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Sample records for neuronal gene transfer

  1. Neuronal expression and regulation of CGRP promoter activity following viral gene transfer into cultured trigeminal ganglia neurons.

    PubMed

    Durham, Paul L; Dong, Penny X; Belasco, Kevin T; Kasperski, Jeffrey; Gierasch, William W; Edvinsson, Lars; Heistad, Donald D; Faraci, Frank M; Russo, Andrew F

    2004-01-30

    We have examined the regulation of calcitonin gene-related peptide (CGRP) promoter activity in primary cultures of rat trigeminal ganglia neurons. A viral vector was used to circumvent the potential complication of examining only a small subpopulation of cells in the heterogeneous cultures. Infection with high titers of recombinant adenovirus containing 1.25 kb of the rat CGRP promoter linked to the beta-galactosidase reporter gene (AdCGRP-lacZ) yielded expression in about 50% of the CGRP-expressing neurons. The CGRP-lacZ reporter gene was preferentially expressed in neurons, with 91% co-expression with endogenous CGRP. In contrast, an adenoviral vector containing a CMV-lacZ reporter was predominantly expressed in non-neuronal cells, with only 29% co-expression with CGRP. We then asked whether the CGRP promoter in the viral vector could be regulated by serotonin receptor type 1 (5-HT(1)) agonists. Promoter activity was decreased two- to threefold by treatment with five 5-HT(1B/D) agonists, including the triptan drugs sumatriptan, eletriptan, and rizatriptan that are used for migraine treatment. As controls, CMV promoter activity was not affected, and 5-HT(1B/D) receptor antagonists blocked the repression caused by sumatriptan and eletriptan. Thus, adenoviral gene transfer can be used in trigeminal ganglia neurons for studying the mechanisms of triptan drug action on CGRP synthesis. PMID:14715155

  2. Optogenetic Dissection of Neuronal Circuits in Zebrafish using Viral Gene Transfer and the Tet System

    PubMed Central

    Zhu, Peixin; Narita, Yuichi; Bundschuh, Sebastian T.; Fajardo, Otto; Schärer, Yan-Ping Zhang; Chattopadhyaya, Bidisha; Bouldoires, Estelle Arn; Stepien, Anna Ewa; Deisseroth, Karl; Arber, Silvia; Sprengel, Rolf; Rijli, Filippo M.; Friedrich, Rainer W.

    2009-01-01

    The conditional expression of transgenes at high levels in sparse and specific populations of neurons is important for high-resolution optogenetic analyses of neuronal circuits. We explored two complementary methods, viral gene delivery and the iTet-Off system, to express transgenes in the brain of zebrafish. High-level gene expression in neurons was achieved by Sindbis and Rabies viruses. The Tet system produced strong and specific gene expression that could be modulated conveniently by doxycycline. Moreover, transgenic lines showed expression in distinct, sparse and stable populations of neurons that appeared to be subsets of the neurons targeted by the promoter driving the Tet-activator. The Tet system therefore provides the opportunity to generate libraries of diverse expression patterns similar to gene trap approaches or the thy-1 promoter in mice, but with the additional possibility to pre-select cell types of interest. In transgenic lines expressing channelrhodopsin-2, action potential firing could be precisely controlled by two-photon stimulation at low laser power, presumably because the expression levels of the Tet-controlled genes were high even in adults. In channelrhodopsin-2-expressing larvae, optical stimulation with a single blue LED evoked distinct swimming behaviors including backward swimming. These approaches provide new opportunities for the optogenetic dissection of neuronal circuit structure and function. PMID:20126518

  3. Subretinal delivery of recombinant AAV serotype 8 vector in dogs results in gene transfer to neurons in the brain.

    PubMed

    Stieger, Knut; Colle, Marie-Anne; Dubreil, Laurence; Mendes-Madeira, Alexandra; Weber, Michel; Le Meur, Guylène; Deschamps, Jack Yves; Provost, Nathalie; Nivard, Delphine; Cherel, Yan; Moullier, Philippe; Rolling, Fabienne

    2008-05-01

    Recombinant adeno-associated virus (rAAV) vectors are among the most efficient gene delivery vehicles for gene transfer to the retina. This study evaluates the behavior of the rAAV8 serotype vector with regard to intraocular delivery in rats and dogs. Subretinal delivery of an AAV2/8.gfp vector results in efficient gene transfer in the retinal pigment epithelium (RPE), the photoreceptors and, surprisingly, in the cells of the inner nuclear layer as well as in ganglion cells. Most importantly, in dogs, gene transfer also occurred distal to the injection site in neurons of the lateral geniculate nucleus of the brain. Because green fluorescent protein (GFP) was detected along the visual pathway within the brain, we analyzed total DNA extracted from various brain slices using PCR. Vector sequences were detected in many parts of the brain, but chiefly in the contralateral hemisphere.

  4. Replication-deficient adenovirus vector transfer of gfp reporter gene into supraoptic nucleus and subfornical organ neurons

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Johnson, R. F.; Beltz, T. G.; Haskell, R. E.; Davidson, B. L.; Johnson, A. K.

    1998-01-01

    The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 x 10(6) pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 x 10(6) to 2 x 10(8) pfu). The expression of GFP in in vitro transfected cells was higher in nonneuronal (approximately 28% in SON and SFO) than neuronal (approximately 4% in SON and 10% in SFO) cells. The expression of GFP was time and viral concentration related. No apparent alterations in cellular morphology of transfected cells were detected and electrophysiological characterization of transfected cells was similar between GFP-expressing and nonexpressing neurons. We conclude that (1) GFP is an effective marker for gene transfer in living SON and SFO cells, (2) Ad infects both neuronal and nonneuronal cells, (3) Ad is taken up by axonal projections from the SON and retrogradely transported to the SFO where it is expressed at detectable levels, and (4) Ad does not adversely affect neuronal viability. These results demonstrate the feasibility of using adenoviral vectors to deliver genes to the SFO-SON axis. Copyright 1998 Academic Press.

  5. Optimizing NTS-polyplex as a tool for gene transfer to cultured dopamine neurons.

    PubMed

    Hernandez-Baltazar, Daniel; Martinez-Fong, Daniel; Trudeau, Louis-Eric

    2012-01-01

    The study of signal transduction in dopamine (DA)-containing neurons as well as the development of new therapeutic approaches for Parkinson's disease requires the selective expression of transgenes in such neurons. Here we describe optimization of the use of the NTS-polyplex, a gene carrier system taking advantage of neurotensin receptor internalization, to transfect mouse DA neurons in primary culture. The plasmids DsRed2 (4.7 kbp) and VGLUT2-Venus (11 kbp) were used to compare the ability of this carrier system to transfect plasmids of different sizes. We examined the impact of age of the neurons (1, 3, 5 and 8 days after seeding), of culture media used during the transfection (Neurobasal with B27 vs. conditioned medium) and of three molar ratios of plasmid DNA to carrier. While the NTS-polyplex successfully transfected both plasmids in a control N1E-115 cell line, only the pDsRed2 plasmid could be transfected in primary cultured DA neurons. We achieved 20% transfection efficiency of pDsRed2 in DA neurons, with 80% cell viability. The transfection was demonstrated pharmacologically to be dependent on activation of neurotensin receptors and to be selective for DA neurons. The presence of conditioned medium for transfection was found to be required to insure cell viability. Highest transfection efficiency was achieved in the most mature neurons. In contrast, transfection with the VGLUT2-Venus plasmid produced cell damage, most likely due to the high molar ratios required, as evidenced by a 15% cell viability of DA neurons at the three molar ratios tested (1:36, 1:39 and 1:42). We conclude that, when used at molar ratios lower than 1:33, the NTS-polyplex can selectively transfect mature cultured DA neurons with only low levels of toxicity. Our results provide evidence that the NTS-polyplex has good potential for targeted gene delivery in cultured DA neurons, an in vitro system of great use for the screening of new therapeutic approaches for Parkinson's disease.

  6. Survival Advantage of Neonatal CNS Gene Transfer for Late Infantile Neuronal Ceroid Lipofuscinosis

    PubMed Central

    Sondhi, Dolan; Peterson, Daniel A.; Edelstein, Andrew M.; del Fierro, Katrina; Hackett, Neil R.; Crystal, Ronald G.

    2009-01-01

    Summary Late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal autosomal recessive neurodegenerative lysosomal storage disorder of childhood, is caused by mutations in the CLN2 gene, resulting in deficiency of the protein tripeptidyl peptidase I (TPP-I). We have previously shown that direct CNS administration of AAVrh.10hCLN2 to adult CLN2 knockout mice, a serotype rh.10 adeno-associated virus expressing the wild type CLN2 cDNA, will partially improve neurological function and survival. In this study, we explore the hypothesis that administration of AAVrh.10hCLN2 to the neonatal brain will significantly improve the results of AAVrh.10hCLN2 therapy. To assess this concept, AAVrh.10hCLN2 vector was administered directly to the CNS of CLN2 knockout mice at 2 days, 3 wk and 7 wk of age. While all treatment groups show a marked increase in total TPP-I activity over wild-type mice, neonatally treated mice displayed high levels of TPP-I activity in the CNS 1 yr after administration which was spread throughout the brain. Using behavioral markers, 2 day treated mice demonstrate marked improvement over 3 wk, 7 wk or untreated mice. Finally, neonatal administration of AAVrh.10hCLN2 was associated with markedly enhanced survival, with a median time of death 376 days for neonatal treated mice, 277 days for 3 wk treated mice, 168 days for 7 wk treated mice, and 121 days for untreated mice. These data suggest that neonatal treatment offers many unique advantages, and that early detection and treatment may be essential for maximal gene therapy for childhood lysosomal storage disorders affecting the CNS. PMID:18639872

  7. Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

    PubMed Central

    Towne, Chris; Pertin, Marie; Beggah, Ahmed T; Aebischer, Patrick; Decosterd, Isabelle

    2009-01-01

    Background Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. Results Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependant manner. More than 90% of transduced cells were small and medium sized neurons (< 700 μm2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (≈ 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell

  8. TrkB gene transfer does not alter hippocampal neuronal loss and cognitive deficits following traumatic brain injury in mice

    PubMed Central

    Conte, Valeria; Raghupathi, Ramesh; Watson, Deborah J.; Fujimoto, Scott; Royo, Nicolas C.; Marklund, Niklas; Stocchetti, Nino; McIntosh, Tracy K.

    2008-01-01

    Purpose The ability of brain-derived neurotrophic factor (BDNF) to attenuate secondary damage and influence behavioral outcome after experimental traumatic brain injury (TBI) remains controversial. Because TBI can result in decreased expression of the trkB receptor, thereby preventing BDNF from exerting potential neuroprotective effects, the contribution of both BDNF and its receptor trkB to hippocampal neuronal loss and cognitive dysfunction were evaluated. Methods Full-length trkB was overexpressed in the left hippocampus of adult C57Bl/6 mice using recombinant adeno-associated virus serotype 2/5 (rAAV 2/5). EGFP (enhanced green fluorescent protein) expression was present at two weeks after AAV-EGFP injection and remained sustained up to four weeks after the injection. At 2 weeks following gene transduction, mice were subjected to parasagittal controlled cortical impact (CCI) brain injury, followed by either BDNF or PBS infusion into the hippocampus. Results No differences were observed in learning ability at two weeks post-injury or in motor function from 48 hours to two weeks among treatment groups. The number of surviving pyramidal neurons in the CA2-CA3 region of the hippocampus was also not different among treatment groups. Conclusions These data suggest that neither overexpression of trkB, BNDF infusion or their combination affects neuronal survival or behavioral outcome following experimental TBI in mice. PMID:18431005

  9. Regulation of Neuronal Gene Expression

    NASA Astrophysics Data System (ADS)

    Thiel, Gerald; Lietz, Michael; Leichter, Michael

    Humans as multicellular organisms contain a variety of different cell types where each cell population must fulfill a distinct function in the interest of the whole organism. The molecular basis for the variations in morphology, biochemistry, molecular biology, and function of the various cell types is the cell-type specific expression of genes. These genes encode proteins necessary for executing the specialized functions of each cell type within an organism. We describe here a regulatory mechanism for the expression of neuronal genes. The zinc finger protein REST binds to the regulatory region of many neuronal genes and represses neuronal gene expression in nonneuronal tissues. A negative regulatory mechanism, involving a transcriptional repressor, seems to play an important role in establishing the neuronal phenotype.

  10. Inferring horizontal gene transfer.

    PubMed

    Ravenhall, Matt; Škunca, Nives; Lassalle, Florent; Dessimoz, Christophe

    2015-05-01

    Horizontal or Lateral Gene Transfer (HGT or LGT) is the transmission of portions of genomic DNA between organisms through a process decoupled from vertical inheritance. In the presence of HGT events, different fragments of the genome are the result of different evolutionary histories. This can therefore complicate the investigations of evolutionary relatedness of lineages and species. Also, as HGT can bring into genomes radically different genotypes from distant lineages, or even new genes bearing new functions, it is a major source of phenotypic innovation and a mechanism of niche adaptation. For example, of particular relevance to human health is the lateral transfer of antibiotic resistance and pathogenicity determinants, leading to the emergence of pathogenic lineages. Computational identification of HGT events relies upon the investigation of sequence composition or evolutionary history of genes. Sequence composition-based ("parametric") methods search for deviations from the genomic average, whereas evolutionary history-based ("phylogenetic") approaches identify genes whose evolutionary history significantly differs from that of the host species. The evaluation and benchmarking of HGT inference methods typically rely upon simulated genomes, for which the true history is known. On real data, different methods tend to infer different HGT events, and as a result it can be difficult to ascertain all but simple and clear-cut HGT events. PMID:26020646

  11. Inferring Horizontal Gene Transfer

    PubMed Central

    Lassalle, Florent; Dessimoz, Christophe

    2015-01-01

    Horizontal or Lateral Gene Transfer (HGT or LGT) is the transmission of portions of genomic DNA between organisms through a process decoupled from vertical inheritance. In the presence of HGT events, different fragments of the genome are the result of different evolutionary histories. This can therefore complicate the investigations of evolutionary relatedness of lineages and species. Also, as HGT can bring into genomes radically different genotypes from distant lineages, or even new genes bearing new functions, it is a major source of phenotypic innovation and a mechanism of niche adaptation. For example, of particular relevance to human health is the lateral transfer of antibiotic resistance and pathogenicity determinants, leading to the emergence of pathogenic lineages [1]. Computational identification of HGT events relies upon the investigation of sequence composition or evolutionary history of genes. Sequence composition-based ("parametric") methods search for deviations from the genomic average, whereas evolutionary history-based ("phylogenetic") approaches identify genes whose evolutionary history significantly differs from that of the host species. The evaluation and benchmarking of HGT inference methods typically rely upon simulated genomes, for which the true history is known. On real data, different methods tend to infer different HGT events, and as a result it can be difficult to ascertain all but simple and clear-cut HGT events. PMID:26020646

  12. Evolutionary genomics: transdomain gene transfers.

    PubMed

    Bordenstein, Seth R

    2007-11-01

    Biologists have until now conceded that bacterial gene transfer to multicellular animals is relatively uncommon in Nature. A new study showing promiscuous insertions of bacterial endosymbiont genes into invertebrate genomes ushers in a shift in this paradigm.

  13. PPARγ-coactivator-1α gene transfer reduces neuronal loss and amyloid-β generation by reducing β-secretase in an Alzheimer’s disease model

    PubMed Central

    Lim, Yau M.; Blondrath, Katrin; Eleftheriadou, Ioanna; Lombardero, Laura; Birch, Amy M.; Mirzaei, Nazanin; Irvine, Elaine E.; Mazarakis, Nicholas D.; Sastre, Magdalena

    2016-01-01

    Current therapies for Alzheimer’s disease (AD) are symptomatic and do not target the underlying Aβ pathology and other important hallmarks including neuronal loss. PPARγ-coactivator-1α (PGC-1α) is a cofactor for transcription factors including the peroxisome proliferator-activated receptor-γ (PPARγ), and it is involved in the regulation of metabolic genes, oxidative phosphorylation, and mitochondrial biogenesis. We previously reported that PGC-1α also regulates the transcription of β-APP cleaving enzyme (BACE1), the main enzyme involved in Aβ generation, and its expression is decreased in AD patients. We aimed to explore the potential therapeutic effect of PGC-1α by generating a lentiviral vector to express human PGC-1α and target it by stereotaxic delivery to hippocampus and cortex of APP23 transgenic mice at the preclinical stage of the disease. Four months after injection, APP23 mice treated with hPGC-1α showed improved spatial and recognition memory concomitant with a significant reduction in Aβ deposition, associated with a decrease in BACE1 expression. hPGC-1α overexpression attenuated the levels of proinflammatory cytokines and microglial activation. This effect was accompanied by a marked preservation of pyramidal neurons in the CA3 area and increased expression of neurotrophic factors. The neuroprotective effects were secondary to a reduction in Aβ pathology and neuroinflammation, because wild-type mice receiving the same treatment were unaffected. These results suggest that the selective induction of PGC-1α gene in specific areas of the brain is effective in targeting AD-related neurodegeneration and holds potential as therapeutic intervention for this disease. PMID:27791018

  14. Physical methods for gene transfer.

    PubMed

    Alsaggar, Mohammad; Liu, Dexi

    2015-01-01

    The key impediment to the successful application of gene therapy in clinics is not the paucity of therapeutic genes. It is rather the lack of nontoxic and efficient strategies to transfer therapeutic genes into target cells. Over the past few decades, considerable progress has been made in gene transfer technologies, and thus far, three different delivery systems have been developed with merits and demerits characterizing each system. Viral and chemical methods of gene transfer utilize specialized carrier to overcome membrane barrier and facilitate gene transfer into cells. Physical methods, on the other hand, utilize various forms of mechanical forces to enforce gene entry into cells. Starting in 1980s, physical methods have been introduced as alternatives to viral and chemical methods to overcome various extra- and intracellular barriers that limit the amount of DNA reaching the intended cells. Accumulating evidence suggests that it is quite feasible to directly translocate genes into cytoplasm or even nuclei of target cells by means of mechanical force, bypassing endocytosis, a common pathway for viral and nonviral vectors. Indeed, several methods have been developed, and the majority of them share the same underlying mechanism of gene transfer, i.e., physically created transient pores in cell membrane through which genes get into cells. Here, we provide an overview of the current status and future research directions in the field of physical methods of gene transfer.

  15. AAV-Dominant Negative Tumor Necrosis Factor (DN-TNF) Gene Transfer to the Striatum Does Not Rescue Medium Spiny Neurons in the YAC128 Mouse Model of Huntington's Disease

    PubMed Central

    Alto, Laura Taylor; Chen, Xi; Ruhn, Kelly A.; Treviño, Isaac; Tansey, Malú G.

    2014-01-01

    CNS inflammation is a hallmark of neurodegenerative disease, and recent studies suggest that the inflammatory response may contribute to neuronal demise. In particular, increased tumor necrosis factor (TNF) signaling is implicated in the pathology of both Parkinson's disease (PD) and Alzheimer's disease (AD). We have previously shown that localized gene delivery of dominant negative TNF to the degenerating brain region can limit pathology in animal models of PD and AD. TNF is upregulated in Huntington's disease (HD), like in PD and AD, but it is unknown whether TNF signaling contributes to neuronal degeneration in HD. We used in vivo gene delivery to test whether selective reduction of soluble TNF signaling could attenuate medium spiny neuron (MSN) degeneration in the YAC128 transgenic (TG) mouse model of Huntington's disease (HD). AAV vectors encoding cDNA for dominant-negative tumor necrosis factor (DN-TNF) or GFP (control) were injected into the striatum of young adult wild type WT and YAC128 TG mice and achieved 30–50% target coverage. Expression of dominant negative TNF protein was confirmed immunohistologically and biochemically and was maintained as mice aged to one year, but declined significantly over time. However, the extent of striatal DN-TNF gene transfer achieved in our studies was not sufficient to achieve robust effects on neuroinflammation, rescue degenerating MSNs or improve motor function in treated mice. Our findings suggest that alternative drug delivery strategies should be explored to determine whether greater target coverage by DN-TNF protein might afford some level of neuroprotection against HD-like pathology and/or that soluble TNF signaling may not be the primary driver of striatal neuroinflammation and MSN loss in YAC128 TG mice. PMID:24824433

  16. Lateral gene transfer in eukaryotes.

    PubMed

    Andersson, J O

    2005-06-01

    Lateral gene transfer -- the transfer of genetic material between species -- has been acknowledged as a major mechanism in prokaryotic genome evolution for some time. Recently accumulating data indicate that the process also occurs in the evolution of eukaryotic genomes. However, there are large rate variations between groups of eukaryotes; animals and fungi seem to be largely unaffected, with a few exceptions, while lateral gene transfer frequently occurs in protists with phagotrophic lifestyles, possibly with rates comparable to prokaryotic organisms. Gene transfers often facilitate the acquisition of functions encoded in prokaryotic genomes by eukaryotic organisms, which may enable them to colonize new environments. Transfers between eukaryotes also occur, mainly into larger phagotrophic eukaryotes that ingest eukaryotic cells, but also between plant lineages. These findings have implications for eukaryotic genomic research in general, and studies of the origin and phylogeny of eukaryotes in particular.

  17. Gene Transfer into Cardiac Myocytes

    PubMed Central

    Lang, Sarah E.; Westfall, Margaret V.

    2016-01-01

    Traditional methods for DNA transfection are often inefficient and toxic for terminally differentiated cells, such as cardiac myocytes. Vector-based gene transfer is an efficient approach for introducing exogenous cDNA into these types of primary cell cultures. In this chapter, separate protocols for adult rat cardiac myocyte isolation and gene transfer with recombinant adenovirus are provided and are routinely utilized for studying the effects of sarcomeric proteins on myofilament function. PMID:25836585

  18. Horizontal gene transfer in choanoflagellates.

    PubMed

    Tucker, Richard P

    2013-01-01

    Horizontal gene transfer (HGT), also known as lateral gene transfer, results in the rapid acquisition of genes from another organism. HGT has long been known to be a driving force in speciation in prokaryotes, and there is evidence for HGT from symbiotic and infectious bacteria to metazoans, as well as from protists to bacteria. Recently, it has become clear that as many as a 1,000 genes in the genome of the choanoflagellate Monosiga brevicollis may have been acquired by HGT. Interestingly, these genes reportedly come from algae, bacteria, and other choanoflagellate prey. Some of these genes appear to have allowed an ancestral choanoflagellate to exploit nutrient-poor environments and were not passed on to metazoan descendents. However, some of these genes are also found in animal genomes, suggesting that HGT into a common ancestor of choanozoans and animals may have contributed to metazoan evolution.

  19. Tetracycline inducible gene manipulation in serotonergic neurons.

    PubMed

    Weber, Tillmann; Renzland, Insa; Baur, Max; Mönks, Simon; Herrmann, Elke; Huppert, Verena; Nürnberg, Frank; Schönig, Kai; Bartsch, Dusan

    2012-01-01

    The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a

  20. Gene transfer: transduction.

    PubMed

    Frangipani, Emanuela

    2014-01-01

    Bacteriophages able to propagate on Pseudomonas strains are very common and can be easily isolated from natural environments or lysogenic strains. The development of transducing systems has allowed bacterial geneticists to perform chromosome analyses and mutation mapping. Moreover, these systems have also been proved to be a successful tool for molecular microbiologists to introduce a foreign gene or a mutation into the chromosome of a bacterial cell. This chapter provides a description of the phage methodology illustrated by Adams in 1959 and applicable to strain PAO1 derivatives. PMID:24818891

  1. Efficient information transfer by Poisson neurons.

    PubMed

    Kostal, Lubomir; Shinomoto, Shigeru

    2016-06-01

    Recently, it has been suggested that certain neurons with Poissonian spiking statistics may communicate by discontinuously switching between two levels of firing intensity. Such a situation resembles in many ways the optimal information transmission protocol for the continuous-time Poisson channel known from information theory. In this contribution we employ the classical information-theoretic results to analyze the efficiency of such a transmission from different perspectives, emphasising the neurobiological viewpoint. We address both the ultimate limits, in terms of the information capacity under metabolic cost constraints, and the achievable bounds on performance at rates below capacity with fixed decoding error probability. In doing so we discuss optimal values of experimentally measurable quantities that can be compared with the actual neuronal recordings in a future effort. PMID:27106184

  2. Panspermia and horizontal gene transfer

    NASA Astrophysics Data System (ADS)

    Klyce, Brig

    2009-08-01

    Evidence that extremophiles are hardy and ubiquitous is helping to make panspermia a respectable theory. But even if life on Earth originally came from space, biologists assume that the subsequent evolution of life is still governed by the darwinian paradigm. In this review we show how panspermia could amend darwinism and point to a cosmic source for, not only extremophiles but, all of life. This version of panspermia can be called "strong panspermia." To support this theory we will discuss recent evidence pertaining to horizontal gene transfer, viruses, genes apparently older than the Earthly evolution of the features they encode, and primate-specific genes without identifiable precursors.

  3. Rich club neurons dominate Information Transfer in local cortical networks

    NASA Astrophysics Data System (ADS)

    Nigam, Sunny; Shimono, Masanori; Sporns, Olaf; Beggs, John

    2015-03-01

    The performance of complex networks depends on how they route their traffic. It is unknown how information is transferred in local cortical networks of hundreds of closely-spaced neurons. To address this, it is necessary to record simultaneously from hundreds of neurons at a spacing that matches typical axonal connection distances, and at a temporal resolution that matches synaptic delays. We used a 512 electrode array (60 μm spacing) to record spontaneous activity at 20 kHz, simultaneously from up to 700 neurons in slice cultures of mouse somatosensory cortex for 1 hr at a time. We used transfer entropy to quantify directed information transfer (IT) between pairs of neurons. We found an approximately lognormal distribution of firing rates as reported in in-vivo. Pairwise information transfer strengths also were nearly lognormally distributed, similar to synaptic strengths. 20% of the neurons accounted for 70% of the total IT coming into, and going out of the network and were defined as rich nodes. These rich nodes were more densely and strongly connected to each other expected by chance, forming a rich club. This highly uneven distribution of IT has implications for the efficiency and robustness of local cortical networks, and gives clues to the plastic processes that shape them. JSPS.

  4. Multifaceted effects of oligodendroglial exosomes on neurons: impact on neuronal firing rate, signal transduction and gene regulation

    PubMed Central

    Fröhlich, Dominik; Kuo, Wen Ping; Frühbeis, Carsten; Sun, Jyh-Jang; Zehendner, Christoph M.; Luhmann, Heiko J.; Pinto, Sheena; Toedling, Joern; Trotter, Jacqueline; Krämer-Albers, Eva-Maria

    2014-01-01

    Exosomes are small membranous vesicles of endocytic origin that are released by almost every cell type. They exert versatile functions in intercellular communication important for many physiological and pathological processes. Recently, exosomes attracted interest with regard to their role in cell–cell communication in the nervous system. We have shown that exosomes released from oligodendrocytes upon stimulation with the neurotransmitter glutamate are internalized by neurons and enhance the neuronal stress tolerance. Here, we demonstrate that oligodendroglial exosomes also promote neuronal survival during oxygen–glucose deprivation, a model of cerebral ischaemia. We show the transfer from oligodendrocytes to neurons of superoxide dismutase and catalase, enzymes which are known to help cells to resist oxidative stress. Additionally, we identify various effects of oligodendroglial exosomes on neuronal physiology. Electrophysiological analysis using in vitro multi-electrode arrays revealed an increased firing rate of neurons exposed to oligodendroglial exosomes. Moreover, gene expression analysis and phosphorylation arrays uncovered differentially expressed genes and altered signal transduction pathways in neurons after exosome treatment. Our study thus provides new insight into the broad spectrum of action of oligodendroglial exosomes and their effects on neuronal physiology. The exchange of extracellular vesicles between neural cells may exhibit remarkable potential to impact brain performance. PMID:25135971

  5. Transfer of mitochondria from astrocytes to neurons after stroke.

    PubMed

    Hayakawa, Kazuhide; Esposito, Elga; Wang, Xiaohua; Terasaki, Yasukazu; Liu, Yi; Xing, Changhong; Ji, Xunming; Lo, Eng H

    2016-07-28

    Neurons can release damaged mitochondria and transfer them to astrocytes for disposal and recycling. This ability to exchange mitochondria may represent a potential mode of cell-to-cell signalling in the central nervous system. Here we show that astrocytes in mice can also release functional mitochondria that enter neurons. Astrocytic release of extracellular mitochondrial particles was mediated by a calcium-dependent mechanism involving CD38 and cyclic ADP ribose signalling. Transient focal cerebral ischaemia in mice induced entry of astrocytic mitochondria into adjacent neurons, and this entry amplified cell survival signals. Suppression of CD38 signalling by short interfering RNA reduced extracellular mitochondria transfer and worsened neurological outcomes. These findings suggest a new mitochondrial mechanism of neuroglial crosstalk that may contribute to endogenous neuroprotective and neurorecovery mechanisms after stroke. PMID:27466127

  6. Ethics of Cancer Gene Transfer Clinical Research.

    PubMed

    Kimmelman, Jonathan

    2015-01-01

    Translation of cancer gene transfer confronts many familiar-and some distinctive-ethical challenges. In what follows, I survey three major ethical dimensions of cancer gene transfer development. Subheading 1 centers on the ethics of planning, designing, and reporting animal studies. Subheading 2 describes basic elements of human subjects protection as pertaining to cancer gene transfer. In Subheading 3, I describe how cancer gene transfer researchers have obligations to downstream consumers of the evidence they produce.

  7. The molecular and gene regulatory signature of a neuron

    PubMed Central

    Hobert, Oliver; Carrera, Inés; Stefanakis, Nikolaos

    2010-01-01

    Neuron-type specific gene batteries define the morphological and functional diversity of cell types in the nervous system. Here, we discuss the composition of neuron-type specific gene batteries and illustrate gene regulatory strategies employed by distinct organisms from C.elegans to higher vertebrates, which are instrumental in determining the unique gene expression profile and molecular composition of individual neuronal cell types. Based on principles learned from prokaryotic gene regulation, we argue that neuronal, terminal gene batteries are functionally grouped into parallel acting “regulons”. The theoretical concepts discussed here provide testable hypotheses for future experimental analysis into the exact gene regulatory mechanisms that are employed in the generation of neuronal diversity and identity. PMID:20663572

  8. Readministration of adenoviral gene delivery to dopamine neurons.

    PubMed

    Gonzalez, Sarah C; McMenamin, Margaret M; Charlton, Harry M; Goodman, James; Lantos, Tibor; Simpson, Christine; Wood, Matthew J A

    2007-10-01

    An approach currently being explored as treatment for Parkinson's disease is gene therapy. An important question concerns the duration of transgene expression in dopamine neurons and the issues of vector persistence, neuronal damage and the feasibility of readministering vector to the same neuronal population. We show, using an adenoviral vector expressing the LacZ reporter gene, that transgene expression declined over time but with minimal loss of dopamine neurons or vector DNA. Readministration of vector resulted in low levels of transgene delivery to the neurons. Moreover, the neurons to which vector had already been delivered were unable to transport the retrograde tracer fluorogold. Our findings indicate that transgene expression declined in dopamine neurons despite the persistence of virus, and the capacity to readminister vector to these neurons was limited. PMID:17885611

  9. Generation of cloned mice from adult neurons by direct nuclear transfer.

    PubMed

    Mizutani, Eiji; Oikawa, Mami; Kassai, Hidetoshi; Inoue, Kimiko; Shiura, Hirosuke; Hirasawa, Ryutaro; Kamimura, Satoshi; Matoba, Shogo; Ogonuki, Narumi; Nagatomo, Hiroaki; Abe, Kuniya; Wakayama, Teruhiko; Aiba, Atsu; Ogura, Atsuo

    2015-03-01

    Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain "unclonable" for unknown reasons. Here, using a combination of two epigenetic approaches, we examined whether neurons from adult mice could be cloned. First, we used a specific antibody to discover cell types with reduced amounts of a repressive histone mark-dimethylated histone H3 lysine 9 (H3K9me2)-and identified CA1 pyramidal cells in the hippocampus and Purkinje cells in the cerebellum as candidates. Second, reconstructed embryos were treated with trichostatin A (TSA), a potent histone deacetylase inhibitor. Using CA1 cells, cloned offspring were obtained at high rates, reaching 10.2% and 4.6% (of embryos transferred) for male and female donors, respectively. Cerebellar Purkinje cell nuclei were too large to maintain their genetic integrity during nuclear transfer, leading to developmental arrest of embryos. However, gene expression analysis using cloned blastocysts corroborated a high rate of genomic reprogrammability of CA1 pyramidal and Purkinje cells. Neurons from the hippocampal dentate gyrus and cerebral cortex, which had higher amounts of H3K9me2, could also be used for producing cloned offspring, but the efficiencies were low. A more thorough analysis revealed that TSA treatment was essential for cloning adult neuronal cells. This study demonstrates, to our knowledge for the first time, that adult neurons can be cloned by nuclear transfer. Furthermore, our data imply that reduced amounts of H3K9me2 and increased histone acetylation appear to act synergistically to improve the development of cloned embryos.

  10. Gene transfer into neural cells in vitro using adenoviral vectors.

    PubMed

    Southgate, T D; Kingston, P A; Castro, M G

    2001-05-01

    Adenoviruses (Ads) have become a very attractive and versatile vector system for delivering genes into brain cells in vitro and in vivo. One of the main attractions of Ads is that they can mediate gene transfer into post-mitotic cells, i.e. neurons. Ads are easy to grow and manipulate, stable, and their biology is very well understood. This unit is designed to help newcomers into the field, to design, prepare and grow replication-defective recombinant adenovirus vectors with the aim of transferring genes into neurons and glial cells in primary culture. It provides step-by-step methods describing the preparation of brain cell cultures, their infection using recombinant adenovirus vectors and also the assessment of transgene expression using a variety of techniques including fluorescence immunocytochemistry and fluorescence activated cell-sorting (FACS) analysis. The methods described will be useful to scientists wishing to enter the adenovirus field to construct adenovirus vectors to be used for gene transfer into neural cells.

  11. Lateral Gene Transfer from the Dead

    PubMed Central

    Szöllősi, Gergely J.; Tannier, Eric; Lartillot, Nicolas; Daubin, Vincent

    2013-01-01

    In phylogenetic studies, the evolution of molecular sequences is assumed to have taken place along the phylogeny traced by the ancestors of extant species. In the presence of lateral gene transfer, however, this may not be the case, because the species lineage from which a gene was transferred may have gone extinct or not have been sampled. Because it is not feasible to specify or reconstruct the complete phylogeny of all species, we must describe the evolution of genes outside the represented phylogeny by modeling the speciation dynamics that gave rise to the complete phylogeny. We demonstrate that if the number of sampled species is small compared with the total number of existing species, the overwhelming majority of gene transfers involve speciation to and evolution along extinct or unsampled lineages. We show that the evolution of genes along extinct or unsampled lineages can to good approximation be treated as those of independently evolving lineages described by a few global parameters. Using this result, we derive an algorithm to calculate the probability of a gene tree and recover the maximum-likelihood reconciliation given the phylogeny of the sampled species. Examining 473 near-universal gene families from 36 cyanobacteria, we find that nearly a third of transfer events (28%) appear to have topological signatures of evolution along extinct species, but only approximately 6% of transfers trace their ancestry to before the common ancestor of the sampled cyanobacteria. [Gene tree reconciliation; lateral gene transfer; macroevolution; phylogeny.] PMID:23355531

  12. Gene transfer from genetically modified food.

    PubMed

    Gasson, M J

    2000-10-01

    The current debate about the safety of genetically modified food includes some important scientific issues where more scientific data would aid the robustness of safety evaluation. One example is the possibility of gene transfer, especially from genetically modified plant material.

  13. Analysis of neuronal gene expression with laser capture microdissection.

    PubMed

    Vincent, Valerie A M; DeVoss, Jason J; Ryan, Heather S; Murphy, Greer M

    2002-09-01

    The brain is a heterogeneous tissue in which the numbers of neurons, glia, and other cell types vary among anatomic regions. Gene expression studies performed on brain homogenates yield results reflecting mRNA abundance in a mixture of cell types. Therefore, a method for quantifying gene expression in individual cell populations would be useful. Laser capture microdissection (LCM) is a new technique for obtaining pure populations of cells from heterogeneous tissues. Most studies thus far have used LCM to detect DNA sequences. We developed a method to quantify gene expression in hippocampal neurons from mouse brain using LCM and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). This method was optimized to permit histochemical or immunocytochemical visualization of nerve cells during LCM while minimizing RNA degradation. As an example, gene expression was quantified in hippocampal neurons from the Tg2576 mouse model for Alzheimer's disease.

  14. Lessons learned from gene transfer approaches.

    PubMed

    Evans, C H; Ghivizzani, S C; Lechman, E R; Mi, Z; Jaffurs, D; Robbins, P D

    1999-01-01

    Recent technological advances allow the transfer of genes to the synovial lining of joints. As well as opening novel opportunities for therapy, these techniques provide valuable new tools for the study of synovitis and other aspects of the biology of joints in health and disease. This article reviews briefly the results of experiments in which selected genes have been transferred to the knee joints of healthy rabbits and rabbits with antigen-induced arthritis.

  15. Transferable neuronal mini-cultures to accelerate screening in primary and induced pluripotent stem cell-derived neurons.

    PubMed

    Niedringhaus, Mark; Dumitru, Raluca; Mabb, Angela M; Wang, Yuli; Philpot, Benjamin D; Allbritton, Nancy L; Taylor, Anne Marion

    2015-01-01

    The effort and cost of obtaining neurons for large-scale screens has limited drug discovery in neuroscience. To overcome these obstacles, we fabricated arrays of releasable polystyrene micro-rafts to generate thousands of uniform, mobile neuron mini-cultures. These mini-cultures sustain synaptically-active neurons which can be easily transferred, thus increasing screening throughput by >30-fold. Compared to conventional methods, micro-raft cultures exhibited significantly improved neuronal viability and sample-to-sample consistency. We validated the screening utility of these mini-cultures for both mouse neurons and human induced pluripotent stem cell-derived neurons by successfully detecting disease-related defects in synaptic transmission and identifying candidate small molecule therapeutics. This affordable high-throughput approach has the potential to transform drug discovery in neuroscience. PMID:25666972

  16. Gene Transfers Between Distantly Related Organisms

    NASA Technical Reports Server (NTRS)

    Doolittle, Russell F.

    2003-01-01

    With the completion of numerous microbial genome sequences, reports of individual gene transfers between distantly related prokaryotes have become commonplace. On the other hand, transfers between prokaryotes and eukaryotes still excite the imagination. Many of these claims may be premature, but some are certainly valid. In this chapter, the kinds of supporting data needed to propose transfers between distantly related organisms and cite some interesting examples are considered.

