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Sample records for domesticated piggybac transposase

  1. A Domesticated PiggyBac Transposase Interacts with Heterochromatin and Catalyzes Reproducible DNA Elimination in Tetrahymena

    PubMed Central

    Vogt, Alexander; Mochizuki, Kazufumi

    2013-01-01

    The somatic genome of the ciliated protist Tetrahymena undergoes DNA elimination of defined sequences called internal eliminated sequences (IESs), which account for ∼30% of the germline genome. During DNA elimination, IES regions are heterochromatinized and assembled into heterochromatin bodies in the developing somatic nucleus. The domesticated piggyBac transposase Tpb2p is essential for the formation of heterochromatin bodies and DNA elimination. In this study, we demonstrate that the activities of Tpb2p involved in forming heterochromatin bodies and executing DNA elimination are genetically separable. The cysteine-rich domain of Tpb2p, which interacts with the heterochromatin-specific histone modifications, is necessary for both heterochromatin body formation and DNA elimination, whereas the endonuclease activity of Tpb2p is only necessary for DNA elimination. Furthermore, we demonstrate that the endonuclease activity of Tpb2p in vitro and the endonuclease activity that executes DNA elimination in vivo have similar substrate sequence preferences. These results strongly indicate that Tpb2p is the endonuclease that directly catalyzes the excision of IESs and that the boundaries of IESs are at least partially determined by the combination of Tpb2p-heterochromatin interaction and relaxed sequence preference of the endonuclease activity of Tpb2p. PMID:24348275

  2. Transposon Invasion of the Paramecium Germline Genome Countered by a Domesticated PiggyBac Transposase and the NHEJ Pathway

    PubMed Central

    Dubois, Emeline; Bischerour, Julien; Marmignon, Antoine; Mathy, Nathalie; Régnier, Vinciane; Bétermier, Mireille

    2012-01-01

    Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus), ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliate Paramecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists between Paramecium IESs and Tc1/mariner transposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticated piggyBac transposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate, Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of a piggyBac transposase might have allowed Tc1/mariner elements to spread throughout the germline genome of Paramecium, without strong counterselection against insertion within genes. PMID:22888464

  3. Mutational analysis of highly conserved aspartate residues essential to the catalytic core of the piggyBac transposase

    PubMed Central

    Keith, James H; Schaeper, Cheryl A; Fraser, Tresa S; Fraser, Malcolm J

    2008-01-01

    Background The piggyBac mobile element is quickly gaining popularity as a tool for the transgenesis of many eukaryotic organisms. By studying the transposase which catalyzes the movement of piggyBac, we may be able to modify this vector system to make it a more effective transgenesis tool. In a previous publication, Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, and Collins FH have proposed the presence of the widespread 'DDE/DDD' motif for piggyBac at amino acid positions D268, D346, and D447. Results This study utilizes directed mutagenesis and plasmid-based mobility assays to assess the importance of these residues as the catalytic core of the piggyBac transposase. We have functionally analyzed individual point-mutations with respect to charge and physical size in all three proposed residues of the 'DDD' motif as well as another nearby, highly conserved aspartate at D450. All of our mutations had a significant effect on excision frequency in S2 cell cultures. We have also aligned the piggyBac transposase to other close family members, both functional and non-functional, in an attempt to identify the most highly conserved regions and position a number of interesting features. Conclusion We found all the designated DDD aspartates reside in clusters of amino acids that conserved among piggyBac family transposase members. Our results indicate that all four aspartates are necessary, to one degree or another, for excision to occur in a cellular environment, but D450 seems to have a tolerance for a glutamate substitution. All mutants tested significantly decreased excision frequency in cell cultures when compared with the wild-type transposase. PMID:18694512

  4. Molecular Evolution of piggyBac Superfamily: From Selfishness to Domestication

    PubMed Central

    Bouallègue, Maryem; Rouault, Jacques-Deric; Hua-Van, Aurélie; Makni, Mohamed

    2017-01-01

    The piggyBac transposable element was originally isolated from the cabbage looper moth, Trichoplusia ni, in the 1980s. Despite its early discovery and specificity compared to the other Class II elements, the diversity and evolution of this superfamily have been only partially analyzed. Two main types of elements can be distinguished: the piggyBac-like elements (PBLE) with terminal inverted repeats, untranslated region, and an open reading frame encoding a transposase, and the piggyBac-derived sequences (PGBD), containing a sequence derived from a piggyBac transposase, and which correspond to domesticated elements. To define the distribution, their structural diversity and phylogenetic relationships, analyses were conducted using known PBLE and PGBD sequences to scan databases. From this data mining, numerous new sequences were characterized (50 for PBLE and 396 for PGBD). Structural analyses suggest that four groups of PBLE can be defined according to the presence/absence of sub-terminal repeats. The transposase is characterized by highly variable catalytic domain and C-terminal region. There is no relationship between the structural groups and the phylogeny of these PBLE elements. The PGBD are clearly structured into nine main groups. A new group of domesticated elements is suspected in Neopterygii and the remaining eight previously described elements have been investigated in more detail. In all cases, these sequences are no longer transposable elements, the catalytic domain of the ancestral transposase is not always conserved, but they are under strong purifying selection. The phylogeny of both PBLE and PGBD suggests multiple and independent domestication events of PGBD from different PBLE ancestors. PMID:28082605

  5. Molecular evolution of piggyBac superfamily: from selfishness to domestication.

    PubMed

    Bouallègue, Maryem; Rouault, Jacques-Deric; Hua-Van, Aurélie; Makni, Mohamed; Capy, Pierre

    2017-01-12

    The piggyBac transposable element was originally isolated from the cabbage looper moth, Trichoplusia ni, in the 1980s. Despite its early discovery and specificity compared to the other Class II elements, the diversity and evolution of this superfamily have been only partially analyzed. Two main types of elements can be distinguished: the piggyBac-like elements (PBLE) with terminal inverted repeats (TIR), UTR and an ORF encoding a transposase, and the piggyBac-derived sequences (PGBD), containing a sequence derived from a piggyBac transposase, and which correspond to domesticated elements. To define the distribution, their structural diversity and phylogenetic relationships, analyses were conducted using known PBLE and PGBD sequences to scan databases. From this data mining, numerous new sequences were characterized (50 for PBLE and 396 for PGBD). Structural analyses suggest that four groups of PBLE can be defined according to the presence/absence of sub-terminal repeats. The transposase is characterized by highly variable catalytic domain and C-terminal region. There is no relationship between the structural groups and the phylogeny of these PBLE elements. The PGBD are clearly structured into nine main groups. A new group of domesticated elements is suspected in Neopterygii and the remaining eight previously described elements have been investigated in more detail. In all cases, these sequences are no longer transposable elements, the catalytic domain of the ancestral transposase is not always conserved, but they are under strong purifying selection. The phylogeny of both PBLE and PGBD suggests multiple and independent domestication events of PGBD from different PBLE ancestors.

  6. Tracking and transforming neocortical progenitors by CRISPR/Cas9 gene targeting and piggyBac transposase lineage labeling

    PubMed Central

    Chen, Fuyi; Rosiene, Joel; Che, Alicia; Becker, Albert; LoTurco, Joseph

    2015-01-01

    The ability to induce targeted mutations in somatic cells in developing organisms and then track the fates of those cells is a powerful approach both for studying neural development and for modeling human disease. The CRISPR/Cas9 system allows for such targeted mutagenesis, and we therefore tested it in combination with a piggyBac transposase lineage labeling system to track the development of neocortical neural progenitors with targeted mutations in genes linked to neurodevelopmental disruptions and tumor formation. We show that sgRNAs designed to target PTEN successfully decreased PTEN expression, and led to neuronal hypertrophy and altered neuronal excitability. Targeting NF1, by contrast, caused increased astrocytogenesis at the expense of neurogenesis, and combined targeting of three tumor suppressors (PTEN, NF1 and P53) resulted in formation of glioblastoma tumors. Our results demonstrate that CRISPR/Cas9 combined with piggyBac transposase lineage labeling can produce unique models of neurodevelopmental disruption and tumors caused by somatic mutation in neural progenitors. PMID:26400094

  7. A Nucleolus-Predominant piggyBac Transposase, NP-mPB, Mediates Elevated Transposition Efficiency in Mammalian Cells

    PubMed Central

    Ku, Amy T.; Fan, Hsiang-Hsuan; Lee, Tung-Lung; Huang, Yung-Hsin; Yang, Tsung-Lin; Su, I-Chang; Yu, I-Shing; Lin, Shu-Wha; Chien, Chung-Liang; Ho, Hong-Nerng; Chen, You-Tzung

    2014-01-01

    PiggyBac is a prevalent transposon system used to deliver transgenes and functionally explore the mammalian untouched genomic territory. The important features of piggyBac transposon are the relatively low insertion site preference and the ability of seamless removal from genome, which allow its potential uses in functional genomics and regenerative medicine. Efforts to increase its transposition efficiency in mammals were made through engineering the corresponding transposase (PBase) codon usage to enhance its expression level and through screening for mutant PBase variants with increased enzyme activity. To improve the safety for its potential use in regenerative medicine applications, site-specific transposition was achieved by using engineered zinc finger- and Gal4-fused PBases. An excision-prone PBase variant has also been successfully developed. Here we describe the construction of a nucleolus-predominant PBase, NP-mPB, by adding a nucleolus-predominant (NP) signal peptide from HIV-1 TAT protein to a mammalian codon-optimized PBase (mPB). Although there is a predominant fraction of the NP-mPB-tGFP fusion proteins concentrated in the nucleoli, an insertion site preference toward nucleolar organizer regions is not detected. Instead a 3–4 fold increase in piggyBac transposition efficiency is reproducibly observed in mouse and human cells. PMID:24586748

  8. Generation of Transgenic Pigs by Cytoplasmic Injection of piggyBac Transposase-Based pmGENIE-3 Plasmids1

    PubMed Central

    Li, Zicong; Zeng, Fang; Meng, Fanming; Xu, Zhiqian; Zhang, Xianwei; Huang, Xiaoling; Tang, Fei; Gao, Wenchao; Shi, Junsong; He, Xiaoyan; Liu, Dewu; Wang, Chong; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

    2014-01-01

    ABSTRACT The process of transgenesis involves the introduction of a foreign gene, the transgene, into the genome of an animal. Gene transfer by pronuclear microinjection (PNI) is the predominant method used to produce transgenic animals. However, this technique does not always result in germline transgenic offspring and has a low success rate for livestock. Alternate approaches, such as somatic cell nuclear transfer using transgenic fibroblasts, do not show an increase in efficiency compared to PNI, while viral-based transgenesis is hampered by issues regarding transgene size and biosafety considerations. We have recently described highly successful transgenesis experiments with mice using a piggyBac transposase-based vector, pmhyGENIE-3. This construct, a single and self-inactivating plasmid, contains all the transpositional elements necessary for successful gene transfer. In this series of experiments, our laboratories have implemented cytoplasmic injection (CTI) of pmGENIE-3 for transgene delivery into in vivo-fertilized pig zygotes. More than 8.00% of the injected embryos developed into transgenic animals containing monogenic and often single transgenes in their genome. However, the CTI technique was unsuccessful during the injection of in vitro-fertilized pig zygotes. In summary, here we have described a method that is not only easy to implement, but also demonstrated the highest efficiency rate for nonviral livestock transgenesis. PMID:24671876

  9. Fate mapping by piggyBac transposase reveals that neocortical GLAST+ progenitors generate more astrocytes than Nestin+ progenitors in rat neocortex.

    PubMed

    Siddiqi, Faez; Chen, Fuyi; Aron, Abraham W; Fiondella, Christopher G; Patel, Komal; LoTurco, Joseph J

    2014-02-01

    Progenitors within the neocortical ventricular zone (VZ) first generate pyramidal neurons and then astrocytes. We applied novel piggyBac transposase lineage tracking methods to fate-map progenitor populations positive for Nestin or glutamate and aspartate transpoter (GLAST) promoter activities in the rat neocortex. GLAST+ and Nestin+ progenitors at embryonic day 13 (E13) produce lineages containing similar rations of neurons and astrocytes. By E15, the GLAST+ progenitor population diverges significantly to produce lineages with 5-10-fold more astrocytes relative to neurons than generated by the Nestin+ population. To determine when birth-dated progeny within GLAST+ and Nestin+ populations diverge, we used a Cre/loxP fate-mapping system in which plasmids are lost after a cell division. By E18, birth-dated progeny of GLAST+ progenitors give rise to 2-3-fold more neocortical astrocytes than do Nestin+ progenitors. Finally, we used a multicolor clonal labeling method to show that the GLAST+ population labeled at E15 generates astrocyte progenitors that produce larger, spatially restricted, clonal clusters than the Nestin+ population. This study provides in vivo evidence that by mid-corticogenesis (E15), VZ progenitor populations have significantly diversified in terms of their potential to generate astrocytes and neurons.

  10. Domesticated transposase Kat1 and its fossil imprints induce sexual differentiation in yeast.

    PubMed

    Rajaei, Naghmeh; Chiruvella, Kishore K; Lin, Feng; Aström, Stefan U

    2014-10-28

    Transposable elements (TEs) have had a major influence on shaping both prokaryotic and eukaryotic genomes, largely through stochastic events following random or near-random insertions. In the mammalian immune system, the recombination activation genes1/2 (Rag1/2) recombinase has evolved from a transposase gene, demonstrating that TEs can be domesticated by the host. In this study, we uncovered a domesticated transposase, Kluyveromyces lactis hobo/Activator/Tam3 (hAT) transposase 1 (Kat1), operating at the fossil imprints of an ancient transposon, that catalyzes the differentiation of cell type. Kat1 induces mating-type switching from mating type a (MATa) to MATα in the yeast K. lactis. Kat1 activates switching by introducing two hairpin-capped DNA double-strand breaks (DSBs) in the MATa1-MATa2 intergenic region, as we demonstrate both in vivo and in vitro. The DSBs stimulate homologous recombination with the cryptic hidden MAT left alpha (HMLα) locus resulting in a switch of the cell type. The sites where Kat1 acts in the MATa locus most likely are ancient remnants of terminal inverted repeats from a long-lost TE. The KAT1 gene is annotated as a pseudogene because it contains two overlapping ORFs. We demonstrate that translation of full-length Kat1 requires a programmed -1 frameshift. The frameshift limited Kat1 activity, because restoring the zero frame causes switching to the MATα genotype. Kat1 also was transcriptionally activated by nutrient limitation via the transcription factor mating type switch 1 (Mts1). A phylogenetic analysis indicated that KAT1 was domesticated specifically in the Kluyveromyces clade of the budding yeasts. We conclude that Kat1 is a highly regulated transposase-derived endonuclease vital for sexual differentiation.

  11. Domesticated transposase Kat1 and its fossil imprints induce sexual differentiation in yeast

    PubMed Central

    Rajaei, Naghmeh; Chiruvella, Kishore K.; Lin, Feng; Åström, Stefan U.

    2014-01-01

    Transposable elements (TEs) have had a major influence on shaping both prokaryotic and eukaryotic genomes, largely through stochastic events following random or near-random insertions. In the mammalian immune system, the recombination activation genes1/2 (Rag1/2) recombinase has evolved from a transposase gene, demonstrating that TEs can be domesticated by the host. In this study, we uncovered a domesticated transposase, Kluyveromyces lactis hobo/Activator/Tam3 (hAT) transposase 1 (Kat1), operating at the fossil imprints of an ancient transposon, that catalyzes the differentiation of cell type. Kat1 induces mating-type switching from mating type a (MATa) to MATα in the yeast K. lactis. Kat1 activates switching by introducing two hairpin-capped DNA double-strand breaks (DSBs) in the MATa1–MATa2 intergenic region, as we demonstrate both in vivo and in vitro. The DSBs stimulate homologous recombination with the cryptic hidden MAT left alpha (HMLα) locus resulting in a switch of the cell type. The sites where Kat1 acts in the MATa locus most likely are ancient remnants of terminal inverted repeats from a long-lost TE. The KAT1 gene is annotated as a pseudogene because it contains two overlapping ORFs. We demonstrate that translation of full-length Kat1 requires a programmed −1 frameshift. The frameshift limited Kat1 activity, because restoring the zero frame causes switching to the MATα genotype. Kat1 also was transcriptionally activated by nutrient limitation via the transcription factor mating type switch 1 (Mts1). A phylogenetic analysis indicated that KAT1 was domesticated specifically in the Kluyveromyces clade of the budding yeasts. We conclude that Kat1 is a highly regulated transposase-derived endonuclease vital for sexual differentiation. PMID:25313032

  12. Functional characterization of sugarcane mustang domesticated transposases and comparative diversity in sugarcane, rice, maize and sorghum

    PubMed Central

    Kajihara, Daniela; de Godoy, Fabiana; Hamaji, Thais Alves; Blanco, Silvia Regina; Van Sluys, Marie-Anne; Rossi, Magdalena

    2012-01-01

    Transposable elements (TEs) account for a large portion of plant genomes, particularly in grasses, in which they correspond to 50%–80% of the genomic content. TEs have recently been shown to be a source of new genes and new regulatory networks. The most striking contribution of TEs is referred as “molecular domestication”, by which the element coding sequence loses its movement capacity and acquires cellular function. Recently, domesticated transposases known as mustang and derived from the Mutator element have been described in sugarcane. In order to improve our understanding of the function of these proteins, we identified mustang genes from Sorghum bicolor and Zea mays and performed a phenetic analysis to assess the diversity and evolutionary history of this gene family. This analysis identified orthologous groups and showed that mustang genes are highly conserved in grass genomes. We also explored the transcriptional activity of sugarcane mustang genes in heterologous and homologous systems. These genes were found to be ubiquitously transcribed, with shoot apical meristem having the highest expression levels, and were downregulated by phytohormones. Together, these findings suggest the possible involvement of mustang proteins in the maintenance of hormonal homeostasis. PMID:23055803

  13. Convergent domestication of pogo-like transposases into centromere-binding proteins in fission yeast and mammals

    PubMed Central

    Casola, Claudio; Hucks, Donald; Feschotte, Cédric

    2007-01-01

    The mammalian centromeric protein CENP-B shares significant sequence similarity with three proteins in fission yeast (Abp1, Cbh1 and Cbh2) that also bind centromeres and have essential function for chromosome segregation and centromeric heterochromatin formation. Each of these proteins displays extensive sequence similarity with pogo-like transposases, which have been previously identified in the genomes of various insects and vertebrates, in the protozoan Entamoeba and in plants. Based on this distribution, it has been proposed that the mammalian and fission yeast centromeric proteins are derived from ‘domesticated’ pogo-like transposons. Here we took advantage of the vast amount of sequence information that has become recently available for a wide range of fungal and animal species to investigate the origin of the mammalian CENP-B and yeast CENP-B-like genes. A highly conserved ortholog of CENP-B was detected in 31 species of mammals, including opossum and platypus, but was absent from all non-mammalian species represented in the databases. Similarly, no ortholog of the fission yeast centromeric proteins was identified in any of the various fungal genomes currently available. In contrast, we discovered a plethora of novel pogo-like transposons in diverse invertebrates and vertebrates and in several filamentous fungi. Phylogenetic analysis revealed that the mammalian and fission yeast CENP-B proteins fall into two distinct monophyletic clades, each of which includes a different set of pogo-like transposons. These results are most parsimoniously explained by independent domestication events of pogo-like transposases into centromeric proteins in the mammalian and fission yeast lineages, a case of ‘convergent domestication’. These findings highlight the propensity of transposases to give rise to new host proteins and the potential of transposons as sources of genetic innovation. PMID:17940212

  14. Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching

    PubMed Central

    Chiruvella, Kishore K.; Rajaei, Naghmeh; Jonna, Venkateswara Rao; Hofer, Anders; Åström, Stefan U.

    2016-01-01

    Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MATα). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons. PMID:26902909

  15. Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching.

    PubMed

    Chiruvella, Kishore K; Rajaei, Naghmeh; Jonna, Venkateswara Rao; Hofer, Anders; Åström, Stefan U

    2016-02-23

    Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MATα). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons.

  16. Excision of the piggyBac transposable element in maize cells is a precise event

    USDA-ARS?s Scientific Manuscript database

    The piggyBac transposable element (TE) from the moth Trichoplusia ni encodes a ‘cut and paste’ DNA transposase that has been used to transform a number of insects, as well as planaria, mammalian cells, and mice. The wild type and a mutated piggyBac TE excised from a DNA vector in transient assays u...

  17. Sequence diversity of a domesticated transposase gene, MUG1, in Oryza species.

    PubMed

    Kwon, Soon-Jae; Park, Kyong-Cheul; Son, Jae-Han; Bureau, Thomas; Park, Cheul-Ho; Kim, Nam-Soo

    2009-04-30

    MUG1 is a MULE transposon-related domesticated gene in plants. We assessed the sequence diversity, neutrality, expression, and phylogenetics of the MUG1 gene among Oryza ssp. We found MUG1 expression in all tissues analyzed, with different levels in O. sativa. There were 408 variation sites in the 3886 bp of MUG1 locus. The nucleotide diversity of the MUG1 was higher than functionally known genes in rice. The nucleotide diversity (pi) in the domains was lower than the average nucleotide diversity in whole coding region. The pi values in nonsynonymous sites were lower than those of synonymous sites. Tajima D and Fu and Li D* values were mostly negative values, suggesting purifying selection in MUG1 sequences of Oryza ssp. Genome-specific variation and phylogenetic analyses show a general grouping of MUG1 sequences congruent with Oryza ssp. biogeography; however, our MUG1 phylogenetic results, in combination with separate B and D genome studies, might suggest an early divergence of the Oryza ssp. by continental drift of Gondwanaland. O. longistaminata MUG1 divergence from other AA diploids suggests that it might not be a direct ancestor of the African rice species.

  18. Kicking against the PRCs – A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2

    PubMed Central

    Perera, Pumi; Mora-García, Santiago; de Leau, Erica; Thornton, Harry; de Alves, Flavia Lima; Rapsilber, Juri; Yang, Suxin; James, Geo Velikkakam; Schneeberger, Korbinian; Finnegan, E. Jean; Turck, Franziska; Goodrich, Justin

    2015-01-01

    G silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity. PMID:26642436

  19. Kicking against the PRCs - A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2.

    PubMed

    Liang, Shih Chieh; Hartwig, Ben; Perera, Pumi; Mora-García, Santiago; de Leau, Erica; Thornton, Harry; de Lima Alves, Flavia; de Alves, Flavia Lima; Rappsilber, Juri; Rapsilber, Juri; Yang, Suxin; James, Geo Velikkakam; Schneeberger, Korbinian; Finnegan, E Jean; Turck, Franziska; Goodrich, Justin

    2015-12-01

    G silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity.

  20. Genome-wide target profiling of piggyBac and Tol2 in HEK 293: pros and cons for gene discovery and gene therapy

    PubMed Central

    2011-01-01

    Background DNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery. Results We have observed that (1) the Tol2 transposase (but not piggyBac) is highly sensitive to molecular engineering; (2) the piggyBac donor with only the 40 bp 3'-and 67 bp 5'-terminal repeat domain is sufficient for effective transposition; and (3) a small amount of piggyBac transposases results in robust transposition suggesting the piggyBac transpospase is highly active. Performing genome-wide target profiling on data sets obtained by retrieving chromosomal targeting sequences from individual clones, we have identified several piggyBac and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear difference in targeting preferences in the human genome. Finally, we have observed that (5) only sites with a particular sequence context can be targeted by either piggyBac or Tol2. Conclusions The non-overlapping targeting preference of piggyBac and Tol2 makes them complementary research tools for manipulating mammalian genomes. PiggyBac is the most promising transposon-based vector system for achieving site-specific targeting of therapeutic genes due to the flexibility of its transposase for being molecularly engineered. Insights from this study will provide a basis for engineering piggyBac transposases to achieve site-specific therapeutic gene targeting. PMID:21447194

  1. Genome-wide target profiling of piggyBac and Tol2 in HEK 293: pros and cons for gene discovery and gene therapy.

    PubMed

    Meir, Yaa-Jyuhn J; Weirauch, Matthew T; Yang, Herng-Shing; Chung, Pei-Cheng; Yu, Robert K; Wu, Sareina C-Y

    2011-03-30

    DNA transposons have emerged as indispensible tools for manipulating vertebrate genomes with applications ranging from insertional mutagenesis and transgenesis to gene therapy. To fully explore the potential of two highly active DNA transposons, piggyBac and Tol2, as mammalian genetic tools, we have conducted a side-by-side comparison of the two transposon systems in the same setting to evaluate their advantages and disadvantages for use in gene therapy and gene discovery. We have observed that (1) the Tol2 transposase (but not piggyBac) is highly sensitive to molecular engineering; (2) the piggyBac donor with only the 40 bp 3'-and 67 bp 5'-terminal repeat domain is sufficient for effective transposition; and (3) a small amount of piggyBac transposases results in robust transposition suggesting the piggyBac transpospase is highly active. Performing genome-wide target profiling on data sets obtained by retrieving chromosomal targeting sequences from individual clones, we have identified several piggyBac and Tol2 hotspots and observed that (4) piggyBac and Tol2 display a clear difference in targeting preferences in the human genome. Finally, we have observed that (5) only sites with a particular sequence context can be targeted by either piggyBac or Tol2. The non-overlapping targeting preference of piggyBac and Tol2 makes them complementary research tools for manipulating mammalian genomes. PiggyBac is the most promising transposon-based vector system for achieving site-specific targeting of therapeutic genes due to the flexibility of its transposase for being molecularly engineered. Insights from this study will provide a basis for engineering piggyBac transposases to achieve site-specific therapeutic gene targeting.

  2. Insertional mutagenesis by a hybrid piggyBac and sleeping beauty transposon in the rat.

    PubMed

    Furushima, Kenryo; Jang, Chuan-Wei; Chen, Diane W; Xiao, Ningna; Overbeek, Paul A; Behringer, Richard R

    2012-12-01

    A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat.

  3. Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat

    PubMed Central

    Furushima, Kenryo; Jang, Chuan-Wei; Chen, Diane W.; Xiao, Ningna; Overbeek, Paul A.; Behringer, Richard R.

    2012-01-01

    A hybrid piggyBac/Sleeping Beauty transposon-based insertional mutagenesis system that can be mobilized by simple breeding was established in the rat. These transposons were engineered to include gene trap sequences and a tyrosinase (Tyr) pigmentation reporter to rescue the albinism of the genetic background used in the mutagenesis strategy. Single-copy transposon insertions were transposed into the rat genome by co-injection of plasmids carrying the transposon and RNA encoding piggyBac transposase into zygotes. The levels of transgenic Tyr expression were influenced by chromosomal context, leading to transgenic rats with different pigmentation that enabled visual genotyping. Transgenic rats designed to ubiquitously express either piggyBac or Sleeping Beauty transposase were generated by standard zygote injection also on an albino background. Bigenic rats carrying single-copy transposons at known loci and transposase transgenes exhibited coat color mosaicism, indicating somatic transposition. PiggyBac or Sleeping Beauty transposase bigenic rats bred with wild-type albino rats yielded offspring with pigmentation distinct from the initial transposon insertions as a consequence of germline transposition to new loci. The germline transposition frequency for Sleeping Beauty and piggyBac was ∼10% or about one new insertion per litter. Approximately 50% of the insertions occurred in introns. Chimeric transcripts containing endogenous and gene trap sequences were identified in Gabrb1 mutant rats. This mutagenesis system based on simple crosses and visual genotyping can be used to generate a collection of single-gene mutations in the rat. PMID:23023007

  4. piggyBac is a flexible and highly active transposon as compared to Sleeping Beauty, Tol2, and Mos1 in mammalian cells

    PubMed Central

    Wu, Sareina Chiung-Yuan; Meir, Yaa-Jyuhn James; Coates, Craig J.; Handler, Alfred M.; Pelczar, Pawel; Moisyadi, Stefan; Kaminski, Joseph M.

    2006-01-01

    A nonviral vector for highly efficient site-specific integration would be desirable for many applications in transgenesis, including gene therapy. In this study we directly compared the genomic integration efficiencies of piggyBac, hyperactive Sleeping Beauty (SB11), Tol2, and Mos1 in four mammalian cell lines. piggyBac demonstrated significantly higher transposition activity in all cell lines whereas Mos1 had no activity. Furthermore, piggyBac transposase coupled to the GAL4 DNA-binding domain retains transposition activity whereas similarly manipulated gene products of Tol2 and SB11 were inactive. The high transposition activity of piggyBac and the flexibility for molecular modification of its transposase suggest the possibility of using it routinely for mammalian transgenesis. PMID:17005721

  5. Hybrid Nonviral/Viral Vector Systems for Improved piggyBac DNA Transposon In Vivo Delivery

    PubMed Central

    Cooney, Ashley L; Singh, Brajesh K; Sinn, Patrick L

    2015-01-01

    The DNA transposon piggyBac is a potential therapeutic agent for multiple genetic diseases such as cystic fibrosis (CF). Recombinant piggyBac transposon and transposase are typically codelivered by plasmid transfection; however, plasmid delivery is inefficient in somatic cells in vivo and is a barrier to the therapeutic application of transposon-based vector systems. Here, we investigate the potential for hybrid piggyBac/viral vectors to transduce cells and support transposase-mediated genomic integration of the transposon. We tested both adenovirus (Ad) and adeno-associated virus (AAV) as transposon delivery vehicles. An Ad vector expressing hyperactive insect piggyBac transposase (iPB7) was codelivered. We show transposase-dependent transposition activity and mapped integrations in mammalian cells in vitro and in vivo from each viral vector platform. We also demonstrate efficient and persistent transgene expression following nasal delivery of piggyBac/viral vectors to mice. Furthermore, using piggyBac/Ad expressing Cystic Fibrosis transmembrane Conductance Regulator (CFTR), we show persistent correction of chloride current in well-differentiated primary cultures of human airway epithelial cells derived from CF patients. Combining the emerging technologies of DNA transposon-based vectors with well-studied adenoviral and AAV delivery provides new tools for in vivo gene transfer and presents an exciting opportunity to increase the delivery efficiency for therapeutic genes such as CFTR. PMID:25557623

  6. Vast potential for using the piggyBac transposon to engineer transgenic plants at specific genomic locations.

    PubMed

    Johnson, Eric T; Owens, Jesse B; Moisyadi, Stefan

    2016-01-01

    The acceptance of bioengineered plants by some nations is hampered by a number of factors, including the random insertion of a transgene into the host genome. Emerging technologies, such as site-specific nucleases, are enabling plant scientists to promote recombination or mutations at specific plant loci. Off target activity of these nucleases may limit widespread use. Insertion of transgenes by transposases engineered with a specific DNA binding domain has been accomplished in a number of organisms, but not in plants. The piggyBac transposon system, originally isolated from an insect, has been utilized to transform a variety of organisms. The piggyBac transposase is amendable to structural modifications, and was able to insert a transgene at a specific human locus through fusion of a DNA binding domain to its N-terminus. Recent developments demonstrating the activity of piggyBac transposase in plants is an important first step toward the potential use of engineered versions of piggyBac transposase for site-specific transgene insertion in plants.

  7. Size matters: versatile use of PiggyBac transposons as a genetic manipulation tool.

    PubMed

    Kim, Adele; Pyykko, Ilmari

    2011-08-01

    Transposons have been promising elements for gene integration, and the Sleeping Beauty (SB) system has been the major one for many years, although there have been several other transposon systems available, for example, Tol2. However, recently another system known as PiggyBac (PB) has been introduced and developed for fulfilling the same purposes, for example, mutagenesis, transgenesis and gene therapy and in some cases with improved transposition efficiency and advantages over the Sleeping Beauty transposon system, although improved hyperactive transposase has highly increased the transposition efficacy for SB. The PB systems have been used in many different scientific research fields; therefore, the purpose of this review is to describe some of these versatile uses of the PiggyBac system to give readers an overview on the usage of PiggyBac system.

  8. Generation of an inducible and optimized piggyBac transposon system†

    PubMed Central

    Cadiñanos, Juan; Bradley, Allan

    2007-01-01

    Genomic studies in the mouse have been slowed by the lack of transposon-mediated mutagenesis. However, since the resurrection of Sleeping Beauty (SB), the possibility of performing forward genetics in mice has been reinforced. Recently, piggyBac (PB), a functional transposon from insects, was also described to work in mammals. As the activity of PB is higher than that of SB11 and SB12, two hyperactive SB transposases, we have characterized and improved the PB system in mouse ES cells. We have generated a mouse codon-optimized version of the PB transposase coding sequence (CDS) which provides transposition levels greater than the original. We have also found that the promoter sequence predicted in the 5′-terminal repeat of the PB transposon is active in the mammalian context. Finally, we have engineered inducible versions of the optimized piggyBac transposase fused with ERT2. One of them, when induced, provides higher levels of transposition than the native piggyBac CDS, whereas in the absence of induction its activity is indistinguishable from background. We expect that these tools, adaptable to perform mouse-germline mutagenesis, will facilitate the identification of genes involved in pathological and physiological processes, such as cancer or ES cell differentiation. PMID:17576687

  9. piggyBac transposon remobilization and enhancer detection in Anopheles mosquitoes.

    PubMed

    O'Brochta, David A; Alford, Robert T; Pilitt, Kristina L; Aluvihare, Channa U; Harrell, Robert A

    2011-09-27

    Technical advances in mosquito biology are enabling the development of new approaches to vector control. Absent are powerful forward-genetics technologies, such as enhancer and gene traps, that permit determination of gene functions from the phenotypes arising from transposon insertion mutations. We show that the piggyBac transposon is highly active in the germline of the human malaria vector Anopheles stephensi. Up to 6% of the progeny from transgenic A. stephensi containing a single 6-kb piggyBac element with a marker gene expressing EGFP had the vector in new genomic locations when piggyBac transposase was provided in trans from a second integrated transgene. The active transposition of piggyBac resulted in the efficient detection of enhancers, with ~10% of the progeny with piggyBac in new locations with novel patterns of EGFP expression in third and fourth instar larvae and in adults. The availability of advanced transgenic capabilities such as efficient transposon-based forward-genetics technologies for Anopheles mosquitoes not only will accelerate our understanding of mosquito functional genomics and the development of novel vector and disease transmission control strategies, but also will enable studies by evolutionary developmental biologists, virologists, and parasitologists.

  10. Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon

    PubMed Central

    Katayama, Hokahiro; Yasuchika, Kentaro; Miyauchi, Yuya; Kojima, Hidenobu; Yamaoka, Ryoya; Kawai, Takayuki; Yukie Yoshitoshi, Elena; Ogiso, Satoshi; Kita, Sadahiko; Yasuda, Katsutaro; Sasaki, Naoya; Fukumitsu, Ken; Komori, Junji; Ishii, Takamichi; Uemoto, Shinji

    2017-01-01

    Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various methods to deliver genes into cells, typically used genome-integrating viral vectors are associated with integration-related adverse events such as mutagenesis, whereas non-integrating viral vectors have low efficiency, making viral vectors unsuitable for clinical application. Therefore, we focused on developing a transposon system to establish a non-viral reprogramming method. Transposons are unique DNA elements that can be integrated into and removed from chromosomes. PiggyBac, a type of transposon, has high transduction efficiency and cargo capacity, and the integrated transgene can be precisely excised in the presence of transposase. This feature enables the piggyBac vector to achieve efficient transgene expression and a transgene-free state, thus making it a promising method for cell reprogramming. Here, we attempted to utilize the piggyBac transposon system to generate iHeps by integrating a transgene consisting of Hnf4a and Foxa3, and successfully obtained functional iHeps. We then demonstrated removal of the transgene to obtain transgene-free iHeps, which still maintained hepatocyte functions. This non-viral, transgene-free reprogramming method using the piggyBac vector may facilitate clinical applications of iHeps in upcoming cell therapy. PMID:28295042

  11. Hyperactive PiggyBac Transposons for Sustained and Robust Liver-targeted Gene Therapy

    PubMed Central

    Di Matteo, Mario; Samara-Kuko, Emira; Ward, Natalie J; Waddingon, Simon N; McVey, John H; Chuah, Marinee KL; VandenDriessche, Thierry

    2014-01-01

    The development of robust nonviral vectors could facilitate clinical gene therapy applications and may overcome some of the immune complications of viral vectors. Nevertheless, most nonviral gene deliver approaches typically yield only transient and/or low gene expression. To address these caveats, we have explored piggyBac transposons to correct hemophilia B by liver-directed factor IX (FIX) gene therapy in hemophilic mice. To achieve this, we combined the use of: (i) a hyperactive codon-optimized piggyBac transposase, (ii) a computationally enhanced liver-specific promoter, (iii) a hyperfunctional codon-optimized FIX transgene (FIX R338L Padua), and (iv) a modification of the transposon terminal repeats. This combination strategy resulted in a robust 400-fold improvement in vector performance in hepatocytes, yielding stable supraphysiologic human FIX activity (>1 year). Liver-specific expression resulted in the induction of FIX-specific immune tolerance. Remarkably, only very low transposon/transposase doses were required to cure the bleeding diathesis. Similarly, PB transposons could be used to express supraphysiologic factor VIII levels using low transposon/transposase doses. PB transposition did not induce tumors in a sensitive hepatocellular carcinoma-prone mouse model. These results underscore the potency and relative safety of the latest generation PB transposons, which constitutes a versatile platform for stable and robust secretion of therapeutic proteins. PMID:25034357

  12. Hyperactive PiggyBac Transposons for Sustained and Robust Liver-targeted Gene Therapy.

    PubMed

    Di Matteo, Mario; Samara-Kuko, Emira; Ward, Natalie J; Waddingon, Simon N; McVey, John H; Chuah, Marinee Kl; VandenDriessche, Thierry

    2014-09-01

    The development of robust nonviral vectors could facilitate clinical gene therapy applications and may overcome some of the immune complications of viral vectors. Nevertheless, most nonviral gene deliver approaches typically yield only transient and/or low gene expression. To address these caveats, we have explored piggyBac transposons to correct hemophilia B by liver-directed factor IX (FIX) gene therapy in hemophilic mice. To achieve this, we combined the use of: (i) a hyperactive codon-optimized piggyBac transposase, (ii) a computationally enhanced liver-specific promoter, (iii) a hyperfunctional codon-optimized FIX transgene (FIX R338L Padua), and (iv) a modification of the transposon terminal repeats. This combination strategy resulted in a robust 400-fold improvement in vector performance in hepatocytes, yielding stable supraphysiologic human FIX activity (>1 year). Liver-specific expression resulted in the induction of FIX-specific immune tolerance. Remarkably, only very low transposon/transposase doses were required to cure the bleeding diathesis. Similarly, PB transposons could be used to express supraphysiologic factor VIII levels using low transposon/transposase doses. PB transposition did not induce tumors in a sensitive hepatocellular carcinoma-prone mouse model. These results underscore the potency and relative safety of the latest generation PB transposons, which constitutes a versatile platform for stable and robust secretion of therapeutic proteins.

  13. Hyperactive piggyBac transposons for sustained and robust liver-targeted gene therapy.

    PubMed

    Di Matteo, Mario; Samara-Kuko, Emira; Ward, Natalie J; Waddington, Simon N; Waddingon, Simon N; McVey, John H; Chuah, Marinee K L; VandenDriessche, Thierry

    2014-09-01

    The development of robust nonviral vectors could facilitate clinical gene therapy applications and may overcome some of the immune complications of viral vectors. Nevertheless, most nonviral gene deliver approaches typically yield only transient and/or low gene expression. To address these caveats, we have explored piggyBac transposons to correct hemophilia B by liver-directed factor IX (FIX) gene therapy in hemophilic mice. To achieve this, we combined the use of: (i) a hyperactive codon-optimized piggyBac transposase, (ii) a computationally enhanced liver-specific promoter, (iii) a hyperfunctional codon-optimized FIX transgene (FIX R338L Padua), and (iv) a modification of the transposon terminal repeats. This combination strategy resulted in a robust 400-fold improvement in vector performance in hepatocytes, yielding stable supraphysiologic human FIX activity (>1 year). Liver-specific expression resulted in the induction of FIX-specific immune tolerance. Remarkably, only very low transposon/transposase doses were required to cure the bleeding diathesis. Similarly, PB transposons could be used to express supraphysiologic factor VIII levels using low transposon/transposase doses. PB transposition did not induce tumors in a sensitive hepatocellular carcinoma-prone mouse model. These results underscore the potency and relative safety of the latest generation PB transposons, which constitutes a versatile platform for stable and robust secretion of therapeutic proteins.

  14. Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition.

    PubMed

    Bire, Solenne; Gosset, David; Jégot, Gwenhael; Midoux, Patrick; Pichon, Chantal; Rouleux-Bonnin, Florence

    2013-09-26

    Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration

  15. Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.

    PubMed

    Owens, Jesse B; Mauro, Damiano; Stoytchev, Ilko; Bhakta, Mital S; Kim, Moon-Soo; Segal, David J; Moisyadi, Stefan

    2013-10-01

    Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.

  16. A piggyBac transposon gene trap for the analysis of gene expression and function in Drosophila.

    PubMed Central

    Bonin, Christopher P; Mann, Richard S

    2004-01-01

    P-element-based gene and enhancer trap strategies have provided a wealth of information on the expression and function of genes in Drosophila melanogaster. Here we present a new vector that utilizes the simple insertion requirements of the piggyBac transposon, coupled to a splice acceptor (SA) site fused to the sequence encoding enhanced green fluorescent protein (EGFP) and a transcriptional terminator. Mobilization of the piggyBac splice site gene trap vector (PBss) was accomplished by heat-shock-induced expression of piggyBac transposase (PBase). We show that insertion of PBss into genes leads to fusions between the gene's mRNA and the PBss-encoded EGFP transcripts. As heterozygotes, these fusions report the normal pattern of expression of the trapped gene. As homozygotes, these fusions can inactivate the gene and lead to lethality. Molecular characterization of PBss insertion events shows that they are single copy, that they always occur at TTAA sequences, and that splicing utilizes the engineered splice site in PBss. In those instances where protein-EGFP fusions are predicted to occur, the subcellular localization of the wild-type protein can be inferred from the localization of the EGFP fusion protein. These experiments highlight the utility of the PBss system for expanding the functional genomics tools that are available in Drosophila. PMID:15342518

  17. piggybac- and PhiC31-mediated genetic transformation of the Asian tiger mosquito, Aedes albopictus (Skuse).

    PubMed

    Labbé, Geneviève M C; Nimmo, Derric D; Alphey, Luke

    2010-08-17

    The Asian tiger mosquito, Aedes albopictus (Skuse), is a vector of several arboviruses including dengue and chikungunya. This highly invasive species originating from Southeast Asia has travelled the world in the last 30 years and is now established in Europe, North and South America, Africa, the Middle East and the Caribbean. In the absence of vaccine or antiviral drugs, efficient mosquito control strategies are crucial. Conventional control methods have so far failed to control Ae. albopictus adequately. Germline transformation of Aedes albopictus was achieved by micro-injection of embryos with a piggyBac-based transgene carrying a 3xP3-ECFP marker and an attP site, combined with piggyBac transposase mRNA and piggyBac helper plasmid. Five independent transgenic lines were established, corresponding to an estimated transformation efficiency of 2-3%. Three lines were re-injected with a second-phase plasmid carrying an attB site and a 3xP3-DsRed2 marker, combined with PhiC31 integrase mRNA. Successful site-specific integration was observed in all three lines with an estimated transformation efficiency of 2-6%. Both piggybac- and site-specific PhiC31-mediated germline transformation of Aedes albopictus were successfully achieved. This is the first report of Ae. albopictus germline transformation and engineering, a key step towards studying and controlling this species using novel molecular techniques and genetic control strategies.

  18. DDE Transposases: Structural Similarity and Diversity

    PubMed Central

    Nesmelova, Irina V.; Hackett, Perry B.

    2010-01-01

    DNA transposons are mobile DNA elements that can move from one DNA molecule to another and thereby deliver genetic information into human chromosomes in order to confer a new function or replace a defective gene. This process requires a transposase enzyme. During transposition DD[E/D]-transposases undergo a series of conformational changes. We summarize the structural features of DD[E/D]-transposases for which three-dimensional structures are available and that relate to transposases, which are being developed for use in mammalian cells. Similar to other members of the polynucleotidyl transferase family, the catalytic domains of DD[E/D]-transposases share a common feature: an RNase H-like fold that draws three catalytically active residues, the DDE motif, into close proximity. Beyond this fold, the structures of catalytic domains vary considerably, and the DD[E/D]-transposases display marked structural diversity within their DNA-binding domains. Yet despite such structural variability, essentially the same end result is achieved. PMID:20615441

  19. Structural Determinants of Sleeping Beauty Transposase Activity

    PubMed Central

    Abrusán, György; Yant, Stephen R; Szilágyi, András; Marsh, Joseph A; Mátés, Lajos; Izsvák, Zsuzsanna; Barabás, Orsolya; Ivics, Zoltán

    2016-01-01

    Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called “sectors”, which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold. PMID:27401040

  20. Structural Determinants of Sleeping Beauty Transposase Activity.

    PubMed

    Abrusán, György; Yant, Stephen R; Szilágyi, András; Marsh, Joseph A; Mátés, Lajos; Izsvák, Zsuzsanna; Barabás, Orsolya; Ivics, Zoltán

    2016-08-01

    Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called "sectors", which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold.

  1. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts

    PubMed Central

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K.

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. PMID:26682797

  2. Suicidal autointegration of sleeping beauty and piggyBac transposons in eukaryotic cells.

    PubMed

    Wang, Yongming; Wang, Jichang; Devaraj, Anatharam; Singh, Manvendra; Jimenez Orgaz, Ana; Chen, Jia-Xuan; Selbach, Matthias; Ivics, Zoltán; Izsvák, Zsuzsanna

    2014-03-01

    Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. 'Cut and paste' DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.

  3. Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.

    PubMed

    Wang, Gang; Yang, Luhan; Grishin, Dennis; Rios, Xavier; Ye, Lillian Y; Hu, Yong; Li, Kai; Zhang, Donghui; Church, George M; Pu, William T

    2017-01-01

    Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.

  4. Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system.

    PubMed

    Cunningham, Sharon C; Siew, Susan M; Hallwirth, Claus V; Bolitho, Christine; Sasaki, Natsuki; Garg, Gagan; Michael, Iacovos P; Hetherington, Nicola A; Carpenter, Kevin; de Alencastro, Gustavo; Nagy, Andras; Alexander, Ian E

    2015-08-01

    Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf(ash) mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration. © 2015 by

  5. piggyBac transposons expressing full-length human dystrophin enable genetic correction of dystrophic mesoangioblasts.

    PubMed

    Loperfido, Mariana; Jarmin, Susan; Dastidar, Sumitava; Di Matteo, Mario; Perini, Ilaria; Moore, Marc; Nair, Nisha; Samara-Kuko, Ermira; Athanasopoulos, Takis; Tedesco, Francesco Saverio; Dickson, George; Sampaolesi, Maurilio; VandenDriessche, Thierry; Chuah, Marinee K

    2016-01-29

    Duchenne muscular dystrophy (DMD) is a genetic neuromuscular disorder caused by the absence of dystrophin. We developed a novel gene therapy approach based on the use of the piggyBac (PB) transposon system to deliver the coding DNA sequence (CDS) of either full-length human dystrophin (DYS: 11.1 kb) or truncated microdystrophins (MD1: 3.6 kb; MD2: 4 kb). PB transposons encoding microdystrophins were transfected in C2C12 myoblasts, yielding 65±2% MD1 and 66±2% MD2 expression in differentiated multinucleated myotubes. A hyperactive PB (hyPB) transposase was then deployed to enable transposition of the large-size PB transposon (17 kb) encoding the full-length DYS and green fluorescence protein (GFP). Stable GFP expression attaining 78±3% could be achieved in the C2C12 myoblasts that had undergone transposition. Western blot analysis demonstrated expression of the full-length human DYS protein in myotubes. Subsequently, dystrophic mesoangioblasts from a Golden Retriever muscular dystrophy dog were transfected with the large-size PB transposon resulting in 50±5% GFP-expressing cells after stable transposition. This was consistent with correction of the differentiated dystrophic mesoangioblasts following expression of full-length human DYS. These results pave the way toward a novel non-viral gene therapy approach for DMD using PB transposons underscoring their potential to deliver large therapeutic genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Suicidal Autointegration of Sleeping Beauty and piggyBac Transposons in Eukaryotic Cells

    PubMed Central

    Devaraj, Anatharam; Singh, Manvendra; Jimenez Orgaz, Ana; Chen, Jia-Xuan; Selbach, Matthias; Ivics, Zoltán; Izsvák, Zsuzsanna

    2014-01-01

    Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. ‘Cut and paste’ DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB. PMID:24625543

  7. A hyperactive sleeping beauty transposase enhances transgenesis in zebrafish embryos.

    PubMed

    Newman, Morgan; Lardelli, Michael

    2010-11-04

    Transposons are useful molecular tools for transgenesis. The 'sleeping beauty' transposon is a synthetic member of the Tc1/mariner transposon family. Davidson et al. (2003) previously described a vector for zebrafish transgenesis consisting of the inverted repeats of 'sleeping beauty' flanking the gene to be transposed. Subsequently, there have been attempts to enhance the transpositional activity of 'sleeping beauty' by increasing the activity of its transposase. Recently, Mates et al. (2009) generated a hyperactive transposase giving a 100-fold increased transposition rate in mouse embryos. The aim of this experiment was to determine whether this novel hyperactive transposase enhances transgenesis in zebrafish embryos. Using our previously characterised mitfa-amyloidβ-GFP transgene, we observed an eight-fold enhancement in transient transgenesis following detection of transgene expression in melanophores by whole mount in-situ hybridisation. However, high rates of defective embryogenesis were also observed. The novel hyperactive 'sleeping beauty' transposase enhances the rate of transgenesis in zebrafish embryos.

  8. Excision Efficiency Is Not Strongly Coupled to Transgenic Rate: Cell Type-Dependent Transposition Efficiency of Sleeping Beauty and piggyBac DNA Transposons

    PubMed Central

    Kolacsek, Orsolya; Erdei, Zsuzsa; Apáti, Ágota; Sándor, Sára; Izsvák, Zsuzsanna; Ivics, Zoltán; Sarkadi, Balázs

    2014-01-01

    Abstract The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared because of differences in applications. In addition, “overproduction inhibition,” a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of transposase dose applied. Therefore, because of lack of comprehensive analysis, researchers are forced to optimize the technology for their own “in-house” platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in various cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2–3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future. PMID:25045962

  9. Excision efficiency is not strongly coupled to transgenic rate: cell type-dependent transposition efficiency of sleeping beauty and piggyBac DNA transposons.

    PubMed

    Kolacsek, Orsolya; Erdei, Zsuzsa; Apáti, Agota; Sándor, Sára; Izsvák, Zsuzsanna; Ivics, Zoltán; Sarkadi, Balázs; Orbán, Tamás I

    2014-08-01

    The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared because of differences in applications. In addition, "overproduction inhibition," a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of transposase dose applied. Therefore, because of lack of comprehensive analysis, researchers are forced to optimize the technology for their own "in-house" platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in various cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2-3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future.

  10. Sperm-mediated transgenesis in chicken using a PiggyBac transposon system

    USDA-ARS?s Scientific Manuscript database

    Towards development of transgenic chickens without the use of viral vectors, factors affecting sperm mediated gene transfer (SMGT) using a piggyBac vector are being studied. The piggyBac pPBCAG-LacZ contains 13bp terminal inverted repeats flanking a LacZ gene driven by the CAG promoter. A helper pla...

  11. A non-autonomous insect piggyBac trasposable element is mobile in tobacco

    USDA-ARS?s Scientific Manuscript database

    The piggyBac transposable element, originally isolated from a virus in an insect cell line, is a valuable molecular tool for transgenesis and mutagenesis of invertebrates. For heterologous transgenesis in a variety of mammals, transfer of the piggyBac transposable element from an ectopic plasmid onl...

