa, Hisashi; Kobayashi, Tetsuo; Kato, Masashi; Haas, Hubertus; Brakhage, Axel A.

    2015-01-01

    The heterotrimeric CCAAT-binding complex (CBC) is evolutionarily conserved in eukaryotic organisms, including fungi, plants, and mammals. The CBC consists of three subunits, which are named in the filamentous fungus Aspergillus nidulans HapB, HapC, and HapE. HapX, a fourth CBC subunit, was identified exclusively in fungi, except for Saccharomyces cerevisiae and the closely related Saccharomycotina species. The CBC-HapX complex acts as the master regulator of iron homeostasis. HapX belongs to the class of basic region leucine zipper transcription factors. We demonstrated that the CBC and HapX bind cooperatively to bipartite DNA motifs with a general HapX/CBC/DNA 2:1:1 stoichiometry in a class of genes that are repressed by HapX-CBC in A. nidulans during iron limitation. This combinatorial binding mode requires protein-protein interaction between the N-terminal domain of HapE and the N-terminal CBC binding domain of HapX as well as sequence-specific DNA binding of both the CBC and HapX. Initial binding of the CBC to CCAAT boxes is mandatory for DNA recognition of HapX. HapX specifically targets the minimal motif 5′-GAT-3′, which is located at a distance of 11–12 bp downstream of the respective CCAAT box. Single nucleotide substitutions at the 5′- and 3′-end of the GAT motif as well as different spacing between the CBC and HapX DNA-binding sites revealed a remarkable promiscuous DNA-recognition mode of HapX. This flexible DNA-binding code may have evolved as a mechanism for fine-tuning the transcriptional activity of CBC-HapX at distinct target promoters. PMID:25589790

  5. Array-based gene expression, CGH and tissue data defines a 12q24 gain in neuroblastic tumors with prognostic implication

    Directory of Open Access Journals (Sweden)

    Kilpinen Sami

    2010-05-01

    Full Text Available Abstract Background Neuroblastoma has successfully served as a model system for the identification of neuroectoderm-derived oncogenes. However, in spite of various efforts, only a few clinically useful prognostic markers have been found. Here, we present a framework, which integrates DNA, RNA and tissue data to identify and prioritize genetic events that represent clinically relevant new therapeutic targets and prognostic biomarkers for neuroblastoma. Methods A single-gene resolution aCGH profiling was integrated with microarray-based gene expression profiling data to distinguish genetic copy number alterations that were strongly associated with transcriptional changes in two neuroblastoma cell lines. FISH analysis using a hotspot tumor tissue microarray of 37 paraffin-embedded neuroblastoma samples and in silico data mining for gene expression information obtained from previously published studies including up to 445 healthy nervous system samples and 123 neuroblastoma samples were used to evaluate the clinical significance and transcriptional consequences of the detected alterations and to identify subsequently activated gene(s. Results In addition to the anticipated high-level amplification and subsequent overexpression of MYCN, MEIS1, CDK4 and MDM2 oncogenes, the aCGH analysis revealed numerous other genetic alterations, including microamplifications at 2p and 12q24.11. Most interestingly, we identified and investigated the clinical relevance of a previously poorly characterized amplicon at 12q24.31. FISH analysis showed low-level gain of 12q24.31 in 14 of 33 (42% neuroblastomas. Patients with the low-level gain had an intermediate prognosis in comparison to patients with MYCN amplification (poor prognosis and to those with no MYCN amplification or 12q24.31 gain (good prognosis (P = 0.001. Using the in silico data mining approach, we identified elevated expression of five genes located at the 12q24.31 amplicon in neuroblastoma (DIABLO, ZCCHC

  6. Cloning and expression of a cDNA covering the complete coding region of the P32 subunit of human pre-mRNA splicing factor SF2

    DEFF Research Database (Denmark)

    Honoré, B; Madsen, Peder; Rasmussen, H H

    1993-01-01

    We have cloned and expressed a cDNA encoding the 32-kDa subunit (P32) of the human pre-mRNA splicing factor, SF2. This cDNA extends beyond the 5'-end of a previously reported cDNA [Krainer et al., Cell 66 (1991) 383-394]. Importantly, our fragment includes an ATG start codon which was absent from...... the previously reported cDNA, where it was suggested that translation might initiate at a CTG codon instead of at an ATG codon. Using the vaccinia virus (Vv) expression system, we demonstrate that translation starts at the conventional ATG start codon and not at the CTG codon. The protein is synthesized as a pro...

  7. Altered DNA methylation patterns of the H19 differentially methylated region and the DAZL gene promoter are associated with defective human sperm.

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    Bo Li

    Full Text Available DNA methylation disturbance is associated with defective human sperm. However, oligozoospermia (OZ and asthenozoospermia (AZ usually present together, and the relationship between the single-phenotype defects in human sperm and DNA methylation is poorly understood. In this study, 20 infertile OZ patients and 20 infertile AZ patients were compared with 20 fertile normozoospermic men. Bisulfate-specific PCR was used to analyze DNA methylation of the H19-DMR and the DAZL promoter in these subjects. A similar DNA methylation pattern of the H19-DMR was detected in AZ and NZ(control, with only complete methylation and mild hypomethylation(0.05. However, the methylation pattern of severe hypomethylation (>50% unmethylated CpGs and complete unmethylation was only detected in 5 OZ patients, and the occurrence of these two methylation patterns was 8.54±10.86% and 9±6.06%, respectively. Loss of DNA methylation of the H19-DMR in the OZ patients was found to mainly occur in CTCF-binding site 6, with occurrence of 18.15±14.71%, which was much higher than that in patients with NZ (0.84±2.05% and AZ (0.58±1.77% (P20% methylated clones in the DAZL promoter only in infertile patients, there was no significant difference between the AZ and OZ patients in the proportion of moderately-to-severely hypermethylated clones (p>0.05. In all cases, global sperm genome methylation analyses, using LINE1 transposon as the indicator, showed that dysregulation of DNA methylation is specifically associated with the H19-DMR and DAZL promoter. Therefore, abnormal DNA methylation status of H19-DMR, especially at the CTCF-binding site 6, is closely associated with OZ. Abnormal DNA methylation of the DAZL promoter might represent an epigenetic marker of male infertility.

  8. Mitochondrial myopathy associated with high levels of mitochondrial DNA harboring a 260 bp tandem duplication in the D-loop region

    Energy Technology Data Exchange (ETDEWEB)

    Manfredi, G.; Shanske, S.; Schon, E.A. [Columbia Univ., NY (United States)] [and others

    1994-09-01

    Low levels of a 260 bp duplication in the D-loop of the mitochondrial DNA (mtDNA) were reported in some patients with mitochondrial disorders harboring large-scale mtDNA deletions. Because the same duplication was observed in unaffected mothers of these patients, it was suggested that the 260 bp duplication predispose mtDNA to deletion. More recently, PCR-levels of this duplication were also observed in a subgroup of normal Caucasions. To test the hypothesis that this genetic abnormality may be prevalent in patients with large-scale deletions of the mitochondrial genome, we used a semi-quantitative PCR protocol to search for the 260 by duplication in 34 patients with, and 35 without mtDNA deletions. Our results do not suppor