  17. Arginine-Rich Polyplexes for Gene Delivery to Neuronal Cells

    PubMed Central

    Morris, Viola B.; Labhasetwar, Vinod

    2015-01-01

    Neuronal gene therapy potentially offers an effective therapeutic intervention to cure or slow the progression of neurological diseases. However, neuronal cells are difficult to transfect with nonviral vectors, and in vivo their transport across the blood-brain barrier (BBB) is inefficient. We synthesized a series of arginine-rich oligopeptides, grafted with polyethyleneimine (PEI) and modified with a short-chain polyethylene glycol (PEG). We hypothesized that the arginine would enhance cellular uptake and transport of these polyplexes across the BBB, with PEG imparting biocompatibility and “stealth” properties and PEI facilitating DNA condensation and gene transfection. The optimized composition of the polyplexes demonstrated hemocompatibility with red blood cells, causing no lysis or aggregation, and showed significantly better cytocompatibility than PEI in vitro. Polyplexes formulated with luciferase-expressing plasmid DNA could transfect rat primary astrocytes and neurons in vitro. Confocal imaging data showed efficient cellular uptake of DNA and its sustained intracellular retention and nuclear localization with polyplexes. Intravenous administration of the optimized polyplexes in mice led to gene expression in the brain, which upon further immunohistochemical analysis demonstrated gene expression in neurons. In conclusion, we have successfully designed a nonviral vector for in vitro and in vivo neuronal gene delivery. PMID:26000961

  18. Lateral gene transfer from the dead.

    PubMed

    Szöllosi, Gergely J; Tannier, Eric; Lartillot, Nicolas; Daubin, Vincent

    2013-05-01

    In phylogenetic studies, the evolution of molecular sequences is assumed to have taken place along the phylogeny traced by the ancestors of extant species. In the presence of lateral gene transfer, however, this may not be the case, because the species lineage from which a gene was transferred may have gone extinct or not have been sampled. Because it is not feasible to specify or reconstruct the complete phylogeny of all species, we must describe the evolution of genes outside the represented phylogeny by modeling the speciation dynamics that gave rise to the complete phylogeny. We demonstrate that if the number of sampled species is small compared with the total number of existing species, the overwhelming majority of gene transfers involve speciation to and evolution along extinct or unsampled lineages. We show that the evolution of genes along extinct or unsampled lineages can to good approximation be treated as those of independently evolving lineages described by a few global parameters. Using this result, we derive an algorithm to calculate the probability of a gene tree and recover the maximum-likelihood reconciliation given the phylogeny of the sampled species. Examining 473 near-universal gene families from 36 cyanobacteria, we find that nearly a third of transfer events (28%) appear to have topological signatures of evolution along extinct species, but only approximately 6% of transfers trace their ancestry to before the common ancestor of the sampled cyanobacteria.

  19. Fate of midbrain dopaminergic neurons controlled by the engrailed genes.

    PubMed

    Simon, H H; Saueressig, H; Wurst, W; Goulding, M D; O'Leary, D D

    2001-05-01

    Deficiencies in neurotransmitter-specific cell groups in the midbrain result in prominent neural disorders, including Parkinson's disease, which is caused by the loss of dopaminergic neurons of the substantia nigra. We have investigated in mice the role of the engrailed homeodomain transcription factors, En-1 and En-2, in controlling the developmental fate of midbrain dopaminergic neurons. En-1 is highly expressed by essentially all dopaminergic neurons in the substantia nigra and ventral tegmentum, whereas En-2 is highly expressed by a subset of them. These neurons are generated and differentiate their dopaminergic phenotype in En-1/En-2 double null mutants, but disappear soon thereafter. Use of an En-1/tau-LacZ knock-in mouse as an autonomous marker for these neurons indicates that they are lost, rather than that they change their neurotransmitter phenotype. A single allele of En-1 on an En-2 null background is sufficient to produce a wild type-like substantia nigra and ventral tegmentum, whereas in contrast a single allele of En-2 on an En-1 null background results in the survival of only a small proportion of these dopaminergic neurons, a finding that relates to the differential expression of En-1 and En-2. Additional findings indicate that En-1 and En-2 regulate expression of alpha-synuclein, a gene that is genetically linked to Parkinson's disease. These findings show that the engrailed genes are expressed by midbrain dopaminergic neurons from their generation to adulthood but are not required for their specification. However, the engrailed genes control the survival of midbrain dopaminergic neurons in a gene dose-dependent manner. Our findings also suggest a link between engrailed and Parkinson's disease.

  20. Fate of midbrain dopaminergic neurons controlled by the engrailed genes.

    PubMed

    Simon, H H; Saueressig, H; Wurst, W; Goulding, M D; O'Leary, D D

    2001-05-01

    Deficiencies in neurotransmitter-specific cell groups in the midbrain result in prominent neural disorders, including Parkinson's disease, which is caused by the loss of dopaminergic neurons of the substantia nigra. We have investigated in mice the role of the engrailed homeodomain transcription factors, En-1 and En-2, in controlling the developmental fate of midbrain dopaminergic neurons. En-1 is highly expressed by essentially all dopaminergic neurons in the substantia nigra and ventral tegmentum, whereas En-2 is highly expressed by a subset of them. These neurons are generated and differentiate their dopaminergic phenotype in En-1/En-2 double null mutants, but disappear soon thereafter. Use of an En-1/tau-LacZ knock-in mouse as an autonomous marker for these neurons indicates that they are lost, rather than that they change their neurotransmitter phenotype. A single allele of En-1 on an En-2 null background is sufficient to produce a wild type-like substantia nigra and ventral tegmentum, whereas in contrast a single allele of En-2 on an En-1 null background results in the survival of only a small proportion of these dopaminergic neurons, a finding that relates to the differential expression of En-1 and En-2. Additional findings indicate that En-1 and En-2 regulate expression of alpha-synuclein, a gene that is genetically linked to Parkinson's disease. These findings show that the engrailed genes are expressed by midbrain dopaminergic neurons from their generation to adulthood but are not required for their specification. However, the engrailed genes control the survival of midbrain dopaminergic neurons in a gene dose-dependent manner. Our findings also suggest a link between engrailed and Parkinson's disease. PMID:11312297

  1. Horizontal gene transfer of stress resistance genes through plasmid transport.

    PubMed

    Shoeb, Erum; Badar, Uzma; Akhter, Jameela; Shams, Hina; Sultana, Maria; Ansari, Maqsood A

    2012-03-01

    The horizontal gene transfer of plasmid-determined stress tolerance was achieved under lab conditions. Bacterial isolates, Enterobacter cloacae (DGE50) and Escherichia coli (DGE57) were used throughout the study. Samples were collected from contaminated marine water and soil to isolate bacterial strains having tolerance against heavy metals and antimicrobial agents. We have demonstrated plasmid transfer, from Amp(+)Cu(+)Zn(-) strain (DGE50) to Amp(-)Cu(-)Zn(+) strain (DGE57), producing Amp(+)Cu(+)Zn(+) transconjugants (DGE(TC50→57)) and Amp(+)Cu(-)Zn(+) transformants (DGE(TF50→57)). DGE57 did not carry any plasmid, therefore, it can be speculated that zinc tolerance gene in DGE57 is located on chromosome. DGE50 was found to carry three plasmids, out of which two were transferred through conjugation into DGE57, and only one was transferred through transformation. Plasmid transferred through transformation was one out of the two transferred through conjugation. Through the results of transformation it was revealed that the genes of copper and ampicillin tolerance in DGE50 were located on separate plasmids, since only ampicillin tolerance genes were transferred through transformation as a result of one plasmid transfer. By showing transfer of plasmids under lab conditions and monitoring retention of respective phenotype via conjugation and transformation, it is very well demonstrated how multiple stress tolerant strains are generated in nature. PMID:22805823

  2. Dopamine receptor gene expression by enkephalin neurons in rat forebrain

    SciTech Connect

    Le Moine, C.; Normand, E.; Guitteny, A.F.; Fouque, B.; Teoule, R.; Bloch, B. )

    1990-01-01

    In situ hybridization experiments were performed with brain sections from normal, control and haloperidol-treated rats to identify and map the cells expressing the D2 dopamine receptor gene. D2 receptor mRNA was detected with radioactive or biotinylated oligonucleotide probes. D2 receptor mRNA was present in glandular cells of the pituitary intermediate lobe and in neurons of the substantia nigra, ventral tegmental area, and forebrain, especially in caudate putamen, nucleus accumbens, olfactory tubercle, and piriform cortex. Hybridization with D2 and preproenkephalin A probes in adjacent sections, as well as combined hybridization with the two probes in the same sections, demonstrated that all detectable enkephalin neurons in the striatum contained the D2 receptor mRNA. Large neurons in caudate putamen, which were unlabeled with the preproenkephalin A probe and which may have been cholinergic, also expressed the D2 receptor gene. Haloperidol treatment (14 or 21 days) provoked an increase in mRNA content for D2 receptor and preproenkephalin A in the striatum. This suggests that the increase in D2 receptor number observed after haloperidol treatment is due to increased activity of the D2 gene. These results indicate that in the striatum, the enkephalin neurons are direct targets for dopamine liberated from mesostriatal neurons.

  3. Gene transfer mediated by alpha2-macroglobulin.

    PubMed Central

    Schneider, H; Huse, K; Birkenmeier, G; Otto, A; Scholz, G H

    1996-01-01

    alpha2-Macroglobulin covalently linked to poly(L)-lysine can be used as a vehicle for receptor-mediated gene transfer. This modified alpha2-macroglobulin maintains its ability to bind to the alpha2-macroglobulin receptor, and was shown to introduce a luciferase reporter gene plasmid into HepG2 human hepatoma cells in vitro. The alpha2-macroglobulin receptor is a very large and multifunctional cell surface receptor, whose rapid and efficient internalization rate makes it attractive for gene therapy, e.g. for hepatic gene targeting via injection into the portal vein. PMID:8871570

  4. Corepressor-dependent silencing of chromosomal regions encoding neuronal genes.

    PubMed

    Lunyak, Victoria V; Burgess, Robert; Prefontaine, Gratien G; Nelson, Charles; Sze, Sing-Hoi; Chenoweth, Josh; Schwartz, Phillip; Pevzner, Pavel A; Glass, Christopher; Mandel, Gail; Rosenfeld, Michael G

    2002-11-29

    The molecular mechanisms by which central nervous system-specific genes are expressed only in the nervous system and repressed in other tissues remain a central issue in developmental and regulatory biology. Here, we report that the zinc-finger gene-specific repressor element RE-1 silencing transcription factor/neuronal restricted silencing factor (REST/NRSF) can mediate extraneuronal restriction by imposing either active repression via histone deacetylase recruitment or long-term gene silencing using a distinct functional complex. Silencing of neuronal-specific genes requires the recruitment of an associated corepressor, CoREST, that serves as a functional molecular beacon for the recruitment of molecular machinery that imposes silencing across a chromosomal interval, including transcriptional units that do not themselves contain REST/NRSF response elements.

  5. AAV-Mediated Gene Transfer to Dorsal Root Ganglion.

    PubMed

    Yu, Hongwei; Fischer, Gregory; Hogan, Quinn H

    2016-01-01

    Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.

  6. RANGE: Gene Transfer of Reversibly Controlled Polycistronic Genes

    PubMed Central

    Chen, Yiwei; Cao, Liji; Luo, Chonglin; Ditzel, Désirée AW; Peter, Jörg; Sprengel, Rolf

    2013-01-01

    We developed a single vector recombinant adeno-associated viral (rAAV) expression system for spatial and reversible control of polycistronic gene expression. Our approach (i) integrates the advantages of the tetracycline (Tet)-controlled transcriptional silencer tTSKid and the self-cleaving 2A peptide bridge, (ii) combines essential regulatory components as an autoregulatory loop, (iii) simplifies the gene delivery scheme, and (iv) regulates multiple genes in a synchronized manner. Controlled by an upstream Tet-responsive element (TRE), both the ubiquitous chicken β-actin promoter (CAG) and the neuron-specific synapsin-1 promoter (Syn) could regulate expression of tTSKid together with two 2A-linked reporter genes. Transduction in vitro exhibited maximally 50-fold regulation by doxycycline (Dox). Determined by gene delivery method as well as promoter, highly specific tissues were transduced in vivo. Bioluminescence imaging (BLI) visualized reversible “ON/OFF” gene switches over repeated “Doxy-Cycling” in living mice. Thus, the reversible rAAV-mediated N-cistronic gene expression system, termed RANGE, may serve as a versatile tool to achieve reversible polycistronic gene regulation for the study of gene function as well as gene therapy. PMID:23571608

  7. Viral Vectors for in Vivo Gene Transfer

    NASA Astrophysics Data System (ADS)

    Thévenot, E.; Dufour, N.; Déglon, N.

    The transfer of DNA into the nucleus of a eukaryotic cell (gene transfer) is a central theme of modern biology. The transfer is said to be somatic when it refers to non-germline organs of a developed individual, and germline when it concerns gametes or the fertilised egg of an animal, with the aim of transmitting the relevant genetic modification to its descendents [1]. The efficient introduction of genetic material into a somatic or germline cell and the control of its expression over time have led to major advances in understanding how genes work in vivo, i.e., in living organisms (functional genomics), but also to the development of innovative therapeutic methods (gene therapy). The efficiency of gene transfer is conditioned by the vehicle used, called the vector. Desirable features for a vector are as follows: Easy to produce high titer stocks of the vector in a reproducible way. Absence of toxicity related to transduction (transfer of genetic material into the target cell, and its expression there) and no immune reaction of the organism against the vector and/or therapeutic protein. Stability in the expression of the relevant gene over time, and the possibility of regulation, e.g., to control expression of the therapeutic protein on the physiological level, or to end expression at the end of treatment. Transduction of quiescent cells should be as efficient as transduction of dividing cells. Vectors currently used fall into two categories: non-viral and viral vectors. In non-viral vectors, the DNA is complexed with polymers, lipids, or cationic detergents (described in Chap. 3). These vectors have a low risk of toxicity and immune reaction. However, they are less efficient in vivo than viral vectors when it comes to the number of cells transduced and long-term transgene expression. (Naked DNA transfer or electroporation is rather inefficient in the organism. This type of gene transfer will not be discussed here, and the interested reader is referred to the

  8. Inducible Gene Manipulations in Brain Serotonergic Neurons of Transgenic Rats

    PubMed Central

    Tews, Björn; Bartsch, Dusan

    2011-01-01

    The serotonergic (5-HT) system has been implicated in various physiological processes and neuropsychiatric disorders, but in many aspects its role in normal and pathologic brain function is still unclear. One reason for this might be the lack of appropriate animal models which can address the complexity of physiological and pathophysiological 5-HT functioning. In this respect, rats offer many advantages over mice as they have been the animal of choice for sophisticated neurophysiological and behavioral studies. However, only recently technologies for the targeted and tissue specific modification of rat genes - a prerequisite for a detailed study of the 5-HT system - have been successfully developed. Here, we describe a rat transgenic system for inducible gene manipulations in 5-HT neurons. We generated a Cre driver line consisting of a tamoxifen-inducible CreERT2 recombinase under the control of mouse Tph2 regulatory sequences. Tissue-specific serotonergic Cre recombinase expression was detected in four transgenic TPH2-CreERT2 rat founder lines. For functional analysis of Cre-mediated recombination, we used a rat Cre reporter line (CAG-loxP.EGFP), in which EGFP is expressed after Cre-mediated removal of a loxP-flanked lacZ STOP cassette. We show an in-depth characterisation of this rat Cre reporter line and demonstrate its applicability for monitoring Cre-mediated recombination in all major neuronal subpopulations of the rat brain. Upon tamoxifen induction, double transgenic TPH2-CreERT2/CAG-loxP.EGFP rats show selective and efficient EGFP expression in 5-HT neurons. Without tamoxifen administration, EGFP is only expressed in few 5-HT neurons which confirms minimal background recombination. This 5-HT neuron specific CreERT2 line allows Cre-mediated, inducible gene deletion or gene overexpression in transgenic rats which provides new opportunities to decipher the complex functions of the mammalian serotonergic system. PMID:22140568

  9. Adenoviral vector-mediated gene transfer for human gene therapy.

    PubMed

    Breyer, B; Jiang, W; Cheng, H; Zhou, L; Paul, R; Feng, T; He, T C

    2001-07-01

    Human gene therapy promises to change the practice of medicine by treating the causes of disease rather than the symptoms. Since the first clinical trial made its debut ten years ago, there are over 400 approved protocols in the United States alone, most of which have failed to show convincing data of clinical efficacy. This setback is largely due to the lack of efficient and adequate gene transfer vehicles. With the recent progress in elucidating the molecular mechanisms of human diseases and the imminent arrival of the post genomic era, there are increasing numbers of therapeutic genes or targets that are available for gene therapy. Therefore, the urgency and need for efficacious gene therapies are greater than ever. Clearly, the current fundamental obstacle is to develop delivery vectors that exhibit high efficacy and specificity of gene transfer. Recombinant adenoviruses have provided a versatile system for gene expression studies and therapeutic applications. Of late, there has been a remarkable increase in adenoviral vector-based clinical trials. Recent endeavors in the development of recombinant adenoviral vectors have focused on modification of virus tropism, accommodation of larger genes, increase in stability and control of transgene expression, and down-modulation of host immune responses. These modifications and continued improvements in adenoviral vectors will provide a great opportunity for human gene therapy to live up to its enormous potential in the second decade.

  10. Specific patterns of defective HSV-1 gene transfer in the adult central nervous system: implications for gene targeting.

    PubMed

    Wood, M J; Byrnes, A P; Kaplitt, M G; Pfaff, D W; Rabkin, S D; Charlton, H M

    1994-11-01

    Viral vectors are a means by which genes can be delivered to specific sites in the adult central nervous system. Nevertheless, the interaction between the viral vector and cells of the nervous system, which forms the basis for specific gene transfer, is not well understood. In this study a nonreplicating defective herpes simplex virus type 1 vector, expressing the marker gene lacZ, was stereotaxically injected at varying titers into the rat central nervous system. Three sites were targeted: the caudate nucleus, dentate gyrus, and cerebellar cortex, and the resulting patterns of beta-galactosidase activity were examined. Many cells of neuronal and glial morphology, and of differing neuronal subtypes, expressed beta-galactosidase at each of the injection sites. However, beta-galactosidase activity was also detected in distant secondary brain areas, the neurons of which make afferent connections with the primary sites. This strongly suggested that the retrograde transport of defective virus was the basis for the enzyme activity observed at a distance. Moreover, retrograde transport to secondary sites was found to be highly selective and restricted to certain retrograde neuroanatomical pathways in a specific and titer dependent fashion. The pathways observed were predominantly, but not exclusively, monoaminergic in origin. This finding is supported by reports of specific tropism by HSV for monoaminergic circuits in experimental encephalitis and transneuronal tracing studies. Our observations suggest that certain functional neuronal populations, which are permissive for the retrograde transfer of defective HSV-1 vectors, might be specifically targeted for gene transfer using this approach. Conversely, a knowledge of the pathways permissive for viral uptake, retrograde transfer, and subsequent gene expression will be essential in order to predict the consequences of gene transfer using viral vectors. PMID:7821388

  11. Tetranectin gene deletion induces Parkinson's disease by enhancing neuronal apoptosis.

    PubMed

    Chen, Zhifeng; Wang, Ersong; Hu, Rong; Sun, Yu; Zhang, Lei; Jiang, Jue; Zhang, Ying; Jiang, Hong

    Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). We previously identified tetranectin (TET) as a potential biomarker for PD whose expression is downregulated in the cerebrospinal fluid of PD patients. In the present study, we investigate the role of TET in neurodegeneration in vitro and in vivo. Our results showed that siRNA knockdown of TET decreased cell viability and the number of tyrosine hydroxylase (TH) positive cells, whereas it increased caspase-3 activity and the Bax/Bcl-2 ratio in cultured primary dopaminergic neurons. Overexpression of TET protected dopaminergic neurons against neuronal apoptosis in 1-methyl-4-phenylpyridinium cell culture model in vitro. In TET knockdown mouse model of PD, TET gene deletion decreased the number of TH positive cells in the SNpc, induced apoptosis via the p53/Bax pathway, and significantly impaired the motor behavior of transgenic mice. The findings suggest that TET plays a neuroprotective role via reducing neuron apoptosis and could be a valuable biomarker or potential therapeutic target for the treatment of patients with PD. PMID:26597345

  12. Lateral gene transfer in the subsurface

    SciTech Connect

    Barkay, Tamar; Sobecky, Patricia

    2007-08-27

    Lateral gene transfer (LGT) is an important adaptive mechanism among prokaryotic organisms. This mechanism is particularly important for the response of microorganisms to changing environmental conditions because it facilitates the transfer of a large number of genes and their rapid expression. Together the transferred genes promote rapid genetic and metabolic changes that may enhance survival to newly established and sometimes hostile environmental conditions. The goal of our project was to examine if and how LGT enhances microbial adaptation to toxic heavy metals in subsurface environments that had been contaminated by mixed wastes due to activities associated with the production of nuclear energy and weapons. This task has been accomplished by dividing the project to several sub-tasks. Thus, we: (1) Determined the level of resistance of subsurface bacterial isolates to several toxic metals, all identified as pollutants of concern in subsurface environments; (2) Designed, tested, and applied, a molecular approach that determined whether metal resistance genes had evolved by LGT among subsurface bacteria; and (3) Developed a DNA hybridization array for the identification of broad host range plasmids and of metal resistance plasmids. The results are briefly summarized below with references to published papers and manuscripts in preparation where details about our research can be found. Additional information may be found in copies of our published manuscripts and conference proceedings, and our yearly reports that were submitted through the RIMS system.

  13. Clinical Applications Involving CNS Gene Transfer

    PubMed Central

    Kantor, Boris; McCown, Thomas; Leone, Paola; Gray, Steven J.

    2015-01-01

    Diseases of the central nervous system (CNS) have traditionally been the most difficult to treat by traditional pharmacological methods, due mostly to the blood–brain barrier and the difficulties associated with repeated drug administration targeting the CNS. Viral vector gene transfer represents a way to permanently provide a therapeutic protein within the nervous system after a single administration, whether this be a gene replacement strategy for an inherited disorder or a disease-modifying protein for a disease such as Parkinson's. Gene therapy approaches for CNS disorders has evolved considerably over the last two decades. Although a breakthrough treatment has remained elusive, current strategies are now considerably safer and potentially much more effective. This chapter will explore the past, current, and future status of CNS gene therapy, focusing on clinical trials utilizing adeno-associated virus and lentiviral vectors. PMID:25311921

  14. Perinatal Gene Transfer to the Liver

    PubMed Central

    McKay, Tristan R; Rahim, Ahad A; Buckley, Suzanne M.K; Ward, Natalie J; Chan, Jerry K.Y; Howe, Steven J; Waddington, Simon N

    2011-01-01

    The liver acts as a host to many functions hence raising the possibility that any one may be compromised by a single gene defect. Inherited or de novo mutations in these genes may result in relatively mild diseases or be so devastating that death within the first weeks or months of life is inevitable. Some diseases can be managed using conventional medicines whereas others are, as yet, untreatable. In this review we consider the application of early intervention gene therapy in neonatal and fetal preclinical studies. We appraise the tools of this technology, including lentivirus, adenovirus and adeno-associated virus (AAV)-based vectors. We highlight the application of these for a range of diseases including hemophilia, urea cycle disorders such as ornithine transcarbamylase deficiency, organic acidemias, lysosomal storage diseases including mucopolysaccharidoses, glycogen storage diseases and bile metabolism. We conclude by assessing the advantages and disadvantages associated with fetal and neonatal liver gene transfer. PMID:21774770

  15. Gene transfer between streptomycetes in soil.

    PubMed

    Wellington, E M; Cresswell, N; Herron, P R

    1992-06-15

    The growth and activity of Streptomyces violaceolatus and Streptomyces lividans was studied in soil under controlled conditions. The life cycle was followed under differing nutrient regimes and the fate of plasmid- and phage-borne genes determined by direct and indirect techniques. Methods were developed for the direct monitoring of plasmid DNA extracted from soil which allowed differentiation of the cellular location of plasmid DNA between mycelium and spores. In a dynamic, nutrient-fed soil microcosm, inoculants survived poorly, but a specific stage was defined by direct and indirect methods when the inoculants were most active and this correlated with the detection of gene transfer events. Plasmid transfer, phage infection and lysogeny only occurred to a significant extent within this stage at days 15-17 during a 60-day incubation. Estimates based on plasmid DNA recovery indicated that viable counts underestimated spore and mycelial propagules by a factor of greater than 100.

  16. Endosymbiotic gene transfer and transcriptional regulation of transferred genes in Paulinella chromatophora.

    PubMed

    Nowack, Eva C M; Vogel, Heiko; Groth, Marco; Grossman, Arthur R; Melkonian, Michael; Glöckner, Gernot

    2011-01-01

    Paulinella chromatophora is a cercozoan amoeba that contains "chromatophores," which are photosynthetic inclusions of cyanobacterial origin. The recent discovery that chromatophores evolved independently of plastids, underwent major genome reduction, and transferred at least two genes to the host nucleus has highlighted P. chromatophora as a model to infer early steps in the evolution of photosynthetic organelles. However, owing to the paucity of nuclear genome sequence data, the extent of endosymbiotic gene transfer (EGT) and host symbiont regulation are currently unknown. A combination of 454 and Illumina next generation sequencing enabled us to generate a comprehensive reference transcriptome data set for P. chromatophora on which we mapped short Illumina cDNA reads generated from cultures from the dark and light phases of a diel cycle. Combined with extensive phylogenetic analyses of the deduced protein sequences, these data revealed that 1) about 0.3-0.8% of the nuclear genes were obtained by EGT compared with 11-14% in the Plantae, 2) transferred genes show a distinct bias in that many encode small proteins involved in photosynthesis and photoacclimation, 3) host cells established control over expression of transferred genes, and 4) not only EGT, but to a minor extent also horizontal gene transfer from organisms that presumably served as food sources, helped to shape the nuclear genome of P. chromatophora. The identification of a significant number of transferred genes involved in photosynthesis and photoacclimation of thylakoid membranes as well as the observed transcriptional regulation of these genes strongly implies import of the encoded gene products into chromatophores, a feature previously thought to be restricted to canonical organelles. Thus, a possible mechanism by which P. chromatophora exerts control over the performance of its newly acquired photosynthetic organelle may involve controlling the expression of nuclear-encoded chromatophore

  17. Regulation of gene expression during early neuronal differentiation: evidence for patterns conserved across neuron populations and vertebrate classes.

    PubMed

    Ernsberger, Uwe

    2012-04-01

    Analysis of transcription factor function during neurogenesis has provided a huge amount of data on the generation and specification of diverse neuron populations in the central and peripheral nervous systems of vertebrates. However, an understanding of the induction of key neuron functions including electrical information processing and synaptic transmission lags seriously behind. Whereas pan-neuronal markers such as neurofilaments, neuron-specific tubulin and RNA-binding proteins have often been included in developmental analysis, the molecular players underlying electrical activity and transmitter release have been neglected in studies addressing gene expression during neuronal induction. Here, I summarize the evidence for a distinct accumulation pattern of mRNAs for synaptic proteins, a pattern that is delayed compared with pan-neuronal gene expression during neurogenesis. The conservation of this pattern across diverse avian and mammalian neuron populations suggests a common mechanism for the regulation of various sets of neuronal genes during initial neuronal differentiation. The co-regulation of genes coding for synaptic proteins from embryonic to postnatal development indicates that the expression of the players required for synaptic transmission shares common regulatory features. For the ion channels involved in neuronal electrical activity, such as voltage-gated sodium channels, the situation is less clear because of the lack of comparative studies early during neurogenesis. Transcription factors have been characterized that regulate the expression of synaptic proteins in vitro and in vivo. They currently do not explain the co-regulation of these genes across different neuron populations. The neuron-restrictive silencing factor NRSF/REST targets a large gene set, but not all of the genes coding for pan-neuronal, synaptic and ion channel proteins. The discrepancy between NRSF/REST loss-of-function and silencer-to-activator-switch studies leaves the full

  18. Adenovirus-mediated gene delivery to hypothalamic magnocellular neurons in mice

    NASA Technical Reports Server (NTRS)

    Vasquez, E. C.; Beltz, T. G.; Meyrelles, S. S.; Johnson, A. K.

    1999-01-01

    Vasopressin is synthesized by magnocellular neurons in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei and released by their axon terminals in the neurohypophysis (NH). With its actions as an antidiuretic hormone and vasoactive agent, vasopressin plays a pivotal role in the control of body fluids and cardiovascular homeostasis. Because of its well-defined neurobiology and functional importance, the SON/PVN-NH system is ideal to establish methods for gene transfer of genetic material into specific pathways in the mouse central nervous system. In these studies, we compared the efficiency of transferring the gene lacZ, encoding for beta-galactosidase (beta-gal), versus a gene encoding for green fluorescent protein by using replication-deficient adenovirus (Ad) vectors in adult mice. Transfection with viral concentrations up to 2 x 10(7) plaque-forming units per coverslip of NH, PVN, and SON in dissociated, cultured cells caused efficient transfection without cytotoxicity. However, over an extended period of time, higher levels (50% to 75% of the cells) of beta-gal expression were detected in comparison with green fluorescent protein (5% to 50% of the cells). With the use of a stereotaxic approach, the pituitary glands of mice were injected with Ad (4 x 10(6) plaque-forming units). In material from these animals, we were able to visualize the expression of the beta-gal gene in the NH and in magnocellular neurons of both the PVN and SON. The results of these experiments indicate that Ad-Rous sarcoma virus promoter-beta-gal is taken up by nerve terminals at the injection site (NH) and retrogradely transported to the soma of the neurons projecting to the NH. We conclude that the application of these experimental approaches will provide powerful tools for physiological studies and potential approaches to deliver therapeutic genes to treat diseases.

  19. Foamy virus vectors for gene transfer

    PubMed Central

    Trobridge, Grant D.

    2009-01-01

    Foamy virus (FV) vectors are efficient gene delivery vehicles that have shown great promise for gene therapy in preclinical animal models. FVs or spumaretroviruses are not endemic in humans, but are prevalent in nonhuman primates and in other mammals. They have evolved means for efficient horizontal transmission in their host species without pathology. FV vectors have several unique properties that make them well-suited for therapeutic gene transfer including a desirable safety profile, a broad tropism, a large transgene capacity, and the ability to persist in quiescent cells. They mediate efficient and stable gene transfer to hematopoietic stem cells (HSCs) in mouse models, and in the canine large animal model. Analysis of FV vector integration sites in vitro and in hematopoietic repopulating cells shows they have a unique integration profile, and suggests they may be safer than gammaretroviruses or lentiviral vectors. Here properties of FVs relevant to the safety and efficacy of FV vectors are discussed. The development of FV vector systems is described, and studies evaluating their potential in vitro, and in small and large animal models is reviewed. PMID:19743892

  20. Horizontal Gene Transfer, Dispersal and Haloarchaeal Speciation

    PubMed Central

    Papke, R. Thane; Corral, Paulina; Ram-Mohan, Nikhil; de la Haba, Rafael R.; Sánchez-Porro, Cristina; Makkay, Andrea; Ventosa, Antonio

    2015-01-01

    The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation) are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria. PMID:25997110

  1. Horizontal gene transfer, dispersal and haloarchaeal speciation.

    PubMed

    Papke, R Thane; Corral, Paulina; Ram-Mohan, Nikhil; Haba, Rafael R de la; Sánchez-Porro, Cristina; Makkay, Andrea; Ventosa, Antonio

    2015-01-01

    The Halobacteria are a well-studied archaeal class and numerous investigations are showing how their diversity is distributed amongst genomes and geographic locations. Evidence indicates that recombination between species continuously facilitates the arrival of new genes, and within species, it is frequent enough to spread acquired genes amongst all individuals in the population. To create permanent independent diversity and generate new species, barriers to recombination are probably required. The data support an interpretation that rates of evolution (e.g., horizontal gene transfer and mutation) are faster at creating geographically localized variation than dispersal and invasion are at homogenizing genetic differences between locations. Therefore, we suggest that recurrent episodes of dispersal followed by variable periods of endemism break the homogenizing forces of intrapopulation recombination and that this process might be the principal stimulus leading to divergence and speciation in Halobacteria. PMID:25997110

  2. Immortalized neural progenitor cells for CNS gene transfer and repair.

    PubMed

    Martínez-Serrano, A; Björklund, A

    1997-11-01

    Immortalized multipotent neural stem and progenitor cells have emerged as a highly convenient source of tissue for genetic manipulation and ex vivo gene transfer to the CNS. Recent studies show that these cells, which can be maintained and genetically transduced as cell lines in culture, can survive, integrate and differentiate into both neurons and glia after transplantation to the intact or damaged brain. Progenitors engineered to secrete trophic factors, or to produce neurotransmitter-related or metabolic enzymes can be made to repopulate diseased or injured brain areas, thus providing a new potential therapeutic tool for the blockade of neurodegenerative processes and reversal of behavioural deficits in animal models of neurodegenerative diseases. With further technical improvements, the use of immortalized neural progenitors may bring us closer to the challenging goal of targeted and effective CNS repair.

  3. Gene transfer in bacteria: speciation without species?

    PubMed

    Lawrence, Jeffrey G

    2002-06-01

    Although Bacteria and Archaea reproduce by binary fission, exchange of genes among lineages has shaped the diversity of their populations and the diversification of their lineages. Gene exchange can occur by two distinct routes, each differentially impacting the recipient genome. First, homologous recombination mediates the exchange of DNA between closely related individuals (those whose sequences are sufficient similarly to allow efficient integration). As a result, homologous recombination mediates the dispersal of advantageous alleles that may rise to high frequency among genetically related individuals via periodic selection events. Second, lateral gene transfer can introduce novel DNA into a genome from completely unrelated lineages via illegitimate recombination. Gene exchange by this route serves to distribute genes throughout distantly related clades and therefore may confer complex abilities--not otherwise found among closely related lineages--onto the recipient organisms. These two mechanisms of gene exchange play complementary roles in the diversification of microbial populations into independent, ecologically distinct lineages. Although the delineation of microbial "species" then becomes difficult--if not impossible--to achieve, a cogent process of speciation can be predicted.

  4. Phenotypic characteristics of hybrid cells generated by transferring neuronal nuclei into bone marrow stromal cell cytoplasts.

    PubMed

    Zhou, Zhujuan; Xu, Yan; Zhong, Qi; Zheng, Jian

    2012-02-10

    Bone marrow stromal cells (BMSCs) are promising donor cells for transplantation therapies for a variety of diseases. However, there still lack efficient ways to induce directional differentiation of BMSCs to promote their practical use in transplantation therapy. In this study, we constructed hybrid cells by transferring neuronal nuclei into BMSC cytoplasts and investigated the proliferative capacity and phenotypic characteristics of the hybrid cells. The neuronal nuclei were labeled with Hoechst 33342 before the transfer process, and the cell membrane antigen CD71 was used as a marker of BMSC cytoplasts. The BMSC cytoplasts and neuronal karyoplasts were separated by Ficoll density gradient ultracentrifugation. The hybrid cells were generated by the polyethylene glycol-mediated fusion of BMSC cytoplasts with neuronal karyoplasts. The hybrid cells exhibited Hoechst 33342 staining in their nuclei and CD71 staining on their cytomembranes, which confirmed the success of cell fusion. The hybrid cells were positive for BrdU immunostaining. Viability analysis of the cultured hybrid cells by the MTT assay demonstrated their proliferative ability. Immunocytochemical staining revealed the expression of the neuron-specific markers NeuN and MAP2 in the third passage hybrid cells, which indicated their neuronal phenotypic characteristics. The results demonstrated that the hybrid cells produced by fusing neuronal karyoplasts with BMSC cytoplasts had proliferative capability and expressed the neuron-specific markers. Further study is required to investigate the phenotype of the hybrid cells both structurally and functionally.

  5. LIS1 Lissencephaly gene CNS expression: Relation to neuronal migration

    SciTech Connect

    Reiner, O. |; Gal-Gerber, O.; Sapir, T.

    1994-09-01

    Lis1 is the murine gene corresponding to human LIS1 gene involved in Miller-Dieker lissencephaly located on chromosome 17p13.3 as demonstrated by cDNA cloning, sequence analysis and genetic mapping. Lis1 expression was studied in developing mouse brain using in situ hybridization. At embryonic day 15, Lis1 expression was most prominently localized in the neuronal layer of the retina, the developing hippocampus, doral root ganglia, cranial ganglia and the thalamus. At postnatal day 5 a unique pattern of expression was detected in the developing cerebellum. Lis1 was expressed at high levels in the Purkinje cell layer when the granule cells were migrating through the Purkinje cell layer inwards. The expression of Lis1 in Purkinje cells in the adult is markedly reduced. Similarly, Lis1 was expressed in the ontogenetically older layers of the neocortex (layers 5 and 6) where younger neurons have to migrate through to settle in the superficial layers. Thus, at both sites a link between expression and neuronal migration was demonstrated. These studies on the expression pattern of Lis1 could be useful in understanding abnormalities in Miller-Dieker lissencephaly syndrome (MDS) patients.

  6. Simple rapid method for gene transfer

    SciTech Connect

    Cockburn, A.F.; Meier, H.

    1990-01-30

    The object of the present invention is to provide methods for gene transfer that reduce or eliminate cellular pretreatment steps, e.g., the removal of cell wall by chemical or enzymatic methods, is rapid and can be practiced without the need of additional expensive equipment. Cells, embryos or tissues selected for genetic manipulation are suspended in an Eppendorf tube in an aliquot of the desired genetic material to be transferred to which the resulting mixture is added and is agitated by vortexing from about 30 to about 90 seconds. The cells, embryos or tissue are sedimented and the DNA supernatant removed. After sedimentation, the injected material is resuspended in or on a growth medium to assay for expression.