  12. Purification, crystallization and preliminary crystallographic analysis of the Hermes transposase

    SciTech Connect

    Perez, Zhanita N.; Musingarimi, Primrose; Craig, Nancy L.; Dyda, Fred; Hickman, Alison Burgess

    2005-06-01

    Upon purification, an N-terminally deleted version of the Hermes transposase exists in solution as a mixture of two species that are approximately hexameric and dimeric. Crystals have been obtained of the smaller species that diffract to 2.1 Å resolution. DNA transposition is the movement of a defined segment of DNA from one location to another. Although the enzymes that catalyze transposition in bacterial systems have been well characterized, much less is known about the families of transposase enzymes that function in higher organisms. Active transposons have been identified in many insect species, providing tools for gene identification and offering the possibility of altering the genotypes of natural insect populations. One of these active transposons is Hermes, a 2749-base-pair element from Musca domestica that encodes its own transposase. An N-terminally deleted version of the Hermes transposase (residues 79–612) has been overexpressed and purified, and crystals that diffract to 2.1 Å resolution have been obtained at 277 K by the hanging-drop method.

  13. The Functionality of Minimal PiggyBac Transposons in Mammalian Cells

    PubMed Central

    Troyanovsky, Boris; Bitko, Vira; Pastukh, Viktor; Fouty, Brian; Solodushko, Victor

    2016-01-01

    Minimal piggyBac vectors are a modified single-plasmid version of the classical piggyBac delivery system that can be used for stable transgene integration. These vectors have a truncated terminal domain in the delivery cassette and thus, integrate significantly less flanking transposon DNA into host cell chromatin than classical piggyBac vectors. Herein, we test various characteristics of this modified transposon. The integration efficiency of minimal piggyBac vectors was inversely related to the size of both the transposon and the entire plasmid, but inserts as large as 15 kb were efficiently integrated. Open and super-coiled vectors demonstrated the same integration efficiency while DNA methylation decreased the integration efficiency and silenced the expression of previously integrated sequences in some cell types. Importantly, the incidence of plasmid backbone integration was not increased above that seen in nontransposon control vectors. In BALB/c mice, we demonstrated prolonged expression of two transgenes (intracellular mCherry and secretable Gaussia luciferase) when delivered by the minimal piggyBac that resulted in a more sustained antibody production against the immunogenic luciferase than when delivered by a transient (nontransposon) vector plasmid. We conclude that minimal piggyBac vectors are an effective alternative to other integrative systems for stable DNA delivery in vitro and in vivo. PMID:27701401

  14. Sleeping Beauty transposase structure allows rational design of hyperactive variants for genetic engineering.

    PubMed

    Voigt, Franka; Wiedemann, Lisa; Zuliani, Cecilia; Querques, Irma; Sebe, Attila; Mátés, Lajos; Izsvák, Zsuzsanna; Ivics, Zoltán; Barabas, Orsolya

    2016-03-30

    Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB's applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases.

  15. Sleeping Beauty transposase structure allows rational design of hyperactive variants for genetic engineering

    PubMed Central

    Voigt, Franka; Wiedemann, Lisa; Zuliani, Cecilia; Querques, Irma; Sebe, Attila; Mátés, Lajos; Izsvák, Zsuzsanna; Ivics, Zoltán; Barabas, Orsolya

    2016-01-01

    Sleeping Beauty (SB) is a prominent Tc1/mariner superfamily DNA transposon that provides a popular genome engineering tool in a broad range of organisms. It is mobilized by a transposase enzyme that catalyses DNA cleavage and integration at short specific sequences at the transposon ends. To facilitate SB's applications, here we determine the crystal structure of the transposase catalytic domain and use it to model the SB transposase/transposon end/target DNA complex. Together with biochemical and cell-based transposition assays, our structure reveals mechanistic insights into SB transposition and rationalizes previous hyperactive transposase mutations. Moreover, our data enables us to design two additional hyperactive transposase variants. Our work provides a useful resource and proof-of-concept for structure-based engineering of tailored SB transposases. PMID:27025571

  16. Germ-line transformation of the Queensland fruit fly, Bactrocera tryoni, using a piggyBac vector in the presence of endogenous piggyBac elements

    USDA-ARS?s Scientific Manuscript database

    We report the stable genetic transformation of the Queensland fruit fly Bactrocera tryoni using a piggyBac vector marked with either the fluorescent protein DsRed or EGFP.A transformation frequency of 5–10% was obtained.Inheritance of the transgenes has remained stable over eight generations despite...

  17. Mutations in the mariner transposase: The D,D(35)E consensus sequence is nonfunctional

    PubMed Central

    Lohe, Allan R.; De Aguiar, Daniel; Hartl, Daniel L.

    1997-01-01

    Genetic analysis of eukaryote transposases and comparison with their prokaryote counterparts have been greatly hindered by difficulty in isolating mutations. We describe a simple eye-color screen that facilitates isolation and analysis of mutations in the mariner transposase in Drosophila melanogaster. Use of ethyl methanesulfonate and site-directed mutagenesis has identified 18 residues that are critical for in vivo excision of a target mariner element. When the mutations were examined in heterozygous mutant/nonmutant genotypes, more than half of the mutant transposase proteins were found to reduce the activity of the wild-type transposase, as assayed by the frequency of germline excision of a target element. Remarkably, transposase function is obliterated when the D,D(34)D acidic, ion-binding domain is replaced with the consensus sequence D,D(34)E found in the nematode Tc1 transposase and in many other transposases in the superfamily. A number of mutations strongly complement wild-type transposase in a dominant-negative manner, suggestive of subunit interactions in the excision reaction; these mutations are located in a small region that includes part of the D,D(34)D motif. Transposase function also is eliminated by a mutation in the inferred initiation codon and by a mutation in a putative nuclear localization signal. PMID:9037046

  18. Transposases are the most abundant, most ubiquitous genes in nature

    PubMed Central

    Aziz, Ramy K.; Breitbart, Mya; Edwards, Robert A.

    2010-01-01

    Genes, like organisms, struggle for existence, and the most successful genes persist and widely disseminate in nature. The unbiased determination of the most successful genes requires access to sequence data from a wide range of phylogenetic taxa and ecosystems, which has finally become achievable thanks to the deluge of genomic and metagenomic sequences. Here, we analyzed 10 million protein-encoding genes and gene tags in sequenced bacterial, archaeal, eukaryotic and viral genomes and metagenomes, and our analysis demonstrates that genes encoding transposases are the most prevalent genes in nature. The finding that these genes, classically considered as selfish genes, outnumber essential or housekeeping genes suggests that they offer selective advantage to the genomes and ecosystems they inhabit, a hypothesis in agreement with an emerging body of literature. Their mobile nature not only promotes dissemination of transposable elements within and between genomes but also leads to mutations and rearrangements that can accelerate biological diversification and—consequently—evolution. By securing their own replication and dissemination, transposases guarantee to thrive so long as nucleic acid-based life forms exist. PMID:20215432

  19. Integrating prokaryotes and eukaryotes: DNA transposases in light of structure

    PubMed Central

    Hickman, Alison Burgess; Chandler, Michael; Dyda, Fred

    2011-01-01

    DNA rearrangements are important in genome function and evolution. Genetic material can be rearranged inadvertently during processes such as DNA repair, or can be moved in a controlled manner by enzymes specifically dedicated to the task. DNA transposases comprise one class of such enzymes. These move DNA segments known as transposons to new locations, without the need for sequence homology between transposon and target site. Several biochemically distinct pathways have evolved for DNA transposition, and genetic and biochemical studies have provided valuable insights into many of these. However, structural information on transposases – particularly with DNA substrates – has proven elusive in most cases. On the other hand, large-scale genome sequencing projects have led to an explosion in the number of annotated prokaryotic and eukaryotic mobile elements. Here, we briefly review biochemical and mechanistic aspects of DNA transposition, and propose that integrating sequence information with structural information using bioinformatics tools such as secondary structure prediction and protein threading can lead not only to an additional level of understanding but possibly also to testable hypotheses regarding transposition mechanisms. Detailed understanding of transposition pathways is a prerequisite for the long-term goal of exploiting DNA transposons as genetic tools and as a basis for genetic medical applications. PMID:20067338

  20. Chromosomal mobilization and reintegration of Sleeping Beauty and PiggyBac transposons.

    PubMed

    Liang, Qi; Kong, Jun; Stalker, James; Bradley, Allan

    2009-06-01

    The Sleeping Beauty and PiggyBac DNA transposon systems have recently been developed as tools for insertional mutagenesis. We have compared the chromosomal mobilization efficiency and insertion site preference of the two transposons mobilized from the same donor site in mouse embryonic stem (ES) cells under conditions in which there were no selective constraints on the transposons' insertion sites. Compared with Sleeping Beauty, PiggyBac exhibits higher transposition efficiencies, no evidence for local hopping and a significant bias toward reintegration in intragenic regions, which demonstrate its utility for insertional mutagenesis. Although Sleeping Beauty had no detectable genomic bias with respect to insertions in genes or intergenic regions, both Sleeping Beauty and PiggyBac transposons displayed preferential integration into actively transcribed loci. Copyright 2009 Wiley-Liss, Inc.

  1. Bioreactor scale up and protein product quality characterization of piggyBac transposon derived CHO pools.

    PubMed

    Rajendra, Yashas; Balasubramanian, Sowmya; Peery, Robert B; Swartling, James R; McCracken, Neil A; Norris, Dawn L; Frye, Christopher C; Barnard, Gavin C

    2017-02-11

    Chinese hamster ovary (CHO) cells remain the most popular host for the production of biopharmaceutical drugs, particularly monoclonal antibodies (mAbs), bispecific antibodies, and Fc-fusion proteins. Creating and characterizing the stable CHO clonally-derived cell lines (CDCLs) needed to manufacture these therapeutic proteins is a lengthy and laborious process. Therefore, CHO pools have increasingly been used to rapidly produce protein to support and enable preclinical drug development. We recently described the generation of CHO pools yielding mAb titers as high as 7.6 g/L in a 16 day bioprocess using piggyBac transposon-mediated gene integration. In this study, we wanted to understand why the piggyBac pool titers were significantly higher (2-10 fold) than the control CHO pools. Higher titers were the result of a combination of increased average gene copy number, significantly higher messenger RNA levels and the homogeneity (i.e. less diverse population distribution) of the piggyBac pools, relative to the control pools. In order to validate the use of piggyBac pools to support preclinical drug development, we then performed an in-depth product quality analysis of purified protein. The product quality of protein obtained from the piggyBac pools was very similar to the product quality profile of protein obtained from the control pools. Finally, we demonstrated the scalability of these pools from shake flasks to 36L bioreactors. Overall, these results suggest that gram quantities of therapeutic protein can be rapidly obtained from piggyBac CHO pools without significantly changing product quality attributes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 2017.

  2. Genetic transformation of the codling moth, Cydia pomonella L., with piggyBac EGFP.

    PubMed

    Ferguson, Holly J; Neven, Lisa G; Thibault, Stephen T; Mohammed, Ahmed; Fraser, Malcolm

    2011-02-01

    Genetic transformation of the codling moth, Cydia pomonella, was accomplished through embryo microinjection with a plasmid-based piggyBac vector containing the enhanced green fluorescent protein (EGFP) gene. Sequencing of the flanking regions around the inserted construct resulted in identification of insect genomic sequences, not plasmid sequences, thus providing evidence that the piggyBac EGFP cassette had integrated into the codling moth genome. EGFP-positive moths were confirmed in the 28th and earlier generations post injection through PCR and Southern blot analyses, indicating heritability of the transgene.

  3. The piggyBac Transposon Displays Local and Distant Reintegration Preferences and Can Cause Mutations at Noncanonical Integration Sites

    PubMed Central

    Li, Meng Amy; Pettitt, Stephen J.; Eckert, Sabine; Ning, Zemin; Rice, Stephen; Cadiñanos, Juan; Yusa, Kosuke; Conte, Nathalie

    2013-01-01

    The DNA transposon piggyBac is widely used as a tool in mammalian experimental systems for transgenesis, mutagenesis, and genome engineering. We have characterized genome-wide insertion site preferences of piggyBac by sequencing a large set of integration sites arising from transposition from two separate genomic loci and a plasmid donor in mouse embryonic stem cells. We found that piggyBac preferentially integrates locally to the excision site when mobilized from a chromosomal location and identified other nonlocal regions of the genome with elevated insertion frequencies. piggyBac insertions were associated with expressed genes and markers of open chromatin structure and were excluded from heterochromatin. At the nucleotide level, piggyBac prefers to insert into TA-rich regions within a broader GC-rich context. We also found that piggyBac can insert into sites other than its known TTAA insertion site at a low frequency (2%). Such insertions introduce mismatches that are repaired with signatures of host cell repair pathways. Transposons could be mobilized from plasmids with the observed noncanonical flanking regions, indicating that piggyBac could generate point mutations in the genome. PMID:23358416

  4. Efficient production of transgenic chickens based on piggyBac.

    PubMed

    Liu, Xiaojuan; Li, Ning; Hu, Xiaoxiang; Zhang, Ran; Li, Qingyuan; Cao, Dainan; Liu, Tongxin; Zhang, Yaqiong; Liu, Xiaofang

    2013-04-01

    Transgenic techniques in chickens have been developed much more slowly than in mammals due to chickens' unique reproduction mechanism. Retroviral methods have been the most successful. piggyBac (PB) is a transposon that has a 13 bp perfect terminal invert repeat sequence. PB can be inserted into TTAA sites and can also be precisely excised in mammals. Therefore, we have selected PB as a candidate to establish a new method to produce transgenic chickens. We constructed three donor vectors (ZGl-neo, ZGm-neo and ZGs-neo) expressing a GFP marker-gene and a neomycin resistant gene based on PB. We co-transfected each donor vector with a helper vector (CAG-PBase). We found that ZGl-neo was the most efficient PB vector. This vector could insert into TTAA sites in DF-1 cells. PB vectors were microinjected into sub-germinal cavity of newly laid eggs, and electroporation was then performed with a 20-V pulse for 5 cycles of 50 ms on and 100 ms off. GFP was expressed in different tissues of the embryos, including the gonads. Twenty-two chickens hatched after microinjection with compounds ZGl-neo and CAG-PBase (3:1). When we screened the blood DNA, 73 % (16/22) of the individuals were positive. Thirteen of the chickens grew to adulthood, 11 of which were males. 40 % (4/10) of the individuals were semen positive, and their copy numbers ranged from 0.05 to 0.21 (0.11, 0.21, 0.05, 0.06). No G1 offspring containing the integrated transposon were produced. We conclude that the PB transposon system is a novel useful tool for the efficient production of transgenic chickens.

  5. Sperm-mediated transgenesis in chicken using a PiggyBac transposon system

    USDA-ARS?s Scientific Manuscript database

    Sperm-mediated transgenesis in chicken using a PiggyBac transposon system Emmanuel Quansah1,2, Julie Long2, David Donovan2, Stephen Becker2, Bhanu Telugu2, Juli Frey2, Nigel Urwin1 1,Charles Sturt University, Graham Center of Agricultural Innovation, Wagga Wagga. Australia and 2Beltsville Agricultu...

  6. Genetic transformation mediated by piggyBac in the Asian corn borer, Ostrinia furnacalis (Lepidoptera: Crambidae)

    USDA-ARS?s Scientific Manuscript database

    The Asian corn borer, Ostrinia furnacalis, is a serious pest of corn, sorghum and cotton in China and other Asian countries. The present study is the first attempt to establish the transgenic line in O. furnacalis using a piggyBac transposon, which will shed light on the future genetic control of O....

  7. Insect transformation with piggyBac: getting the number of injections just right

    PubMed Central

    Morrison, N. I.; Shimeld, S. M.

    2016-01-01

    Abstract The insertion of exogenous genetic cargo into insects using transposable elements is a powerful research tool with potential applications in meeting food security and public health challenges facing humanity. piggyBac is the transposable element most commonly utilized for insect germline transformation. The described efficiency of this process is variable in the published literature, and a comprehensive review of transformation efficiency in insects is lacking. This study compared and contrasted all available published data with a comprehensive data set provided by a biotechnology group specializing in insect transformation. Based on analysis of these data, with particular focus on the more complete observational data from the biotechnology group, we designed a decision tool to aid researchers' decision‐making when using piggyBac to transform insects by microinjection. A combination of statistical techniques was used to define appropriate summary statistics of piggyBac transformation efficiency by species and insect order. Publication bias was assessed by comparing the data sets. The bias was assessed using strategies co‐opted from the medical literature. The work culminated in building the Goldilocks decision tool, a Markov‐Chain Monte‐Carlo simulation operated via a graphical interface and providing guidance on best practice for those seeking to transform insects using piggyBac. PMID:27027400

  8. Putative transposases conserved in Exiguobacterium isolates from ancient Siberian permafrost and from contemporary surface habitats.

    PubMed

    Vishnivetskaya, Tatiana A; Kathariou, Sophia

    2005-11-01

    Gram-positive bacteria of the genus Exiguobacterium have been repeatedly isolated from Siberian permafrost ranging in age from 20,000 to 2 to 3 million years and have been sporadically recovered from markedly diverse habitats, including microbial mats in Lake Fryxell (Antarctic), surface water, and food-processing environments. However, there is currently no information on genomic diversity of this microorganism or on the physiological strategies that have allowed its survival under prolonged freezing in the permafrost. Analysis of the genome sequence of the most ancient available Exiguobacterium isolate (Exiguobacterium sp. strain 255-15, from 2 to 3 million-year-old Siberian permafrost) revealed numerous putative transposase sequences, primarily of the IS200/IS605, IS30, and IS3 families, with four transposase families identified. Several of the transposase genes appeared to be part of insertion sequences. Southern blots with different transposase probes yielded high-resolution genomic fingerprints which differentiated the different permafrost isolates from each other and from the Exiguobacterium spp. type strains which have been derived from diverse surface habitats. Each of the Exiguobacterium sp. strain 255-15 transposases that were used as probes had highly conserved homologs in the genome of other Exiguobacterium strains, both from permafrost and from modern sites. These findings suggest that, prior to their entrapment in permafrost, Exiguobacterium isolates had acquired transposases and that conserved transposases are present in Exiguobacterium spp., which now can be isolated from various modern surface habitats.

  9. Mariner-like transposases are widespread and diverse in flowering plants.

    PubMed

    Feschotte, Cédric; Wessler, Susan R

    2002-01-08

    Complete and partial sequences of mariner-like elements (MLEs) have been reported for hundreds of species of animals, but only two have been identified in plants. On the basis of these two plant MLEs and several related sequences identified by database searches, plant-specific degenerate primers were derived and used to amplify a conserved region of MLE transposase genes from a variety of plant genomes. Positive products were obtained for 6 dicots and 31 monocots of 54 plant species tested. Phylogenetic analysis of 68 distinct MLE transposase sequences from 25 grass species is consistent with vertical transmission and rapid diversification of multiple lineages of transposases. Surprisingly, the evolution of MLEs in grasses was accompanied by repeated and independent acquisition of introns in a localized region of the transposase gene.

  10. Abundant transposases encoded by the metagenome of a hydrothermal chimney biofilm.

    PubMed

    Brazelton, William J; Baross, John A

    2009-12-01

    The carbonate chimneys of the Lost City Hydrothermal Field on the Mid-Atlantic Ridge are coated in thick microbial biofilms consisting of just a few dominant species. We report a preliminary analysis of a biofilm metagenome that revealed a remarkable abundance and diversity of genes potentially involved in lateral gene transfer (LGT). More than 8% of all metagenomic reads showed significant sequence similarity to transposases; all available metagenomic data sets from other environments contained at least an order of magnitude fewer transposases. Furthermore, the sequence diversity of transposase genes in the biofilm was much greater than that of 16S rRNA genes. The small size and high sequencing coverage of contigs containing transposases indicate that they are located on small but abundant extragenomic molecules. These results suggest that rampant LGT among members of the Lost City biofilm may serve as a generator of phenotypic diversity in a community with very low organismal diversity.

  11. Transposase interaction with the β sliding clamp: effects on insertion sequence proliferation and transposition rate

    PubMed Central

    Díaz-Maldonado, Héctor; Gómez, Manuel J.; Moreno-Paz, Mercedes; San Martín-Úriz, Patxi; Amils, Ricardo; Parro, Víctor; López de Saro, Francisco J.

    2015-01-01

    Insertion sequences (ISs) are ubiquitous and abundant mobile genetic elements in prokaryotic genomes. ISs often encode only one protein, the transposase, which catalyzes their transposition. Recent studies have shown that transposases of many different IS families interact with the β sliding clamp, a DNA replication factor of the host. However, it was unclear to what extent this interaction limits or favors the ability of ISs to colonize a chromosome from a phylogenetically-distant organism, or if the strength of this interaction affects the transposition rate. Here we describe the proliferation of a member of the IS1634 family in Acidiphilium over ~600 generations of cultured growth. We demonstrate that the purified transposase binds to the β sliding clamp of Acidiphilium, Leptospirillum and E. coli. Further, we also demonstrate that the Acidiphilium IS1634 transposase binds to the archaeal sliding clamp (PCNA) from Methanosarcina, and that the transposase encoded by Methanosarcina IS1634 binds to Acidiphilium β. Finally, we demonstrate that increasing the strength of the interaction between β and transposase results in a higher transposition rate in vivo. Our results suggest that the interaction could determine the potential of ISs to be mobilized in bacterial populations and also their ability to proliferate within chromosomes. PMID:26306550

  12. Crystal Structure of the Human Hsmar1-Derived Transposase Domain in the DNA Repair Enzyme Metnase

    SciTech Connect

    Goodwin, Kristie D.; He, Hongzhen; Imasaki, Tsuyoshi; Lee, Suk-Hee; Georgiadis, Millie M.

    2010-08-12

    Although the human genome is littered with sequences derived from the Hsmar1 transposon, the only intact Hsmar1 transposase gene exists within a chimeric SET-transposase fusion protein referred to as Metnase or SETMAR. Metnase retains many of the transposase activities including terminal inverted repeat (TIR) specific DNA-binding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein that is specifically involved in nonhomologous end joining. Here, we present two crystal structures of the transposase catalytic domain of Metnase revealing a dimeric enzyme with unusual active site plasticity that may be involved in modulating metal binding. We show through characterization of a dimerization mutant, F460K, that the dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. Of significance is the conservation of F460 along with residues that we propose may be involved in the modulation of metal binding in both the predicted ancestral Hsmar1 transposase sequence as well as in the modern enzyme. The Metnase transposase has been remarkably conserved through evolution; however, there is a clustering of substitutions located in alpha helices 4 and 5 within the putative DNA-binding site, consistent with loss of transposition specific DNA cleavage activity and acquisition of DNA repair specific cleavage activity.

  13. Genome-wide Profiling Reveals Remarkable Parallels Between Insertion Site Selection Properties of the MLV Retrovirus and the piggyBac Transposon in Primary Human CD4+ T Cells

    PubMed Central

    Gogol-Döring, Andreas; Ammar, Ismahen; Gupta, Saumyashree; Bunse, Mario; Miskey, Csaba; Chen, Wei; Uckert, Wolfgang; Schulz, Thomas F; Izsvák, Zsuzsanna; Ivics, Zoltán

    2016-01-01

    The inherent risks associated with vector insertion in gene therapy need to be carefully assessed. We analyzed the genome-wide distributions of Sleeping Beauty (SB) and piggyBac (PB) transposon insertions as well as MLV retrovirus and HIV lentivirus insertions in human CD4+ T cells with respect to a panel of 40 chromatin states. The distribution of SB transposon insertions displayed the least deviation from random, while the PB transposon and the MLV retrovirus showed unexpected parallels across all chromatin states. Both MLV and PB insertions are enriched at transcriptional start sites (TSSs) and co-localize with BRD4-associated sites. We demonstrate physical interaction between the PB transposase and bromodomain and extraterminal domain proteins (including BRD4), suggesting convergent evolution of a tethering mechanism that directs integrating genetic elements into TSSs. We detect unequal biases across the four systems with respect to targeting genes whose deregulation has been previously linked to serious adverse events in gene therapy clinical trials. The SB transposon has the highest theoretical chance of targeting a safe harbor locus in the human genome. The data underscore the significance of vector choice to reduce the mutagenic load on cells in clinical applications. PMID:26755332

  14. Mobilization of giant piggyBac transposons in the mouse genome.

    PubMed

    Li, Meng Amy; Turner, Daniel J; Ning, Zemin; Yusa, Kosuke; Liang, Qi; Eckert, Sabine; Rad, Lena; Fitzgerald, Tomas W; Craig, Nancy L; Bradley, Allan

    2011-12-01

    The development of technologies that allow the stable delivery of large genomic DNA fragments in mammalian systems is important for genetic studies as well as for applications in gene therapy. DNA transposons have emerged as flexible and efficient molecular vehicles to mediate stable cargo transfer. However, the ability to carry DNA fragments >10 kb is limited in most DNA transposons. Here, we show that the DNA transposon piggyBac can mobilize 100-kb DNA fragments in mouse embryonic stem (ES) cells, making it the only known transposon with such a large cargo capacity. The integrity of the cargo is maintained during transposition, the copy number can be controlled and the inserted giant transposons express the genomic cargo. Furthermore, these 100-kb transposons can also be excised from the genome without leaving a footprint. The development of piggyBac as a large cargo vector will facilitate a wider range of genetic and genomic applications.

  15. Hyperactive Himar1 transposase mediates transposition in cell culture and enhances gene expression in vivo.

    PubMed

    Keravala, Annahita; Liu, Dexi; Lechman, Eric R; Wolfe, Darren; Nash, Joan A; Lampe, David J; Robbins, Paul D

    2006-10-01

    The use of nonviral delivery systems results in transient gene expression, in part because of the low efficiency of DNA integration. Previously, vectors based on transposon systems such as Sleeping Beauty have been shown to be able to increase stable transfection efficiencies in cell culture and in animal models. Himar1, a reconstructed active transposon belonging to the Tc1/mariner superfamily, also has been used as a vector for stable gene delivery, but the rate of transposition after transfection is low. In this paper, we evaluate the potential of the hyperactive Himar1 transposase C9, in combination with the Himar1 inverted repeat transposon, as a gene delivery vector. The C9 transposase is a hyperactive mutant of Himar1 with two amino acid substitutions, Q131R and E137K, that result in an increase in activity relative to wild type. Here we demonstrate that cotransfection of the C9 transposase with a Himar1-based vector increases the frequency of stable gene expression in human cells in a transposase concentration-dependent manner. In addition, we establish that C9 transposase mediates integration of the transgene in mammalian cells at a frequency similar to that of Sleeping Beauty under some of the conditions tested. Last, we show significantly higher levels of reporter gene expression in vivo in mouse liver and in synovium of rabbit knee joints after injection of the transposon plasmid expressing the transgene and the C9 transposase. These data suggest that vectors based on the Himar1 transposable element, in conjunction with the hyperactive mutant transposase C9, may be suitable vectors for gene therapy applications.

  16. Temporal self-regulation of transposition through host-independent transposase rodlet formation

    PubMed Central

    Woodard, Lauren E.; Downes, Laura M.; Lee, Yi-Chien; Kaja, Aparna; Terefe, Eyuel S.; Wilson, Matthew H.

    2017-01-01

    Transposons are highly abundant in eukaryotic genomes, but their mobilization must be finely tuned to maintain host organism fitness and allow for transposon propagation. Forty percent of the human genome is comprised of transposable element sequences, and the most abundant cut-and-paste transposons are from the hAT superfamily. We found that the hAT transposase TcBuster from Tribolium castaneum formed filamentous structures, or rodlets, in human tissue culture cells, after gene transfer to adult mice, and ex vivo in cell-free conditions, indicating that host co-factors or cellular structures were not required for rodlet formation. Time-lapsed imaging of GFP-laced rodlets in human cells revealed that they formed quickly in a dynamic process involving fusion and fission. We delayed the availability of the transposon DNA and found that transposition declined after transposase concentrations became high enough for visible transposase rodlets to appear. In combination with earlier findings for maize Ac elements, these results give insight into transposase overproduction inhibition by demonstrating that the appearance of transposase protein structures and the end of active transposition are simultaneous, an effect with implications for genetic engineering and horizontal gene transfer. PMID:27899587

  17. Active Site Sharing and Subterminal Hairpin Recognition in a New Class of DNA Transposases

    SciTech Connect

    Ronning, Donald R.; Guynet, Catherine; Ton-Hoang, Bao; Perez, Zhanita N.; Ghirlando, Rodolfo; Chandler, Michael; Dyda, Fred

    2010-07-20

    Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg{sup 2+} and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalent intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.

  18. Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase

    PubMed Central

    Cocchiarella, Fabienne; Latella, Maria Carmela; Basile, Valentina; Miselli, Francesca; Galla, Melanie; Imbriano, Carol; Recchia, Alessandra

    2016-01-01

    The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raises increasing interest for gene therapy application, including genome modification and, more recently, induced pluripotent stem cells (iPS) reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by an integrating retroviral vector (iRV), has been circumvented by the transient delivery of SB100X using retroviral mRNA transfer. In this study, we developed an alternative, safe, and efficient transposase delivery system based on a tetracycline-ON regulated expression cassette and the rtTA2S-M2 transactivator gene transiently delivered by integration-defective lentiviral vectors (IDLVs). Compared with iRV-mediated delivery, expression of tetracycline-induced SB100X delivered by an IDLV results in more efficient integration of a GFP transposon and reduced toxicity. Tightly regulated expression and reactivation of the transposase was achieved in HeLa cells as wells as in human primary keratinocytes. Based on these properties, the regulated transposase-IDLV vectors may represent a valuable tool for genetic engineering and therapeutic gene transfer. PMID:27574698

  19. Transcriptionally regulated and nontoxic delivery of the hyperactive Sleeping Beauty Transposase.

    PubMed

    Cocchiarella, Fabienne; Latella, Maria Carmela; Basile, Valentina; Miselli, Francesca; Galla, Melanie; Imbriano, Carol; Recchia, Alessandra

    2016-01-01

    The Sleeping Beauty (SB) transposase and, in particular, its hyperactive variant SB100X raises increasing interest for gene therapy application, including genome modification and, more recently, induced pluripotent stem cells (iPS) reprogramming. The documented cytotoxicity of the transposase, when constitutively expressed by an integrating retroviral vector (iRV), has been circumvented by the transient delivery of SB100X using retroviral mRNA transfer. In this study, we developed an alternative, safe, and efficient transposase delivery system based on a tetracycline-ON regulated expression cassette and the rtTA2(S)-M2 transactivator gene transiently delivered by integration-defective lentiviral vectors (IDLVs). Compared with iRV-mediated delivery, expression of tetracycline-induced SB100X delivered by an IDLV results in more efficient integration of a GFP transposon and reduced toxicity. Tightly regulated expression and reactivation of the transposase was achieved in HeLa cells as wells as in human primary keratinocytes. Based on these properties, the regulated transposase-IDLV vectors may represent a valuable tool for genetic engineering and therapeutic gene transfer.

  20. ATP-dependent specific binding of Tn3 transposase to Tn3 inverted repeats

    NASA Astrophysics Data System (ADS)

    Wishart, W. L.; Broach, J. R.; Ohtsubo, E.

    1985-04-01

    Transposons are discrete segments of DNA which are capable of moving from one site in a genome to many different sites1,2. Tn3 is a prokaryotic transposon which is 4,957 base pairs (bp) long and encodes a transposase protein which is essential for transposition3-7. We report here a simple method for purifying Tn3 transposase and demonstrate that the transposase protein binds specifically to the ends of the Tn3 transposon in an ATP-dependent manner. The transposase protein binds to linear double-stranded DNA both nonspecifically and specifically; the nonspecific DNA binding activity is sensitive to challenge with heparin. Site-specific DNA binding to the ends (inverted repeats) of Tn3 is observed only when binding is performed in the presence of ATP; this ATP-dependent site-specific DNA binding activity is resistant to heparin challenge. Our results indicate that ATP qualitatively alters the DNA binding activity of the transposase protein so that the protein is able to bind specifically to the ends of the Tn3 transposon.

  1. High abundance and expression of transposases in bacteria from the Baltic Sea.

    PubMed

    Vigil-Stenman, Theoden; Ininbergs, Karolina; Bergman, Birgitta; Ekman, Martin

    2017-07-21

    Transposases are mobile genetic elements suggested to have an important role in bacterial genome plasticity and host adaptation but their transcriptional activity in natural bacterial communities is largely unexplored. Here we analyzed metagenomes and -transcriptomes of size fractionated (0.1-0.8, 0.8-3.0 and 3.0-200 μm) bacterial communities from the brackish Baltic Sea, and adjacent marine waters. The Baltic Sea transposase levels, up to 1.7% of bacterial genes and 2% of bacterial transcripts, were considerably higher than in marine waters and similar to levels reported for extreme environments. Large variations in expression were found between transposase families and groups of bacteria, with a two-fold higher transcription in Cyanobacteria than in any other phylum. The community-level results were corroborated at the genus level by Synechococcus transposases reaching up to 5.2% of genes and 6.9% of transcripts, which is in contrast to marine Synechococcus that largely lack these genes. Levels peaked in Synechococcus from the largest size fraction, suggesting high frequencies of lateral gene transfer and high genome plasticity in colony-forming picocyanobacteria. Together, the results support an elevated rate of transposition-based genome change and adaptation in bacterial populations of the Baltic Sea, and possibly also of other highly dynamic estuarine waters.The ISME Journal advance online publication, 21 July 2017; doi:10.1038/ismej.2017.114.

  2. Sequence analysis and quantification of transposase cDNAs of transposon TCp3.2 in Cydia pomonella larvae.

    PubMed

    Arends, Hugo M; Jehle, Johannes A

    2006-11-01

    The Tc1-like transposable element TCp3.2 was previously found to be horizontally transferred from the genome of Cydia pomonella to the C. pomonella granulovirus (CpGV). In this study, the transcription of transposase genes of endogenous TCp3.2 copies in the insect host genome was investigated. Cloning and sequencing of cDNAs prepared from TCp3.2 transposase transcripts resulted in the identification of a 199-bp-long intron. Sequence heterogeneities among different cDNA clones suggested that multiple copies of the transposase are transcribed, but that a part of these copies encode a defective transposase. The actin gene of C. pomonella was cloned and sequenced, and used to standardise quantitative real time PCR on prepared cDNA of the TCp3.2 transposase. Comparison of cDNA levels of TCp3.2 transposase prepared from mock and CpGV-infected C. pomonella larvae did not provide evidence that CpGV infection influenced the transcription level of TCp3.2 transposase. (c) 2006 Wiley-Liss, Inc.

  3. piggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens

    PubMed Central

    Park, Tae Sub; Han, Jae Yong

    2012-01-01

    Transgenic birds embody one of the most potent and exciting research tools in biotechnology for agriculture, medicine, and model animals. To date, retrovirus- or lentivirus-mediated transgenesis has been established in chickens and quail. However, despite having a valid technique for viral transduction to achieve transgenic birds, many obstacles exist for practical applications because of relatively low and variable rates of germ-line transmission and transgenic offspring showing transgene silencing, as well as safety issues related to viral vector use. Thus, the generation of transgenic poultry by nonviral integration is a prerequisite for the introduction of biotechnology to practical applications. Herein, we show that a germ-line–competent chicken primordial germ-cell (PGC) line was established with high efficiency of transmission to offspring and that piggyBac transposition into PGCs improved the efficiency of transgenic chicken production and led to high-level transgene expression. GFP transgene-expressing donor PGC-transferred recipient chickens produced donor-derived progenies, and the germ-line transmission efficiency of donor PGCs was 95.2% on average. Subsequently, half of the donor-derived offspring (52.2%) were transgenic chicks because GFP-expressing donor PGCs, in which a transgene was inserted into one chromosome 20, were heterozygous. In all of the transgenic chickens, GFP expression was constant and strong, regardless of age. Our results demonstrate that piggyBac transposition into the chicken PGC line could be the surest way to generate transgenic chickens safely for practical applications. PMID:22645326

  4. NMR solution structure of the RED subdomain of the Sleeping Beauty transposase.

    PubMed

    Konnova, Tatiana A; Singer, Christopher M; Nesmelova, Irina V

    2017-06-01

    DNA transposons can be employed for stable gene transfer in vertebrates. The Sleeping Beauty (SB) DNA transposon has been recently adapted for human application and is being evaluated in clinical trials, however its molecular mechanism is not clear. SB transposition is catalyzed by the transposase enzyme, which is a multi-domain protein containing the catalytic and the DNA-binding domains. The DNA-binding domain of the SB transposase contains two structurally independent subdomains, PAI and RED. Recently, the structures of the catalytic domain and the PAI subdomain have been determined, however no structural information on the RED subdomain and its interactions with DNA has been available. Here, we used NMR spectroscopy to determine the solution structure of the RED subdomain and characterize its interactions with the transposon DNA. © 2017 The Protein Society.

  5. Reprogramming the purine nucleotide cofactor requirement of Drosophila P element transposase in vivo.

    PubMed Central

    Mul, Y M; Rio, D C

    1997-01-01

    Guanosine triphosphate (GTP)-binding proteins are involved in controlling a wide range of fundamental cellular processes. In vitro studies have indicated a role for GTP during Drosophila P element transposition. Here we show that P element transposase contains a non-canonical GTP-binding domain that is critical for its ability to mediate transposition in Drosophila cells. Moreover, a single amino acid substitution could switch the nucleotide binding-specificity of transposase from GTP to xanthosine triphosphate (XTP). Importantly, this mutant protein could no longer function effectively in transposition in vivo but required addition of exogenous xanthine or xanthosine for reactivation. These results suggest that transposition may be controlled by physiological GTP levels and demonstrate that a single mutation can switch the nucleotide specificity for a complex cellular process in vivo. PMID:9250688

  6. Drosophila P-element transposase is a novel site-specific endonuclease

    PubMed Central

    Beall, Eileen L.; Rio, Donald C.

    1997-01-01

    We developed in vitro assays to study the first step of the P-element transposition reaction: donor DNA cleavage. We found that P-element transposase required both 5′ and 3′ P-element termini for efficient DNA cleavage to occur, suggesting that a synaptic complex forms prior to cleavage. Transposase made a staggered cleavage at the P-element termini that is novel for all known site-specific endonucleases: the 3′ cleavage site is at the end of the P-element, whereas the 5′ cleavage site is 17 bp within the P-element 31-bp inverted repeats. The P-element termini were protected from exonucleolytic degradation following the cleavage reaction, suggesting that a stable protein complex remains bound to the element termini after cleavage. These data are consistent with a cut-and-paste mechanism for P-element transposition and may explain why P elements predominantly excise imprecisely in vivo. PMID:9284052

  7. Piggy-BACing the human genome I: constructing a porcine BAC physical map through comparative genomics.

    PubMed

    Rogatcheva, Margarita B; Chen, Kefei; Larkin, Denis M; Meyers, Stacey N; Marron, Brandy M; He, Weisong; Schook, Lawrence B; Beever, Jonathan E

    2008-01-01

    Availability of the human genome sequence and high similarity between humans and pigs at the molecular level provides an opportunity to use a comparative mapping approach to piggy-BAC the human genome. In order to advance the pig genome sequencing initiative, sequence similarity between large-scale porcine BAC-end sequences (BESs) and human genome sequence was used to construct a comparatively-anchored porcine physical map that is a first step towards sequencing the pig genome. A total of 50,300 porcine BAC clones were end-sequenced, yielding 76,906 BESs after trimming with an average read length of 538 bp. To anchor the porcine BACs on the human genome, these BESs were subjected to BLAST analysis using the human draft sequence, revealing 31.5% significant hits (E < e(-5)). Both genic and non-genic regions of homology contributed to the alignments between the human and porcine genomes. Porcine BESs with unique homology matches within the human genome provided a source of markers spaced approximately 70 to 300 kb along each human chromosome. In order to evaluate the utility of piggy-BACing human genome sequences, and confirm predictions of orthology, 193 evenly spaced BESs with similarity to HSA3 and HSA21 were selected and then utilized for developing a high-resolution (1.22 Mb) comparative radiation hybrid map of SSC13 that represents a fusion of HSA3 and HSA21. Resulting RH mapping of SSC13 covers 99% and 97% of HSA3 and HSA21, respectively. Seven evolutionary conserved blocks were identified including six on HSA3 and a single syntenic block corresponding to HSA21. The strategy of piggy-BACing the human genome described in this study demonstrates that through a directed, targeted comparative genomics approach construction of a high-resolution anchored physical map of the pig genome can be achieved. This map supports the selection of BACs to construct a minimal tiling path for genome sequencing and targeted gap filling. Moreover, this approach is highly relevant

  8. Microbial co-habitation and lateral gene transfer: what transposases can tell us

    SciTech Connect

    Hooper, Sean D.; Mavromatis, Konstantinos; Kyrpides, Nikos C.

    2009-03-01

    Determining the habitat range for various microbes is not a simple, straightforward matter, as habitats interlace, microbes move between habitats, and microbial communities change over time. In this study, we explore an approach using the history of lateral gene transfer recorded in microbial genomes to begin to answer two key questions: where have you been and who have you been with? All currently sequenced microbial genomes were surveyed to identify pairs of taxa that share a transposase that is likely to have been acquired through lateral gene transfer. A microbial interaction network including almost 800 organisms was then derived from these connections. Although the majority of the connections are between closely related organisms with the same or overlapping habitat assignments, numerous examples were found of cross-habitat and cross-phylum connections. We present a large-scale study of the distributions of transposases across phylogeny and habitat, and find a significant correlation between habitat and transposase connections. We observed cases where phylogenetic boundaries are traversed, especially when organisms share habitats; this suggests that the potential exists for genetic material to move laterally between diverse groups via bridging connections. The results presented here also suggest that the complex dynamics of microbial ecology may be traceable in the microbial genomes.

  9. Duration of Expression and Activity of Sleeping Beauty Transposase in mouse liver following hydrodynamic DNA delivery

    PubMed Central

    Bell, Jason B; Aronovich, Elena L; Schreifels, Jeffrey M; Beadnell, Thomas C; Hackett, Perry B

    2010-01-01

    The Sleeping Beauty (SB) transposon system can direct integration of DNA sequences into mammalian genomes. The SB system comprises a transposon and transposase that “cuts” the transposon from a plasmid and “pastes” it into a recipient genome. The transposase gene may integrate very rarely and randomly into genomes, which has led to concerns that continued expression might support continued remobilization of transposons and genomic instability. Consequently, we measured the duration of SB11 transposase expression needed for remobilization to determine whether continued expression might be a problem. The SB11 gene was expressed from the plasmid pT2/mCAGGS-Luc//UbC-SB11 that contained a luciferase expression cassette in a hyperactive SB transposon. Mice were imaged and killed at periodic intervals out to 24 weeks. Over the first 2 weeks, the number of plasmids with SB11 genes and SB11 mRNA dropped about 90 and 99.9%, respectively. Expression of the luciferase reporter gene in the transposon declined about 99% and stabilized for 5 months at nearly 1,000-fold above background. In stark contrast, transposition-supporting levels of SB11 mRNA lasted only about 4 days postinfusion. Thus, within the limits of current technology, we show that SB transposons appear to be as stably integrated as their viral counterparts. PMID:20628359

  10. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.).

    PubMed

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef; Hehl, Reinhard

    2010-09-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT-PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain.

  11. Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.)

    PubMed Central

    Lisson, Ralph; Hellert, Jan; Ringleb, Malte; Machens, Fabian; Kraus, Josef

    2010-01-01

    The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT–PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain. PMID:20512402

  12. The diversity of prokaryotic DDE transposases of the mutator superfamily, insertion specificity, and association with conjugation machineries.

    PubMed

    Guérillot, Romain; Siguier, Patricia; Gourbeyre, Edith; Chandler, Michael; Glaser, Philippe

    2014-02-01

    Transposable elements (TEs) are major components of both prokaryotic and eukaryotic genomes and play a significant role in their evolution. In this study, we have identified new prokaryotic DDE transposase families related to the eukaryotic Mutator-like transposases. These genes were retrieved by cascade PSI-Blast using as initial query the transposase of the streptococcal integrative and conjugative element (ICE) TnGBS2. By combining secondary structure predictions and protein sequence alignments, we predicted the DDE catalytic triad and the DNA-binding domain recognizing the terminal inverted repeats. Furthermore, we systematically characterized the organization and the insertion specificity of the TEs relying on these prokaryotic Mutator-like transposases (p-MULT) for their mobility. Strikingly, two distant TE families target their integration upstream σA dependent promoters. This allowed us to identify a transposase sequence signature associated with this unique insertion specificity and to show that the dissymmetry between the two inverted repeats is responsible for the orientation of the insertion. Surprisingly, while DDE transposases are generally associated with small and simple transposons such as insertion sequences (ISs), p-MULT encoding TEs show an unprecedented diversity with several families of IS, transposons, and ICEs ranging in size from 1.1 to 52 kb.

  13. Domestic Violence

    MedlinePlus

    ... Press Room Careers OVW FOIA Contact the Office Domestic Violence Get Help | What is Domestic Violence? Get Help ... 7233) 1-800-787-3224 (TTY) What is Domestic Violence? We define domestic violence as a pattern of ...

  14. Generation of a transgenic cashmere goat using the piggyBac transposition system.

    PubMed

    Bai, Ding-Ping; Yang, Ming-Ming; Qu, Lei; Chen, Yu-Lin

    2017-04-15

    The development of transgenic technologies in the Cashmere goat (Capra hircus) has the potential to improve the quality of the meat and wool. The piggyBac (PB) transposon system is highly efficient and can be used to transpose specific target genes into the genome. Here, we developed a PB transposon system to produce transgenic Cashmere goat fetal fibroblasts (GFFs) with the enhanced green fluorescent protein (EGFP). We then used the genetically modified GFFs as nuclear donors to generate transgenic embryos by somatic cell nuclear transfer (SCNT). The embryos (n = 40) were implanted into female goats (n = 20). One transgenic kid that expressed EGFP throughout the surface features of its body was born. This result demonstrated the usefulness of PB transposon system in generating transgenic Cashmere goats. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Pig transgenesis by piggyBac transposition in combination with somatic cell nuclear transfer.

    PubMed

    Wu, Zhenfang; Xu, Zhiqian; Zou, Xian; Zeng, Fang; Shi, Junsong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Li, Zicong

    2013-12-01

    The production of animals by somatic cell nuclear transfer (SCNT) is inefficient, with approximately 2% of micromanipulated oocytes going to term and resulting in live births. However, it is the most commonly used method for the generation of cloned transgenic livestock as it facilitates the attainment of transgenic animals once the nuclear donor cells are stably transfected and more importantly as alternatives methods of transgenesis in farm animals have proven even less efficient. Here we describe piggyBac-mediated transposition of a transgene into porcine primary cells and use of these genetically modified cells as nuclear donors for the generation of transgenic pigs by SCNT. Gene transfer by piggyBac transposition serves to provide an alternative approach for the transfection of nuclear donor cells used in SCNT.

  16. piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice.

    PubMed

    Xu, Chunlong; Qi, Xiaolan; Du, Xuguang; Zou, Huiying; Gao, Fei; Feng, Tao; Lu, Hengxing; Li, Shenglan; An, Xiaomeng; Zhang, Lijun; Wu, Yuanyuan; Liu, Ying; Li, Ning; Capecchi, Mario R; Wu, Sen

    2017-01-24

    CRISPR/Cas9 is becoming an increasingly important tool to functionally annotate genomes. However, because genome-wide CRISPR libraries are mostly constructed in lentiviral vectors, in vivo applications are severely limited as a result of difficulties in delivery. Here, we examined the piggyBac (PB) transposon as an alternative vehicle to deliver a guide RNA (gRNA) library for in vivo screening. Although tumor induction has previously been achieved in mice by targeting cancer genes with the CRISPR/Cas9 system, in vivo genome-scale screening has not been reported. With our PB-CRISPR libraries, we conducted an in vivo genome-wide screen in mice and identified genes mediating liver tumorigenesis, including known and unknown tumor suppressor genes (TSGs). Our results demonstrate that PB can be a simple and nonviral choice for efficient in vivo delivery of CRISPR libraries.

  17. Soluble expression, purification and characterization of the full length IS2 Transposase

    PubMed Central

    2011-01-01

    Background The two-step transposition pathway of insertion sequences of the IS3 family, and several other families, involves first the formation of a branched figure-of-eight (F-8) structure by an asymmetric single strand cleavage at one optional donor end and joining to the flanking host DNA near the target end. Its conversion to a double stranded minicircle precedes the second insertional step, where both ends function as donors. In IS2, the left end which lacks donor function in Step I acquires it in Step II. The assembly of two intrinsically different protein-DNA complexes in these F-8 generating elements has been intuitively proposed, but a barrier to testing this hypothesis has been the difficulty of isolating a full length, soluble and active transposase that creates fully formed synaptic complexes in vitro with protein bound to both binding and catalytic domains of the ends. We address here a solution to expressing, purifying and structurally analyzing such a protein. Results A soluble and active IS2 transposase derivative with GFP fused to its C-terminus functions as efficiently as the native protein in in vivo transposition assays. In vitro electrophoretic mobility shift assay data show that the partially purified protein prepared under native conditions binds very efficiently to cognate DNA, utilizing both N- and C-terminal residues. As a precursor to biophysical analyses of these complexes, a fluorescence-based random mutagenesis protocol was developed that enabled a structure-function analysis of the protein with good resolution at the secondary structure level. The results extend previous structure-function work on IS3 family transposases, identifying the binding domain as a three helix H + HTH bundle and explaining the function of an atypical leucine zipper-like motif in IS2. In addition gain- and loss-of-function mutations in the catalytic active site define its role in regional and global binding and identify functional signatures that are common

  18. RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration

    PubMed Central

    Rauschhuber, Christina; Ehrhardt, Anja

    2012-01-01

    Background Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB) transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi) machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions. Principal Findings To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU) after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. Conclusion In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system. PMID:22570690

  19. A proposed superfamily of transposase genes: transposon-like elements in ciliated protozoa and a common "D35E" motif.

    PubMed Central

    Doak, T G; Doerder, F P; Jahn, C L; Herrick, G

    1994-01-01

    The transposon-like elements TBE1, Tec1, and Tec2 of hypotrichous ciliated protozoa appear to encode a protein that belongs to the IS630-Tc1 family of transposases. The Anabaena IS895 transposase also is placed in this family. We note that most family members transpose into the dinucleotide target, TA, and that members with eukaryotic hosts have a tendency for somatic excision that is carried to an extreme by the ciliate elements. Alignments including the additional members, and also mariner elements, show that transposases of this family share strongly conserved residues in a large C-terminal portion, including a fully conserved dipeptide, Asp-Glu (DE), and a block consisting of a fully conserved Asp and highly conserved Glu, separated by 34 or 35 residues (D35E). This D35E motif likely is homologous to the previously characterized D35E motif of the family of retroviral-retrotransposon integrases and IS3-like transposases. Because it is known that the IS3-retroposon D35E region is a critical portion of a domain capable of various in vitro transposition-related reactions, the results suggest that the two families share homologous catalytic transposase domains and that members of both families may share a common transposition mechanism. PMID:8302872

  20. A High-Capacity Adenoviral Hybrid Vector System Utilizing the Hyperactive Sleeping Beauty Transposase SB100X for Enhanced Integration.

    PubMed

    Boehme, Philip; Zhang, Wenli; Solanki, Manish; Ehrke-Schulz, Eric; Ehrhardt, Anja

    2016-07-19

    For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose.

  1. A High-Capacity Adenoviral Hybrid Vector System Utilizing the Hyperactive Sleeping Beauty Transposase SB100X for Enhanced Integration

    PubMed Central

    Boehme, Philip; Zhang, Wenli; Solanki, Manish; Ehrke-Schulz, Eric; Ehrhardt, Anja

    2016-01-01

    For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose. PMID:27434682

  2. A High-Capacity Adenoviral Hybrid Vector System Utilizing the Hyperactive Sleeping Beauty Transposase SB100X for Enhanced Integration.