  7. Astrocyte-to-neuron intercellular prion transfer is mediated by cell-cell contact

    PubMed Central

    Victoria, Guiliana Soraya; Arkhipenko, Alexander; Zhu, Seng; Syan, Sylvie; Zurzolo, Chiara

    2016-01-01

    Prion diseases are caused by misfolding of the cellular protein PrPC to an infectious conformer, PrPSc. Intercellular PrPSc transfer propagates conversion and allows infectivity to move from the periphery to the brain. However, how prions spread between cells of the central nervous system is unclear. Astrocytes are specialized non-neuronal cells within the brain that have a number of functions indispensable for brain homeostasis. Interestingly, they are one of the earliest sites of prion accumulation in the brain. A fundamental question arising from this observation is whether these cells are involved in intercellular prion transfer and thereby disease propagation. Using co-culture systems between primary infected astrocytes and granule neurons or neuronal cell lines, we provide direct evidence that prion-infected astrocytes can disseminate prion to neurons. Though astrocytes are capable of secreting PrP, this is an inefficient method of transferring prion infectivity. Efficient transfer required co-culturing and direct cell contact. Astrocytes form numerous intercellular connections including tunneling nanotubes, containing PrPSc, often colocalized with endolysosomal vesicles, which may constitute the major mechanism of transfer. Because of their role in intercellular transfer of prions astrocytes may influence progression of the disease. PMID:26857744

  8. Gene transfer and expression in plants.

    PubMed

    Lorence, Argelia; Verpoorte, Robert

    2004-01-01

    Until recently, agriculture and plant breeding relied solely on the accumulated experience of generations of farmers and breeders that is, on sexual transfer of genes between plant species. However, recent developments in plant molecular biology and genomics now give us access to knowledge and understanding of plant genomes and the possibility of modifying them. This chapter presents an updated overview of the two most powerful technologies for transferring genetic material (DNA) into plants: Agrobacterium-mediated transformation and microparticle bombardment (biolistics). Some of the topics that are discussed in detail are the main variables controlling the transformation efficiency that can be achieved using each one of these approaches; the advantages and limitations of each methodology; transient versus stable transformation approaches; the potential of some in planta transformation systems; alternatives to developing transgenic plants without selection markers; the availability of diverse genetic tools generated as part of the genome sequencing of different plant species; transgene expression, gene silencing, and their association with regulatory elements; and prospects and ways to possibly overcome some transgene expression difficulties, in particular the use of matrix-attachment regions (MARs).

  9. Benefits of Neuronal Preferential Systemic Gene Therapy for Neurotransmitter Deficiency.

    PubMed

    Lee, Ni-Chung; Muramatsu, Shin-Ichi; Chien, Yin-Hsiu; Liu, Wen-Shin; Wang, Wei-Hua; Cheng, Chia-Hao; Hu, Meng-Kai; Chen, Pin-Wen; Tzen, Kai-Yuan; Byrne, Barry J; Hwu, Wuh-Liang

    2015-10-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disease that impairs synthesis of dopamine and serotonin. Children with AADC deficiency exhibit severe motor, behavioral, and autonomic dysfunctions. We previously generated an IVS6+4A>T knock-in mouse model of AADC deficiency (Ddc(KI) mice) and showed that gene therapy at the neonatal stage can rescue this phenotype. In the present study, we extended this treatment to systemic therapy on young mice. After intraperitoneal injection of AADC viral vectors into 7-day-old Ddc(KI) mice, the treated mice exhibited improvements in weight gain, survival, motor function, autonomic function, and behavior. The yfAAV9/3-Syn-I-mAADC-treated mice showed greater neuronal transduction and higher brain dopamine levels than AAV9-CMV-hAADC-treated mice, whereas AAV9-CMV-hAADC-treated mice exhibited hyperactivity. Therefore, neurotransmitter-deficient animals can be rescued at a young age using systemic gene therapy, although a vector for preferential neuronal expression may be necessary to avoid hyperactivity caused by this treatment. PMID:26137853

  10. Benefits of Neuronal Preferential Systemic Gene Therapy for Neurotransmitter Deficiency.

    PubMed

    Lee, Ni-Chung; Muramatsu, Shin-Ichi; Chien, Yin-Hsiu; Liu, Wen-Shin; Wang, Wei-Hua; Cheng, Chia-Hao; Hu, Meng-Kai; Chen, Pin-Wen; Tzen, Kai-Yuan; Byrne, Barry J; Hwu, Wuh-Liang

    2015-10-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disease that impairs synthesis of dopamine and serotonin. Children with AADC deficiency exhibit severe motor, behavioral, and autonomic dysfunctions. We previously generated an IVS6+4A>T knock-in mouse model of AADC deficiency (Ddc(KI) mice) and showed that gene therapy at the neonatal stage can rescue this phenotype. In the present study, we extended this treatment to systemic therapy on young mice. After intraperitoneal injection of AADC viral vectors into 7-day-old Ddc(KI) mice, the treated mice exhibited improvements in weight gain, survival, motor function, autonomic function, and behavior. The yfAAV9/3-Syn-I-mAADC-treated mice showed greater neuronal transduction and higher brain dopamine levels than AAV9-CMV-hAADC-treated mice, whereas AAV9-CMV-hAADC-treated mice exhibited hyperactivity. Therefore, neurotransmitter-deficient animals can be rescued at a young age using systemic gene therapy, although a vector for preferential neuronal expression may be necessary to avoid hyperactivity caused by this treatment.

  11. Benefits of Neuronal Preferential Systemic Gene Therapy for Neurotransmitter Deficiency

    PubMed Central

    Lee, Ni-Chung; Muramatsu, Shin-Ichi; Chien, Yin-Hsiu; Liu, Wen-Shin; Wang, Wei-Hua; Cheng, Chia-Hao; Hu, Meng-Kai; Chen, Pin-Wen; Tzen, Kai-Yuan; Byrne, Barry J; Hwu, Wuh-Liang

    2015-01-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive disease that impairs synthesis of dopamine and serotonin. Children with AADC deficiency exhibit severe motor, behavioral, and autonomic dysfunctions. We previously generated an IVS6+4A>T knock-in mouse model of AADC deficiency (DdcKI mice) and showed that gene therapy at the neonatal stage can rescue this phenotype. In the present study, we extended this treatment to systemic therapy on young mice. After intraperitoneal injection of AADC viral vectors into 7-day-old DdcKI mice, the treated mice exhibited improvements in weight gain, survival, motor function, autonomic function, and behavior. The yfAAV9/3-Syn-I-mAADC-treated mice showed greater neuronal transduction and higher brain dopamine levels than AAV9-CMV-hAADC-treated mice, whereas AAV9-CMV-hAADC-treated mice exhibited hyperactivity. Therefore, neurotransmitter-deficient animals can be rescued at a young age using systemic gene therapy, although a vector for preferential neuronal expression may be necessary to avoid hyperactivity caused by this treatment. PMID:26137853

  12. Direct Transfer of Viral and Cellular Proteins from Varicella-Zoster Virus-Infected Non-Neuronal Cells to Human Axons

    PubMed Central

    Grigoryan, Sergei; Yee, Michael B; Glick, Yair; Gerber, Doron; Kepten, Eldad; Garini, Yuval; Yang, In Hong; Kinchington, Paul R.; Goldstein, Ronald S.

    2015-01-01

    Varicella Zoster Virus (VZV), the alphaherpesvirus that causes varicella upon primary infection and Herpes zoster (shingles) following reactivation in latently infected neurons, is known to be fusogenic. It forms polynuclear syncytia in culture, in varicella skin lesions and in infected fetal human ganglia xenografted to mice. After axonal infection using VZV expressing green fluorescent protein (GFP) in compartmentalized microfluidic cultures there is diffuse filling of axons with GFP as well as punctate fluorescence corresponding to capsids. Use of viruses with fluorescent fusions to VZV proteins reveals that both proteins encoded by VZV genes and those of the infecting cell are transferred in bulk from infecting non-neuronal cells to axons. Similar transfer of protein to axons was observed following cell associated HSV1 infection. Fluorescence recovery after photobleaching (FRAP) experiments provide evidence that this transfer is by diffusion of proteins from the infecting cells into axons. Time-lapse movies and immunocytochemical experiments in co-cultures demonstrate that non-neuronal cells fuse with neuronal somata and proteins from both cell types are present in the syncytia formed. The fusogenic nature of VZV therefore may enable not only conventional entry of virions and capsids into axonal endings in the skin by classical entry mechanisms, but also by cytoplasmic fusion that permits viral protein transfer to neurons in bulk. PMID:25973990

  13. Gene therapy: Biological pacemaker created by gene transfer

    NASA Astrophysics Data System (ADS)

    Miake, Junichiro; Marbán, Eduardo; Nuss, H. Bradley

    2002-09-01

    The pacemaker cells of the heart initiate the heartbeat, sustain the circulation, and dictate the rate and rhythm of cardiac contraction. Circulatory collapse ensues when these specialized cells are damaged by disease, a situation that currently necessitates the implantation of an electronic pacemaker. Here we report the use of viral gene transfer to convert quiescent heart-muscle cells into pacemaker cells, and the successful generation of spontaneous, rhythmic electrical activity in the ventricle in vivo. Our results indicate that genetically engineered pacemakers could be developed as a possible alternative to implantable electronic devices.

  14. Gene duplication and transfer events in plant mitochondria genome

    SciTech Connect

    Xiong Aisheng Peng Rihe; Zhuang Jing; Gao Feng; Zhu Bo; Fu Xiaoyan; Xue Yong; Jin Xiaofen; Tian Yongsheng; Zhao Wei; Yao Quanhong

    2008-11-07

    Gene or genome duplication events increase the amount of genetic material available to increase the genomic, and thereby phenotypic, complexity of organisms during evolution. Gene duplication and transfer events have been important to molecular evolution in all three domains of life, and may be the first step in the emergence of new gene functions. Gene transfer events have been proposed as another accelerator of evolution. The duplicated gene or genome, mainly nuclear, has been the subject of several recent reviews. In addition to the nuclear genome, organisms have organelle genomes, including mitochondrial genome. In this review, we briefly summarize gene duplication and transfer events in the plant mitochondrial genome.

  15. Information transfer between neurons in the motor cortex triggered by visual cues.

    PubMed

    Kim, Sanggyun; Takahashi, Kazutaka; Hatsopoulos, Nicholas G; Coleman, Todd P

    2011-01-01

    It was previously shown that beta oscillations of local field potentials in the arm area of the primary motor cortex (MI) of nonhuman primates propagate as travelling waves across MI of monkeys during movement preparation and execution and are believed to subserve cortical information transfer. To investigate the information transfer and its change over time at the single-cell level, we analyzed simultaneously recorded multiple MI neural spike trains of a monkey using a Granger causality measure for point process models before and after visual cues instructing the onset of reaching movements. In this analysis, we found that more pairs of neurons showed information transfer between them after appearances of upcoming movement targets than before, and the directions of the information transfer across neurons in MI were coincident with the directions of the propagating waves. These results suggest that the neuron pairs identified in the current study are the candidates of neurons that travel with spatiotemporal dynamics of beta oscillations in the MI.

  16. Mitochondrial Transfer from Astrocytes to Neurons following Ischemic Insult: Guilt by Association?

    PubMed

    Berridge, Michael V; Schneider, Remy T; McConnell, Melanie J

    2016-09-13

    Intercellular mitochondrial transfer has been shown in tumor models, following lung injury and in xenotransplants of leukemic cells, but trafficking between cells in the brain remains unexplored. A suggestion that mitochondria move from astrocytes to neurons in a model of ischemia in a recent article in Nature by Hayakawa et al. (2016) should be interpreted with caution. PMID:27626198

  17. Horizontal gene transfer: A critical view

    PubMed Central

    Kurland, C. G.; Canback, B.; Berg, Otto G.

    2003-01-01

    It has been suggested that horizontal gene transfer (HGT) is the “essence of phylogeny.” In contrast, much data suggest that this is an exaggeration resulting in part from a reliance on inadequate methods to identify HGT events. In addition, the assumption that HGT is a ubiquitous influence throughout evolution is questionable. Instead, rampant global HGT is likely to have been relevant only to primitive genomes. In modern organisms we suggest that both the range and frequencies of HGT are constrained most often by selective barriers. As a consequence those HGT events that do occur most often have little influence on genome phylogeny. Although HGT does occur with important evolutionary consequences, classical Darwinian lineages seem to be the dominant mode of evolution for modern organisms. PMID:12902542

  18. Targeting single neuronal networks for gene expression and cell labeling in vivo.

    PubMed

    Marshel, James H; Mori, Takuma; Nielsen, Kristina J; Callaway, Edward M

    2010-08-26

    To understand fine-scale structure and function of single mammalian neuronal networks, we developed and validated a strategy to genetically target and trace monosynaptic inputs to a single neuron in vitro and in vivo. The strategy independently targets a neuron and its presynaptic network for specific gene expression and fine-scale labeling, using single-cell electroporation of DNA to target infection and monosynaptic retrograde spread of a genetically modifiable rabies virus. The technique is highly reliable, with transsynaptic labeling occurring in every electroporated neuron infected by the virus. Targeting single neocortical neuronal networks in vivo, we found clusters of both spiny and aspiny neurons surrounding the electroporated neuron in each case, in addition to intricately labeled distal cortical and subcortical inputs. This technique, broadly applicable for probing and manipulating single neuronal networks with single-cell resolution in vivo, may help shed new light on fundamental mechanisms underlying circuit development and information processing by neuronal networks throughout the brain.

  19. Optical gene transfer by femtosecond laser pulses

    NASA Astrophysics Data System (ADS)

    Konig, Karsten; Riemann, Iris; Tirlapur, Uday K.

    2003-07-01

    Targeted transfection of cells is an important technique for gene therapy and related biomedical applications. We delineate how high-intensity (1012 W/cm2) near-infrared (NIR) 80 MHz nanojoule femtosecond laser pulses can create highly localised membrane perforations within a minute focal volume, enabling non-invasive direct transfection of mammalian cells with DNA. We suspended Chinese hamster ovarian (CHO), rat kangaroo kidney epithelial (PtK2) and rat fibroblast cells in 0.5 ml culture medium in a sterile miniaturized cell chamber (JenLab GmbH, Jena, Germany) containing 0.2 μg plasmid DNA vector pEGFP-N1 (4.7 kb), which codes for green fluorescent protein (GFP). The NIR laser beam was introduced into a femtosecond laser scanning microscope (JenLab GmbH, Jena, Germany; focussed on the edge of the cell membrane of a target cell for 16 ms. The integration and expression efficiency of EGFP were assessed in situ by two-photon fluorescence-lifetime imaging using time-correlated single photon counting. The unique capability to transfer foreign DNA safely and efficiently into specific cell types (including stem cells), circumventing mechanical, electrical or chemical means, will have many applications, such as targeted gene therapy and DNA vaccination.

  20. Lentiviral Vector Gene Transfer to Porcine Airways

    PubMed Central

    Sinn, Patrick L; Cooney, Ashley L; Oakland, Mayumi; Dylla, Douglas E; Wallen, Tanner J; Pezzulo, Alejandro A; Chang, Eugene H; McCray, Paul B

    2012-01-01

    In this study, we investigated lentiviral vector development and transduction efficiencies in well-differentiated primary cultures of pig airway epithelia (PAE) and wild-type pigs in vivo. We noted gene transfer efficiencies similar to that observed for human airway epithelia (HAE). Interestingly, feline immunodeficiency virus (FIV)-based vectors transduced immortalized pig cells as well as pig primary cells more efficiently than HIV-1–based vectors. PAE express TRIM5α, a well-characterized species-specific lentiviral restriction factor. We contrasted the restrictive properties of porcine TRIM5α against FIV- and HIV-based vectors using gain and loss of function approaches. We observed no effect on HIV-1 or FIV conferred transgene expression in response to porcine TRIM5α overexpression or knockdown. To evaluate the ability of GP64-FIV to transduce porcine airways in vivo, we delivered vector expressing mCherry to the tracheal lobe of the lung and the ethmoid sinus of 4-week-old pigs. One week later, epithelial cells expressing mCherry were readily detected. Our findings indicate that pseudotyped FIV vectors confer similar tropisms in porcine epithelia as observed in human HAE and provide further support for the selection of GP64 as an appropriate envelope pseudotype for future preclinical gene therapy studies in the porcine model of cystic fibrosis (CF). PMID:23187455

  1. Plant transformation via pollen tube-mediated gene transfer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic transformation using foreign genes and the subsequent development of transgenic plants has been employed to develop enhanced elite germplasm. Although some skepticism exits regarding pollen tube-mediated gene transfer (PTT), reports demonstrating improved transformation efficiency with PTT ...

  2. Gene Transfer & Hybridization Studies in Hyperthermophilic Species

    SciTech Connect

    Nelson, Karen E.

    2005-10-14

    A. ABSTRACT The importance of lateral gene transfer (LGT) in the evolution of microbial species has become increasingly evident with each completed microbial genome sequence. Most significantly, the genome of Thermotoga maritima MSB8, a hyperthermophilic bacterium isolated by Karl Stetter and workers from Vulcano Italy in 1986, and sequenced at The Institute for Genomic Research (TIGR) in Rockville Maryland in 1999, revealed extensive LGT between % . this bacterium and members of the archaeal domain (in particular Archaeoglobus fulgidus, and Pyracoccus frcriosus species). Based on whole genome comparisons, it was estimated that 24% of the genetic information in this organism was acquired by genetic exchange with archaeal species, Independent analyses including periodicity analysis of the T. maritimu genomic DNA sequence, phylogenetic reconstruction based on genes that appear archaeal-like, and codon and amino acid usage, have provided additional evidence for LGT between T. maritima and the archaea. More recently, DiRuggiero and workers have identified a very recent LGT event between two genera of hyperthermophilic archaea, where a nearly identical DNA fragment of 16 kb in length flanked by insertion sequence (IS) elements, exists. Undoubtedly, additional examples of LGT will be identified as more microbial genomes are completed. For the present moment however, the genome sequence of T. maritima and other hyperthermophiles including P. furiosus, Pyrococcus horikoshii, Pyrococcus abyssi, A. fulgidus, and Aquifex aeolicus, have significantly increased out awareness of evolution being a web of life rather than a tree of life, as suggested by single gene phylogenies. In this proposal, we will aim to determine the extent of LGT across the hyperthemophiles, employing iY maritima as the model organism. A variety of biochemical techniques and phylogenetic reconstructions will allow for a detailed and thorough characterization of the extent of LGT in this species. The

  3. Widespread Central Nervous System Gene Transfer and Silencing After Systemic Delivery of Novel AAV-AS Vector.

    PubMed

    Choudhury, Sourav R; Harris, Anne F; Cabral, Damien J; Keeler, Allison M; Sapp, Ellen; Ferreira, Jennifer S; Gray-Edwards, Heather L; Johnson, Jacob A; Johnson, Aime K; Su, Qin; Stoica, Lorelei; DiFiglia, Marian; Aronin, Neil; Martin, Douglas R; Gao, Guangping; Sena-Esteves, Miguel

    2016-04-01

    Effective gene delivery to the central nervous system (CNS) is vital for development of novel gene therapies for neurological diseases. Adeno-associated virus (AAV) vectors have emerged as an effective platform for in vivo gene transfer, but overall neuronal transduction efficiency of vectors derived from naturally occurring AAV capsids after systemic administration is relatively low. Here, we investigated the possibility of improving CNS transduction of existing AAV capsids by genetically fusing peptides to the N-terminus of VP2 capsid protein. A novel vector AAV-AS, generated by the insertion of a poly-alanine peptide, is capable of extensive gene transfer throughout the CNS after systemic administration in adult mice. AAV-AS is 6- and 15-fold more efficient than AAV9 in spinal cord and cerebrum, respectively. The neuronal transduction profile varies across brain regions but is particularly high in the striatum where AAV-AS transduces 36% of striatal neurons. Widespread neuronal gene transfer was also documented in cat brain and spinal cord. A single intravenous injection of an AAV-AS vector encoding an artificial microRNA targeting huntingtin (Htt) resulted in 33-50% knockdown of Htt across multiple CNS structures in adult mice. This novel AAV-AS vector is a promising platform to develop new gene therapies for neurodegenerative disorders.

  4. Unbiased View of Synaptic and Neuronal Gene Complement in Ctenophores: Are There Pan-neuronal and Pan-synaptic Genes across Metazoa?

    PubMed

    Moroz, Leonid L; Kohn, Andrea B

    2015-12-01

    Hypotheses of origins and evolution of neurons and synapses are controversial, mostly due to limited comparative data. Here, we investigated the genome-wide distribution of the bilaterian "synaptic" and "neuronal" protein-coding genes in non-bilaterian basal metazoans (Ctenophora, Porifera, Placozoa, and Cnidaria). First, there are no recognized genes uniquely expressed in neurons across all metazoan lineages. None of the so-called pan-neuronal genes such as embryonic lethal abnormal vision (ELAV), Musashi, or Neuroglobin are expressed exclusively in neurons of the ctenophore Pleurobrachia. Second, our comparative analysis of about 200 genes encoding canonical presynaptic and postsynaptic proteins in bilaterians suggests that there are no true "pan-synaptic" genes or genes uniquely and specifically attributed to all classes of synapses. The majority of these genes encode receptive and secretory complexes in a broad spectrum of eukaryotes. Trichoplax (Placozoa) an organism without neurons and synapses has more orthologs of bilaterian synapse-related/neuron-related genes than do ctenophores-the group with well-developed neuronal and synaptic organization. Third, the majority of genes encoding ion channels and ionotropic receptors are broadly expressed in unicellular eukaryotes and non-neuronal tissues in metazoans. Therefore, they cannot be viewed as neuronal markers. Nevertheless, the co-expression of multiple types of ion channels and receptors does correlate with the presence of neural and synaptic organization. As an illustrative example, the ctenophore genomes encode a greater diversity of ion channels and ionotropic receptors compared with the genomes of the placozoan Trichoplax and the demosponge Amphimedon. Surprisingly, both placozoans and sponges have a similar number of orthologs of "synaptic" proteins as we identified in the genomes of two ctenophores. Ctenophores have a distinct synaptic organization compared with other animals. Our analysis of

  5. Unbiased View of Synaptic and Neuronal Gene Complement in Ctenophores: Are There Pan-neuronal and Pan-synaptic Genes across Metazoa?

    PubMed

    Moroz, Leonid L; Kohn, Andrea B

    2015-12-01

    Hypotheses of origins and evolution of neurons and synapses are controversial, mostly due to limited comparative data. Here, we investigated the genome-wide distribution of the bilaterian "synaptic" and "neuronal" protein-coding genes in non-bilaterian basal metazoans (Ctenophora, Porifera, Placozoa, and Cnidaria). First, there are no recognized genes uniquely expressed in neurons across all metazoan lineages. None of the so-called pan-neuronal genes such as embryonic lethal abnormal vision (ELAV), Musashi, or Neuroglobin are expressed exclusively in neurons of the ctenophore Pleurobrachia. Second, our comparative analysis of about 200 genes encoding canonical presynaptic and postsynaptic proteins in bilaterians suggests that there are no true "pan-synaptic" genes or genes uniquely and specifically attributed to all classes of synapses. The majority of these genes encode receptive and secretory complexes in a broad spectrum of eukaryotes. Trichoplax (Placozoa) an organism without neurons and synapses has more orthologs of bilaterian synapse-related/neuron-related genes than do ctenophores-the group with well-developed neuronal and synaptic organization. Third, the majority of genes encoding ion channels and ionotropic receptors are broadly expressed in unicellular eukaryotes and non-neuronal tissues in metazoans. Therefore, they cannot be viewed as neuronal markers. Nevertheless, the co-expression of multiple types of ion channels and receptors does correlate with the presence of neural and synaptic organization. As an illustrative example, the ctenophore genomes encode a greater diversity of ion channels and ionotropic receptors compared with the genomes of the placozoan Trichoplax and the demosponge Amphimedon. Surprisingly, both placozoans and sponges have a similar number of orthologs of "synaptic" proteins as we identified in the genomes of two ctenophores. Ctenophores have a distinct synaptic organization compared with other animals. Our analysis of

  6. Simultaneous identification of duplications and lateral gene transfers.

    PubMed

    Tofigh, Ali; Hallett, Michael; Lagergren, Jens

    2011-01-01

    The incongruency between a gene tree and a corresponding species tree can be attributed to evolutionary events such as gene duplication and gene loss. This paper describes a combinatorial model where so-called DTL-scenarios are used to explain the differences between a gene tree and a corresponding species tree taking into account gene duplications, gene losses, and lateral gene transfers (also known as horizontal gene transfers). The reasonable biological constraint that a lateral gene transfer may only occur between contemporary species leads to the notion of acyclic DTL-scenarios. Parsimony methods are introduced by defining appropriate optimization problems. We show that finding most parsimonious acyclic DTL-scenarios is NP-hard. However, by dropping the condition of acyclicity, the problem becomes tractable, and we provide a dynamic programming algorithm as well as a fixed-parameter tractable algorithm for finding most parsimonious DTL-scenarios.

  7. Methods for Gene Transfer to the Central Nervous System

    PubMed Central

    Kantor, Boris; Bailey, Rachel M.; Wimberly, Keon; Kalburgi, Sahana N.; Gray, Steven J.

    2015-01-01

    Gene transfer is an increasingly utilized approach for research and clinical applications involving the central nervous system (CNS). Vectors for gene transfer can be as simple as an unmodified plasmid, but more commonly involve complex modifications to viruses to make them suitable gene delivery vehicles. This chapter will explain how tools for CNS gene transfer have been derived from naturally occurring viruses. The current capabilities of plasmid, retroviral, adeno-associated virus, adenovirus, and herpes simplex virus vectors for CNS gene delivery will be described. These include both focal and global CNS gene transfer strategies, with short- or long-term gene expression. As is described in this chapter, an important aspect of any vector is the cis-acting regulatory elements incorporated into the vector genome that control when, where, and how the transgene is expressed. PMID:25311922

  8. Multiplexed transposon-mediated stable gene transfer in human cells

    PubMed Central

    Kahlig, Kristopher M.; Saridey, Sai K.; Kaja, Aparna; Daniels, Melissa A.; George, Alfred L.; Wilson, Matthew H.

    2010-01-01

    Generation of cultured human cells stably expressing one or more recombinant gene sequences is a widely used approach in biomedical research, biotechnology, and drug development. Conventional methods are not efficient and have severe limitations especially when engineering cells to coexpress multiple transgenes or multiprotein complexes. In this report, we harnessed the highly efficient, nonviral, and plasmid-based piggyBac transposon system to enable concurrent genomic integration of multiple independent transposons harboring distinct protein-coding DNA sequences. Flow cytometry of cell clones derived from a single multiplexed transfection demonstrated approximately 60% (three transposons) or approximately 30% (four transposons) stable coexpression of all delivered transgenes with selection for a single marker transposon. We validated multiplexed piggyBac transposon delivery by coexpressing large transgenes encoding a multisubunit neuronal voltage-gated sodium channel (SCN1A) containing a pore-forming subunit and two accessory subunits while using two additional genes for selection. Previously unobtainable robust sodium current was demonstrated through 38 passages, suitable for use on an automated high-throughput electrophysiology platform. Cotransfection of three large (up to 10.8 kb) piggyBac transposons generated a heterozygous SCN1A stable cell line expressing two separate alleles of the pore-forming subunit and two accessory subunits (total of four sodium channel subunits) with robust functional expression. We conclude that the piggyBac transposon system can be used to perform multiplexed stable gene transfer in cultured human cells, and this technology may be valuable for applications requiring concurrent expression of multiprotein complexes. PMID:20080581

  9. Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus

    PubMed Central

    Chua, Streamson; Jo, Young-Hwan

    2016-01-01

    The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC), plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT)-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th) mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat. PMID:27611685

  10. Single-Cell Gene Expression Analysis of Cholinergic Neurons in the Arcuate Nucleus of the Hypothalamus.

    PubMed

    Jeong, Jae Hoon; Woo, Young Jae; Chua, Streamson; Jo, Young-Hwan

    2016-01-01

    The cholinoceptive system in the hypothalamus, in particular in the arcuate nucleus (ARC), plays a role in regulating food intake. Neurons in the ARC contain multiple neuropeptides, amines, and neurotransmitters. To study molecular and neurochemical heterogeneity of ARC neurons, we combine single-cell qRT-PCR and single-cell whole transcriptome amplification methods to analyze expression patterns of our hand-picked 60 genes in individual neurons in the ARC. Immunohistochemical and single-cell qRT-PCR analyses show choline acetyltransferase (ChAT)-expressing neurons in the ARC. Gene expression patterns are remarkably distinct in each individual cholinergic neuron. Two-thirds of cholinergic neurons express tyrosine hydroxylase (Th) mRNA. A large subset of these Th-positive cholinergic neurons is GABAergic as they express the GABA synthesizing enzyme glutamate decarboxylase and vesicular GABA transporter transcripts. Some cholinergic neurons also express the vesicular glutamate transporter transcript gene. POMC and POMC-processing enzyme transcripts are found in a subpopulation of cholinergic neurons. Despite this heterogeneity, gene expression patterns in individual cholinergic cells appear to be highly regulated in a cell-specific manner. In fact, membrane receptor transcripts are clustered with their respective intracellular signaling and downstream targets. This novel population of cholinergic neurons may be part of the neural circuitries that detect homeostatic need for food and control the drive to eat. PMID:27611685

  11. Genetic ablation of Rest leads to in vitro-specific derepression of neuronal genes during neurogenesis.

    PubMed

    Aoki, Hitomi; Hara, Akira; Era, Takumi; Kunisada, Takahiro; Yamada, Yasuhiro

    2012-02-01

    Rest (RE1-silencing transcription factor, also called Nrsf) is involved in the maintenance of the undifferentiated state of neuronal stem/progenitor cells in vitro by preventing precocious expression of neuronal genes. However, the function of Rest during neurogenesis in vivo remains to be elucidated because of the early embryonic lethal phenotype of conventional Rest knockout mice. In the present study, we have generated Rest conditional knockout mice, which allow the effect of genetic ablation of Rest during embryonic neurogenesis to be examined in vivo. We show that Rest plays a role in suppressing the expression of neuronal genes in cultured neuronal cells in vitro, as well as in non-neuronal cells outside of the central nervous system, but that it is dispensable for embryonic neurogenesis in vivo. Our findings highlight the significance of extrinsic signals for the proper intrinsic regulation of neuronal gene expression levels in the specification of cell fate during embryonic neurogenesis in vivo. PMID:22241837

  12. In utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons.

    PubMed

    Rice, Heather; Suth, Seiyam; Cavanaugh, William; Bai, Jilin; Young-Pearse, Tracy L

    2010-10-08

    In vitro study of primary neuronal cultures allows for quantitative analyses of neurite outgrowth. In order to study how genetic alterations affect neuronal process outgrowth, shRNA or cDNA constructs can be introduced into primary neurons via chemical transfection or viral transduction. However, with primary cortical cells, a heterogeneous pool of cell types (glutamatergic neurons from different layers, inhibitory neurons, glial cells) are transfected using these methods. The use of in utero electroporation to introduce DNA constructs in the embryonic rodent cortex allows for certain subsets of cells to be targeted: while electroporation of early embryonic cortex targets deep layers of the cortex, electroporation at late embryonic timepoints targets more superficial layers. Further, differential placement of electrodes across the heads of individual embryos results in the targeting of dorsal-medial versus ventral-lateral regions of the cortex. Following electroporation, transfected cells can be dissected out, dissociated, and plated in vitro for quantitative analysis of neurite outgrowth. Here, we provide a step-by-step method to quantitatively measure neuronal process outgrowth in subsets of cortical cells. The basic protocol for in utero electroporation has been described in detail in two other JoVE articles from the Kriegstein lab. We will provide an overview of our protocol for in utero electroporation, focusing on the most important details, followed by a description of our protocol that applies in utero electroporation to the study of gene function in neuronal process outgrowth.

  13. Problems associated with gene transfer and opportunities for microgravity environments

    NASA Astrophysics Data System (ADS)

    Tennessen, Daniel J.

    1997-01-01

    The method of crop improvement by gene transfer is becoming increasingly routine with transgenic foods and ornamental crops now being marketed to consumers. However, biological processes of plants, and the physical barriers of current protocols continue to limit the application of gene transfer in many commercial crops. The goal of this paper is to outline the current limitations of gene transfer and to hypothesize possible opportunities for use of microgravity to overcome such limitations. The limitations detailed in this paper include host-range specificity of Agrobacterium mediated transformation, probability of gene insertion, position effects of the inserted genes, gene copy number, stability of foreign gene expression in host plants, and regeneration of recalcitrant plant species. Microgravity offers an opportunity for gene transfer where cell growth kinetics, DNA synthesis, and genetic recombination rates can be altered. Such biological conditions may enhance the ability for recombination of reporter genes and other genes of interest to agriculture. Proposed studies would be useful for understanding instability of foreign gene expression and may lead to stable transformed plants. Other aspects of gene transfer in microgravity are discussed.

  14. Problems associated with gene transfer and opportunities for microgravity environments

    SciTech Connect

    Tennessen, D.J.

    1997-01-01

    The method of crop improvement by gene transfer is becoming increasingly routine with transgenic foods and ornamental crops now being marketed to consumers. However, biological processes of plants, and the physical barriers of current protocols continue to limit the application of gene transfer in many commercial crops. The goal of this paper is to outline the current limitations of gene transfer and to hypothesize possible opportunities for use of microgravity to overcome such limitations. The limitations detailed in this paper include host-range specificity of {ital Agrobacterium} mediated transformation, probability of gene insertion, position effects of the inserted genes, gene copy number, stability of foreign gene expression in host plants, and regeneration of recalcitrant plant species. Microgravity offers an opportunity for gene transfer where cell growth kinetics, DNA synthesis, and genetic recombination rates can be altered. Such biological conditions may enhance the ability for recombination of reporter genes and other genes of interest to agriculture. Proposed studies would be useful for understanding instability of foreign gene expression and may lead to stable transformed plants. Other aspects of gene transfer in microgravity are discussed. {copyright} {ital 1997 American Institute of Physics.}

  15. Gene transfer for congestive heart failure: update 2013.

    PubMed

    Tang, Tong; Hammond, H Kirk

    2013-04-01

    Congestive heart failure is a major cause of morbidity and mortality with increasing social and economic costs. There have been no new high impact therapeutic agents for this devastating disease for more than a decade. However, many pivotal regulators of cardiac function have been identified using cardiac-directed transgene expression and gene deletion in preclinical studies. Some of these increase function of the failing heart. Altering the expression of these pivotal regulators using gene transfer is now either being tested in clinical gene transfer trials, or soon will be. In this review, we summarize recent progress in cardiac gene transfer for clinical congestive heart failure.

  16. Microbubbles and ultrasound increase intraventricular polyplex gene transfer to the brain.

    PubMed

    Tan, James-Kevin Y; Pham, Binhan; Zong, Yujin; Perez, Camilo; Maris, Don O; Hemphill, Ashton; Miao, Carol H; Matula, Thomas J; Mourad, Pierre D; Wei, Hua; Sellers, Drew L; Horner, Philip J; Pun, Suzie H

    2016-06-10

    Neurons in the brain can be damaged or lost from neurodegenerative disease, stroke, or traumatic injury. Although neurogenesis occurs in mammalian adult brains, the levels of natural neurogenesis are insufficient to restore function in these cases. Gene therapy has been pursued as a promising strategy to induce differentiation of neural progenitor cells into functional neurons. Non-viral vectors are a preferred method of gene transfer due to potential safety and manufacturing benefits but suffer from lower delivery efficiencies compared to viral vectors. Since the neural stem and progenitor cells reside in the subventricular zone of the brain, intraventricular injection has been used as an administration route for gene transfer to these cells. However, the choroid plexus epithelium remains an obstacle to delivery. Recently, transient disruption of the blood-brain barrier by microbubble-enhanced ultrasound has been used to successfully improve drug delivery to the brain after intravenous injection. In this work, we demonstrate that microbubble-enhanced ultrasound can similarly improve gene transfer to the subventricular zone after intraventricular injection. Microbubbles of different surface charges (neutral, slightly cationic, and cationic) were prepared, characterized by acoustic flow cytometry, and evaluated for their ability to increase the permeability of immortalized choroid plexus epithelium monolayers in vitro. Based on these results, slightly cationic microbubbles were evaluated for microbubble and ultrasound-mediated enhancement of non-viral gene transfer in vivo. When coupled with our previously reported gene delivery vehicles, the slightly cationic microbubbles significantly increased ultrasound-mediated transfection of the murine brain when compared to commercially available Definity® microbubbles. Temporary disruption of the choroid plexus by microbubble-enhanced ultrasound is therefore a viable way of enhancing gene delivery to the brain and merits

  17. Regulatory logic of pan-neuronal gene expression in C. elegans

    PubMed Central

    Stefanakis, Nikolaos; Carrera, Ines; Hobert, Oliver

    2015-01-01

    While neuronal cell types display an astounding degree of phenotypic diversity, most if not all neuron types share a core panel of terminal features. However, little is known about how pan-neuronal expression patterns are genetically programmed. Through an extensive analysis of the cis-regulatory control regions of a battery of pan-neuronal C.elegans genes, including genes involved in synaptic vesicle biology and neuropeptide signaling, we define a common organizational principle in the regulation of pan-neuronal genes in the form of a surprisingly complex array of seemingly redundant, parallel-acting cis-regulatory modules that direct expression to broad, overlapping domains throughout the nervous system. These parallel-acting cis-regulatory modules are responsive to a multitude of distinct trans-acting factors. Neuronal gene expression programs therefore fall into two fundamentally distinct classes. Neuron type-specific genes are generally controlled by discrete and non-redundantly acting regulatory inputs, while pan-neuronal gene expression is controlled by diverse, coincident and seemingly redundant regulatory inputs. PMID:26291158

  18. A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and in vivo: Polyethylenimine

    NASA Astrophysics Data System (ADS)

    Boussif, Otmane; Lezoualc'h, Frank; Zanta, Maria Antonietta; Djavaheri Mergny, Mojgan; Scherman, Daniel; Demeneix, Barbara; Behr, Jean-Paul

    1995-08-01

    Several polycations possessing substantial buffering capacity below physiological pH, such as lipopolyamines and polyamidoamine polymers, are efficient transfection agents per se-i.e., without the addition of cell targeting or membrane-disruption agents. This observation led us to test the cationic polymer polyethylenimine (PEI) for its genedelivery potential. Indeed, every third atom of PEI is a protonable amino nitrogen atom, which makes the polymeric network an effective "proton sponge" at virtually any pH. Luciferase reporter gene transfer with this polycation into a variety of cell lines and primary cells gave results comparable to, or even better than, lipopolyamines. Cytotoxicity was low and seen only at concentrations well above those required for optimal transfection. Delivery of oligonucleotides into embryonic neurons was followed by using a fluorescent probe. Virtually all neurons showed nuclear labeling, with no toxic effects. The optimal PEI cation/anion balance for in vitro transfection is only slightly on the cationic side, which is advantageous for in vivo delivery. Indeed, intracerebral luciferase gene transfer into newborn mice gave results comparable (for a given amount of DNA) to the in vitro transfection of primary rat brain endothelial cells or chicken embryonic neurons. Together, these properties make PEI a promising vector for gene therapy and an outstanding core for the design of more sophisticated devices. Our hypothesis is that its efficiency relies on extensive lysosome buffering that protects DNA from nuclease degradation, and consequent lysosomal swelling and rupture that provide an escape mechanism for the PEI/DNA particles.

  19. Transfer Entropy Reconstruction and Labeling of Neuronal Connections from Simulated Calcium Imaging

    PubMed Central

    Orlandi, Javier G.; Stetter, Olav; Soriano, Jordi; Geisel, Theo; Battaglia, Demian

    2014-01-01

    Neuronal dynamics are fundamentally constrained by the underlying structural network architecture, yet much of the details of this synaptic connectivity are still unknown even in neuronal cultures in vitro. Here we extend a previous approach based on information theory, the Generalized Transfer Entropy, to the reconstruction of connectivity of simulated neuronal networks of both excitatory and inhibitory neurons. We show that, due to the model-free nature of the developed measure, both kinds of connections can be reliably inferred if the average firing rate between synchronous burst events exceeds a small minimum frequency. Furthermore, we suggest, based on systematic simulations, that even lower spontaneous inter-burst rates could be raised to meet the requirements of our reconstruction algorithm by applying a weak spatially homogeneous stimulation to the entire network. By combining multiple recordings of the same in silico network before and after pharmacologically blocking inhibitory synaptic transmission, we show then how it becomes possible to infer with high confidence the excitatory or inhibitory nature of each individual neuron. PMID:24905689

  20. Phenotypical characterization of the rat striatal neurons expressing the D1 dopamine receptor gene.

    PubMed Central

    Le Moine, C; Normand, E; Bloch, B

    1991-01-01

    In situ hybridization experiments were performed in rat brain sections from normal and 6-hydroxydopamine-treated rats in order to map and identify the neurons expressing the D1 receptor gene in the striatum and the substantia nigra. Procedures of combined in situ hybridization, allowing the simultaneous detection of two mRNAs in the same section or in adjacent sections, were used to characterize the phenotypes of the neurons expressing the D1 receptor gene. D1 receptor mRNA was found in neurons all over the caudate-putamen, the accumbens nucleus, and the olfactory tubercle but not in the substantia nigra. In the caudate-putamen and accumbens nucleus, most of the neurons containing D1 receptor mRNA were characterized as medium-sized substance P neurons and distinct from those containing D2 receptor mRNA. Nevertheless, 15-20% of the substance P neurons did not contain D1 receptor mRNA. The neurons containing preproenkephalin A mRNA did not contain D1 receptor mRNA but contained D2 receptor mRNA. A small number of cholinergic and somatostatinergic neurons exhibited a weak reaction for D1 receptor mRNA. These results demonstrate that dopamine acts on efferent striatal neurons through expression of distinct receptors--namely, D1 and D2 in separate cell populations (substance P and preproenkephalin A neurons, respectively)--and can also act on nonprojecting neurons through D1 receptor expression. Images PMID:1827915

  1. Guanidinylated block copolymers for gene transfer: A comparison with amine-based materials for in vitro and in vivo gene transfer efficiency

    PubMed Central

    Choi, Jennifer L.; Tan, James-Kevin Y.; Sellers, Drew L.; Wei, Hua; Horner, Philip J.; Pun, Suzie H.

    2015-01-01

    There is currently no cure for neuron loss in the brain, which can occur due to traumatic injury or neurodegenerative disease. One method proposed to enhance neurogenesis in the brain is gene transfer to neural progenitor cells. In this work, a guanidine-based copolymer was synthesized and compared to an amine-based copolymer analog previously shown to effectively deliver genes in the murine brain. The guanidine-based copolymer was more efficient at gene transfer to immortalized, cultured cell lines; however, the amine-based copolymer was more effective at gene transfer in the brain. DNA condensation studies revealed that the nucleic acid complexes formed with the guanidine-based copolymer were more susceptible to unpackaging in the presence of heparin sulfate proteoglycans compared to complexes formed with the amine-based copolymer. Therefore, polyplexes formed from the amine-based copolymer may be more resistant to destabilization by the heparan sulfate proteoglycans present in the stem cell niches of the brain. PMID:25907042

  2. Stable and Efficient Gene Transfer into the Retina Using an HIV-Based Lentiviral Vector

    NASA Astrophysics Data System (ADS)

    Miyoshi, Hiroyuki; Takahashi, Masayo; Gage, Fred H.; Verma, Inder M.

    1997-09-01

    The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.

  3. A recently transferred cluster of bacterial genes in Trichomonas vaginalis - lateral gene transfer and the fate of acquired genes

    PubMed Central

    2014-01-01

    Background Lateral Gene Transfer (LGT) has recently gained recognition as an important contributor to some eukaryote proteomes, but the mechanisms of acquisition and fixation in eukaryotic genomes are still uncertain. A previously defined norm for LGTs in microbial eukaryotes states that the majority are genes involved in metabolism, the LGTs are typically localized one by one, surrounded by vertically inherited genes on the chromosome, and phylogenetics shows that a broad collection of bacterial lineages have contributed to the transferome. Results A unique 34 kbp long fragment with 27 clustered genes (TvLF) of prokaryote origin was identified in the sequenced genome of the protozoan parasite Trichomonas vaginalis. Using a PCR based approach we confirmed the presence of the orthologous fragment in four additional T. vaginalis strains. Detailed sequence analyses unambiguously suggest that TvLF is the result of one single, recent LGT event. The proposed donor is a close relative to the firmicute bacterium Peptoniphilus harei. High nucleotide sequence similarity between T. vaginalis strains, as well as to P. harei, and the absence of homologs in other Trichomonas species, suggests that the transfer event took place after the radiation of the genus Trichomonas. Some genes have undergone pseudogenization and degradation, indicating that they may not be retained in the future. Functional annotations reveal that genes involved in informational processes are particularly prone to degradation. Conclusions We conclude that, although the majority of eukaryote LGTs are single gene occurrences, they may be acquired in clusters of several genes that are subsequently cleansed of evolutionarily less advantageous genes. PMID:24898731

  4. Widespread horizontal transfer of mitochondrial genes in flowering plants.

    PubMed

    Bergthorsson, Ulfar; Adams, Keith L; Thomason, Brendan; Palmer, Jeffrey D

    2003-07-10

    Horizontal gene transfer--the exchange of genes across mating barriers--is recognized as a major force in bacterial evolution. However, in eukaryotes it is prevalent only in certain phagotrophic protists and limited largely to the ancient acquisition of bacterial genes. Although the human genome was initially reported to contain over 100 genes acquired during vertebrate evolution from bacteria, this claim was immediately and repeatedly rebutted. Moreover, horizontal transfer is unknown within the evolution of animals, plants and fungi except in the special context of mobile genetic elements. Here we show, however, that standard mitochondrial genes, encoding ribosomal and respiratory proteins, are subject to evolutionarily frequent horizontal transfer between distantly related flowering plants. These transfers have created a variety of genomic outcomes, including gene duplication, recapture of genes lost through transfer to the nucleus, and chimaeric, half-monocot, half-dicot genes. These results imply the existence of mechanisms for the delivery of DNA between unrelated plants, indicate that horizontal transfer is also a force in plant nuclear genomes, and are discussed in the contexts of plant molecular phylogeny and genetically modified plants.

  5. Peripheral Nervous System Genes Expressed in Central Neurons Induce Growth on Inhibitory Substrates

    PubMed Central

    Buchser, William J.; Smith, Robin P.; Pardinas, Jose R.; Haddox, Candace L.; Hutson, Thomas; Moon, Lawrence; Hoffman, Stanley R.; Bixby, John L.; Lemmon, Vance P.

    2012-01-01

    Trauma to the spinal cord and brain can result in irreparable loss of function. This failure of recovery is in part due to inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans (CSPGs). Peripheral nervous system (PNS) neurons exhibit increased regenerative ability compared to central nervous system neurons, even in the presence of inhibitory environments. Previously, we identified over a thousand genes differentially expressed in PNS neurons relative to CNS neurons. These genes represent intrinsic differences that may account for the PNS’s enhanced regenerative ability. Cerebellar neurons were transfected with cDNAs for each of these PNS genes to assess their ability to enhance neurite growth on inhibitory (CSPG) or permissive (laminin) substrates. Using high content analysis, we evaluated the phenotypic profile of each neuron to extract meaningful data for over 1100 genes. Several known growth associated proteins potentiated neurite growth on laminin. Most interestingly, novel genes were identified that promoted neurite growth on CSPGs (GPX3, EIF2B5, RBMX). Bioinformatic approaches also uncovered a number of novel gene families that altered neurite growth of CNS neurons. PMID:22701605

  6. Direct transfer of IL-12 gene into growing Renca tumors.

    PubMed

    Budryk, M; Wilczyńska, U; Szary, J; Szala, S

    2000-01-01

    We investigated the feasibility of transferring naked plasmid DNA containing a therapeutic gene (IL-12) into mice harboring growing Renca tumors. We found that naked DNA transferred into growing Renca and B16(F10) tumors gives higher expression level of reporter gene than complexes of DNA with DDAB/DOPE or DC-Chol/DOPE. Transfer of naked DNA carrying the IL-12 gene into growing Renca tumors causes a distinct therapeutic effect that depends on the time span between inoculation of mice with cancer cells and the beginning of the therapy. Therapy started on day 3 resulted in total cure (100%) of mice. PMID:11051203

  7. LATERAL GENE TRANSFER AND THE HISTORY OF BACTERIAL GENOMES

    SciTech Connect

    Howard Ochman

    2006-02-22

    The aims of this research were to elucidate the role and extent of lateral transfer in the differentiation of bacterial strains and species, and to assess the impact of gene transfer on the evolution of bacterial genomes. The ultimate goal of the project is to examine the dynamics of a core set of protein-coding genes (i.e., those that are distributed universally among Bacteria) by developing conserved primers that would allow their amplification and sequencing in any bacterial taxa. In addition, we adopted a bioinformatic approach to elucidate the extent of lateral gene transfer in sequenced genome.

  8. Intracellular gene transfer: Reduced hydrophobicity facilitates gene transfer for subunit 2 of cytochrome c oxidase

    PubMed Central

    Daley, Daniel O.; Clifton, Rachel; Whelan, James

    2002-01-01

    Subunit 2 of cytochrome c oxidase (Cox2) in legumes offers a rare opportunity to investigate factors necessary for successful gene transfer of a hydrophobic protein that is usually mitochondrial-encoded. We found that changes in local hydrophobicity were necessary to allow import of this nuclear-encoded protein into mitochondria. All legume species containing both a mitochondrial and nuclear encoded Cox2 displayed a similar pattern, with a large decrease in hydrophobicity evident in the first transmembrane region of the nuclear encoded protein compared with the organelle-encoded protein. Mitochondrial-encoded Cox2 could not be imported into mitochondria under the direction of the mitochondrial targeting sequence that readily supports the import of nuclear encoded Cox2. Removal of the first transmembrane region promotes import ability of the mitochondrial-encoded Cox2. Changing just two amino acids in the first transmembrane region of mitochondrial-encoded Cox2 to the corresponding amino acids in the nuclear encoded Cox2 also promotes import ability, whereas changing the same two amino acids in the nuclear encoded Cox2 to what they are in the mitochondrial-encoded copy prevents import. Therefore, changes in amino acids in the mature protein were necessary and sufficient for gene transfer to allow import under the direction of an appropriate signal to achieve the functional topology of Cox2. PMID:12142462

  9. Age-associated bidirectional modulation of gene expression in single identified R15 neuron of Aplysia

    PubMed Central

    2013-01-01

    Background Despite the advances in our understanding of aging-associated behavioral decline, relatively little is known about how aging affects neural circuits that regulate specific behaviors, particularly the expression of genes in specific neural circuits during aging. We have addressed this by exploring a peptidergic neuron R15, an identified neuron of the marine snail Aplysia californica. R15 is implicated in reproduction and osmoregulation and responds to neurotransmitters such as acetylcholine, serotonin and glutamate and is characterized by its action potential bursts. Results We examined changes in gene expression in R15 neurons during aging by microarray analyses of RNAs from two different age groups, mature and old animals. Specifically we find that 1083 ESTs are differentially regulated in mature and old R15 neurons. Bioinformatics analyses of these genes have identified specific biological pathways that are up or downregulated in mature and old neurons. Comparison with human signaling networks using pathway analyses have identified three major networks [(1) cell signaling, cell morphology, and skeletal muscular system development (2) cell death and survival, cellular function maintenance and embryonic development and (3) neurological diseases, developmental and hereditary disorders] altered in old R15 neurons. Furthermore, qPCR analysis of single R15 neurons to quantify expression levels of candidate regulators involved in transcription (CREB1) and translation (S6K) showed that aging is associated with a decrease in expression of these regulators, and similar analysis in three other neurons (L7, L11 and R2) showed that gene expression change during aging could be bidirectional. Conclusions We find that aging is associated with bidirectional changes in gene expression. Detailed bioinformatics analyses and human homolog searches have identified specific biological processes and human-relevant signaling pathways in R15 that are affected during aging

  10. Inflammatory effects of gene transfer into the CNS with defective HSV-1 vectors.

    PubMed

    Wood, M J; Byrnes, A P; Pfaff, D W; Rabkin, S D; Charlton, H M

    1994-09-01

    The use of viral vectors which infect and express genes in post-mitotic neurons is a potential strategy for the treatment of disorders affecting the central nervous system (CNS). However, the inflammatory consequences of such strategies have yet to be systematically examined. Preparations of non-replicating defective herpes simplex virus type 1 (HSV-1) amplicon vectors containing the lacZ gene were obtained by standard methods and stereotaxically injected into the adult rat dentate gyrus (DG). The consequent gene expression and inflammatory effects following microinjection were investigated. beta-Galactosidase activity was detected in neurons of the DG from 24 h to at least 12 days after vector injection. A strong inflammatory response developed within 2 days, characterized by diffuse up-regulation of major histocompatibility complex (MHC) class I antigens and the activation of microglia. After 4 days the recruitment of MHC class II+ cells, activated T lymphocytes and macrophages was detected. These features persisted for at least 31 days. Of importance was the finding of beta-galactosidase activity in a bilateral group of neurons in the supramammillary nuclei (SMN) of the posterior hypothalamus, known to send afferent projections to the DG. The onset of inflammation at this secondary site was delayed, but its cellular characteristics resembled those found at the primary site of injection. Thus, the use of preparations of defective HSV-1 vectors for gene transfer in the CNS has immunological implications both at primary and secondary sites within the CNS.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7584093

  11. Enhancement of primary neuronal cell proliferation using printing-transferred carbon nanotube sheets.

    PubMed

    Kang, Dong-Wan; Sun, Fangfang; Choi, Yoon Ji; Zou, Fengming; Cho, Won-Ho; Choi, Byung-Kwan; Koh, Kwangnak; Lee, Jaebeom; Han, In Ho

    2015-05-01

    Artificial nerve guidance conduits (aNGCs) prepared from polymer scaffolds and carbon nanotubes (CNTs) possess unique chemical and physical properties, and have been widely used in preclinical trials to promote neuronal differentiation and growth. However, there have been only a few reports on the clinical applicability of CNT sheets for proliferation of primary neuronal cells due to safety concerns. The present study assesses the ability and potential applicability of multiwalled CNTs (MWNTs) composited with polydimethylsiloxane (PDMS) sheets to promote and enhance the proliferation of primary neuronal cells. In this study, the aqueous MWNT dispersion was filtered, and the PDMS/MWNT sheets were prepared using a simple printing transfer method. Characterization of PDMS/MWNT sheets demonstrated their unique physical properties such as superior mechanical strength and electroconductivity when compared with PDMS sheets. The effect of the PDMS/MWNT sheets on the neural cell proliferation and cytotoxicity was evaluated using MTT and alamar blue assays. Our results indicate the viability and proliferation of primary neuronal cells and Schwann cells in PDMS/MWNT sheets increased over twice when compared with a noncoated dish that is not usual in the primary neuronal cell growth control (p < 0.05). In addition, PDMS/MWNT sheets enhanced the adhesion and viability of the cells compared with poly-l-lysine coated dishes, which are most commonly used for improving cell adherence. Additionally, the PDMS/MWNT sheets exhibited excellent biocompatibility for culturing neuronal and Schwann cells. Overall, all assessments indicate that PDMS/MWNT sheets are ideal candidates for the development of artificial nerve conduits for clinical use following peripheral nerve injury. PMID:25087551

  12. Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms.

    PubMed

    Mundell, Nathan A; Beier, Kevin T; Pan, Y Albert; Lapan, Sylvain W; Göz Aytürk, Didem; Berezovskii, Vladimir K; Wark, Abigail R; Drokhlyansky, Eugene; Bielecki, Jan; Born, Richard T; Schier, Alexander F; Cepko, Constance L

    2015-08-01

    Current limitations in technology have prevented an extensive analysis of the connections among neurons, particularly within nonmammalian organisms. We developed a transsynaptic viral tracer originally for use in mice, and then tested its utility in a broader range of organisms. By engineering the vesicular stomatitis virus (VSV) to encode a fluorophore and either the rabies virus glycoprotein (RABV-G) or its own glycoprotein (VSV-G), we created viruses that can transsynaptically label neuronal circuits in either the retrograde or anterograde direction, respectively. The vectors were investigated for their utility as polysynaptic tracers of chicken and zebrafish visual pathways. They showed patterns of connectivity consistent with previously characterized visual system connections, and revealed several potentially novel connections. Further, these vectors were shown to infect neurons in several other vertebrates, including Old and New World monkeys, seahorses, axolotls, and Xenopus. They were also shown to infect two invertebrates, Drosophila melanogaster, and the box jellyfish, Tripedalia cystophora, a species previously intractable for gene transfer, although no clear evidence of transsynaptic spread was observed in these species. These vectors provide a starting point for transsynaptic tracing in most vertebrates, and are also excellent candidates for gene transfer in organisms that have been refractory to other methods. PMID:25688551

  13. Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms.

    PubMed

    Mundell, Nathan A; Beier, Kevin T; Pan, Y Albert; Lapan, Sylvain W; Göz Aytürk, Didem; Berezovskii, Vladimir K; Wark, Abigail R; Drokhlyansky, Eugene; Bielecki, Jan; Born, Richard T; Schier, Alexander F; Cepko, Constance L

    2015-08-01

    Current limitations in technology have prevented an extensive analysis of the connections among neurons, particularly within nonmammalian organisms. We developed a transsynaptic viral tracer originally for use in mice, and then tested its utility in a broader range of organisms. By engineering the vesicular stomatitis virus (VSV) to encode a fluorophore and either the rabies virus glycoprotein (RABV-G) or its own glycoprotein (VSV-G), we created viruses that can transsynaptically label neuronal circuits in either the retrograde or anterograde direction, respectively. The vectors were investigated for their utility as polysynaptic tracers of chicken and zebrafish visual pathways. They showed patterns of connectivity consistent with previously characterized visual system connections, and revealed several potentially novel connections. Further, these vectors were shown to infect neurons in several other vertebrates, including Old and New World monkeys, seahorses, axolotls, and Xenopus. They were also shown to infect two invertebrates, Drosophila melanogaster, and the box jellyfish, Tripedalia cystophora, a species previously intractable for gene transfer, although no clear evidence of transsynaptic spread was observed in these species. These vectors provide a starting point for transsynaptic tracing in most vertebrates, and are also excellent candidates for gene transfer in organisms that have been refractory to other methods.

  14. Vesicular stomatitis virus enables gene transfer and transsynaptic tracing in a wide range of organisms

    PubMed Central

    Mundell, Nathan A.; Beier, Kevin T.; Pan, Y. Albert; Lapan, Sylvain W.; Göz Aytürk, Didem; Berezovskii, Vladimir K.; Wark, Abigail R.; Drokhlyansky, Eugene; Bielecki, Jan; Born, Richard T.; Schier, Alexander F.

    2015-01-01

    Current limitations in technology have prevented an extensive analysis of the connections among neurons, particularly within nonmammalian organisms. We developed a transsynaptic viral tracer originally for use in mice, and then tested its utility in a broader range of organisms. By engineering the vesicular stomatitis virus (VSV) to encode a fluorophore and either the rabies virus glycoprotein (RABV‐G) or its own glycoprotein (VSV‐G), we created viruses that can transsynaptically label neuronal circuits in either the retrograde or anterograde direction, respectively. The vectors were investigated for their utility as polysynaptic tracers of chicken and zebrafish visual pathways. They showed patterns of connectivity consistent with previously characterized visual system connections, and revealed several potentially novel connections. Further, these vectors were shown to infect neurons in several other vertebrates, including Old and New World monkeys, seahorses, axolotls, and Xenopus. They were also shown to infect two invertebrates, Drosophila melanogaster, and the box jellyfish, Tripedalia cystophora, a species previously intractable for gene transfer, although no clear evidence of transsynaptic spread was observed in these species. These vectors provide a starting point for transsynaptic tracing in most vertebrates, and are also excellent candidates for gene transfer in organisms that have been refractory to other methods. J. Comp. Neurol. 523:1639–1663, 2015. © 2015 Wiley Periodicals, Inc. PMID:25688551

  15. Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer

    PubMed Central

    Gubbels, Samuel. P.; Woessner, David W.; Mitchell, John C.; Ricci, Anthony J.; Brigande, John V.

    2010-01-01

    Sensory hair cells in the mammalian cochlea convert mechanical stimuli into electrical impulses that subserve audition1,2. Loss of hair cells and their innervating neurons is the most frequent cause of hearing impairment3. Atonal homolog 1 (Atoh1, also known as Math1) is a basic helix-loop-helix transcription factor required for hair cell development4-6 and its misexpression in vitro7,8 and in vivo9,10 generates hair-cell-like cells. Atoh1-based gene therapy to ameliorate auditory10 and vestibular11 dysfunction has been proposed. However, the biophysical properties of putative hair cells induced by Atoh1 misexpression have not been characterized. Here we show that in utero gene transfer of Atoh1 produces functional supernumerary hair cells in the mouse cochlea. The induced hair cells display stereociliary bundles, attract neuronal processes, and express the ribbon synapse marker C-terminal binding protein 2 (Ctbp2)12,13. Moreover, the hair cells are capable of mechanoelectrical transduction1,2 and display basolateral conductances with age-appropriate specializations. Our results demonstrate that manipulation of cell fate by transcription factor misexpression produces functional sensory cells in the postnatal mammalian cochlea. We anticipate that our in utero gene transfer paradigm will enable the design and validation of gene therapies to ameliorate hearing loss in mouse models of human deafness14,15. PMID:18754012

  16. An early history of gene transfer and therapy.

    PubMed

    Wolff, J A; Lederberg, J

    1994-04-01

    The term "gene therapy" was coined to distinguish it from the Orwellian connotations of "human genetic engineering," which, in turn, was derived from the term "genetic engineering." Genetic engineering was first used at the Sixth International Congress of Genetics held in 1932 and was taken to mean "the application of genetic principles to animal and plant breeding." Once the basics of molecular genetics and gene transfer in bacteria were established in the 1960s, gene transfer into animals and humans using either viral vectors and/or genetically modified cultured cells became inevitable. Despite the early exposition of the concept of gene therapy, progress awaited the advent of recombinant DNA technology. The lack of trustworthy techniques did not stop many researchers from attempting to transfer genes into cells in culture, animals, and humans. Viral genomes were used for the development of the first relatively efficient methods for gene transfer into mammalian cells in culture. In the late 1970s, early transfection techniques were combined with selection systems for cultured cells and recombinant DNA technology. With the development of retroviral vectors in the early 1980s, the possibility of efficient gene transfer into mammalian cells for the purpose of gene therapy became widely accepted.

  17. Gene transfer in inner ear cells: a challenging race.

    PubMed

    Sacheli, R; Delacroix, L; Vandenackerveken, P; Nguyen, L; Malgrange, B

    2013-03-01

    Recent advances in human genomics led to the identification of numerous defective genes causing deafness, which represent novel putative therapeutic targets. Future gene-based treatment of deafness resulting from genetic or acquired sensorineural hearing loss may include strategies ranging from gene therapy to antisense delivery. For successful development of gene therapies, a minimal requirement involves the engineering of appropriate gene carrier systems. Transfer of exogenous genetic material into the mammalian inner ear using viral or non-viral vectors has been characterized over the last decade. The nature of inner ear cells targeted, as well as the transgene expression level and duration, are highly dependent on the vector type, the route of administration and the strength of the promoter driving expression. This review summarizes and discusses recent advances in inner ear gene-transfer technologies aimed at examining gene function or identifying new treatment for inner ear disorders.

  18. Adenoviral-vector-mediated gene transfer to dendritic cells.

    PubMed

    Song, W; Crystal, R G

    2001-01-01

    Dendritic cells (DC) are the most potent antigen presenting cells capable of initiating T-cell-dependent immune responses (1-5). This biologic potential can be harnessed to elicit effective antigen-specific immune responses by transferring the relevant antigens to the DC. Once the DC have been mobilized and purified, the relevant antigens can be transferred to the DC as intact proteins, or as peptides representing specific epitopes, or with gene transfer using sequences of DNA or RNA coding for the pertinent antigen(s) (6-15). Theoretically, genetically modifying DC with genes coding for specific antigens has potential advantages over pulsing the DC with peptides repeating the antigen or antigen fragment. First, the genetically modified DC may present previously unknown epitopes in association with different MHC molecules. Second, gene transfer to DC ensures that the gene product is endogenously processed, leading to the generation of MHC class I-restricted cytotoxic T lymphocytes (CTL), the effector arm of cell-mediated immune responses. Finally, in addition to genes coding for the antigen(s), genetic modification of the DC can induce genes coding for mediators relevant to generation of the immune response to the antigen(s), further boosting host responses to the antigens presented by the modified DC. Different gene transfer approaches have been explored to genetically modify DC, including retroviral vectors (16-18), recombinant vaccinia virus vectors (19), and recombinant adenovirus (Ad) vectors (19-23). The focus of this chapter is on using recombinant Ad vectors to transfer genes to murine DC. We have used a similar strategy to transfer genes to human DC (24). As an example of the power of this technology, we will describe the use of Ad-vector-modified DC to suppress the growth of tumor cells modified to express a specific antigen.

  19. Rates of Lateral Gene Transfer in Prokaryotes: High but Why?

    PubMed

    Vos, Michiel; Hesselman, Matthijn C; te Beek, Tim A; van Passel, Mark W J; Eyre-Walker, Adam

    2015-10-01

    Lateral gene transfer is of fundamental importance to the evolution of prokaryote genomes and has important practical consequences, as evidenced by the rapid dissemination of antibiotic resistance and virulence determinants. Relatively little effort has so far been devoted to explicitly quantifying the rate at which accessory genes are taken up and lost, but it is possible that the combined rate of lateral gene transfer and gene loss is higher than that of point mutation. What evolutionary forces underlie the rate of lateral gene transfer are not well understood. We here use theory developed to explain the evolution of mutation rates to address this question and explore its consequences for the study of prokaryote evolution.

  20. Interneuronal Transfer and Distal Action of Tetanus Toxin and Botulinum Neurotoxins A and D in Central Neurons.

    PubMed

    Bomba-Warczak, Ewa; Vevea, Jason D; Brittain, Joel M; Figueroa-Bernier, Annette; Tepp, William H; Johnson, Eric A; Yeh, Felix L; Chapman, Edwin R

    2016-08-16

    Recent reports suggest that botulinum neurotoxin (BoNT) A, which is widely used clinically to inhibit neurotransmission, can spread within networks of neurons to have distal effects, but this remains controversial. Moreover, it is not known whether other members of this toxin family are transferred between neurons. Here, we investigate the potential distal effects of BoNT/A, BoNT/D, and tetanus toxin (TeNT), using central neurons grown in microfluidic devices. Toxins acted upon the neurons that mediated initial entry, but all three toxins were also taken up, via an alternative pathway, into non-acidified organelles that mediated retrograde transport to the somato-dendritic compartment. Toxins were then released into the media, where they entered and exerted their effects upon upstream neurons. These findings directly demonstrate that these agents undergo transcytosis and interneuronal transfer in an active form, resulting in long-distance effects. PMID:27498860

  1. “Seq-ing” Insights into the Epigenetics of Neuronal Gene Regulation

    PubMed Central

    Telese, Francesca; Gamliel, Amir; Skowronska-Krawczyk, Dorota; Garcia-Bassets, Ivan; Rosenfeld, Michael G.

    2013-01-01

    The epigenetic control of neuronal gene expression patterns has emerged as an underlying regulatory mechanism for neuronal function, identity and plasticity, where short-to long-lasting adaptation is required to dynamically respond and process external stimuli. To achieve a comprehensive understanding of the physiology and pathology of the brain, it becomes essential to understand the mechanisms that regulate the epigenome and transcriptome in neurons. Here, we review recent advances in the study of regulated neuronal gene expression, which are dramatically expanding as a result of the development of new and powerful contemporary methodologies, based on the power of next-generation sequencing. This flood of new information has already transformed our understanding of the biology of cells, and is now driving discoveries elucidating the molecular mechanisms of brain function in cognition, behavior and disease, and may also inform the study of neuronal identity, diversity and cell reprogramming. PMID:23439116

  2. Particle-mediated gene transfer into murine livers using a newly developed gene gun.

    PubMed

    Kuriyama, S; Mitoro, A; Tsujinoue, H; Nakatani, T; Yoshiji, H; Tsujimoto, T; Yamazaki, M; Fukui, H

    2000-07-01

    Although particle-mediated gene transfer using gene gun technology has been applied for gene transfer into epidermis, applications of this technology to visceral tissues have not been well investigated. Although all helium gas-driven gene gun instruments have used macrocarriers to discharge DNA-coated microprojectiles so far, we used a newly developed gene gun instrument, in which a hammering bullet is used to discharge microprojectiles. With the gene gun, gold particles coated with lacZ expression plasmid were discharged to murine livers. LacZ expression was induced much more profoundly in the liver by particle-mediated gene transfer than by simple plasmid injection and electroporation-mediated gene transfer. LacZ expression was broadly and randomly distributed throughout the bombarded livers, indicating that particle-mediated gene transfer can induce transgene expression even at relatively distant areas from the surface of the bombarded tissue. Furthermore, although transgene expression was at its peak on day 2 after the bombardment, it was still detectable even on day 28. These results indicate that particle-mediated gene transfer with a newly developed gene gun may provide a new approach to gene therapy for human diseases.

  3. The weaver gene expression affects neuronal generation patterns depending on age and encephalic region.

    PubMed

    Martí, Joaquín; Carmen Santa-Cruz, M; Bayer, Shirley A; Hervás, José P

    2006-04-01

    Cell generation and survival are investigated in three different neuronal populations of weaver mice: Purkinje and fastigial neurons in the cerebellum, and dopaminergic neurons in the substantia nigra pars compacta. Tritiated thymidine was supplied to pregnant females at the time that these neurons were being produced. Autoradiography was then applied on brain sections obtained from the control and weaver offspring at postnatal (P) day 8 and 90. This makes it possible to assess the differential survival of neurons that were born at distinct embryonic times on the basis of the proportion of labeled cells at two postnatal ages. When labeling profiles were measured at P8, the inferred time of origin was similar between +/+ and wv/wv genotypes for each neuronal population considered. The same occurred at P90 for Purkinje or fastigial neurons, but the labeling profiles of midbrain neurons were different between wild type and weaver homozygotes. There is already a substantial reduction in the number of Purkinje and fastigial cells at P8, but loss of dopaminergic neurons was only detected in 90-day-old weavers and, therefore, vulnerability is built into this midbrain neural system during its late postnatal development. Our results show that depletion of Purkinje and fastigial cells is random with respect to the time of their birth, whereas the weaver gene seems to be specifically targeting the late-generated dopaminergic neurons.

  4. Parkinson's disease candidate gene prioritization based on expression profile of midbrain dopaminergic neurons

    PubMed Central

    2010-01-01

    Background Parkinson's disease is the second most common neurodegenerative disorder. The pathological hallmark of the disease is degeneration of midbrain dopaminergic neurons. Genetic association studies have linked 13 human chromosomal loci to Parkinson's disease. Identification of gene(s), as part of the etiology of Parkinson's disease, within the large number of genes residing in these loci can be achieved through several approaches, including screening methods, and considering appropriate criteria. Since several of the indentified Parkinson's disease genes are expressed in substantia nigra pars compact of the midbrain, expression within the neurons of this area could be a suitable criterion to limit the number of candidates and identify PD genes. Methods In this work we have used the combination of findings from six rodent transcriptome analysis studies on the gene expression profile of midbrain dopaminergic neurons and the PARK loci in OMIM (Online Mendelian Inheritance in Man) database, to identify new candidate genes for Parkinson's disease. Results Merging the two datasets, we identified 20 genes within PARK loci, 7 of which are located in an orphan Parkinson's disease locus and one, which had been identified as a disease gene. In addition to identifying a set of candidates for further genetic association studies, these results show that the criteria of expression in midbrain dopaminergic neurons may be used to narrow down the number of genes in PARK loci for such studies. PMID:20716345

  5. Global Analysis of Horizontal Gene Transfer in Fusarium verticillioides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The co-occurrence of microbes within plants and other specialized niches may facilitate horizontal gene transfer (HGT) affecting host-pathogen interactions. We recently identified fungal-to-fungal HGTs involving metabolic gene clusters. For a global analysis of HGTs in the maize pathogen Fusarium ve...

  6. Gene transfer into mammalian somatic cells in vivo.

    PubMed

    Yang, N S

    1992-01-01

    Direct gene transfer into mammalian somatic tissues in vivo is a developing technology with potential application for human gene therapy. During the past 2 years, extensive progress and numerous breakthroughs have been made in this area of research. Genetically engineered retroviral vectors have been used successfully to infect live animals, effecting foreign gene expression in liver, blood vessels, and mammary tissues. Recombinant adenovirus and herpes simplex virus vectors have been utilized effectively for in vivo gene transfer into lung and brain tissues, respectively. Direct injection or particle bombardment of DNA has been demonstrated to provide a physical means for in situ gene transfer, while carrier-mediated DNA delivery techniques have been extended to target specific organs for gene expression. These technological developments in conjunction with the initiation of the NIH human gene therapy trials have marked a milestone in developing new medical treatments for various genetic diseases and cancer. Various in vivo gene transfer techniques should also provide new tools for basic research in molecular and developmental genetics.

  7. Evolution of and horizontal gene transfer in the Endornavirus genus.

    PubMed

    Song, Dami; Cho, Won Kyong; Park, Sang-Ho; Jo, Yeonhwa; Kim, Kook-Hyung

    2013-01-01

    The transfer of genetic information between unrelated species is referred to as horizontal gene transfer. Previous studies have demonstrated that both retroviral and non-retroviral sequences have been integrated into eukaryotic genomes. Recently, we identified many non-retroviral sequences in plant genomes. In this study, we investigated the evolutionary origin and gene transfer of domains present in endornaviruses which are double-stranded RNA viruses. Using the available sequences for endornaviruses, we found that Bell pepper endornavirus-like sequences homologous to the glycosyltransferase 28 domain are present in plants, fungi, and bacteria. The phylogenetic analysis revealed the glycosyltransferase 28 domain of Bell pepper endornavirus may have originated from bacteria. In addition, two domains of Oryza sativa endornavirus, a glycosyltransferase sugar-binding domain and a capsular polysaccharide synthesis protein, also exhibited high similarity to those of bacteria. We found evidence that at least four independent horizontal gene transfer events for the glycosyltransferase 28 domain have occurred among plants, fungi, and bacteria. The glycosyltransferase sugar-binding domains of two proteobacteria may have been horizontally transferred to the genome of Thalassiosira pseudonana. Our study is the first to show that three glycome-related viral genes in the genus Endornavirus have been acquired from marine bacteria by horizontal gene transfer.

  8. Evolution of and horizontal gene transfer in the Endornavirus genus.

    PubMed

    Song, Dami; Cho, Won Kyong; Park, Sang-Ho; Jo, Yeonhwa; Kim, Kook-Hyung

    2013-01-01

    The transfer of genetic information between unrelated species is referred to as horizontal gene transfer. Previous studies have demonstrated that both retroviral and non-retroviral sequences have been integrated into eukaryotic genomes. Recently, we identified many non-retroviral sequences in plant genomes. In this study, we investigated the evolutionary origin and gene transfer of domains present in endornaviruses which are double-stranded RNA viruses. Using the available sequences for endornaviruses, we found that Bell pepper endornavirus-like sequences homologous to the glycosyltransferase 28 domain are present in plants, fungi, and bacteria. The phylogenetic analysis revealed the glycosyltransferase 28 domain of Bell pepper endornavirus may have originated from bacteria. In addition, two domains of Oryza sativa endornavirus, a glycosyltransferase sugar-binding domain and a capsular polysaccharide synthesis protein, also exhibited high similarity to those of bacteria. We found evidence that at least four independent horizontal gene transfer events for the glycosyltransferase 28 domain have occurred among plants, fungi, and bacteria. The glycosyltransferase sugar-binding domains of two proteobacteria may have been horizontally transferred to the genome of Thalassiosira pseudonana. Our study is the first to show that three glycome-related viral genes in the genus Endornavirus have been acquired from marine bacteria by horizontal gene transfer. PMID:23667703

  9. ENDOSYMBIOTIC AND HORIZONTAL GENE TRANSFER IN CHROMALVEOLATES(1).

    PubMed

    Bhattacharya, Debashish; Nosenko, Tetyana

    2008-02-01

    Chromalveolates include photosynthetic and nonphotosynthetic (some plastid-lacking) algae and protists that define a vast swath of eukaryotic diversity. These taxa are masters of gene acquisition through serial endosymbiosis (endosymbiotic gene transfer, EGT) and horizontal gene transfer (HGT). Understanding the contribution of these sources to nuclear genomes is key to elucidating chromalveolate evolution and to identifying suitable phylogenetic markers to place this lineage in the tree of life. Here we briefly review recent findings in our lab with regard to EGT and HGT in chromalveolates.

  10. Vector-mediated antibody gene transfer for infectious diseases.

    PubMed

    Schnepp, Bruce C; Johnson, Philip R

    2015-01-01

    This chapter discusses the emerging field of vector-mediated antibody gene transfer as an alternative vaccine for infectious disease, with a specific focus on HIV. However, this methodology need not be confined to HIV-1; the general strategy of vector-mediated antibody gene transfer can be applied to other difficult vaccine targets like hepatitis C virus, malaria, respiratory syncytial virus, and tuberculosis. This approach is an improvement over classical passive immunization strategies that administer antibody proteins to the host to provide protection from infection. With vector-mediated gene transfer, the antibody gene is delivered to the host, via a recombinant adeno-associated virus (rAAV) vector; this in turn results in long-term endogenous antibody expression from the injected muscle that confers protective immunity. Vector-mediated antibody gene transfer can rapidly move existing, potent broadly cross-neutralizing HIV-1-specific antibodies into the clinic. The gene transfer products demonstrate a potency and breadth identical to the original product. This strategy eliminates the need for immunogen design and interaction with the adaptive immune system to generate protection, a strategy that so far has shown limited promise.