    PubMed

    Boehme, Philip; Zhang, Wenli; Solanki, Manish; Ehrke-Schulz, Eric; Ehrhardt, Anja

    2016-01-01

    For efficient delivery of required genetic elements we utilized high-capacity adenoviral vectors in the past allowing high transgene capacities of up to 36 kb. Previously we explored the hyperactive Sleeping Beauty (SB) transposase (HSB5) for somatic integration from the high-capacity adenoviral vectors genome. To further improve this hybrid vector system we hypothesized that the previously described hyperactive SB transposase SB100X will result in significantly improved efficacies after transduction of target cells. Plasmid based delivery of the SB100X system revealed significantly increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5. After optimizing experimental setups for high-capacity adenoviral vectors-based delivery of the SB100X system we observed up to eightfold and 100-fold increased integration efficiencies compared with the previously published hyperactive SB transposase HSB5 and the inactive transposase mSB, respectively. Furthermore, transposon copy numbers per cell were doubled with SB100X compared with HSB5 when using the identical multiplicity of infection. We believe that this improved hybrid vector system represents a valuable tool for achieving stabilized transgene expression in cycling cells and for treatment of numerous genetic disorders. Especially for in vivo approaches this improved adenoviral hybrid vector system will be advantageous because it may potentially allow reduction of the applied viral dose.

  3. Characterization of Growth and Reproduction Performance, Transgene Integration, Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer.

    PubMed

    Zeng, Fang; Li, Zicong; Cai, Gengyuan; Gao, Wenchao; Jiang, Gelong; Liu, Dewu; Urschitz, Johann; Moisyadi, Stefan; Wu, Zhenfang

    2016-10-01

    Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance and characterized the transgene insertion, transmission, and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression, and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favorable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition.

  4. Domestic violence

    MedlinePlus

    ... Spousal abuse; Elder abuse; Child abuse; Sexual abuse - domestic violence ... Domestic violence can include any of these behaviors: Physical abuse, including hitting, kicking, biting, slapping, choking, or attacking ...

  5. Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly

    PubMed Central

    Nicolas, Emilien; Oger, Cédric A.; Lambin, Michaël; Draime, Amandine; Leterme, Sébastien C.; Hallet, Bernard F. J.

    2017-01-01

    The Tn3 family is a widespread group of replicative transposons that are notorious for their contribution to the dissemination of antibiotic resistance and the emergence of multiresistant pathogens worldwide. The TnpA transposase of these elements catalyzes DNA breakage and rejoining reactions required for transposition. It also is responsible for target immunity, a phenomenon that prevents multiple insertions of the transposon into the same genomic region. However, the molecular mechanisms whereby TnpA acts in both processes remain unknown. Here, we have developed sensitive biochemical assays for the TnpA transposase of the Tn3-family transposon Tn4430 and used these assays to characterize previously isolated TnpA mutants that are selectively affected in immunity. Compared with wild-type TnpA, these mutants exhibit deregulated activities. They spontaneously assemble a unique asymmetric synaptic complex in which one TnpA molecule simultaneously binds two transposon ends. In this complex, TnpA is in an activated state competent for DNA cleavage and strand transfer. Wild-type TnpA can form this complex only on precleaved ends mimicking the initial step of transposition. The data suggest that transposition is controlled at an early stage of transpososome assembly, before DNA cleavage, and that mutations affecting immunity have unlocked TnpA by stabilizing the protein in a monomeric activated synaptic configuration. We propose an asymmetric pathway for coupling active transpososome assembly with proper target recruitment and discuss this model with respect to possible immunity mechanisms. PMID:28096365

  6. Targeting the Transposase Domain of the DNA Repair Component Metnase to Enhance Chemotherapy

    PubMed Central

    Williamson, Elizabeth A.; Damiani, Leah; Leitao, Andrei; Hu, Chelin; Hathaway, Helen; Oprea, Tudor; Sklar, Larry; Shaheen, Montaser; Bauman, Julie; Wang, Wei; Nickoloff, Jac A.; Lee, Suk-Hee; Hromas, Robert

    2014-01-01

    Previous studies have shown that the DNA repair component Metnase (SETMAR) mediates resistance to DNA damaging cancer chemotherapy. Metnase has a nuclease domain that shares homology with the Transposase family. We therefore virtually screened the tertiary Metnase structure against the 550,000 compound ChemDiv library to identify small molecules that might dock in the active site of the transposase nuclease domain of Metnase. We identified eight compounds as possible Metnase inhibitors. Interestingly, among these candidate inhibitors were quinolone antibiotics and HIV integrase inhibitors, which share common structural features. Previous reports have described possible activity of quinolones as antineoplastic agents. Therefore, we chose the quinolone ciprofloxacin for further study, based on its wide clinical availability and low toxicity. We found that ciprofloxacin inhibits the ability of Metnase to cleave DNA and inhibits Metnase-dependent DNA repair. Ciprofloxacin on its own did not induce DNA damage, but it did reduce repair of chemotherapy-induced DNA damage. Ciprofloxacin increased the sensitivity of cancer cell lines and a xenograft tumor model to clinically relevant chemotherapy. These studies provide a mechanism for the previously postulated antineoplastic activity of quinolones, and suggest that ciprofloxacin might be a simple yet effective adjunct to cancer chemotherapy. PMID:23090115

  7. Ablation of RNAi and retrotransposons accompany acquisition and evolution of transposases to heterochromatin protein CENPB.

    PubMed

    Upadhyay, Udita; Srivastava, Suchita; Khatri, Indu; Nanda, Jagpreet Singh; Subramanian, Srikrishna; Arora, Amit; Singh, Jagmohan

    2017-02-22

    Inactivation of retrotransposons is accompanied by the emergence of CENPB (centromere binding protein-B) in Schizosaccharomyces as well as in metazoans. The RNAi-induced Transcriptional Silencing (RITS) Complex, comprising Chp1 (chromodomain protein-1), Tas3 (protein with unknown function) and Ago1 (Argonaute), plays an important role in RNAi-mediated heterochromatinization. We find that while the Ago1 subunit of the RITS complex is highly conserved, Tas3 is lost and Chp1 is truncated in S. cryophilus and S. octosporus We show that truncated Chp1 has lost the property of heterochromatin localization and silencing when transformed in S. pombe Furthermore, multiple copies of CENPB, related to Tc1/mariner and Tc5 transposons, occur in all Schizosaccharomyces species as well as in humans, but with loss of transposase function (except S. japonicus). We propose that acquisition of Tc1/mariner and Tc5 elements by horizontal transfer in S. pombe (and humans), is accompanied by alteration of their function from a transposase/ endonuclease to a heterochromatin protein, designed to suppress transposon expression and recombination. The resulting redundancy of RITS may have eased the selection pressure resulting in progressive loss or truncation of tas3 and chp1 genes in S. octosporus and S. cryophilus and triggered similar evolutionary dynamics in the metazoan orthologs.

  8. A systematic identification of Kolobok superfamily transposons in Trichomonas vaginalis and sequence analysis on related transposases.

    PubMed

    Meng, Qingshu; Chen, Kaifu; Ma, Lina; Hu, Songnian; Yu, Jun

    2011-02-01

    Transposons are sequence elements widely distributed among genomes of all three kingdoms of life, providing genomic changes and playing significant roles in genome evolution. Trichomonas vaginalis is an excellent model system for transposon study since its genome (~160 Mb) has been sequenced and is composed of ~65% transposons and other repetitive elements. In this study, we primarily report the identification of Kolobok-type transposons (termed tvBac) in T. vaginalis and the results of transposase sequence analysis. We categorized 24 novel subfamilies of the Kolobok element, including one autonomous subfamily and 23 non-autonomous subfamilies. We also identified a novel H2CH motif in tvBac transposases based on multiple sequence alignment. In addition, we supposed that tvBac and Mutator transposons may have evolved independently from a common ancestor according to our phylogenetic analysis. Our results provide basic information for the understanding of the function and evolution of tvBac transposons in particular and other related transposon families in general.

  9. A piggyBac insertion disrupts Foxl2 expression that mimics BPES syndrome in mice.

    PubMed

    Shi, Fubiao; Ding, Sheng; Zhao, Shimin; Han, Min; Zhuang, Yuan; Xu, Tian; Wu, Xiaohui

    2014-07-15

    Blepharophimosis, ptosis, epicanthus inversus syndrome (BPES) is an autosomal dominant genetic disorder characterized by small palpebral fissures and other craniofacial malformations, often with (type I) but could also without (type II) premature ovarian failure. While mutations of the forkhead transcription factor FOXL2 are associated with and likely be responsible for many BPES cases, how FOXL2 affects craniofacial development remain to be understood. Through a large-scale piggyBac (PB) insertion mutagenesis, we have identified a mouse mutant carrying a PB insertion ∼160 kb upstream of the transcription start site (TSS) of Foxl2. The insertion reduces, but not eliminates, the expression of Foxl2. This mutant, but not its revertant, displays BPES-like conditions such as midface hypoplasia, eyelid abnormalities and female subfertility. Further analysis indicates that the mutation does not affect mandible, but causes premature fusion of the premaxilla-maxilla suture, smaller premaxilla and malformed maxilla during midface development. We further identified an evolutionarily conserved fragment near the insertion site and observed enhancer activity of this element in tissue culture cells. Analyses using DNase I hypersensitivity assay and chromosome conformation capture assay in developing maxillary and periocular tissues suggest that the DNA region near the insertion site likely interacts with Foxl2 TSS. Therefore, this mutant presents an excellent animal model for mechanistic study of BPES and regulation of Foxl2. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  10. Multigene expression in stable CHO cell pools generated with the piggyBac transposon system.

    PubMed

    Balasubramanian, Sowmya; Wurm, Florian M; Hacker, David L

    2016-09-01

    Heterogenous populations of recombinant cells (cell pools) stably expressing 1-4 transgenes were generated from Chinese hamster overy (CHO) cells with the piggyBac (PB) transposon system. The cell pools produced different combinations of three model proteins-enhanced green fluorescent protein (EGFP), secreted alkaline phosphatase (SEAP), and a monoclonal IgG1 antibody. Each transgene was present on a separate PB donor plasmid with either the same or a different selection gene. In both cases, we obtained PB-derived cell pools with higher recombinant protein yields than from cell pools generated by conventional gene delivery. In PB-derived cell pools generated using a single selection agent, both protein production and the number of integrated copies of each transgene declined as the number of transfected transgenes increased. However, the total number of integrated transgenes was similar regardless of the number of different transgenes transfected. For PB-derived cell pools generated by selection of each transgene with a different selection agent, the total number of integrated transgenes increased with the number of transfected transgenes. The results suggest that the generation of cell pools producing multiple recombinant proteins is feasible and that the method is more efficient when each individual transgene is selected with a different marker. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1308-1317, 2016. © 2016 American Institute of Chemical Engineers.

  11. PiggyBac as a novel vector in cancer gene therapy: current perspective.

    PubMed

    Mirzaei, H; Sahebkar, A; Jaafari, M R; Hadjati, J; Javanmard, S H; Mirzaei, H R; Salehi, R

    2016-01-01

    Selection of suitable delivery system is one of the crucial aspects in gene therapy that determines the efficiency of gene therapy. The past two decades have witnessed extensive efforts for finding safe and efficient vectors to overcome the limitations of viral vectors. The utilization of DNA transposon-based vectors for gene therapy has emerged as a promising non-viral alternative. DNA 'cut-and-paste' is one of the main mechanisms of genome engineering by transposon elements. However, the lack of an efficient transposition system has limited the utilization of transposon vectors in mice and mammalian systems. PiggyBac (PB) is known as a highly efficient DNA transposon originally isolated from Trichoplusia ni as an alternative to Sleeping Beauty (SB). It has been shown that PB can be functional in various species including mammalian systems. This vector could overcome some limitations of other vectors in cancer gene therapy. Some advantages of PB include the capacity for integration into the genome and providing a stable expression, capacity to harbor 10 and 9.1 kb of foreign DNA into the host genome, without a significant reduction in their transposition activity and display non-overlapping targeting preferences. However, to advance PB to clinical applications, some obstacles still require to be overcome to improve its safety and efficiency. Hence, it seems that this vector could open new horizons in gene and cancer therapy.

  12. Simple piggyBac transposon-based mammalian cell expression system for inducible protein production

    PubMed Central

    Li, Zhijie; Michael, Iacovos P.; Zhou, Dongxia; Nagy, Andras; Rini, James M.

    2013-01-01

    Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible, stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover, the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures, thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction, protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format, a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally, we demonstrate the utility of the system through the large-scale production (140–750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents. PMID:23476064

  13. Germline transformation of the diamondback moth, Plutella xylostella L., using the piggyBac transposable element.

    PubMed

    Martins, S; Naish, N; Walker, A S; Morrison, N I; Scaife, S; Fu, G; Dafa'alla, T; Alphey, L

    2012-08-01

    The diamondback moth, Plutella xylostella, is one of the most economically important agricultural pests. The larvae of this moth cause damage by feeding on the foliage of cruciferous vegetables such as cabbage, broccoli, cauliflower and rapeseed. Control generally comprises chemical treatment; however, the diamondback moth is renowned for rapid development of resistance to pesticides. Other methods, such as biological control, have not been able to provide adequate protection. Germline transformation of pest insects has become available in recent years as an enabling technology for new genetics-based control methods, such as the Release of Insects carrying a Dominant Lethal (RIDL(®) ). In the present study, we report the first transformation of the diamondback moth, using the piggyBac transposable element, by embryo microinjection. In generating transgenic strains using four different constructs, the function of three regulatory sequences in this moth was demonstrated in driving expression of fluorescent proteins. The transformation rates achieved, 0.48-0.68%, are relatively low compared with those described in other Lepidoptera, but not prohibitive, and are likely to increase with experience. We anticipate that germline transformation of the diamondback moth will permit the development of RIDL strains for use against this pest and facilitate the wider use of this species as a model organism for basic studies.

  14. Efficient Production of Fluorescent Transgenic Rats using the piggyBac Transposon

    PubMed Central

    Li, Tianda; Shuai, Ling; Mao, Junjie; Wang, Xuepeng; Wang, Mei; Zhang, Xinxin; Wang, Leyun; Li, Yanni; Li, Wei; Zhou, Qi

    2016-01-01

    Rats with fluorescent markers are of great value for studies that trace lineage-specific development, particularly those assessing the differentiation potential of embryonic stem cells (ESCs). The piggyBac (PB) transposon is widely used for the efficient introduction of genetic modifications into genomes, and has already been successfully used to produce transgenic mice and rats. Here, we generated transgenic rats carrying either the desRed fluorescent protein (RFP) gene or the enhanced green fluorescent protein (eGFP) gene by injecting pronuclei with PB plasmids. We showed that the transgenic rats expressed the RFP or eGFP gene in many organs and had the capability to transmit the marker gene to the next generation through germline integration. In addition, rat embryonic stem cells (ESCs) carrying an RFP reporter gene can be derived from the blastocysts of the transgenic rats. Moreover, the RFP gene can be detected in chimeras derived from RFP ESCs via blastocyst injection. This work suggests that PB-mediated transgenesis is a powerful tool to generate transgenic rats expressing fluorescent proteins with high efficiency, and this technique can be used to derive rat ESCs expressing a reporter protein. PMID:27624004

  15. Domestic Violence

    MedlinePlus

    ... f AQ FREQUENTLY ASKED QUESTIONS FAQ083 WOMEN’S HEALTH Domestic Violence • What is domestic violence? • What are the types of abuse? • How ... are available to help abused women? What is domestic violence? Domestic violence is a pattern of threatening ...

  16. Domestic Violence

    MedlinePlus

    f AQ FREQUENTLY ASKED QUESTIONS FAQ083 WOMEN’S HEALTH Domestic Violence • What is domestic violence? • What are the types of abuse? • How can ... are available to help abused women? What is domestic violence? Domestic violence is a pattern of threatening or ...

  17. Conformational Diversity of Single-Stranded DNA from Bacterial Repetitive Extragenic Palindromes: Implications for the DNA Recognition Elements of Transposases

    PubMed Central

    Charnavets, Tatsiana; Nunvar, Jaroslav; Nečasová, Iva; Völker, Jens; Breslauer, Kenneth J; Schneider, Bohdan

    2015-01-01

    Repetitive extragenic palindrome (REP)—associated tyrosine transposase enzymes (RAYTs) bind REP DNA domains and catalyze their cleavage. Genomic sequence analyses identify potential noncoding REP sequences associated with RAYT-encoding genes. To probe the conformational space of potential RAYT DNA binding domains, we report here spectroscopic and calorimetric measurements that detect and partially characterize the solution conformational heterogeneity of REP oligonucleotides from six bacterial species. Our data reveal most of these REP oligonucleotides adopt multiple conformations, suggesting that RAYTs confront a landscape of potential DNA substrates in dynamic equilibrium that could be selected, enriched, and/or induced via differential binding. Thus, the transposase-bound DNA motif may not be the predominant conformation of the isolated REP domain. Intriguingly, for several REPs, the circular dichroism spectra suggest guanine tetraplexes as potential alternative or additional RAYT recognition elements, an observation consistent with these REP domains being highly nonrandom, with tetraplex-favoring 5′-G and 3′-C-rich segments. In fact, the conformational heterogeneity of REP domains detected and reported here, including the formation of noncanonical DNA secondary structures, may reflect a general feature required for recognition by RAYT transposases. Based on our biophysical data, we propose guanine tetraplexes as an additional DNA recognition element for binding by RAYT transposase enzymes. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 585–596, 2015. PMID:25951997

  18. One to rule them all: A highly conserved motif in mariner transposase controls multiple steps of transposition.

    PubMed

    Bouuaert, Corentin Claeys; Tellier, Michael; Chalmers, Ronald

    2014-01-01

    The development of transposon-based genome manipulation tools can benefit greatly from understanding transposons' inherent regulatory mechanisms. The Tc1-mariner transposons, which are being widely used in biotechnological applications, are subject to a self-inhibitory mechanism whereby increasing transposase expression beyond a certain point decreases the rate of transposition. In a recent paper, Liu and Chalmers performed saturating mutagenesis on the highly conserved WVPHEL motif in the mariner-family transposase from the Hsmar1 element. Curiously, they found that the majority of all possible single mutations were hyperactive. Biochemical characterizations of the mutants revealed that the hyperactivity is due to a defect in communication between transposase subunits, which normally regulates transposition by reducing the rate of synapsis. This provides important clues for improving transposon-based tools. However, some WVPHEL mutants also showed features that would be undesirable for most biotechnological applications: they showed uncontrolled DNA cleavage activities and defects in the coordination of cleavage between the two transposon ends. The study illustrates how the knowledge of inhibitory mechanisms can help improve transposon tools but also highlights an important challenge, which is to specifically target a regulatory mechanism without affecting other important functions of the transposase.

  19. Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats.

    PubMed

    Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

    2013-08-01

    Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats☆

    PubMed Central

    Marzo, Mar; Liu, Danxu; Ruiz, Alfredo; Chalmers, Ronald

    2013-01-01

    Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity. PMID:23648487

  1. Lens Epithelium-derived Growth Factor/p75 Interacts with the Transposase-derived DDE Domain of PogZ*S⃞

    PubMed Central

    Bartholomeeusen, Koen; Christ, Frauke; Hendrix, Jelle; Rain, Jean-Christophe; Emiliani, Stéphane; Benarous, Richard; Debyser, Zeger; Gijsbers, Rik; De Rijck, Jan

    2009-01-01

    Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular interaction partner of human immunodeficiency virus-1 (HIV-1) integrase, tethering the preintegration complex to the host chromosome. In light of the development of LEDGF/p75-integrase interaction inhibitors, it is essential to understand the cell biology of LEDGF/p75. We identified pogZ as new cellular interaction partner of LEDGF/p75. Analogous to lentiviral integrase, pogZ, a domesticated transposase, carries a DDE domain, the major determinant for LEDGF/p75 interaction. Using different in vitro and in vivo approaches, we corroborated the interaction between the C terminus of LEDGF/p75 and the DDE domain of pogZ, revealing an overlap in the binding of pogZ and HIV-1 integrase. Competition experiments showed that integrase is efficient in displacing pogZ from LEDGF/p75. Moreover, pogZ does not seem to play a role as a restriction factor of HIV. The finding that LEDGF/p75 is capable of interacting with a DDE domain protein that is not a lentiviral integrase points to a profound role of LEDGF/p75 in DDE domain protein function. PMID:19244240

  2. The SET and transposase domain protein Metnase enhances chromosome decatenation: regulation by automethylation.

    PubMed

    Williamson, Elizabeth A; Rasila, Kanwaldeep Kaur; Corwin, Lori Kwan; Wray, Justin; Beck, Brian D; Severns, Virginia; Mobarak, Charlotte; Lee, Suk-Hee; Nickoloff, Jac A; Hromas, Robert

    2008-10-01

    Metnase is a human SET and transposase domain protein that methylates histone H3 and promotes DNA double-strand break repair. We now show that Metnase physically interacts and co-localizes with Topoisomerase IIalpha (Topo IIalpha), the key chromosome decatenating enzyme. Metnase promotes progression through decatenation and increases resistance to the Topo IIalpha inhibitors ICRF-193 and VP-16. Purified Metnase greatly enhanced Topo IIalpha decatenation of kinetoplast DNA to relaxed circular forms. Nuclear extracts containing Metnase decatenated kDNA more rapidly than those without Metnase, and neutralizing anti-sera against Metnase reversed that enhancement of decatenation. Metnase automethylates at K485, and the presence of a methyl donor blocked the enhancement of Topo IIalpha decatenation by Metnase, implying an internal regulatory inhibition. Thus, Metnase enhances Topo IIalpha decatenation, and this activity is repressed by automethylation. These results suggest that cancer cells could subvert Metnase to mediate clinically relevant resistance to Topo IIalpha inhibitors.

  3. TnpPred: A Web Service for the Robust Prediction of Prokaryotic Transposases

    PubMed Central

    Riadi, Gonzalo; Medina-Moenne, Cristobal; Holmes, David S.

    2012-01-01

    Transposases (Tnps) are enzymes that participate in the movement of insertion sequences (ISs) within and between genomes. Genes that encode Tnps are amongst the most abundant and widely distributed genes in nature. However, they are difficult to predict bioinformatically and given the increasing availability of prokaryotic genomes and metagenomes, it is incumbent to develop rapid, high quality automatic annotation of ISs. This need prompted us to develop a web service, termed TnpPred for Tnp discovery. It provides better sensitivity and specificity for Tnp predictions than given by currently available programs as determined by ROC analysis. TnpPred should be useful for improving genome annotation. The TnpPred web service is freely available for noncommercial use. PMID:23251097

  4. Analysis of Tc1-Mariner elements in Sclerotinia sclerotiorum suggests recent activity and flexible transposases.

    PubMed

    Santana, Mateus F; Silva, José C F; Mizubuti, Eduardo S G; Araújo, Elza F; Queiroz, Marisa V

    2014-10-03

    Sclerotinia sclerotiorum is a necrotrophic fungus that is pathogenic to many plants. Genomic analysis of its revealed transposable element expansion that has strongly influenced the evolutionary trajectory of several species. Transposons from the Tc1-Mariner superfamily are thought to be ubiquitous components of fungal genomes and are generally found in low copy numbers with large numbers of deleterious mutations in their transposase coding sequence. This study shows that the genome of S. sclerotiorum has a large number of copies of Tc1-Mariner transposons, and in silico analysis shows evidence that they were recently active. This finding was confirmed by expressed sequence tag (EST) analysis. Fourteen new Tc1-Mariner transposon families that were distributed throughout the genome were identified, and in some cases, due to the excision/retention of introns, different transcripts were observed for the same family, which might be the result of an efficient strategy to circumvent mutations that generate premature stop codons in the RNA sequence. In addition, the presence of these introns shows that the transposase protein has a flexible coding sequence and, consequently, conformation. No evidence for RIP-like gene silencing mechanisms, which are commonly found in fungi, was found in the identified Tc1-Mariner elements, and analysis of the genomic insertion sites of these elements showed that they were widely distributed throughout the genome with some copies located near the 3' regions of genes. In particular, EST analysis demonstrated that one of these copies was co-expressed with a gene, which showed the potential for these elements to undergo exaptation. Fourteen novel Tc1-Mariner families were characterized. Some families had evidence of introns, which might or might not be excised depending on the family or element in question, and this finding demonstrates a possible strategy for overcoming possible mutations that generate premature stop codons in a RNA sequence

  5. Transposon Domestication versus Mutualism in Ciliate Genome Rearrangements

    PubMed Central

    Vogt, Alexander; Goldman, Aaron David; Mochizuki, Kazufumi; Landweber, Laura F.

    2013-01-01

    Ciliated protists rearrange their genomes dramatically during nuclear development via chromosome fragmentation and DNA deletion to produce a trimmer and highly reorganized somatic genome. The deleted portion of the genome includes potentially active transposons or transposon-like sequences that reside in the germline. Three independent studies recently showed that transposase proteins of the DDE/DDD superfamily are indispensible for DNA processing in three distantly related ciliates. In the spirotrich Oxytricha trifallax, high copy-number germline-limited transposons mediate their own excision from the somatic genome but also contribute to programmed genome rearrangement through a remarkable transposon mutualism with the host. By contrast, the genomes of two oligohymenophorean ciliates, Tetrahymena thermophila and Paramecium tetraurelia, encode homologous PiggyBac-like transposases as single-copy genes in both their germline and somatic genomes. These domesticated transposases are essential for deletion of thousands of different internal sequences in these species. This review contrasts the events underlying somatic genome reduction in three different ciliates and considers their evolutionary origins and the relationships among their distinct mechanisms for genome remodeling. PMID:23935529

  6. Derivation of Induced Trophoblast Cell Lines in Cattle by Doxycycline-Inducible piggyBac Vectors

    PubMed Central

    Hayashi, Masafumi; Tsukiyama, Tomoyuki; Kimura, Koji; Matsuyama, Shuichi; Minami, Naojiro; Yamada, Masayasu; Imai, Hiroshi

    2016-01-01

    Trophectoderm lineage specification is one of the earliest differentiation events in mammalian development. The trophoblast lineage, which is derived from the trophectoderm, mediates implantation and placental formation. However, the processes involved in trophoblastic differentiation and placental formation in cattle remain unclear due to interspecies differences when compared with other model systems and the small repertoire of available trophoblast cell lines. Here, we describe the generation of trophoblast cell lines (biTBCs) from bovine amnion-derived cells (bADCs) using an induced pluripotent stem cell technique. bADCs were introduced with piggyBac vectors containing doxycycline (Dox)-inducible transcription factors (Oct3⁄4(POU5F1), Sox2, Klf4, and c-Myc). Colonies that appeared showed a flattened epithelial-like morphology similar to cobblestones, had a more definite cell boundary between cells, and frequently formed balloon-like spheroids similar to trophoblastic vesicles (TVs). biTBCs were propagated for over 60 passages and expressed trophoblast-related (CDX2, ELF5, ERRβ, and IFN-τ) and pluripotency-related genes (endogenous OCT3/4, SOX2, KLF4, and c-MYC). Furthermore, when biTBCs were induced to differentiate by removing Dox from culture, they formed binucleate cells and began to express pregnancy-related genes (PL, PRP1, and PAG1). This is the first report demonstrating that the induction of pluripotency in bovine amniotic cells allows the generation of trophoblastic cell lines that possess trophoblast stem cell-like characteristics and have the potential to differentiate into the extra-embryonic cell lineage. These cell lines can be a new cell source as a model for studying trophoblast cell lineages and implantation processes in cattle. PMID:27907214

  7. Low-copy piggyBac transposon mutagenesis in mice identifies genes driving melanoma.

    PubMed

    Ni, Thomas K; Landrette, Sean F; Bjornson, Robert D; Bosenberg, Marcus W; Xu, Tian

    2013-09-17

    Despite considerable efforts to sequence hypermutated cancers such as melanoma, distinguishing cancer-driving genes from thousands of recurrently mutated genes remains a significant challenge. To circumvent the problematic background mutation rates and identify new melanoma driver genes, we carried out a low-copy piggyBac transposon mutagenesis screen in mice. We induced eleven melanomas with mutation burdens that were 100-fold lower relative to human melanomas. Thirty-eight implicated genes, including two known drivers of human melanoma, were classified into three groups based on high, low, or background-level mutation frequencies in human melanomas, and we further explored the functional significance of genes in each group. For two genes overlooked by prevailing discovery methods, we found that loss of membrane associated guanylate kinase, WW and PDZ domain containing 2 and protein tyrosine phosphatase, receptor type, O cooperated with the v-raf murine sarcoma viral oncogene homolog B (BRAF) recurrent V600E mutation to promote cellular transformation. Moreover, for infrequently mutated genes often disregarded by current methods, we discovered recurrent mitogen-activated protein kinase kinase kinase 1 (Map3k1)-activating insertions in our screen, mirroring recurrent MAP3K1 up-regulation in human melanomas. Aberrant expression of Map3k1 enabled growth factor-autonomous proliferation and drove BRAF-independent ERK signaling, thus shedding light on alternative means of activating this prominent signaling pathway in melanoma. In summary, our study contributes several previously undescribed genes involved in melanoma and establishes an important proof-of-principle for the utility of the low-copy transposon mutagenesis approach for identifying cancer-driving genes, especially those masked by hypermutation.

  8. Domestic Violence

    MedlinePlus

    Domestic violence is a type of abuse. It usually involves a spouse or partner, but it can also ... a child, elderly relative, or other family member. Domestic violence may include Physical violence that can lead to ...

  9. Use of the piggyBac transposon to create stable packaging cell lines for the production of clinical-grade self-inactivating γ-retroviral vectors.

    PubMed

    Feldman, Steven A; Xu, Hui; Black, Mary A; Park, Tristen S; Robbins, Paul F; Kochenderfer, James N; Morgan, Richard A; Rosenberg, Steven A

    2014-08-01

    Efforts to improve the biosafety of γ-retroviral-mediated gene therapy have resulted in a shift toward the use of self-inactivating (SIN) γ-retroviral vectors. However, scale-up and manufacturing of such vectors requires significant optimization of transient transfection-based processes or development of novel platforms for the generation of stable producer cell clones. To that end, we describe the use of the piggybac transposon to generate stable producer cell clones for the production of SIN γ-retroviral vectors. The piggybac transposon is a universal tool allowing for the stable integration of SIN γ-retroviral constructs into murine (PG13) and human 293-based Phoenix (GALV and RD114, respectively) packaging cell lines without reverse transcription. Following transposition, a high-titer clone is selected for manufacture of a master cell bank and subsequent γ-retroviral vector supernatant production. Packaging cell clones created using the piggybac transposon have comparable titers to non-SIN vectors generated via conventional methods. We describe herein the use of the piggybac transposon for the production of stable packaging cell clones for the manufacture of clinical-grade SIN γ-retroviral vectors for ex vivo gene therapy clinical trials.

  10. piggyBac transposon-derived targeting shRNA interference against the Bombyx mori nucleopolyhedrovirus (BmNPV).

    PubMed

    Zhou, Fang; Chen, Rui-ting; Lu, Yan; Liang, Shuang; Wang, Mei-xian; Miao, Yun-gen

    2014-12-01

    The Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most destructive diseases in silkworm, which has caused the main damage to sericulture industry. In this study, we developed a system of RNAi to prevent the BmNPV infection using the piggyBac transposon-derived targeting short hairpin RNA (shRNA) interference. The shRNAs targeting the genes of i.e.-1, lef-1, lef-2 and lef-3 of BmNPV were designed and used to inhibit the intracellular replication or multiplication of BmNPV in Bm cells. The highest activity was presented in the shRNA targeting the i.e.-1c of BmNPV, of which the inhibition rate reached 94.5 % in vitro. Further a stable Bm cell line of piggyBac transposon-derived targeting shRNA interference against BmNPV was established, which has a highly efficacious suppression on virus proliferation. These results indicated that the recombinant shRNA expression system was a useful tool for resistance to BmNPV in vitro. The approach by recombinant shRNAs opens a door of RNAi technology as a strategy that offering technically simpler, cheaper, and quicker gene knockdown for promising research and biotechnology application on silkworm lethal diseases.

  11. Generation of Naïve Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors.

    PubMed

    Kawaguchi, Takamasa; Tsukiyama, Tomoyuki; Kimura, Koji; Matsuyama, Shuichi; Minami, Naojiro; Yamada, Masayasu; Imai, Hiroshi

    2015-01-01

    Generation of pluripotent stem cells (PSCs) in large domestic animals has achieved only limited success; most of the PSCs obtained to date have been classified as primed PSCs, which possess very little capacity to produce chimeric offspring. By contrast, mouse PSCs have been classified as naïve PSCs that can contribute to most of the tissues of chimeras, including germ cells. Here, we describe the generation of two different types of bovine induced pluripotent stem cells (biPSCs) from amnion cells, achieved through introduction of piggyBac vectors containing doxycycline-inducible transcription factors (Oct3/4, Sox2, Klf4, and c-Myc). One type of biPSCs, cultured in medium supplemented with knockout serum replacement (KSR), FGF2, and bovine leukemia inhibitory factor (bLIF), had a flattened morphology like human PSCs; these were classified as primed-type. The other type biPSCs, cultured in KSR, bLIF, Mek/Erk inhibitor, GSK3 inhibitor and forskolin, had a compact morphology like mouse PSCs; these were classified as naïve-type. Cells could easily be switched between these two types of biPSCs by changing the culture conditions. Both types of biPSCs had strong alkaline phosphatase activity, expressed pluripotent markers (OCT3/4, NANOG, REX1, ESRRβ, STELLA, and SOCS3), and formed embryoid bodies that gave rise to differentiated cells from all three embryonic germ layers. However, only naïve-type biPSCs showed the hallmarks of naïve mouse PSCs, such as LIF-dependent proliferation, lack of FGF5 expression, and active XIST expression with two active X chromosomes. Furthermore, naïve-type biPSCs could contribute to the inner cell mass (ICM) of host blastocysts and most tissues within chimeric embryos. This is the first report of generation of biPSCs with several characteristics similar to those of naïve mouse PSCs and a demonstrated potential to contribute to chimeras.

  12. Generation of Naïve Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors

    PubMed Central

    Kawaguchi, Takamasa; Tsukiyama, Tomoyuki; Kimura, Koji; Matsuyama, Shuichi; Minami, Naojiro; Yamada, Masayasu; Imai, Hiroshi

    2015-01-01

    Generation of pluripotent stem cells (PSCs) in large domestic animals has achieved only limited success; most of the PSCs obtained to date have been classified as primed PSCs, which possess very little capacity to produce chimeric offspring. By contrast, mouse PSCs have been classified as naïve PSCs that can contribute to most of the tissues of chimeras, including germ cells. Here, we describe the generation of two different types of bovine induced pluripotent stem cells (biPSCs) from amnion cells, achieved through introduction of piggyBac vectors containing doxycycline-inducible transcription factors (Oct3/4, Sox2, Klf4, and c-Myc). One type of biPSCs, cultured in medium supplemented with knockout serum replacement (KSR), FGF2, and bovine leukemia inhibitory factor (bLIF), had a flattened morphology like human PSCs; these were classified as primed-type. The other type biPSCs, cultured in KSR, bLIF, Mek/Erk inhibitor, GSK3 inhibitor and forskolin, had a compact morphology like mouse PSCs; these were classified as naïve-type. Cells could easily be switched between these two types of biPSCs by changing the culture conditions. Both types of biPSCs had strong alkaline phosphatase activity, expressed pluripotent markers (OCT3/4, NANOG, REX1, ESRRβ, STELLA, and SOCS3), and formed embryoid bodies that gave rise to differentiated cells from all three embryonic germ layers. However, only naïve-type biPSCs showed the hallmarks of naïve mouse PSCs, such as LIF-dependent proliferation, lack of FGF5 expression, and active XIST expression with two active X chromosomes. Furthermore, naïve-type biPSCs could contribute to the inner cell mass (ICM) of host blastocysts and most tissues within chimeric embryos. This is the first report of generation of biPSCs with several characteristics similar to those of naïve mouse PSCs and a demonstrated potential to contribute to chimeras. PMID:26287611

  13. Transposase-Mediated Excision, Conjugative Transfer, and Diversity of ICE6013 Elements in Staphylococcus aureus.

    PubMed

    Sansevere, Emily A; Luo, Xiao; Park, Joo Youn; Yoon, Sunghyun; Seo, Keun Seok; Robinson, D Ashley

    2017-04-15

    ICE6013 represents one of two families of integrative conjugative elements (ICEs) identified in the pan-genome of the human and animal pathogen Staphylococcus aureus Here we investigated the excision and conjugation functions of ICE6013 and further characterized the diversity of this element. ICE6013 excision was not significantly affected by growth, temperature, pH, or UV exposure and did not depend on recA The IS30-like DDE transposase (Tpase; encoded by orf1 and orf2) of ICE6013 must be uninterrupted for excision to occur, whereas disrupting three of the other open reading frames (ORFs) on the element significantly affects the level of excision. We demonstrate that ICE6013 conjugatively transfers to different S. aureus backgrounds at frequencies approaching that of the conjugative plasmid pGO1. We found that excision is required for conjugation, that not all S. aureus backgrounds are successful recipients, and that transconjugants acquire the ability to transfer ICE6013 Sequencing of chromosomal integration sites in serially passaged transconjugants revealed a significant integration site preference for a 15-bp AT-rich palindromic consensus sequence, which surrounds the 3-bp target site that is duplicated upon integration. A sequence analysis of ICE6013 from different host strains of S. aureus and from eight other species of staphylococci identified seven divergent subfamilies of ICE6013 that include sequences previously classified as a transposon, a plasmid, and various ICEs. In summary, these results indicate that the IS30-like Tpase functions as the ICE6013 recombinase and that ICE6013 represents a diverse family of mobile genetic elements that mediate conjugation in staphylococci.IMPORTANCE Integrative conjugative elements (ICEs) encode the abilities to integrate into and excise from bacterial chromosomes and plasmids and mediate conjugation between bacteria. As agents of horizontal gene transfer, ICEs may affect bacterial evolution. ICE6013 represents one

  14. The folding of the specific DNA recognition subdomain of the sleeping beauty transposase is temperature-dependent and is required for its binding to the transposon DNA.

    PubMed

    Leighton, Gage O; Konnova, Tatiana A; Idiyatullin, Bulat; Hurr, Sophia H; Zuev, Yuriy F; Nesmelova, Irina V

    2014-01-01

    The reaction of DNA transposition begins when the transposase enzyme binds to the transposon DNA. Sleeping Beauty is a member of the mariner family of DNA transposons. Although it is an important tool in genetic applications and has been adapted for human gene therapy, its molecular mechanism remains obscure. Here, we show that only the folded conformation of the specific DNA recognition subdomain of the Sleeping Beauty transposase, the PAI subdomain, binds to the transposon DNA. Furthermore, we show that the PAI subdomain is well folded at low temperatures, but the presence of unfolded conformation gradually increases at temperatures above 15°C, suggesting that the choice of temperature may be important for the optimal transposase activity. Overall, the results provide a molecular-level insight into the DNA recognition by the Sleeping Beauty transposase.

  15. The Folding of the Specific DNA Recognition Subdomain of the Sleeping Beauty Transposase Is Temperature-Dependent and Is Required for Its Binding to the Transposon DNA

    PubMed Central

    Leighton, Gage O.; Konnova, Tatiana A.; Idiyatullin, Bulat; Hurr, Sophia H.; Zuev, Yuriy F.; Nesmelova, Irina V.

    2014-01-01

    The reaction of DNA transposition begins when the transposase enzyme binds to the transposon DNA. Sleeping Beauty is a member of the mariner family of DNA transposons. Although it is an important tool in genetic applications and has been adapted for human gene therapy, its molecular mechanism remains obscure. Here, we show that only the folded conformation of the specific DNA recognition subdomain of the Sleeping Beauty transposase, the PAI subdomain, binds to the transposon DNA. Furthermore, we show that the PAI subdomain is well folded at low temperatures, but the presence of unfolded conformation gradually increases at temperatures above 15°C, suggesting that the choice of temperature may be important for the optimal transposase activity. Overall, the results provide a molecular-level insight into the DNA recognition by the Sleeping Beauty transposase. PMID:25375127

  16. Integration profile and safety of an adenovirus hybrid-vector utilizing hyperactive sleeping beauty transposase for somatic integration.

    PubMed

    Zhang, Wenli; Muck-Hausl, Martin; Wang, Jichang; Sun, Chuanbo; Gebbing, Maren; Miskey, Csaba; Ivics, Zoltan; Izsvak, Zsuzsanna; Ehrhardt, Anja

    2013-01-01

    We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB) transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We found that in all mice which received the hyperactive SB transposase, transgene expression levels were stabilized in a dose-dependent manner and that the highest vector dose was accompanied by fatalities in mice. To analyze potential genotoxic side-effects due to somatic integration into host chromosomes, we performed a genome-wide integration site analysis using linker-mediated PCR (LM-PCR) and linear amplification-mediated PCR (LAM-PCR). Analysis of genomic DNA samples obtained from HSB5 treated female and male mice revealed a total of 1327 unique transposition events. Overall the chromosomal distribution pattern was close-to-random and we observed a random integration profile with respect to integration into gene and non-gene areas. Notably, when using the LM-PCR protocol, 27 extra-chromosomal integration events were identified, most likely caused by transposon excision and subsequent transposition into the delivered adenoviral vector genome. In total, this study provides a careful evaluation of the safety profile of adenovirus/Sleeping Beauty transposase hybrid-vectors. The obtained information will be useful when designing future preclinical studies utilizing hybrid-vectors in small and large animal models.

  17. Integration Profile and Safety of an Adenovirus Hybrid-Vector Utilizing Hyperactive Sleeping Beauty Transposase for Somatic Integration

    PubMed Central

    Zhang, Wenli; Muck-Hausl, Martin; Wang, Jichang; Sun, Chuanbo; Gebbing, Maren; Miskey, Csaba; Ivics, Zoltan; Izsvak, Zsuzsanna; Ehrhardt, Anja

    2013-01-01

    We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB) transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and genotoxicity remains to be investigated. Herein, we evaluated this hybrid-vector system in C57Bl/6 mice with escalating vector dose settings. We found that in all mice which received the hyperactive SB transposase, transgene expression levels were stabilized in a dose-dependent manner and that the highest vector dose was accompanied by fatalities in mice. To analyze potential genotoxic side-effects due to somatic integration into host chromosomes, we performed a genome-wide integration site analysis using linker-mediated PCR (LM-PCR) and linear amplification-mediated PCR (LAM-PCR). Analysis of genomic DNA samples obtained from HSB5 treated female and male mice revealed a total of 1327 unique transposition events. Overall the chromosomal distribution pattern was close-to-random and we observed a random integration profile with respect to integration into gene and non-gene areas. Notably, when using the LM-PCR protocol, 27 extra-chromosomal integration events were identified, most likely caused by transposon excision and subsequent transposition into the delivered adenoviral vector genome. In total, this study provides a careful evaluation of the safety profile of adenovirus/Sleeping Beauty transposase hybrid-vectors. The obtained information will be useful when designing future preclinical studies utilizing hybrid-vectors in small and large animal models. PMID:24124483

  18. Flexibility in MuA transposase family protein structures: functional mapping with scanning mutagenesis and sequence alignment of protein homologues.

    PubMed

    Rasila, Tiina S; Vihinen, Mauno; Paulin, Lars; Haapa-Paananen, Saija; Savilahti, Harri

    2012-01-01

    MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIβ, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIβ were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.

  19. A green fluorescent protein solubility screen in E. coli reveals domain boundaries of the GTP-binding domain in the P element transposase

    PubMed Central

    Sabogal, Alex; Rio, Donald C

    2010-01-01

    Guanosine triphosphate (GTP) binding and hydrolysis events often act as molecular switches in proteins, modulating conformational changes between active and inactive states in many signaling molecules and transport systems. The P element transposase of Drosophila melanogaster requires GTP binding to proceed along its reaction pathway, following initial site-specific DNA binding. GTP binding is unique to P elements and may represent a novel form of transpositional regulation, allowing the bound transposase to find a second site, looping the transposon DNA for strand cleavage and excision. The GTP-binding activity has been previously mapped to the central portion of the transposase protein; however, the P element transposase contains little sequence identity with known GTP-binding folds. To identify soluble, active transposase domains, a GFP solubility screen was used testing the solubility of random P element gene fragments in E. coli. The screen produced a single clone spanning known GTP-binding residues in the central portion of the transposase coding region. This clone, amino acids 275–409 in the P element transposase, was soluble, highly expressed in E.coli and active for GTP-binding activity, therefore is a candidate for future biochemical and structural studies. In addition, the chimeric screen revealed a minimal N-terminal THAP DNA-binding domain attached to an extended leucine zipper coiled-coil dimerization domain in the P element transposase, precisely delineating the DNA-binding and dimerization activities on the primary sequence. This study highlights the use of a GFP-based solubility screen on a large multidomain protein to identify highly expressed, soluble truncated domain subregions. PMID:20842711

  20. Assembly of the Tc1 and mariner transposition initiation complexes depends on the origins of their transposase DNA binding domains.

    PubMed

    Brillet, Benjamin; Benjamin, Brillet; Bigot, Yves; Yves, Bigot; Augé-Gouillou, Corinne; Corinne, Augé-Gouillou

    2007-06-01

    In this review, we focus on the assembly of DNA/protein complexes that trigger transposition in eukaryotic members of the IS630-Tc1-mariner (ITm) super-family, the Tc1- and mariner-like elements (TLEs and MLEs). Elements belonging to this super-family encode transposases with DNA binding domains of different origins, and recent data indicate that the chimerization of functional domains has been an important evolutionary aspect in the generation of new transposons within the ITm super-family. These data also reveal that the inverted terminal repeats (ITRs) at the ends of transposons contain three kinds of motif within their sequences. The first two are well known and correspond to the cleavage site on the outer ITR extremities, and the transposase DNA binding site. The organization of ITRs and of the transposase DNA binding domains implies that differing pathways are used by MLEs and TLEs to regulate transposition initiation. These differences imply that the ways ITRs are recognized also differ leading to the formation of differently organized synaptic complexes. The third kind of motif is the transposition enhancers, which have been found in almost all the functional MLEs and TLEs analyzed to date. Finally, in vitro and in vivo assays of various elements all suggest that the transposition initiation complex is not formed randomly, but involves a mechanism of oriented transposon scanning.

  1. Treponema denticola biofilm-induced expression of a bacteriophage, toxin-antitoxin systems and transposases.

    PubMed

    Mitchell, Helen L; Dashper, Stuart G; Catmull, Deanne V; Paolini, Rita A; Cleal, Steven M; Slakeski, Nada; Tan, Kheng H; Reynolds, Eric C

    2010-03-01

    Treponema denticola is an oral spirochaete that has been strongly associated with chronic periodontitis. The bacterium exists as part of a dense biofilm (subgingival dental plaque) accreted to the tooth. To determine T. denticola gene products important for persistence as a biofilm we developed a continuous-culture biofilm model and conducted a genome-wide transcriptomic analysis of biofilm and planktonic cells. A total of 126 genes were differentially expressed with a fold change of 1.5 or greater. This analysis identified the upregulation of putative prophage genes in the T. denticola 35405 genome. Intact bacteriophage particles were isolated from T. denticola and circular phage DNA was detected by PCR analysis. This represents the first, to our knowledge, functional bacteriophage isolated from T. denticola, which we have designated varphitd1. In biofilm cells there was also an upregulation of genes encoding several virulence factors, toxin-antitoxin systems and a family of putative transposases. Together, these data indicate that there is a higher potential for genetic mobility in T. denticola when growing as a biofilm and that these systems are important for the biofilm persistence and therefore virulence of this bacterium.

  2. Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle

    PubMed Central

    Escobar, Helena; Schöwel, Verena; Spuler, Simone; Marg, Andreas; Izsvák, Zsuzsanna

    2016-01-01

    Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysfprmd Prkdcscid/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy. PMID:26784637

  3. Full-length Dysferlin Transfer by the Hyperactive Sleeping Beauty Transposase Restores Dysferlin-deficient Muscle.

    PubMed

    Escobar, Helena; Schöwel, Verena; Spuler, Simone; Marg, Andreas; Izsvák, Zsuzsanna

    2016-01-19

    Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysf(prmd) Prkdc(scid)/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy.

  4. Purification of the Caenorhabditis elegans transposase Tc1A refolded during gel filtration chromatography.

    PubMed

    García-Sáez, I; Plasterk, R H

    2000-08-01

    Full-length recombinant transposase Tc1A from Caenorhabditis elegans (343 amino acids) expressed in Escherichia coli BL21 in inclusion bodies has been purified in a high yield in a soluble form. The procedure includes denaturation of the inclusion bodies followed by refolding of the Tc1A protein by gel filtration. This last step is absolutely crucial to give a high yield of soluble and active protein since it allows the physical separation of the aggregates from intermediates that give rise to correctly refolded protein. This step is very sensitive to the concentration of protein. Good yields of refolded protein are obtained by refolding 2 to 12 mg of denatured protein. The other purification steps involve the initial use of gel filtration under denaturing conditions and a final step of ion-exchange chromatography. Biological activity of the purified protein was confirmed in an in vitro transposon excision assay and its DNA-binding capacity by UV crosslinking. This new Tc1A purification procedure gives a yield of 12-16 mg/liter E. coli culture, in a form suitable for crystallization studies.

  5. An atypical AAA+ ATPase assembly controls efficient transposition through DNA remodeling and transposase recruitment

    PubMed Central

    Arias-Palomo, Ernesto; Berger, James M.

    2015-01-01

    Transposons are ubiquitous genetic elements that drive genome rearrangements, evolution, and the spread of infectious disease and drug-resistance. Many transposons, such as Mu, Tn7 and IS21, require regulatory AAA+ ATPases for function. We use x-ray crystallography and cryo-electron microscopy to show that the ATPase subunit of IS21, IstB, assembles into a clamshell-shaped decamer that sandwiches DNA between two helical pentamers of ATP-associated AAA+ domains, sharply bending the duplex into a 180° U-turn. Biochemical studies corroborate key features of the structure, and further show that the IS21 transposase, IstA, recognizes the IstB•DNA complex and promotes its disassembly by stimulating ATP hydrolysis. Collectively, these studies reveal a distinct manner of higher-order assembly and client engagement by a AAA+ ATPase and suggest a mechanistic model where IstB binding and subsequent DNA bending primes a selected insertion site for efficient transposition. PMID:26276634

  6. Assessing fitness costs for transgenic Aedes aegypti expressing the GFP marker and transposase genes.

    PubMed

    Irvin, Nic; Hoddle, Mark S; O'Brochta, David A; Carey, Bryan; Atkinson, Peter W

    2004-01-20

    The development of transgenic mosquitoes that are refractory to the transmission of human diseases such as malaria, dengue, and yellow fever has received much interest due to the ability to transform a number of vector mosquito species with transposable elements. Transgenic strains of mosquitoes have been generated with molecular techniques that exhibit a reduced capacity to transmit pathogens. These advancements have led to questions regarding the fitness of transgenic mosquitoes and the ability of transformed mosquitoes to compete and effectively spread beneficial genes through nontransformed field populations, the core requirement of a genetically based control strategy aimed at reducing the spread of mosquito-borne human disease. Here we examine the impact of transgenesis on the fitness of Aedes aegypti, a mosquito that transmits yellow fever. Mosquitoes were altered with two types of transgene, the enhanced GFP gene and two transposase genes from the Hermes and MOS1 transposable elements. We examined the effects of these elements on the survivorship, longevity, fecundity, sex ratio, and sterility of transformed mosquitoes and compared results to the nontransformed laboratory strain. We show that demographic parameters are significantly diminished in transgenic mosquitoes relative to the untransformed laboratory strain. Reduced fitness in transgenic mosquitoes has important implications for the development and utilization of this technology for control programs based on manipulative molecular modification.

  7. Domestic violence.

    PubMed

    Bennett, B

    1997-01-01

    This article explores the usefulness of white feminist domestic intervention to Aboriginal women who are victims of domestic violence. The discussion opens with a brief history of Aboriginal society before the age of colonization, followed by a summary of feminist intervention to fight domestic violence and the reactions of the Aboriginal women and communities towards this. It also presents a brief description of works conducted overseas where more appropriate frameworks are being used in intervening against domestic violence such as anti-racist social work. Towards the end, this paper explores some future possibilities for workers, in both Aboriginal and non-Aboriginal societies.