  11. Rabies Virus CVS-N2c(ΔG) Strain Enhances Retrograde Synaptic Transfer and Neuronal Viability.

    PubMed

    Reardon, Thomas R; Murray, Andrew J; Turi, Gergely F; Wirblich, Christoph; Croce, Katherine R; Schnell, Matthias J; Jessell, Thomas M; Losonczy, Attila

    2016-02-17

    Virally based transsynaptic tracing technologies are powerful experimental tools for neuronal circuit mapping. The glycoprotein-deletion variant of the SAD-B19 vaccine strain rabies virus (RABV) has been the reagent of choice in monosynaptic tracing, since it permits the mapping of synaptic inputs to genetically marked neurons. Since its introduction, new helper viruses and reagents that facilitate complementation have enhanced the efficiency of SAD-B19(ΔG) transsynaptic transfer, but there has been little focus on improvements to the core RABV strain. Here we generate a new deletion mutant strain, CVS-N2c(ΔG), and examine its neuronal toxicity and efficiency in directing retrograde transsynaptic transfer. We find that by comparison with SAD-B19(ΔG), the CVS-N2c(ΔG) strain exhibits a reduction in neuronal toxicity and a marked enhancement in transsynaptic neuronal transfer. We conclude that the CVS-N2c(ΔG) strain provides a more effective means of mapping neuronal circuitry and of monitoring and manipulating neuronal activity in vivo in the mammalian CNS. PMID:26804990

  12. Neuronal Remodeling During Metamorphosis Is Regulated by the alan shepard (shep) Gene in Drosophila melanogaster

    PubMed Central

    Chen, Dahong; Qu, Chunjing; Bjorum, Sonia M.; Beckingham, Kathleen M.; Hewes, Randall S.

    2014-01-01

    Peptidergic neurons are a group of neuronal cells that synthesize and secrete peptides to regulate a variety of biological processes. To identify genes controlling the development and function of peptidergic neurons, we conducted a screen of 545 splice-trap lines and identified 28 loci that drove expression in peptidergic neurons when crossed to a GFP reporter transgene. Among these lines, an insertion in the alan shepard (shep) gene drove expression specifically in most peptidergic neurons. shep transcripts and SHEP proteins were detected primarily and broadly in the central nervous system (CNS) in embryos, and this expression continued into the adult stage. Loss of shep resulted in late pupal lethality, reduced adult life span, wing expansion defects, uncoordinated adult locomotor activities, rejection of males by virgin females, and reduced neuropil area and reduced levels of multiple presynaptic markers throughout the adult CNS. Examination of the bursicon neurons in shep mutant pharate adults revealed smaller somata and fewer axonal branches and boutons, and all of these cellular phenotypes were fully rescued by expression of the most abundant wild-type shep isoform. In contrast to shep mutant animals at the pharate adult stage, shep mutant larvae displayed normal bursicon neuron morphologies. Similarly, shep mutant adults were uncoordinated and weak, while shep mutant larvae displayed largely, although not entirely, normal locomotor behavior. Thus, shep played an important role in the metamorphic development of many neurons. PMID:24931409

  13. Role of Immediate-Early Genes in Synaptic Plasticity and Neuronal Ensembles Underlying the Memory Trace

    PubMed Central

    Minatohara, Keiichiro; Akiyoshi, Mika; Okuno, Hiroyuki

    2016-01-01

    In the brain, neuronal gene expression is dynamically changed in response to neuronal activity. In particular, the expression of immediate-early genes (IEGs) such as egr-1, c-fos, and Arc is rapidly and selectively upregulated in subsets of neurons in specific brain regions associated with learning and memory formation. IEG expression has therefore been widely used as a molecular marker for neuronal populations that undergo plastic changes underlying formation of long-term memory. In recent years, optogenetic and pharmacogenetic studies of neurons expressing c-fos or Arc have revealed that, during learning, IEG-positive neurons encode and store information that is required for memory recall, suggesting that they may be involved in formation of the memory trace. However, despite accumulating evidence for the role of IEGs in synaptic plasticity, the molecular and cellular mechanisms associated with this process remain unclear. In this review, we first summarize recent literature concerning the role of IEG-expressing neuronal ensembles in organizing the memory trace. We then focus on the physiological significance of IEGs, especially Arc, in synaptic plasticity, and describe our hypotheses about the importance of Arc expression in various types of input-specific circuit reorganization. Finally, we offer perspectives on Arc function that would unveil the role of IEG-expressing neurons in the formation of memory traces in the hippocampus and other brain areas. PMID:26778955

  14. Role of Immediate-Early Genes in Synaptic Plasticity and Neuronal Ensembles Underlying the Memory Trace.

    PubMed

    Minatohara, Keiichiro; Akiyoshi, Mika; Okuno, Hiroyuki

    2015-01-01

    In the brain, neuronal gene expression is dynamically changed in response to neuronal activity. In particular, the expression of immediate-early genes (IEGs) such as egr-1, c-fos, and Arc is rapidly and selectively upregulated in subsets of neurons in specific brain regions associated with learning and memory formation. IEG expression has therefore been widely used as a molecular marker for neuronal populations that undergo plastic changes underlying formation of long-term memory. In recent years, optogenetic and pharmacogenetic studies of neurons expressing c-fos or Arc have revealed that, during learning, IEG-positive neurons encode and store information that is required for memory recall, suggesting that they may be involved in formation of the memory trace. However, despite accumulating evidence for the role of IEGs in synaptic plasticity, the molecular and cellular mechanisms associated with this process remain unclear. In this review, we first summarize recent literature concerning the role of IEG-expressing neuronal ensembles in organizing the memory trace. We then focus on the physiological significance of IEGs, especially Arc, in synaptic plasticity, and describe our hypotheses about the importance of Arc expression in various types of input-specific circuit reorganization. Finally, we offer perspectives on Arc function that would unveil the role of IEG-expressing neurons in the formation of memory traces in the hippocampus and other brain areas. PMID:26778955

  15. Lateral Transfer of Genes and Gene Fragments in Staphylococcus Extends beyond Mobile Elements ▿ †

    PubMed Central

    Chan, Cheong Xin; Beiko, Robert G.; Ragan, Mark A.

    2011-01-01

    The widespread presence of antibiotic resistance and virulence among Staphylococcus isolates has been attributed in part to lateral genetic transfer (LGT), but little is known about the broader extent of LGT within this genus. Here we report the first systematic study of the modularity of genetic transfer among 13 Staphylococcus genomes covering four distinct named species. Using a topology-based phylogenetic approach, we found, among 1,354 sets of homologous genes examined, strong evidence of LGT in 368 (27.1%) gene sets, and weaker evidence in another 259 (19.1%). Within-gene and whole-gene transfer contribute almost equally to the topological discordance of these gene sets against a reference phylogeny. Comparing genetic transfer in single-copy and in multicopy gene sets, we observed a higher frequency of LGT in the latter, and a substantial functional bias in cases of whole-gene transfer (little such bias was observed in cases of fragmentary genetic transfer). We found evidence that lateral transfer, particularly of entire genes, impacts not only functions related to antibiotic, drug, and heavy-metal resistance, as well as membrane transport, but also core informational and metabolic functions not associated with mobile elements. Although patterns of sequence similarity support the cohesion of recognized species, LGT within S. aureus appears frequently to disrupt clonal complexes. Our results demonstrate that LGT and gene duplication play important parts in functional innovation in staphylococcal genomes. PMID:21622749

  16. Evidence for evolutionary divergence of activity-dependent gene expression in developing neurons

    PubMed Central

    Qiu, Jing; McQueen, Jamie; Bilican, Bilada; Dando, Owen; Magnani, Dario; Punovuori, Karolina; Selvaraj, Bhuvaneish T; Livesey, Matthew; Haghi, Ghazal; Heron, Samuel; Burr, Karen; Patani, Rickie; Rajan, Rinku; Sheppard, Olivia; Kind, Peter C; Simpson, T Ian; Tybulewicz, Victor LJ; Wyllie, David JA; Fisher, Elizabeth MC; Lowell, Sally; Chandran, Siddharthan; Hardingham, Giles E

    2016-01-01

    Evolutionary differences in gene regulation between humans and lower mammalian experimental systems are incompletely understood, a potential translational obstacle that is challenging to surmount in neurons, where primary tissue availability is poor. Rodent-based studies show that activity-dependent transcriptional programs mediate myriad functions in neuronal development, but the extent of their conservation in human neurons is unknown. We compared activity-dependent transcriptional responses in developing human stem cell-derived cortical neurons with those induced in developing primary- or stem cell-derived mouse cortical neurons. While activity-dependent gene-responsiveness showed little dependence on developmental stage or origin (primary tissue vs. stem cell), notable species-dependent differences were observed. Moreover, differential species-specific gene ortholog regulation was recapitulated in aneuploid mouse neurons carrying human chromosome-21, implicating promoter/enhancer sequence divergence as a factor, including human-specific activity-responsive AP-1 sites. These findings support the use of human neuronal systems for probing transcriptional responses to physiological stimuli or indeed pharmaceutical agents. DOI: http://dx.doi.org/10.7554/eLife.20337.001 PMID:27692071

  17. Duplication is more common among laterally transferred genes than among indigenous genes

    PubMed Central

    Hooper, Sean D; Berg, Otto G

    2003-01-01

    Background Recent developments in the understanding of paralogous evolution have prompted a focus not only on obviously advantageous genes, but also on genes that can be considered to have a weak or sporadic impact on the survival of the organism. Here we examine the duplicative behavior of a category of genes that can be considered to be mostly transient in the genome, namely laterally transferred genes. Using both a compositional method and a gene-tree approach, we identify a number of proposed laterally transferred genes and study their nucleotide composition and frequency of duplication. Results It is found that duplications are significantly overrepresented among potential laterally transferred genes compared to the indigenous ones. Furthermore, the GC3 distribution of potential laterally transferred genes was found to be largely uniform in some genomes, suggesting an import from a broad range of donors. Conclusions The results are discussed not in a context of strongly optimized established genes, but rather of genes with weak or ancillary functions. The importance of duplication may therefore depend on the variability and availability of weak genes for which novel functions may be discovered. Therefore, lateral transfer may accelerate the evolutionary process of duplication by bringing foreign genes that have mainly weak or no function into the genome. PMID:12914657

  18. Gene expression of muscular and neuronal pathways is cooperatively dysregulated in patients with idiopathic achalasia.

    PubMed

    Palmieri, Orazio; Mazza, Tommaso; Merla, Antonio; Fusilli, Caterina; Cuttitta, Antonello; Martino, Giuseppina; Latiano, Tiziana; Corritore, Giuseppe; Bossa, Fabrizio; Palumbo, Orazio; Muscarella, Lucia Anna; Carella, Massimo; Graziano, Paolo; Andriulli, Angelo; Latiano, Anna

    2016-01-01

    Idiopathic achalasia is characterized by the absence of peristalsis secondary to loss of neurons in the myenteric plexus that hampers proper relaxation of the lower esophageal sphincter. Achalasia can be considered a multifactorial disorder as it occurs in related individuals and is associated with HLA class II genes, thereby suggesting genetic influence. We used microarray technology and advanced in-silico functional analyses to perform the first genome-wide expression profiling of mRNA in tissue samples from 12 achalasia and 5 control patients. It revealed 1,728 differentially expressed genes, of these, 837 (48.4%) were up-regulated in cases. In particular, genes participating to the smooth muscle contraction biological function were mostly up-regulated. Functional analysis revealed a significant enrichment of neuronal/muscular and neuronal/immunity processes. Upstream regulatory analysis of 180 genes involved in these processes suggested TLR4 and IL18 as critical key-players. Two functional gene networks were significantly over-represented: one involved in organ morphology, skeletal muscle system development and function, and neurological diseases, and the other participating in cell morphology, humoral immune response and cellular movement. These results highlight on pivotal genes that may play critical roles in neuronal/muscular and neuronal/immunity processes, and that may contribute to the onset and development of achalasia. PMID:27511445

  19. Gene expression of muscular and neuronal pathways is cooperatively dysregulated in patients with idiopathic achalasia

    PubMed Central

    Palmieri, Orazio; Mazza, Tommaso; Merla, Antonio; Fusilli, Caterina; Cuttitta, Antonello; Martino, Giuseppina; Latiano, Tiziana; Corritore, Giuseppe; Bossa, Fabrizio; Palumbo, Orazio; Muscarella, Lucia Anna; Carella, Massimo; Graziano, Paolo; Andriulli, Angelo; Latiano, Anna

    2016-01-01

    Idiopathic achalasia is characterized by the absence of peristalsis secondary to loss of neurons in the myenteric plexus that hampers proper relaxation of the lower esophageal sphincter. Achalasia can be considered a multifactorial disorder as it occurs in related individuals and is associated with HLA class II genes, thereby suggesting genetic influence. We used microarray technology and advanced in-silico functional analyses to perform the first genome-wide expression profiling of mRNA in tissue samples from 12 achalasia and 5 control patients. It revealed 1,728 differentially expressed genes, of these, 837 (48.4%) were up-regulated in cases. In particular, genes participating to the smooth muscle contraction biological function were mostly up-regulated. Functional analysis revealed a significant enrichment of neuronal/muscular and neuronal/immunity processes. Upstream regulatory analysis of 180 genes involved in these processes suggested TLR4 and IL18 as critical key-players. Two functional gene networks were significantly over-represented: one involved in organ morphology, skeletal muscle system development and function, and neurological diseases, and the other participating in cell morphology, humoral immune response and cellular movement. These results highlight on pivotal genes that may play critical roles in neuronal/muscular and neuronal/immunity processes, and that may contribute to the onset and development of achalasia. PMID:27511445

  20. Physiological Transgene Regulation and Functional Complementation of a Neurological Disease Gene Deficiency in Neurons

    PubMed Central

    Peruzzi, Pier Paolo; Lawler, Sean E; Senior, Steve L; Dmitrieva, Nina; Edser, Pauline AH; Gianni, Davide; Chiocca, E Antonio; Wade-Martins, Richard

    2009-01-01

    The microtubule-associated protein tau (MAPT) and α-synuclein (SNCA) genes play central roles in neurodegenerative disorders. Mutations in each gene cause familial disease, whereas common genetic variation at both loci contributes to susceptibility to sporadic neurodegenerative disease. Here, we demonstrate exquisite gene regulation of the human MAPT and SNCA transgene loci and functional complementation in neuronal cell cultures and organotypic brain slices using the herpes simplex virus type 1 (HSV-1) amplicon-based infectious bacterial artificial chromosome (iBAC) vector to express complete loci >100 kb. Cell cultures transduced by iBAC vectors carrying a 143 kb MAPT or 135 kb SNCA locus expressed the human loci similar to the endogenous gene. We focused on analysis of the iBAC-MAPT vector carrying the complete MAPT locus. On transduction into neuronal cultures, multiple MAPT transcripts were expressed from iBAC-MAPT under strict developmental and cell type–specific control. In primary neurons from Mapt−/− mice, the iBAC-MAPT vector expressed the human tau protein, as detected by enzyme-linked immunosorbent assay and immunocytochemistry, and restored sensitivity of Mapt−/− neurons to Aβ peptide treatment in dissociated neuronal cultures and in organotypic slice cultures. The faithful retention of gene expression and phenotype complementation by the system provides a novel method to analyze neurological disease genes. PMID:19352323

  1. Using Expression Profiles of Caenorhabditis elegans Neurons To Identify Genes That Mediate Synaptic Connectivity

    PubMed Central

    Baruch, Leehod; Itzkovitz, Shalev; Golan-Mashiach, Michal; Shapiro, Ehud; Segal, Eran

    2008-01-01

    Synaptic wiring of neurons in Caenorhabditis elegans is largely invariable between animals. It has been suggested that this feature stems from genetically encoded molecular markers that guide the neurons in the final stage of synaptic formation. Identifying these markers and unraveling the logic by which they direct synapse formation is a key challenge. Here, we address this task by constructing a probabilistic model that attempts to explain the neuronal connectivity diagram of C. elegans as a function of the expression patterns of its neurons. By only considering neuron pairs that are known to be connected by chemical or electrical synapses, we focus on the final stage of synapse formation, in which neurons identify their designated partners. Our results show that for many neurons the neuronal expression map of C. elegans can be used to accurately predict the subset of adjacent neurons that will be chosen as its postsynaptic partners. Notably, these predictions can be achieved using the expression patterns of only a small number of specific genes that interact in a combinatorial fashion. PMID:18711638

  2. Progranulin Gene Delivery Protects Dopaminergic Neurons in a Mouse Model of Parkinson’s Disease

    PubMed Central

    Van Kampen, Jackalina M.; Baranowski, David; Kay, Denis G.

    2014-01-01

    Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by tremor, rigidity and akinesia/bradykinesia resulting from the progressive loss of nigrostriatal dopaminergic neurons. To date, only symptomatic treatment is available for PD patients, with no effective means of slowing or stopping the progression of the disease. Progranulin (PGRN) is a 593 amino acid multifunction protein that is widely distributed throughout the CNS, localized primarily in neurons and microglia. PGRN has been demonstrated to be a potent regulator of neuroinflammation and also acts as an autocrine neurotrophic factor, important for long-term neuronal survival. Thus, enhancing PGRN expression may strengthen the cells resistance to disease. In the present study, we have used the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD to investigate the possible use of PGRN gene delivery as a therapy for the prevention or treatment of PD. Viral vector delivery of the PGRN gene was an effective means of elevating PGRN expression in nigrostriatal neurons. When PGRN expression was elevated in the SNC, nigrostriatal neurons were protected from MPTP toxicity in mice, along with a preservation of striatal dopamine content and turnover. Further, protection of nigrostriatal neurons by PGRN gene therapy was accompanied by reductions in markers of MPTP-induced inflammation and apoptosis as well as a complete preservation of locomotor function. We conclude that PGRN gene therapy may have beneficial effects in the treatment of PD. PMID:24804730

  3. Horizontal functional gene transfer from bacteria to fishes

    PubMed Central

    Sun, Bao-Fa; Li, Tong; Xiao, Jin-Hua; Jia, Ling-Yi; Liu, Li; Zhang, Peng; Murphy, Robert W.; He, Shun-Min; Huang, Da-Wei

    2015-01-01

    Invertebrates can acquire functional genes via horizontal gene transfer (HGT) from bacteria but fishes are not known to do so. We provide the first reliable evidence of one HGT event from marine bacteria to fishes. The HGT appears to have occurred after emergence of the teleosts. The transferred gene is expressed and regulated developmentally. Its successful integration and expression may change the genetic and metabolic repertoire of fishes. In addition, this gene contains conserved domains and similar tertiary structures in fishes and their putative donor bacteria. Thus, it may function similarly in both groups. Evolutionary analyses indicate that it evolved under purifying selection, further indicating its conserved function. We document the first likely case of HGT of functional gene from prokaryote to fishes. This discovery certifies that HGT can influence vertebrate evolution. PMID:26691285

  4. Transferred interbacterial antagonism genes augment eukaryotic innate immune function.

    PubMed

    Chou, Seemay; Daugherty, Matthew D; Peterson, S Brook; Biboy, Jacob; Yang, Youyun; Jutras, Brandon L; Fritz-Laylin, Lillian K; Ferrin, Michael A; Harding, Brittany N; Jacobs-Wagner, Christine; Yang, X Frank; Vollmer, Waldemar; Malik, Harmit S; Mougous, Joseph D

    2015-02-01

    Horizontal gene transfer allows organisms to rapidly acquire adaptive traits. Although documented instances of horizontal gene transfer from bacteria to eukaryotes remain rare, bacteria represent a rich source of new functions potentially available for co-option. One benefit that genes of bacterial origin could provide to eukaryotes is the capacity to produce antibacterials, which have evolved in prokaryotes as the result of eons of interbacterial competition. The type VI secretion amidase effector (Tae) proteins are potent bacteriocidal enzymes that degrade the cell wall when delivered into competing bacterial cells by the type VI secretion system. Here we show that tae genes have been transferred to eukaryotes on at least six occasions, and that the resulting domesticated amidase effector (dae) genes have been preserved for hundreds of millions of years through purifying selection. We show that the dae genes acquired eukaryotic secretion signals, are expressed within recipient organisms, and encode active antibacterial toxins that possess substrate specificity matching extant Tae proteins of the same lineage. Finally, we show that a dae gene in the deer tick Ixodes scapularis limits proliferation of Borrelia burgdorferi, the aetiologic agent of Lyme disease. Our work demonstrates that a family of horizontally acquired toxins honed to mediate interbacterial antagonism confers previously undescribed antibacterial capacity to eukaryotes. We speculate that the selective pressure imposed by competition between bacteria has produced a reservoir of genes encoding diverse antimicrobial functions that are tailored for co-option by eukaryotic innate immune systems. PMID:25470067

  5. Immune Clearance of Attenuated Rabies Virus Results in Neuronal Survival with Altered Gene Expression

    PubMed Central

    Gomme, Emily A.; Wirblich, Christoph; Addya, Sankar; Rall, Glenn F.; Schnell, Matthias J.

    2012-01-01

    Rabies virus (RABV) is a highly neurotropic pathogen that typically leads to mortality of infected animals and humans. The precise etiology of rabies neuropathogenesis is unknown, though it is hypothesized to be due either to neuronal death or dysfunction. Analysis of human brains post-mortem reveals surprisingly little tissue damage and neuropathology considering the dramatic clinical symptomology, supporting the neuronal dysfunction model. However, whether or not neurons survive infection and clearance and, provided they do, whether they are functionally restored to their pre-infection phenotype has not been determined in vivo for RABV, or any neurotropic virus. This is due, in part, to the absence of a permanent “mark” on once-infected cells that allow their identification long after viral clearance. Our approach to study the survival and integrity of RABV-infected neurons was to infect Cre reporter mice with recombinant RABV expressing Cre-recombinase (RABV-Cre) to switch neurons constitutively expressing tdTomato (red) to expression of a Cre-inducible EGFP (green), permanently marking neurons that had been infected in vivo. We used fluorescence microscopy and quantitative real-time PCR to measure the survival of neurons after viral clearance; we found that the vast majority of RABV-infected neurons survive both infection and immunological clearance. We were able to isolate these previously infected neurons by flow cytometry and assay their gene expression profiles compared to uninfected cells. We observed transcriptional changes in these “cured” neurons, predictive of decreased neurite growth and dysregulated microtubule dynamics. This suggests that viral clearance, though allowing for survival of neurons, may not restore them to their pre-infection functionality. Our data provide a proof-of-principle foundation to re-evaluate the etiology of human central nervous system diseases of unknown etiology: viruses may trigger permanent neuronal damage that

  6. Neuron-specific splicing of the Alzheimer amyloid precursor protein gene in a mini-gene system.

    PubMed

    Yamada, T; Goto, I; Sakaki, Y

    1993-08-31

    Several forms of Alzheimer amyloid precursor protein (APP) mRNA are generated by alternative splicing. Among them, the APP695 mRNA skipping the exon 7 and 8 is expressed specifically in neurons, suggesting that this alternative splicing is regulated in a neuron-specific manner. As the first step for investigating the mechanism of the neuron-specific splicing, a mini-gene system was developed, in which mini-APP genes consisting of the exon 6, 7, 8, 9 and their flanking regions were introduced into neuronal and nonneuronal cultured cell lines to see their expression profiles. In the system the exon 7 and 8 of the mini-gene were significantly skipped in the neuronal cell, and the deletion study indicated that cis-acting elements for skipping the exons existed in the corresponding skipped-exon and its flanking regions. A small deletion upstream of the exon 8 suppressed the skipping of the exon 8 in the neuronal cell, suggesting that one of the regulatory sequence(s) for the exon skipping exists in a small region upstream of the skipped exon. PMID:8363619

  7. Shock wave induced sonoporation and gene transfer

    NASA Astrophysics Data System (ADS)

    Miller, Douglas L.

    2003-10-01

    During shockwave (SW) treatment, cavitation activity can be applied for cell killing. A bonus is that some surviving cells appear to be briefly permeabilized, or sonoporated, allowing them to take up large molecules including DNA. In vitro research has indicated that as the number of SW increased, survival declined exponentially but the number of sonoporated cells increased to better than 50% of survivors for 1000 SW. In vivo tests have demonstrated SW-induced tumor ablation could indeed be accompanied by the transfection of marker plasmids into mouse B16 melanoma tumors in vivo. With intratumor injection of plasmid DNA and air bubbles, significant results were obtained for only 400 SW. In a trial of cancer therapy, the effects of 500 SW combined with interleukin-12 immuno-gene therapy was observed on the progression of two mouse tumors, B16 melanoma and RENCA renal carcinoma. The combination of SW and IL-12 plasmid injection provided a statistically significant inhibition of tumor growth relative to SW alone for both tumor models, demonstrating feasibility for this treatment method. In the future, the development of intravenous gene delivery and improved transfection, together with image-guided ultrasound treatment, should lead to the clinical application of ultrasound enhanced gene therapy. [Work supported by NIH Grant No. EB002782.

  8. From Green to Red: Horizontal Gene Transfer of the Phycoerythrin Gene Cluster between Planktothrix Strains

    PubMed Central

    Sogge, Hanne; Rounge, Trine Ballestad; Nederbragt, Alexander J.; Lagesen, Karin; Glöckner, Gernot; Hayes, Paul K.; Rohrlack, Thomas

    2013-01-01

    Horizontal gene transfer is common in cyanobacteria, and transfer of large gene clusters may lead to acquisition of new functions and conceivably niche adaption. In the present study, we demonstrate that horizontal gene transfer between closely related Planktothrix strains can explain the production of the same oligopeptide isoforms by strains of different colors. Comparison of the genomes of eight Planktothrix strains revealed that strains producing the same oligopeptide isoforms are closely related, regardless of color. We have investigated genes involved in the synthesis of the photosynthetic pigments phycocyanin and phycoerythrin, which are responsible for green and red appearance, respectively. Sequence comparisons suggest the transfer of a functional phycoerythrin gene cluster generating a red phenotype in a strain that is otherwise more closely related to green strains. Our data show that the insertion of a DNA fragment containing the 19.7-kb phycoerythrin gene cluster has been facilitated by homologous recombination, also replacing a region of the phycocyanin operon. These findings demonstrate that large DNA fragments spanning entire functional gene clusters can be effectively transferred between closely related cyanobacterial strains and result in a changed phenotype. Further, the results shed new light on the discussion of the role of horizontal gene transfer in the sporadic distribution of large gene clusters in cyanobacteria, as well as the appearance of red and green strains. PMID:23995927

  9. [Gene doping: gene transfer and possible molecular detection].

    PubMed

    Argüelles, Carlos Francisco; Hernández-Zamora, Edgar

    2007-01-01

    The use of illegal substances in sports to enhance athletic performance during competition has caused international sports organizations such as the COI and WADA to take anti doping measures. A new doping method know as gene doping is defined as "the non-therapeutic use of genes, genetic elements and/or cells that have the capacity to enhance athletic performance". However, gene doping in sports is not easily identified and can cause serious consequences. Molecular biology techniques are needed in order to distinguish the difference between a "normal" and an "altered" genome. Further, we need to develop new analytic methods and biological molecular techniques in anti-doping laboratories, and design programs that avoid the non therapeutic use of genes.

  10. Recent events dominate interdomain lateral gene transfers between prokaryotes and eukaryotes and, with the exception of endosymbiotic gene transfers, few ancient transfer events persist.

    PubMed

    Katz, Laura A

    2015-09-26

    While there is compelling evidence for the impact of endosymbiotic gene transfer (EGT; transfer from either mitochondrion or chloroplast to the nucleus) on genome evolution in eukaryotes, the role of interdomain transfer from bacteria and/or archaea (i.e. prokaryotes) is less clear. Lateral gene transfers (LGTs) have been argued to be potential sources of phylogenetic information, particularly for reconstructing deep nodes that are difficult to recover with traditional phylogenetic methods. We sought to identify interdomain LGTs by using a phylogenomic pipeline that generated 13 465 single gene trees and included up to 487 eukaryotes, 303 bacteria and 118 archaea. Our goals include searching for LGTs that unite major eukaryotic clades, and describing the relative contributions of LGT and EGT across the eukaryotic tree of life. Given the difficulties in interpreting single gene trees that aim to capture the approximately 1.8 billion years of eukaryotic evolution, we focus on presence-absence data to identify interdomain transfer events. Specifically, we identify 1138 genes found only in prokaryotes and representatives of three or fewer major clades of eukaryotes (e.g. Amoebozoa, Archaeplastida, Excavata, Opisthokonta, SAR and orphan lineages). The majority of these genes have phylogenetic patterns that are consistent with recent interdomain LGTs and, with the notable exception of EGTs involving photosynthetic eukaryotes, we detect few ancient interdomain LGTs. These analyses suggest that LGTs have probably occurred throughout the history of eukaryotes, but that ancient events are not maintained unless they are associated with endosymbiotic gene transfer among photosynthetic lineages.

  11. Recent events dominate interdomain lateral gene transfers between prokaryotes and eukaryotes and, with the exception of endosymbiotic gene transfers, few ancient transfer events persist

    PubMed Central

    Katz, Laura A.

    2015-01-01

    While there is compelling evidence for the impact of endosymbiotic gene transfer (EGT; transfer from either mitochondrion or chloroplast to the nucleus) on genome evolution in eukaryotes, the role of interdomain transfer from bacteria and/or archaea (i.e. prokaryotes) is less clear. Lateral gene transfers (LGTs) have been argued to be potential sources of phylogenetic information, particularly for reconstructing deep nodes that are difficult to recover with traditional phylogenetic methods. We sought to identify interdomain LGTs by using a phylogenomic pipeline that generated 13 465 single gene trees and included up to 487 eukaryotes, 303 bacteria and 118 archaea. Our goals include searching for LGTs that unite major eukaryotic clades, and describing the relative contributions of LGT and EGT across the eukaryotic tree of life. Given the difficulties in interpreting single gene trees that aim to capture the approximately 1.8 billion years of eukaryotic evolution, we focus on presence–absence data to identify interdomain transfer events. Specifically, we identify 1138 genes found only in prokaryotes and representatives of three or fewer major clades of eukaryotes (e.g. Amoebozoa, Archaeplastida, Excavata, Opisthokonta, SAR and orphan lineages). The majority of these genes have phylogenetic patterns that are consistent with recent interdomain LGTs and, with the notable exception of EGTs involving photosynthetic eukaryotes, we detect few ancient interdomain LGTs. These analyses suggest that LGTs have probably occurred throughout the history of eukaryotes, but that ancient events are not maintained unless they are associated with endosymbiotic gene transfer among photosynthetic lineages. PMID:26323756

  12. Gene Therapy: The Potential Applicability of Gene Transfer Technology to the Human Germline

    PubMed Central

    2004-01-01

    The theoretical possibility of applying gene transfer methodologies to the human germline is explored. Transgenic methods for genetically manipulating embryos may in principle be applied to humans. In particular, microinjection of retroviral vector appears to hold the greatest promise, with transgenic primates already obtained from this approach. Sperm-mediated gene transfer offers potentially the easiest route to the human germline, however the requisite methodology is presently underdeveloped. Nuclear transfer (cloning) offers an alternative approach to germline genetic modification, however there are major health concerns associated with current nuclear transfer methods. It is concluded that human germline gene therapy remains for all practical purposes a future possibility that must await significant and important advances in gene transfer technology. PMID:15912200

  13. Topoisomerase 1 Regulates Gene Expression in Neurons through Cleavage Complex-Dependent and -Independent Mechanisms

    PubMed Central

    Mabb, Angela M.; Simon, Jeremy M.; King, Ian F.; Lee, Hyeong-Min; An, Lin-Kun; Philpot, Benjamin D.; Zylka, Mark J.

    2016-01-01

    Topoisomerase 1 (TOP1) inhibitors, including camptothecin and topotecan, covalently trap TOP1 on DNA, creating cleavage complexes (cc’s) that must be resolved before gene transcription and DNA replication can proceed. We previously found that topotecan reduces the expression of long (>100 kb) genes and unsilences the paternal allele of Ube3a in neurons. Here, we sought to evaluate overlap between TOP1cc-dependent and -independent gene regulation in neurons. To do this, we utilized Top1 conditional knockout mice, Top1 knockdown, the CRISPR-Cas9 system to delete Top1, TOP1 catalytic inhibitors that do not generate TOP1cc’s, and a TOP1 mutation (T718A) that stabilizes TOP1cc’s. We found that topotecan treatment significantly alters the expression of many more genes, including long neuronal genes, immediate early genes, and paternal Ube3a, when compared to Top1 deletion. Our data show that topotecan has a stronger effect on neuronal transcription than Top1 deletion, and identifies TOP1cc-dependent and -independent contributions to gene expression. PMID:27231886

  14. A Gene in the Process of Endosymbiotic Transfer

    PubMed Central

    Jiroutová, Kateřina; Kořený, Luděk; Bowler, Chris; Oborník, Miroslav

    2010-01-01

    Background The endosymbiotic birth of organelles is accompanied by massive transfer of endosymbiont genes to the eukaryotic host nucleus. In the centric diatom Thalassiosira pseudonana the Psb28 protein is encoded in the plastid genome while a second version is nuclear-encoded and possesses a bipartite N-terminal presequence necessary to target the protein into the diatom complex plastid. Thus it can represent a gene captured during endosymbiotic gene transfer. Methodology/Principal Findings To specify the origin of nuclear- and plastid-encoded Psb28 in T. pseudonana we have performed extensive phylogenetic analyses of both mentioned genes. We have also experimentally tested the intracellular location of the nuclear-encoded Psb28 protein (nuPsb28) through transformation of the diatom Phaeodactylum tricornutum with the gene in question fused to EYFP. Conclusions/Significance We show here that both versions of the psb28 gene in T. pseudonana are transcribed. We also provide experimental evidence for successful targeting of the nuPsb28 fused with EYFP to the diatom complex plastid. Extensive phylogenetic analyses demonstrate that nucleotide composition of the analyzed genes deeply influences the tree topology and that appropriate methods designed to deal with a compositional bias of the sequences and the long branch attraction artefact (LBA) need to be used to overcome this obstacle. We propose that nuclear psb28 in T. pseudonana is a duplicate of a plastid localized version, and that it has been transferred from its endosymbiont. PMID:20949086

  15. Evolution of gene fusions: horizontal transfer versus independent events

    PubMed Central

    Yanai, Itai; Wolf, Yuri I; Koonin, Eugene V

    2002-01-01

    Background Gene fusions can be used as tools for functional prediction and also as evolutionary markers. Fused genes often show a scattered phyletic distribution, which suggests a role for processes other than vertical inheritance in their evolution. Results The evolutionary history of gene fusions was studied by phylogenetic analysis of the domains in the fused proteins and the orthologous domains that form stand-alone proteins. Clustering of fusion components from phylogenetically distant species was construed as evidence of dissemination of the fused genes by horizontal transfer. Of the 51 examined gene fusions that are represented in at least two of the three primary kingdoms (Bacteria, Archaea and Eukaryota), 31 were most probably disseminated by cross-kingdom horizontal gene transfer, whereas 14 appeared to have evolved independently in different kingdoms and two were probably inherited from the common ancestor of modern life forms. On many occasions, the evolutionary scenario also involves one or more secondary fissions of the fusion gene. For approximately half of the fusions, stand-alone forms of the fusion components are encoded by juxtaposed genes, which are known or predicted to belong to the same operon in some of the prokaryotic genomes. This indicates that evolution of gene fusions often, if not always, involves an intermediate stage, during which the future fusion components exist as juxtaposed and co-regulated, but still distinct, genes within operons. Conclusion These findings suggest a major role for horizontal transfer of gene fusions in the evolution of protein-domain architectures, but also indicate that independent fusions of the same pair of domains in distant species is not uncommon, which suggests positive selection for the multidomain architectures. PMID:12049665

  16. Important aspects of placental-specific gene transfer.

    PubMed

    Kaufman, Melissa R; Albers, Renee E; Keoni, Chanel; Kulkarni-Datar, Kashmira; Natale, David R; Brown, Thomas L

    2014-10-15

    The placenta is a unique and highly complex organ that develops only during pregnancy and is essential for growth and survival of the developing fetus. The placenta provides the vital exchange of gases and wastes, the necessary nutrients for fetal development, acts as immune barrier that protects against maternal rejection, and produces numerous hormones and growth factors that promote fetal maturity to regulate pregnancy until parturition. Abnormal placental development is a major underlying cause of pregnancy-associated disorders that often result in preterm birth. Defects in placental stem cell propagation, growth, and differentiation are the major factors that affect embryonic and fetal well-being and dramatically increase the risk of pregnancy complications. Understanding the processes that regulate placentation is important in determining the underlying factors behind abnormal placental development. The ability to manipulate genes in a placenta-specific manner provides a unique tool to analyze development and eliminates potentially confounding results that can occur with traditional gene knockouts. Trophoblast stem cells and mouse embryos are not overly amenable to traditional gene transfer techniques. Most viral vectors, however, have a low infection rate and often lead to mosaic transgenesis. Although the traditional method of embryo transfer is intrauterine surgical implantation, the methodology reported here, combining lentiviral blastocyst infection and nonsurgical embryo transfer, leads to highly efficient and placental-specific gene transfer. Numerous advantages of our optimized procedures include increased investigator safety, a reduction in animal stress, rapid and noninvasive embryo transfer, and higher a rate of pregnancy and live birth.

  17. Bacteriophage-associated gene transfer in pneumococcus: transduction or pseudotransduction?

    PubMed Central

    Porter, R D; Shoemaker, N B; Rampe, G; Guild, W R

    1979-01-01

    Lysates of pneumococcal phage PG24 transferred genes from one host to another in a process with many of the properties of generalized transduction, in that the host genes were packaged in DNase-resistant particles that closely resembled infectious phage in physical properties, adsorbed to the recipient cells like phage, and were inhibited by antisera to the phage and by trypsin. However, phage processes did not complete the transfer of host DNA as they did phage DNA. Instead, gene transfer required development of competence and entry of the host DNA by the endonuclease-dependent pathway used for transforming and transfecting DNA. This process often occurred on the assay plate hours after adsorption of the particles to the cells, and the transfer was DNase sensitive if challenged at this time. Phenotypic expression was therefore also delayed. The product of entry was like that in transformation, a single strand of DNA that integrates by formation of a hex-sensitive donor-recipient heteroduplex. Whether this gene transfer process is unique to this system or is only the first one described is not clear. The term "pseudotransduction" may be useful in calling attention to its unexpected features. The DNA of PG24 phage has anomalous physical properties reflecting unusual bases. Images PMID:33154

  18. The interconnection between biofilm formation and horizontal gene transfer.

    PubMed

    Madsen, Jonas Stenløkke; Burmølle, Mette; Hansen, Lars Hestbjerg; Sørensen, Søren Johannes

    2012-07-01

    Recent research has revealed that horizontal gene transfer and biofilm formation are connected processes. Although published research investigating this interconnectedness is still limited, we will review this subject in order to highlight the potential of these observations because of their believed importance in the understanding of the adaptation and subsequent evolution of social traits in bacteria. Here, we discuss current evidence for such interconnectedness centred on plasmids. Horizontal transfer rates are typically higher in biofilm communities compared with those in planktonic states. Biofilms, furthermore, promote plasmid stability and may enhance the host range of mobile genetic elements that are transferred horizontally. Plasmids, on the other hand, are very well suited to promote the evolution of social traits such as biofilm formation. This, essentially, transpires because plasmids are independent replicons that enhance their own success by promoting inter-bacterial interactions. They typically also carry genes that heighten their hosts' direct fitness. Furthermore, current research shows that the so-called mafia traits encoded on mobile genetic elements can enforce bacteria to maintain stable social interactions. It also indicates that horizontal gene transfer ultimately enhances the relatedness of bacteria carrying the mobile genetic elements of the same origin. The perspective of this review extends to an overall interconnectedness between horizontal gene transfer, mobile genetic elements and social evolution of bacteria.