  8. Scanning of Transposable Elements and Analyzing Expression of Transposase Genes of Sweet Potato [Ipomoea batatas

    PubMed Central

    Tao, Xiang; Lai, Xian-Jun; Zhang, Yi-Zheng; Tan, Xue-Mei; Wang, Haiyan

    2014-01-01

    Background Transposable elements (TEs) are the most abundant genomic components in eukaryotes and affect the genome by their replications and movements to generate genetic plasticity. Sweet potato performs asexual reproduction generally and the TEs may be an important genetic factor for genome reorganization. Complete identification of TEs is essential for the study of genome evolution. However, the TEs of sweet potato are still poorly understood because of its complex hexaploid genome and difficulty in genome sequencing. The recent availability of the sweet potato transcriptome databases provides an opportunity for discovering and characterizing the expressed TEs. Methodology/Principal Findings We first established the integrated-transcriptome database by de novo assembling four published sweet potato transcriptome databases from three cultivars in China. Using sequence-similarity search and analysis, a total of 1,405 TEs including 883 retrotransposons and 522 DNA transposons were predicted and categorized. Depending on mapping sets of RNA-Seq raw short reads to the predicted TEs, we compared the quantities, classifications and expression activities of TEs inter- and intra-cultivars. Moreover, the differential expressions of TEs in seven tissues of Xushu 18 cultivar were analyzed by using Illumina digital gene expression (DGE) tag profiling. It was found that 417 TEs were expressed in one or more tissues and 107 in all seven tissues. Furthermore, the copy number of 11 transposase genes was determined to be 1–3 copies in the genome of sweet potato by Real-time PCR-based absolute quantification. Conclusions/Significance Our result provides a new method for TE searching on species with transcriptome sequences while lacking genome information. The searching, identification and expression analysis of TEs will provide useful TE information in sweet potato, which are valuable for the further studies of TE-mediated gene mutation and optimization in asexual reproduction

  9. In vitro, long-range sequence information for de novo genome assembly via transposase contiguity.

    PubMed

    Adey, Andrew; Kitzman, Jacob O; Burton, Joshua N; Daza, Riza; Kumar, Akash; Christiansen, Lena; Ronaghi, Mostafa; Amini, Sasan; Gunderson, Kevin L; Steemers, Frank J; Shendure, Jay

    2014-12-01

    We describe a method that exploits contiguity preserving transposase sequencing (CPT-seq) to facilitate the scaffolding of de novo genome assemblies. CPT-seq is an entirely in vitro means of generating libraries comprised of 9216 indexed pools, each of which contains thousands of sparsely sequenced long fragments ranging from 5 kilobases to > 1 megabase. These pools are "subhaploid," in that the lengths of fragments contained in each pool sums to ∼5% to 10% of the full genome. The scaffolding approach described here, termed fragScaff, leverages coincidences between the content of different pools as a source of contiguity information. Specifically, CPT-seq data is mapped to a de novo genome assembly, followed by the identification of pairs of contigs or scaffolds whose ends disproportionately co-occur in the same indexed pools, consistent with true adjacency in the genome. Such candidate "joins" are used to construct a graph, which is then resolved by a minimum spanning tree. As a proof-of-concept, we apply CPT-seq and fragScaff to substantially boost the contiguity of de novo assemblies of the human, mouse, and fly genomes, increasing the scaffold N50 of de novo assemblies by eight- to 57-fold with high accuracy. We also demonstrate that fragScaff is complementary to Hi-C-based contact probability maps, providing midrange contiguity to support robust, accurate chromosome-scale de novo genome assemblies without the need for laborious in vivo cloning steps. Finally, we demonstrate CPT-seq as a means of anchoring unplaced novel human contigs to the reference genome as well as for detecting misassembled sequences.

  10. In vitro, long-range sequence information for de novo genome assembly via transposase contiguity

    PubMed Central

    Adey, Andrew; Kitzman, Jacob O.; Burton, Joshua N.; Daza, Riza; Kumar, Akash; Christiansen, Lena; Ronaghi, Mostafa; Amini, Sasan; L. Gunderson, Kevin; Steemers, Frank J.

    2014-01-01

    We describe a method that exploits contiguity preserving transposase sequencing (CPT-seq) to facilitate the scaffolding of de novo genome assemblies. CPT-seq is an entirely in vitro means of generating libraries comprised of 9216 indexed pools, each of which contains thousands of sparsely sequenced long fragments ranging from 5 kilobases to >1 megabase. These pools are “subhaploid,” in that the lengths of fragments contained in each pool sums to ∼5% to 10% of the full genome. The scaffolding approach described here, termed fragScaff, leverages coincidences between the content of different pools as a source of contiguity information. Specifically, CPT-seq data is mapped to a de novo genome assembly, followed by the identification of pairs of contigs or scaffolds whose ends disproportionately co-occur in the same indexed pools, consistent with true adjacency in the genome. Such candidate “joins” are used to construct a graph, which is then resolved by a minimum spanning tree. As a proof-of-concept, we apply CPT-seq and fragScaff to substantially boost the contiguity of de novo assemblies of the human, mouse, and fly genomes, increasing the scaffold N50 of de novo assemblies by eight- to 57-fold with high accuracy. We also demonstrate that fragScaff is complementary to Hi-C-based contact probability maps, providing midrange contiguity to support robust, accurate chromosome-scale de novo genome assemblies without the need for laborious in vivo cloning steps. Finally, we demonstrate CPT-seq as a means of anchoring unplaced novel human contigs to the reference genome as well as for detecting misassembled sequences. PMID:25327137

  11. "Domestic violence".

    PubMed

    Davidhizar, Ruth; Giger, Joy Newman

    2002-01-01

    Domestic violence is no respector of persons but may involve spouses or cohabiting adults across all socioeconomic, ethnic, and religious groups. It is no respect or of community. The abuse can be physical, sexual, or emotional/psychological. It can be economic and be demonstrated by neglect. Nursing care for the problem of domestic abuse needs to be directed at primary, secondary, and tertiary prevention. Care for victims and victimizers of domestic violence involves a collaborative relationship with other professionals as well as interagency cross referrals that involve health, welfare, refuge, and judicial protective services.

  12. Delivery of full-length factor VIII using a piggyBac transposon vector to correct a mouse model of hemophilia A.

    PubMed

    Matsui, Hideto; Fujimoto, Naoko; Sasakawa, Noriko; Ohinata, Yasuhide; Shima, Midori; Yamanaka, Shinya; Sugimoto, Mitsuhiko; Hotta, Akitsu

    2014-01-01

    Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice. piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII.

  13. The N-terminal zinc finger domain of Tgf2 transposase contributes to DNA binding and to transposition activity

    PubMed Central

    Jiang, Xia-Yun; Hou, Fei; Shen, Xiao-Dan; Du, Xue-Di; Xu, Hai-Li; Zou, Shu-Ming

    2016-01-01

    Active Hobo/Activator/Tam3 (hAT) transposable elements are rarely found in vertebrates. Previously, goldfish Tgf2 was found to be an autonomously active vertebrate transposon that is efficient at gene-transfer in teleost fish. However, little is known about Tgf2 functional domains required for transposition. To explore this, we first predicted in silico a zinc finger domain in the N-terminus of full length Tgf2 transposase (L-Tgf2TPase). Two truncated recombinant Tgf2 transposases with deletions in the N-terminal zinc finger domain, S1- and S2-Tgf2TPase, were expressed in bacteria from goldfish cDNAs. Both truncated Tgf2TPases lost their DNA-binding ability in vitro, specifically at the ends of Tgf2 transposon than native L-Tgf2TPase. Consequently, S1- and S2-Tgf2TPases mediated gene transfer in the zebrafish genome in vivo at a significantly (p < 0.01) lower efficiency (21%–25%), in comparison with L-Tgf2TPase (56% efficiency). Compared to L-Tgf2TPase, truncated Tgf2TPases catalyzed imprecise excisions with partial deletion of TE ends and/or plasmid backbone insertion/deletion. The gene integration into the zebrafish genome mediated by truncated Tgf2TPases was imperfect, creating incomplete 8-bp target site duplications at the insertion sites. These results indicate that the zinc finger domain in Tgf2 transposase is involved in binding to Tgf2 terminal sequences, and loss of those domains has effects on TE transposition. PMID:27251101

  14. Tc1-like Transposase Thm3 of Silver Carp (Hypophthalmichthys molitrix) Can Mediate Gene Transposition in the Genome of Blunt Snout Bream (Megalobrama amblycephala).

    PubMed

    Guo, Xiu-Ming; Zhang, Qian-Qian; Sun, Yi-Wen; Jiang, Xia-Yun; Zou, Shu-Ming

    2015-10-04

    Tc1-like transposons consist of an inverted repeat sequence flanking a transposase gene that exhibits similarity to the mobile DNA element, Tc1, of the nematode, Caenorhabditis elegans. They are widely distributed within vertebrate genomes including teleost fish; however, few active Tc1-like transposases have been discovered. In this study, 17 Tc1-like transposon sequences were isolated from 10 freshwater fish species belonging to the families Cyprinidae, Adrianichthyidae, Cichlidae, and Salmonidae. We conducted phylogenetic analyses of these sequences using previously isolated Tc1-like transposases and report that 16 of these elements comprise a new subfamily of Tc1-like transposons. In particular, we show that one transposon, Thm3 from silver carp (Hypophthalmichthys molitrix; Cyprinidae), can encode a 335-aa transposase with apparently intact domains, containing three to five copies in its genome. We then coinjected donor plasmids harboring 367 bp of the left end and 230 bp of the right end of the nonautonomous silver carp Thm1 cis-element along with capped Thm3 transposase RNA into the embryos of blunt snout bream (Megalobrama amblycephala; one- to two-cell embryos). This experiment revealed that the average integration rate could reach 50.6% in adult fish. Within the blunt snout bream genome, the TA dinucleotide direct repeat, which is the signature of Tc1-like family of transposons, was created adjacent to both ends of Thm1 at the integration sites. Our results indicate that the silver carp Thm3 transposase can mediate gene insertion by transposition within the genome of blunt snout bream genome, and that this occurs with a TA position preference.

  15. Sense overlapping transcripts in IS1341-type transposase genes are functional non-coding RNAs in archaea

    PubMed Central

    Gomes-Filho, José Vicente; Zaramela, Livia Soares; Italiani, Valéria Cristina da Silva; Baliga, Nitin S; Vêncio, Ricardo Z N; Koide, Tie

    2015-01-01

    The existence of sense overlapping transcripts that share regulatory and coding information in the same genomic sequence shows an additional level of prokaryotic gene expression complexity. Here we report the discovery of ncRNAs associated with IS1341-type transposase (tnpB) genes, at the 3'-end of such elements, with examples in archaea and bacteria. Focusing on the model haloarchaeon Halobacterium salinarum NRC-1, we show the existence of sense overlapping transcripts (sotRNAs) for all its IS1341-type transposases. Publicly available transcriptome compendium show condition-dependent differential regulation between sotRNAs and their cognate genes. These sotRNAs allowed us to find a UUCA tetraloop motif that is present in other archaea (ncRNA family HgcC) and in a H. salinarum intergenic ncRNA derived from a palindrome associated transposable elements (PATE). Overexpression of one sotRNA and the PATE-derived RNA harboring the tetraloop motif improved H. salinarum growth, indicating that these ncRNAs are functional. PMID:25806405

  16. Escherichia coli DNA Topoisomerase I and Suppression of Killing by Tn5 Transposase Overproduction: Topoisomerase I Modulates Tn5 Transposition

    PubMed Central

    Yigit, Hesna; Reznikoff, William S.

    1998-01-01

    Tn5 transposase (Tnp) overproduction is lethal to Escherichia coli. The overproduction causes cell filamentation and abnormal chromosome segregation. Here we present three lines of evidence strongly suggesting that Tnp overproduction killing is due to titration of topoisomerase I. First, a suppressor mutation of transposase overproduction killing, stkD10, is localized in topA (the gene for topoisomerase I). The stkD10 mutant has the following characteristics: first, it has an increased abundance of topoisomerase I protein, the topoisomerase I is defective for the DNA relaxation activity, and DNA gyrase activity is reduced; second, the suppressor phenotype of a second mutation localized in rpoH, stkA14 (H. Yigit and W. S. Reznikoff, J. Bacteriol. 179:1704–1713, 1997), can be explained by an increase in topA expression; and third, overexpression of wild-type topA partially suppresses the killing. Finally, topoisomerase I was found to enhance Tn5 transposition up to 30-fold in vivo. PMID:9811643

  17. Chromatin states shape insertion profiles of the piggyBac, Tol2 and Sleeping Beauty transposons and murine leukemia virus

    PubMed Central

    Yoshida, Junko; Akagi, Keiko; Misawa, Ryo; Kokubu, Chikara; Takeda, Junji; Horie, Kyoji

    2017-01-01

    DNA transposons and retroviruses are versatile tools in functional genomics and gene therapy. To facilitate their application, we conducted a genome-wide insertion site profiling of the piggyBac (PB), Tol2 and Sleeping Beauty (SB) transposons and the murine leukemia virus (MLV) in mouse embryonic stem cells (ESCs). PB and MLV preferred highly expressed genes, whereas Tol2 and SB preferred weakly expressed genes. However, correlations with DNase I hypersensitive sites were different for all vectors, indicating that chromatin accessibility is not the sole determinant. Therefore, we analysed various chromatin states. PB and MLV highly correlated with Cohesin, Mediator and ESC-specific transcription factors. Notably, CTCF sites were correlated with PB but not with MLV, suggesting MLV prefers smaller promoter–enhancer loops, whereas PB insertion encompasses larger chromatin loops termed topologically associating domains. Tol2 also correlated with Cohesin and CTCF. However, correlations with ESC-specific transcription factors were weaker, suggesting that Tol2 prefers transcriptionally weak chromatin loops. Consistently, Tol2 insertions were associated with bivalent histone modifications characteristic of silent and inducible loci. SB showed minimum preference to all chromatin states, suggesting the least adverse effect on adjacent genes. These results will be useful for vector selection for various applications. PMID:28252665

  18. Chromatin states shape insertion profiles of the piggyBac, Tol2 and Sleeping Beauty transposons and murine leukemia virus.

    PubMed

    Yoshida, Junko; Akagi, Keiko; Misawa, Ryo; Kokubu, Chikara; Takeda, Junji; Horie, Kyoji

    2017-03-02

    DNA transposons and retroviruses are versatile tools in functional genomics and gene therapy. To facilitate their application, we conducted a genome-wide insertion site profiling of the piggyBac (PB), Tol2 and Sleeping Beauty (SB) transposons and the murine leukemia virus (MLV) in mouse embryonic stem cells (ESCs). PB and MLV preferred highly expressed genes, whereas Tol2 and SB preferred weakly expressed genes. However, correlations with DNase I hypersensitive sites were different for all vectors, indicating that chromatin accessibility is not the sole determinant. Therefore, we analysed various chromatin states. PB and MLV highly correlated with Cohesin, Mediator and ESC-specific transcription factors. Notably, CTCF sites were correlated with PB but not with MLV, suggesting MLV prefers smaller promoter-enhancer loops, whereas PB insertion encompasses larger chromatin loops termed topologically associating domains. Tol2 also correlated with Cohesin and CTCF. However, correlations with ESC-specific transcription factors were weaker, suggesting that Tol2 prefers transcriptionally weak chromatin loops. Consistently, Tol2 insertions were associated with bivalent histone modifications characteristic of silent and inducible loci. SB showed minimum preference to all chromatin states, suggesting the least adverse effect on adjacent genes. These results will be useful for vector selection for various applications.

  19. Non-viral reprogramming of fibroblasts into induced pluripotent stem cells by Sleeping Beauty and piggyBac transposons.

    PubMed

    Talluri, Thirumala R; Kumar, Dharmendra; Glage, Silke; Garrels, Wiebke; Ivics, Zoltan; Debowski, Katharina; Behr, Rüdiger; Kues, Wilfried A

    2014-07-18

    The generation of induced pluripotent stem (iPS) cells represents a promising approach for innovative cell therapies. The original method requires viral transduction of several reprogramming factors, which may be associated with an increased risk of tumorigenicity. Transposition of reprogramming cassettes represents a recent alternative to viral approaches. Since binary transposons can be produced as common plasmids they provide a safe and cost-efficient alternative to viral delivery methods. Here, we compared the efficiency of two different transposon systems, Sleeping Beauty (SB) and piggyBac (PB), for the generation of murine iPS. Murine fibroblasts derived from an inbred BL/6 mouse line carrying a pluripotency reporter, Oct4-EGFP, and fibroblasts derived from outbred NMRI mice were employed for reprogramming. Both transposon systems resulted in the successful isolation of murine iPS cell lines. The reduction of the core reprogramming factors to omit the proto-oncogene c-Myc was compatible with iPS cell line derivation, albeit with reduced reprogramming efficiencies. The transposon-derived iPS cells featured typical hallmarks of pluripotency, including teratoma growth in immunodeficient mice. Thus SB and PB transposons represent a promising non-viral approach for iPS cell derivation. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. A Hybrid Adenoviral Vector System Achieves Efficient Long-Term Gene Expression in the Liver via piggyBac Transposition

    PubMed Central

    Smith, Ryan P.; Riordan, Jesse D.; Feddersen, Charlotte R.

    2015-01-01

    Abstract Much research has gone into the development of hybrid gene delivery systems that combine the broad tropism and efficient transduction of adenoviral vectors with the ability to achieve stable expression of cargo genes. In addition to gene therapy applications, such a system has considerable advantages for studies of gene function in vivo, permitting fine-tuned genetic manipulation with higher throughput than can be achieved using standard transgenic and DNA targeting techniques. Existing strategies are limited, however, by low integration efficiencies, small cargo capacity, and/or a dependence on target cell division. The utility of this approach could be enhanced by a system that provides all of the following: (1) efficient delivery, (2) stable expression in a high percentage of target cells (whether mitotic or not), (3) large cargo capacity, (4) flexibility to use with a wide range of additional experimental conditions, and (5) simple experimental technique. Here we report the initial characterization of a hybrid system that meets these criteria by utilizing piggyBac (PB) transposition to achieve genomic integration from adenoviral vectors. We demonstrate stable expression of an adenovirus (Ad)-PB-delivered reporter gene in ∼20–40% of hepatocytes following standard tail vein injection. Its high efficiency and flexibility relative to existing hybrid adenoviral gene delivery approaches indicate a considerable potential utility of the Ad-PB system for therapeutic gene delivery and in vivo studies of gene function. PMID:25808258

  1. Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons.

    PubMed

    Macdonald, Joni; Taylor, Lorna; Sherman, Adrian; Kawakami, Koichi; Takahashi, Yoshiko; Sang, Helen M; McGrew, Michael J

    2012-06-05

    The derivation of germ-line competent avian primordial germ cells establishes a cell-based model system for the investigation of germ cell differentiation and the production of genetically modified animals. Current methods to modify primordial germ cells using DNA or retroviral vectors are inefficient and prone to epigenetic silencing. Here, we validate the use of transposable elements for the genetic manipulation of primordial germ cells. We demonstrate that chicken primordial germ cells can be modified in vitro using transposable elements. Both piggyBac and Tol2 transposons efficiently transpose primordial germ cells. Tol2 transposon integration sites were spread throughout both the macro- and microchromosomes of the chicken genome and were more prevalent in gene transcriptional units and intronic regions, consistent with transposon integrations observed in other species. We determined that the presence of insulator elements was not required for reporter gene expression from the integrated transposon. We further demonstrate that a gene-trap cassette carried in the Tol2 transposon can trap and mutate endogenous transcripts in primordial germ cells. Finally, we observed that modified primordial germ cells form functional gametes as demonstrated by the generation of transgenic offspring that correctly expressed a reporter gene carried in the transposon. Transposable elements are therefore efficient vectors for the genetic manipulation of primordial germ cells and the chicken genome.

  2. Inducible Transgene Expression in Human iPS Cells Using Versatile All-in-One piggyBac Transposons.

    PubMed

    Kim, Shin-Il; Oceguera-Yanez, Fabian; Sakurai, Chiho; Nakagawa, Masato; Yamanaka, Shinya; Woltjen, Knut

    2016-01-01

    Transgenics is a mainstay of functional genomics. Conditionally overexpressing genes of interest (GOIs) helps to reveal their roles in the control of complex biological processes. Complemented by findings in classic animal model systems, recent advances in human embryonic stem cell (hESC) and patient-specific induced pluripotent stem cell (hiPSC) differentiation have led to sophisticated in vitro models of human development and disease. Yet, as transgenic elements encoding inducible systems must be introduced de novo into each genetically unique human stem cell line, robust and straightforward solutions to gene delivery are required. Transposons are a family of mobile DNA elements that have been adapted as experimental tools for stable genomic integration of transgenes. The piggyBac (PB) transposon from Trichoplusia ni presents a number of benefits over classic viral or BAC transgenesis: ease of application, simple integration-site mapping, and the unique capacity for traceless excision. Moreover, their large capacity permits the consolidation of multiple transgene components in a single vector system. In this chapter, we outline the features of a panel of "All-in-One" PB transposons designed for drug-inducible gene expression and provide guidelines to establish and validate populations or clones of transgenic hiPSCs.

  3. One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes.

    PubMed

    Li, Shenglan; Zhang, Anqi; Xue, Haipeng; Li, Dali; Liu, Ying

    2017-09-15

    Neural cell fate is determined by a tightly controlled transcription regulatory network during development. The ability to manipulate the expression of multiple transcription factors simultaneously is required to delineate the complex picture of neural cell development. Because of the limited carrying capacity of the commonly used viral vectors, such as lentiviral or retroviral vectors, it is often challenging to perform perturbation experiments on multiple transcription factors. Here we have developed a piggyBac (PB) transposon-based CRISPR activation (CRISPRa) all-in-one system, which allows for simultaneous and stable endogenous transactivation of multiple transcription factors and long non-coding RNAs. As a proof of principle, we showed that the PB-CRISPRa system could accelerate the differentiation of human induced pluripotent stem cells into neurons and astrocytes by triggering endogenous expression of different sets of transcription factors. The PB-CRISPRa system has the potential to become a convenient and robust tool in neuroscience, which can meet the needs of a variety of in vitro and in vivo gain-of-function applications. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  4. Slingshot: a PiggyBac based transposon system for tamoxifen-inducible 'self-inactivating' insertional mutagenesis.

    PubMed

    Kong, Jun; Wang, Feng; Brenton, James D; Adams, David J

    2010-10-01

    We have developed a self-inactivating PiggyBac transposon system for tamoxifen inducible insertional mutagenesis from a stably integrated chromosomal donor. This system, which we have named 'Slingshot', utilizes a transposon carrying elements for both gain- and loss-of-function screens in vitro. We show that the Slingshot transposon can be efficiently mobilized from a range of chromosomal loci with high inducibility and low background generating insertions that are randomly dispersed throughout the genome. Furthermore, we show that once the Slingshot transposon has been mobilized it is not remobilized producing stable clonal integrants in all daughter cells. To illustrate the efficacy of Slingshot as a screening tool we set out to identify mediators of resistance to puromycin and the chemotherapeutic drug vincristine by performing genetrap screens in mouse embryonic stem cells. From these genome-wide screens we identified multiple independent insertions in the multidrug resistance transporter genes Abcb1a/b and Abcg2 conferring resistance to drug treatment. Importantly, we also show that the Slingshot transposon system is functional in other mammalian cell lines such as human HEK293, OVCAR-3 and PE01 cells suggesting that it may be used in a range of cell culture systems. Slingshot represents a flexible and potent system for genome-wide transposon-mediated mutagenesis with many potential applications.

  5. PBmice: an integrated database system of piggyBac (PB) insertional mutations and their characterizations in mice

    PubMed Central

    Sun, Ling V.; Jin, Ke; Liu, Yiming; Yang, Wenwei; Xie, Xing; Ye, Lin; Wang, Li; Zhu, Lin; Ding, Sheng; Su, Yi; Zhou, Jie; Han, Min; Zhuang, Yuan; Xu, Tian; Wu, Xiaohui; Gu, Ning; Zhong, Yang

    2008-01-01

    DNA transposon piggyBac (PB) is a newly established mutagen for large-scale mutagenesis in mice. We have designed and implemented an integrated database system called PBmice (PB Mutagenesis Information CEnter) for storing, retrieving and displaying the information derived from PB insertions (INSERTs) in the mouse genome. This system is centered on INSERTs with information including their genomic locations and flanking genomic sequences, the expression levels of the hit genes, and the expression patterns of the trapped genes if a trapping vector was used. It also archives mouse phenotyping data linked to INSERTs, and allows users to conduct quick and advanced searches for genotypic and phenotypic information relevant to a particular or a set of INSERT(s). Sequence-based information can be cross-referenced with other genomic databases such as Ensembl, BLAST and GBrowse tools used in PBmice offer enhanced search and display for additional information relevant to INSERTs. The total number and genomic distribution of PB INSERTs, as well as the availability of each PB insertional LINE can also be viewed with user-friendly interfaces. PBmice is freely available at http://www.idmshanghai.cn/PBmice or http://www.scbit.org/PBmice/. PMID:17932058

  6. The hyperactive Sleeping Beauty transposase SB100X improves the genetic modification of T cells to express a chimeric antigen receptor1

    PubMed Central

    Jin, Zeming; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Olivares, Simon; Mátés, Lajos; Izsvák, Zsuzsanna; Ivics, Zoltán; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J.N.

    2014-01-01

    Sleeping Beauty (SB3) transposon and transposase constitute a DNA plasmid system used for therapeutic human cell genetic engineering. Here we report a comparison of SB100X, a newly developed hyperactive SB transposase, to a previous generation SB11 transposase to achieve stable expression of a CD19-specific chimeric antigen receptor (CAR3) in primary human T cells. The electro-transfer of SB100X expressed from a DNA plasmid or as an introduced mRNA species had superior transposase activity in T cells based on measurement of excision circles released after transposition and emergence of CAR expression on T cells selectively propagated upon CD19+ artificial antigen presenting cells. Given that T cells modified with SB100X and SB11 integrate on average one copy of the CAR transposon in each T-cell genome, the improved transposition mediated by SB100X apparently leads to an augmented founder effect of electroporated T cells with durable integration of CAR. In aggregate, SB100X improves SB transposition in primary human T cells and can be titrated with a SB transposon plasmid to improve the generation of CD19-specific CAR+ T cells. PMID:21451576

  7. Conserved amino acid motifs from the novel Piv/MooV family of transposases and site-specific recombinases are required for catalysis of DNA inversion by Piv.

    PubMed

    Tobiason, D M; Buchner, J M; Thiel, W H; Gernert, K M; Karls, A C

    2001-02-01

    Piv, a site-specific invertase from Moraxella lacunata, exhibits amino acid homology with the transposases of the IS110/IS492 family of insertion elements. The functions of conserved amino acid motifs that define this novel family of both transposases and site-specific recombinases (Piv/MooV family) were examined by mutagenesis of fully conserved amino acids within each motif in Piv. All Piv mutants altered in conserved residues were defective for in vivo inversion of the M. lacunata invertible DNA segment, but competent for in vivo binding to Piv DNA recognition sequences. Although the primary amino acid sequences of the Piv/MooV recombinases do not contain a conserved DDE motif, which defines the retroviral integrase/transposase (IN/Tnps) family, the predicted secondary structural elements of Piv align well with those of the IN/Tnps for which crystal structures have been determined. Molecular modelling of Piv based on these alignments predicts that E59, conserved as either E or D in the Piv/MooV family, forms a catalytic pocket with the conserved D9 and D101 residues. Analysis of Piv E59G confirms a role for E59 in catalysis of inversion. These results suggest that Piv and the related IS110/IS492 transposases mediate DNA recombination by a common mechanism involving a catalytic DED or DDD motif.

  8. THE HUMAN SET AND TRANSPOSASE DOMAIN PROTEIN METNASE INTERACTS WITH DNA LIGASE IV AND ENHANCES THE EFFICIENCY AND ACCURACY OF NON-HOMOLOGOUS END JOINING

    PubMed Central

    Hromas, Robert; Wray, Justin; Lee, Suk-Hee; Martinez, Leah; Farrington, Jacqueline; Corwin, Lori Kwan; Ramsey, Heather; Nickoloff, Jac A.; Williamson, Elizabeth A.

    2008-01-01

    Transposase domain proteins mediate DNA movement from one location in the genome to another in lower organisms. However, in human cells such DNA mobility would be deleterious, and therefore the vast majority of transposase-related sequences in humans are pseudogenes. We recently isolated and characterized a SET and Transposase domain protein termed Metnase that promotes DNA double strand break (DSB) repair by non-homologous end-joining (NHEJ). Both the SET and Transposase domain were required for its NHEJ activity. In this study we found that Metnase interacts with DNA Ligase IV, an important component of the classical NHEJ pathway. We investigated whether Metnase had structural requirements of the free DNA ends for NHEJ repair, and found that Metnase assists in joining all types of free DNA ends equally well. Metnase also prevents long deletions from processing of the free DNA ends, and improves the accuracy of NHEJ. Metnase levels correlate with the speed of disappearance of γ-H2Ax sites after ionizing radiation. However, Metnase has little effect on homologous recombination repair of a single DSB. Altogether, these results fit a model where Metnase plays a role in the fate of free DNA ends during NHEJ repair of DSBs. PMID:18773976

  9. The human set and transposase domain protein Metnase interacts with DNA Ligase IV and enhances the efficiency and accuracy of non-homologous end-joining.

    PubMed

    Hromas, Robert; Wray, Justin; Lee, Suk-Hee; Martinez, Leah; Farrington, Jacqueline; Corwin, Lori Kwan; Ramsey, Heather; Nickoloff, Jac A; Williamson, Elizabeth A

    2008-12-01

    Transposase domain proteins mediate DNA movement from one location in the genome to another in lower organisms. However, in human cells such DNA mobility would be deleterious, and therefore the vast majority of transposase-related sequences in humans are pseudogenes. We recently isolated and characterized a SET and transposase domain protein termed Metnase that promotes DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). Both the SET and transposase domain were required for its NHEJ activity. In this study we found that Metnase interacts with DNA Ligase IV, an important component of the classical NHEJ pathway. We investigated whether Metnase had structural requirements of the free DNA ends for NHEJ repair, and found that Metnase assists in joining all types of free DNA ends equally well. Metnase also prevents long deletions from processing of the free DNA ends, and improves the accuracy of NHEJ. Metnase levels correlate with the speed of disappearance of gamma-H2Ax sites after ionizing radiation. However, Metnase has little effect on homologous recombination repair of a single DSB. Altogether, these results fit a model where Metnase plays a role in the fate of free DNA ends during NHEJ repair of DSBs.

  10. Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats

    PubMed Central

    Jung, Chris J.; Ménoret, Séverine; Brusselle, Lucas; Tesson, Laurent; Usal, Claire; Chenouard, Vanessa; Remy, Séverine; Ouisse, Laure-Hélène; Poirier, Nicolas; Vanhove, Bernard; de Jong, Pieter J.; Anegon, Ignacio

    2016-01-01

    BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC transgenesis, resulting in complete BAC integration with predictable end sequences, thereby permitting precise assessment of the integration site. Neither CRISPR/Cas9 nor TALEN increased BAC transgenesis. Therefore, an efficient generation of human SIRPA transgenic rats using piggyBac opens opportunities for expansion of humanized transgenic rat models in the future to advance biomedical research and therapeutic applications. PMID:27530248

  11. A hyperactive piggyBac transposon system is an easy-to-implement method for introducing foreign genes into mouse preimplantation embryos.

    PubMed

    Suzuki, Shinnosuke; Tsukiyama, Tomoyuki; Kaneko, Takehito; Imai, Hiroshi; Minami, Naojiro

    2015-01-01

    Transgenic mice are important tools for genetic analysis. A current prominent method for producing transgenic mice involves pronuclear microinjection into 1-cell embryos. However, the total transgenic efficiency obtained using this method is less than 10%. Here, we demonstrate that highly efficient transgenesis in mice can be achieved by cytoplasmic microinjection using a hyperactive piggyBac system. In embryos in which hyPBase mRNA and pPB-CAG-TagRFP DNA were co-injected into the cytoplasm, TagRFP fluorescence was observed after the 2-cell stage; when 30 ng/µl pPB-CAG-TagRFP DNA and 30 ng/µl hyPBase mRNA were co-injected, 94.4% of blastocysts were TagRFP positive. Furthermore, a high concentration of hyPBase mRNA resulted in creation of mosaic embryos in which the TagRFP signals partially disappeared. However, suitable concentrations of injected DNA and hyPBase mRNA produced embryos in which almost all blastomeres were TagRFP positive. Thus, the hyperactive piggyBac transposon system is an easy-to-implement and highly effective method that can contribute to production of transgenic mice.

  12. Delivery of Full-Length Factor VIII Using a piggyBac Transposon Vector to Correct a Mouse Model of Hemophilia A

    PubMed Central

    Matsui, Hideto; Fujimoto, Naoko; Sasakawa, Noriko; Ohinata, Yasuhide; Shima, Midori; Yamanaka, Shinya; Sugimoto, Mitsuhiko; Hotta, Akitsu

    2014-01-01

    Viral vectors have been used for hemophilia A gene therapy. However, due to its large size, full-length Factor VIII (FVIII) cDNA has not been successfully delivered using conventional viral vectors. Moreover, viral vectors may pose safety risks, e.g., adverse immunological reactions or virus-mediated cytotoxicity. Here, we took advantages of the non-viral vector gene delivery system based on piggyBac DNA transposon to transfer the full-length FVIII cDNA, for the purpose of treating hemophilia A. We tested the efficiency of this new vector system in human 293T cells and iPS cells, and confirmed the expression of the full-length FVIII in culture media using activity-sensitive coagulation assays. Hydrodynamic injection of the piggyBac vectors into hemophilia A mice temporally treated with an immunosuppressant resulted in stable production of circulating FVIII for over 300 days without development of anti-FVIII antibodies. Furthermore, tail-clip assay revealed significant improvement of blood coagulation time in the treated mice.piggyBac transposon vectors can facilitate the long-term expression of therapeutic transgenes in vitro and in vivo. This novel gene transfer strategy should provide safe and efficient delivery of FVIII. PMID:25126862

  13. Phenotypic Screens Identify Parasite Genetic Factors Associated with Malarial Fever Response in Plasmodium falciparum piggyBac Mutants

    PubMed Central

    Thomas, Phaedra; Sedillo, Jennifer; Oberstaller, Jenna; Li, Suzanne; Zhang, Min; Singh, Naresh; Wang, Chengqi C. Q.; Udenze, Kenneth; Jiang, Rays H. Y.

    2016-01-01

    ABSTRACT Malaria remains one of the most devastating parasitic diseases worldwide, with 90% of the malaria deaths in Africa in 2013 attributable to Plasmodium falciparum. The clinical symptoms of malaria include cycles of fever, corresponding to parasite rupture from red blood cells every 48 h. Parasite pathways involved in the parasite’s ability to survive the host fever response, and indeed, the functions of ~40% of P. falciparum genes as a whole, are still largely unknown. Here, we evaluated the potential of scalable forward-genetic screening methods to identify genes involved in the host fever response. We performed a phenotypic screen for genes linked to the parasite response to febrile temperatures by utilizing a selection of single-disruption P. falciparum mutants generated via random piggyBac transposon mutagenesis in a previous study. We identified several mutants presenting significant phenotypes in febrile response screens compared to the wild type, indicating possible roles for the disrupted genes in this process. We present these initial studies as proof that forward genetics can be used to gain insight into critical factors associated with parasite biology. IMPORTANCE Though the P. falciparum genome sequence has been available for many years, ~40% of its genes do not have informative annotations, as they show no detectable homology to those of studied organisms. More still have not been evaluated via genetic methods. Scalable forward-genetic approaches that allow interrogation of gene function without any pre-existing knowledge are needed to hasten understanding of parasite biology, which will expedite the identification of drug targets and the development of future interventions in the face of spreading resistance to existing frontline drugs. In this work, we describe a new approach to pursue forward-genetic phenotypic screens for P. falciparum to identify factors associated with virulence. Future large-scale phenotypic screens developed to

  14. From Crop Domestication to Super-domestication

    PubMed Central

    Vaughan, D. A.; Balázs, E.; Heslop-Harrison, J. S.

    2007-01-01

    Research related to crop domestication has been transformed by technologies and discoveries in the genome sciences as well as information-related sciences that are providing new tools for bioinformatics and systems' biology. Rapid progress in archaeobotany and ethnobotany are also contributing new knowledge to understanding crop domestication. This sense of rapid progress is encapsulated in this Special Issue, which contains 18 papers by scientists in botanical, crop sciences and related disciplines on the topic of crop domestication. One paper focuses on current themes in the genetics of crop domestication across crops, whereas other papers have a crop or geographic focus. One feature of progress in the sciences related to crop domestication is the availability of well-characterized germplasm resources in the global network of genetic resources centres (genebanks). Germplasm in genebanks is providing research materials for understanding domestication as well as for plant breeding. In this review, we highlight current genetic themes related to crop domestication. Impressive progress in this field in recent years is transforming plant breeding into crop engineering to meet the human need for increased crop yield with the minimum environmental impact – we consider this to be ‘super-domestication’. While the time scale of domestication of 10 000 years or less is a very short evolutionary time span, the details emerging of what has happened and what is happening provide a window to see where domestication might – and can – advance in the future. PMID:17940074

  15. Domestic Violence - Multiple Languages

    MedlinePlus

    ... Are Here: Home → Multiple Languages → All Health Topics → Domestic Violence URL of this page: https://medlineplus.gov/languages/ ... V W XYZ List of All Topics All Domestic Violence - Multiple Languages To use the sharing features on ...

  16. Clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated mutagenesis and phenotype rescue by piggyBac transgenesis in a nonmodel Drosophila species.

    PubMed

    Tanaka, R; Murakami, H; Ote, M; Yamamoto, D

    2016-08-01

    How behavioural diversity emerged in evolution is an unexplored subject in biology. To tackle this problem, genes and circuits for a behaviour need to be determined in different species for phylogenetic comparisons. The recently developed clustered regulatory interspaced short palindromic repeats/CRISPR associated protein9 (CRISPR/Cas9) system made such a challenge possible by providing the means to induce mutations in a gene of interest in any organism. Aiming at elucidating diversification in genetic and neural networks for courtship behaviour, we attempted to generate a genetic tool kit in Drosophila subobscura, a nonmodel species distantly related to the genetic model Drosophila melanogaster. Here we report the generation of yellow (y) and white mutations with the aid of the CRISPR/Cas9 system, and the rescue of the y mutant phenotype by germline transformation of the newly established y mutant fly line with a y(+) -marked piggyBac vector. This successful mutagenesis and transformation in D. subobscura open up an avenue for comprehensive genetic analyses of higher functions in this and other nonmodel Drosophila species, representing a key step toward systematic comparisons of genes and circuitries underlying behaviour amongst species. © 2016 The Royal Entomological Society.

  17. Gene Transfer Efficiency and Genome-Wide Integration Profiling of Sleeping Beauty, Tol2, and PiggyBac Transposons in Human Primary T Cells

    PubMed Central

    Huang, Xin; Guo, Hongfeng; Tammana, Syam; Jung, Yong-Chul; Mellgren, Emil; Bassi, Preetinder; Cao, Qing; Tu, Zheng Jin; Kim, Yeong C; Ekker, Stephen C; Wu, Xiaolin; Wang, San Ming; Zhou, Xianzheng

    2010-01-01

    In this study, we compared the genomic integration efficiencies and transposition site preferences of Sleeping Beauty (SB or SB11), Tol2, and piggyBac (PB) transposon systems in primary T cells derived from peripheral blood lymphocytes (PBL) and umbilical cord blood (UCB). We found that PB demonstrated the highest efficiency of stable gene transfer in PBL-derived T cells, whereas SB11 and Tol2 mediated intermediate and lowest efficiencies, respectively. Southern hybridization analysis demonstrated that PB generated the highest number of integrants when compared to SB and Tol2 in both PBL and UCB T cells. Tol2 and PB appeared more likely to promote clonal expansion than SB, which may be in part due to the dysregulated expression of cancer-related genes near the insertion sites. Genome-wide integration analysis demonstrated that SB, Tol2, and PB integrations occurred in all the chromosomes without preference. Additionally, Tol2 and PB integration sites were mainly localized near transcriptional start sites (TSSs), CpG islands and DNaseI hypersensitive sites, whereas SB integrations were randomly distributed. These results suggest that SB may be a preferential choice of the delivery vector in T cells due to its random integration site preference and relatively high efficiency, and support continuing development of SB-mediated T-cell phase I trials. PMID:20606646

  18. Development of an optimized cytotoxicity assay system for CYP3A4-mediated metabolic activation via modified piggyBac transposition.

    PubMed

    Huang, Lizhen; Zou, Shuxiang; Deng, Jifeng; Dai, Tianming; Jiang, Jingwei; Jia, Ying; Dai, Renke; Xie, Shuilin

    2016-04-01

    Drug-induced hepatotoxicity is often caused by cytochrome P450 (CYP)-dependent metabolism of drugs into reactive metabolites. Assessment of hepatotoxicity induced by bioactive compounds is hampered by low CYP expression within in vitro cell lines. To overcome this limitation, piggyBac transposition and monoclonal expansion were used to generate a HepG2 cell line with stable and homogenously high expression of CYP3A4, a prominent CYP isoform. Our studies demonstrate the generated line's constant CYP3A4 expression and activity for over 40 cell passages; to date, it has been in subculture for more than a year without addition of Puromycin. This cell line was utilized to evaluate cytotoxicity of two bioactive (troglitazone and acetaminophen) and two non-bioactive (citrate and galactosamine) compounds by MTT assay. Cell viability significantly decreased upon treatment with bioactive drugs. Moreover, cell lines used in the present study were more sensitive to toxic effects of troglitazone than previously reported. Therefore, this HepG2 cell-based assay system may provide a suitable hepatic model for predicting CYP3A4-mediated hepatotoxicity during preclinical drug development.

  19. Combination of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 technique with the piggybac transposon system for mouse in utero electroporation to study cortical development.

    PubMed

    Cheng, Man; Jin, Xubin; Mu, Lili; Wang, Fangyu; Li, Wei; Zhong, Xiaoling; Liu, Xuan; Shen, Wenchen; Liu, Ying; Zhou, Yan

    2016-09-01

    In utero electroporation (IUE) is commonly used to study cortical development of cerebrum by downregulating or overexpressing genes of interest in neural progenitor cells (NPCs) of small mammals. However, exogenous plasmids are lost or diluted over time. Furthermore, gene knockdown based on short-hairpin RNAs may exert nonspecific effects that lead to aberrant neuronal migration. Genomic engineering by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has great research and therapeutic potentials. Here we integrate the CRISPR/Cas9 components into the piggyBac (PB) transposon system (the CRISPR/Cas9-PB toolkit) for cortical IUEs. The mouse Sry-related HMG box-2 (Sox2) gene was selected as the target for its application. Most transduced cortical NPCs were depleted of SOX2 protein as early as 3 days post-IUE, whereas expressions of SOX1 and PAX6 remained intact. Furthermore, both the WT Cas9 and the D10A nickase mutant Cas9n showed comparable knockout efficiency. Transduced cortical cells were purified with fluorescence-activated cell sorting, and effective gene editing at the Sox2 loci was confirmed. Thus, application of the CRISPR/Cas9-PB toolkit in IUE is a promising strategy to study gene functions in cortical NPCs and their progeny. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  20. PIF-like transposons are common in drosophila and have been repeatedly domesticated to generate new host genes.

    PubMed

    Casola, Claudio; Lawing, A Michelle; Betrán, Esther; Feschotte, Cédric

    2007-08-01

    The P instability factor or PIF superfamily of DNA transposons constitutes an important group of transposable elements (TEs) in plants, but it is still poorly characterized in metazoans. Taking advantage of the availability of draft genome sequences for twelve Drosophila species, we discovered 4 different lineages of Drosophila PIF-like transposons, named DPLT1-4. These lineages have experienced a complex evolutionary history during the Drosophila radiation, involving differential amplification and retention among species and probable events of horizontal transmission. Like previously described plant and animal PIF transposons, full-length DPLTs encode a putative transposase as well as a second predicted protein containing a Myb/SANT domain. In DPLTs, this domain is most closely related to the MADF DNA-binding domain found in several Drosophila transcription factors. In addition, we identified 7 distinct genes distributed across the Drosophila genus that encode proteins related to PIF transposases, but lack the hallmarks of transposons. Instead, these sequences show features of functional genes, such as an intact coding region evolving under purifying selection, the presence of orthologs in at least 2 Drosophila species, and the conservation of intron/exon structure across orthologs. We also provide evidence that most of these genes are transcribed and that some are developmentally regulated. Together the data indicate that these genes derived from PIF-transposons that have been "domesticated" to serve cellular functions. In one instance the recruitment of the transposase gene was accompanied by the co-recruitment of the adjacent second PIF gene, which raises the hypothesis that both proteins now function in the same pathway. The second PIF gene has retained the capacity to encode a protein with an intact MADF domain, suggesting that it may function as a transcription factor. We conclude that PIF transposons are common in the Drosophila lineage and have been a

  1. Domestic violence against men.

    PubMed

    Barber, Christopher F

    This article reviews the literature relating to domestic violence against men and examines some of the reasons why men are reluctant to report violent episodes. The article focuses on men as the victims and women as the perpetrators of domestic violence and identifies gaps in service provision. The role of the nurse in supporting male victims is also discussed.

  2. The Tn7 transposition regulator TnsC interacts with the transposase subunit TnsB and target selector TnsD

    PubMed Central

    Choi, Ki Young; Spencer, Jeanelle M.; Craig, Nancy L.

    2014-01-01

    The excision of transposon Tn7 from a donor site and its insertion into its preferred target site, attachment site attTn7, is mediated by four Tn7-encoded transposition proteins: TnsA, TnsB, TnsC, and TnsD. Transposition requires the assembly of a nucleoprotein complex containing all four Tns proteins and the DNA substrates, the donor site containing Tn7, and the preferred target site attTn7. TnsA and TnsB together form the heteromeric Tn7 transposase, and TnsD is a target-selecting protein that binds specifically to attTn7. TnsC is the key regulator of transposition, interacting with both the TnsAB transposase and TnsD-attTn7. We show here that TnsC interacts directly with TnsB, and identify the specific region of TnsC involved in the TnsB–TnsC interaction during transposition. We also show that a TnsC mutant defective in interaction with TnsB is defective for Tn7 transposition both in vitro and in vivo. Tn7 displays cis-acting target immunity, which blocks Tn7 insertion into a target DNA that already contains Tn7. We provide evidence that the direct TnsB–TnsC interaction that we have identified also mediates cis-acting Tn7 target immunity. We also show that TnsC interacts directly with the target selector protein TnsD. PMID:24982178

  3. Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System.

    PubMed

    Deniger, Drew C; Pasetto, Anna; Tran, Eric; Parkhurst, Maria R; Cohen, Cyrille J; Robbins, Paul F; Cooper, Laurence Jn; Rosenberg, Steven A

    2016-06-01

    Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAK(S2580F) or ERBB2(H473Y) or the HLA-DQB*0601-restricted neoantigen ERBB2IP(E805G) were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ(+) T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27(-)CD45RA(-)) and less-differentiated (CD27(+)CD45RA(+)) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens.

  4. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase.

    PubMed

    Podetz-Pedersen, Kelly M; Olson, Erik R; Somia, Nikunj V; Russell, Stephen J; McIvor, R Scott

    2016-01-19

    The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1-2.5 µg) conferring optimal levels of long-term expression (>10(11) photons/second/cm(2)). At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>10(10) photons/second/cm(2)) was achieved at a transposon dose of 5-125 μg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver.

  5. Stable, Nonviral Expression of Mutated Tumor Neoantigen-specific T-cell Receptors Using the Sleeping Beauty Transposon/Transposase System

    PubMed Central

    Deniger, Drew C; Pasetto, Anna; Tran, Eric; Parkhurst, Maria R; Cohen, Cyrille J; Robbins, Paul F; Cooper, Laurence JN; Rosenberg, Steven A

    2016-01-01

    Neoantigens unique to each patient's tumor can be recognized by autologous T cells through their T-cell receptor (TCR) but the low frequency and/or terminal differentiation of mutation-specific T cells in tumors can limit their utility as adoptive T-cell therapies. Transfer of TCR genes into younger T cells from peripheral blood with a high proliferative potential could obviate this problem. We generated a rapid, cost-effective strategy to genetically engineer cancer patient T cells with TCRs using the clinical Sleeping Beauty transposon/transposase system. Patient-specific TCRs reactive against HLA-A*0201-restriced neoantigens AHNAKS2580F or ERBB2H473Y or the HLA-DQB*0601-restricted neoantigen ERBB2IPE805G were assembled with murine constant chains and cloned into Sleeping Beauty transposons. Patient peripheral blood lymphocytes were coelectroporated with SB11 transposase and Sleeping Beauty transposon, and transposed T cells were enriched by sorting on murine TCRβ (mTCRβ) expression. Rapid expansion of mTCRβ+ T cells with irradiated allogeneic peripheral blood lymphocytes feeders, OKT3, interleukin-2 (IL-2), IL-15, and IL-21 resulted in a preponderance of effector (CD27−CD45RA−) and less-differentiated (CD27+CD45RA+) T cells. Transposed T cells specifically mounted a polyfunctional response against cognate mutated neoantigens and tumor cell lines. Thus, Sleeping Beauty transposition of mutation-specific TCRs can facilitate the use of personalized T-cell therapy targeting unique neoantigens. PMID:26945006

  6. A Broad Range of Dose Optima Achieve High-level, Long-term Gene Expression After Hydrodynamic Delivery of Sleeping Beauty Transposons Using Hyperactive SB100x Transposase

    PubMed Central

    Podetz-Pedersen, Kelly M; Olson, Erik R; Somia, Nikunj V; Russell, Stephen J; McIvor, R Scott

    2016-01-01

    The Sleeping Beauty (SB) transposon system has been shown to enable long-term gene expression by integrating new sequences into host cell chromosomes. We found that the recently reported SB100x hyperactive transposase conferred a surprisingly high level of long-term expression after hydrodynamic delivery of luciferase-encoding reporter transposons in the mouse. We conducted dose-ranging studies to determine the effect of varying the amount of SB100x transposase-encoding plasmid (pCMV-SB100x) at a set dose of luciferase transposon and of varying the amount of transposon-encoding DNA at a set dose of pCMV-SB100x in hydrodynamically injected mice. Animals were immunosuppressed using cyclophosphamide in order to prevent an antiluciferase immune response. At a set dose of transposon DNA (25 µg), we observed a broad range of pCMV-SB100x doses (0.1–2.5 µg) conferring optimal levels of long-term expression (>1011 photons/second/cm2). At a fixed dose of 0.5 μg of pCMV-SB100x, maximal long-term luciferase expression (>1010 photons/second/cm2) was achieved at a transposon dose of 5–125 μg. We also found that in the linear range of transposon doses (100 ng), co-delivering the CMV-SB100x sequence on the same plasmid was less effective in achieving long-term expression than delivery on separate plasmids. These results show marked flexibility in the doses of SB transposon plus pCMV-SB100x that achieve maximal SB-mediated gene transfer efficiency and long-term gene expression after hydrodynamic DNA delivery to mouse liver. PMID:26784638

  7. Tumor-directed gene therapy in mice using a composite nonviral gene delivery system consisting of the piggyBac transposon and polyethylenimine

    PubMed Central

    2009-01-01

    Background Compared with viral vectors, nonviral vectors are less immunogenic, more stable, safer and easier to replication for application in cancer gene therapy. However, nonviral gene delivery system has not been extensively used because of the low transfection efficiency and the short transgene expression, especially in vivo. It is desirable to develop a nonviral gene delivery system that can support stable genomic integration and persistent gene expression in vivo. Here, we used a composite nonviral gene delivery system consisting of the piggyBac (PB) transposon and polyethylenimine (PEI) for long-term transgene expression in mouse ovarian tumors. Methods A recombinant plasmid PB [Act-RFP, HSV-tk] encoding both the herpes simplex thymidine kinase (HSV-tk) and the monomeric red fluorescent protein (mRFP1) under PB transposon elements was constructed. This plasmid and the PBase plasmid were injected into ovarian cancer tumor xenografts in mice by in vivo PEI system. The antitumor effects of HSV-tk/ganciclovir (GCV) system were observed after intraperitoneal injection of GCV. Histological analysis and TUNEL assay were performed on the cryostat sections of the tumor tissue. Results Plasmid construction was confirmed by PCR analysis combined with restrictive enzyme digestion. mRFP1 expression could be visualized three weeks after the last transfection of pPB/TK under fluorescence microscopy. After GCV admission, the tumor volume of PB/TK group was significantly reduced and the tumor inhibitory rate was 81.96% contrasted against the 43.07% in the TK group. Histological analysis showed that there were extensive necrosis and lymphocytes infiltration in the tumor tissue of the PB/TK group but limited in the tissue of control group. TUNEL assays suggested that the transfected cells were undergoing apoptosis after GCV admission in vivo. Conclusion Our results show that the nonviral gene delivery system coupling PB transposon with PEI can be used as an efficient tool for

  8. Resistance mechanisms to TP53-MDM2 inhibition identified by in vivo piggyBac transposon mutagenesis screen in an Arf(-/-) mouse model.

    PubMed

    Chapeau, Emilie A; Gembarska, Agnieszka; Durand, Eric Y; Mandon, Emeline; Estadieu, Claire; Romanet, Vincent; Wiesmann, Marion; Tiedt, Ralph; Lehar, Joseph; de Weck, Antoine; Rad, Roland; Barys, Louise; Jeay, Sebastien; Ferretti, Stephane; Kauffmann, Audrey; Sutter, Esther; Grevot, Armelle; Moulin, Pierre; Murakami, Masato; Sellers, William R; Hofmann, Francesco; Jensen, Michael Rugaard

    2017-03-21

    Inhibitors of double minute 2 protein (MDM2)-tumor protein 53 (TP53) interaction are predicted to be effective in tumors in which the TP53 gene is wild type, by preventing TP53 protein degradation. One such setting is represented by the frequent CDKN2A deletion in human cancer that, through inactivation of p14ARF, activates MDM2 protein, which in turn degrades TP53 tumor suppressor. Here we used piggyBac (PB) transposon insertional mutagenesis to anticipate resistance mechanisms occurring during treatment with the MDM2-TP53 inhibitor HDM201. Constitutive PB mutagenesis in Arf(-/-) mice provided a collection of spontaneous tumors with characterized insertional genetic landscapes. Tumors were allografted in large cohorts of mice to assess the pharmacologic effects of HDM201. Sixteen out of 21 allograft models were sensitive to HDM201 but ultimately relapsed under treatment. A comparison of tumors with acquired resistance to HDM201 and untreated tumors identified 87 genes that were differentially and significantly targeted by the PB transposon. Resistant tumors displayed a complex clonality pattern suggesting the emergence of several resistant subclones. Among the most frequent alterations conferring resistance, we observed somatic and insertional loss-of-function mutations in transformation-related protein 53 (Trp53) in 54% of tumors and transposon-mediated gain-of-function alterations in B-cell lymphoma-extra large (Bcl-xL), Mdm4, and two TP53 family members, resulting in expression of the TP53 dominant negative truncations ΔNTrp63 and ΔNTrp73. Enhanced BCL-xL and MDM4 protein expression was confirmed in resistant tumors, as well as in HDM201-resistant patient-derived tumor xenografts. Interestingly, concomitant inhibition of MDM2 and BCL-xL demonstrated significant synergy in p53 wild-type cell lines in vitro. Collectively, our findings identify several potential mechanisms by which TP53 wild-type tumors may escape MDM2-targeted therapy.

  9. Domestic Communication Satellites

    ERIC Educational Resources Information Center

    Horowitz, Andrew

    1974-01-01

    A discussion of the Federal Communications Commission's new policy on domestic satellites in light of our 1) military and economic history; 2) corporate interests; 3) citizen surveillance; and 4) media control. (HB)

  10. Domestic Communication Satellites

    ERIC Educational Resources Information Center

    Horowitz, Andrew

    1974-01-01

    A discussion of the Federal Communications Commission's new policy on domestic satellites in light of our 1) military and economic history; 2) corporate interests; 3) citizen surveillance; and 4) media control. (HB)

  11. Domestic violence in America.

    PubMed

    Bash, K L; Jones, F

    1994-09-01

    Domestic violence is an underrecognized problem of immense cost. It is a crime; its victims must be identified and protected. The medical and judicial communities share responsibility in addressing this issue and providing support for victims. The role of health care workers in recognizing and preventing domestic violence cannot be overestimated. Direct questioning of patients, especially about the source of any injuries and about safety at home, is the first step in uncovering abuse. Educational programs for health care providers and the general public can change society's view and tolerance of this problem. Physicians must take an active role in changing community attitudes about domestic violence and in instituting programs to reduce its incidence. Medical treatment of the injuries resulting from domestic violence is not sufficient. Abused women need the care of a team of professionals who can address psychological, emotional, and physical injuries. They must also be provided with safe housing and financial and legal assistance in order to escape the abusive relationship. Physicians and legislators must work together to effect change. Domestic violence is a public health menace. We need to break the cycle of abuse that has become an integral part of our society.

  12. Efficient transformation of the yellow fever mosquito Aedes aegypti using the piggyBac transposable element vector pBac[3xP3-EGFP afm].

    PubMed

    Kokoza, V; Ahmed, A; Wimmer, E A; Raikhel, A S

    2001-11-01

    We report efficient germ-line transformation in the yellow fever mosquito Aedes aegypti accomplished using the piggyBac transposable element vector pBac[3xP3-EGFP afm]. Two transgenic lines were established and characterized; each contained the Vg-Defensin A transgene with strong eye-specific expression of the enhanced green fluorescent protein (EGFP) marker gene regulated by the artificial 3xP3 promoter. Southern blot hybridization and inverse PCR analyses of genomic DNA demonstrated a precise piggyBac-mediated, single copy insertion of the pBac[3xP3-EGFP afm,Vg-DefA] transposon in each transgenic line. For each line, genetic analysis confirmed stability and integrity of the entire transposon construct in the mosquito genome through the G2-G6 generations. Successful establishment of homozygous transgenic lines indicated that in both cases a non-lethal integration of the transposon into the mosquito genome had occurred. The 3xP3-EGFP marker was tested in mosquitoes with different genetic backgrounds. In white-eyed transgenic mosquitoes, the strong eye-specific expression of GFP was observed throughout all stages of development, starting from newly hatched first instar larvae to adults. A similar level and pattern of fluorescence was observed in red-eyed mosquitoes that were generated by crossing the 3xP3-EGFP transformants with the kh(w) white-eye mosquitoes transformed with the Drosophila cinnabar gene. Importantly, the utility of the 3xP3-EGFP, as marker gene for transformation of wild type mosquitoes, was demonstrated by strong eye-specific GFP expression in larval and pupal stages of black-eyed hybrids of the 3xP3-EGFP white-eye transformants and the wild type Rockefeller/UGAL strain. Finally, analysis of the Vg-DefA transgene expression in transformants from two established lines demonstrated strong blood-meal activation and fat-body-specific expression regulated by the Vg 1.8-kb 5' upstream region.

  13. Assay for transposase-accessible chromatin and circularized chromosome conformation capture, two methods to explore the regulatory landscapes of genes in zebrafish.

    PubMed

    Fernández-Miñán, A; Bessa, J; Tena, J J; Gómez-Skarmeta, J L

    2016-01-01

    Accurate transcriptional control of genes is fundamental for the correct functioning of organs and developmental processes. This control depends on the interplay between the promoter of genes and other noncoding sequences, whose interaction is mediated by 3D chromatin arrangements. Thus, the detailed description of transcriptional regulatory landscapes is essential to understand the mechanisms of transcriptional regulation. However, to achieve that, two important challenges have to be faced: (1) the identification of the noncoding sequences that contribute to gene transcription and (2) the association of these sequences to the respective genes they control. In this chapter, we describe two protocols that allow overcoming these important challenges: the assay for transposase-accessible chromatin using sequencing (ATAC-seq) and circularized chromosome conformation capture (4C-seq). ATAC-seq is a very efficient technique that, using a very low number of cells as starting material, allows the identification of active chromatin regions genome wide, whereas 4C-seq detects the subset of sequences that interact specifically with the promoter of a given gene. When combined, both techniques provide a comprehensive snapshot of the regulatory landscapes of developmental genes. The protocols we present here have been optimized for teleost fish samples, zebrafish and medaka, allowing the in-depth study of transcriptional regulation in these two emerging animal models. Given the amenability and easy genetic manipulation of these two experimental systems, we anticipate that they will be important in revealing general principles of the vertebrate regulatory genome.

  14. Nuclear translocation of Acinetobacter baumannii transposase induces DNA methylation of CpG regions in the promoters of E-cadherin gene.

    PubMed

    Moon, Dong Chan; Choi, Chul Hee; Lee, Su Man; Lee, Jung Hwa; Kim, Seung Il; Kim, Dong Sun; Lee, Je Chul

    2012-01-01

    Nuclear targeting of bacterial proteins has emerged as a pathogenic mechanism whereby bacterial proteins induce host cell pathology. In this study, we examined nuclear targeting of Acinetobacter baumannii transposase (Tnp) and subsequent epigenetic changes in host cells. Tnp of A. baumannii ATCC 17978 possesses nuclear localization signals (NLSs), (225)RKRKRK(230). Transient expression of A. baumannii Tnp fused with green fluorescent protein (GFP) resulted in the nuclear localization of these proteins in COS-7 cells, whereas the truncated Tnp without NLSs fused with GFP were exclusively localized in the cytoplasm. A. baumannii Tnp was found in outer membrane vesicles, which delivered this protein to the nucleus of host cells. Nuclear expression of A. baumannii Tnp fused with GFP in A549 cells induced DNA methylation of CpG regions in the promoters of E-cadherin (CDH1) gene, whereas the cytoplasmic localization of the truncated Tnp without NLSs fused with GFP did not induce DNA methylation. DNA methylation in the promoters of E-cadherin gene induced by nuclear targeting of A. baumannii Tnp resulted in down-regulation of gene expression. In conclusion, our data show that nuclear traffic of A. baumannii Tnp induces DNA methylation of CpG regions in the promoters of E-cadherin gene, which subsequently down-regulates gene expression. This study provides a new insight into the epigenetic control of host genes by bacterial proteins.

  15. Cloning of a CACTA transposon-like insertion in intron I of tomato invertase Lin5 gene and identification of transposase-like sequences of Solanaceae species.

    PubMed

    Proels, Reinhard K; Roitsch, Thomas

    2006-03-01

    Very few CACTA transposon-like sequences have been described in Solanaceae species. Sequence information has been restricted to partial transposase (TPase)-like fragments, and no target gene of CACTA-like transposon insertion has been described in tomato to date. In this manuscript, we report on a CACTA transposon-like insertion in intron I of tomato (Lycopersicon esculentum) invertase gene Lin5 and TPase-like sequences of several Solanaceae species. Consensus primers deduced from the TPase region of the tomato CACTA transposon-like element allowed the amplification of similar sequences from various Solanaceae species of different subfamilies including Solaneae (Solanum tuberosum), Cestreae (Nicotiana tabacum) and Datureae (Datura stramonium). This demonstrates the ubiquitous presence of CACTA-like elements in Solanaceae genomes. The obtained partial sequences are highly conserved, and allow further detection and detailed analysis of CACTA-like transposons throughout Solanaceae species. CACTA-like transposon sequences make possible the evaluation of their use for genome analysis, functional studies of genes and the evolutionary relationships between plant species.

  16. Domestic Research at Rand.

    ERIC Educational Resources Information Center

    Rand Corp., Santa Monica, CA.

    This third descriptive inventory of the Rand Corporation's domestic non-military research covers all projects since 1972, as well as its special 1969-1975 program for New York City. For each of over 300 research projects in nine program areas, brief summaries identify the project title and sponsor, beginning and ending dates, person-years of work…

  17. Domestic Research at Rand.

    ERIC Educational Resources Information Center

    Rand Corp., Santa Monica, CA.

    This third descriptive inventory of the Rand Corporation's domestic non-military research covers all projects since 1972, as well as its special 1969-1975 program for New York City. For each of over 300 research projects in nine program areas, brief summaries identify the project title and sponsor, beginning and ending dates, person-years of work…

  18. Domestic Communications Satellites.

    ERIC Educational Resources Information Center

    Network Project Notebook, 1972

    1972-01-01

    The June, 1972 Federal Communications Commission's (FCC) decision allowed an "open skies" policy in regard to domestic communication satellites and raised Liberal opposition to a situation where exclusive and unchecked communications power is now in the hands of private entrepreneurs, primarily the big Defense Department oriented…

  19. Challenging Domestic Violence.

    ERIC Educational Resources Information Center

    Scarlett, Chris

    2002-01-01

    In Britain, a Women's Aid program offers practical support and assistance to abused women. Survivors of domestic abuse can benefit from the opportunity afforded by an objective appraisal of the social context of their personal experiences, facilitated by trained volunteers. (JOW)

  20. Signs of domestic abuse.

    PubMed

    Pearce, Lynne

    2017-07-13

    Essential facts The government's definition of domestic violence and abuse, published in 2016 by the Home Office, is: 'Any incident or pattern of incidents of controlling, coercive, threatening behaviour, violence or abuse between those 16 years or over who are, or have been, intimate partners or family members, regardless of gender or sexuality.'

  1. Domestic Communications Satellites.

    ERIC Educational Resources Information Center

    Network Project Notebook, 1972

    1972-01-01

    The June, 1972 Federal Communications Commission's (FCC) decision allowed an "open skies" policy in regard to domestic communication satellites and raised Liberal opposition to a situation where exclusive and unchecked communications power is now in the hands of private entrepreneurs, primarily the big Defense Department oriented…

  2. Know Your Rights: Domestic Violence

    MedlinePlus

    ... of work • harassing telephone calls • spying on you • child abuse Domestic violence can be handled in three different ... or visitation privileges) Domestic violence might also involve child abuse and neglect and might impact on other areas ...

  3. Know Your Rights: Domestic Violence

    MedlinePlus

    ... of work • harassing telephone calls • spying on you • child abuse Domestic violence can be handled in three different ... or visitation privileges) Domestic violence might also involve child abuse and neglect and might impact on other areas ...

  4. Low-cost generation of Good Manufacturing Practice-grade CD19-specific chimeric antigen receptor-expressing T cells using piggyBac gene transfer and patient-derived materials.

    PubMed

    Ramanayake, Saumya; Bilmon, Ian; Bishop, David; Dubosq, Ming-Celine; Blyth, Emily; Clancy, Leighton; Gottlieb, David; Micklethwaite, Kenneth

    2015-09-01

    Protocols for the production of CD19-specific chimeric antigen receptor (CAR19) T cells are often complex and expensive because of the use of retroviral and lentiviral vectors or the need for CAR19 T-cell enrichment. We aimed to simplify the generation of CAR19 T cells from the peripheral blood of normal donors and patients using the piggyBac transposon system of gene modification. We varied electroporation voltage, cytokines and stimulation conditions for the generation and expansion of CAR19 T cells over a 3-week culture period. Using optimized electroporation voltage, interleukin-15 alone and co-culturing CAR T cells with peripheral blood mononuclear cells, we were able to expand CAR19 T-cell cultures by up to 765-fold over 3 weeks in normal donors and 180-fold in patients with B-cell malignancies. Final median CAR19 expression of 72% was seen in normal donors, and 81% was seen in patients with acute lymphoblastic leukaemia, chronic lymphocytic leukemia or non-Hodgkin lymphoma. CAR19 T cells produced interferon gamma on stimulation with CD19(+) cell lines and efficiently lysed both CD19(+) cell lines and primary leukemia cells. In addition, combining CAR expression with an inducible caspase safety switch allowed elimination of CAR19 T cells by the application of a small molecule dimerizer. We have produced a simple, inexpensive and easily adoptable protocol for the generation of CAR19 T cells suitable for use in clinical trials using the piggyBac transposon system. This provides a robust platform for further enhancing the T-cell product and testing new CAR technologies. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  5. Handbook for Domestic Violence Victims.

    ERIC Educational Resources Information Center

    Illinois Coalition Against Domestic Violence, Springfield.

    This handbook provides guidance for women in Illinois who are victims of domestic violence and spouse abuse. It consists of facts about domestic violence, a survival sheet telling what to do before, during, and after incidents of domestic violence, and advice on seeking emergency assistance and shelter. It then provides advice and resources on…

  6. Domestic fuelwood use in Louisiana

    Treesearch

    Victor A. Rudis

    1986-01-01

    A telephone survey of Louisiana households and commercial vendors of domestic fuelwood was conducted in 1984 to assess domestic fuelwood use and sources of production.Twenty-two percent of the households surveyed used fuelwood during the 1983-84 heating season.Domestic fuelwood production amounted to 981,000 m3, an amount comparable to 10% of Louisiana's 1982...

  7. Handbook for Domestic Violence Victims.

    ERIC Educational Resources Information Center

    Illinois Coalition Against Domestic Violence, Springfield.

    This handbook provides guidance for women in Illinois who are victims of domestic violence and spouse abuse. It consists of facts about domestic violence, a survival sheet telling what to do before, during, and after incidents of domestic violence, and advice on seeking emergency assistance and shelter. It then provides advice and resources on…

  8. Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine.

    PubMed

    Mancilla, Marcos; Grilló, María-Jesús; de Miguel, María-Jesús; López-Goñi, Ignacio; San-Román, Beatriz; Zabalza-Baranguá, Ana; Moriyón, Ignacio

    2013-10-31

    Brucella melitensis Rev 1 is the best vaccine available for the prophylaxis of small ruminant brucellosis and, indirectly, for reducing human brucellosis. However, Rev 1 shows anomalously high rates of spontaneous dissociation from smooth (S) to rough (R) bacteria, the latter being inefficacious as vaccines. This S-R instability results from the loss of the O-polysaccharide. To overcome this problem, we investigated whether some recently described mechanisms promoting mutations in O-polysaccharide genes were involved in Rev 1 S-R dissociation. We found that a proportion of Rev 1 R mutants result from genome rearrangements affecting the wbo O-polysaccharide loci of genomic island GI-2 and the wbkA O-polysaccharide glycosyltransferase gene of the wbk region. Accordingly, we mutated the GI-2 int gene and the wbk IS transposase involved in those arrangements, and found that these Rev 1 mutants maintained the S phenotype and showed lower dissociation levels. Combining these two mutations resulted in a strain (Rev 2) displaying a 95% decrease in dissociation with respect to parental Rev 1 under conditions promoting dissociation. Rev 2 did not differ from Rev 1 in the characteristics used in Rev 1 typing (growth rate, colonial size, reactivity with O-polysaccharide antibodies, phage, dye and antibiotic susceptibility). Moreover, Rev 2 and Rev 1 showed similar attenuation and afforded similar protection in the mouse model of brucellosis vaccines. We conclude that mutations targeting genes and DNA sequences involved in spontaneous O-polysaccharide loss enhance the stability of a critical vaccine phenotype and complement the empirical stabilization precautions taken during S Brucella vaccine production.

  9. [Domestic violence in Chile].

    PubMed

    León, Tomás; Grez, Marcela; Prato, Juan Andrés; Torres, Rafael; Ruiz, Sergio

    2014-08-01

    According to recent surveys, there is a high prevalence of domestic violence (DV) in Chile. A systematic review was conducted in PubMed, Scielo, and Lilacs with the MesH terms "Chile", "Mental Health", "Health", "Domestic Violence", to explore the impact of DV on health in Chile. Eleven studies fulfilled the inclusion criteria. Two studies were prospective, exploring the influence of DV on maternal-infant health. Nine studies explored the influence of DV on mental health in adults. DV was associated with deranged mental health indicators specially anxiety and depressive symptoms and suicidal ideation. Similar results were observed among mothers who were victims of violence and their children. It is concluded that DV is a complex phenomenon with serious effects on health. However the number of studies on the subject is low and new follow up studies are required. Predictive models for DV and effective preventive measures are urgently needed.

  10. Satellite Surveillance: Domestic Issues

    DTIC Science & Technology

    2008-06-27

    Council in Los Angeles, California, July 25, 1996. 9 Robert B Murrett, “NGA — Then and Now; Celebrating 10 Years of GEOINT,” Pathfinder , September/October...Domestic Spy Satellites before the House Committee on Homeland Security, September 6, 2007; also, Statement of Lisa Graves, Deputy Director of the Center...United States House of Representatives, September 1, 2007. 24 Lisa Graves, Deputy Director of the Center for National Security Studies, Statement before

  11. Dermatophytoses in domesticated animals.

    PubMed

    Nweze, Emeka I

    2011-01-01

    Dermatophytes are among the most frequent causes of ringworm infections in domesticated animals. They are known to serve as reservoirs of the zoophilic dermatophytes and these infections have important zoonotic implication. In Nigeria and probably West Africa, there are not many studies on the incidence of dermatophytosis in domesticated animals. In the current study, 538 domesticated animals with clinically suggestive lesions were investigated for dermatophytes. Identification of dermatophyte species was performed by macro- and micro morphological examination of colonies and by biochemical methods. In the cases of isolates that had atypical morphology and/or biochemical test results, the rDNA internal transcribed spacer region 2 (ITS 2) sequencing was performed. Out of this number, 214 (39.8%) were found to be colonized by a variety of ten species of dermatophytes. M. canis was the most frequently isolated species (37.4%), followed by T. mentagrophytes (22.9%) and T. verrucosum (15.9%). M. persicolor and T. gallinae were jointly the least species isolated with a frequency of 0.55% respectively. The recovery of dermatophyte isolates previously shown to be common etiological agents of dermatophytosis especially from children in the same region suggests that animal to human transmission may be common. Possible implications and recommendations are discussed.

  12. Genetic perspectives on crop domestication

    PubMed Central

    Gross, Briana L.; Olsen, Kenneth M.

    2010-01-01

    The process of crop domestication has long been a topic of active research for biologists, anthropologists and others. Genetic data have proved a powerful resource for drawing inferences on questions regarding the geographical origins of crops, the numbers of independent domestication events for a given crop species, the specific molecular changes underlying domestication traits, and the nature of artificial selection during domestication and subsequent crop improvement. We would argue that these genetic inferences are fundamentally compatible with recent archaeological data that support a view of domestication as a geographically diffuse, gradual process. In this review, we summarize methodologies ranging from QTL mapping to resequencing used in genetic analyses of crop evolution. We also highlight recent major insights regarding the timing and spatial patterning of crop domestication and the distinct genetic underpinnings of domestication, diversification, and improvement traits. PMID:20541451

  13. Animal evolution during domestication: the domesticated fox as a model.

    PubMed

    Trut, Lyudmila; Oskina, Irina; Kharlamova, Anastasiya

    2009-03-01

    We review the evolution of domestic animals, emphasizing the effect of the earliest steps of domestication on its course. Using the first domesticated species, the dog (Canis familiaris), for illustration, we describe the evolutionary peculiarities during the historical domestication, such as the high level and wide range of diversity. We suggest that the process of earliest domestication via unconscious and later conscious selection of human-defined behavioral traits may accelerate phenotypic variations. The review is based on the results of a long-term experiment designed to reproduce early mammalian domestication in the silver fox (Vulpes vulpes) selected for tameability or amenability to domestication. We describe changes in behavior, morphology and physiology that appeared in the fox during its selection for tameability, which were similar to those observed in the domestic dog. Based on the data of the fox experiment and survey of relevant data, we discuss the developmental, genetic and possible molecular genetic mechanisms underlying these changes. We ascribe the causative role in evolutionary transformation of domestic animals to the selection for behavior and to the neurospecific regulatory genes it affects.

  14. Diverse faces of domestic violence.

    PubMed

    Anderson, Tanya R; Aviles, Ann M

    2006-01-01

    Domestic violence is one of the most common causes of serious injury among women. Domestic violence victims endure physical and psychological sequelae that often go undetected by the health care professionals they encounter. There are many barriers women who are victims of domestic violence face. Women of color encounter additional barriers such as stereotypes that construct domestic violence as a "minority" issue. This article surveys the relevant literature to provide the reader with a review of the current state of knowledge for this special sub-population of domestic violence victims. Health care professionals need to be aware of the issues of this sub-population and be appropriately educated and trained to actively screen them. In addition, health care professionals need to be culturally sensitive to the needs of women of color who may experience domestic violence in order to appropriately screen and refer women for services that meet their needs.

  15. Selection signature in domesticated animals.

    PubMed

    Zhangyuan, Pan; Xiaoyun, He; Xiangyu, Wang; Xiaofei, Guo; Xiaohan, Cao; Wenping, Hu; Ran, Di; Qiuyue, Liu; Mingxing, Chu

    2016-12-20

    Domesticated animals play an important role in the life of humanity. All these domesticated animals undergo same process, first domesticated from wild animals, then after long time natural and artificial selection, formed various breeds that adapted to the local environment and human needs. In this process, domestication, natural and artificial selection will leave the selection signal in the genome. The research on these selection signals can find functional genes directly, is one of the most important strategies in screening functional genes. The current studies of selection signal have been performed in pigs, chickens, cattle, sheep, goats, dogs and other domestic animals, and found a great deal of functional genes. This paper provided an overview of the types and the detected methods of selection signal, and outlined researches of selection signal in domestic animals, and discussed the key issues in selection signal analysis and its prospects.

  16. Core questions in domestication research

    PubMed Central

    Zeder, Melinda A.

    2015-01-01

    The domestication of plants and animals is a key transition in human history, and its profound and continuing impacts are the focus of a broad range of transdisciplinary research spanning the physical, biological, and social sciences. Three central aspects of domestication that cut across and unify this diverse array of research perspectives are addressed here. Domestication is defined as a distinctive coevolutionary, mutualistic relationship between domesticator and domesticate and distinguished from related but ultimately different processes of resource management and agriculture. The relative utility of genetic, phenotypic, plastic, and contextual markers of evolving domesticatory relationships is discussed. Causal factors are considered, and two leading explanatory frameworks for initial domestication of plants and animals, one grounded in optimal foraging theory and the other in niche-construction theory, are compared. PMID:25713127

  17. Domestic and Domesticating Education in the Late Victorian City

    ERIC Educational Resources Information Center

    Heggie, Vanessa

    2011-01-01

    This article explores the various types of domestic education, particularly cookery, available in Manchester between 1870 and 1902. The work of the two local School Boards and the Manchester School of Domestic Economy are shown as part of a complicated network of provision--a mixed economy of welfare, including enthusiastic philanthropists and…

  18. Domestic and Domesticating Education in the Late Victorian City

    ERIC Educational Resources Information Center

    Heggie, Vanessa

    2011-01-01

    This article explores the various types of domestic education, particularly cookery, available in Manchester between 1870 and 1902. The work of the two local School Boards and the Manchester School of Domestic Economy are shown as part of a complicated network of provision--a mixed economy of welfare, including enthusiastic philanthropists and…

  19. [Domestic violence: any progress?].

    PubMed

    Henrion, Roger

    2014-01-01

    Since the publication of the French national survey of violence against women in 2000, the fight against domestic violence has made steady progress. Knowledge of the phenomenon has significantly improved. A nationwide study of murders and manslaughters perpetrated by one partner of a couple against the other has been published annually since 2006. In 2012, domestic violence resulted in the deaths of 314 persons: 166 women, 31 men, 25 children, 9 collateral victims, 14 rivals, and two former spouses killed by their ex-fathers in law. In addition, 67 perpetrators committed suicide (51 men and3 women). The number of victims fluctuates from year to year but has remained fairly stable since 2006 (n=168). Legislation has improved significantly: eight new laws have been passed since 2004, all designed to protect women and to ensure that violent men are restrained and treated. New measures to inform and protect women have been implemented and others have been improved, such as the anonymous helpline (phone no 3919, "domestic violence information"). An inter-ministerial committee on the protection of women from violence and the prevention of human trafficking (MIPROF) was created on 3 January 2013. A website entitled "Stop violence against women " (Stop violences faites aux femmes) is now available. The "Imminent Danger" mobile phone system, designed to alert police if a suspected or known perpetrator breaches restraint conditions, will be extended to the entire country from January 2014. Referees charged with coordinating comprehensive long-tern care of women victims have been deployed at the county level. Information centers on the rights of women and families (CIDFF) now form a local nationwide network. Routine interviews with a midwife during the fourth month of pregnancy, focusing on the woman's emotional, economic and social conditions, have been implemented in 21 % of maternity units and should gradually be generalized. The authorities who have enforced the law have

  20. The Complexities of Domestic Violence

    ERIC Educational Resources Information Center

    Dutton, Donald G.

    2007-01-01

    Comments on the article by Robert Bornstein, "The complex relationship between dependency and domestic violence: Converging psychological factors and social forces." Although a more focused examination of the psychological factors involved in domestic violence is welcome, there are some factual errors in Bornstein's article that need attention and…

  1. The Complexities of Domestic Violence

    ERIC Educational Resources Information Center

    Dutton, Donald G.

    2007-01-01

    Comments on the article by Robert Bornstein, "The complex relationship between dependency and domestic violence: Converging psychological factors and social forces." Although a more focused examination of the psychological factors involved in domestic violence is welcome, there are some factual errors in Bornstein's article that need attention and…

  2. Plant domestication slows pest evolution.

    PubMed

    Turcotte, Martin M; Lochab, Amaneet K; Turley, Nash E; Johnson, Marc T J

    2015-09-01

    Agricultural practices such as breeding resistant varieties and pesticide use can cause rapid evolution of pest species, but it remains unknown how plant domestication itself impacts pest contemporary evolution. Using experimental evolution on a comparative phylogenetic scale, we compared the evolutionary dynamics of a globally important economic pest - the green peach aphid (Myzus persicae) - growing on 34 plant taxa, represented by 17 crop species and their wild relatives. Domestication slowed aphid evolution by 13.5%, maintained 10.4% greater aphid genotypic diversity and 5.6% higher genotypic richness. The direction of evolution (i.e. which genotypes increased in frequency) differed among independent domestication events but was correlated with specific plant traits. Individual-based simulation models suggested that domestication affects aphid evolution directly by reducing the strength of selection and indirectly by increasing aphid density and thus weakening genetic drift. Our results suggest that phenotypic changes during domestication can alter pest evolutionary dynamics.

  3. The piggyBac transposon-derived genes TPB1 and TPB6 mediate essential transposon-like excision during the developmental rearrangement of key genes in Tetrahymena thermophila.

    PubMed

    Cheng, Chao-Yin; Young, Janet M; Lin, Chih-Yi Gabriela; Chao, Ju-Lan; Malik, Harmit S; Yao, Meng-Chao

    2016-12-15

    Ciliated protozoans perform extreme forms of programmed somatic DNA rearrangement during development. The model ciliate Tetrahymena thermophila removes 34% of its germline micronuclear genome from somatic macronuclei by excising thousands of internal eliminated sequences (IESs), a process that shares features with transposon excision. Indeed, piggyBac transposon-derived genes are necessary for genome-wide IES excision in both Tetrahymena (TPB2 [Tetrahymena piggyBac-like 2] and LIA5) and Paramecium tetraurelia (PiggyMac). T. thermophila has at least three other piggyBac-derived genes: TPB1, TPB6, and TPB7 Here, we show that TPB1 and TPB6 excise a small, distinct set of 12 unusual IESs that disrupt exons. TPB1-deficient cells complete mating, but their progeny exhibit slow growth, giant vacuoles, and osmotic shock sensitivity due to retention of an IES in the vacuolar gene DOP1 (Dopey domain-containing protein). Unlike most IESs, TPB1-dependent IESs have piggyBac-like terminal inverted motifs that are necessary for excision. Transposon-like excision mediated by TPB1 and TPB6 provides direct evidence for a transposon origin of not only IES excision machinery but also IESs themselves. Our study highlights a division of labor among ciliate piggyBac-derived genes, which carry out mutually exclusive categories of excision events mediated by either transposon-like features or RNA-directed heterochromatin. © 2016 Cheng et al.; Published by Cold Spring Harbor Laboratory Press.

  4. Isolation and characterization of lymphoid enhancer factor-1-positive deciduous dental pulp stem-like cells after transfection with a piggyBac vector containing LEF1 promoter-driven selection markers.

    PubMed

    Murakami, Tomoya; Saitoh, Issei; Sato, Masahiro; Inada, Emi; Soda, Miki; Oda, Masataka; Domon, Hisanori; Iwase, Yoko; Sawami, Tadashi; Matsueda, Kazunari; Terao, Yutaka; Ohshima, Hayato; Noguchi, Hirofumi; Hayasaki, Haruaki

    2017-09-01

    Lymphoid enhancer-binding factor-1 (LEF1) is a 48-kD nuclear protein that is expressed in pre-B and T cells. LEF1 is also an important member of the Wnt/β-catenin signaling pathway that plays important roles in the self-renewal and differentiation of embryonic stem cells. We speculated that LEF1 might function in the stem cells from human exfoliated deciduous teeth (SHED). In this study, we attempted to isolate such LEF1-positive cells from human deciduous dental pulp cells (HDDPCs) by genetic engineering technology, using the human LEF1 promoter. A piggyBac transposon plasmid (pTA-LEN) was introduced into HDDPCs, using the Neon(®) transfection system. After G418 selection, the emerging colonies were assessed for EGFP-derived fluorescence by fluorescence microscopy. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using RNA isolated from these colonies to examine stem cell-specific transcript expression. Osteoblastic or neuronal differentiation was induced by cultivating the LEF1-positive cells with differentiation-inducing medium. RT-PCR analysis confirmed the expression of several stem cell markers, including OCT3/4, SOX2, REX1, and NANOG, in LEF1-positive HDDPCs, which could be differentiated into osteoblasts and neuronal cells. The isolated LEF1-positive HDDPCs exhibited the properties of stem cells, suggesting that LEF1 might serve as a marker for SHED. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  5. Thermal biology of domestic animals.

    PubMed

    Collier, Robert J; Gebremedhin, Kifle G

    2015-01-01

    The thermal environment is the most important ecological factor determining the growth, development, and productivity of domestic animals. Routes of energy exchange (sensible heat and latent heat) between animals and their environment are greatly influenced by body weight, fat deposition, hair-coat properties, functional activity, and number of sweat glands, as well as the presence or absence of anatomical respiratory countercurrent heat exchange capability. Differences in these anatomical features across species have led to specialization of heat exchange. Thermal plasticity and degree of acclimation are critical factors determining the ability of animals to respond to environmental change. Increases in productive capability of domestic animals can compromise thermal acclimation and plasticity, requiring greater investments in housing systems that reduce variability of the thermal environment. The combination of steadily increasing metabolic heat production as domestic animal productivity increases and a rising world temperature poses ongoing and future challenges to maintaining health and well-being of domestic animals.

  6. How to make a domesticate.

    PubMed

    Stetter, Markus G; Gates, Daniel J; Mei, Wenbin; Ross-Ibarra, Jeffrey

    2017-09-11

    The Neolithic Revolution brought about the transition from hunting and gathering to sedentary societies, laying the foundation for the development of modern civilizations. The primary innovation that facilitated these changes was the domestication of plants and animals. In the case of plants, this involved the cultivation and selection of individuals with larger edible parts, easier harvesting, and decreased defenses, traits that allowed for the production of a food surplus and occupational specialization. Plant domestication is a process which started approximately 10,000 years ago and has thereafter been repeated independently in many locales around the world. Here, we offer a perspective that seeks to predict what factors influence the success of domestication, how many genes contributed to the process, where these genes originated and the implications for de novo domestication. Published by Elsevier Ltd.

  7. [Mycoses in domestic animals].

    PubMed

    García, M E; Blanco, J L

    2000-03-01

    In the present paper we will present a general view of the main mycoses affecting domestic animals. In the dog, we show the importance of the dermatophytoses, increased by its zoonosic character and the problem of the false negatives in the traditional microbiological culture. Under the general term of systemic mycoses we include a series of conditions considered usually as aspergillosis, bat with more and more fungal species implicated as possible etiological agents. In addition, fungi, especially yeasts, are being implicated in canine otitis; in our laboratory 86 % of canine chronic otitis involve a yeast etiology, alone or in collaboration with bacteria. In the cat, dermatophytes are more common than in the dog, and are the main source of infection in man, with the description of a high percentage of healthy carrier animals. Cryptococcosis is a severe disease, usually secondary to other process, especially feline immunodeficiency. In cows we refer to fungal abortion, with three main fungi implicated: Aspergillus, Candida and Zygomycetes. In some areas of our country the percentage of fungal abortion is around 10 %. A consequence of the multiple use of antibiotics in mastitis is selection of yeasts, especially those included in the genera Candida and Cryptococcus. Bovine dermatophytoses is an extensively disseminated disease in our country, with a commercial specific vaccine available. In small ruminants, Cryptococcus causes severe pneumonic processes that could be confused clinically with other conditions. An additional important question is the description of isolation of this fungus from tree leaves. In poultry, aspergillosis is a known and controlled disease, but with more importance in captive wild birds with an ecological value. In horses, we emphasize the lung infections by different fungi, specially Pneumocystis carinii, and arthritis by yeasts as consequence of wound contamination or surgery.

  8. Mutualism breakdown in breadfruit domestication

    PubMed Central

    Xing, Xiaoke; Koch, Alexander M.; Jones, A. Maxwell P.; Ragone, Diane; Murch, Susan; Hart, Miranda M.

    2012-01-01

    During the process of plant domestication, below-ground communities are rarely considered. Some studies have attempted to understand the changes in root symbionts owing to domestication, but little is known about how it influences mycorrhizal response in domesticated crops. We hypothesized that selection for above-ground traits may also result in decreased mycorrhizal abundance in roots. Breadfruit (Artocarpus sp.) has a long domestication history, with a strong geographical movement of cultivars from west to east across the Melanesian and Polynesian islands. Our results clearly show a decrease in arbuscular mycorrhizas (AMs) along a domestication gradient from wild to recently derived cultivars. We showed that the vesicular and arbuscular colonization rate decreased significantly in more recently derived breadfruit cultivars. In addition, molecular analyses of breadfruit roots indicated that AM fungal species richness also responded along the domestication gradient. These results suggest that human-driven selection for plant cultivars can have unintended effects on below-ground mutualists, with potential impacts on the stress tolerance of crops and long-term food security. PMID:21920983

  9. Mutualism breakdown in breadfruit domestication.

    PubMed

    Xing, Xiaoke; Koch, Alexander M; Jones, A Maxwell P; Ragone, Diane; Murch, Susan; Hart, Miranda M

    2012-03-22

    During the process of plant domestication, below-ground communities are rarely considered. Some studies have attempted to understand the changes in root symbionts owing to domestication, but little is known about how it influences mycorrhizal response in domesticated crops. We hypothesized that selection for above-ground traits may also result in decreased mycorrhizal abundance in roots. Breadfruit (Artocarpus sp.) has a long domestication history, with a strong geographical movement of cultivars from west to east across the Melanesian and Polynesian islands. Our results clearly show a decrease in arbuscular mycorrhizas (AMs) along a domestication gradient from wild to recently derived cultivars. We showed that the vesicular and arbuscular colonization rate decreased significantly in more recently derived breadfruit cultivars. In addition, molecular analyses of breadfruit roots indicated that AM fungal species richness also responded along the domestication gradient. These results suggest that human-driven selection for plant cultivars can have unintended effects on below-ground mutualists, with potential impacts on the stress tolerance of crops and long-term food security.

  10. Domestic violence: an educational imperative?

    PubMed

    Chambliss, L R; Bay, R C; Jones, R F

    1995-03-01

    In the US, domestic violence is the most common cause of injury to women, and, because they are women's primary care physicians, obstetrician-gynecologists (OB-GYNs) are in a position to identify battered women. With legislation pending that would require federally-funded medical education to include domestic violence in its curriculum, a survey was conducted of all OB-GYN residencies to determine their current status in the provision of domestic violence education. With 83% of the programs responding (n = 264), it was found that 28% had at least one faculty member with expertise in domestic violence, that the incidence of domestic violence among clients was underestimated, and that 75% of respondents did not recognize at least 1 of the 10 common clinical scenarios as suggestive of battering (at least 44% failed to recognize the risk factors of no prenatal care, preterm labor, emergency room visits, and psychiatric diagnoses). Only 28% of respondents reported that they teach residents to ask all or almost all patients about battering. Most respondents expressed dissatisfaction with their coverage of this topic and asked for help in curriculum development. In addition, 40% were unaware of the pending federal legislation. This study concluded that there is a strong need to develop an OB-GYN curriculum that deals with domestic violence but that further study is needed to determine the optimal teaching methods.

  11. The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

    PubMed

    Arnaiz, Olivier; Mathy, Nathalie; Baudry, Céline; Malinsky, Sophie; Aury, Jean-Marc; Denby Wilkes, Cyril; Garnier, Olivier; Labadie, Karine; Lauderdale, Benjamin E; Le Mouël, Anne; Marmignon, Antoine; Nowacki, Mariusz; Poulain, Julie; Prajer, Malgorzata; Wincker, Patrick; Meyer, Eric; Duharcourt, Sandra; Duret, Laurent; Bétermier, Mireille; Sperling, Linda

    2012-01-01

    Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the

  12. The Paramecium Germline Genome Provides a Niche for Intragenic Parasitic DNA: Evolutionary Dynamics of Internal Eliminated Sequences

    PubMed Central

    Arnaiz, Olivier; Mathy, Nathalie; Baudry, Céline; Malinsky, Sophie; Aury, Jean-Marc; Denby Wilkes, Cyril; Garnier, Olivier; Labadie, Karine; Lauderdale, Benjamin E.; Le Mouël, Anne; Marmignon, Antoine; Nowacki, Mariusz; Poulain, Julie; Prajer, Malgorzata; Wincker, Patrick; Meyer, Eric; Duharcourt, Sandra; Duret, Laurent; Bétermier, Mireille; Sperling, Linda

    2012-01-01

    Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of ∼45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a ∼10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the

  13. 31 CFR 500.320 - Domestic bank.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Domestic bank. 500.320 Section 500... § 500.320 Domestic bank. The term domestic bank shall mean any branch or office within the United States... Secretary of the Treasury may also authorize any other banking institution to be treated as a “domestic...

  14. 31 CFR 515.320 - Domestic bank.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Domestic bank. 515.320 Section 515.320... Domestic bank. The term domestic bank shall mean any branch or office within the United States of any of... Treasury may also authorize any other banking institution to be treated as a “domestic bank” for...

  15. 37 CFR 3.61 - Domestic representative.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2010-07-01 2010-07-01 false Domestic representative. 3.61... COMMERCE GENERAL ASSIGNMENT, RECORDING AND RIGHTS OF ASSIGNEE Domestic Representative § 3.61 Domestic... is not domiciled in the United States, the assignee may designate a domestic representative in...

  16. 31 CFR 535.320 - Domestic bank.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 31 Money and Finance:Treasury 3 2011-07-01 2011-07-01 false Domestic bank. 535.320 Section 535.320....320 Domestic bank. (a) The term domestic bank shall mean any branch or office within the United States... Treasury may also authorize any other banking institution to be treated as a “domestic bank” for...

  17. 31 CFR 535.320 - Domestic bank.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Domestic bank. 535.320 Section 535... § 535.320 Domestic bank. (a) The term domestic bank shall mean any branch or office within the United... Treasury may also authorize any other banking institution to be treated as a “domestic bank” for...

  18. 31 CFR 515.320 - Domestic bank.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Domestic bank. 515.320 Section 515... § 515.320 Domestic bank. The term domestic bank shall mean any branch or office within the United States... Secretary of the Treasury may also authorize any other banking institution to be treated as a “domestic...

  19. 37 CFR 3.61 - Domestic representative.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 37 Patents, Trademarks, and Copyrights 1 2011-07-01 2011-07-01 false Domestic representative. 3.61... COMMERCE GENERAL ASSIGNMENT, RECORDING AND RIGHTS OF ASSIGNEE Domestic Representative § 3.61 Domestic... is not domiciled in the United States, the assignee may designate a domestic representative in...

  20. Child Domestic Work. Innocenti Digest 5.

    ERIC Educational Resources Information Center

    Black, Maggie, Comp.; Blagbrough, Jonathan, Comp.

    Child "domestics" or "domestic workers" are defined here as children under the age of 18 who work in other people's households, doing domestic chores, caring for children and running errands, among other tasks. This digest focuses mainly on the situation of live-in child domestics, that is, children who work full time in…

  1. Child Domestic Work. Innocenti Digest 5.

    ERIC Educational Resources Information Center

    Black, Maggie, Comp.; Blagbrough, Jonathan, Comp.

    Child "domestics" or "domestic workers" are defined here as children under the age of 18 who work in other people's households, doing domestic chores, caring for children and running errands, among other tasks. This digest focuses mainly on the situation of live-in child domestics, that is, children who work full time in…

  2. Multi-perpetrator domestic violence.

    PubMed

    Salter, Michael

    2014-04-01

    A significant proportion of reports of domestic violence against women involve multiple perpetrators. Although the number of perpetrators has been consistently identified as a measure of abuse severity, only a minority of studies of domestic violence examine the role of multiple offenders. Data on multi-perpetrator domestic violence (MDV) is frequently removed from analysis in domestic violence studies, or multi-perpetrator incidents are treated as single-perpetrator incidents. However, the available research links MDV to negative mental and physical health outcomes, intimate partner homicide, homelessness among women, and severe mental illness and suicidality. This article reviews the available prevalence data on MDV and draws together research on the contexts in which MDV takes place. It highlights two groups that are particularly vulnerable to MDV: (1) girls and women partnered to members of gangs and organized crime groups and (2) girls and women in some ethnic minority communities. While discussions of honor in relation to domestic violence are often racialized in Western media, this article highlights the cross-cultural role of masculine honor in collective violence against women in the working class and impoverished communities of majority cultures as well as in migrant and ethnic minority communities. It is clear that such complex forms of violence present a range of challenges for intervention and treatment and the article emphasizes the need for specialized and coordinated modes of investigation, support, and care.

  3. PiggyBac transposon-mediated gene delivery efficiently generates stable transfectants derived from cultured primary human deciduous tooth dental pulp cells (HDDPCs) and HDDPC-derived iPS cells

    PubMed Central

    Inada, Emi; Saitoh, Issei; Watanabe, Satoshi; Aoki, Reiji; Miura, Hiromi; Ohtsuka, Masato; Murakami, Tomoya; Sawami, Tadashi; Yamasaki, Youichi; Sato, Masahiro

    2015-01-01

    The ability of human deciduous tooth dental pulp cells (HDDPCs) to differentiate into odontoblasts that generate mineralized tissue holds immense potential for therapeutic use in the field of tooth regenerative medicine. Realization of this potential depends on efficient and optimized protocols for the genetic manipulation of HDDPCs. In this study, we demonstrate the use of a PiggyBac (PB)-based gene transfer system as a method for introducing nonviral transposon DNA into HDDPCs and HDDPC-derived inducible pluripotent stem cells. The transfection efficiency of the PB-based system was significantly greater than previously reported for electroporation-based transfection of plasmid DNA. Using the neomycin resistance gene as a selection marker, HDDPCs were stably transfected at a rate nearly 40-fold higher than that achieved using conventional methods. Using this system, it was also possible to introduce two constructs simultaneously into a single cell. The resulting stable transfectants, expressing tdTomato and enhanced green fluorescent protein, exhibited both red and green fluorescence. The established cell line did not lose the acquired phenotype over three months of culture. Based on our results, we concluded that PB is superior to currently available methods for introducing plasmid DNA into HDDPCs. There may be significant challenges in the direct clinical application of this method for human dental tissue engineering due to safety risks and ethical concerns. However, the high level of transfection achieved with PB may have significant advantages in basic scientific research for dental tissue engineering applications, such as functional studies of genes and proteins. Furthermore, it is a useful tool for the isolation of genetically engineered HDDPC-derived stem cells for studies in tooth regenerative medicine. PMID:26208039

  4. Cohabitation Duration and Transient Domesticity

    PubMed Central

    GOLUB, ANDREW; REID, MEGAN; STRICKLER, JENNIFER; DUNLAP, ELOISE

    2013-01-01

    Research finds that many impoverished urban Black adults engage in a pattern of partnering and family formation involving a succession of short cohabitations yielding children, a paradigm referred to as transient domesticity. Researchers have identified socioeconomic status, cultural adaptations, and urbanicity as explanations for aspects of this pattern. We used longitudinal data from the 2001 Survey of Income and Program Participation to analyze variation in cohabitation and marriage duration by race/ethnicity, income, and urban residence. Proportional hazards regression indicated that separation risk is greater among couples that are cohabiting, below 200% of the federal poverty line, and Black but is not greater among urban dwellers. This provides empirical demographic evidence to support the emerging theory of transient domesticity and suggests that both socioeconomic status and race explain this pattern. We discuss the implications of these findings for understanding transient domesticity and make recommendations for using the Survey of Income and Program Participation to further study this family formation paradigm. PMID:24273358

  5. State Support of Domestic Production

    SciTech Connect

    Amy Wright

    2007-12-30

    This project was developed in response to a cooperative agreement offering by the U.S. Department of Energy (DOE) and the National Energy Technology Laboratory (NETL) under the State Support of Domestic Production DE-FC26-04NT15456. The Interstate Oil and Gas Compact Commission (IOGCC) performed efforts in support of State programs related to the security, reliability and growth if our nation's domestic production of oil and natural gas. The project objectives were to improve the States ability to monitor the security of oil and gas operations; to maximize the production of domestic oil and natural gas thereby minimizing the threat to national security posed by interruptions in energy imports; to assist States in developing and maintaining high standards of environmental protection; to assist in addressing issues that limit the capacity of the industry; to promote the deployment of the appropriate application of technology for regulatory efficiency; and to inform the public about emerging energy issues.

  6. Cohabitation Duration and Transient Domesticity.

    PubMed

    Golub, Andrew; Reid, Megan; Strickler, Jennifer; Dunlap, Eloise

    2013-01-01

    Research finds that many impoverished urban Black adults engage in a pattern of partnering and family formation involving a succession of short cohabitations yielding children, a paradigm referred to as transient domesticity. Researchers have identified socioeconomic status, cultural adaptations, and urbanicity as explanations for aspects of this pattern. We used longitudinal data from the 2001 Survey of Income and Program Participation to analyze variation in cohabitation and marriage duration by race/ethnicity, income, and urban residence. Proportional hazards regression indicated that separation risk is greater among couples that are cohabiting, below 200% of the federal poverty line, and Black but is not greater among urban dwellers. This provides empirical demographic evidence to support the emerging theory of transient domesticity and suggests that both socioeconomic status and race explain this pattern. We discuss the implications of these findings for understanding transient domesticity and make recommendations for using the Survey of Income and Program Participation to further study this family formation paradigm.

  7. Domestic violence documentation project 2012.

    PubMed

    Nittis, Maria; Hughes, Rod; Gray, Cecile; Ashton, Mandy

    2013-08-01

    One in four women presenting to Emergency Departments in Australia have experienced domestic violence in their lives but there are no specialist services for victims of domestic violence in the state of New South Wales, population of 7.25 million. Fundamental forensic medical and nursing skills developed for the comprehensive assessment of complainants of sexual assault were utilised in the examination of victims of domestic violence in a trial project at Nepean Hospital, Sydney. The project was then reviewed via a series of qualitative patient and police interviews along with an analysis of court outcomes. Assessment by specialists in forensic documentation and interpretation of injuries with the provision of balanced expert opinions for court purposes can result in a number of benefits for the victims and the criminal justice system, including an increase in the rate of successful prosecutions. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  8. Superheroes and masterminds of plant domestication.

    PubMed

    Martínez-Ainsworth, Natalia E; Tenaillon, Maud I

    2016-01-01

    Domestication is one of the most fundamental changes in the evolution of human societies. The geographical origins of domesticated plants are inferred from archaeology, ecology and genetic data. Scenarios vary among species and include single, diffuse or multiple independent domestications. Cultivated plants present a panel of traits, the "domestication syndrome" that distinguish them from their wild relatives. It encompasses yield-, food usage-, and cultivation-related traits. Most genes underlying those traits are "masterminds" affecting the regulation of gene networks. Phenotypic convergence of domestication traits across species or within species between independently domesticated forms rarely coincides with convergence at the gene level. We review here current data/models that propose a protracted transition model for domestication and investigate the impact of mating system, life cycle and gene flow on the pace of domestication. Finally, we discuss the cost of domestication, pointing to the importance of characterizing adaptive functional variation in wild resources.