  19. Gene transfer strategies in animal transgenesis.

    PubMed

    Montoliu, Lluís

    2002-01-01

    Position effects in animal transgenesis have prevented the reproducible success and limited the initial expectations of this technique in many biotechnological projects. Historically, several strategies have been devised to overcome such position effects, including the progressive addition of regulatory elements belonging to the same or to a heterologous expression domain. An expression domain is thought to contain all regulatory elements that are needed to specifically control the expression of a given gene in time and space. The lack of profound knowledge on the chromatin structure of expression domains of biotechnological interest, such as mammary gland-specific genes, explains why most standard expression vectors have failed to drive high-level, position-independent, and copy-number-dependent expression of transgenes in a reproducible manner. In contrast, the application of artificial chromosome-type constructs to animal transgenesis usually ensures optimal expression levels. YACs, BACs, and PACs have become crucial tools in animal transgenesis, allowing the inclusion of distant key regulatory sequences, previously unknown, that are characteristic for each expression domain. These elements contribute to insulating the artificial chromosome-type constructs from chromosomal position effects and are fundamental in order to guarantee the correct expression of transgenes.

  20. Characterization of claustral neurons by comparative gene expression profiling and dye-injection analyses

    PubMed Central

    Watakabe, Akiya; Ohsawa, Sonoko; Ichinohe, Noritaka; Rockland, Kathleen S.; Yamamori, Tetsuo

    2014-01-01

    The identity of the claustrum as a part of cerebral cortex, and in particular of the adjacent insular cortex, has been investigated by connectivity features and patterns of gene expression. In the present paper, we mapped the cortical and claustral expression of several cortical genes in rodent and macaque monkey brains (nurr1, latexin, cux2, and netrinG2) to further assess shared features between cortex and claustrum. In mice, these genes were densely expressed in the claustrum, but very sparsely in the cortex and not present in the striatum. To test whether the cortical vs. claustral cell types can be distinguished by co-expression of these genes, we performed a panel of double ISH in mouse and macaque brain. NetrinG2 and nurr1 genes were co-expressed across entire cortex and claustrum, but cux2 and nurr1 were co-expressed only in the insular cortex and claustrum. Latexin was expressed, in the macaque, only in the claustrum. The nurr1+ claustral neurons expressed VGluT1, a marker for cortical glutamatergic cells and send cortical projections. Taken together, our data suggest a partial commonality between claustral neurons and a subtype of cortical neurons in the monkey brain. Moreover, in the embryonic (E110) macaque brain, many nurr1+ neurons were scattered in the white matter between the claustrum and the insular cortex, possibly representing their migratory history. In a second set of experiments, we injected Lucifer Yellow intracellularly in mouse and rat slices to investigate whether dendrites of insular and claustral neurons can cross the border of the two brain regions. Dendrites of claustral neurons did not invade the overlying insular territory. In summary, gene expression profile of the claustrum is similar to that of the neocortex, in both rodent and macaque brains, but with modifications in density of expression and cellular co-localization of specific genes. PMID:24904319

  1. The multiple sclerosis drug fingolimod (FTY720) stimulates neuronal gene expression, axonal growth and regeneration.

    PubMed

    Anastasiadou, Sofia; Knöll, Bernd

    2016-05-01

    Fingolimod (FTY720) is a new generation oral treatment for multiple sclerosis (MS). So far, FTY720 was mainly considered to target trafficking of immune cells but not brain cells such as neurons. Herein, we analyzed FTY720's potential to directly alter neuronal function. In CNS neurons, we identified a FTY720 governed gene expression response. FTY720 upregulated immediate early genes (IEGs) encoding for neuronal activity associated transcription factors such as c-Fos, FosB, Egr1 and Egr2 and induced actin cytoskeleton associated genes (actin isoforms, tropomyosin, calponin). Stimulation of primary neurons with FTY720 enhanced neurite growth and altered growth cone morphology. In accordance, FTY720 enhanced axon regeneration in mice upon facial nerve axotomy. We identified components of a FTY720 engaged signaling cascade including S1P receptors, G12/13G-proteins, RhoA-GTPases and the transcription factors SRF/MRTF. In summary, we uncovered a broader cellular and therapeutic operation mode of FTY720, suggesting beneficial FTY720 effects also on CNS neurons during MS therapy and for treatment of other neurodegenerative diseases requiring neuroprotective and neurorestorative processes. PMID:26980486

  2. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain

    PubMed Central

    Deverman, Benjamin E.; Pravdo, Piers L.; Simpson, Bryan P.; Kumar, Sripriya Ravindra; Chan, Ken Y.; Banerjee, Abhik; Wu, Wei-Li; Yang, Bin; Huber, Nina; Pasca, Sergiu P.; Gradinaru, Viviana

    2015-01-01

    Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer1-6. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics7-13. Here we describe a capsid selection method, called Cre-recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV914-17, and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications. PMID:26829320

  3. Antibacterial gene transfer across the tree of life

    PubMed Central

    Metcalf, Jason A; Funkhouser-Jones, Lisa J; Brileya, Kristen; Reysenbach, Anna-Louise; Bordenstein, Seth R

    2014-01-01

    Though horizontal gene transfer (HGT) is widespread, genes and taxa experience biased rates of transferability. Curiously, independent transmission of homologous DNA to archaea, bacteria, eukaryotes, and viruses is extremely rare and often defies ecological and functional explanations. Here, we demonstrate that a bacterial lysozyme family integrated independently in all domains of life across diverse environments, generating the only glycosyl hydrolase 25 muramidases in plants and archaea. During coculture of a hydrothermal vent archaeon with a bacterial competitor, muramidase transcription is upregulated. Moreover, recombinant lysozyme exhibits broad-spectrum antibacterial action in a dose-dependent manner. Similar to bacterial transfer of antibiotic resistance genes, transfer of a potent antibacterial gene across the universal tree seemingly bestows a niche-transcending adaptation that trumps the barriers against parallel HGT to all domains. The discoveries also comprise the first characterization of an antibacterial gene in archaea and support the pursuit of antibiotics in this underexplored group. DOI: http://dx.doi.org/10.7554/eLife.04266.001 PMID:25422936

  4. Development and morphology of the clock-gene-expressing lateral neurons of Drosophila melanogaster.

    PubMed

    Helfrich-Förster, Charlotte; Shafer, Orie T; Wülbeck, Corinna; Grieshaber, Eva; Rieger, Dirk; Taghert, Paul

    2007-01-01

    The clock-gene-expressing lateral neurons are essential for the locomotor activity rhythm of Drosophila melanogaster. Traditionally, these neurons are divided into three groups: the dorsal lateral neurons (LN(d)), the large ventral lateral neurons (l-LN(v)), and the small ventral lateral neurons (s-LN(v)), whereby the latter group consists of four neurons that express the neuropeptide pigment-dispersing factor (PDF) and a fifth PDF-negative neuron. So far, only the l-LN(v) and the PDF-positive s-LN(v) have been shown to project into the accessory medulla, a small neuropil that contains the circadian pacemaker center in several insects. We show here that the other lateral neurons also arborize in the accessory medulla, predominantly forming postsynaptic sites. Both the l-LN(v) and LN(d) are anatomically well suited to connect the accessory medullae. Whereas the l-LN(v) may receive ipsilateral photic input from the Hofbauer-Buchner eyelet, the LN(d) invade mainly the contralateral accessory medulla and thus may receive photic input from the contralateral side. Both the LN(d) and the l-LN(v) differentiate during midmetamorphosis. They do so in close proximity to one another and the fifth PDF-negative s-LN(v), suggesting that these cell groups may derive from common precursors. PMID:17099895

  5. Functionalized organic nanotubes as tubular nonviral gene transfer vector.

    PubMed

    Ding, Wuxiao; Wada, Momoyo; Kameta, Naohiro; Minamikawa, Hiroyuki; Shimizu, Toshimi; Masuda, Mitsutoshi

    2011-11-30

    Tubular nanomaterials are expected to represent a novel nonviral gene transfer vectors due to the unique morphology and potential biological functionalities. Here we rationally constructed functionalized organic nanotubes (ONTs) for gene delivery through the co-assembly of bipolar glycolipid, arginine-lipid and PEG-lipid. The arginine- and PEG-functionalized ONTs efficiently formed complexes with plasmid DNA without aggregation, and protect DNA from enzymatic degradation; while the arginine-functionalized ONTs aggregated with DNA as large bundles. Long ONTs exceeding 1μm in length was rarely taken up into the cells, while those with a length of 400-800nm could effectively deliver plasmid DNA into cells and induce high transgene expression of green fluorescense protein. This study demonstrated the usefulness of functionalized ONT in gene delivery, and the functionalized ONT represents a novel type of tubular nonviral gene transfer vector.

  6. A short upstream promoter region mediates transcriptional regulation of the mouse doublecortin gene in differentiating neurons

    PubMed Central

    2010-01-01

    Background Doublecortin (Dcx), a MAP (Microtubule-Associated Protein), is transiently expressed in migrating and differentiating neurons and thereby characterizes neuronal precursors and neurogenesis in developing and adult neurogenesis. In addition, reduced Dcx expression during development has been related to appearance of brain pathologies. Here, we attempt to unveil the molecular mechanisms controlling Dcx gene expression by studying its transcriptional regulation during neuronal differentiation. Results To determine and analyze important regulatory sequences of the Dcx promoter, we studied a putative regulatory region upstream from the mouse Dcx coding region (pdcx2kb) and several deletions thereof. These different fragments were used in vitro and in vivo to drive reporter gene expression. We demonstrated, using transient expression experiments, that pdcx2kb is sufficient to control specific reporter gene expression in cerebellar cells and in the developing brain (E14.5). We determined the temporal profile of Dcx promoter activity during neuronal differentiation of mouse embryonic stem cells (mESC) and found that transcriptional activation of the Dcx gene varies along with neuronal differentiation of mESC. Deletion experiments and sequence comparison of Dcx promoters across rodents, human and chicken revealed the importance of a highly conserved sequence in the proximal region of the promoter required for specific and strong expression in neuronal precursors and young neuronal cells. Further analyses revealed the presence in this short sequence of several conserved, putative transcription factor binding sites: LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor) which are effectors of the canonical Wnt pathway; HNF6/OC2 (Hepatocyte Nuclear Factor-6/Oncecut-2) members of the ONECUT family and NF-Y/CAAT (Nuclear Factor-Y). Conclusions Studies of Dcx gene regulatory sequences using native, deleted and mutated constructs suggest that fragments located upstream of the

  7. Gene transfer techniques in whole embryo cultured post-implantation mouse embryos.

    PubMed

    Sakai, Daisuke; Trainor, Paul A

    2014-01-01

    Gene transfer techniques such as electroporation and lipofection are powerful systems for investigating gene function. In this chapter we focus on the methods and applications of gene transfer into specific cells and tissues of post-implantation mouse embryos.

  8. Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes

    PubMed Central

    Huddleston, Jennifer R

    2014-01-01

    Bacterial infections are becoming increasingly difficult to treat due to widespread antibiotic resistance among pathogens. This review aims to give an overview of the major horizontal transfer mechanisms and their evolution and then demonstrate the human lower gastrointestinal tract as an environment in which horizontal gene transfer of resistance determinants occurs. Finally, implications for antibiotic usage and the development of resistant infections and persistence of antibiotic resistance genes in populations as a result of horizontal gene transfer in the large intestine will be discussed. PMID:25018641

  9. Replacing and Additive Horizontal Gene Transfer in Streptococcus

    PubMed Central

    Choi, Sang Chul; Rasmussen, Matthew D.; Hubisz, Melissa J.; Gronau, Ilan; Stanhope, Michael J.; Siepel, Adam

    2012-01-01

    The prominent role of Horizontal Gene Transfer (HGT) in the evolution of bacteria is now well documented, but few studies have differentiated between evolutionary events that predominantly cause genes in one lineage to be replaced by homologs from another lineage (“replacing HGT”) and events that result in the addition of substantial new genomic material (“additive HGT”). Here in, we make use of the distinct phylogenetic signatures of replacing and additive HGTs in a genome-wide study of the important human pathogen Streptococcus pyogenes (SPY) and its close relatives S. dysgalactiae subspecies equisimilis (SDE) and S. dysgalactiae subspecies dysgalactiae (SDD). Using recently developed statistical models and computational methods, we find evidence for abundant gene flow of both kinds within each of the SPY and SDE clades and of reduced levels of exchange between SPY and SDD. In addition, our analysis strongly supports a pronounced asymmetry in SPY–SDE gene flow, favoring the SPY-to-SDE direction. This finding is of particular interest in light of the recent increase in virulence of pathogenic SDE. We find much stronger evidence for SPY–SDE gene flow among replacing than among additive transfers, suggesting a primary influence from homologous recombination between co-occurring SPY and SDE cells in human hosts. Putative virulence genes are correlated with transfer events, but this correlation is found to be driven by additive, not replacing, HGTs. The genes affected by additive HGTs are enriched for functions having to do with transposition, recombination, and DNA integration, consistent with previous findings, whereas replacing HGTs seen to influence a more diverse set of genes. Additive transfers are also found to be associated with evidence of positive selection. These findings shed new light on the manner in which HGT has shaped pathogenic bacterial genomes. PMID:22617954

  10. Horizontal gene transfer in eukaryotes: the weak-link model.

    PubMed

    Huang, Jinling

    2013-10-01

    The significance of horizontal gene transfer (HGT) in eukaryotic evolution remains controversial. Although many eukaryotic genes are of bacterial origin, they are often interpreted as being derived from mitochondria or plastids. Because of their fixed gene pool and gene loss, however, mitochondria and plastids alone cannot adequately explain the presence of all, or even the majority, of bacterial genes in eukaryotes. Available data indicate that no insurmountable barrier to HGT exists, even in complex multicellular eukaryotes. In addition, the discovery of both recent and ancient HGT events in all major eukaryotic groups suggests that HGT has been a regular occurrence throughout the history of eukaryotic evolution. A model of HGT is proposed that suggests both unicellular and early developmental stages as likely entry points for foreign genes into multicellular eukaryotes.

  11. Estimating the Frequency of Horizontal Gene Transfer Using Phylogenetic Models of Gene Gain and Loss.

    PubMed

    Zamani-Dahaj, Seyed Alireza; Okasha, Mohamed; Kosakowski, Jakub; Higgs, Paul G

    2016-07-01

    We analyze patterns of gene presence and absence in a maximum likelihood framework with rate parameters for gene gain and loss. Standard methods allow independent gains and losses in different parts of a tree. While losses of the same gene are likely to be frequent, multiple gains need to be considered carefully. A gene gain could occur by horizontal transfer or by origin of a gene within the lineage being studied. If a gene is gained more than once, then at least one of these gains must be a horizontal transfer. A key parameter is the ratio of gain to loss rates, a/v We consider the limiting case known as the infinitely many genes model, where a/v tends to zero and a gene cannot be gained more than once. The infinitely many genes model is used as a null model in comparison to models that allow multiple gains. Using genome data from cyanobacteria and archaea, it is found that the likelihood is significantly improved by allowing for multiple gains, but the average a/v is very small. The fraction of genes whose presence/absence pattern is best explained by multiple gains is only 15% in the cyanobacteria and 20% and 39% in two data sets of archaea. The distribution of rates of gene loss is very broad, which explains why many genes follow a treelike pattern of vertical inheritance, despite the presence of a significant minority of genes that undergo horizontal transfer.

  12. The core clock gene Per1 phases molecular and electrical circadian rhythms in SCN neurons

    PubMed Central

    Jones, Jeff R.

    2016-01-01

    The brain’s biological clock, the suprachiasmatic nucleus (SCN), exhibits endogenous 24-hour rhythms in gene expression and spontaneous firing rate; however, the functional relationship between these neuronal rhythms is not fully understood. Here, we used a Per1::GFP transgenic mouse line that allows for the simultaneous quantification of molecular clock state and firing rate in SCN neurons to examine the relationship between these key components of the circadian clock. We find that there is a stable, phased relationship between E-box-driven clock gene expression and spontaneous firing rate in SCN neurons and that these relationships are independent of light input onto the system or of GABAA receptor-mediated synaptic activity. Importantly, the concordant phasing of gene and neural rhythms is disrupted in the absence of the homologous clock gene Per1, but persists in the absence of the core clock gene Per2. These results suggest that Per1 plays a unique, non-redundant role in phasing gene expression and firing rate rhythms in SCN neurons to increase the robustness of cellular timekeeping. PMID:27602274

  13. The core clock gene Per1 phases molecular and electrical circadian rhythms in SCN neurons.

    PubMed

    Jones, Jeff R; McMahon, Douglas G

    2016-01-01

    The brain's biological clock, the suprachiasmatic nucleus (SCN), exhibits endogenous 24-hour rhythms in gene expression and spontaneous firing rate; however, the functional relationship between these neuronal rhythms is not fully understood. Here, we used a Per1::GFP transgenic mouse line that allows for the simultaneous quantification of molecular clock state and firing rate in SCN neurons to examine the relationship between these key components of the circadian clock. We find that there is a stable, phased relationship between E-box-driven clock gene expression and spontaneous firing rate in SCN neurons and that these relationships are independent of light input onto the system or of GABAA receptor-mediated synaptic activity. Importantly, the concordant phasing of gene and neural rhythms is disrupted in the absence of the homologous clock gene Per1, but persists in the absence of the core clock gene Per2. These results suggest that Per1 plays a unique, non-redundant role in phasing gene expression and firing rate rhythms in SCN neurons to increase the robustness of cellular timekeeping. PMID:27602274

  14. The core clock gene Per1 phases molecular and electrical circadian rhythms in SCN neurons

    PubMed Central

    Jones, Jeff R.

    2016-01-01

    The brain’s biological clock, the suprachiasmatic nucleus (SCN), exhibits endogenous 24-hour rhythms in gene expression and spontaneous firing rate; however, the functional relationship between these neuronal rhythms is not fully understood. Here, we used a Per1::GFP transgenic mouse line that allows for the simultaneous quantification of molecular clock state and firing rate in SCN neurons to examine the relationship between these key components of the circadian clock. We find that there is a stable, phased relationship between E-box-driven clock gene expression and spontaneous firing rate in SCN neurons and that these relationships are independent of light input onto the system or of GABAA receptor-mediated synaptic activity. Importantly, the concordant phasing of gene and neural rhythms is disrupted in the absence of the homologous clock gene Per1, but persists in the absence of the core clock gene Per2. These results suggest that Per1 plays a unique, non-redundant role in phasing gene expression and firing rate rhythms in SCN neurons to increase the robustness of cellular timekeeping.

  15. Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment.

    PubMed

    Jacobs, William R

    2014-04-01

    Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents. These remarkable successes in the control of infections came from knowing the causative agents of the diseases, followed by serendipitous discoveries of attenuated viruses and antibiotics. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids-chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages-was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis research.

  16. Gene Transfer in Mycobacterium tuberculosis: Shuttle Phasmids to Enlightenment

    PubMed Central

    JACOBS, WILLIAM R.

    2016-01-01

    Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents. These remarkable successes in the control of infections came from knowing the causative agents of the diseases, followed by serendipitous discoveries of attenuated viruses and antibiotics. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids—chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages—was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis research. “I had to know my enemy in order to prevail against him.”Nelson Mandela PMID:26105819

  17. Connexin Gene Transfer Preserves Conduction Velocity and Prevents Atrial Fibrillation

    PubMed Central

    Igarashi, Tomonori; Finet, J. Emanuel; Takeuchi, Ayano; Fujino, Yoshihisa; Strom, Maria; Greener, Ian D.; Rosenbaum, David S.; Donahue, J. Kevin

    2011-01-01

    Background Several lines of evidence have suggested that maintenance of atrial fibrillation (AF) depends on reentrant mechanisms. Maintenance of reentry necessitates a sufficiently short refractory period and/or delayed conduction, and AF has been associated with both alterations. Fibrosis, cellular dysfunction and gap junction protein alterations occur in AF and cause conduction delay. We performed this study to test the hypothesis that gap junction protein overexpression would improve conduction and prevent AF. Methods and Results Thirty Yorkshire swine were randomized into 2 groups (sinus rhythm (SR) and AF), and within each group into 3 subgroups: sham-operated control, gene therapy with adenovirus expressing connexin (Cx) 40 and Cx43 (n=5 per subgroup). All animals had epicardial gene painting; the AF group had burst atrial pacing. All animals underwent terminal study 7 days after gene transfer. SR animals had strong transgene expression but no atrial conduction changes. In AF animals, controls had reduced and lateralized Cx43 expression, and Cx43 gene transfer restored expression and cellular location to SR control levels. In the AF group, both Cx40 and Cx43 gene transfer improved conduction and reduced AF relative to controls. Conclusions Connexin gene therapy preserved atrial conduction and prevented AF. PMID:22158756

  18. The Drosophila Shaker gene codes for a distinctive K+ current in a subset of neurons.

    PubMed

    Baker, K; Salkoff, L

    1990-01-01

    A transient K+ current coded by the Shaker gene was identified in muscle and expressed in Xenopus oocytes by injecting cRNA transcribed from a cloned cDNA. The Shaker current has not previously been identified in neurons. Mutational analysis now reveals that in neurons, Shaker is required for expression of a very rapidly inactivating K+ current with a depolarized steady-state inactivation curve. Together, these properties distinguish the Shaker-coded current from similar fast transient K+ currents coded by other genes. The Sh5 mutation further enhanced the depolarization of the Shaker current steady-state inactivation curve. Deletion of the Shaker gene completely removes the transient K+ current from a small percentage of neurons (15%) in a mixed population, and removes a portion of the whole-cell current in about 35% of neurons. The remaining 50% of neurons were apparently unaffected by deletion of the Shaker gene. The unique combination of rapid inactivation and depolarized steady-state inactivation of the Shaker current may reflect a unique functional role for this current in the nervous system such as the rapid repolarization of action potentials.

  19. Bacterial Genes in the Aphid Genome: Absence of Functional Gene Transfer from Buchnera to Its Host

    PubMed Central

    Nikoh, Naruo; McCutcheon, John P.; Kudo, Toshiaki; Miyagishima, Shin-ya; Moran, Nancy A.; Nakabachi, Atsushi

    2010-01-01

    Genome reduction is typical of obligate symbionts. In cellular organelles, this reduction partly reflects transfer of ancestral bacterial genes to the host genome, but little is known about gene transfer in other obligate symbioses. Aphids harbor anciently acquired obligate mutualists, Buchnera aphidicola (Gammaproteobacteria), which have highly reduced genomes (420–650 kb), raising the possibility of gene transfer from ancestral Buchnera to the aphid genome. In addition, aphids often harbor other bacteria that also are potential sources of transferred genes. Previous limited sampling of genes expressed in bacteriocytes, the specialized cells that harbor Buchnera, revealed that aphids acquired at least two genes from bacteria. The newly sequenced genome of the pea aphid, Acyrthosiphon pisum, presents the first opportunity for a complete inventory of genes transferred from bacteria to the host genome in the context of an ancient obligate symbiosis. Computational screening of the entire A. pisum genome, followed by phylogenetic and experimental analyses, provided strong support for the transfer of 12 genes or gene fragments from bacteria to the aphid genome: three LD–carboxypeptidases (LdcA1, LdcA2,ψLdcA), five rare lipoprotein As (RlpA1-5), N-acetylmuramoyl-L-alanine amidase (AmiD), 1,4-beta-N-acetylmuramidase (bLys), DNA polymerase III alpha chain (ψDnaE), and ATP synthase delta chain (ψAtpH). Buchnera was the apparent source of two highly truncated pseudogenes (ψDnaE and ψAtpH). Most other transferred genes were closely related to genes from relatives of Wolbachia (Alphaproteobacteria). At least eight of the transferred genes (LdcA1, AmiD, RlpA1-5, bLys) appear to be functional, and expression of seven (LdcA1, AmiD, RlpA1-5) are highly upregulated in bacteriocytes. The LdcAs and RlpAs appear to have been duplicated after transfer. Our results excluded the hypothesis that genome reduction in Buchnera has been accompanied by gene transfer to the host

  20. DETECTING EVOLUTIONARY TRANSFER OF GENES USING PhIGs(1).

    PubMed

    Boore, Jeffrey L

    2008-02-01

    Organisms have acquired plastids by convoluted paths that have provided multiple opportunities for gene transfer into a host nucleus from intracellular organisms, including the cyanobacterial ancestor of plastids, the proteobacterial ancestor of mitochondria, and both green and red algae whose engulfment has led to secondary acquisition of plastids. These gene movements are most accurately demonstrated by building phylogenetic trees that identify the evolutionary origin of each gene, and one effective tool for this is "PhIGs" (Phylogenetically Inferred Groups; http://PhIGs.org), a set of databases and computer tools with a Web interface for whole-genome evolutionary analysis. PhIGs takes as input gene sets of completely sequenced genomes, builds clusters of genes using a novel, graph-based approach, and reconstructs the evolutionary relationships among all gene families. The user can view and download the sequence alignments, compare intron-exon structures, and follow links to functional genomic databases. Currently, PhIGs contains 652,756 genes from 45 genomes grouped into 61,059 gene families. Graphical displays show the relative positions of these genes among genomes. PhIGs has been used to detect the evolutionary transfer of hundreds of genes from cyanobacteria and red algae into oömycete nuclear genomes, revealing that even though they have no plastids, their ancestors did, having secondarily acquired them from an intracellular red alga. A great number of genomes are soon to become available that are relevant to our broader understanding of the movement of genes among intracellular compartments after engulfing other organisms, and PhIGs will be an effective tool to interpret these gene movements.

  1. Disrupting astrocyte–neuron lactate transfer persistently reduces conditioned responses to cocaine

    PubMed Central

    Boury-Jamot, B; Carrard, A; Martin, J L; Halfon, O; Magistretti, P J; Boutrel, B

    2016-01-01

    A central problem in the treatment of drug addiction is the high risk of relapse often precipitated by drug-associated cues. The transfer of glycogen-derived lactate from astrocytes to neurons is required for long-term memory. Whereas blockade of drug memory reconsolidation represents a potential therapeutic strategy, the role of astrocyte–neuron lactate transport in long-term conditioning has received little attention. By infusing an inhibitor of glycogen phosphorylase into the basolateral amygdala of rats, we report that disruption of astrocyte-derived lactate not only transiently impaired the acquisition of a cocaine-induced conditioned place preference but also persistently disrupted an established conditioning. The drug memory was rescued by L-Lactate co-administration through a mechanism requiring the synaptic plasticity-related transcription factor Zif268 and extracellular signal-regulated kinase (ERK) signalling pathway but not the brain-derived neurotrophic factor (Bdnf). The long-term amnesia induced by glycogenolysis inhibition and the concomitant decreased expression of phospho-ERK were both restored with L-Lactate co-administration. These findings reveal a critical role for astrocyte-derived lactate in positive memory formation and highlight a novel amygdala-dependent reconsolidation process, whose disruption may offer a novel therapeutic target to reduce the long-lasting conditioned responses to cocaine. PMID:26503760

  2. Disrupting astrocyte-neuron lactate transfer persistently reduces conditioned responses to cocaine.

    PubMed

    Boury-Jamot, B; Carrard, A; Martin, J L; Halfon, O; Magistretti, P J; Boutrel, B

    2016-08-01

    A central problem in the treatment of drug addiction is the high risk of relapse often precipitated by drug-associated cues. The transfer of glycogen-derived lactate from astrocytes to neurons is required for long-term memory. Whereas blockade of drug memory reconsolidation represents a potential therapeutic strategy, the role of astrocyte-neuron lactate transport in long-term conditioning has received little attention. By infusing an inhibitor of glycogen phosphorylase into the basolateral amygdala of rats, we report that disruption of astrocyte-derived lactate not only transiently impaired the acquisition of a cocaine-induced conditioned place preference but also persistently disrupted an established conditioning. The drug memory was rescued by L-Lactate co-administration through a mechanism requiring the synaptic plasticity-related transcription factor Zif268 and extracellular signal-regulated kinase (ERK) signalling pathway but not the brain-derived neurotrophic factor (Bdnf). The long-term amnesia induced by glycogenolysis inhibition and the concomitant decreased expression of phospho-ERK were both restored with L-Lactate co-administration. These findings reveal a critical role for astrocyte-derived lactate in positive memory formation and highlight a novel amygdala-dependent reconsolidation process, whose disruption may offer a novel therapeutic target to reduce the long-lasting conditioned responses to cocaine. PMID:26503760

  3. Detecting Horizontal Gene Transfer between Closely Related Taxa.

    PubMed

    Adato, Orit; Ninyo, Noga; Gophna, Uri; Snir, Sagi

    2015-10-01

    Horizontal gene transfer (HGT), the transfer of genetic material between organisms, is crucial for genetic innovation and the evolution of genome architecture. Existing HGT detection algorithms rely on a strong phylogenetic signal distinguishing the transferred sequence from ancestral (vertically derived) genes in its recipient genome. Detecting HGT between closely related species or strains is challenging, as the phylogenetic signal is usually weak and the nucleotide composition is normally nearly identical. Nevertheless, there is a great importance in detecting HGT between congeneric species or strains, especially in clinical microbiology, where understanding the emergence of new virulent and drug-resistant strains is crucial, and often time-sensitive. We developed a novel, self-contained technique named Near HGT, based on the synteny index, to measure the divergence of a gene from its native genomic environment and used it to identify candidate HGT events between closely related strains. The method confirms candidate transferred genes based on the constant relative mutability (CRM). Using CRM, the algorithm assigns a confidence score based on "unusual" sequence divergence. A gene exhibiting exceptional deviations according to both synteny and mutability criteria, is considered a validated HGT product. We first employed the technique to a set of three E. coli strains and detected several highly probable horizontally acquired genes. We then compared the method to existing HGT detection tools using a larger strain data set. When combined with additional approaches our new algorithm provides richer picture and brings us closer to the goal of detecting all newly acquired genes in a particular strain. PMID:26439115

  4. Antibiotics and gene transfer in swine gut bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The mammalian gastrointestinal (GI) tract hosts a diverse collection bacteria, most of which are beneficial for host health. This bacterial community also supports a community of viruses that infect bacteria (called bacteriophages or phages). Phages transfer genes between bacteria, and phage-media...

  5. Physical methods for gene transfer: improving the kinetics of gene delivery into cells.

    PubMed

    Mehier-Humbert, Sophie; Guy, Richard H

    2005-04-01

    One factor critical to successful gene therapy is the development of efficient delivery systems. Although advances in gene transfer technology, including viral and non-viral vectors, have been made, an ideal vector system has not yet been constructed. This review describes the basic principles behind various physical methods for gene transfer and assesses the advantages and performance of such approaches, compared to other transfection systems. In particular, the kinetics and efficiency of gene delivery, the toxicity, in vivo feasibility, and targeting ability of different physical methodologies are discussed and evaluated.

  6. Specific Gene Repression by CRISPRi System Transferred through Bacterial Conjugation

    PubMed Central

    2014-01-01

    In microbial communities, bacterial populations are commonly controlled using indiscriminate, broad range antibiotics. There are few ways to target specific strains effectively without disrupting the entire microbiome and local environment. Here, we use conjugation, a natural DNA horizontal transfer process among bacterial species, to deliver an engineered CRISPR interference (CRISPRi) system for targeting specific genes in recipient Escherichia coli cells. We show that delivery of the CRISPRi system is successful and can specifically repress a reporter gene in recipient cells, thereby establishing a new tool for gene regulation across bacterial cells and potentially for bacterial population control. PMID:25409531

  7. Familial Dysautonomia (FD) Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by IKBKAP Downregulation

    PubMed Central

    Kantor, Gal; Cheishvili, David; Even, Aviel; Birger, Anastasya; Turetsky, Tikva; Gil, Yaniv; Even-Ram, Sharona; Aizenman, Einat; Bashir, Nibal; Maayan, Channa; Razin, Aharon; Reubinoff, Benjamim E.; Weil, Miguel

    2015-01-01

    A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD. PMID:26437462

  8. Controlled plasmid gene transfer to murine renal carcinoma by hexadecylphosphocholine.

    PubMed

    Settelen, Nathalie; Roch, Olivier; Bock, David; Rooke, Ronald; Braun, Serge; Meyer, Olivier

    2004-01-01

    We report here that the anticancer drug hexadecylphosphocholine (HPC) can control plasmid DNA-mediated gene transfer to renal carcinoma following intratumoral administration. Significant improvement of gene expression levels could be achieved depending on HPC dose administered. Optimal concentration of HPC co-injected with plasmid DNA was found to be 0.2% (w/v) showing up to a 10-fold increase in reporter gene expression levels when compared to DNA administered alone. In vivo gene transfer activity of HPC was not affected by the nature of the diluent used, i.e. glucose-based or saline-based isotonic solutions. Although in vitro transfection activity of HPC formulations could not be evidenced, a liposome leakage assay revealed that HPC could significantly destabilize stable lipid membranes suggesting that a possible membrane permeation enhancer activity of HPC combined to the physical stress induced by the intratumor injection may facilitate plasmid DNA entry inside the cells resulting in increased gene expression. HPC/plasmid formulations represent new and attractive non-viral gene delivery systems with potential in cancer gene therapy and vaccination. PMID:14684287

  9. Adult rat bone marrow stromal cells express genes associated with dopamine neurons

    SciTech Connect

    Kramer, Brian C.; Woodbury, Dale . E-mail: WOODBURYDL@AOL.COM; Black, Ira B.

    2006-05-19

    An intensive search is underway to identify candidates to replace the cells that degenerate in Parkinson's disease (PD). To date, no suitable substitute has been found. We have recently found that adult rat bone marrow stromal cells (MSCs) can be induced to assume a neuronal phenotype in vitro. These findings may have particular relevance to the treatment of PD. We now report that adult MSCs express multiple dopaminergic genes, suggesting that they are potential candidates for cell therapy. Using RT-PCR, we have examined families of genes that are associated with the development and/or survival of dopaminergic neurons. MSCs transcribe a variety of dopaminergic genes including patched and smoothened (components of the Shh receptor), Gli-1 (downstream mediator of Shh), and Otx-1, a gene associated with formation of the mesencephalon during development. Furthermore, Shh treatment elicits a 1.5-fold increase in DNA synthesis in cultured MSCs, suggesting the presence of a functional Shh receptor complex. We have also found that MSCs transcribe and translate Nurr-1, a nuclear receptor essential for the development of dopamine neurons. In addition, MSCs express a variety of growth factor receptors including the glycosyl-phosphatidylinositol-anchored ligand-binding subunit of the GDNF receptor, GFR{alpha}1, as well as fibroblast growth factor receptors one and four. The expression of genes that are associated with the development and survival of dopamine neurons suggests a potential role for these cells in the treatment of Parkinson's disease.

  10. Detection of Horizontal Gene Transfers from Phylogenetic Comparisons

    PubMed Central

    Pylro, Victor Satler; Vespoli, Luciano de Souza; Duarte, Gabriela Frois; Yotoko, Karla Suemy Clemente

    2012-01-01

    Bacterial phylogenies have become one of the most important challenges for microbial ecology. This field started in the mid-1970s with the aim of using the sequence of the small subunit ribosomal RNA (16S) tool to infer bacterial phylogenies. Phylogenetic hypotheses based on other sequences usually give conflicting topologies that reveal different evolutionary histories, which in some cases may be the result of horizontal gene transfer events. Currently, one of the major goals of molecular biology is to understand the role that horizontal gene transfer plays in species adaptation and evolution. In this work, we compared the phylogenetic tree based on 16S with the tree based on dszC, a gene involved in the cleavage of carbon-sulfur bonds. Bacteria of several genera perform this survival task when living in environments lacking free mineral sulfur. The biochemical pathway of the desulphurization process was extensively studied due to its economic importance, since this step is expensive and indispensable in fuel production. Our results clearly show that horizontal gene transfer events could be detected using common phylogenetic methods with gene sequences obtained from public sequence databases. PMID:22675653

  11. Clathrin light chain B: gene structure and neuron-specific splicing.

    PubMed Central

    Stamm, S; Casper, D; Dinsmore, J; Kaufmann, C A; Brosius, J; Helfman, D M

    1992-01-01

    The clathrin light chains are components of clathrin coated vesicles, structural constituents involved in endocytosis and membrane recycling. The clathrin light chain B (LCB) gene encodes two isoforms, termed LCB2 and LCB3, via an alternative RNA splicing mechanism. We have determined the structure of the rat clathrin light chain B gene. The gene consists of six exons that extend over 11.9 kb. The first four exons and the last exon are common to the LCB2 and LCB3 isoforms. The fifth exon, termed EN, is included in the mRNA in brain, giving rise to the brain specific form LCB2 but is excluded in other tissues, generating the LCB3 isoform. Primary rat neuronal cell cultures express predominantly the brain specific LCB2 isoform, whereas primary rat cultures of glia express only the LCB3 isoform, suggesting that expression of the brain-specific LCB2 form is limited to neurons. Further evidence for neuronal localization of the LCB2 form is provided using a teratocarcinoma cell line, P19, which can be induced by retinoic acid to express a neuronal phenotype, concomitant with the induction of the LCB2 form. In order to determine the sequences involved in alternative splice site selection, we constructed a minigene containing the alternative spliced exon EN and its flanking intron and exon sequences. This minigene reflects the splicing pattern of the endogenous gene upon transfection in HeLa cell and primary neuronal cell cultures, indicating that this region of the LCB gene contains all the necessary information for neuron-specific splicing. Images PMID:1408826

  12. [Experimental approach to the gene therapy of motor neuron disease with the use of genes hypoxia-inducible factors].