  9. Assessment of Domestic Appliance Noise.

    NASA Astrophysics Data System (ADS)

    Brooks, Jeanette Rosamond

    Available from UMI in association with The British Library. The aims of this study were: (i) to identify the factors involved in eliciting a subjective reaction to domestic appliance noise, (ii) to identify the noise index (or indices) that correlate highly with a subjective reaction to the noise, and (iii) to investigate the contribution of domestic appliance noise to an individual's daily noise dose. Two series of experimental studies were carried out using several examples of each of five types of domestic appliances. One determined the index values of domestic appliance noise--namely L_{WA} (using ISO 3741), L_{pA} , L_{pD}, L _{p}, PNL, L_{Aeq, 30 sec}, L_{Amax} and L_{AX}; the other determined subjective reactions to domestic appliance noise (judgements of noisiness, annoyance, the acceptability of the appliance noise and appraisals of usefulness). The success or failure of the research hypotheses was assessed statistically by analysis of variance, regression analysis, log linear analysis, Hotelling test, bootstrapping, t-test and post-hoc comparisons. Ratings of annoyance, noisiness and the acceptability of the noise of the appliance were found to be interrelated and interdependent, and not influenced by appraisals of usefulness of the appliances. Noisiness ratings were the most consistent of the subjective ratings investigated, and were influenced by the duration of the exposure, and the actual appliance type under investigation. Significant correlations were obtained between noisiness ratings and all the noise indices under investigation. However, statistical analysis demonstrated that L_ {WA} correlated less successfully with noisiness ratings than all other indices. L_ {Amax},L_{Aeq,30 sec }, and L_{AX} indices were the most successful. It is therefore suggested that the labelling of domestic appliance noise consist of L_{WA} and L _{Aeq} as measured in a standardised test environment. The percentage contribution of domestic appliance noise to the total

  10. The Japanese Domestic Labor Debate.

    ERIC Educational Resources Information Center

    Ueno, Chizuko

    The changing role of Japanese women can be seen in the stages of a domestic labor debate which occurred at three different times in the past 30 years. The first debate began with Ayako Ishigaki's (1955) insistence that women should have a job outside the home. Wartime production helped break down traditional divisions of labor by encouraging women…

  11. Domestic Violence. Technical Assistance Packet.

    ERIC Educational Resources Information Center

    Join Together, Boston, MA.

    Substance abuse has long been recognized as a precipitating factor in many domestic violence incidents. The main type of substance abuse is alcohol usage. Forty-six percent of the offenders reported being dependent on or abusing alcohol, while another 28% were found to be dependent on opiates, cocaine, marijuana, or inhalants. Nearly two-fifths of…

  12. Girl domestic workers in Kenya.

    PubMed

    Mzungu, M

    1999-03-01

    This article exposes the conditions among children who are forced by their poor families to assume domestic work in households in Kenya. It is an accepted practice for parents to place daughters in households to help with housework and baby-sitting. The Sinaga Women and Child Labor Resource Center in Nairobi finds this exploitative and part of a wider practice that institutionalizes violence against women. The Center was established in 1995 to challenge the practice of child domestic labor. The Center's research reveals that child domestic workers tend to come from large, poor, and rural families or from urban slums. Wages are low or exchanged for shoes, clothes, and food. The hours of work are long. Mistreatment may include sexual molestation by male household members, beatings, verbal abuse, and mistrust. There is little recourse. Complaints from child workers or others outside the household can result in further mistreatment. Action against mistreatment is complicated by the prevailing image of activists as frustrated women with vendettas against men. The Center focuses on rehabilitation, literacy training, marketable skill development, and awareness creation. Counseling includes parents, children, and employers. Public awareness campaigns have resulted in employer referrals of youth workers for training. Other groups are joining the effort to improve conditions for child domestic workers.

  13. Collaborating to Eliminate Domestic Violence

    ERIC Educational Resources Information Center

    Berger, Ami

    2004-01-01

    On June 16, 2004, the College of Human Ecology (CHE), University of Minnesota partnered with a Twin Cities community service agency to host an event that raised awareness of domestic violence and also raised funds. "The Sheila Shawl Extravaganza," was a celebration of Sheila Wellstone's life (she was the wife of Minnesota senator Paul…

  14. Bringing attention to domestic abuse.

    PubMed

    Pearce, Lynne

    2014-06-03

    Nursing skills are vital to the roles of those working with victims of domestic abuse in Nottinghamshire. Specialist nurses at Nottingham University Hospitals NHS Trust are involved in referring patients on to further support services and training hospital staff to identify and respond to signs of abuse.

  15. Effect of domestic violence training

    PubMed Central

    Zaher, Eman; Keogh, Kelly; Ratnapalan, Savithiri

    2014-01-01

    Abstract Objective To describe and evaluate the effectiveness of domestic violence education in improving physicians’ knowledge, recognition, and management of abused women. Data sources The Cochrane Database of Systematic Reviews, MEDLINE, PubMed, PsycINFO, ERIC, and EMBASE were searched for articles published between January 1, 2000, and November 1, 2012. This search was supplemented by manual searches for relevant articles using a combined text-word and MeSH-heading search strategy. Study selection Randomized controlled trials were selected that used educational interventions among physicians and provided data on the effects of the interventions. Synthesis Nine randomized controlled trials were included that described different educational approaches with various outcome measures. Three studies examined the effects of educational interventions among postgraduate trainee physicians and found an increase in knowledge but no change in behaviour with regard to identifying victims of domestic violence. Six studies examined educational interventions for practising physicians. Three of these studies used multifaceted physician training that combined education with system support interventions to change physician behaviour, such as increasing general awareness of domestic violence with brochures and posters, providing aids to remind physicians how to identify victims, facilitating physician access to victim support services, and providing audits and feedback. Multifaceted educational interventions included interactive workshops, Web-based learning, and experiential training. Another study used focus-group discussions and training, and showed improved domestic violence reporting among physicians. The remaining 2 studies showed improved perceptions of practising physicians’ self-efficacy using problem-based online learning. Conclusion It was difficult to determine the most effective educational strategy, as the educational interventions and the outcome measures varied

  16. Rice domestication: histories and mysteries.

    PubMed

    Gross, Briana L

    2012-09-01

    Domesticated rice (Oryza sativa) is one of the world's most important food crops, culturally, nutritionally and economically (Khush 1997). Thus, it is no surprise that there is intense curiosity about its genetic and geographical origins, its response to selection under domestication, and the genetic structure of its wild relative, Oryza rufipogon. Studies of Oryza attempting to answer these questions have accompanied each stage of the development of molecular markers, starting with allozymes and continuing to genome sequencing. While many of these studies have been restricted to small sample sizes, in terms of either the number of markers used or the number and distribution of the accessions, costs are now low enough that researchers are including large numbers of molecular markers and accessions. How will these studies relate to previous findings and long-held assumptions about rice domestication and evolution? If the paper in this issue of Molecular Ecology (Huang et al. 2012) is any indication, there will be some considerable surprises in store. In this study, a geographically and genomically thorough sampling of O. rufipogon and O. sativa revealed two genetically distinct groups of wild rice and also indicated that only one of these groups appears to be related to domesticated rice. While this fits well with previous studies indicating that there are genetic subdivisions within O. rufipogon, it stands in contrast to previous findings that the two major varieties of O. sativa (indica and japonica) were domesticated from two (or more) subpopulations of wild rice.

  17. Genetic analysis of safflower domestication

    PubMed Central

    2014-01-01

    Background Safflower (Carthamus tinctorius L.) is an oilseed crop in the Compositae (a.k.a. Asteraceae) that is valued for its oils rich in unsaturated fatty acids. Here, we present an analysis of the genetic architecture of safflower domestication and compare our findings to those from sunflower (Helianthus annuus L.), an independently domesticated oilseed crop within the same family. We mapped quantitative trait loci (QTL) underlying 24 domestication-related traits in progeny from a cross between safflower and its wild progenitor, Carthamus palaestinus Eig. Also, we compared QTL positions in safflower against those that have been previously identified in cultivated x wild sunflower crosses to identify instances of colocalization. Results We mapped 61 QTL, the vast majority of which (59) exhibited minor or moderate phenotypic effects. The two large-effect QTL corresponded to one each for flower color and leaf spininess. A total of 14 safflower QTL colocalized with previously reported sunflower QTL for the same traits. Of these, QTL for three traits (days to flower, achene length, and number of selfed seed) had cultivar alleles that conferred effects in the same direction in both species. Conclusions As has been observed in sunflower, and unlike many other crops, our results suggest that the genetics of safflower domestication is quite complex. Moreover, our comparative mapping results indicate that safflower and sunflower exhibit numerous instances of QTL colocalization, suggesting that parallel trait transitions during domestication may have been driven, at least in part, by parallel genotypic evolution at some of the same underlying genes. PMID:24502326

  18. 48 CFR 2129.401 - Domestic contracts.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Domestic contracts. 2129.401 Section 2129.401 Federal Acquisition Regulations System OFFICE OF PERSONNEL MANAGEMENT, FEDERAL... Contract Clauses 2129.401 Domestic contracts....

  19. 48 CFR 2129.401 - Domestic contracts.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Domestic contracts. 2129.401 Section 2129.401 Federal Acquisition Regulations System OFFICE OF PERSONNEL MANAGEMENT, FEDERAL... Contract Clauses 2129.401 Domestic contracts....

  20. 7 CFR 993.21 - Domestic.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 8 2011-01-01 2011-01-01 false Domestic. 993.21 Section 993.21 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Order Regulating Handling Definitions § 993.21 Domestic. Domestic means the United States, Canal...

  1. 12 CFR 613.3100 - Domestic lending.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 6 2011-01-01 2011-01-01 false Domestic lending. 613.3100 Section 613.3100... Financing for Banks Operating Under Title III of the Farm Credit Act § 613.3100 Domestic lending. (a... disposal facilities in rural areas. (e) Domestic lessors. A bank for cooperatives or agricultural...

  2. 7 CFR 993.21 - Domestic.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Domestic. 993.21 Section 993.21 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements... Order Regulating Handling Definitions § 993.21 Domestic. Domestic means the United States, Canal...

  3. 7 CFR 983.12 - Domestic shipments.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 8 2011-01-01 2011-01-01 false Domestic shipments. 983.12 Section 983.12 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements..., ARIZONA, AND NEW MEXICO Definitions § 983.12 Domestic shipments. Domestic shipments means shipments to...

  4. 78 FR 14913 - Domestic Baggage Liability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-03-08

    ... Office of the Secretary 14 CFR Part 254 RIN 2105-AE21 Domestic Baggage Liability AGENCY: Office of the... existing regulations, this final rule raises the minimum limit on domestic baggage liability applicable to... applicable to domestic air service. Section 254.6 of this rule requires the Department to review every...

  5. 19 CFR 146.43 - Domestic status.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 2 2010-04-01 2010-04-01 false Domestic status. 146.43 Section 146.43 Customs... (CONTINUED) FOREIGN TRADE ZONES Status of Merchandise in a Zone § 146.43 Domestic status. (a) General. Domestic status may be granted to merchandise: (1) The growth, product, or manufacture of the U.S. on...

  6. 12 CFR 613.3100 - Domestic lending.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Domestic lending. 613.3100 Section 613.3100... Financing for Banks Operating Under Title III of the Farm Credit Act § 613.3100 Domestic lending. (a... disposal facilities in rural areas. (e) Domestic lessors. A bank for cooperatives or agricultural...

  7. 7 CFR 983.12 - Domestic shipments.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 8 2010-01-01 2010-01-01 false Domestic shipments. 983.12 Section 983.12 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements..., ARIZONA, AND NEW MEXICO Definitions § 983.12 Domestic shipments. Domestic shipments means shipments to...

  8. Risk Factors for Domestic Violence in Curacao

    ERIC Educational Resources Information Center

    van Wijk, N. Ph. L.; de Bruijn, J. G. M.

    2012-01-01

    One out of three people (25% of men, 38% of women) in Curacao have experienced some form of domestic violence at some point in their adult lives. The most significant risk factors for domestic violence in Curacao are the female gender, a young age, low education, and experiencing domestic violence victimization in childhood. Divorce, single…

  9. 19 CFR 146.43 - Domestic status.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 2 2011-04-01 2011-04-01 false Domestic status. 146.43 Section 146.43 Customs... (CONTINUED) FOREIGN TRADE ZONES Status of Merchandise in a Zone § 146.43 Domestic status. (a) General. Domestic status may be granted to merchandise: (1) The growth, product, or manufacture of the U.S. on...

  10. Risk Factors for Domestic Violence in Curacao

    ERIC Educational Resources Information Center

    van Wijk, N. Ph. L.; de Bruijn, J. G. M.

    2012-01-01

    One out of three people (25% of men, 38% of women) in Curacao have experienced some form of domestic violence at some point in their adult lives. The most significant risk factors for domestic violence in Curacao are the female gender, a young age, low education, and experiencing domestic violence victimization in childhood. Divorce, single…

  11. 25 CFR 11.454 - Domestic violence.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 25 Indians 1 2014-04-01 2014-04-01 false Domestic violence. 11.454 Section 11.454 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAW AND ORDER COURTS OF INDIAN OFFENSES AND LAW AND ORDER CODE Criminal Offenses § 11.454 Domestic violence. (a) A person who commits domestic violence by...

  12. 25 CFR 11.454 - Domestic violence.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 25 Indians 1 2011-04-01 2011-04-01 false Domestic violence. 11.454 Section 11.454 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAW AND ORDER COURTS OF INDIAN OFFENSES AND LAW AND ORDER CODE Criminal Offenses § 11.454 Domestic violence. (a) A person who commits domestic violence by...

  13. Domestic Violence against Men: Know the Signs

    MedlinePlus

    Healthy Lifestyle Adult health Domestic violence against men isn't always easy to identify, but it can be a serious threat. Know how to recognize if ... Staff Women aren't the only victims of domestic violence. Understand the signs of domestic violence against men, ...

  14. 25 CFR 11.454 - Domestic violence.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 25 Indians 1 2010-04-01 2010-04-01 false Domestic violence. 11.454 Section 11.454 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAW AND ORDER COURTS OF INDIAN OFFENSES AND LAW AND ORDER CODE Criminal Offenses § 11.454 Domestic violence. (a) A person who commits domestic violence by...

  15. 25 CFR 11.454 - Domestic violence.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 25 Indians 1 2013-04-01 2013-04-01 false Domestic violence. 11.454 Section 11.454 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAW AND ORDER COURTS OF INDIAN OFFENSES AND LAW AND ORDER CODE Criminal Offenses § 11.454 Domestic violence. (a) A person who commits domestic violence by...

  16. 25 CFR 11.454 - Domestic violence.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 25 Indians 1 2012-04-01 2011-04-01 true Domestic violence. 11.454 Section 11.454 Indians BUREAU OF INDIAN AFFAIRS, DEPARTMENT OF THE INTERIOR LAW AND ORDER COURTS OF INDIAN OFFENSES AND LAW AND ORDER CODE Criminal Offenses § 11.454 Domestic violence. (a) A person who commits domestic violence by inflicting...

  17. Domestic competitiveness in secondary wood industries

    Treesearch

    Matthew Bumgardner; Urs Buehlmann; Albert Schuler; Rich Christianson

    2004-01-01

    As imports capture a substantial portion of the domestic wood furniture market, there is much speculation and concern as to the future of this and related industries. This study sought to obtain an industry perspective of trends in domestic manufacturing and importing, and to identify factors that might enhance domestic competitiveness. A mail survey was conducted...

  18. Genetics and consequences of crop domestication

    USDA-ARS?s Scientific Manuscript database

    Phenotypic variation has been manipulated by humans during crop domestication, which occurred primarily between 3,000 and 10,000 years ago in the various centers of origin around the world. The process of domestication has profound consequences on crops, where the domesticate has moderately reduced ...

  19. A pipeline strategy for grain crop domestication

    USDA-ARS?s Scientific Manuscript database

    In recent decades, in the interest of diversifying the global food system, improving human nutrition, or making agriculture more sustainable, there have been many proposals for domesticating or completing the domestication of wild plants or semi-domesticated “orphan” crops. However, very few new cro...

  20. 19 CFR 123.13 - Foreign repairs to domestic locomotives and other domestic railroad equipment.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Foreign repairs to domestic locomotives and other domestic railroad equipment. 123.13 Section 123.13 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION... International Traffic § 123.13 Foreign repairs to domestic locomotives and other domestic railroad equipment....

  1. 26 CFR 1.904(g)-1 - Overall domestic loss and the overall domestic loss account.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 9 2011-04-01 2011-04-01 false Overall domestic loss and the overall domestic loss account. 1.904(g)-1 Section 1.904(g)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF... the United States § 1.904(g)-1 Overall domestic loss and the overall domestic loss account....

  2. 26 CFR 1.904(g)-1 - Overall domestic loss and the overall domestic loss account.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 9 2010-04-01 2010-04-01 false Overall domestic loss and the overall domestic loss account. 1.904(g)-1 Section 1.904(g)-1 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF... States § 1.904(g)-1 Overall domestic loss and the overall domestic loss account. For further...

  3. 49 CFR 526.3 - Transfer of vehicle from non-domestic to domestic fleet.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 6 2011-10-01 2011-10-01 false Transfer of vehicle from non-domestic to domestic... UNDER THE AUTOMOBILE FUEL EFFICIENCY ACT OF 1980 § 526.3 Transfer of vehicle from non-domestic to domestic fleet. Each plan submitted under section 503(b)(4) of the Motor Vehicle Information and...

  4. 19 CFR 123.13 - Foreign repairs to domestic locomotives and other domestic railroad equipment.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 1 2010-04-01 2010-04-01 false Foreign repairs to domestic locomotives and other domestic railroad equipment. 123.13 Section 123.13 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION... International Traffic § 123.13 Foreign repairs to domestic locomotives and other domestic railroad equipment....

  5. 49 CFR 526.3 - Transfer of vehicle from non-domestic to domestic fleet.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 6 2010-10-01 2010-10-01 false Transfer of vehicle from non-domestic to domestic... UNDER THE AUTOMOBILE FUEL EFFICIENCY ACT OF 1980 § 526.3 Transfer of vehicle from non-domestic to domestic fleet. Each plan submitted under section 503(b)(4) of the Motor Vehicle Information and...

  6. 19 CFR 123.13 - Foreign repairs to domestic locomotives and other domestic railroad equipment.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... domestic railroad equipment. 123.13 Section 123.13 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION... International Traffic § 123.13 Foreign repairs to domestic locomotives and other domestic railroad equipment. A report of the first arrival in the United States of a domestic locomotive or other railroad...

  7. 19 CFR 123.13 - Foreign repairs to domestic locomotives and other domestic railroad equipment.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... domestic railroad equipment. 123.13 Section 123.13 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION... International Traffic § 123.13 Foreign repairs to domestic locomotives and other domestic railroad equipment. A report of the first arrival in the United States of a domestic locomotive or other railroad...

  8. 19 CFR 123.13 - Foreign repairs to domestic locomotives and other domestic railroad equipment.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... domestic railroad equipment. 123.13 Section 123.13 Customs Duties U.S. CUSTOMS AND BORDER PROTECTION... International Traffic § 123.13 Foreign repairs to domestic locomotives and other domestic railroad equipment. A report of the first arrival in the United States of a domestic locomotive or other railroad...

  9. Frustration behaviors in domestic dogs.

    PubMed

    Jakovcevic, Adriana; Elgier, Angel M; Mustaca, Alba E; Bentosela, Mariana

    2013-01-01

    During extinction a previously learned behavior stops being reinforced. In addition to the decrease in the rate of the instrumental response, it produces an aversive emotional state known as frustration. This state can be assimilated with the fear reactions that occur after aversive stimuli are introduced at both the physiological and behavioral levels. This study evaluated frustration reactions of domestic dogs (Canis familiaris) during a communicative situation involving interactions with a human. The task included the reinforcement and extinction of the gaze response toward the experimenter's face when the dogs tried to obtain inaccessible food. The dog's frustration reactions during extinction involved an increase in withdrawal and side orientation to the location of the human as well as lying down, ambulation, sniffing, and vocalizations compared with the last acquisition trial. These results are especially relevant for domestic dog training situations in which the extinction technique is commonly used to discourage undesirable behaviors.

  10. Ferrochromium from Domestic Lateritic Chromites

    NASA Astrophysics Data System (ADS)

    Nafziger, R. H.

    1988-09-01

    As part of its effort to devise suitable technology for processing low-grade domestic materials and recycling wastes, the U.S. Bureau of Mines has evaluated the feasibility of smelting a chromite concentrate derived from residues generated by the processing of nickel and cobalt from domestic later-ites. The product sought was a high-carbon ferrochromium suitable for stainless- and alloy-steel production. The concentrate was blended with re-ductants and fluxing constituents and was smelted under submerged arc conditions in a laboratory-scale, single-phase ac electric arc furnace. The results revealed that metallurgical-grade coke provides the best quality product. High-carbon ferrochromium, which met ASTM specifications except for phosphorus and sulfur, was obtained. Agglomeration of the charge materials was not required.

  11. Effects of domestication on animal behaviour.

    PubMed

    Kretchmer, K R; Fox, M W

    1975-02-01

    Centuries of domestication of animals by civilised man have had many measurable effects on the various species involved, but only in relatively recent history has scientific curiosity been directed to assessing their extent. The process of domestication is analysed and its known effects on animals reviewed through observations of and experiments with various species of domesticated animals conducted by researchers into animal psychology and biology. The known facts on the effects of domestication in animals are extrapolated in an attempt to determine to what extent modern man himself has been domesticated in the urban environment.

  12. Artificial cloning of domestic animals.

    PubMed

    Keefer, Carol L

    2015-07-21

    Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research.

  13. Artificial cloning of domestic animals

    PubMed Central

    Keefer, Carol L.

    2015-01-01

    Domestic animals can be cloned using techniques such as embryo splitting and nuclear transfer to produce genetically identical individuals. Although embryo splitting is limited to the production of only a few identical individuals, nuclear transfer of donor nuclei into recipient oocytes, whose own nuclear DNA has been removed, can result in large numbers of identical individuals. Moreover, clones can be produced using donor cells from sterile animals, such as steers and geldings, and, unlike their genetic source, these clones are fertile. In reality, due to low efficiencies and the high costs of cloning domestic species, only a limited number of identical individuals are generally produced, and these clones are primarily used as breed stock. In addition to providing a means of rescuing and propagating valuable genetics, somatic cell nuclear transfer (SCNT) research has contributed knowledge that has led to the direct reprogramming of cells (e.g., to induce pluripotent stem cells) and a better understanding of epigenetic regulation during embryonic development. In this review, I provide a broad overview of the historical development of cloning in domestic animals, of its application to the propagation of livestock and transgenic animal production, and of its scientific promise for advancing basic research. PMID:26195770

  14. A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase.

    PubMed

    Mizuuchi, K; Nobbs, T J; Halford, S E; Adzuma, K; Qin, J

    1999-04-06

    A new method was developed for tracking the stereochemical path of enzymatic cleavage of DNA. DNA with a phosphorothioate of known chirality at the scissile bond is cleaved by the enzyme in H218O. The cleavage produces a DNA molecule with the 5'-[16O,18O, S]-thiophosphoryl group, whose chirality depends on whether the cleavage reaction proceeds by a single-step hydrolysis mechanism or by a two-step mechanism involving a protein-DNA covalent intermediate. To determine this chirality, the cleaved DNA is joined to an oligonucleotide by DNA ligase. Given the strict stereochemistry of the DNA ligase reaction, determined here, the original chirality of the phosphorothioate dictates whether the 18O is retained or lost in the ligation product, which can be determined by mass spectrometry. This method has advantages over previous methods in that it is not restricted to particular DNA sequences, requires substantially less material, and avoids purification of the products at intermediate stages in the procedure. The method was validated by confirming that DNA cleavage by the EcoRI restriction endonuclease causes inversion of configuration at the scissile phosphate. It was then applied to the reactions of the SfiI and HpaII endonucleases and the MuA transposase. In all three cases, DNA cleavage proceeded with inversion of configuration, indicating direct hydrolysis of the phosphodiester bond by water as opposed to a reaction involving a covalent enzyme-DNA intermediate.

  15. Transposase-Derived Proteins FHY3/FAR1 Interact with PHYTOCHROME-INTERACTING FACTOR1 to Regulate Chlorophyll Biosynthesis by Modulating HEMB1 during Deetiolation in Arabidopsis[W

    PubMed Central

    Tang, Weijiang; Wang, Wanqing; Chen, Dongqin; Ji, Qiang; Jing, Yanjun; Wang, Haiyang; Lin, Rongcheng

    2012-01-01

    Successful chlorophyll biosynthesis during initial light exposure is critical for plant survival and growth, as excess accumulation of chlorophyll precursors in darkness can cause photooxidative damage to cells. Therefore, efficient mechanisms have evolved to precisely regulate chlorophyll biosynthesis in plants. Here, we identify FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and FAR-RED IMPAIRED RESPONSE1 (FAR1), two transposase-derived transcription factors, as positive regulators of chlorophyll biosynthesis in Arabidopsis thaliana. We show that null mutations in FHY3 and FAR1 cause reduced protochlorophyllide (a precursor of chlorophyll) levels in darkness and less photobleaching in the light. We find that FHY3 directly binds to the promoter and activates expression of HEMB1, which encodes 5-aminolevulinic acid dehydratase in the chlorophyll biosynthetic pathway. We reveal that PHYTOCHROME-INTERACTING FACTOR1 physically interacts with the DNA binding domain of FHY3, thereby partly repressing FHY3/FAR1-activated HEMB1 expression. Strikingly, FHY3 expression is upregulated by white light. In addition, our genetic data indicate that overexpression, severe reduction, or lack of HEMB1 impairs plant growth and development. Together, our findings reveal a crucial role of FHY3/FAR1 in regulating chlorophyll biosynthesis, thus uncovering a new layer of regulation by which light promotes plant dark–light transition in early seedling development. PMID:22634759

  16. 26 CFR 1.904(g)-1T - Overall domestic loss and the overall domestic loss account (temporary).

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 9 2011-04-01 2011-04-01 false Overall domestic loss and the overall domestic... Without the United States § 1.904(g)-1T Overall domestic loss and the overall domestic loss account... domestic losses, for establishing overall domestic loss accounts, and for making additions to...

  17. Evolutionary genomics of dog domestication.

    PubMed

    Wayne, Robert K; vonHoldt, Bridgett M

    2012-02-01

    We review the underlying principles and tools used in genomic studies of domestic dogs aimed at understanding the genetic changes that have occurred during domestication. We show that there are two principle modes of evolution within dogs. One primary mode that accounts for much of the remarkable diversity of dog breeds is the fixation of discrete mutations of large effect in individual lineages that are then crossed to various breed groupings. This transfer of mutations across the dog evolutionary tree leads to the appearance of high phenotypic diversity that in actuality reflects a small number of major genes. A second mechanism causing diversification involves the selective breeding of dogs within distinct phenotypic or functional groups, which enhances specific group attributes such as heading or tracking. Such progressive selection leads to a distinct genetic structure in evolutionary trees such that functional and phenotypic groups cluster genetically. We trace the origin of the nuclear genome in dogs based on haplotype-sharing analyses between dogs and gray wolves and show that contrary to previous mtDNA analyses, the nuclear genome of dogs derives primarily from Middle Eastern or European wolves, a result more consistent with the archeological record. Sequencing analysis of the IGF1 gene, which has been the target of size selection in small breeds, further supports this conclusion. Finally, we discuss how a black coat color mutation that evolved in dogs has transformed North American gray wolf populations, providing a first example of a mutation that appeared under domestication and selectively swept through a wild relative.

  18. [Atopic dermatitis and domestic animals].

    PubMed

    Song, M

    2000-09-01

    Several arguments are raised attributing to aeroallergens an important role in atopic dermatitis. The aeroallergens that penetrate the epidermis could be fixed by IgE on the Langerhans cells and then induce a cellular mediator reaction comparable to that of allergic contact eczema. Patch tests have been developed to evaluate the role of aeroallergens (dust mites, animal dander, etc.). Preventive anti-dust mites measures in the home of atopic patients are recommended. Eviction of domestic animals (cat, dog, etc.) or avoidance measures for animal dander in the home can produce improvement in atopic dermatitis. Oral specific immunotherapy is being validated as a treatment for this disease.

  19. Deciphering the genetic basis of animal domestication

    PubMed Central

    Wiener, Pamela; Wilkinson, Samantha

    2011-01-01

    Genomic technologies for livestock and companion animal species have revolutionized the study of animal domestication, allowing an increasingly detailed description of the genetic changes accompanying domestication and breed development. This review describes important recent results derived from the application of population and quantitative genetic approaches to the study of genetic changes in the major domesticated species. These include findings of regions of the genome that show between-breed differentiation, evidence of selective sweeps within individual genomes and signatures of demographic events. Particular attention is focused on the study of the genetics of behavioural traits and the implications for domestication. Despite the operation of severe bottlenecks, high levels of inbreeding and intensive selection during the history of domestication, most domestic animal species are genetically diverse. Possible explanations for this phenomenon are discussed. The major insights from the surveyed studies are highlighted and directions for future study are suggested. PMID:21885467

  20. Wheat domestication: lessons for the future.

    PubMed

    Charmet, Gilles

    2011-03-01

    Wheat was one of the first crops to be domesticated more than 10,000 years ago in the Middle East. Molecular genetics and archaeological data have allowed the reconstruction of plausible domestication scenarios leading to modern cultivars. For diploid einkorn and tetraploid durum wheat, a single domestication event has likely occurred in the Karacadag Mountains, Turkey. Following a cross between tetraploid durum and diploid T. tauschii, the resultant hexaploid bread wheat was domesticated and disseminated around the Caucasian region. These polyploidisation events facilitated wheat domestication and created genetic bottlenecks, which excluded potentially adaptive alleles. With the urgent need to accelerate genetic progress to confront the challenges of climate change and sustainable agriculture, wild ancestors and old landraces represent a reservoir of underexploited genetic diversity that may be utilized through modern breeding methods. Understanding domestication processes may thus help identifying new strategies. Copyright © 2011 Académie des sciences. Published by Elsevier SAS. All rights reserved.

  1. Genetic testing in domestic cats

    PubMed Central

    Lyons, Leslie A.

    2012-01-01

    Varieties of genetic tests are currently available for the domestic cat that support veterinary health care, breed management, species identification, and forensic investigations. Approximately thirty-five genes contain over fifty mutations that cause feline health problems or alterations in the cat’s appearance. Specific genes, such as sweet and drug receptors, have been knocked-out of Felidae during evolution and can be used along with mtDNA markers for species identification. Both STR and SNP panels differentiate cat race, breed, and individual identity, as well as gender-specific markers to determine sex of an individual. Cat genetic tests are common offerings for commercial laboratories, allowing both the veterinary clinician and the private owner to obtain DNA test results. This article will review the genetic tests for the domestic cat, and their various applications in different fields of science. Highlighted are genetic tests specific to the individual cat, which are a part of the cat’s genome. PMID:22546621

  2. Domestic Abuse in Behshahr, Iran

    PubMed Central

    Rahmatian, Ali Akbar; Hosseini, Seyyed Ali Asghar

    2015-01-01

    Background: The United Nations in a resolution defined abuse as any violent act that is primarily or exclusively committed against females and results in physical, sexual and psychological harm. Objectives: The aim of this research was to study the contributing factors of husband’s violence against females residing in the city of Behshahr, Iran. Materials and Methods: We distributed a specifically designed questionnaire among 380 married females aged between 15 and 65 years. According to the Morgan table, the subjects were randomly selected from a list of 301000 females. Demographic data and data on spouse abuse were then analyzed using the SPSS software, Spearman and Pearson correlation coefficients. According to Cronbach’s alpha, the reliability of the questionnaire was 0.96. Results: All of the females reported at least one form of violence within the past year, with R square 0.20, indicating that the independent variable can explain 20% of the violence against females. years of marriage, female’s education, male’s addiction and the number of children each had their share in the explanation of violence against females. Conclusions: This study revealed a high prevalence of domestic violence in the sample population. Violence existed among all ages, social categories and male occupational groups, and also involved both employed and unemployed females. The situation regarding domestic abuse is similar worldwide. PMID:26834799

  3. Germline modification of domestic animals.

    PubMed

    Tang, L; González, R; Dobrinski, I

    2015-01-01

    Genetically-modified domestic animal models are of increasing significance in biomedical research and agriculture. As authentic ES cells derived from domestic animals are not yet available, the prevailing approaches for engineering genetic modifications in those animals are pronuclear microinjection and somatic cell nuclear transfer (SCNT, also known as cloning). Both pronuclear microinjection and SCNT are inefficient, costly, and time-consuming. In animals produced by pronuclear microinjection, the exogenous transgene is usually inserted randomly into the genome, which results in highly variable expression patterns and levels in different founders. Therefore, significant efforts are required to generate and screen multiple founders to obtain animals with optimal transgene expression. For SCNT, specific genetic modifications (both gain-of-function and loss-of-function) can be engineered and carefully selected in the somatic cell nucleus before nuclear transfer. SCNT has been used to generate a variety of genetically modified animals such as goats, pigs, sheep and cattle; however, animals resulting from SCNT frequently suffer from developmental abnormalities associated with incomplete nuclear reprogramming. Other strategies to generate genetically-modified animals rely on the use of the spermatozoon as a natural vector to introduce genetic material into the female gamete. This sperm mediated DNA transfer (SMGT) combined with intracytoplasmatic sperm injection (ICSI) has relatively high efficiency and allows the insertion of large DNA fragments, which, in turn, enhance proper gene expression. An approach currently being developed to complement SCNT for producing genetically modified animals is germ cell transplantation using genetically modified male germline stem cells (GSCs). This approach relies on the ability of GSCs that are genetically modified in vitro to colonize the recipient testis and produce donor derived sperm upon transplantation. As the genetic change

  4. Germline modification of domestic animals

    PubMed Central

    Tang, L.; González, R.; Dobrinski, I.

    2016-01-01

    Genetically-modified domestic animal models are of increasing significance in biomedical research and agriculture. As authentic ES cells derived from domestic animals are not yet available, the prevailing approaches for engineering genetic modifications in those animals are pronuclear microinjection and somatic cell nuclear transfer (SCNT, also known as cloning). Both pronuclear microinjection and SCNT are inefficient, costly, and time-consuming. In animals produced by pronuclear microinjection, the exogenous transgene is usually inserted randomly into the genome, which results in highly variable expression patterns and levels in different founders. Therefore, significant efforts are required to generate and screen multiple founders to obtain animals with optimal transgene expression. For SCNT, specific genetic modifications (both gain-of-function and loss-of-function) can be engineered and carefully selected in the somatic cell nucleus before nuclear transfer. SCNT has been used to generate a variety of genetically modified animals such as goats, pigs, sheep and cattle; however, animals resulting from SCNT frequently suffer from developmental abnormalities associated with incomplete nuclear reprogramming. Other strategies to generate genetically-modified animals rely on the use of the spermatozoon as a natural vector to introduce genetic material into the female gamete. This sperm mediated DNA transfer (SMGT) combined with intracytoplasmatic sperm injection (ICSI) has relatively high efficiency and allows the insertion of large DNA fragments, which, in turn, enhance proper gene expression. An approach currently being developed to complement SCNT for producing genetically modified animals is germ cell transplantation using genetically modified male germline stem cells (GSCs). This approach relies on the ability of GSCs that are genetically modified in vitro to colonize the recipient testis and produce donor derived sperm upon transplantation. As the genetic change

  5. International labor migration and domestic labor supply.

    PubMed

    Kochhar, R

    1992-04-01

    "This paper constructs a dynamic, general equilibrium framework to study the relationship between international labor migration and domestic labor supply. The general equilibrium nature of the model enables us to endogenize the pattern of labor migration. The effect of labor migration on domestic wage rates and labor supply is shown to depend on the pattern of labor migration. If the substitution effect dominates the income effect in labor supply, the domestic supply of labor necessarily decreases in response to an inflow of migrants....Similarly, if the dominant effect is the income effect, the immigration of labor necessarily increases the domestic supply of labor."

  6. Mothers, domestic violence, and child protection.

    PubMed

    Douglas, Heather; Walsh, Tamara

    2010-05-01

    This article explores the relationship between understandings of domestic violence and the child protection response drawing on material gathered in focus groups with workers who support mothers dealing with both domestic violence and child protection issues. The interviewees expressed concern that the dynamics of domestic violence are often misunderstood and inappropriately responded to by child protection workers. This article critically examines the interviewees' concerns and concludes that to properly protect children, it is crucial that child protection workers have a clear understanding of the dynamics of and issues related to domestic violence.

  7. 76 FR 36512 - USDA Increases the Domestic Sugar Overall Allotment Quantity, Reassigns Domestic Cane Sugar...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-06-22

    ... Office of the Secretary USDA Increases the Domestic Sugar Overall Allotment Quantity, Reassigns Domestic Cane Sugar Allotments, and Increases the Fiscal Year 2011 Raw Sugar Tariff-Rate Quota AGENCY: Office of... in the domestic sugar Overall Allotment Quantity (OAQ); a reassignment of surplus sugar under...

  8. Asthma and domestic air quality.

    PubMed

    Jones, A P

    1998-09-01

    In recent years, there has been a global increase in the prevalence of asthma. This has coincided with many modifications to the home environment, resulting in changes to the quality of indoor air. This article considers the links between indoor air pollution and asthma. Exposure to a range of pollutants is examined. Airborne allergens such as those from house dust mites and cockroaches, domestic pets and moulds and fungal spores may be important. Pollution from particulate materials associated with bio-fuel combustion and smoking is discussed, as is the role of chemical vapours and gases including nitrogen dioxide, formaldehyde and volatile organic compounds. The efficacy of various environmental controls to limit the impact of these pollutants is explored. It is concluded that indoor air pollution may be an important risk for asthma and the health impacts of building design and management require greater recognition and further research.

  9. Domestic Options to Offshore Oil and Gas.

    ERIC Educational Resources Information Center

    Kash, Don E.

    1983-01-01

    The continuing controversey over offshore oil/gas has given impetus to searching for domestic energy alternatives. The need for and types of several alternative sources are discussed. Indicates that the United States needs to pursue both offshore and other domestic liquid-fuel sources if it is to avoid becoming increasingly dependent on imports.…

  10. 48 CFR 629.401 - Domestic contracts.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 4 2011-10-01 2011-10-01 false Domestic contracts. 629.401 Section 629.401 Federal Acquisition Regulations System DEPARTMENT OF STATE GENERAL CONTRACTING REQUIREMENTS TAXES Contract Clauses 629.401 Domestic contracts....

  11. 48 CFR 529.401 - Domestic contracts.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 4 2011-10-01 2011-10-01 false Domestic contracts. 529.401 Section 529.401 Federal Acquisition Regulations System GENERAL SERVICES ADMINISTRATION GENERAL CONTRACTING REQUIREMENTS TAXES Contract Clauses 529.401 Domestic contracts....

  12. 48 CFR 1529.401 - Domestic contracts.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 6 2010-10-01 2010-10-01 true Domestic contracts. 1529.401 Section 1529.401 Federal Acquisition Regulations System ENVIRONMENTAL PROTECTION AGENCY GENERAL CONTRACTING REQUIREMENTS TAXES Contract Clauses 1529.401 Domestic contracts....

  13. 48 CFR 1529.401 - Domestic contracts.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 6 2011-10-01 2011-10-01 false Domestic contracts. 1529.401 Section 1529.401 Federal Acquisition Regulations System ENVIRONMENTAL PROTECTION AGENCY GENERAL CONTRACTING REQUIREMENTS TAXES Contract Clauses 1529.401 Domestic contracts....

  14. 48 CFR 529.401 - Domestic contracts.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Domestic contracts. 529.401 Section 529.401 Federal Acquisition Regulations System GENERAL SERVICES ADMINISTRATION GENERAL CONTRACTING REQUIREMENTS TAXES Contract Clauses 529.401 Domestic contracts....

  15. 48 CFR 629.401 - Domestic contracts.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Domestic contracts. 629.401 Section 629.401 Federal Acquisition Regulations System DEPARTMENT OF STATE GENERAL CONTRACTING REQUIREMENTS TAXES Contract Clauses 629.401 Domestic contracts....

  16. 7 CFR 1260.310 - Domestic assessments.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Domestic assessments. 1260.310 Section 1260.310 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING... Rules and Regulations § 1260.310 Domestic assessments. (a) A $1.00 per head assessment on cattle...

  17. 7 CFR 1260.310 - Domestic assessments.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Domestic assessments. 1260.310 Section 1260.310 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING... Rules and Regulations § 1260.310 Domestic assessments. (a) A $1.00 per head assessment on cattle...

  18. Domestic Violence and Poverty: Some Women's Experiences

    ERIC Educational Resources Information Center

    Slabbert, Ilze

    2017-01-01

    Purpose: Domestic violence poses a major challenge to social workers. Low-income families are significantly more likely to have to contend with domestic violence, as poverty can act as a fuelling factor in this type of conflict. The objective of this study was to explore and describe the experiences of low-income abused women. Method: A…

  19. 48 CFR 29.401 - Domestic contracts.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false Domestic contracts. 29.401 Section 29.401 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS TAXES Contract Clauses 29.401 Domestic contracts....

  20. 48 CFR 29.401 - Domestic contracts.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Domestic contracts. 29.401 Section 29.401 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION GENERAL CONTRACTING REQUIREMENTS TAXES Contract Clauses 29.401 Domestic contracts....

  1. Domestic Violence against Married Women in Cambodia

    ERIC Educational Resources Information Center

    Yount, Kathryn M.; Carrera, Jennifer S.

    2006-01-01

    We evaluate the effects of marital resources and early-life experiences on recent domestic violence and attitudes about wife abuse among 2,074 married Cambodian women. Household standard of living was negatively associated with physical domestic violence. Women with 8-13 fewer years of schooling than their husbands more often experienced physical…

  2. Evolutionary genomics of peach and almond domestication

    USDA-ARS?s Scientific Manuscript database

    The domesticated almond [Prunus dulcis (L.) Batsch] and peach [P. persica (Mill.) D. A. Webb] originate on opposite sides of Asia and were independently domesticated approximately 5000 years ago. While interfertile, they possess alternate mating systems and di er in a number of morphological and ph...

  3. The Effect of Divorce on Domestic Crime

    ERIC Educational Resources Information Center

    Stolzenberg, Lisa; D'Alessio, Stewart J.

    2007-01-01

    Social scientists remain unsure as to whether divorce acts to alleviate domestic violence or whether ex-spouses become the targets of the displaced violence. Using data from the National Incident-Based Reporting System and the Census, this study investigates the relationship between the divorce rate and the domestic crime rate. The study…

  4. Toxoplasmosis in wild and domestic animals

    USDA-ARS?s Scientific Manuscript database

    Toxoplasma gondii is widely distributed in wild and domestic animals. The present chapter reviews toxoplasmosis in wild and domestic animals. Coverage in wild animal species is limited to confirmed cases of toxoplasmosis, cases with parasite isolation, cases with parasite detection by PCR, and exper...

  5. Domestic Violence and Poverty: Some Women's Experiences

    ERIC Educational Resources Information Center

    Slabbert, Ilze

    2017-01-01

    Purpose: Domestic violence poses a major challenge to social workers. Low-income families are significantly more likely to have to contend with domestic violence, as poverty can act as a fuelling factor in this type of conflict. The objective of this study was to explore and describe the experiences of low-income abused women. Method: A…

  6. Empowering Women with Domestic Violence Experience

    ERIC Educational Resources Information Center

    Anczewska, Marta; Roszczynska-Michta, Joanna; Waszkiewicz, Justyna; Charzynska, Katarzyna; Czabala, Czeslaw

    2012-01-01

    It is generally held that it has been only recently that domestic violence gained appropriate attention as a major social problem. However several approaches, drawn from different theories are applicable in explaining the origin of this negative phenomenon. It is well recognized that trauma of domestic violence has destructive impact on somatic…

  7. Domestic Violence during Pregnancy in India

    ERIC Educational Resources Information Center

    Mahapatro, Meerambika; Gupta, R. N.; Gupta, Vinay; Kundu, A. S.

    2011-01-01

    Domestic violence can result in many negative health consequences for women's health and well-being. Studies on domestic violence illustrate that abused women in various settings had increased health problems such as injury, chronic pain, gastrointestinal, and gynecological signs including sexually transmitted diseases, depression, and…

  8. Culture and Domestic Violence: Transforming Knowledge Development

    ERIC Educational Resources Information Center

    Bent-Goodley, Tricia B.

    2005-01-01

    Cultural competence continues to receive limited attention in domestic violence service provision from research to the evaluation of programs. Yet with changing demographics reflecting larger numbers of people of color and increasing needs for more effective responses, it is critical that we change the way we think about domestic violence. Using a…

  9. Domestic Violence against Married Women in Edirne

    ERIC Educational Resources Information Center

    Tokuc, Burcu; Ekuklu, Galip; Avcioglu, Serap

    2010-01-01

    The aim of this study is to assess the prevalence and risk factors of domestic violence against married women in Edirne, Turkey. This is a cross-sectional study which included a representative sample of the married women living in the Provincial Center of Edirne. The total past year prevalence of some forms of physical domestic violence is 34% in…

  10. Domestic Violence against Married Women in Cambodia

    ERIC Educational Resources Information Center

    Yount, Kathryn M.; Carrera, Jennifer S.

    2006-01-01

    We evaluate the effects of marital resources and early-life experiences on recent domestic violence and attitudes about wife abuse among 2,074 married Cambodian women. Household standard of living was negatively associated with physical domestic violence. Women with 8-13 fewer years of schooling than their husbands more often experienced physical…

  11. Service Learning in Domestic and International Settings

    ERIC Educational Resources Information Center

    Miller, Kari Knutson; Gonzalez, Amber M.

    2009-01-01

    This paper examines pre-service teacher outcomes associated with service learning in domestic and international settings. One group of upper-division undergraduate level pre service teachers participated in supervised experiences in domestic settings. A second group of upper-division undergraduate level pre-service teachers participated in…

  12. Domestic Options to Offshore Oil and Gas.

    ERIC Educational Resources Information Center

    Kash, Don E.

    1983-01-01

    The continuing controversey over offshore oil/gas has given impetus to searching for domestic energy alternatives. The need for and types of several alternative sources are discussed. Indicates that the United States needs to pursue both offshore and other domestic liquid-fuel sources if it is to avoid becoming increasingly dependent on imports.…

  13. Teaching about Domestic Violence: Strategies for Empowerment.

    ERIC Educational Resources Information Center

    Gardner, Saundra

    1993-01-01

    Offers the author's experiences in teaching a college-level domestic violence sociology course, presenting specific strategies and a description of the syllabus. The course presents a feminist analysis of domestic violence and examines how the patriarchal structure and ideology of society create and perpetuate violence. (SLD)

  14. Culture and Domestic Violence: Transforming Knowledge Development

    ERIC Educational Resources Information Center

    Bent-Goodley, Tricia B.

    2005-01-01

    Cultural competence continues to receive limited attention in domestic violence service provision from research to the evaluation of programs. Yet with changing demographics reflecting larger numbers of people of color and increasing needs for more effective responses, it is critical that we change the way we think about domestic violence. Using a…

  15. Domestic Violence against Married Women in Edirne

    ERIC Educational Resources Information Center

    Tokuc, Burcu; Ekuklu, Galip; Avcioglu, Serap

    2010-01-01

    The aim of this study is to assess the prevalence and risk factors of domestic violence against married women in Edirne, Turkey. This is a cross-sectional study which included a representative sample of the married women living in the Provincial Center of Edirne. The total past year prevalence of some forms of physical domestic violence is 34% in…

  16. Domestic Violence during Pregnancy in India

    ERIC Educational Resources Information Center

    Mahapatro, Meerambika; Gupta, R. N.; Gupta, Vinay; Kundu, A. S.

    2011-01-01

    Domestic violence can result in many negative health consequences for women's health and well-being. Studies on domestic violence illustrate that abused women in various settings had increased health problems such as injury, chronic pain, gastrointestinal, and gynecological signs including sexually transmitted diseases, depression, and…

  17. Pathobiology of avian influenza in domestic ducks

    USDA-ARS?s Scientific Manuscript database

    Domestic ducks are an important source of food and income in many parts of the world. The susceptibility of domestic ducks to avian influenza (AI) viruses varies depending on many factors, including the species and the age of the ducks, the virus strain, and management practices. Although wild wat...

  18. The Effect of Divorce on Domestic Crime

    ERIC Educational Resources Information Center

    Stolzenberg, Lisa; D'Alessio, Stewart J.

    2007-01-01

    Social scientists remain unsure as to whether divorce acts to alleviate domestic violence or whether ex-spouses become the targets of the displaced violence. Using data from the National Incident-Based Reporting System and the Census, this study investigates the relationship between the divorce rate and the domestic crime rate. The study…

  19. Responding to Domestic Violence against Women.

    ERIC Educational Resources Information Center

    Stalans, Loretta J.; Lurigio, Arthur J.

    1995-01-01

    Gives an overview of issues related to domestic violence against women as a social problem: changing responses from the legal system and the community over the course of history, possible causes of domestic violence against women, current perspectives and trends, prevalence, seriousness, and our response as a society. (LKS)

  20. The Near Eastern origin of cat domestication.

    PubMed

    Driscoll, Carlos A; Menotti-Raymond, Marilyn; Roca, Alfred L; Hupe, Karsten; Johnson, Warren E; Geffen, Eli; Harley, Eric H; Delibes, Miguel; Pontier, Dominique; Kitchener, Andrew C; Yamaguchi, Nobuyuki; O'brien, Stephen J; Macdonald, David W

    2007-07-27

    The world's domestic cats carry patterns of sequence variation in their genome that reflect a history of domestication and breed development. A genetic assessment of 979 domestic cats and their wild progenitors-Felis silvestris silvestris (European wildcat), F. s. lybica (Near Eastern wildcat), F. s. ornata (central Asian wildcat), F. s. cafra (southern African wildcat), and F. s. bieti (Chinese desert cat)-indicated that each wild group represents a distinctive subspecies of Felis silvestris. Further analysis revealed that cats were domesticated in the Near East, probably coincident with agricultural village development in the Fertile Crescent. Domestic cats derive from at least five founders from across this region, whose descendants were transported across the world by human assistance.

  1. The Near Eastern Origin of Cat Domestication

    PubMed Central

    Driscoll, Carlos A.; Menotti-Raymond, Marilyn; Roca, Alfred L.; Hupe, Karsten; Johnson, Warren E.; Geffen, Eli; Harley, Eric H.; Delibes, Miguel; Pontier, Dominique; Kitchener, Andrew C.; Yamaguchi, Nobuyuki; O’Brien, Stephen J.; Macdonald, David W.

    2017-01-01

    The world’s domestic cats carry patterns of sequence variation in their genome that reflect a history of domestication and breed development. A genetic assessment of 979 domestic cats and their wild progenitors—Felis silvestris silvestris (European wildcat), F. s. lybica (Near Eastern wildcat), F. s. ornata (central Asian wildcat), F. s. cafra (southern African wildcat), and F. s. bieti (Chinese desert cat)—indicated that each wild group represents a distinctive subspecies of Felis silvestris. Further analysis revealed that cats were domesticated in the Near East, probably coincident with agricultural village development in the Fertile Crescent. Domestic cats derive from at least five founders from across this region, whose descendants were transported across the world by human assistance. PMID:17600185

  2. Behavior genetics and the domestication of animals.

    PubMed

    Jensen, Per

    2014-02-01

    Across species, a similar suite of traits tends to develop in response to domestication, including modifications in behavior. Reduced fear and increased stress tolerance were central in early domestication, and many domestication-related behaviors may have developed as traits correlated to reduced fear. Genetic mechanisms involved in domestication of behavior can be investigated by using top-down or bottom-up approaches, either starting from the behavior variation and searching for underlying genes or finding selected loci and then attempting to identify the associated phenotypes. Combinations of these approaches have proven powerful, and examples of results from such studies are presented and discussed. This includes loci associated with tameness in foxes and dogs, as well as loci correlated with reduced aggression and increased sociality in chickens. Finally, some examples are provided on epigenetic mechanisms in behavior, and it is suggested that selection of favorable epigenetic variants may have been an important mechanism in domestication.

  3. 26 CFR 1.904(g)-1T - Overall domestic loss and the overall domestic loss account (temporary).

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 9 2010-04-01 2010-04-01 false Overall domestic loss and the overall domestic... United States § 1.904(g)-1T Overall domestic loss and the overall domestic loss account (temporary). (a) Overview of regulations. This section provides rules for determining a taxpayer's overall domestic...

  4. Domestic violence and child nutrition in Liberia.

    PubMed

    Sobkoviak, Rudina M; Yount, Kathryn M; Halim, Nafisa

    2012-01-01

    Domestic violence against women is endemic globally and is an important social problem in its own right. A compounding concern is the impact of domestic violence against mothers on the nutritional status of their children. Liberia is an apt setting to examine this understudied topic, given the poor nutritional status of young children, high rate of domestic violence against women, and prolonged period of conflict that included systematic sexual violence against women. We expected that maternal exposure to domestic violence would predict lower anthropometric z-scores and higher odds of stunting, wasting, and underweight in children less than five years. Using data from 2467 mother-child dyads in the 2007 Liberia Demographic and Health Survey (LDHS) undertaken between December 24, 2006 and April 19, 2007, we conducted descriptive and multivariate analyses to examine the total, unadjusted and adjusted associations of maternal exposure to domestic violence with these anthropometric measures in children. Maternal reports of sexual domestic violence in the prior year predicted lower adjusted z-scores for height-for-age and weight-for-height as well as higher odds of stunting and underweight. The findings underscore the needs to (1) enhance and enforce conventional and customary laws to prevent the occurrence of domestic violence; (2) treat maternal survivors of domestic violence and screen their children for nutritional deficits; (3) heighten awareness of the intergenerational implications especially of recent sexual domestic violence; and (4) clarify the biological and behavior pathways by which domestic violence may influence child growth, thereby mitigating early growth failure and its adverse implications into adulthood.

  5. Genetic architecture of domestication-related traits in maize

    USDA-ARS?s Scientific Manuscript database

    Strong directional selection occurred during the domestication of maize from its wild ancestor teosinte, reducing its genetic diversity, particularly at genes controlling domestication-related traits. Nevertheless, variability for some domestication-related traits is maintained in maize. The genet...

  6. Alcohol consumption and domestic violence against mothers.

    PubMed

    Sabia, Joseph J

    2004-12-01

    A recent high-profile murder case and the passage of the Unborn Victims of Violence Act have focused policymakers' attention on domestic violence against pregnant women and new mothers. The link between men's alcohol consumption and spousal abuse has led some to suggest that more stringent alcohol regulations could ameliorate domestic violence. (i) To examine the correlation between men's alcohol consumption and domestic violence against new mothers and test how sensitive the correlation is to assumptions about unobserved heterogeneity, (ii) To test whether higher liquor taxes and more stringent alcohol control regulations are associated with a lower incidence of domestic abuse. Using the Fragile Families and Child Wellbeing Study, I estimate ordinary least squares, bivariate probit, two-stage least squares, and fixed effects models to test the relationship between alcohol consumption and domestic violence. My findings suggest that while there is a strong positive association between men's alcohol consumption and the commission of domestic violence against new mothers, this correlation is highly sensitive to assumptions about unobservables. There is little evidence that higher liquor taxes or more stringent alcohol regulations will significantly reduce domestic violence. The empirical results suggest evidence for an 'unobserved bum hypothesis' That is, unobservable characteristics of the father may be correlated with both the likelihood that he abuses pregnant women (or new mothers) and that he drinks heavily. While the findings of this paper cannot rule out the possibility that men's alcohol consumption has some effect on domestic violence, there is little evidence to suggest that the impact is large in magnitude. Moreover, there is little evidence that higher liquor taxes or stricter alcohol supply regulations reduce the incidence of domestic abuse. However, greater policy heterogeneity across states and over time would be beneficial in further exploring this

  7. Counting Child Domestic Servants in Latin America

    PubMed Central

    Levison, Deborah; Langer, Anna

    2012-01-01

    This paper summarizes trends in the use of child domestic servants in six Latin American countries using IPUMS-International census samples for 1960 to 2000. Child domestics are among the most vulnerable of child workers, and the most invisible. They may be treated kindly and allowed to attend school, or they may be secluded in their employers’ home, overworked, verbally abused, beaten, and unable to leave or report their difficulties to kin. Estimates and imputations are based on labor force and relationship-to-head variables. We find that domestic service makes up a substantial fraction of girls’ employment in some countries. We also analyze trends in live-in versus live-out status and school enrollment of child domestic servants. While all child workers are disadvantaged in enrollment relative to non-workers, domestics are sometimes better off than non-domestic workers. In some samples, live-ins are more likely to go to school than live-out child domestics. In others, they are substantially worse off. PMID:22844164

  8. Resilience in Women who Experience Domestic Violence.

    PubMed

    Tsirigotis, Konstantinos; Łuczak, Joanna

    2017-08-12

    Violence in the family constitutes a serious social and psychological problem with harmful consequences leading, among others, to changes in the psychological functioning of the victim and, secondarily, also the perpetrator. The aim of this study was to examine resilience in women experiencing domestic violence. The "Ego Resiliency Scale" (ERS) was used to study the group of women suffering domestic violence. The study group included 52 women aged 30-65 years (mean age: 40.15) using assistance of the Crisis Intervention Centre due to experienced domestic violence. They most often reported suffering psychological and physical violence, with the husband or intimate partner being the most common perpetrator. Study women experiencing domestic violence obtained significantly lower scores on the ERS. The lowest scores on the ERS were achieved by women suffering paternal violence, while the highest - by women experiencing violence on the part of the intimate partner. Resilience of study women suffering domestic violence was lower than resilience of the general population, i.e. individuals not experiencing domestic violence. Suffered violence inflicted by the father exerted the greatest adverse impact on resilience. It seems advisable to consider resilience in the process of providing women experiencing domestic violence with psychosocial help.

  9. Phylogeography and Domestication of Chinese Swamp Buffalo

    PubMed Central

    Xie, Wen-Mei; Xu, Ping; Chang, Ti-Cheng; Liu, Li; Cheng, Feng; Zhang, Run-Feng; Lan, Xian-Yong; Chen, Hong; Lei, Chu-Zhao

    2013-01-01

    To further probe into whether swamp buffaloes were domesticated once or multiple times in China, this survey examined the mitochondrial DNA (mtDNA) Control Region (D-loop) diversity of 471 individuals representing 22 populations of 455 Chinese swamp buffaloes and 16 river buffaloes. Phylogenetic analysis revealed that Chinese swamp buffaloes could be divided into two distinct lineages, A and B, which were defined previously. Of the two lineages, lineage A was predominant across all populations. For predominant lineage A, Southwestern buffalo populations possess the highest genetic diversity among the three hypothesized domestication centers (Southeastern, Central, and Southwestern China), suggesting Southwestern China as the most likely location for the domestication of lineage A. However, a complex pattern of diversity is detected for the lineage B, preventing the unambiguous pinpointing of the exact place of domestication center and suggesting the presence of a long-term, strong gene flow among swamp buffalo populations caused by extensive migrations of buffaloes and frequent human movements along the Yangtze River throughout history. Our current study suggests that Southwestern China is the most likely domestication center for lineage A, and may have been a primary center of swamp buffalo domestication. More archaeological and genetic evidence is needed to show the process of domestication. PMID:23437167

  10. Phylogeography and domestication of Chinese swamp buffalo.

    PubMed

    Yue, Xiang-Peng; Li, Ran; Xie, Wen-Mei; Xu, Ping; Chang, Ti-Cheng; Liu, Li; Cheng, Feng; Zhang, Run-Feng; Lan, Xian-Yong; Chen, Hong; Lei, Chu-Zhao

    2013-01-01

    To further probe into whether swamp buffaloes were domesticated once or multiple times in China, this survey examined the mitochondrial DNA (mtDNA) Control Region (D-loop) diversity of 471 individuals representing 22 populations of 455 Chinese swamp buffaloes and 16 river buffaloes. Phylogenetic analysis revealed that Chinese swamp buffaloes could be divided into two distinct lineages, A and B, which were defined previously. Of the two lineages, lineage A was predominant across all populations. For predominant lineage A, Southwestern buffalo populations possess the highest genetic diversity among the three hypothesized domestication centers (Southeastern, Central, and Southwestern China), suggesting Southwestern China as the most likely location for the domestication of lineage A. However, a complex pattern of diversity is detected for the lineage B, preventing the unambiguous pinpointing of the exact place of domestication center and suggesting the presence of a long-term, strong gene flow among swamp buffalo populations caused by extensive migrations of buffaloes and frequent human movements along the Yangtze River throughout history. Our current study suggests that Southwestern China is the most likely domestication center for lineage A, and may have been a primary center of swamp buffalo domestication. More archaeological and genetic evidence is needed to show the process of domestication.

  11. The Genetic Architecture of Domestication in Animals

    PubMed Central

    Wright, Dominic

    2015-01-01

    Domestication has been essential to the progress of human civilization, and the process itself has fascinated biologists for hundreds of years. Domestication has led to a series of remarkable changes in a variety of plants and animals, in what is termed the “domestication phenotype.” In domesticated animals, this general phenotype typically consists of similar changes in tameness, behavior, size/morphology, color, brain composition, and adrenal gland size. This domestication phenotype is seen in a range of different animals. However, the genetic basis of these associated changes is still puzzling. The genes for these different traits tend to be grouped together in clusters in the genome, though it is still not clear whether these clusters represent pleiotropic effects, or are in fact linked clusters. This review focuses on what is currently known about the genetic architecture of domesticated animal species, if genes of large effect (often referred to as major genes) are prevalent in driving the domestication phenotype, and whether pleiotropy can explain the loci underpinning these diverse traits being colocated. PMID:26512200

  12. The Genetic Structure of Domestic Rabbits

    PubMed Central

    Carneiro, Miguel; Afonso, Sandra; Geraldes, Armando; Garreau, Hervé; Bolet, Gerard; Boucher, Samuel; Tircazes, Aurélie; Queney, Guillaume; Nachman, Michael W.; Ferrand, Nuno

    2011-01-01

    Understanding the genetic structure of domestic species provides a window into the process of domestication and motivates the design of studies aimed at making links between genotype and phenotype. Rabbits exhibit exceptional phenotypic diversity, are of great commercial value, and serve as important animal models in biomedical research. Here, we provide the first comprehensive survey of nucleotide polymorphism and linkage disequilibrium (LD) within and among rabbit breeds. We resequenced 16 genomic regions in population samples of both wild and domestic rabbits and additional 35 fragments in 150 rabbits representing six commonly used breeds. Patterns of genetic variation suggest a single origin of domestication in wild populations from France, supporting historical records that place rabbit domestication in French monasteries. Levels of nucleotide diversity both within and among breeds were ∼0.2%, but only 60% of the diversity present in wild populations from France was captured by domestic rabbits. Despite the recent origin of most breeds, levels of population differentiation were high (FST = 17.9%), but the majority of polymorphisms were shared and thus transferable among breeds. Coalescent simulations suggest that domestication began with a small founding population of less than 1,200 individuals. Taking into account the complex demographic history of domestication with two successive bottlenecks, two loci showed deviations that were consistent with artificial selection, including GPC4, which is known to be associated with growth rates in humans. Levels of diversity were not significantly different between autosomal and X-linked loci, providing no evidence for differential contributions of males and females to the domesticated gene pool. The structure of LD differed substantially within and among breeds. Within breeds, LD extends over large genomic distances. Markers separated by 400 kb typically showed r2 higher than 0.2, and some LD extended up to 3,200 kb

  13. Endogenous retroviruses in domestic animals.

    PubMed

    Garcia-Etxebarria, Koldo; Sistiaga-Poveda, Maialen; Jugo, Begoña Marina

    2014-08-01

    Endogenous retroviruses (ERVs) are genomic elements that are present in a wide range of vertebrates. Although the study of ERVs has been carried out mainly in humans and model organisms, recently, domestic animals have become important, and some species have begun to be analyzed to gain further insight into ERVs. Due to the availability of complete genomes and the development of new computer tools, ERVs can now be analyzed from a genome-wide viewpoint. In addition, more experimental work is being carried out to analyze the distribution, expression and interplay of ERVs within a host genome. Cats, cattle, chicken, dogs, horses, pigs and sheep have been scrutinized in this manner, all of which are interesting species in health and economic terms. Furthermore, several studies have noted differences in the number of endogenous retroviruses and in the variability of these elements among different breeds, as well as their expression in different tissues and the effects of their locations, which, in some cases, are near genes. These findings suggest a complex, intriguing relationship between ERVs and host genomes. In this review, we summarize the most important in silico and experimental findings, discuss their implications and attempt to predict future directions for the study of these genomic elements.

  14. Myofasciitis in the domestic ferret.

    PubMed

    Garner, M M; Ramsell, K; Schoemaker, N J; Sidor, I F; Nordhausen, R W; Bolin, S; Evermann, J F; Kiupel, M

    2007-01-01

    Since late 2003, an inflammatory disease of muscle and fascia has been diagnosed in several ferrets at Northwest ZooPath, and this report describes the condition in 17 ferrets. It is a disease of young ferrets, characterized by rapid onset of clinical signs, high fever, neutrophilic leukocytosis, treatment failure, and death (or euthanasia). Gross lesions include atrophy of skeletal muscle; red and white mottling and dilatation of the esophagus; and splenomegaly. Histologically, moderate to severe suppurative to pyogranulomatous inflammation is in the skeletal muscle and the fascia at multiple sites, including esophagus, heart, limbs, body wall, head, and lumbar regions. Myeloid hyperplasia of spleen and/or bone marrow also is a prominent feature. Ultrastructural lesions include mitochondrial swelling, intracellular edema, disruption of myofibrils and Z bands. Bacterial and viral cultures, electron microscopy, immunohistochemistry, and polymerase chain reaction were negative for a variety of infectious agents. The clinical presentation and distribution of lesions suggests that polymyositis in domestic ferrets is likely a distinct entity. The etiopathogenesis if this condition is not known.

  15. Comparative methylomics between domesticated and wild silkworms implies possible epigenetic influences on silkworm domestication.

    PubMed

    Xiang, Hui; Li, Xin; Dai, Fangyin; Xu, Xun; Tan, Anjiang; Chen, Lei; Zhang, Guojie; Ding, Yun; Li, Qiye; Lian, Jinmin; Willden, Andrew; Guo, Qiuhong; Xia, Qingyou; Wang, Jun; Wang, Wen

    2013-09-23

    In contrast to wild species, which have typically evolved phenotypes over long periods of natural selection, domesticates rapidly gained human-preferred agronomic traits in a relatively short-time frame via artificial selection. Under domesticated conditions, many traits can be observed that cannot only be due to environmental alteration. In the case of silkworms, aside from genetic divergence, whether epigenetic divergence played a role in domestication is an unanswered question. The silkworm is still an enigma in that it has two DNA methyltransferases (DNMT1 and DNMT2) but their functionality is unknown. Even in particular the functionality of the widely distributed DNMT1 remains unknown in insects in general. By embryonic RNA interference, we reveal that knockdown of silkworm Dnmt1 caused decreased hatchability, providing the first direct experimental evidence of functional significance of insect Dnmt1. In the light of this fact and those that DNA methylation is correlated with gene expression in silkworms and some agronomic traits in domesticated organisms are not stable, we comprehensively compare silk gland methylomes of 3 domesticated (Bombyx mori) and 4 wild (Bombyx mandarina) silkworms to identify differentially methylated genes between the two. We observed 2-fold more differentiated methylated cytosinces (mCs) in domesticated silkworms as compared to their wild counterparts, suggesting a trend of increasing DNA methylation during domestication. Further study of more domesticated and wild silkworms narrowed down the domesticates' epimutations, and we were able to identify a number of differential genes. One such gene showing demethyaltion in domesticates correspondently displays lower gene expression, and more interestingly, has experienced selective sweep. A methylation-increased gene seems to result in higher expression in domesticates and the function of its Drosophila homolog was previously found to be essential for cell volume regulation, indicating a

  16. Domestic violence: caring for a colleague.

    PubMed

    Weiss, Elaine

    2005-08-01

    Domestic abuse is a devastating social, legal, and health care problem of enormous proportions. Routine and multiple screenings by skilled health care providers, when conducted face-to-face, markedly increase the identification of physical, psychological, and sexual abuse. Patients are not the only ones who can benefit from domestic violence screening and intervention. This article describes the subtle signs of domestic abuse that might be observed in a colleague, explains how to initiate a conversation with a colleague, and suggests appropriate ways to offer support.

  17. Helping Children Exposed to Domestic Violence

    MedlinePlus

    ... is domestic violence between partners, there is often child abuse as well. Sometimes children get hurt accidentally. Children ... Definition Sleep Problems Bedwetting Posttraumatic Stress Disorder (PTSD) Child Abuse - The Hidden Bruises Depression in Children and Teens ...

  18. Domestic Violence: Protecting Yourself and Your Children

    MedlinePlus

    ... broken teeth.Ruptured eardrum.Cigarette burns.Bite marks.Rope burns.Welts (raised, red marks) on the body ... let known abuse go unreported. Contact your local law enforcement officials. Many communities have shelters for domestic ...

  19. Congenital and Genetic Disease in Domestic Animals

    ERIC Educational Resources Information Center

    Mulvihill, John J.

    1972-01-01

    Reviews observations on domestic animals that have led to the identification of environmental teratogens, and have provided insight into the pathogenesis of congenital defects and genetic diseases in man." (Author/AL)

  20. [Domestic animals in heraldry, volume1].

    PubMed

    Mache, Christa

    2003-01-01

    The author presents in the first part of her paper the historical background for the heraldic. In the coming seven papers she will discuss the use and position of different domestic animals in the heraldic.

  1. Plant domestication through an ecological lens.

    PubMed

    Milla, Rubén; Osborne, Colin P; Turcotte, Martin M; Violle, Cyrille

    2015-08-01

    Our understanding of domestication comes largely from archeology and genetics. Here, we advocate using current ecological theory and methodologies to provide novel insights into the causes and limitations of evolution under cultivation, as well as into the wider ecological impacts of domestication. We discuss the importance of natural selection under cultivation, that is, the forces promoting differences in Darwinian fitness between plants in crop populations and of constraints, that is, limitations of diverse nature that, given values for trait X, shorten the range of variation of trait Y, during the domestication process. Throughout this opinion paper, we highlight how ecology can yield insight into the effects of domestication on plant traits, on crop feedback over ecological processes, and on how species interactions develop in croplands.

  2. Whole mitochondrial genome sequencing of domestic horses reveals incorporation of extensive wild horse diversity during domestication.

    PubMed

    Lippold, Sebastian; Matzke, Nicholas J; Reissmann, Monika; Hofreiter, Michael

    2011-11-14

    DNA target enrichment by micro-array capture combined with high throughput sequencing technologies provides the possibility to obtain large amounts of sequence data (e.g. whole mitochondrial DNA genomes) from multiple individuals at relatively low costs. Previously, whole mitochondrial genome data for domestic horses (Equus caballus) were limited to only a few specimens and only short parts of the mtDNA genome (especially the hypervariable region) were investigated for larger sample sets. In this study we investigated whole mitochondrial genomes of 59 domestic horses from 44 breeds and a single Przewalski horse (Equus przewalski) using a recently described multiplex micro-array capture approach. We found 473 variable positions within the domestic horses, 292 of which are parsimony-informative, providing a well resolved phylogenetic tree. Our divergence time estimate suggests that the mitochondrial genomes of modern horse breeds shared a common ancestor around 93,000 years ago and no later than 38,000 years ago. A Bayesian skyline plot (BSP) reveals a significant population expansion beginning 6,000-8,000 years ago with an ongoing exponential growth until the present, similar to other domestic animal species. Our data further suggest that a large sample of wild horse diversity was incorporated into the domestic population; specifically, at least 46 of the mtDNA lineages observed in domestic horses (73%) already existed before the beginning of domestication about 5,000 years ago. Our study provides a window into the maternal origins of extant domestic horses and confirms that modern domestic breeds present a wide sample of the mtDNA diversity found in ancestral, now extinct, wild horse populations. The data obtained allow us to detect a population expansion event coinciding with the beginning of domestication and to estimate both the minimum number of female horses incorporated into the domestic gene pool and the time depth of the domestic horse mtDNA gene pool.

  3. Community-based domestic violence services.

    PubMed

    Saathoff, A J; Stoffel, E A

    1999-01-01

    Community-based domestic violence services have grown significantly since their emergence in the 1970s. Now more than 2,000 in number, domestic violence organizations have expanded their range of programs. In addition to crisis-oriented services, such as telephone hot lines and temporary shelter, many of these agencies provide legal, health, mental health, or vocational services or referrals, and assistance in finding housing, relocating, and planning for safety. Most recently, in response to increasing knowledge about the deleterious effects of exposure to domestic violence on children, community-based service providers have developed programs addressing children's mental health, health, educational, and safety needs. This article describes and analyzes trends in service delivery by these community-based organizations to children affected by domestic violence. It concludes that, although there has been significant growth in services, substantial segments of the target population still are not reached, and most organizations do not yet have a sufficient range of services to meet children's diverse needs. Challenges posed by inadequate funding, needs for specialized staffing, and a dearth of data on the efficacy of current intervention programs hamper domestic violence service providers' ability to meet children's needs. However, this article highlights promising new directions in service delivery. Community-based domestic violence organizations increasingly are using innovative strategies to address children's service needs. These agencies are expanding community outreach efforts and attempts to educate the public and professionals about domestic violence and children. In addition, these organizations are building important collaborative relationships with other agencies concerned with children's welfare, such as child protective services, law enforcement, schools, and health care facilities. These and related developments suggest cautious optimism that future

  4. Domestic Preference Policies in Federal Procurement

    DTIC Science & Technology

    1990-01-01

    PROGRAM PROJECT TASK WORK UNIT ELEMENT NO. NO. NO. ACCESSION NO. 11. TITLE (Include Security Classification) (UNCLAS SIFIED) Domestic Preference Policies ...obsolete SECURF:Y CLASSIcICATION O. THIS PAGE DOMESTIC PREFERENCE POLICIES IN FEDERAL PROCUREMENT By David Roy Francis B.A. May 1977, State University of...authored by Captain Charles W. Trainor). 3 such, they directly conflict with the liberal international trade policies advocated by the United States

  5. Domestic Violence: Battered Women Who Kill

    DTIC Science & Technology

    1988-08-15

    domestic violence within American society indicates a problem of serious dimensions. Spouse abuse has become the "norm" rather than the "exception" due to...pressing charges against their abusers and continue to participate in a violent marital relationship. Others decide the only solution to ending the violence ...domestic disturbance or arrest an abuser perpetuates the violence . Although there are laws to protect a woman against her abuser , she is often discouraged

  6. Domestic violence awareness in the perianesthesia setting.

    PubMed

    Scales, Barbara A

    2004-02-01

    Identification of victims of domestic violence is the first phase in the intervention of stopping the cycle of violence. Phase two is acting on that information and assisting the victims to help themselves. Nurses have the unique opportunity of being "present" at the bedside for the victim of abuse. This article reviews the signs and symptoms of domestic violence. Keen patient assessment tools and support can make a world of difference in the outcome of abuse.

  7. Electric Energy Security in the Domestic Theater

    DTIC Science & Technology

    2010-06-01

    Electric Energy Security in the Domestic Theater Dr Bill Waugaman N-NC Energy Security WG June 2010 This Briefing is Classified UNCLASSIFIED Report...2. REPORT TYPE 3. DATES COVERED 00-00-2010 to 00-00-2010 4. TITLE AND SUBTITLE Electric Energy Security in the Domestic Theater 5a. CONTRACT... Security & Sustainability (E2S2) Symposium & Exhibition held 14-17 June 2010 in Denver, CO. 14. ABSTRACT 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION

  8. Domestic violence: the challenge for nursing.

    PubMed

    Draucker, Claire Burke

    2002-01-01

    Domestic violence is a serious public health and human rights concern and an on-going challenge for nursing. This article provides an overview of the three major types of domestic violence: intimate partner abuse, child abuse, elder abuse. The scope, history, and health consequences of each type of violence are addressed. Despite advances in research, public awareness, legislative initiatives, and public policy, these types of interpersonal violence continue to affect millions worldwide.

  9. Domestic violence and the trauma surgeon.

    PubMed

    Guth, A A; Pachter, L

    2000-02-01

    Domestic violence has become increasingly recognized as a public health problem, and was declared a national epidemic by C. Everett Koop in 1992. In the United States, 1 to million women yearly suffer injuries due to domestic violence, and 30% to 50% of female homicides are committed by a present or former partner. The majority of these murder victims had either been seen in emergency rooms for prior domestic violence-related injuries, or had reported these injuries to the police. It is estimated that 50% of all acute injuries and 21% of all injuries in women requiring urgent surgery ar the result of partner abuse. Medline and current literature review. Health care professionals in the emergency room are an important contact with the victims of domestic violence, and timely identification and intervention can save lives. Overall, upwards of 35% of all emergency room visits by women are the result of domestic violence, whether due to acute injury, problems during pregnancy, or stress-related complaints. Unfortunately, domestic abuse is infrequently disclosed voluntarily by the patient, and often overlooked by the treating physician. Thus, the purpose of this review is to familiarize surgeons with the presentation and management of victims of this hidden epidemic.

  10. The domestication of Amazonia before European conquest.

    PubMed

    Clement, Charles R; Denevan, William M; Heckenberger, Michael J; Junqueira, André Braga; Neves, Eduardo G; Teixeira, Wenceslau G; Woods, William I

    2015-08-07

    During the twentieth century, Amazonia was widely regarded as relatively pristine nature, little impacted by human history. This view remains popular despite mounting evidence of substantial human influence over millennial scales across the region. Here, we review the evidence of an anthropogenic Amazonia in response to claims of sparse populations across broad portions of the region. Amazonia was a major centre of crop domestication, with at least 83 native species containing populations domesticated to some degree. Plant domestication occurs in domesticated landscapes, including highly modified Amazonian dark earths (ADEs) associated with large settled populations and that may cover greater than 0.1% of the region. Populations and food production expanded rapidly within land management systems in the mid-Holocene, and complex societies expanded in resource-rich areas creating domesticated landscapes with profound impacts on local and regional ecology. ADE food production projections support estimates of at least eight million people in 1492. By this time, highly diverse regional systems had developed across Amazonia where subsistence resources were created with plant and landscape domestication, including earthworks. This review argues that the Amazonian anthrome was no less socio-culturally diverse or populous than other tropical forested areas of the world prior to European conquest.

  11. The domestication of Amazonia before European conquest

    PubMed Central

    Clement, Charles R.; Denevan, William M.; Heckenberger, Michael J.; Junqueira, André Braga; Neves, Eduardo G.; Teixeira, Wenceslau G.; Woods, William I.

    2015-01-01

    During the twentieth century, Amazonia was widely regarded as relatively pristine nature, little impacted by human history. This view remains popular despite mounting evidence of substantial human influence over millennial scales across the region. Here, we review the evidence of an anthropogenic Amazonia in response to claims of sparse populations across broad portions of the region. Amazonia was a major centre of crop domestication, with at least 83 native species containing populations domesticated to some degree. Plant domestication occurs in domesticated landscapes, including highly modified Amazonian dark earths (ADEs) associated with large settled populations and that may cover greater than 0.1% of the region. Populations and food production expanded rapidly within land management systems in the mid-Holocene, and complex societies expanded in resource-rich areas creating domesticated landscapes with profound impacts on local and regional ecology. ADE food production projections support estimates of at least eight million people in 1492. By this time, highly diverse regional systems had developed across Amazonia where subsistence resources were created with plant and landscape domestication, including earthworks. This review argues that the Amazonian anthrome was no less socio-culturally diverse or populous than other tropical forested areas of the world prior to European conquest. PMID:26202998

  12. 20 CFR 404.1038 - Domestic employees under age 18.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 2 2011-04-01 2011-04-01 false Domestic employees under age 18. 404.1038... from Employment § 404.1038 Domestic employees under age 18. Domestic services you perform in a private... which you are under age 18 if domestic service is not your principal occupation. The exclusion...

  13. 26 CFR 1.1503(d)-2 - Domestic use.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 26 Internal Revenue 12 2010-04-01 2010-04-01 false Domestic use. 1.1503(d)-2 Section 1.1503(d)-2...) INCOME TAXES Administrative Provisions and Other Rules § 1.1503(d)-2 Domestic use. A domestic use of a... offset, directly or indirectly, the income of a domestic affiliate (other than the dual...

  14. 26 CFR 1.1503(d)-2 - Domestic use.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 26 Internal Revenue 12 2011-04-01 2011-04-01 false Domestic use. 1.1503(d)-2 Section 1.1503(d)-2...) INCOME TAXES (CONTINUED) Administrative Provisions and Other Rules § 1.1503(d)-2 Domestic use. A domestic... available to offset, directly or indirectly, the income of a domestic affiliate (other than the...

  15. 45 CFR 7.3 - Determination as to domestic rights.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 1 2011-10-01 2011-10-01 false Determination as to domestic rights. 7.3 Section 7... § 7.3 Determination as to domestic rights. The determination of the ownership of the domestic right... domestic right, title and interest in and to all inventions made by any Government employee (1)...

  16. 20 CFR 404.1038 - Domestic employees under age 18.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 2 2010-04-01 2010-04-01 false Domestic employees under age 18. 404.1038... from Employment § 404.1038 Domestic employees under age 18. Domestic services you perform in a private... which you are under age 18 if domestic service is not your principal occupation. The exclusion...

  17. 19 CFR 175.3 - Domestic interested party.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 2 2011-04-01 2011-04-01 false Domestic interested party. 175.3 Section 175.3... TREASURY (CONTINUED) PETITIONS BY DOMESTIC INTERESTED PARTIES Request for Classification, Appraised Value and Rate of Duty § 175.3 Domestic interested party. “Domestic interested party”, when used in...

  18. 19 CFR 122.88 - Aircraft carrying domestic (stopover) passengers.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 1 2011-04-01 2011-04-01 false Aircraft carrying domestic (stopover) passengers... Passengers § 122.88 Aircraft carrying domestic (stopover) passengers. Airlines that commingle domestic... domestic (stopover) passengers must be transported on U.S.-registered aircraft, or...

  19. 34 CFR 7.3 - Determination as to domestic rights.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Determination as to domestic rights. 7.3 Section 7.3... domestic rights. The determination of the ownership of the domestic right, title, and interest in and to an... follows: (a) The Government as represented by the Secretary shall obtain the entire domestic right,...

  20. 19 CFR 175.3 - Domestic interested party.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 2 2010-04-01 2010-04-01 false Domestic interested party. 175.3 Section 175.3... TREASURY (CONTINUED) PETITIONS BY DOMESTIC INTERESTED PARTIES Request for Classification, Appraised Value and Rate of Duty § 175.3 Domestic interested party. “Domestic interested party”, when used in...

  1. Domestic Violence: Issues and Dynamics. Informal Series No. 7.

    ERIC Educational Resources Information Center

    D'Oyley, Vincent, Ed.

    The proceedings from the Toronto Conference present an overview of domestic violence, the roles of the police and judicial system, male/female relationships in domestic violence, the clinical treatment of domestic violence, domestic violence and education, social services, and a bibliography on battered wives. The multi-dimensionality of, and the…

  2. Domestic Violence: Issues and Dynamics. Informal Series No. 7.

    ERIC Educational Resources Information Center

    D'Oyley, Vincent, Ed.

    The proceedings from the Toronto Conference present an overview of domestic violence, the roles of the police and judicial system, male/female relationships in domestic violence, the clinical treatment of domestic violence, domestic violence and education, social services, and a bibliography on battered wives. The multi-dimensionality of, and the…

  3. Wild genius - domestic fool? Spatial learning abilities of wild and domestic guinea pigs.

    PubMed

    Lewejohann, Lars; Pickel, Thorsten; Sachser, Norbert; Kaiser, Sylvia

    2010-03-25

    Domestic animals and their wild relatives differ in a wide variety of aspects. The process of domestication of the domestic guinea pig (Cavia aperea f. porcellus), starting at least 4500 years ago, led to changes in the anatomy, physiology, and behaviour compared with their wild relative, the wild cavy, Cavia aperea. Although domestic guinea pigs are widely used as a laboratory animal, learning and memory capabilities are often disregarded as being very scarce. Even less is known about learning and memory of wild cavies. In this regard, one striking domestic trait is a reduction in relative brain size, which in the domesticated form of the guinea pig amounts to 13%. However, the common belief, that such a reduction of brain size in the course of domestication of different species is accomplished by less learning capabilities is not at all very well established in the literature. Indeed, domestic animals might also even outperform their wild conspecifics taking advantage of their adaptation to a man-made environment.In our study we compared the spatial learning abilities of wild and domestic guinea pigs. We expected that the two forms are different regarding their learning performance possibly related to the process of domestication. Therefore wild cavies as well as domestic guinea pigs of both sexes, aged 35 to 45 days, were tested in the Morris water maze to investigate their ability of spatial learning. Both, wild cavies and domestic guinea pigs were able to learn the task, proving the water maze to be a suitable test also for wild cavies. Regarding the speed of learning, male as well as female domestic guinea pigs outperformed their wild conspecifics significantly. Interestingly, only domestic guinea pigs showed a significant spatial association of the platform position, while other effective search strategies were used by wild cavies. The results demonstrate that domestic guinea pigs do not at all perform worse than their wild relatives in tests of spatial

  4. Wild genius - domestic fool? Spatial learning abilities of wild and domestic guinea pigs

    PubMed Central

    2010-01-01

    Background Domestic animals and their wild relatives differ in a wide variety of aspects. The process of domestication of the domestic guinea pig (Cavia aperea f. porcellus), starting at least 4500 years ago, led to changes in the anatomy, physiology, and behaviour compared with their wild relative, the wild cavy, Cavia aperea. Although domestic guinea pigs are widely used as a laboratory animal, learning and memory capabilities are often disregarded as being very scarce. Even less is known about learning and memory of wild cavies. In this regard, one striking domestic trait is a reduction in relative brain size, which in the domesticated form of the guinea pig amounts to 13%. However, the common belief, that such a reduction of brain size in the course of domestication of different species is accomplished by less learning capabilities is not at all very well established in the literature. Indeed, domestic animals might also even outperform their wild conspecifics taking advantage of their adaptation to a man-made environment. In our study we compared the spatial learning abilities of wild and domestic guinea pigs. We expected that the two forms are different regarding their learning performance possibly related to the process of domestication. Therefore wild cavies as well as domestic guinea pigs of both sexes, aged 35 to 45 days, were tested in the Morris water maze to investigate their ability of spatial learning. Results Both, wild cavies and domestic guinea pigs were able to learn the task, proving the water maze to be a suitable test also for wild cavies. Regarding the speed of learning, male as well as female domestic guinea pigs outperformed their wild conspecifics significantly. Interestingly, only domestic guinea pigs showed a significant spatial association of the platform position, while other effective search strategies were used by wild cavies. Conclusion The results demonstrate that domestic guinea pigs do not at all perform worse than their wild

  5. Domesticated animals and human infectious diseases of zoonotic origins: domestication time matters.

    PubMed

    Morand, Serge; McIntyre, K Marie; Baylis, Matthew

    2014-06-01

    The rate of emergence for emerging infectious diseases has increased dramatically over the last century, and research findings have implicated wildlife as an importance source of novel pathogens. However, the role played by domestic animals as amplifiers of pathogens emerging from the wild could also be significant, influencing the human infectious disease transmission cycle. The impact of domestic hosts on human disease emergence should therefore be ascertained. Here, using three independent datasets we showed positive relationships between the time since domestication of the major domesticated mammals and the total number of parasites or infectious diseases they shared with humans. We used network analysis, to better visualize the overall interactions between humans and domestic animals (and amongst animals) and estimate which hosts are potential sources of parasites/pathogens for humans (and for all other hosts) by investigating the network architecture. We used centrality, a measure of the connection amongst each host species (humans and domestic animals) in the network, through the sharing of parasites/pathogens, where a central host (i.e. high value of centrality) is the one that is infected by many parasites/pathogens that infect many other hosts in the network. We showed that domesticated hosts that were associated a long time ago with humans are also the central ones in the network and those that favor parasites/pathogens transmission not only to humans but also to all other domesticated animals. These results urge further investigation of the diversity and origin of the infectious diseases of domesticated animals in their domestication centres and the dispersal routes associated with human activities. Such work may help us to better understand how domesticated animals have bridged the epidemiological gap between humans and wildlife.

  6. Domestic violence in Iranian infertile women

    PubMed Central

    Sheikhan, Zohre; Ozgoli, Giti; Azar, Mahyar; Alavimajd, Hamid

    2014-01-01

    Background: Millions of men and women suffer from infertility worldwide. In many cultures, infertile women are at risk of social and emotional problems. Infertility may affect the public health in many countries. Domestic violence is the intentional use of physical force, power or threat against oneself, another person or another group or community which leads to injury, death, mental harm, lack of development or deprivation. This study aimed to assess the prevalence of domestic violence against infertile women who referred to the infertility centres of Tehran, Iran in 2011. Methods: This was cross- sectional descriptive study conducted on 400 infertile women who were selected through convenient sampling method. The questionnaire used in this study included two sections: a demographic section with questions about demographic characteristics of the infertile women and their husbands; and the domestic violence questionnaire with questions about physical, emotional and sexual violence. Data were analysed by SPSS16; descriptive statistics, Spearman’s test, t- test, one-way analysis of variance (ANOVA) and logistic regression were used for data analysis. Results: Four hundred women with the average age of 30.50 ± 6.16 years participated in the study; of whom, 34.7% experienced domestic violence physical violence (5.3%), emotional violence (74.3%) and sexual violence (47.3%). Domestic violence was significantly associated with unwanted marriage, number of IVFs, drug abuse, emotional status of the women, smoking and addiction or drug abuse of the spouse, mental and physical diseases of the husband (p< 0.05). Conclusion: Many of the current problems in this society, particularly in families are due to the transition of the society from a traditional model to a modern one. The majority of the infertile women experience violence in Iran. Domestic violence against infertile women is a problem that should not be ignored. Clinicians should identify abused women. Providing

  7. Evolutionary Genomics of Peach and Almond Domestication.

    PubMed

    Velasco, Dianne; Hough, Josh; Aradhya, Mallikarjuna; Ross-Ibarra, Jeffrey

    2016-12-07

    The domesticated almond [Prunus dulcis (L.) Batsch] and peach [P. persica (Mill.) D. A. Webb] originated on opposite sides of Asia and were independently domesticated ∼5000 yr ago. While interfertile, they possess alternate mating systems and differ in a number of morphological and physiological traits. Here, we evaluated patterns of genome-wide diversity in both almond and peach to better understand the impacts of mating system, adaptation, and domestication on the evolution of these taxa. Almond has around seven times the genetic diversity of peach, and high genome-wide [Formula: see text] values support their status as separate species. We estimated a divergence time of ∼8 MYA (million years ago), coinciding with an active period of uplift in the northeast Tibetan Plateau and subsequent Asian climate change. We see no evidence of a bottleneck during domestication of either species, but identify a number of regions showing signatures of selection during domestication and a significant overlap in candidate regions between peach and almond. While we expected gene expression in fruit to overlap with candidate selected regions, instead we find enrichment for loci highly differentiated between the species, consistent with recent fossil evidence suggesting fruit divergence long preceded domestication. Taken together, this study tells us how closely related tree species evolve and are domesticated, the impact of these events on their genomes, and the utility of genomic information for long-lived species. Further exploration of this data will contribute to the genetic knowledge of these species and provide information regarding targets of selection for breeding application, and further the understanding of evolution in these species. Copyright © 2016 Velasco et al.

  8. Evolutionary Genomics of Peach and Almond Domestication

    PubMed Central

    Velasco, Dianne; Hough, Josh; Aradhya, Mallikarjuna; Ross-Ibarra, Jeffrey

    2016-01-01

    The domesticated almond [Prunus dulcis (L.) Batsch] and peach [P. persica (Mill.) D. A. Webb] originated on opposite sides of Asia and were independently domesticated ∼5000 yr ago. While interfertile, they possess alternate mating systems and differ in a number of morphological and physiological traits. Here, we evaluated patterns of genome-wide diversity in both almond and peach to better understand the impacts of mating system, adaptation, and domestication on the evolution of these taxa. Almond has around seven times the genetic diversity of peach, and high genome-wide FST values support their status as separate species. We estimated a divergence time of ∼8 MYA (million years ago), coinciding with an active period of uplift in the northeast Tibetan Plateau and subsequent Asian climate change. We see no evidence of a bottleneck during domestication of either species, but identify a number of regions showing signatures of selection during domestication and a significant overlap in candidate regions between peach and almond. While we expected gene expression in fruit to overlap with candidate selected regions, instead we find enrichment for loci highly differentiated between the species, consistent with recent fossil evidence suggesting fruit divergence long preceded domestication. Taken together, this study tells us how closely related tree species evolve and are domesticated, the impact of these events on their genomes, and the utility of genomic information for long-lived species. Further exploration of this data will contribute to the genetic knowledge of these species and provide information regarding targets of selection for breeding application, and further the understanding of evolution in these species. PMID:27707802

  9. [Intracranial allometry and craniologic changes during domestication of silver foxes].

    PubMed

    Trut, L N; Dzerzhinskiĭ, F Ia; Nikol'skiĭ, V S

    1991-09-01

    Skull proportions and pattern of intracranial allometry in farm foxes of bred domesticated populations are compared. Changes in skull measurements and bivariate allometry are observed in domesticated males, which in this respect become closer to domesticated females. The changes in the allometry pattern in the domesticated population points to deep modifications of regulatory ontogenetic processes which underlie the allometric growth. However, morphological changes in domesticated foxes cannot be only accounted for by allometry.

  10. From wild animals to domestic pets, an evolutionary view of domestication.

    PubMed

    Driscoll, Carlos A; Macdonald, David W; O'Brien, Stephen J

    2009-06-16

    Artificial selection is the selection of advantageous natural variation for human ends and is the mechanism by which most domestic species evolved. Most domesticates have their origin in one of a few historic centers of domestication as farm animals. Two notable exceptions are cats and dogs. Wolf domestication was initiated late in the Mesolithic when humans were nomadic hunter-gatherers. Those wolves less afraid of humans scavenged nomadic hunting camps and over time developed utility, initially as guards warning of approaching animals or other nomadic bands and soon thereafter as hunters, an attribute tuned by artificial selection. The first domestic cats had limited utility and initiated their domestication among the earliest agricultural Neolithic settlements in the Near East. Wildcat domestication occurred through a self-selective process in which behavioral reproductive isolation evolved as a correlated character of assortative mating coupled to habitat choice for urban environments. Eurasian wildcats initiated domestication and their evolution to companion animals was initially a process of natural, rather than artificial, selection over time driven during their sympatry with forbear wildcats.

  11. From wild animals to domestic pets, an evolutionary view of domestication

    PubMed Central

    Driscoll, Carlos A.; Macdonald, David W.; O'Brien, Stephen J.

    2009-01-01

    Artificial selection is the selection of advantageous natural variation for human ends and is the mechanism by which most domestic species evolved. Most domesticates have their origin in one of a few historic centers of domestication as farm animals. Two notable exceptions are cats and dogs. Wolf domestication was initiated late in the Mesolithic when humans were nomadic hunter-gatherers. Those wolves less afraid of humans scavenged nomadic hunting camps and over time developed utility, initially as guards warning of approaching animals or other nomadic bands and soon thereafter as hunters, an attribute tuned by artificial selection. The first domestic cats had limited utility and initiated their domestication among the earliest agricultural Neolithic settlements in the Near East. Wildcat domestication occurred through a self-selective process in which behavioral reproductive isolation evolved as a correlated character of assortative mating coupled to habitat choice for urban environments. Eurasian wildcats initiated domestication and their evolution to companion animals was initially a process of natural, rather than artificial, selection over time driven during their sympatry with forbear wildcats. PMID:19528637

  12. The dead end of domestic violence: spotlight on children's narratives during forensic investigations following domestic homicide.

    PubMed

    Katz, Carmit

    2014-12-01

    The current study provides an in-depth exploration of the narratives of children who witnessed their father killing their mother. This exploration was conducted using a thematic analysis of the children's forensic interviews based on seven investigative interviews that were conducted with children following the domestic homicide. Investigative interviews were selected for study only for substantiated cases and only if the children disclosed the domestic homicide. All of the investigative interviews were conducted within 24h of the domestic homicide. Thematic analysis revealed the following four key categories: the domestic homicide as the dead end of domestic violence, what I did when daddy killed mommy, that one time that daddy killed mommy, and mommy will feel better and will go back home. The discussion examines the multiple layers of this phenomenon as revealed in the children's narratives and its consequences for professionals within the legal and clinical contexts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Human tuberculosis predates domestication in ancient Syria.

    PubMed

    Baker, Oussama; Lee, Oona Y-C; Wu, Houdini H T; Besra, Gurdyal S; Minnikin, David E; Llewellyn, Gareth; Williams, Christopher M; Maixner, Frank; O'Sullivan, Niall; Zink, Albert; Chamel, Bérénice; Khawam, Rima; Coqueugniot, Eric; Helmer, Daniel; Le Mort, Françoise; Perrin, Pascale; Gourichon, Lionel; Dutailly, Bruno; Pálfi, György; Coqueugniot, Hélène; Dutour, Olivier

    2015-06-01

    The question of pre-neolithic tuberculosis is still open in paleopathological perspective. One of the major interests is to explore what type of infection could have existed around the early stage of animal domestication. Paleopathological lesions evoking skeletal TB were observed on five human skeletons coming from two PPNB sites in Syria, which belongs to the geographical cradle of agriculture. These sites represent respectively pre-domestication phase (Dja'de el Mughara, Northern Syria, 8800-8300 BCE cal.) and early domestication phase (Tell Aswad, Southern Syria, 8200-7600 BCE cal.). MicroCT scan analyses were performed on two specimens (one per site) and revealed microscopic changes in favor of TB infection. Detection of lipid biomarkers is positive for two specimens (one per site). Initial molecular analysis further indicates the presence of TB in one individual from Dja'de. Interestingly, no morphological evidence of TB was observed on animal remains of wild and newly domesticated species, discovered in these sites. These observations strongly suggest the presence of human tuberculosis before domestication and at its early stages.

  14. Early Holocene chicken domestication in northern China

    PubMed Central

    Xiang, Hai; Gao, Jianqiang; Yu, Baoquan; Zhou, Hui; Cai, Dawei; Zhang, Youwen; Chen, Xiaoyong; Wang, Xi; Hofreiter, Michael; Zhao, Xingbo

    2014-01-01

    Chickens represent by far the most important poultry species, yet the number, locations, and timings of their domestication have remained controversial for more than a century. Here we report ancient mitochondrial DNA sequences from the earliest archaeological chicken bones from China, dating back to ∼10,000 B.P. The results clearly show that all investigated bones, including the oldest from the Nanzhuangtou site, are derived from the genus Gallus, rather than any other related genus, such as Phasianus. Our analyses also suggest that northern China represents one region of the earliest chicken domestication, possibly dating as early as 10,000 y B.P. Similar to the evidence from pig domestication, our results suggest that these early domesticated chickens contributed to the gene pool of modern chicken populations. Moreover, our results support the idea that multiple members of the genus Gallus, specifically Gallus gallus and Gallus sonneratii contributed to the gene pool of the modern domestic chicken. Our results provide further support for the growing evidence of an early mixed agricultural complex in northern China. PMID:25422439

  15. Reshaping of the maize transcriptome by domestication

    PubMed Central

    Swanson-Wagner, Ruth; Briskine, Roman; Schaefer, Robert; Hufford, Matthew B.; Ross-Ibarra, Jeffrey; Myers, Chad L.; Tiffin, Peter; Springer, Nathan M.

    2012-01-01

    Through domestication, humans have substantially altered the morphology of Zea mays ssp. parviglumis (teosinte) into the currently recognizable maize. This system serves as a model for studying adaptation, genome evolution, and the genetics and evolution of complex traits. To examine how domestication has reshaped the transcriptome of maize seedlings, we used expression profiling of 18,242 genes for 38 diverse maize genotypes and 24 teosinte genotypes. We detected evidence for more than 600 genes having significantly different expression levels in maize compared with teosinte. Moreover, more than 1,100 genes showed significantly altered coexpression profiles, reflective of substantial rewiring of the transcriptome since domestication. The genes with altered expression show a significant enrichment for genes previously identified through population genetic analyses as likely targets of selection during maize domestication and improvement; 46 genes previously identified as putative targets of selection also exhibit altered expression levels and coexpression relationships. We also identified 45 genes with altered, primarily higher, expression in inbred relative to outcrossed teosinte. These genes are enriched for functions related to biotic stress and may reflect responses to the effects of inbreeding. This study not only documents alterations in the maize transcriptome following domestication, identifying several genes that may have contributed to the evolution of maize, but highlights the complementary information that can be gained by combining gene expression with population genetic analyses. PMID:22753482

  16. Domestic utility attitudes toward foreign uranium supply

    SciTech Connect

    Not Available

    1981-06-01

    The current embargo on the enrichment of foreign-origin uranium for use in domestic utilization facilities is scheduled to be removed in 1984. The pending removal of this embargo, complicated by a depressed worldwide market for uranium, has prompted consideration of a new or extended embargo within the US Government. As part of its on-going data collection activities, Nuclear Resources International (NRI) has surveyed 50 domestic utility/utility holding companies (representing 60 lead operator-utilities) on their foreign uranium purchase strategies and intentions. The most recent survey was conducted in early May 1981. A number of qualitative observations were made during the course of the survey. The major observations are: domestic utility views toward foreign uranium purchase are dynamic; all but three utilities had some considered foreign purchase strategy; some utilities have problems with buying foreign uranium from particular countries; an inducement is often required by some utilities to buy foreign uranium; opinions varied among utilities concerning the viability of the domestic uranium industry; and many utilities could have foreign uranium fed through their domestic uranium contracts (indirect purchases). The above observations are expanded in the final section of the report. However, it should be noted that two of the observations are particularly important and should be seriously considered in formulation of foreign uranium import restrictions. These important observations are the dynamic nature of the subject matter and the potentially large and imbalanced effect the indirect purchases could have on utility foreign uranium procurement.

  17. Storytelling and story testing in domestication

    PubMed Central

    Gerbault, Pascale; Allaby, Robin G.; Boivin, Nicole; Rudzinski, Anna; Grimaldi, Ilaria M.; Pires, J. Chris; Climer Vigueira, Cynthia; Dobney, Keith; Gremillion, Kristen J.; Barton, Loukas; Arroyo-Kalin, Manuel; Purugganan, Michael D.; Rubio de Casas, Rafael; Bollongino, Ruth; Burger, Joachim; Fuller, Dorian Q.; Bradley, Daniel G.; Balding, David J.; Richerson, Peter J.; Gilbert, M. Thomas P.; Larson, Greger; Thomas, Mark G.