    PubMed

    Ismailov, Sh M; Barykova, Iu A; Shmarov, M M; Tarantul, V Z; Barskov, I V; Kucherianu, V G; Brylev, L V; Logunov, D Iu; Tutykhina, I L; Bocharov, E V; Zakharova, M N; Naroditskiĭ, B S; Illarioshkin, S N

    2014-05-01

    Motor neuron disease (MND), or amyotrophic lateral sclerosis, is a fatal neurodegenerative disorder characterized by a progressive loss of motor neurons in the spinal cord and the brain. Several angiogenic and neurogenic growth factors, such as the vascular endothelial growth factor (VEGF), angiogenin (ANG), insulin-like growth factor (IGF) and others, have been shown to promote survival of the spinal motor neurons during ischemia. We constructed recombinant vectors using human adenovirus 5 (Ad5) carrying the VEGF, ANG or IGF genes under the control of the cytomegalovirus promoter. As a model for MND, we employed a transgenic mice strain, B6SJL-Tg (SOD1*G93A)d11 Gur/J that develops a progressive degeneration of the spinal motor neurons caused by the expression of a mutated Cu/Zn superoxide dismutase gene SOD1. Delivery of the therapeutic genes to the spinal motor neurons was done using the effect of the retrograde axonal transport after multiple injections of the Ad5-VEGF, Ad5-ANG and Ad5-IGF vectors and their combinations into the limbs and back muscles of the SOD1(G93A) mice. Viral transgene expression in the spinal cord motor neurons was confirmed by immunocytochemistry and RT-RCR. We assessed the neurological status, motor activity and lifespan of experimental and control animal groups. We discovered that SOD1(G93A) mice injected with the Ad5-VEGF + Ad5-ANG combination showed a 2-3 week delay in manifestation of the disease, higher motor activity at the advanced stages of the disease, and at least a 10% increase in the lifespan compared to the control and other experimental groups. These results support the safety and therapeutic efficacy of the tested recombinant treatment. We propose that the developed experimental MND treatment based on viral delivery of VEGF + ANG can be used as a basis for gene therapy drug development and testing in the preclinical and clinical trials of the MND.

  13. Rescuing the Failing Heart by Targeted Gene Transfer

    PubMed Central

    Kawase, Yoshiaki; Ladage, Dennis; Hajjar, Roger J.

    2011-01-01

    Congestive heart failure is a major cause of morbidity and mortality in the US. While progress in conventional treatments is making steady and incremental gains to reduce heart failure mortality, there is a critical need to explore new therapeutic approaches. Gene therapy was initially applied in the clinical setting for inherited monogenic disorders. It is now apparent that gene therapy has broader potential that also includes acquired polygenic diseases, such as congestive heart failure. Recent advances in understanding of the molecular basis of myocardial dysfunction, together with the evolution of increasingly efficient gene transfer technology, has placed heart failure within reach of gene-based therapy. Furthermore, the recent successful and safe completion of a phase 2 trial targeting the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) along with the start of more recent phase 1 trials usher a new era for gene therapy for the treatment of heart failure. PMID:21371634

  14. Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System

    PubMed Central

    Jung, Jae-Young; Lee, Seung Eun; Hwang, Eun Mi

    2016-01-01

    Assessing the cell-type expression pattern of a certain gene can be achieved by using cell-type-specific gene manipulation. Recently, cre-recombinase-dependent gene-silencing tool, pSico has become popular in neuroscientific research. However, pSico has a critical limitation that gene-silenced cell cannot be identified by fluorescence, due to an excision of the reporter gene for green fluorescence protein (GFP). To overcome this limitation, we newly developed pSico-Red, with mCherry gene as a reporter outside two loxP sites, so that red mCherry signal is detected in all transfected cells. When a cell expresses cre, GFP is excised and shRNA is enabled, resulting in disappearance of GFP. This feature of pSico-Red provides not only cell-type-specific gene-silencing but also identification of cre expressing cells. Using this system, we demonstrated for the first time the neuronal expression of the Bestrophin-1 (Best1) in thalamic reticular nucleus (TRN) and TRN-neuron-specific gene-silencing of Best1. We combined adeno-associated virus (AAV) carrying Best1-shRNA in pSico-Red vector and transgenic mouse expressing cre under the promoter of distal-less homeobox 5/6 (DLX5/6), a marker for inhibitory neurons. Firstly, we found that almost all of inhibitory neurons in TRN express Best1 by immunohistochemistry. Using pSico-Red virus, we found that 80% of infected TRN neurons were DLX5/6-cre positive but parvalbumin negative. Finally, we found that Best1 in DLX5/6-cre positive neurons were significantly reduced by Best1-shRNA. Our study demonstrates that TRN neurons strongly express Best1 and that pSico-Red is a valuable tool for cell-type-specific gene manipulation and identification of specific cell population. PMID:27358580

  15. Neuronal Expression and Cell-Type-Specific Gene-Silencing of Best1 in Thalamic Reticular Nucleus Neurons Using pSico-Red System.

    PubMed

    Jung, Jae-Young; Lee, Seung Eun; Hwang, Eun Mi; Lee, C Justin

    2016-06-01

    Assessing the cell-type expression pattern of a certain gene can be achieved by using cell-type-specific gene manipulation. Recently, cre-recombinase-dependent gene-silencing tool, pSico has become popular in neuroscientific research. However, pSico has a critical limitation that gene-silenced cell cannot be identified by fluorescence, due to an excision of the reporter gene for green fluorescence protein (GFP). To overcome this limitation, we newly developed pSico-Red, with mCherry gene as a reporter outside two loxP sites, so that red mCherry signal is detected in all transfected cells. When a cell expresses cre, GFP is excised and shRNA is enabled, resulting in disappearance of GFP. This feature of pSico-Red provides not only cell-type-specific gene-silencing but also identification of cre expressing cells. Using this system, we demonstrated for the first time the neuronal expression of the Bestrophin-1 (Best1) in thalamic reticular nucleus (TRN) and TRN-neuron-specific gene-silencing of Best1. We combined adeno-associated virus (AAV) carrying Best1-shRNA in pSico-Red vector and transgenic mouse expressing cre under the promoter of distal-less homeobox 5/6 (DLX5/6), a marker for inhibitory neurons. Firstly, we found that almost all of inhibitory neurons in TRN express Best1 by immunohistochemistry. Using pSico-Red virus, we found that 80% of infected TRN neurons were DLX5/6-cre positive but parvalbumin negative. Finally, we found that Best1 in DLX5/6-cre positive neurons were significantly reduced by Best1-shRNA. Our study demonstrates that TRN neurons strongly express Best1 and that pSico-Red is a valuable tool for cell-type-specific gene manipulation and identification of specific cell population. PMID:27358580

  16. Identification of Neuronal Enhancers of the Proopiomelanocortin Gene by Transgenic Mouse Analysis and Phylogenetic Footprinting

    PubMed Central

    de Souza, Flávio S. J.; Santangelo, Andrea M.; Bumaschny, Viviana; Avale, María Elena; Smart, James L.; Low, Malcolm J.; Rubinstein, Marcelo

    2005-01-01

    The proopiomelanocortin (POMC) gene is expressed in the pituitary and arcuate neurons of the hypothalamus. POMC arcuate neurons play a central role in the control of energy homeostasis, and rare loss-of-function mutations in POMC cause obesity. Moreover, POMC is the prime candidate gene within a highly significant quantitative trait locus on chromosome 2 associated with obesity traits in several human populations. Here, we identify two phylogenetically conserved neuronal POMC enhancers designated nPE1 (600 bp) and nPE2 (150 bp) located approximately 10 to 12 kb upstream of mammalian POMC transcriptional units. We show that mouse or human genomic regions containing these enhancers are able to direct reporter gene expression to POMC hypothalamic neurons, but not the pituitary of transgenic mice. Conversely, deletion of nPE1 and nPE2 in the context of the entire transcriptional unit of POMC abolishes transgene expression in the hypothalamus without affecting pituitary expression. Our results indicate that the nPEs are necessary and sufficient for hypothalamic POMC expression and that POMC expression in the brain and pituitary is controlled by independent sets of enhancers. Our study advances the understanding of the molecular nature of hypothalamic POMC neurons and will be useful to determine whether polymorphisms in POMC regulatory regions play a role in the predisposition to obesity. PMID:15798195

  17. FACS array profiling identifies Ecto-5' nucleotidase as a striatopallidal neuron-specific gene involved in striatal-dependent learning.

    PubMed

    Ena, Sabrina L; De Backer, Jean-François; Schiffmann, Serge N; de Kerchove d'Exaerde, Alban

    2013-05-15

    The striatopallidal (STP) and striatonigral (STN) neurons constitute the main neuronal populations of the striatum. Despite the increasing knowledge concerning their involvement in multiple tasks associated with the striatum, it is still challenging to understand the precise differential functions of these two neuronal populations and to identify and study new genes involved in these functions. Here, we describe a reliable approach, applied on adult mouse brain, to generate specific STP and STN neuron gene profiles. STP and STN neurons were identified in the same animal using the transgenic Adora2A-Cre × Z/EG mouse model combined with retrograde labeling, respectively. Gene profiling was generated from FACS-purified neurons leading to the identification of new STP and STN neuron-specific genes. Knock-down models based on Cre-dependent lentiviral vector were developed to investigate their function either in striatal or in STP neurons. Thereby, we demonstrate that ecto-5'-nucleotidase (NT5e) is specifically expressed in STP neurons and is at the origin of most of the extracellular adenosine produced in the striatum. Behavioral analysis of striatal and STP neuron knock-down mouse models as well as NT5e knock-out mice demonstrates the implication of this STP neuron enzyme in motor learning.

  18. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes

    PubMed Central

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F.

    2015-01-01

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners—the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)—and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic—and plant and algal—lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller’s ratchet—the origin of eukaryotic recombination, or sex—might have required surprisingly little evolutionary innovation. PMID:25733873

  19. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

    PubMed

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F

    2015-08-18

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation.

  20. Endosymbiotic gene transfer from prokaryotic pangenomes: Inherited chimerism in eukaryotes.

    PubMed

    Ku, Chuan; Nelson-Sathi, Shijulal; Roettger, Mayo; Garg, Sriram; Hazkani-Covo, Einat; Martin, William F

    2015-08-18

    Endosymbiotic theory in eukaryotic-cell evolution rests upon a foundation of three cornerstone partners--the plastid (a cyanobacterium), the mitochondrion (a proteobacterium), and its host (an archaeon)--and carries a corollary that, over time, the majority of genes once present in the organelle genomes were relinquished to the chromosomes of the host (endosymbiotic gene transfer). However, notwithstanding eukaryote-specific gene inventions, single-gene phylogenies have never traced eukaryotic genes to three single prokaryotic sources, an issue that hinges crucially upon factors influencing phylogenetic inference. In the age of genomes, single-gene trees, once used to test the predictions of endosymbiotic theory, now spawn new theories that stand to eventually replace endosymbiotic theory with descriptive, gene tree-based variants featuring supernumerary symbionts: prokaryotic partners distinct from the cornerstone trio and whose existence is inferred solely from single-gene trees. We reason that the endosymbiotic ancestors of mitochondria and chloroplasts brought into the eukaryotic--and plant and algal--lineage a genome-sized sample of genes from the proteobacterial and cyanobacterial pangenomes of their respective day and that, even if molecular phylogeny were artifact-free, sampling prokaryotic pangenomes through endosymbiotic gene transfer would lead to inherited chimerism. Recombination in prokaryotes (transduction, conjugation, transformation) differs from recombination in eukaryotes (sex). Prokaryotic recombination leads to pangenomes, and eukaryotic recombination leads to vertical inheritance. Viewed from the perspective of endosymbiotic theory, the critical transition at the eukaryote origin that allowed escape from Muller's ratchet--the origin of eukaryotic recombination, or sex--might have required surprisingly little evolutionary innovation. PMID:25733873

  1. Evidence for a novel regulatory pathway for herpes simplex virus gene expression in trigeminal ganglion neurons.

    PubMed Central

    Kosz-Vnenchak, M; Jacobson, J; Coen, D M; Knipe, D M

    1993-01-01

    Thymidine kinase (TK)-negative (TK-) mutant strains of herpes simplex virus type 1 (HSV-1) show reduced expression of alpha and beta viral genes during acute infection of trigeminal ganglion neurons following corneal infection (M. Kosz-Vnenchak, D. M. Coen, and D. M. Knipe, J. Virol. 64:5396-5402, 1990). It was surprising that a defect in a beta gene product would lead to decreased alpha and beta gene expression, given the regulatory pathways demonstrated for HSV infection of cultured cells. In this study, we have examined viral gene expression during reactivation from latent infection in explanted trigeminal ganglion tissue. In explant reactivation studies with wild-type virus, we observed viral productive gene expression over the first 48 h of explant incubation occurring in a temporal order (alpha, beta, gamma) similar to that in cultured cells. This occurred predominantly in latency-associated transcript-positive neurons but was limited to a fraction of these cells. In contrast, TK- mutant viruses showed greatly reduced alpha and beta gene expression upon explant of latently infected trigeminal ganglion tissue. An inhibitor of viral TK or an inhibitor of viral DNA polymerase greatly decreased viral lytic gene expression in trigeminal ganglion tissue latently infected with wild-type virus and explanted in culture. These results indicate that the regulatory mechanisms governing HSV gene expression are different in trigeminal ganglion neurons and cultured cells. We present a new model for viral gene expression in trigeminal ganglion neurons with implications for the nature of the decision process between latent infection and productive infection by HSV. Images PMID:8394454

  2. y-Synuclein is an Adipocyte-Neuron Gene Coordinately-Expressed with Leptin & Increased in Obesity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Recently, we characterized tumor suppressor candidate 5 (Tusc5) as an adipocyte-neuron peroxisome proliferator activated receptor-y (PPARy) target gene (1). Our objective herein was to identify additional candidate genes that play shared roles in neuron-fat physiology. Research Methods an...

  3. The neuronal transporter gene SLC6A15 confers risk to major depression

    PubMed Central

    Kohli, Martin A.; Lucae, Susanne; Saemann, Philipp G.; Schmidt, Mathias V.; Demirkan, Ayse; Hek, Karin; Czamara, Darina; Alexander, Michael; Salyakina, Daria; Ripke, Stephan; Hoehn, David; Specht, Michael; Menke, Andreas; Hennings, Johannes; Heck, Angela; Wolf, Christiane; Ising, Marcus; Schreiber, Stefan; Czisch, Michael; Müller, Marianne B.; Uhr, Manfred; Bettecken, Thomas; Becker, Albert; Schramm, Johannes; Rietschel, Marcella; Maier, Wolfgang; Bradley, Bekh; Ressler, Kerry J.; Nöthen, Markus M.; Cichon, Sven; Craig, Ian W.; Breen, Gerome; Lewis, Cathryn M.; Hofman, Albert; Tiemeier, Henning; van Duijn, Cornelia M.; Holsboer, Florian; Müller-Myhsok, Bertram; Binder, Elisabeth B.

    2011-01-01

    Major depression (MD) is one of the most prevalent psychiatric disorders and a leading cause of loss in work productivity. A combination of genetic and environmental risk factors likely contributes to MD. We present data from a genome-wide association study revealing a neuron-specific neutral amino acid transporter (SLC6A15) as a novel susceptibility gene for MD. Risk allele carrier status in humans and chronic stress in mice were associated with a downregulation of the expression of this gene in the hippocampus, a brain region implicated in the pathophysiology of MD. The same polymorphisms also showed associations with alterations in hippocampal volume and neuronal integrity. Thus, decreased SLC6A15 expression, due to genetic or environmental factors might alter neuronal circuits related to the susceptibility for MD. Our convergent data from human genetics, expression studies, brain imaging and animal models suggest a novel pathophysiological mechanism for MD that may be accessible to drug targeting. PMID:21521612

  4. Neuronal plasticity after spinal cord injury: identification of a gene cluster driving neurite outgrowth.

    PubMed

    Di Giovanni, Simone; Faden, Alan I; Yakovlev, Alexander; Duke-Cohan, Jonathan S; Finn, Tom; Thouin, Melissa; Knoblach, Susan; De Biase, Andrea; Bregman, Barbara S; Hoffman, Eric P

    2005-01-01

    Functional recovery after spinal cord injury (SCI) may result in part from axon outgrowth and related plasticity through coordinated changes at the molecular level. We employed microarray analysis to identify a subset of genes the expression patterns of which were temporally coregulated and correlated to functional recovery after SCI. Steady-state mRNA levels of this synchronously regulated gene cluster were depressed in both ventral and dorsal horn neurons within 24 h after injury, followed by strong re-induction during the following 2 wk, which paralleled functional recovery. The identified cluster includes neuritin, attractin, microtubule-associated protein 1a, and myelin oligodendrocyte protein genes. Transcriptional and protein regulation of this novel gene cluster was also evaluated in spinal cord tissue and in single neurons and was shown to play a role in axonal plasticity. Finally, in vitro transfection experiments in primary dorsal root ganglion cells showed that cluster members act synergistically to drive neurite outgrowth. PMID:15522907

  5. Immunotherapy of Malignancy by in vivo Gene Transfer into Tumors

    NASA Astrophysics Data System (ADS)

    Plautz, Gregory E.; Yang, Zhi-Yong; Wu, Bei-Yue; Gao, Xiang; Huang, Leaf; Nabel, Gary J.

    1993-05-01

    The immune system confers protection against a variety of pathogens and contributes to the surveillance and destruction of neoplastic cells. Several cell types participate in the recognition and lysis of tumors, and appropriate immune stimulation provides therapeutic effects in malignancy. Foreign major histocompatibility complex (MHC) proteins also serve as a potent stimulus to the immune system. In this report, a foreign MHC gene was introduced directly into malignant tumors in vivo in an effort to stimulate tumor rejection. In contrast to previous attempts to induce tumor immunity by cell-mediated gene transfer, the recombinant gene was introduced directly into tumors in vivo. Expression of the murine class I H-2K^s gene within the CT26 mouse colon adenocarcinoma (H-2K^d) or the MCA 106 fibrosarcoma (H-2K^b) induced a cytotoxic T-cell response to H-2K^s and, more importantly, to other antigens present on unmodified tumor cells. This immune response attenuated tumor growth and caused complete tumor regression in many cases. Direct gene transfer in vivo can therefore induce cell-mediated immunity against specific gene products, which provides an immunotherapeutic effect for malignancy, and potentially can be applied to the treatment of cancer and infectious diseases in man.

  6. Modulation of adenovirus-mediated gene transfer by nitric oxide.

    PubMed

    Haddad, I Y; Sorscher, E J; Garver, R I; Hong, J; Tzeng, E; Matalon, S

    1997-05-01

    We assessed the role of .NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 microM), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and beta-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N(G)-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin-mediated induction of .NO and restored gene transfer to baseline values. Incubation of NIH3T3/iNOS with 8-bromo-cGMP (400 microM) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of .NO, did not alter the efficacy of gene transfer. .NO produced by NIH3T3/iNOS cells also suppressed beta-galactosidase expression in NIH3T3 cocultured cells stably transfected with beta-galactosidase gene, suggesting .NO inhibited gene expression at either the transriptional or posttranscriptional levels. To investigate the effects of inhaled .NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10(9) pfu in 80 microl of saline) and exposed to .NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n > or = 8). We concluded that

  7. Human gene transfer: Characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man

    SciTech Connect

    Kasid, A.; Morecki, S.; Aebersold, P.; Cornetta, K.; Culver, K.; Freeman, S.; Director, E.; Lotze, M.T.; Blaese, R.M.; Anderson, W.F.; Rosenberg, S.A. )

    1990-01-01

    Tumor-infiltrating lymphocytes (TILs) are cells generated from tumor suspensions cultured in interleukin 2 that can mediate cancer regression when adoptively transferred into mice or humans. Since TILs proliferate rapidly in vitro, recirculate, and preferentially localize at the tumor site in vivo, they provide an attractive model for delivery of exogenous genetic material into man. To determine whether efficient gene transfer into TILs is feasible. The authors transduced human TILs with the bacterial gene for neomycin-resistance (Neo{sup R}) using the retroviral vector N2. The transduced TIL populations were stable and polyclonal with respect to the intact Neo{sup R} gene integration and expressed high levels of neomycin phosphotransferase activity. The Neo{sup R} gene insertion did not alter the in vitro growth pattern and interleukin 2 dependence of the transduced TILs. Analyses of T-cell receptor gene rearrangement for {beta}- and {gamma}-chain genes revealed the oligoclonal nature of the TIL populations with no major change in the DNA rearrangement patterns or the levels of mRNA expression of the {beta} and {gamma} chains following transduction and selection of TILs in the neomycin analog G418. Human TILs expressed mRNA for tumor necrosis factors ({alpha} and {beta}) and interleukin 2 receptor P55. This pattern of cytokine-mRNA expression was not significantly altered following the transduction of TILs. The studies demonstrate the feasibility of TILs as suitable cellular vehicles for the introduction of therapeutic genes into patients receiving autologous TILs.

  8. Wolbachia genome integrated in an insect chromosome: Evolution and fate of laterally transferred endosymbiont genes

    PubMed Central

    Nikoh, Naruo; Tanaka, Kohjiro; Shibata, Fukashi; Kondo, Natsuko; Hizume, Masahiro; Shimada, Masakazu; Fukatsu, Takema

    2008-01-01

    Recent accumulation of microbial genome data has demonstrated that lateral gene transfers constitute an important and universal evolutionary process in prokaryotes, while those in multicellular eukaryotes are still regarded as unusual, except for endosymbiotic gene transfers from mitochondria and plastids. Here we thoroughly investigated the bacterial genes derived from a Wolbachia endosymbiont on the nuclear genome of the beetle Callosobruchus chinensis. Exhaustive PCR detection and Southern blot analysis suggested that ∼30% of Wolbachia genes, in terms of the gene repertoire of wMel, are present on the insect nuclear genome. Fluorescent in situ hybridization located the transferred genes on the proximal region of the basal short arm of the X chromosome. Molecular evolutionary and other lines of evidence indicated that the transferred genes are probably derived from a single lateral transfer event. The transferred genes were, for the length examined, structurally disrupted, freed from functional constraints, and transcriptionally inactive. Hence, most, if not all, of the transferred genes have been pseudogenized. Notwithstanding this, the transferred genes were ubiquitously detected from Japanese and Taiwanese populations of C. chinensis, while the number of the transferred genes detected differed between the populations. The transferred genes were not detected from congenic beetle species, indicating that the transfer event occurred after speciation of C. chinensis, which was estimated to be one or several million years ago. These features of the laterally transferred endosymbiont genes are compared with the evolutionary patterns of mitochondrial and plastid genome fragments acquired by nuclear genomes through recent endosymbiotic gene transfers. PMID:18073380

  9. Synaptic protein and pan-neuronal gene expression and their regulation by Dicer-dependent mechanisms differ between neurons and neuroendocrine cells

    PubMed Central

    2013-01-01

    Background Neurons in sympathetic ganglia and neuroendocrine cells in the adrenal medulla share not only their embryonic origin from sympathoadrenal precursors in the neural crest but also a range of functional features. These include the capacity for noradrenaline biosynthesis, vesicular storage and regulated release. Yet the regulation of neuronal properties in early neuroendocrine differentiation is a matter of debate and the developmental expression of the vesicle fusion machinery, which includes components found in both neurons and neuroendocrine cells, is not resolved. Results Analysis of synaptic protein and pan-neuronal marker mRNA expression during mouse development uncovers profound differences between sympathetic neurons and adrenal chromaffin cells, which result in qualitatively similar but quantitatively divergent transcript profiles. In sympathetic neurons embryonic upregulation of synaptic protein mRNA follows early and persistent induction of pan-neuronal marker transcripts. In adrenal chromaffin cells pan-neuronal marker expression occurs only transiently and synaptic protein messages remain at distinctly low levels throughout embryogenesis. Embryonic induction of synaptotagmin I (Syt1) in sympathetic ganglia and postnatal upregulation of synaptotagmin VII (Syt7) in adrenal medulla results in a cell type-specific difference in isoform prevalence. Dicer 1 inactivation in catecholaminergic cells reduces high neuronal synaptic protein mRNA levels but not their neuroendocrine low level expression. Pan-neuronal marker mRNAs are induced in chromaffin cells to yield a more neuron-like transcript pattern, while ultrastructure is not altered. Conclusions Our study demonstrates that remarkably different gene regulatory programs govern the expression of synaptic proteins in the neuronal and neuroendocrine branch of the sympathoadrenal system. They result in overlapping but quantitatively divergent transcript profiles. Dicer 1-dependent regulation is required

  10. Kisspeptin Activates Ankrd 26 Gene Expression in Migrating Embryonic GnRH Neurons.

    PubMed

    Soga, Tomoko; Lim, Wei Ling; Khoo, Alan Soo-Beng; Parhar, Ishwar S

    2016-01-01

    Kisspeptin, a newly discovered neuropeptide, regulates gonadotropin-releasing hormone (GnRH). Kisspeptins are a large RF-amide family of peptides. The kisspeptin coded by KiSS-1 gene is a 145-amino acid protein that is cleaved to C-terminal peptide kisspeptin-10. G-protein-coupled receptor 54 (GPR54) has been identified as a kisspeptin receptor, and it is expressed in GnRH neurons and in a variety of cancer cells. In this study, enhanced green fluorescent protein (EGFP) labeled GnRH cells with migratory properties, which express GPR54, served as a model to study the effects of kisspeptin on cell migration. We monitored EGFP-GnRH neuronal migration in brain slide culture of embryonic day 14 transgenic rat by live cell imaging system and studied the effects of kisspeptin-10 (1 nM) treatment for 36 h on GnRH migration. Furthermore, to determine kisspeptin-induced molecular pathways related with apoptosis and cytoskeletal changes during neuronal migration, we studied the expression levels of candidate genes in laser-captured EGFP-GnRH neurons by real-time PCR. We found that there was no change in the expression level of genes related to cell proliferation and apoptosis. The expression of ankyrin repeat domain-containing protein (ankrd) 26 in EGFP-GnRH neurons was upregulated by the exposure to kisspeptin. These studies suggest that ankrd 26 gene plays an unidentified role in regulating neuronal movement mediated by kisspeptin-GPR54 signaling, which could be a potential pathway to suppress cell migration. PMID:26973595

  11. Kisspeptin Activates Ankrd 26 Gene Expression in Migrating Embryonic GnRH Neurons

    PubMed Central

    Soga, Tomoko; Lim, Wei Ling; Khoo, Alan Soo-Beng; Parhar, Ishwar S.

    2016-01-01

    Kisspeptin, a newly discovered neuropeptide, regulates gonadotropin-releasing hormone (GnRH). Kisspeptins are a large RF-amide family of peptides. The kisspeptin coded by KiSS-1 gene is a 145-amino acid protein that is cleaved to C-terminal peptide kisspeptin-10. G-protein-coupled receptor 54 (GPR54) has been identified as a kisspeptin receptor, and it is expressed in GnRH neurons and in a variety of cancer cells. In this study, enhanced green fluorescent protein (EGFP) labeled GnRH cells with migratory properties, which express GPR54, served as a model to study the effects of kisspeptin on cell migration. We monitored EGFP–GnRH neuronal migration in brain slide culture of embryonic day 14 transgenic rat by live cell imaging system and studied the effects of kisspeptin-10 (1 nM) treatment for 36 h on GnRH migration. Furthermore, to determine kisspeptin-induced molecular pathways related with apoptosis and cytoskeletal changes during neuronal migration, we studied the expression levels of candidate genes in laser-captured EGFP–GnRH neurons by real-time PCR. We found that there was no change in the expression level of genes related to cell proliferation and apoptosis. The expression of ankyrin repeat domain-containing protein (ankrd) 26 in EGFP–GnRH neurons was upregulated by the exposure to kisspeptin. These studies suggest that ankrd 26 gene plays an unidentified role in regulating neuronal movement mediated by kisspeptin–GPR54 signaling, which could be a potential pathway to suppress cell migration. PMID:26973595

  12. Higher vulnerability and stress sensitivity of neuronal precursor cells carrying an alpha-synuclein gene triplication.

    PubMed

    Flierl, Adrian; Oliveira, Luís M A; Falomir-Lockhart, Lisandro J; Mak, Sally K; Hesley, Jayne; Soldner, Frank; Arndt-Jovin, Donna J; Jaenisch, Rudolf; Langston, J William; Jovin, Thomas M; Schüle, Birgitt

    2014-01-01

    Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson's disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this "stem cell pathology" could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new tool for

  13. Higher Vulnerability and Stress Sensitivity of Neuronal Precursor Cells Carrying an Alpha-Synuclein Gene Triplication

    PubMed Central

    Flierl, Adrian; Oliveira, Luís M. A.; Falomir-Lockhart, Lisandro J.; Mak, Sally K.; Hesley, Jayne; Soldner, Frank; Arndt-Jovin, Donna J.; Jaenisch, Rudolf; Langston, J. William; Jovin, Thomas M.; Schüle, Birgitt

    2014-01-01

    Parkinson disease (PD) is a multi-factorial neurodegenerative disorder with loss of dopaminergic neurons in the substantia nigra and characteristic intracellular inclusions, called Lewy bodies. Genetic predisposition, such as point mutations and copy number variants of the SNCA gene locus can cause very similar PD-like neurodegeneration. The impact of altered α-synuclein protein expression on integrity and developmental potential of neuronal stem cells is largely unexplored, but may have wide ranging implications for PD manifestation and disease progression. Here, we investigated if induced pluripotent stem cell-derived neuronal precursor cells (NPCs) from a patient with Parkinson’s disease carrying a genomic triplication of the SNCA gene (SNCA-Tri). Our goal was to determine if these cells these neuronal precursor cells already display pathological changes and impaired cellular function that would likely predispose them when differentiated to neurodegeneration. To achieve this aim, we assessed viability and cellular physiology in human SNCA-Tri NPCs both under normal and environmentally stressed conditions to model in vitro gene-environment interactions which may play a role in the initiation and progression of PD. Human SNCA-Tri NPCs displayed overall normal cellular and mitochondrial morphology, but showed substantial changes in growth, viability, cellular energy metabolism and stress resistance especially when challenged by starvation or toxicant challenge. Knockdown of α-synuclein in the SNCA-Tri NPCs by stably expressed short hairpin RNA (shRNA) resulted in reversal of the observed phenotypic changes. These data show for the first time that genetic alterations such as the SNCA gene triplication set the stage for decreased developmental fitness, accelerated aging, and increased neuronal cell loss. The observation of this “stem cell pathology” could have a great impact on both quality and quantity of neuronal networks and could provide a powerful new

  14. Ethanol upregulates NMDA receptor subunit gene expression in human embryonic stem cell-derived cortical neurons.

    PubMed

    Xiang, Yangfei; Kim, Kun-Yong; Gelernter, Joel; Park, In-Hyun; Zhang, Huiping

    2015-01-01

    Chronic alcohol consumption may result in sustained gene expression alterations in the brain, leading to alcohol abuse or dependence. Because of ethical concerns of using live human brain cells in research, this hypothesis cannot be tested directly in live human brains. In the present study, we used human embryonic stem cell (hESC)-derived cortical neurons as in vitro cellular models to investigate alcohol-induced expression changes of genes involved in alcohol metabolism (ALDH2), anti-apoptosis (BCL2 and CCND2), neurotransmission (NMDA receptor subunit genes: GRIN1, GRIN2A, GRIN2B, and GRIN2D), calcium channel activity (ITPR2), or transcriptional repression (JARID2). hESCs were differentiated into cortical neurons, which were characterized by immunostaining using antibodies against cortical neuron-specific biomarkers. Ethanol-induced gene expression changes were determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR). After a 7-day ethanol (50 mM) exposure followed by a 24-hour ethanol withdrawal treatment, five of the above nine genes (including all four NMDA receptor subunit genes) were highly upregulated (GRIN1: 1.93-fold, P = 0.003; GRIN2A: 1.40-fold, P = 0.003; GRIN2B: 1.75-fold, P = 0.002; GRIN2D: 1.86-fold, P = 0.048; BCL2: 1.34-fold, P = 0.031), and the results of GRIN1, GRIN2A, and GRIN2B survived multiple comparison correction. Our findings suggest that alcohol responsive genes, particularly NMDA receptor genes, play an important role in regulating neuronal function and mediating chronic alcohol consumption-induced neuroadaptations.

  15. A knowledge driven supervised learning approach to identify gene network of differentially up-regulated genes during neuronal senescence in Rattus norvegicus.

    PubMed

    Dholaniya, Pankaj Singh; Ghosh, Soumitra; Surampudi, Bapi Raju; Kondapi, Anand K

    2015-09-01

    Various approaches have been described to infer the gene interaction network from expression data. Several models based on computational and mathematical methods are available. The fundamental thing in the identification of the gene interaction is their biological relevance. Two genes belonging to the same pathway are more likely to affect the expression of each other than the genes of two different pathways. In the present study, interaction network of genes is described based on upregulated genes during neuronal senescence in the Cerebellar granule neurons of rat. We have adopted a supervised learning method and used it in combination with biological pathway information of the genes to develop a gene interaction network. Further modular analysis of the network has been done to identify senescence-related marker genes. Currently there is no adequate information available about the genes implicated in neuronal senescence. Thus identifying multipath genes belonging to the pathway affected by senescence might be very useful in studying the senescence process. PMID:26163927

  16. Gene transfer to subdermal tissues via a new gene gun design.

    PubMed

    Dileo, John; Miller, Theodore E; Chesnoy, Sophie; Huang, Leaf

    2003-01-01

    Although particle-mediated gene transfer technology (gene gun) has been applied for gene transfer to external tissues, the application of this technology to other tissues has met with limited success. Here we report the development of a new design of a gene gun that uses helium discharge to propel DNA-coated gold beads that are suspended in liquid. Higher discharge pressures allow for the delivery of DNA to deeper tissues. Using the new gene gun to deliver a luciferase expression plasmid resulted in higher levels of gene expression in the skin than observed with conventional guns, as well as in subdermal tissues, including subcutaneous tumors. Even when using as little as 125 ng of DNA, gene expression in skin and muscle reached its peak level at 24 hr postbombardment and remained for at least 1 week. The use of a LacZ expression plasmid showed that gene expression was distributed throughout the skin with no observable pathology. The new gene gun was used to deliver a model tumor rejection antigen (a modified human papilloma virus [HPV] E7 gene) to mice. All of the treated animals developed protective immunity against HPV-positive tumors. These results demonstrate that our new design can be used in standard gene gun applications and extends the reach of gene gun technology to tissues that were previously unavailable.

  17. Nano-Sized Sunflower Polycations As Effective Gene Transfer Vehicles.

    PubMed

    Cheng, Yilong; Wei, Hua; Tan, James-Kevin Y; Peeler, David J; Maris, Don O; Sellers, Drew L; Horner, Philip J; Pun, Suzie H

    2016-05-01

    The architecture of polycations plays an important role in both gene transfection efficiency and cytotoxicity. In this work, a new polymer, sunflower poly(2-dimethyl amino)ethyl methacrylate) (pDMAEMA), is prepared by atom transfer radical polymerization and employed as nucleic acid carriers compared to linear pDMAEMA homopolymer and comb pDMAEMA. The sunflower pDMAEMAs show higher IC50 , greater buffering capacity, and stronger binding capacity toward plasmid DNA than their linear and comb counterparts. In vitro transfection studies demonstrate that sunflower pDMAEMAs exhibit high transfection efficiency as well as relatively low cytotoxicity in complete growth medium. In vivo gene delivery by intraventricular injection to the brain shows that sunflower polymer delivers plasmid DNA more effectively than comb polymer. This study provides a new insight into the relationship between polymeric architecture and gene delivery capability, and as well as a useful means to design potent vectors for successful gene delivery. PMID:27061622

  18. Lactate promotes plasticity gene expression by potentiating NMDA signaling in neurons

    PubMed Central

    Yang, Jiangyan; Ruchti, Evelyne; Petit, Jean-Marie; Jourdain, Pascal; Grenningloh, Gabriele; Allaman, Igor; Magistretti, Pierre J.

    2014-01-01

    l-lactate is a product of aerobic glycolysis that can be used by neurons as an energy substrate. Here we report that in neurons l-lactate stimulates the expression of synaptic plasticity-related genes such as Arc, c-Fos, and Zif268 through a mechanism involving NMDA receptor activity and its downstream signaling cascade Erk1/2. l-lactate potentiates NMDA receptor-mediated currents and the ensuing increase in intracellular calcium. In parallel to this, l-lactate increases intracellular levels of NADH, thereby modulating the redox state of neurons. NADH mimics all of the effects of l-lactate on NMDA signaling, pointing to NADH increase as a primary mediator of l-lactate effects. The induction of plasticity genes is observed both in mouse primary neurons in culture and in vivo in the mouse sensory-motor cortex. These results provide insights for the understanding of the molecular mechanisms underlying the critical role of astrocyte-derived l-lactate in long-term memory and long-term potentiation in vivo. This set of data reveals a previously unidentified action of l-lactate as a signaling molecule for neuronal plasticity. PMID:25071212

  19. Dissecting the kinetics of the NADP(+)-FADH2 charge transfer complex and flavin semiquinones in neuronal nitric oxide synthase.

    PubMed

    Li, Huiying; Jamal, Joumana; Chreifi, Georges; Venkatesh, Vikram; Abou-Ziab, Hoda; Poulos, Thomas L

    2013-07-01

    Electron flow within the neuronal nitric oxide synthase reductase domain (nNOSrd) includes hydride transfer from NADPH to FAD followed by two one-electron transfer reactions from FAD to FMN. We have used stopped flow spectrometry to closely monitor these electron transfer steps for both the wild type and the ΔG810 mutant of nNOSrd using a protocol involving both global analyses of the photodiode array spectral scans and curve fittings of single wavelength kinetic traces. The charge transfer complex and interflavin electron transfer events recorded at 750nm and 600nm, respectively, show the kinetics in different time frames. All electron transfer events are slow enough at 4°C to enable measurements of rate constants even for the fast charge transfer event. To our knowledge this is the first time the rate constants for the charge transfer between NADP(+) and FADH2 have been determined for NOS. These procedures allow us to conclude that (1) binding of the second NADPH is necessary to drive the full reduction of FMN and; (2) charge transfer and the subsequent interflavin electron transfer have distinct spectral features that can be monitored separately with stopped flow spectroscopy. These studies also enable us to conclude that interflavin electron transfer reported at 600nm is not limiting in NOS catalysis.