    2014-01-01

    The domestication of plants and animals marks one of the most significant transitions in human, and indeed global, history. Traditionally, study of the domestication process was the exclusive domain of archaeologists and agricultural scientists; today it is an increasingly multidisciplinary enterprise that has come to involve the skills of evolutionary biologists and geneticists. Although the application of new information sources and methodologies has dramatically transformed our ability to study and understand domestication, it has also generated increasingly large and complex datasets, the interpretation of which is not straightforward. In particular, challenges of equifinality, evolutionary variance, and emergence of unexpected or counter-intuitive patterns all face researchers attempting to infer past processes directly from patterns in data. We argue that explicit modeling approaches, drawing upon emerging methodologies in statistics and population genetics, provide a powerful means of addressing these limitations. Modeling also offers an approach to analyzing datasets that avoids conclusions steered by implicit biases, and makes possible the formal integration of different data types. Here we outline some of the modeling approaches most relevant to current problems in domestication research, and demonstrate the ways in which simulation modeling is beginning to reshape our understanding of the domestication process. PMID:24753572

  18. Prevalence of Domestic Violence Among Trauma Patients.

    PubMed

    Joseph, Bellal; Khalil, Mazhar; Zangbar, Bardiya; Kulvatunyou, Narong; Orouji, Tahereh; Pandit, Viraj; O'Keeffe, Terence; Tang, Andrew; Gries, Lynn; Friese, Randall S; Rhee, Peter; Davis, James W

    2015-12-01

    Domestic violence is an extremely underreported crime and a growing social problem in the United States. However, the true burden of the problem remains unknown. To assess the reported prevalence of domestic violence among trauma patients. A 6-year (2007-2012) retrospective analysis of the prospectively maintained National Trauma Data Bank. Trauma patients who experienced domestic violence and who presented to trauma centers participating in the National Trauma Data Bank were identified using International Classification of Diseases, Ninth Revision diagnosis codes (995.80-995.85, 995.50, 995.52-995.55, and 995.59) and E codes (E967.0-E967.9). Patients were stratified by age into 3 groups: children (≤18 years), adults (19-54 years), and elderly patients (≥55 years). Trend analysis was performed on April 10, 2014, to assess the reported prevalence of domestic violence over the years. Trauma patients presenting to trauma centers participating in the National Trauma Data Bank. To assess the reported prevalence of domestic violence among trauma patients. A total of 16 575 trauma patients who experienced domestic violence were included. Of these trauma patients, 10 224 (61.7%) were children, 5503 (33.2%) were adults, and 848 (5.1%) were elderly patients. The mean (SD) age was 15.9 (20.6), the mean (SD) Injury Severity Score was 10.9 (9.6), and 8397 (50.7%) were male patients. Head injuries (46.8% of patients) and extremity fractures (31.2% of patients) were the most common injuries. A total of 12 515 patients (75.1%) were discharged home, and the overall mortality rate was 5.9% (n = 980). The overall reported prevalence of domestic violence among trauma patients was 5.7 cases per 1000 trauma center discharges. The prevalence of domestic violence increased among children (14.0 cases per 1000 trauma center discharges in 2007 to 18.5 case per 1000 trauma center discharges in 2012; P = .001) and adults (3.2 cases per 1000 discharges in 2007 to 4.5 cases per

  19. Domestic violence surveillance system:a model

    PubMed Central

    Espinosa, Rafael; Gutiérrez, María Isabel; Mena-Muñoz, Jorge Humberto; Córdoba, Patricia

    2010-01-01

    Objective To develop a domestic violence surveillance system. Material and Methods The strategies included implementation of a standard digitalized reporting and analysis system along with advocacy with community decision makers, strengthening inter-institutional attention networks, consultation for constructing internal flow charts, sensitizing and training network teams in charge of providing health care in cases of domestic violence and supporting improved public policy prevention initiatives. Results A total of 6 893 cases were observed using 2004 and 2005 surveillance system data. The system reports that 80% of the affected were women, followed by 36% children under 14 years. The identified aggressors were mainly females' partners. The system was useful for improving victim services. Conclusions Findings indicate that significant gains were made in facilitating the attention and treatment of victims of domestic violence, improving the procedural response process and enhancing the quality of information provided to policy-making bodies. PMID:18373003

  20. The domestication and evolutionary ecology of apples.

    PubMed

    Cornille, Amandine; Giraud, Tatiana; Smulders, Marinus J M; Roldán-Ruiz, Isabel; Gladieux, Pierre

    2014-02-01

    The cultivated apple is a major fruit crop in temperate zones. Its wild relatives, distributed across temperate Eurasia and growing in diverse habitats, represent potentially useful sources of diversity for apple breeding. We review here the most recent findings on the genetics and ecology of apple domestication and its impact on wild apples. Genetic analyses have revealed a Central Asian origin for cultivated apple, together with an unexpectedly large secondary contribution from the European crabapple. Wild apple species display strong population structures and high levels of introgression from domesticated apple, and this may threaten their genetic integrity. Recent research has revealed a major role of hybridization in the domestication of the cultivated apple and has highlighted the value of apple as an ideal model for unraveling adaptive diversification processes in perennial fruit crops. We discuss the implications of this knowledge for apple breeding and for the conservation of wild apples. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Embryo transfer in domestic South American camelids.

    PubMed

    Sumar, Julio B

    2013-01-10

    Intraspecific and interspecific embryo transfer in domestic South American camelids is developing into a well-established technique. Reports reveal many benefits of using reproductive biotechnologies to allow rapid propagation of alpacas and llamas of high genetic merit (e.g., high fiber quality, preserve color variation). The objective of this review is to provide up-to-date information about embryo transfer in domestic South American camelids. Specific information is provided on criteria for male selection, donor and recipient synchronization, the practice of single- vs. super-ovulation protocols, embryo recovery and transfer techniques, advances in cryopreservation of embryos, results of intra- and inter-specific transfer, and the future of the embryo transfer in domestic South American camelids.

  2. Dealing with the effects of domestic violence.

    PubMed

    Gibbons, Lynda

    2011-07-01

    Domestic violence has a lasting and damaging effect on the lives of thousands of women, men and children in Ireland and the UK. Yet, healthcare services are il equipped to deal with the victims of physical, sexual, and psychological abuse, many of whom present to emergency departments because they need help and support. As this article discusses, healthcare staff have a responsibility of care for such people. They must be able to recognise and respond to the signs of domestic abuse, and refer people who experience it to the appropriate organisations.

  3. The Gender Wage Gap and Domestic Violence.

    PubMed

    Aizer, Anna

    2010-09-01

    Three quarters of all violence against women is perpetrated by domestic partners. This study exploits exogenous changes in the demand for labor in female-dominated industries to estimate the impact of the male-female wage gap on domestic violence. Decreases in the wage gap reduce violence against women, consistent with a household bargaining model. These findings shed new light on the health production process as well as observed income gradients in health and suggest that in addition to addressing concerns of equity and efficiency, pay parity can also improve the health of American women via reductions in violence.

  4. Domestic violence among Iraqi refugees in Syria.

    PubMed

    Tappis, Hannah; Biermann, Elizabeth; Glass, Nancy; Tileva, Margarita; Doocy, Shannon

    2012-01-01

    A domestic violence questionnaire was administered to 701 adult females in a sample of 813 Iraqi households in Syria; unmarried women and women whose husbands were away were excluded, yielding a final sample of 486. Lifetime physical, verbal, or emotional abuse was reported by 30%, and approximately 20% experienced abuse within the past year. Non-Damascus residence, children <18 years in the household, no financial challenges upon arrival, and borrowing money in Syria were associated with increased risk of domestic violence within the past year. Support services are inadequate and should be expanded; and longer-term prevention measures also should be implemented.

  5. Multidisciplinary perspectives on banana (Musa spp.) domestication

    PubMed Central

    Perrier, Xavier; De Langhe, Edmond; Donohue, Mark; Lentfer, Carol; Vrydaghs, Luc; Bakry, Frédéric; Carreel, Françoise; Hippolyte, Isabelle; Horry, Jean-Pierre; Jenny, Christophe; Lebot, Vincent; Risterucci, Ange-Marie; Tomekpe, Kodjo; Doutrelepont, Hugues; Ball, Terry; Manwaring, Jason; de Maret, Pierre; Denham, Tim

    2011-01-01

    Original multidisciplinary research hereby clarifies the complex geodomestication pathways that generated the vast range of banana cultivars (cvs). Genetic analyses identify the wild ancestors of modern-day cvs and elucidate several key stages of domestication for different cv groups. Archaeology and linguistics shed light on the historical roles of people in the movement and cultivation of bananas from New Guinea to West Africa during the Holocene. The historical reconstruction of domestication processes is essential for breeding programs seeking to diversify and improve banana cvs for the future. PMID:21730145

  6. Multidisciplinary perspectives on banana (Musa spp.) domestication.

    PubMed

    Perrier, Xavier; De Langhe, Edmond; Donohue, Mark; Lentfer, Carol; Vrydaghs, Luc; Bakry, Frédéric; Carreel, Françoise; Hippolyte, Isabelle; Horry, Jean-Pierre; Jenny, Christophe; Lebot, Vincent; Risterucci, Ange-Marie; Tomekpe, Kodjo; Doutrelepont, Hugues; Ball, Terry; Manwaring, Jason; de Maret, Pierre; Denham, Tim

    2011-07-12

    Original multidisciplinary research hereby clarifies the complex geodomestication pathways that generated the vast range of banana cultivars (cvs). Genetic analyses identify the wild ancestors of modern-day cvs and elucidate several key stages of domestication for different cv groups. Archaeology and linguistics shed light on the historical roles of people in the movement and cultivation of bananas from New Guinea to West Africa during the Holocene. The historical reconstruction of domestication processes is essential for breeding programs seeking to diversify and improve banana cvs for the future.

  7. The Gender Wage Gap and Domestic Violence

    PubMed Central

    Aizer, Anna

    2014-01-01

    Three quarters of all violence against women is perpetrated by domestic partners. This study exploits exogenous changes in the demand for labor in female-dominated industries to estimate the impact of the male-female wage gap on domestic violence. Decreases in the wage gap reduce violence against women, consistent with a household bargaining model. These findings shed new light on the health production process as well as observed income gradients in health and suggest that in addition to addressing concerns of equity and efficiency, pay parity can also improve the health of American women via reductions in violence. PMID:25110354

  8. Sunflower domestication alleles support single domestication center in eastern North America

    PubMed Central

    Blackman, Benjamin K.; Scascitelli, Moira; Kane, Nolan C.; Luton, Harry H.; Rasmussen, David A.; Bye, Robert A.; Lentz, David L.; Rieseberg, Loren H.

    2011-01-01

    Phylogenetic analyses of genes with demonstrated involvement in evolutionary transitions can be an important means of resolving conflicting hypotheses about evolutionary history or process. In sunflower, two genes have previously been shown to have experienced selective sweeps during its early domestication. In the present study, we identified a third candidate early domestication gene and conducted haplotype analyses of all three genes to address a recent, controversial hypothesis about the origin of cultivated sunflower. Although the scientific consensus had long been that sunflower was domesticated once in eastern North America, the discovery of pre-Columbian sunflower remains at archaeological sites in Mexico led to the proposal of a second domestication center in southern Mexico. Previous molecular studies with neutral markers were consistent with the former hypothesis. However, only two indigenous Mexican cultivars were included in these studies, and their provenance and genetic purity have been questioned. Therefore, we sequenced regions of the three candidate domestication genes containing SNPs diagnostic for domestication from large, newly collected samples of Mexican sunflower landraces and Mexican wild populations from a broad geographic range. The new germplasm also was genotyped for 12 microsatellite loci. Our evidence from multiple evolutionarily important loci and from neutral markers supports a single domestication event for extant cultivated sunflower in eastern North America. PMID:21844335

  9. When domestic goes capital: Juror decision making in capital murder trials involving domestic homicide.

    PubMed

    Richards, Tara N; Smith, M Dwayne; Fogel, Sondra J; Bjerregaard, Beth

    2015-08-01

    Prior research suggests that homicide cases involving familial offenders and victims are subject to a "domestic discount" that reduces sentencing severity. However, the operation of a domestic discount in regard to death penalty sentencing has been rarely examined. The current research uses a near-population of jury decisions in capital murder trials conducted in North Carolina from 1991 to 2009 (n = 800), and a series of logistic regression analyses to determine whether there is (a) a direct effect between offender-victim relationship (e.g., domestic, friend/acquaintance, and stranger) and jury decision making, and/or (b) whether domestic offender-victim relationship (as well as other offender-victim relationships) moderates the effect of legal and extralegal case characteristics on jury assessment of the death penalty. Our findings revealed no empirical support for a "domestic discount" whereby juries are less likely to impose death sentences in cases involving domestic homicides. However, substantial differences in predictors of death sentencing were found across offender-victim dyads; most notably, domestic homicide cases demonstrated the most legalistic model of jury decisions to impose death sentences.

  10. Evidence of long-term gene flow and selection during domestication from analyses of Eurasian wild and domestic pig genomes.

    PubMed

    Frantz, Laurent A F; Schraiber, Joshua G; Madsen, Ole; Megens, Hendrik-Jan; Cagan, Alex; Bosse, Mirte; Paudel, Yogesh; Crooijmans, Richard P M A; Larson, Greger; Groenen, Martien A M

    2015-10-01

    Traditionally, the process of domestication is assumed to be initiated by humans, involve few individuals and rely on reproductive isolation between wild and domestic forms. We analyzed pig domestication using over 100 genome sequences and tested whether pig domestication followed a traditional linear model or a more complex, reticulate model. We found that the assumptions of traditional models, such as reproductive isolation and strong domestication bottlenecks, are incompatible with the genetic data. In addition, our results show that, despite gene flow, the genomes of domestic pigs have strong signatures of selection at loci that affect behavior and morphology. We argue that recurrent selection for domestic traits likely counteracted the homogenizing effect of gene flow from wild boars and created 'islands of domestication' in the genome. Our results have major ramifications for the understanding of animal domestication and suggest that future studies should employ models that do not assume reproductive isolation.

  11. 78 FR 55124 - Domestic Mail Contract

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-09-09

    ... From the Federal Register Online via the Government Publishing Office POSTAL REGULATORY COMMISSION Domestic Mail Contract AGENCY: Postal Regulatory Commission. ACTION: Notice. SUMMARY: The Commission is noticing a recent Postal Service filing seeking to add Parcel Select Contract 7 to the competitive...

  12. Domestic Violence Encountered among Kurdish Women

    ERIC Educational Resources Information Center

    Ali, Sirwan Kamil

    2015-01-01

    Background and objective; There is growing recognition that violence against women has a large public health impact, in addition to being a gross violation of women's human rights. The study's aims were: To show the types of domestic abuse encountered by Kurdish women, and study the relationship between them. Methods; The study conducted in the…

  13. Displaying Geographically-Based Domestic Statistics

    NASA Technical Reports Server (NTRS)

    Quann, J.; Dalton, J.; Banks, M.; Helfer, D.; Szczur, M.; Winkert, G.; Billingsley, J.; Borgstede, R.; Chen, J.; Chen, L.; Fuh, J.; Cyprych, E.

    1982-01-01

    Decision Information Display System (DIDS) is rapid-response information-retrieval and color-graphics display system. DIDS transforms tables of geographically-based domestic statistics (such as population or unemployment by county, energy usage by county, or air-quality figures) into high-resolution, color-coded maps on television display screen.

  14. Interstitial Spaces: Visual Culture, Domesticity, and Metaphor

    ERIC Educational Resources Information Center

    Sanders, James H., III; Buenger, Allison

    2010-01-01

    Allison Buenger graduated from the Ohio State University with a bachelor of fine arts degree in ceramics in December 2009. Her works morph domestic objects into vessels of meaning, sculptures displayed in larger installations of textiles and found objects. Originally from Northeast Ohio, Allison resides in Columbus, Ohio, and works at Open Door…

  15. Domestic Violence and Children: Analysis and Recommendations.

    ERIC Educational Resources Information Center

    Carter, Lucy Salcido; Weithorn, Lois A.; Behrman, Richard E.

    1999-01-01

    Summarizes current knowledge about the prevalence and effects of childhood exposure to domestic violence. Describes responses to this problem by the services systems with which children exposed to violence have contact and explores what is known about how well these responses work. (Contains 97 references.) (SLD)

  16. Workshop on Children and Domestic Abuse.

    ERIC Educational Resources Information Center

    National Academy of Sciences - National Research Council, Washington, DC. Board on Children, Youth, and Families.

    This workshop coordinates with the publication of a volume of "The Future of Children." The goal of the workshop is to bring together researchers, policymakers, health providers, and law enforcement to review available research literature on children and domestic violence. Topics that were addressed include prevalence and effect of…

  17. Children witnessing domestic violence: a developmental approach.

    PubMed

    Mahony, D L; Campbell, J M

    1998-11-01

    Over the past 30 years, domestic violence has been brought to the forefront of society's consciousness as a number of studies have defined it, identified prevalence and causal factors, and described intervention initiatives. Children who witness domestic violence are at risk for developing behavioral and emotional problems. These behaviors depend on the age of the child, the duration of the violence, whether the child is also being directly abused, the fierceness of the violence, and if the child remains in the abusive environment. The purpose of this paper was to review the literature and theoretical frameworks associated with children who are witnessing domestic violence from a developmental perspective and to determine if in their clinical practices expert pediatric nurse practitioners (PNPs) are seeing these behaviors in witnessing children. Twenty-eight PNPs returned a four-question survey tool describing behaviors in the four developmental categories: infancy, preschool, school-aged, and adolescence. Their descriptions were similar to data recorded in the literature, with extreme behaviors of aggression and withdrawal observed in all age groups. PNPs are in a position to identify the presence of domestic violence if they are sensitive to the behaviors displayed by witnessing children.

  18. Police Attitudes toward Domestic Violence Offenders

    ERIC Educational Resources Information Center

    Logan, T. K.; Shannon, Lisa; Walker, Robert

    2006-01-01

    Police attitudes are important in facilitating a sense of safety and comfort in women seeking justice-system support for protection from partner violence. This study examined police attitudes toward sanctions and treatment for domestic violence offenders compared with other violent and nonviolent offenders. In addition, police attitudes toward…

  19. Domestic Violence, Personal Control, and Gender.

    ERIC Educational Resources Information Center

    Umberson, Debra; Anderson, Kristin; Glick, Jennifer; Shapiro, Adam

    1998-01-01

    Explores how domestic violence is related to personal control. Finds that individuals who have initiated violence against a partner do not differ from individuals who have nonviolent relationships in feelings of personal control. Experiencing violence at the hands of a partner has more significant adverse effects on sense of personal control for…

  20. Two maternal origins of Chinese domestic goose.

    PubMed

    Li, H F; Zhu, W Q; Chen, K W; H, Y; Xu, W J; Song, W

    2011-12-01

    China is particularly rich in goose genetic resources. Systematic study of the genetic diversity and origin of Chinese domestic geese will provide an important scientific basis for the conservation and utilization of these resources and for human history. The 521-bp control region (D-loop) of mitochondrial DNA from 26 goose breeds and 6 Landaise geese were sequenced. The results showed that the average haplotype diversity and nucleotide diversity of Chinese domestic geese were 0.1384 and 0.00029, respectively. Shared haplotype analysis and systematic evolution analysis revealed that Chinese domestic geese had 2 maternal origins. The Yili goose breed originated from the Greylag goose (Anser anser), and the other 25 domestic goose breeds originated from the swan goose (Anser cygnoides). An interesting finding was that 1 Linxian white goose and 1 Wanxi white goose shared the same H4 haplotype with the Rhine goose and the Landaise goose, which originated from the Greylag goose (A. anser). Further research on this finding is planned.

  1. Children's Actions when Experiencing Domestic Violence

    ERIC Educational Resources Information Center

    Overlien, Carolina; Hyden, Margareta

    2009-01-01

    The aim of this article is, by analysing children's discourses, to investigate their actions or absence of actions during a domestic violence episode. The empirical data are recorded group therapy sessions and individual interviews with children who have grown up experiencing their fathers' violence against their mothers. The analysis shows that…

  2. Psychopathology in Women Arrested for Domestic Violence

    ERIC Educational Resources Information Center

    Stuart, Gregory L.; Moore, Todd M.; Gordon, Kristina Coop; Ramsey, Susan E.; Kahler, Christopher W.

    2006-01-01

    This study examined the prevalence of psychopathology among women arrested for violence and whether the experience of intimate partner violence (IPV) was associated with Axis I psychopathology. Women who were arrested for domestic violence perpetration and court referred to violence intervention programs (N=103) completed measures of IPV…

  3. 7 CFR 983.12 - Domestic shipments.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 8 2012-01-01 2012-01-01 false Domestic shipments. 983.12 Section 983.12 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (Marketing Agreements and Orders; Fruits, Vegetables, Nuts), DEPARTMENT OF AGRICULTURE PISTACHIOS GROWN IN...

  4. 7 CFR 983.12 - Domestic shipments.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 8 2013-01-01 2013-01-01 false Domestic shipments. 983.12 Section 983.12 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE PISTACHIOS GROWN IN...

  5. 7 CFR 983.12 - Domestic shipments.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 8 2014-01-01 2014-01-01 false Domestic shipments. 983.12 Section 983.12 Agriculture Regulations of the Department of Agriculture (Continued) AGRICULTURAL MARKETING SERVICE (MARKETING AGREEMENTS AND ORDERS; FRUITS, VEGETABLES, NUTS), DEPARTMENT OF AGRICULTURE PISTACHIOS GROWN IN...

  6. Psychopathology in Women Arrested for Domestic Violence

    ERIC Educational Resources Information Center

    Stuart, Gregory L.; Moore, Todd M.; Gordon, Kristina Coop; Ramsey, Susan E.; Kahler, Christopher W.

    2006-01-01

    This study examined the prevalence of psychopathology among women arrested for violence and whether the experience of intimate partner violence (IPV) was associated with Axis I psychopathology. Women who were arrested for domestic violence perpetration and court referred to violence intervention programs (N=103) completed measures of IPV…

  7. The magnetic compass of domestic chickens.

    PubMed

    Denzau, Susanne; Nießner, Christine; Rogers, Lesley J; Wiltschko, Wolfgang

    2013-11-01

    In a recent paper, we showed that domestic chickens can be trained to search for a social stimulus in specific magnetic directions. Chickens can hardly fly and have only small home ranges, hence their having a functional magnetic compass may seem rather surprising. Yet considering the natural habitat of their ancestors and their lifestyle until recently, the advantages of a magnetic compass become evident.

  8. Police Attitudes toward Domestic Violence Offenders

    ERIC Educational Resources Information Center

    Logan, T. K.; Shannon, Lisa; Walker, Robert

    2006-01-01

    Police attitudes are important in facilitating a sense of safety and comfort in women seeking justice-system support for protection from partner violence. This study examined police attitudes toward sanctions and treatment for domestic violence offenders compared with other violent and nonviolent offenders. In addition, police attitudes toward…

  9. Novel Corynebacterium diphtheriae in Domestic Cats

    PubMed Central

    Cassiday, Pamela K.; Bernard, Kathryn A.; Bolt, Frances; Steigerwalt, Arnold G.; Bixler, Danae; Pawloski, Lucia C.; Whitney, Anne M.; Iwaki, Masaaki; Baldwin, Adam; Dowson, Christopher G.; Komiya, Takako; Takahashi, Motohide; Hinrikson, Hans P.; Tondella, Maria L.

    2010-01-01

    Novel nontoxigenic Corynebacterium diphtheriae was isolated from a domestic cat with severe otitis. Contact investigation and carrier study of human and animal contacts yielded 3 additional, identical isolates from cats, although no evidence of zoonotic transmission was identified. Molecular methods distinguished the feline isolates from known C. diphtheriae. PMID:20350389

  10. [Parasitism by Amblyomma triste in domestic cat].

    PubMed

    da Silva, Aleksandro S; da Silva, Marcos K; Monteiro, Silvia G

    2007-01-01

    Amblyomma triste is an ixodidae, ectoparasite of several mammals' species, with occurrence reported in Argentina, Brazil, Colombia, Ecuador, Peru, Uruguay and Venezuela. The aim of this work was to register the parasitism by A. triste in domestic cat (Felis catus) in Rio Grande do Sul state, Brazil.

  11. Timber rivet connections in US domestic species

    Treesearch

    Marshall Begel; Ronald W. Wolfe; Douglas C. Stahl

    2004-01-01

    This paper discusses results of tests conducted to expand the data base on the performance of timber rivet connections in U.S. domestic species to verify existing and proposed design procedures. Eight-, 80-, and 200-rivet connections were tested. The 8-rivet joint tests illustrate the effects of material type, rivet length, and load direction on the behavior of rivet...

  12. Attitudes toward Domestic Violence: A Cultural Perspective

    ERIC Educational Resources Information Center

    Wallach, Helene S.; Weingram, Ziv; Avitan, Orli

    2010-01-01

    This study examines the effect of acculturation on the attitudes held by Ethiopian Jews in Israel toward domestic violence (DV). The study findings revealed the following: Ethiopians who immigrated to Israel (n = 31) held more lenient attitudes toward DV than Israeli born Jews (n = 62), which supported the hypothesis that culture influences…

  13. Domestic Violence, Emotional Competence, and Child Adjustment

    ERIC Educational Resources Information Center

    Katz, Lynn Fainsilber; Hessler, Danielle M.; Annest, Amalia

    2007-01-01

    This article examined emotion competence in children exposed to domestic violence (DV). It also examined the hypothesis that children's emotional competence mediates relations between DV and children's later difficulties with peers and behavioral adjustment. DV was assessed when children were at the age of five, emotional competence was assessed…

  14. The Economics of a Domestic Approval Plan

    ERIC Educational Resources Information Center

    Axford, H. William

    1971-01-01

    The efficiency and effectiveness of a domestic blanket approval plan is compared with traditional acquisitions procedures based upon a unit cost study carried out at four libraries in the state university system of Florida. Comparative costs are measured. (2 references) (Author/NH)

  15. Preventing Domestic Violence against Women. Special Report.

    ERIC Educational Resources Information Center

    Langan, Patrick A.; Innes, Christopher A.

    This report focuses on domestic violence victims, most of whom are women victimized by their spouses or ex-spouses, and examines the issue of whether calling the police increases or decreases a victim's chance of being victimized again. It was written as part of an effort by the Bureau of Justice Statistics to provide relevant information for the…

  16. The problem of disease when domesticating bees

    USDA-ARS?s Scientific Manuscript database

    When disease strikes a hive of bees, it can devastate the colony and spread to the entire beekeeping operation. All bees are susceptible to diseases, and when they are domesticated, their population densities increase to suit human needs, making them more susceptible. Most attempts at disease contro...

  17. Attitudes toward Domestic Violence: A Cultural Perspective

    ERIC Educational Resources Information Center

    Wallach, Helene S.; Weingram, Ziv; Avitan, Orli

    2010-01-01

    This study examines the effect of acculturation on the attitudes held by Ethiopian Jews in Israel toward domestic violence (DV). The study findings revealed the following: Ethiopians who immigrated to Israel (n = 31) held more lenient attitudes toward DV than Israeli born Jews (n = 62), which supported the hypothesis that culture influences…

  18. Domestic exotics and the perception of invasibility

    Treesearch

    Qinfeng Guo; Robert Ricklefs

    2010-01-01

    Susceptibility of an area to invasion by exotic species is often judged by the fraction of introduced species in the local biota. However, the degree of invasion, particularly in mainland areas, has often been underestimated because of the exclusion of ‘domestic exotics’ (those introduced to internal units from within the national border) in calculations. Because all...

  19. Sarcocystis species lethal for domestic pigeons.

    PubMed

    Olias, Philipp; Gruber, Achim D; Kohls, Andrea; Hafez, Hafez M; Heydorn, Alfred Otto; Mehlhorn, Heinz; Lierz, Michael

    2010-03-01

    A large number of Sarcocystis spp. infect birds as intermediate hosts, but pigeons are rarely affected. We identified a novel Sarcocystis sp. that causes lethal neurologic disease in domestic pigeons in Germany. Experimental infections indicated transmission by northern goshawks, and sequence analyses indicated transnational distribution. Worldwide spread is possible.

  20. Interstitial Spaces: Visual Culture, Domesticity, and Metaphor

    ERIC Educational Resources Information Center

    Sanders, James H., III; Buenger, Allison

    2010-01-01

    Allison Buenger graduated from the Ohio State University with a bachelor of fine arts degree in ceramics in December 2009. Her works morph domestic objects into vessels of meaning, sculptures displayed in larger installations of textiles and found objects. Originally from Northeast Ohio, Allison resides in Columbus, Ohio, and works at Open Door…

  1. 19 CFR 146.43 - Domestic status.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... (CONTINUED) FOREIGN TRADE ZONES Status of Merchandise in a Zone § 146.43 Domestic status. (a) General... and tax has been paid; or (3) Previously entered free of duty and tax. (b) Application. No application... and repair material, to a zone, except upon order of the Commissioner of Customs. No application...

  2. 19 CFR 146.43 - Domestic status.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... (CONTINUED) FOREIGN TRADE ZONES Status of Merchandise in a Zone § 146.43 Domestic status. (a) General... and tax has been paid; or (3) Previously entered free of duty and tax. (b) Application. No application... and repair material, to a zone, except upon order of the Commissioner of Customs. No application...

  3. 19 CFR 146.43 - Domestic status.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... (CONTINUED) FOREIGN TRADE ZONES Status of Merchandise in a Zone § 146.43 Domestic status. (a) General... and tax has been paid; or (3) Previously entered free of duty and tax. (b) Application. No application... and repair material, to a zone, except upon order of the Commissioner of Customs. No application...

  4. Displaying Geographically-Based Domestic Statistics

    NASA Technical Reports Server (NTRS)

    Quann, J.; Dalton, J.; Banks, M.; Helfer, D.; Szczur, M.; Winkert, G.; Billingsley, J.; Borgstede, R.; Chen, J.; Chen, L.; hide

    1982-01-01

    Decision Information Display System (DIDS) is rapid-response information-retrieval and color-graphics display system. DIDS transforms tables of geographically-based domestic statistics (such as population or unemployment by county, energy usage by county, or air-quality figures) into high-resolution, color-coded maps on television display screen.

  5. Children's Actions when Experiencing Domestic Violence

    ERIC Educational Resources Information Center

    Overlien, Carolina; Hyden, Margareta

    2009-01-01

    The aim of this article is, by analysing children's discourses, to investigate their actions or absence of actions during a domestic violence episode. The empirical data are recorded group therapy sessions and individual interviews with children who have grown up experiencing their fathers' violence against their mothers. The analysis shows that…

  6. Quantity Discrimination in Domestic Rats, Rattus norvegicus

    PubMed Central

    Cox, Laura; Montrose, V. Tamara

    2016-01-01

    Simple Summary Quantity discrimination involves distinguishing which of two quantities is greater. This discrimination between larger and smaller quantities has only been demonstrated in rats post extensive training. We tested whether domestic rats could perform quantity discrimination without explicit training. We found that rats could distinguish the greater amount in comparisons of 1 vs. 2, 2 vs. 3, 3 vs. 5, 3 vs. 8, 4 vs. 6, and 4 vs. 8. Rats could not distinguish between 3 vs. 4, 4 vs. 5 and 5 vs. 6. We also found that as the ratio between quantities became finer the choice of the larger quantity decreased. We conclude that rats can perform quantity discrimination without extensive training and that their quantity discrimination ability is influenced by the ratio between quantities. Abstract Quantity discrimination is a basic form of numerical competence where an animal distinguishes which of two amounts is greater in size. Whilst quantity discrimination in rats has been investigated via training paradigms, rats’ natural quantity discrimination abilities without explicit training for a desired response have not been explored. This study investigated domestic rats’ ability to perform quantity discrimination. Domestic rats (n = 12) were examined for their ability to distinguish the larger amount under nine quantity comparisons. One-sample t-tests identified a significant preference for the larger quantity in comparisons of 1 vs. 2, 2 vs. 3, 3 vs. 5, 3 vs. 8, 4 vs. 6, and 4 vs. 8. No preference between quantities was found for comparisons of 3 vs. 4, 4 vs. 5 and 5 vs. 6. Overall, this study drew two key conclusions. Firstly, that domestic rats are capable of performing quantity discrimination without extensive training. Secondly, as subjects adhered to Weber’s law, it was concluded that the approximate number system underpins domestic rats’ ability to perform spontaneous quantity discrimination. PMID:27527223

  7. 49 CFR 526.3 - Transfer of vehicle from non-domestic to domestic fleet.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... UNDER THE AUTOMOBILE FUEL EFFICIENCY ACT OF 1980 § 526.3 Transfer of vehicle from non-domestic to... the plan and for each model type of automobile sought to be included by the submitter in its...

  8. 49 CFR 526.3 - Transfer of vehicle from non-domestic to domestic fleet.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... UNDER THE AUTOMOBILE FUEL EFFICIENCY ACT OF 1980 § 526.3 Transfer of vehicle from non-domestic to... the plan and for each model type of automobile sought to be included by the submitter in its...

  9. 49 CFR 526.3 - Transfer of vehicle from non-domestic to domestic fleet.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... UNDER THE AUTOMOBILE FUEL EFFICIENCY ACT OF 1980 § 526.3 Transfer of vehicle from non-domestic to... the plan and for each model type of automobile sought to be included by the submitter in its...

  10. Domestic Violence and Aging: Teaching about Their Intersection.

    ERIC Educational Resources Information Center

    Wilke, Dina J.; Vinton, Linda

    2003-01-01

    Discusses how women's issues, domestic violence, aging, and elder abuse all relate to social work education and then suggests how they can be integrated together into the social work curriculum through teaching about elder domestic violence. (EV)

  11. International and domestic regulator issues facing the Canadian MSAT system

    NASA Technical Reports Server (NTRS)

    Bahman, Azarbar; Langlois, Jacques R.; Frank, Christopher J.

    1988-01-01

    International and domestic regulatory issues which affect the implementation of a mobile satellite system (MSAT) over North America are addressed. WARC-MOB-87, MSAT frequency co-ordination, frequency sharing and key Canadian domestic issues are discussed.

  12. Engineering genomes of domestic pigs for agricultural applications

    USDA-ARS?s Scientific Manuscript database

    The breeding of domestic animals has a longstanding and successful history, starting with domestication several thousand years ago. Modern animal breeding strategies predominantly based on population genetics, artificial insemination (AI) and embryo transfer (ET) technologies have led to significan...

  13. The goat domestication process inferred from large-scale mitochondrial DNA analysis of wild and domestic individuals.

    PubMed

    Naderi, Saeid; Rezaei, Hamid-Reza; Pompanon, François; Blum, Michael G B; Negrini, Riccardo; Naghash, Hamid-Reza; Balkiz, Ozge; Mashkour, Marjan; Gaggiotti, Oscar E; Ajmone-Marsan, Paolo; Kence, Aykut; Vigne, Jean-Denis; Taberlet, Pierre

    2008-11-18

    The emergence of farming during the Neolithic transition, including the domestication of livestock, was a critical point in the evolution of human kind. The goat (Capra hircus) was one of the first domesticated ungulates. In this study, we compared the genetic diversity of domestic goats to that of the modern representatives of their wild ancestor, the bezoar, by analyzing 473 samples collected over the whole distribution range of the latter species. This partly confirms and significantly clarifies the goat domestication scenario already proposed by archaeological evidence. All of the mitochondrial DNA haplogroups found in current domestic goats have also been found in the bezoar. The geographic distribution of these haplogroups in the wild ancestor allowed the localization of the main domestication centers. We found no haplotype that could have been domesticated in the eastern half of the Iranian Plateau, nor further to the east. A signature of population expansion in bezoars of the C haplogroup suggests an early domestication center on the Central Iranian Plateau (Yazd and Kerman Provinces) and in the Southern Zagros (Fars Province), possibly corresponding to the management of wild flocks. However, the contribution of this center to the current domestic goat population is rather low (1.4%). We also found a second domestication center covering a large area in Eastern Anatolia, and possibly in Northern and Central Zagros. This last domestication center is the likely origin of almost all domestic goats today. This finding is consistent with archaeological data identifying Eastern Anatolia as an important domestication center.

  14. 29 CFR 552.3 - Domestic service employment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 3 2010-07-01 2010-07-01 false Domestic service employment. 552.3 Section 552.3 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION, DEPARTMENT OF LABOR REGULATIONS APPLICATION OF THE FAIR LABOR STANDARDS ACT TO DOMESTIC SERVICE General Regulations § 552.3 Domestic...

  15. 29 CFR 552.101 - Domestic service employment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 3 2010-07-01 2010-07-01 false Domestic service employment. 552.101 Section 552.101 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION, DEPARTMENT OF LABOR REGULATIONS APPLICATION OF THE FAIR LABOR STANDARDS ACT TO DOMESTIC SERVICE Interpretations § 552.101 Domestic...

  16. 29 CFR 552.3 - Domestic service employment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 3 2011-07-01 2011-07-01 false Domestic service employment. 552.3 Section 552.3 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION, DEPARTMENT OF LABOR REGULATIONS APPLICATION OF THE FAIR LABOR STANDARDS ACT TO DOMESTIC SERVICE General Regulations § 552.3 Domestic...

  17. Outsourcing of Domestic Tasks and Time-Saving Effects

    ERIC Educational Resources Information Center

    van der Lippe, Tanja; Tijdens, Kea; de Ruijter, Esther

    2004-01-01

    The increased participation of women in paid labor has changed the organization of domestic work. This article deals with a strategy to cope with remaining domestic duties; to what extent are domestic tasks outsourced, what are the main determinants, and does it indeed save time spent on housework? Five outsourcing options are investigated:…

  18. 29 CFR 552.101 - Domestic service employment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 29 Labor 3 2011-07-01 2011-07-01 false Domestic service employment. 552.101 Section 552.101 Labor Regulations Relating to Labor (Continued) WAGE AND HOUR DIVISION, DEPARTMENT OF LABOR REGULATIONS APPLICATION OF THE FAIR LABOR STANDARDS ACT TO DOMESTIC SERVICE Interpretations § 552.101 Domestic...

  19. 7 CFR 1427.103 - Upland cotton Domestic User Agreement.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Upland cotton Domestic User Agreement. 1427.103... Assistance to Users of Upland Cotton § 1427.103 Upland cotton Domestic User Agreement. (a) Payments specified in this subpart will be made available to eligible domestic users who have entered into an...

  20. 14 CFR 121.593 - Dispatching authority: Domestic operations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 3 2011-01-01 2011-01-01 false Dispatching authority: Domestic operations... OPERATING REQUIREMENTS: DOMESTIC, FLAG, AND SUPPLEMENTAL OPERATIONS Dispatching and Flight Release Rules § 121.593 Dispatching authority: Domestic operations. Except when an airplane lands at an...

  1. 14 CFR 121.639 - Fuel supply: All domestic operations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Fuel supply: All domestic operations. 121... OPERATING REQUIREMENTS: DOMESTIC, FLAG, AND SUPPLEMENTAL OPERATIONS Dispatching and Flight Release Rules § 121.639 Fuel supply: All domestic operations. No person may dispatch or take off an airplane unless...

  2. 17 CFR 240.16a-12 - Domestic relations orders.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Domestic relations orders. 240... Dealers § 240.16a-12 Domestic relations orders. The acquisition or disposition of equity securities pursuant to a domestic relations order, as defined in the Internal Revenue Code or Title I of the...

  3. 48 CFR 252.206-7000 - Domestic source restriction.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 3 2010-10-01 2010-10-01 false Domestic source... of Provisions And Clauses 252.206-7000 Domestic source restriction. As prescribed at 206.302-3-70, use the following provision: Domestic Source Restriction (DEC 1991) This solicitation is restricted...

  4. 48 CFR 452.247-72 - Packing for Domestic Shipment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 4 2011-10-01 2011-10-01 false Packing for Domestic... Packing for Domestic Shipment. As prescribed in 447.305-10(b), insert the following clause: Packing for Domestic Shipment (FEB 1988) Material shall be packed for shipment in such a manner that will...

  5. 46 CFR Sec. 6 - Disbursements at other domestic ports.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 8 2011-10-01 2011-10-01 false Disbursements at other domestic ports. Sec. 6 Section 6 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURAL... domestic ports. Disbursements at domestic ports other than the principal office of the agent for...

  6. 7 CFR 30.42 - Class 7; miscellaneous domestic types.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 2 2011-01-01 2011-01-01 false Class 7; miscellaneous domestic types. 30.42 Section... and Groups of Grades § 30.42 Class 7; miscellaneous domestic types. No group divisions are established... disposition of such tobacco. For tobacco stocks reporting purposes, all miscellaneous domestic tobacco...

  7. 7 CFR 1427.102 - Eligible domestic users.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Eligible domestic users. 1427.102 Section 1427.102... of Upland Cotton § 1427.102 Eligible domestic users. (a) For purposes of this subpart, a person... with CCC to participate in the upland cotton user program, will be considered an eligible domestic...

  8. 48 CFR 252.206-7000 - Domestic source restriction.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 3 2011-10-01 2011-10-01 false Domestic source... of Provisions And Clauses 252.206-7000 Domestic source restriction. As prescribed at 206.302-3-70, use the following provision: Domestic Source Restriction (DEC 1991) This solicitation is restricted...

  9. 7 CFR 1427.103 - Upland cotton Domestic User Agreement.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 10 2010-01-01 2010-01-01 false Upland cotton Domestic User Agreement. 1427.103... Assistance to Users of Upland Cotton § 1427.103 Upland cotton Domestic User Agreement. (a) Payments specified in this subpart will be made available to eligible domestic users who have entered into an...

  10. 46 CFR Sec. 6 - Disbursements at other domestic ports.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 8 2010-10-01 2010-10-01 false Disbursements at other domestic ports. Sec. 6 Section 6 Shipping MARITIME ADMINISTRATION, DEPARTMENT OF TRANSPORTATION A-NATIONAL SHIPPING AUTHORITY PROCEDURAL... domestic ports. Disbursements at domestic ports other than the principal office of the agent for...

  11. Domestic Violence among the Black Poor: Intersectionality and Social Justice

    ERIC Educational Resources Information Center

    Conwill, William Louis

    2010-01-01

    There are striking gender, race, and class variations in rates of domestic violence. Some leading family theorists called for an intersectional analysis of how gender, race and class systems interact to improve domestic violence theory. This article improves domestic violence theory by: 1) using the discourse, or language, of intersectionality; 2)…

  12. Losing out on Both Counts: Disabled Women and Domestic Violence

    ERIC Educational Resources Information Center

    Thiara, Ravi K.; Hague, Gill; Mullender, Audrey

    2011-01-01

    The links between disability and domestic violence have been under-examined to date, leading to the marginalisation of disabled women affected by domestic violence in theory, politics, and practice. This paper draws on the findings from the first national study in the United Kingdom of the needs of disabled women experiencing domestic violence and…

  13. 28 CFR 80.4 - Issuer or domestic concern.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Issuer or domestic concern. 80.4 Section 80.4 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) FOREIGN CORRUPT PRACTICES ACT OPINION PROCEDURE § 80.4 Issuer or domestic concern. The request must be submitted by an issuer or domestic...

  14. 14 CFR 121.557 - Emergencies: Domestic and flag operations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Emergencies: Domestic and flag operations... OPERATING REQUIREMENTS: DOMESTIC, FLAG, AND SUPPLEMENTAL OPERATIONS Flight Operations § 121.557 Emergencies: Domestic and flag operations. (a) In an emergency situation that requires immediate decision and action...

  15. 19 CFR 351.502 - Specificity of domestic subsidies.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 19 Customs Duties 3 2010-04-01 2010-04-01 false Specificity of domestic subsidies. 351.502 Section... domestic subsidies. (a) Sequential analysis. In determining whether a subsidy is de facto specific, the... agricultural sector (domestic subsidy). (e) Subsidies to small-and medium-sized businesses. The Secretary...

  16. 12 CFR 614.4232 - Loans to domestic lessors.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 12 Banks and Banking 6 2011-01-01 2011-01-01 false Loans to domestic lessors. 614.4232 Section 614... Terms and Conditions § 614.4232 Loans to domestic lessors. Loans and financial assistance extended by banks for cooperatives and agricultural credit banks to domestic lessors to finance equipment...

  17. 14 CFR 121.593 - Dispatching authority: Domestic operations.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 14 Aeronautics and Space 3 2010-01-01 2010-01-01 false Dispatching authority: Domestic operations... OPERATING REQUIREMENTS: DOMESTIC, FLAG, AND SUPPLEMENTAL OPERATIONS Dispatching and Flight Release Rules § 121.593 Dispatching authority: Domestic operations. Except when an airplane lands at an...

  18. 14 CFR 121.639 - Fuel supply: All domestic operations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 3 2011-01-01 2011-01-01 false Fuel supply: All domestic operations. 121... OPERATING REQUIREMENTS: DOMESTIC, FLAG, AND SUPPLEMENTAL OPERATIONS Dispatching and Flight Release Rules § 121.639 Fuel supply: All domestic operations. No person may dispatch or take off an airplane unless...

  19. 48 CFR 452.247-72 - Packing for Domestic Shipment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 4 2010-10-01 2010-10-01 false Packing for Domestic... Packing for Domestic Shipment. As prescribed in 447.305-10(b), insert the following clause: Packing for Domestic Shipment (FEB 1988) Material shall be packed for shipment in such a manner that will...

  20. 17 CFR 240.16a-12 - Domestic relations orders.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 17 Commodity and Securities Exchanges 3 2011-04-01 2011-04-01 false Domestic relations orders. 240... Dealers § 240.16a-12 Domestic relations orders. The acquisition or disposition of equity securities pursuant to a domestic relations order, as defined in the Internal Revenue Code or Title I of the...

  1. 7 CFR 1427.102 - Eligible domestic users.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 10 2011-01-01 2011-01-01 false Eligible domestic users. 1427.102 Section 1427.102... of Upland Cotton § 1427.102 Eligible domestic users. (a) For purposes of this subpart, a person... with CCC to participate in the upland cotton user program, will be considered an eligible domestic...

  2. 12 CFR 614.4232 - Loans to domestic lessors.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 12 Banks and Banking 6 2010-01-01 2010-01-01 false Loans to domestic lessors. 614.4232 Section 614... Terms and Conditions § 614.4232 Loans to domestic lessors. Loans and financial assistance extended by banks for cooperatives and agricultural credit banks to domestic lessors to finance equipment...

  3. 45 CFR 1160.5 - Eligibility for domestic exhibitions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 3 2011-10-01 2011-10-01 false Eligibility for domestic exhibitions. 1160.5... AND ARTIFACTS INDEMNITY ACT § 1160.5 Eligibility for domestic exhibitions. An indemnity agreement for a domestic exhibition made under these regulations shall cover eligible items from the United...

  4. 45 CFR 1160.5 - Eligibility for domestic exhibitions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false Eligibility for domestic exhibitions. 1160.5... AND ARTIFACTS INDEMNITY ACT § 1160.5 Eligibility for domestic exhibitions. An indemnity agreement for a domestic exhibition made under these regulations shall cover eligible items from the United...

  5. 7 CFR 30.42 - Class 7; miscellaneous domestic types.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 2 2010-01-01 2010-01-01 false Class 7; miscellaneous domestic types. 30.42 Section... and Groups of Grades § 30.42 Class 7; miscellaneous domestic types. No group divisions are established... disposition of such tobacco. For tobacco stocks reporting purposes, all miscellaneous domestic tobacco...

  6. 77 FR 37576 - Treatment of Overall Foreign and Domestic Losses

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-06-22

    ... Internal Revenue Service 26 CFR Part 1 RIN 1545-BH13 Treatment of Overall Foreign and Domestic Losses... Revenue Code (Code) relating to the recapture of overall domestic losses that was enacted as part of the... limitation, and section 904(g), which addresses overall domestic losses and the recapture of such...

  7. 19 CFR 351.502 - Specificity of domestic subsidies.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 19 Customs Duties 3 2011-04-01 2011-04-01 false Specificity of domestic subsidies. 351.502 Section... domestic subsidies. (a) Sequential analysis. In determining whether a subsidy is de facto specific, the... agricultural sector (domestic subsidy). (e) Subsidies to small-and medium-sized businesses. The Secretary...

  8. 14 CFR 121.557 - Emergencies: Domestic and flag operations.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 14 Aeronautics and Space 3 2011-01-01 2011-01-01 false Emergencies: Domestic and flag operations... OPERATING REQUIREMENTS: DOMESTIC, FLAG, AND SUPPLEMENTAL OPERATIONS Flight Operations § 121.557 Emergencies: Domestic and flag operations. (a) In an emergency situation that requires immediate decision and action...

  9. 28 CFR 80.4 - Issuer or domestic concern.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Issuer or domestic concern. 80.4 Section 80.4 Judicial Administration DEPARTMENT OF JUSTICE (CONTINUED) FOREIGN CORRUPT PRACTICES ACT OPINION PROCEDURE § 80.4 Issuer or domestic concern. The request must be submitted by an issuer or domestic...

  10. Domestic Disasters and Geospatial Technology for the Defense Logistics Agency

    DTIC Science & Technology

    2014-12-01

    NAVAL POSTGRADUATE SCHOOL MONTEREY, CALIFORNIA MBA PROFESSIONAL REPORT DOMESTIC DISASTERS AND GEOSPATIAL TECHNOLOGY FOR THE...REPORT DATE December 2014 3. REPORT TYPE AND DATES COVERED MBA Professional Report 4. TITLE AND SUBTITLE DOMESTIC DISASTERS AND GEOSPATIAL...ANSI Std. 239–18 ii THIS PAGE INTENTIONALLY LEFT BLANK iii Approved for public release; distribution is unlimited DOMESTIC DISASTERS AND

  11. Longitudinal Effects of Domestic Violence on Employment and Welfare Outcomes

    ERIC Educational Resources Information Center

    Lindhorst, Taryn; Oxford, Monica; Gillmore, Mary Rogers

    2007-01-01

    This study uses longitudinal data spanning 13 years from a study of 234 adolescent mothers to evaluate the effects of cumulative domestic violence on employment and welfare use before and after welfare reform. Domestic violence increased the odds of unemployment after welfare reform, but not before; domestic violence had no effect on welfare use…

  12. Perceptions of Domestic Violence: A Dialogue with African American Women

    ERIC Educational Resources Information Center

    Bent-Goodley, Tricia B.

    2004-01-01

    Although empirical research has accumulated over the past 20 years regarding African Americans and domestic violence, many questions remain about African American perceptions of domestic violence. This article explores African American women's perceptions about domestic violence through three focus groups held at a New York social services agency.…

  13. Domestic Violence against Women: Recognize Patterns, Seek Help

    MedlinePlus

    Healthy Lifestyle Adult health Domestic violence is a serious threat for many women. Know the signs of an abusive relationship and how to leave a dangerous ... If this sounds familiar, you might be experiencing domestic violence. Domestic violence — also called intimate partner violence — occurs ...

  14. Domestic Violence among the Black Poor: Intersectionality and Social Justice

    ERIC Educational Resources Information Center

    Conwill, William Louis

    2010-01-01

    There are striking gender, race, and class variations in rates of domestic violence. Some leading family theorists called for an intersectional analysis of how gender, race and class systems interact to improve domestic violence theory. This article improves domestic violence theory by: 1) using the discourse, or language, of intersectionality; 2)…

  15. 32 CFR 635.29 - Domestic violence and protection orders.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 4 2014-07-01 2013-07-01 true Domestic violence and protection orders. 635.29... ENFORCEMENT AND CRIMINAL INVESTIGATIONS LAW ENFORCEMENT REPORTING Offense Reporting § 635.29 Domestic violence... permitted by law and regulation. AR 608-18 contains additional information about domestic violence and...

  16. 32 CFR 635.29 - Domestic violence and protection orders.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 4 2010-07-01 2010-07-01 true Domestic violence and protection orders. 635.29... ENFORCEMENT AND CRIMINAL INVESTIGATIONS LAW ENFORCEMENT REPORTING Offense Reporting § 635.29 Domestic violence... permitted by law and regulation. AR 608-18 contains additional information about domestic violence and...

  17. Perceptions of Domestic Violence: A Dialogue with African American Women

    ERIC Educational Resources Information Center

    Bent-Goodley, Tricia B.

    2004-01-01

    Although empirical research has accumulated over the past 20 years regarding African Americans and domestic violence, many questions remain about African American perceptions of domestic violence. This article explores African American women's perceptions about domestic violence through three focus groups held at a New York social services agency.…

  18. Longitudinal Effects of Domestic Violence on Employment and Welfare Outcomes

    ERIC Educational Resources Information Center

    Lindhorst, Taryn; Oxford, Monica; Gillmore, Mary Rogers

    2007-01-01

    This study uses longitudinal data spanning 13 years from a study of 234 adolescent mothers to evaluate the effects of cumulative domestic violence on employment and welfare use before and after welfare reform. Domestic violence increased the odds of unemployment after welfare reform, but not before; domestic violence had no effect on welfare use…

  19. 32 CFR 635.29 - Domestic violence and protection orders.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 4 2011-07-01 2011-07-01 false Domestic violence and protection orders. 635.29... ENFORCEMENT AND CRIMINAL INVESTIGATIONS LAW ENFORCEMENT REPORTING Offense Reporting § 635.29 Domestic violence... permitted by law and regulation. AR 608-18 contains additional information about domestic violence and...

  20. 32 CFR 635.29 - Domestic violence and protection orders.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 4 2013-07-01 2013-07-01 false Domestic violence and protection orders. 635.29... ENFORCEMENT AND CRIMINAL INVESTIGATIONS LAW ENFORCEMENT REPORTING Offense Reporting § 635.29 Domestic violence... permitted by law and regulation. AR 608-18 contains additional information about domestic violence and...