  20. A role for neural determination genes in specifying the dorsoventral identity of telencephalic neurons

    PubMed Central

    Fode, Carol; Ma, Qiufu; Casarosa, Simona; Ang, Siew-Lan; Anderson, David J.; Guillemot, François

    2000-01-01

    Neurogenin1 (Ngn1), Neurogenin2 (Ngn2), and Mash1 encode bHLH transcription factors with neuronal determination functions. In the telencephalon, the Ngns and Mash1 are expressed at high levels in complementary dorsal and ventral domains, respectively. We found that Ngn function is required to maintain these two separate expression domains, as Mash1 expression is up-regulated in the dorsal telencephalon of Ngn mutant embryos. We have taken advantage of the replacement of the Ngns by Mash1 in dorsal progenitors to address the role of the neural determination genes in neuronal-type specification in the telencephalon. In Ngn2 single and Ngn1; Ngn2 double mutants, a population of early born cortical neurons lose expression of dorsal-specific markers and ectopically express a subset of ventral telencephalic-specific markers. Analysis of Mash1; Ngn2 double mutant embryos and of embryos carrying a Ngn2 to Mash1 replacement mutation demonstrated that ectopic expression of Mash1 is required and sufficient to confer these ventral characteristics to cortical neurons. Our results indicate that in addition to acting as neuronal determinants, Mash1 and Ngns play a role in the specification of dorsal-ventral neuronal identity, directly linking pathways of neurogenesis and regional patterning in the forebrain. PMID:10640277

  1. Ccm3, a gene associated with cerebral cavernous malformations, is required for neuronal migration.

    PubMed

    Louvi, Angeliki; Nishimura, Sayoko; Günel, Murat

    2014-03-01

    Loss of function of cerebral cavernous malformation 3 (CCM3) results in an autosomal dominant cerebrovascular disorder. Here, we uncover a developmental role for CCM3 in regulating neuronal migration in the neocortex. Using cell type-specific gene inactivation in mice, we show that CCM3 has both cell autonomous and cell non-autonomous functions in neural progenitors and is specifically required in radial glia and newly born pyramidal neurons migrating through the subventricular zone, but not in those migrating through the cortical plate. Loss of CCM3 function leads to RhoA activation, alterations in the actin and microtubule cytoskeleton affecting neuronal morphology, and abnormalities in laminar positioning of primarily late-born neurons, indicating CCM3 involvement in radial glia-dependent locomotion and possible interaction with the Cdk5/RhoA pathway. Thus, we identify a novel cytoplasmic regulator of neuronal migration and demonstrate that its inactivation in radial glia progenitors and nascent neurons produces severe malformations of cortical development. PMID:24595293

  2. Electroporation-mediated gene transfer directly to the swine heart.

    PubMed

    Hargrave, B; Downey, H; Strange, R; Murray, L; Cinnamond, C; Lundberg, C; Israel, A; Chen, Y-J; Marshall, W; Heller, R

    2013-02-01

    In vivo gene transfer to the ischemic heart via electroporation holds promise as a potential therapeutic approach for the treatment of heart disease. In the current study, we investigated the use of in vivo electroporation for gene transfer using three different penetrating electrodes and one non-penetrating electrode. The hearts of adult male swine were exposed through a sternotomy. Eight electric pulses synchronized to the rising phase of the R wave of the electrocardiogram were administered at varying pulse widths and field strengths following an injection of either a plasmid encoding luciferase or one encoding green fluorescent protein. Four sites on the anterior wall of the left ventricle were treated. Animals were killed 48 h after injection and electroporation and gene expression was determined. Results were compared with sites in the heart that received plasmid injection but no electric pulses or were not treated. Gene expression was higher in all electroporated sites when compared with injection only sites demonstrating the robustness of this approach. Our results provide evidence that in vivo electroporation can be a safe and effective non-viral method for delivering genes to the heart, in vivo.

  3. Endosymbiotic gene transfer in tertiary plastid-containing dinoflagellates.

    PubMed

    Burki, Fabien; Imanian, Behzad; Hehenberger, Elisabeth; Hirakawa, Yoshihisa; Maruyama, Shinichiro; Keeling, Patrick J

    2014-02-01

    Plastid establishment involves the transfer of endosymbiotic genes to the host nucleus, a process known as endosymbiotic gene transfer (EGT). Large amounts of EGT have been shown in several photosynthetic lineages but also in present-day plastid-lacking organisms, supporting the notion that endosymbiotic genes leave a substantial genetic footprint in the host nucleus. Yet the extent of this genetic relocation remains debated, largely because the long period that has passed since most plastids originated has erased many of the clues to how this process unfolded. Among the dinoflagellates, however, the ancestral peridinin-containing plastid has been replaced by tertiary plastids on several more recent occasions, giving us a less ancient window to examine plastid origins. In this study, we evaluated the endosymbiotic contribution to the host genome in two dinoflagellate lineages with tertiary plastids. We generated the first nuclear transcriptome data sets for the "dinotoms," which harbor diatom-derived plastids, and analyzed these data in combination with the available transcriptomes for kareniaceans, which harbor haptophyte-derived plastids. We found low level of detectable EGT in both dinoflagellate lineages, with only 9 genes and 90 genes of possible tertiary endosymbiotic origin in dinotoms and kareniaceans, respectively, suggesting that tertiary endosymbioses did not heavily impact the host dinoflagellate genomes.

  4. Characterization of an Ancient Lepidopteran Lateral Gene Transfer

    PubMed Central

    Wheeler, David; Redding, Amanda J.; Werren, John H.

    2013-01-01

    Bacteria to eukaryote lateral gene transfers (LGT) are an important potential source of material for the evolution of novel genetic traits. The explosion in the number of newly sequenced genomes provides opportunities to identify and characterize examples of these lateral gene transfer events, and to assess their role in the evolution of new genes. In this paper, we describe an ancient lepidopteran LGT of a glycosyl hydrolase family 31 gene (GH31) from an Enterococcus bacteria. PCR amplification between the LGT and a flanking insect gene confirmed that the GH31 was integrated into the Bombyx mori genome and was not a result of an assembly error. Database searches in combination with degenerate PCR on a panel of 7 lepidopteran families confirmed that the GH31 LGT event occurred deep within the Order approximately 65–145 million years ago. The most basal species in which the LGT was found is Plutella xylostella (superfamily: Yponomeutoidea). Array data from Bombyx mori shows that GH31 is expressed, and low dN/dS ratios indicates the LGT coding sequence is under strong stabilizing selection. These findings provide further support for the proposition that bacterial LGTs are relatively common in insects and likely to be an underappreciated source of adaptive genetic material. PMID:23533610

  5. Glucocerebrosidase gene therapy prevents α-synucleinopathy of midbrain dopamine neurons.

    PubMed

    Rocha, Emily M; Smith, Gaynor A; Park, Eric; Cao, Hongmei; Brown, Eilish; Hayes, Melissa A; Beagan, Jonathan; McLean, Jesse R; Izen, Sarah C; Perez-Torres, Eduardo; Hallett, Penelope J; Isacson, Ole

    2015-10-01

    Diminished lysosomal function can lead to abnormal cellular accumulation of specific proteins, including α-synuclein, contributing to disease pathogenesis of vulnerable neurons in Parkinson's disease (PD) and related α-synucleinopathies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase (GCase), and mutations in GBA1 are a prominent genetic risk factor for PD. Previous studies showed that in sporadic PD, and in normal aging, GCase brain activity is reduced and levels of corresponding glycolipid substrates are increased. The present study tested whether increasing GCase through AAV-GBA1 intra-cerebral gene delivery in two PD rodent models would reduce the accumulation of α-synuclein and protect midbrain dopamine neurons from α-synuclein-mediated neuronal damage. In the first model, transgenic mice overexpressing wildtype α-synuclein throughout the brain (ASO mice) were used, and in the second model, a rat model of selective dopamine neuron degeneration was induced by AAV-A53T mutant α-synuclein. In ASO mice, intra-cerebral AAV-GBA1 injections into several brain regions increased GCase activity and reduced the accumulation of α-synuclein in the substantia nigra and striatum. In rats, co-injection of AAV-GBA1 with AAV-A53T α-synuclein into the substantia nigra prevented α-synuclein-mediated degeneration of nigrostriatal dopamine neurons by 6 months. These neuroprotective effects were associated with altered protein expression of markers of autophagy. These experiments demonstrate, for the first time, the neuroprotective effects of increasing GCase against dopaminergic neuron degeneration, and support the development of therapeutics targeting GCase or other lysosomal genes to improve neuronal handling of α-synuclein.

  6. Tau deficiency leads to the upregulation of BAF-57, a protein involved in neuron-specific gene repression.

    PubMed

    de Barreda, Elena Gómez; Dawson, Hana N; Vitek, Michel P; Avila, Jesús

    2010-06-01

    Although tau is mainly located in the cell cytoplasm, mostly bound to tubulin, it may also be found in the nucleus of neurons. Hence, we tested whether tau might play a role in regulating the expression of certain genes by comparing gene expression in mice containing or lacking the tau protein. Our results identified a significant difference in the expression of the smarce1 gene, which codes for the BAF-57 protein, a protein involved in the repression of neuron specific genes. These data suggest a role for tau in neuron maturation.

  7. Gene transfer from a parasitic flowering plant to a fern

    PubMed Central

    Davis, Charles C; Anderson, William R; Wurdack, Kenneth J

    2005-01-01

    The rattlesnake fern (Botrychium virginianum (L.) Sw.) is obligately mycotrophic and widely distributed across the northern hemisphere. Three mitochondrial gene regions place this species with other ferns in Ophioglossaceae, while two regions place it as a member of the largely parasitic angiosperm order Santalales (sandalwoods and mistletoes). These discordant phylogenetic placements suggest that part of the genome in B. virginianum was acquired by horizontal gene transfer (HGT), perhaps from root-parasitic Loranthaceae. These transgenes are restricted to B. virginianum and occur across the range of the species. Molecular and life-history traits indicate that the transfer preceded the global expansion of B. virginianum, and that the latter may have happened very rapidly. This is the first report of HGT from an angiosperm to a fern, through either direct parasitism or the mediation of interconnecting fungal symbionts. PMID:16191635

  8. Adenovirus serotype 5 hexon mediates liver gene transfer.

    PubMed

    Waddington, Simon N; McVey, John H; Bhella, David; Parker, Alan L; Barker, Kristeen; Atoda, Hideko; Pink, Rebecca; Buckley, Suzanne M K; Greig, Jenny A; Denby, Laura; Custers, Jerome; Morita, Takashi; Francischetti, Ivo M B; Monteiro, Robson Q; Barouch, Dan H; van Rooijen, Nico; Napoli, Claudio; Havenga, Menzo J E; Nicklin, Stuart A; Baker, Andrew H

    2008-02-01

    Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo. PMID:18267072

  9. Neuronal RARβ Signaling Modulates PTEN Activity Directly in Neurons and via Exosome Transfer in Astrocytes to Prevent Glial Scar Formation and Induce Spinal Cord Regeneration

    PubMed Central

    Goncalves, Maria B.; Malmqvist, Tony; Clarke, Earl; Hubens, Chantal J.; Grist, John; Hobbs, Carl; Trigo, Diogo; Risling, Mårten; Angeria, Maria; Damberg, Peter; Carlstedt, Thomas P.

    2015-01-01

    Failure of axonal regeneration in the central nervous system (CNS) is mainly attributed to a lack of intrinsic neuronal growth programs and an inhibitory environment from a glial scar. Phosphatase and tensin homolog (PTEN) is a major negative regulator of neuronal regeneration and, as such, inhibiting its activity has been considered a therapeutic target for spinal cord (SC) injuries (SCIs). Using a novel model of rat cervical avulsion, we show that treatment with a retinoic acid receptor β (RARβ) agonist results in locomotor and sensory recovery. Axonal regeneration from the severed roots into the SC could be seen by biotinylated dextran amine labeling. Light micrographs of the dorsal root entry zone show the peripheral nervous system (PNS)–CNS transition of regrown axons. RARβ agonist treatment also resulted in the absence of scar formation. Mechanism studies revealed that, in RARβ-agonist-treated neurons, PTEN activity is decreased by cytoplasmic phosphorylation and increased secretion in exosomes. These are taken up by astrocytes, resulting in hampered proliferation and causing them to arrange in a normal-appearing scaffold around the regenerating axons. Attribution of the glial modulation to neuronal PTEN in exosomes was demonstrated by the use of an exosome inhibitor in vivo and PTEN siRNA in vitro assays. The dual effect of RARβ signaling, both neuronal and neuronal–glial, results in axonal regeneration into the SC after dorsal root neurotmesis. Targeting this pathway may open new avenues for the treatment of SCIs. SIGNIFICANCE STATEMENT Spinal cord injuries (SCIs) often result in permanent damage in the adult due to the very limited capacity of axonal regeneration. Intrinsic neuronal programs and the formation of a glial scar are the main obstacles. Here, we identify a single target, neuronal retinoic acid receptor β (RARβ), which modulates these two aspects of the postinjury physiological response. Activation of RARβ in the neuron inactivates

  10. Detection of homologous horizontal gene transfer in SNP data

    2012-07-23

    We study the detection of mutations, sequencing errors, and homologous horizontal gene transfers (HGT) in a set of closely related microbial genomes. We base the model on single nucleotide polymorphisms (SNP's) and break the genomes into blocks to handle the rearrangement problem. Then we apply a synamic programming algorithm to model whether changes within each block are likely a result of mutations, sequencing errors, or HGT.

  11. Horizontal gene transfer is a significant driver of gene innovation in dinoflagellates.

    PubMed

    Wisecaver, Jennifer H; Brosnahan, Michael L; Hackett, Jeremiah D

    2013-01-01

    The dinoflagellates are an evolutionarily and ecologically important group of microbial eukaryotes. Previous work suggests that horizontal gene transfer (HGT) is an important source of gene innovation in these organisms. However, dinoflagellate genomes are notoriously large and complex, making genomic investigation of this phenomenon impractical with currently available sequencing technology. Fortunately, de novo transcriptome sequencing and assembly provides an alternative approach for investigating HGT. We sequenced the transcriptome of the dinoflagellate Alexandrium tamarense Group IV to investigate how HGT has contributed to gene innovation in this group. Our comprehensive A. tamarense Group IV gene set was compared with those of 16 other eukaryotic genomes. Ancestral gene content reconstruction of ortholog groups shows that A. tamarense Group IV has the largest number of gene families gained (314-1,563 depending on inference method) relative to all other organisms in the analysis (0-782). Phylogenomic analysis indicates that genes horizontally acquired from bacteria are a significant proportion of this gene influx, as are genes transferred from other eukaryotes either through HGT or endosymbiosis. The dinoflagellates also display curious cases of gene loss associated with mitochondrial metabolism including the entire Complex I of oxidative phosphorylation. Some of these missing genes have been functionally replaced by bacterial and eukaryotic xenologs. The transcriptome of A. tamarense Group IV lends strong support to a growing body of evidence that dinoflagellate genomes are extraordinarily impacted by HGT.

  12. Gene dosage-dependent rescue of HSP neurite defects in SPG4 patients’ neurons

    PubMed Central

    Havlicek, Steven; Kohl, Zacharias; Mishra, Himanshu K.; Prots, Iryna; Eberhardt, Esther; Denguir, Naime; Wend, Holger; Plötz, Sonja; Boyer, Leah; Marchetto, Maria C.N.; Aigner, Stefan; Sticht, Heinrich; Groemer, Teja W.; Hehr, Ute; Lampert, Angelika; Schlötzer-Schrehardt, Ursula; Winkler, Jürgen; Gage, Fred H.; Winner, Beate

    2014-01-01

    The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients. PMID:24381312

  13. Risks from GMOs due to horizontal gene transfer.

    PubMed

    Keese, Paul

    2008-01-01

    Horizontal gene transfer (HGT) is the stable transfer of genetic material from one organism to another without reproduction or human intervention. Transfer occurs by the passage of donor genetic material across cellular boundaries, followed by heritable incorporation to the genome of the recipient organism. In addition to conjugation, transformation and transduction, other diverse mechanisms of DNA and RNA uptake occur in nature. The genome of almost every organism reveals the footprint of many ancient HGT events. Most commonly, HGT involves the transmission of genes on viruses or mobile genetic elements. HGT first became an issue of public concern in the 1970s through the natural spread of antibiotic resistance genes amongst pathogenic bacteria, and more recently with commercial production of genetically modified (GM) crops. However, the frequency of HGT from plants to other eukaryotes or prokaryotes is extremely low. The frequency of HGT to viruses is potentially greater, but is restricted by stringent selection pressures. In most cases the occurrence of HGT from GM crops to other organisms is expected to be lower than background rates. Therefore, HGT from GM plants poses negligible risks to human health or the environment.

  14. Neuronal cell adhesion genes and antidepressant response in three independent samples.

    PubMed

    Fabbri, C; Crisafulli, C; Gurwitz, D; Stingl, J; Calati, R; Albani, D; Forloni, G; Calabrò, M; Martines, R; Kasper, S; Zohar, J; Juven-Wetzler, A; Souery, D; Montgomery, S; Mendlewicz, J; Girolamo, G D; Serretti, A

    2015-12-01

    Drug-effect phenotypes in human lymphoblastoid cell lines recently allowed to identify CHL1 (cell adhesion molecule with homology to L1CAM), GAP43 (growth-associated protein 43) and ITGB3 (integrin beta 3) as new candidates for involvement in the antidepressant effect. CHL1 and ITGB3 code for adhesion molecules, while GAP43 codes for a neuron-specific cytosolic protein expressed in neuronal growth cones; all the three gene products are involved in synaptic plasticity. Sixteen polymorphisms in these genes were genotyped in two samples (n=369 and 90) with diagnosis of major depressive episode who were treated with antidepressants in a naturalistic setting. Phenotypes were response, remission and treatment-resistant depression. Logistic regression including appropriate covariates was performed. Genes associated with outcomes were investigated in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) genome-wide study (n=1861) as both individual genes and through a pathway analysis (Reactome and String databases). Gene-based analysis suggested CHL1 rs4003413, GAP43 rs283393 and rs9860828, ITGB3 rs3809865 as the top candidates due to their replication across the largest original sample and the STAR*D cohort. GAP43 molecular pathway was associated with both response and remission in the STAR*D, with ELAVL4 representing the gene with the highest percentage of single nucleotide polymorphisms (SNPs) associated with outcomes. Other promising genes emerging from the pathway analysis were ITGB1 and NRP1. The present study was the first to analyze cell adhesion genes and their molecular pathways in antidepressant response. Genes and biomarkers involved in neuronal adhesion should be considered by further studies aimed to identify predictors of antidepressant response. PMID:25850031

  15. The study of the Bithorax-complex genes in patterning CCAP neurons reveals a temporal control of neuronal differentiation by Abd-B

    PubMed Central

    Moris-Sanz, M.; Estacio-Gómez, A.; Sánchez-Herrero, E.; Díaz-Benjumea, F. J.

    2015-01-01

    ABSTRACT During development, HOX genes play critical roles in the establishment of segmental differences. In the Drosophila central nervous system, these differences are manifested in the number and type of neurons generated by each neuroblast in each segment. HOX genes can act either in neuroblasts or in postmitotic cells, and either early or late in a lineage. Additionally, they can be continuously required during development or just at a specific stage. Moreover, these features are generally segment-specific. Lately, it has been shown that contrary to what happens in other tissues, where HOX genes define domains of expression, these genes are expressed in individual cells as part of the combinatorial codes involved in cell type specification. In this report we analyse the role of the Bithorax-complex genes – Ultrabithorax, abdominal-A and Abdominal-B – in sculpting the pattern of crustacean cardioactive peptide (CCAP)-expressing neurons. These neurons are widespread in invertebrates, express CCAP, Bursicon and MIP neuropeptides and play major roles in controlling ecdysis. There are two types of CCAP neuron: interneurons and efferent neurons. Our results indicate that Ultrabithorax and Abdominal-A are not necessary for specification of the CCAP-interneurons, but are absolutely required to prevent the death by apoptosis of the CCAP-efferent neurons. Furthermore, Abdominal-B controls by repression the temporal onset of neuropeptide expression in a subset of CCAP-efferent neurons, and a peak of ecdysone hormone at the end of larval life counteracts this repression. Thus, Bithorax complex genes control the developmental appearance of these neuropeptides both temporally and spatially. PMID:26276099

  16. Dynamic monitoring of horizontal gene transfer in soil

    NASA Astrophysics Data System (ADS)

    Cheng, H. Y.; Masiello, C. A.; Silberg, J. J.; Bennett, G. N.

    2015-12-01

    Soil microbial gene expression underlies microbial behaviors (phenotypes) central to many aspects of C, N, and H2O cycling. However, continuous monitoring of microbial gene expression in soils is challenging because genetically-encoded reporter proteins widely used in the lab are difficult to deploy in soil matrices: for example, green fluorescent protein cannot be easily visualized in soils, even in the lab. To address this problem we have developed a reporter protein that releases small volatile gases. Here, we applied this gas reporter in a proof-of-concept soil experiment, monitoring horizontal gene transfer, a microbial activity that alters microbial genotypes and phenotypes. Horizontal gene transfer is central to bacterial evolution and adaptation and is relevant to problems such as the spread of antibiotic resistance, increasing metal tolerance in superfund sites, and bioremediation capability of bacterial consortia. This process is likely to be impacted by a number of matrix properties not well-represented in the petri dish, such as microscale variations in water, nutrients, and O2, making petri-dish experiments a poor proxy for environmental processes. We built a conjugation system using synthetic biology to demonstrate the use of gas-reporting biosensors in safe, lab-based biogeochemistry experiments, and here we report the use of these sensors to monitor horizontal gene transfer in soils. Our system is based on the F-plasmid conjugation in Escherichia coli. We have found that the gas signal reports on the number of cells that acquire F-plasmids (transconjugants) in a loamy Alfisol collected from Kellogg Biological Station. We will report how a gas signal generated by transconjugants varies with the number of F-plasmid donor and acceptor cells seeded in a soil, soil moisture, and soil O2 levels.

  17. Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene.

    PubMed Central

    von der Leyen, H E; Gibbons, G H; Morishita, R; Lewis, N P; Zhang, L; Nakajima, M; Kaneda, Y; Cooke, J P; Dzau, V J

    1995-01-01

    It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as beta-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessels. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia. Images Fig. 1 Fig. 2 Fig. 5 PMID:7532305

  18. Gene Therapy Inhibiting Neointimal Vascular Lesion: In vivo Transfer of Endothelial Cell Nitric Oxide Synthase Gene

    NASA Astrophysics Data System (ADS)

    von der Leyen, Heiko E.; Gibbons, Gary H.; Morishita, Ryuichi; Lewis, Neil P.; Zhang, Lunan; Nakajima, Masatoshi; Kaneda, Yasufumi; Cooke, John P.; Dzau, Victor J.

    1995-02-01

    It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as β-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessel. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.

  19. Enhanced Excitability of Primary Sensory Neurons and Altered Gene Expression of Neuronal Ion Channels in Dorsal Root Ganglion in Paclitaxel-Induced Peripheral Neuropathy

    PubMed Central

    Zhang, Haijun; Dougherty, Patrick M.

    2014-01-01

    Background The mechanism of chemotherapy-induced peripheral neuropathy after paclitaxel treatment is not well understood. Given the poor penetration of paclitaxel into central nervous system, peripheral nervous system is most at risk. Methods Intrinsic membrane properties of dorsal root ganglion (DRG) neurons were studied by intracellular recordings. Multiple-gene real-time Polymerase Chain Reaction array was used to investigate gene expression of DRG neuronal ion channels. Results Paclitaxel increased the incidence of spontaneous activity from 4.8% to 27.1% in large and from 0% to 33.3% in medium-sized neurons. Paclitaxel decreased the rheobase (nA) from 1.6 ± 0.1 to 0.8 ± 0.1 in large, from 1.5 ± 0.2 to 0.6 ± 0.1 in medium-sized, and from 1.6 ± 0.2 to 1.0 ± 0.1 in small neurons. After paclitaxel, other characteristics of membrane properties in each group remained the same except that Aδ neurons showed shorter action potential fall time (ms) (1.0 ± 0.2, n = 10 vs. 1.8 ± 0.3, n = 9, paclitaxel vs. vehicle). Meanwhile, real-time polymerase chain reaction array revealed an alteration in expression of some neuronal ion channel genes including upregulation of HCN1 (fold change 1.76 ± 0.06) and Nav1.7 (1.26 ± 0.02) and downregulation of Kir channels (Kir1.1, 0.73 ± 0.05, Kir3.4, 0.66 ± 0.06) in paclitaxel-treated animals. Conclusions The increased neuronal excitability and the changes in gene expression of some neuronal ion channels in DRG may provide insight into the molecular and cellular basis of paclitaxel neuropathy, which may lead to novel therapeutic strategies. PMID:24534904

  20. Horizontal Gene Transfer Contributes to the Evolution of Arthropod Herbivory

    PubMed Central

    Wybouw, Nicky; Pauchet, Yannick; Heckel, David G.; Van Leeuwen, Thomas

    2016-01-01

    Within animals, evolutionary transition toward herbivory is severely limited by the hostile characteristics of plants. Arthropods have nonetheless counteracted many nutritional and defensive barriers imposed by plants and are currently considered as the most successful animal herbivores in terrestrial ecosystems. We gather a body of evidence showing that genomes of various plant feeding insects and mites possess genes whose presence can only be explained by horizontal gene transfer (HGT). HGT is the asexual transmission of genetic information between reproductively isolated species. Although HGT is known to have great adaptive significance in prokaryotes, its impact on eukaryotic evolution remains obscure. Here, we show that laterally transferred genes into arthropods underpin many adaptations to phytophagy, including efficient assimilation and detoxification of plant produced metabolites. Horizontally acquired genes and the traits they encode often functionally diversify within arthropod recipients, enabling the colonization of more host plant species and organs. We demonstrate that HGT can drive metazoan evolution by uncovering its prominent role in the adaptations of arthropods to exploit plants. PMID:27307274

  1. A Sympathetic Neuron Autonomous Role for Egr3-Mediated Gene Regulation in Dendrite Morphogenesis and Target Tissue Innervation

    PubMed Central

    Quach, David H.; Oliveira-Fernandes, Michelle; Gruner, Katherine A.; Tourtellotte, Warren G.

    2013-01-01

    Egr3 is a nerve growth factor (NGF)-induced transcriptional regulator that is essential for normal sympathetic nervous system development. Mice lacking Egr3 in the germline have sympathetic target tissue innervation abnormalities and physiologic sympathetic dysfunction similar to humans with dysautonomia. However, since Egr3 is widely expressed and has pleiotropic function, it has not been clear whether it has a role within sympathetic neurons and if so, what target genes it regulates to facilitate target tissue innervation. Here, we show that Egr3 expression within sympathetic neurons is required for their normal innervation since isolated sympathetic neurons lacking Egr3 have neurite outgrowth abnormalities when treated with NGF and mice with sympathetic neuron-restricted Egr3 ablation have target tissue innervation abnormalities similar to mice lacking Egr3 in all tissues. Microarray analysis performed on sympathetic neurons identified many target genes deregulated in the absence of Egr3, with some of the most significantly deregulated genes having roles in axonogenesis, dendritogenesis, and axon guidance. Using a novel genetic technique to visualize axons and dendrites in a subpopulation of randomly labeled sympathetic neurons, we found that Egr3 has an essential role in regulating sympathetic neuron dendrite morphology and terminal axon branching, but not in regulating sympathetic axon guidance to their targets. Together, these results indicate that Egr3 has a sympathetic neuron autonomous role in sympathetic nervous system development that involves modulating downstream target genes affecting the outgrowth and branching of sympathetic neuron dendrites and axons. PMID:23467373

  2. HGTree: database of horizontally transferred genes determined by tree reconciliation

    PubMed Central

    Jeong, Hyeonsoo; Sung, Samsun; Kwon, Taehyung; Seo, Minseok; Caetano-Anollés, Kelsey; Choi, Sang Ho; Cho, Seoae; Nasir, Arshan; Kim, Heebal

    2016-01-01

    The HGTree database provides putative genome-wide horizontal gene transfer (HGT) information for 2472 completely sequenced prokaryotic genomes. This task is accomplished by reconstructing approximate maximum likelihood phylogenetic trees for each orthologous gene and corresponding 16S rRNA reference species sets and then reconciling the two trees under parsimony framework. The tree reconciliation method is generally considered to be a reliable way to detect HGT events but its practical use has remained limited because the method is computationally intensive and conceptually challenging. In this regard, HGTree (http://hgtree.snu.ac.kr) represents a useful addition to the biological community and enables quick and easy retrieval of information for HGT-acquired genes to better understand microbial taxonomy and evolution. The database is freely available and can be easily scaled and updated to keep pace with the rapid rise in genomic information. PMID:26578597

  3. Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity.

    PubMed

    Yonehara, Keisuke; Fiscella, Michele; Drinnenberg, Antonia; Esposti, Federico; Trenholm, Stuart; Krol, Jacek; Franke, Felix; Scherf, Brigitte Gross; Kusnyerik, Akos; Müller, Jan; Szabo, Arnold; Jüttner, Josephine; Cordoba, Francisco; Reddy, Ashrithpal Police; Németh, János; Nagy, Zoltán Zsolt; Munier, Francis; Hierlemann, Andreas; Roska, Botond

    2016-01-01

    Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease. PMID:26711119

  4. Congenital Nystagmus Gene FRMD7 Is Necessary for Establishing a Neuronal Circuit Asymmetry for Direction Selectivity

    PubMed Central

    Yonehara, Keisuke; Fiscella, Michele; Drinnenberg, Antonia; Esposti, Federico; Trenholm, Stuart; Krol, Jacek; Franke, Felix; Scherf, Brigitte Gross; Kusnyerik, Akos; Müller, Jan; Szabo, Arnold; Jüttner, Josephine; Cordoba, Francisco; Reddy, Ashrithpal Police; Németh, János; Nagy, Zoltán Zsolt; Munier, Francis; Hierlemann, Andreas; Roska, Botond

    2016-01-01

    Summary Neuronal circuit asymmetries are important components of brain circuits, but the molecular pathways leading to their establishment remain unknown. Here we found that the mutation of FRMD7, a gene that is defective in human congenital nystagmus, leads to the selective loss of the horizontal optokinetic reflex in mice, as it does in humans. This is accompanied by the selective loss of horizontal direction selectivity in retinal ganglion cells and the transition from asymmetric to symmetric inhibitory input to horizontal direction-selective ganglion cells. In wild-type retinas, we found FRMD7 specifically expressed in starburst amacrine cells, the interneuron type that provides asymmetric inhibition to direction-selective retinal ganglion cells. This work identifies FRMD7 as a key regulator in establishing a neuronal circuit asymmetry, and it suggests the involvement of a specific inhibitory neuron type in the pathophysiology of a neurological disease. Video Abstract PMID:26711119

  5. Neurons That Underlie Drosophila melanogaster Reproductive Behaviors: Detection of a Large Male-Bias in Gene Expression in fruitless-Expressing Neurons.

    PubMed

    Newell, Nicole R; New, Felicia N; Dalton, Justin E; McIntyre, Lauren M; Arbeitman, Michelle N

    2016-01-01

    Male and female reproductive behaviors in Drosophila melanogaster are vastly different, but neurons that express sex-specifically spliced fruitless transcripts (fru P1) underlie these behaviors in both sexes. How this set of neurons can generate such different behaviors between the two sexes is an unresolved question. A particular challenge is that fru P1-expressing neurons comprise only 2-5% of the adult nervous system, and so studies of adult head tissue or whole brain may not reveal crucial differences. Translating Ribosome Affinity Purification (TRAP) identifies the actively translated pool of mRNAs from fru P1-expressing neurons, allowing a sensitive, cell-type-specific assay. We find four times more male-biased than female-biased genes in TRAP mRNAs from fru P1-expressing neurons. This suggests a potential mechanism to generate dimorphism in behavior. The male-biased genes may direct male behaviors by establishing cell fate in a similar context of gene expression observed in females. These results suggest a possible global mechanism for how distinct behaviors can arise from a shared set of neurons. PMID:27247289

  6. Neurons That Underlie Drosophila melanogaster Reproductive Behaviors: Detection of a Large Male-Bias in Gene Expression in fruitless-Expressing Neurons

    PubMed Central

    Newell, Nicole R.; New, Felicia N.; Dalton, Justin E.; McIntyre, Lauren M.; Arbeitman, Michelle N.

    2016-01-01

    Male and female reproductive behaviors in Drosophila melanogaster are vastly different, but neurons that express sex-specifically spliced fruitless transcripts (fru P1) underlie these behaviors in both sexes. How this set of neurons can generate such different behaviors between the two sexes is an unresolved question. A particular challenge is that fru P1-expressing neurons comprise only 2–5% of the adult nervous system, and so studies of adult head tissue or whole brain may not reveal crucial differences. Translating Ribosome Affinity Purification (TRAP) identifies the actively translated pool of mRNAs from fru P1-expressing neurons, allowing a sensitive, cell-type-specific assay. We find four times more male-biased than female-biased genes in TRAP mRNAs from fru P1-expressing neurons. This suggests a potential mechanism to generate dimorphism in behavior. The male-biased genes may direct male behaviors by establishing cell fate in a similar context of gene expression observed in females. These results suggest a possible global mechanism for how distinct behaviors can arise from a shared set of neurons. PMID:27247289

  7. CHD5, a brain-specific paralog of Mi2 chromatin remodeling enzymes, regulates expression of neuronal genes.

    PubMed

    Potts, Rebecca Casaday; Zhang, Peisu; Wurster, Andrea L; Precht, Patricia; Mughal, Mohamed R; Wood, William H; Zhang, Yonqing; Becker, Kevin G; Mattson, Mark P; Pazin, Michael J

    2011-01-01

    CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease.

  8. CHD5, a Brain-Specific Paralog of Mi2 Chromatin Remodeling Enzymes, Regulates Expression of Neuronal Genes

    PubMed Central

    Potts, Rebecca Casaday; Zhang, Peisu; Wurster, Andrea L.; Precht, Patricia; Mughal, Mohamed R.; Wood, William H.; Zhang, Yonqing; Becker, Kevin G.; Mattson, Mark P.; Pazin, Michael J.

    2011-01-01

    CHD5 is frequently deleted in neuroblastoma and is a tumor suppressor gene. However, little is known about the role of CHD5 other than it is homologous to chromatin remodeling ATPases. We found CHD5 mRNA was restricted to the brain; by contrast, most remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66ß, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Expression of gene sets linked to aging and Alzheimer's disease were strongly altered by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 protein is found in a NuRD-like multi-protein complex. CHD5 expression is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of genes implicated in aging and Alzheimer's disease. PMID:21931736

  9. Altered Expression of Genes Encoding Neurotransmitter Receptors in GnRH Neurons of Proestrous Mice

    PubMed Central

    Vastagh, Csaba; Rodolosse, Annie; Solymosi, Norbert; Liposits, Zsolt

    2016-01-01

    Gonadotropin-releasing hormone (GnRH) neurons play a key role in the central regulation of reproduction. In proestrous female mice, estradiol triggers the pre-ovulatory GnRH surge, however, its impact on the expression of neurotransmitter receptor genes in GnRH neurons has not been explored yet. We hypothesized that proestrus is accompanied by substantial changes in the expression profile of genes coding for neurotransmitter receptors in GnRH neurons. We compared the transcriptome of GnRH neurons obtained from intact, proestrous, and metestrous female GnRH-GFP transgenic mice, respectively. About 1500 individual GnRH neurons were sampled from both groups and their transcriptome was analyzed using microarray hybridization and real-time PCR. In this study, changes in mRNA expression of genes involved in neurotransmitter signaling were investigated. Differential gene expression was most apparent in GABA-ergic (Gabbr1, Gabra3, Gabrb3, Gabrb2, Gabrg2), glutamatergic (Gria1, Gria2, Grin1, Grin3a, Grm1, Slc17a6), cholinergic (Chrnb2, Chrm4) and dopaminergic (Drd3, Drd4), adrenergic (Adra1b, Adra2a, Adra2c), adenosinergic (Adora2a, Adora2b), glycinergic (Glra), purinergic (P2rx7), and serotonergic (Htr1b) receptors. In concert with these events, expression of genes in the signaling pathways downstream to the receptors, i.e., G-proteins (Gnai1, Gnai2, Gnas), adenylate-cyclases (Adcy3, Adcy5), protein kinase A (Prkaca, Prkacb) protein kinase C (Prkca) and certain transporters (Slc1a4, Slc17a6, Slc6a17) were also changed. The marked differences found in the expression of genes involved in neurotransmitter signaling of GnRH neurons at pro- and metestrous stages of the ovarian cycle indicate the differential contribution of these neurotransmitter systems to the induction of the pre-ovulatory GnRH surge, the known prerequisite of the subsequent hormonal cascade inducing ovulation. PMID:27774052

  10. Downregulation of immediate-early genes linking to suppression of neuronal plasticity in rats after 28-day exposure to glycidol

    SciTech Connect

    Akane, Hirotoshi; Saito, Fumiyo; Shiraki, Ayako; Takeyoshi, Masahiro; Imatanaka, Nobuya; Itahashi, Megu; Murakami, Tomoaki; Shibutani, Makoto

    2014-09-01

    We previously found that the 28-day oral toxicity study of glycidol at 200 mg/kg/day in rats resulted in axonopathy in both the central and peripheral nervous systems and aberrations in the late-stage of hippocampal neurogenesis targeting the process of neurite extension. To capture the neuronal parameters in response to glycidol toxicity, these animals were subjected to region-specific global gene expression profiling in four regions of cerebral and cerebellar architectures, followed by immunohistochemical analysis of selected gene products. Expression changes of genes related to axonogenesis and synaptic transmission were observed in the hippocampal dentate gyrus, cingulate cortex and cerebellar vermis at 200 mg/kg showing downregulation in most genes. In the corpus callosum, genes related to growth, survival and functions of glial cells fluctuated their expression. Immunohistochemically, neurons expressing gene products of immediate-early genes, i.e., Arc, Fos and Jun, decreased in their number in the dentate granule cell layer, cingulate cortex and cerebellar vermis. We also applied immunohistochemical analysis in rat offspring after developmental exposure to glycidol through maternal drinking water. The results revealed increases of Arc{sup +} neurons at 1000 ppm and Fos{sup +} neurons at ≥ 300 ppm in the dentate granule cell layer of offspring only at the adult stage. These results suggest that glycidol suppressed neuronal plasticity in the brain after 28-day exposure to young adult animals, in contrast to the operation of restoration mechanism to increase neuronal plasticity at the adult stage in response to aberrations in neurogenesis after developmental exposure. - Highlights: • Neuronal toxicity parameters after 28-day glycidol treatment were examined in rats. • Region-specific global gene expression profiling was conducted in brain regions. • Cortical tissues downregulated genes on axonogenesis and synaptic transmission. • Cortical tissues

  11. Bacterial gene transfer by natural genetic transformation in the environment.

    PubMed Central

    Lorenz, M G; Wackernagel, W

    1994-01-01

    Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation. PMID:7968924

  12. Gene Transfer into Rat Brain Using Adenoviral Vectors

    PubMed Central

    Puntel, Mariana; Kroeger, Kurt M.; Sanderson, Nicholas S.R.; Thomas, Clare E.; Castro, Maria G.; Lowenstein, Pedro R.

    2010-01-01

    Viral vector–mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose—efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described. Curr. Protoc. Neurosci. 50:4.24.1–4.24.49. © 2010 by John Wiley & Sons, Inc. PMID:20066657

  13. Successful PGD for late infantile neuronal ceroid lipofuscinosis achieved by combined chromosome and TPP1 gene analysis.

    PubMed

    Shen, Jiandong; Cram, David Stephen; Wu, Wei; Cai, Lingbo; Yang, Xiaoyu; Sun, Xueping; Cui, Yugui; Liu, Jiayin

    2013-08-01

    Late infantile neuronal ceroid lipofuscinosis (NCL-2) is a severe debilitating autosomal recessive disease caused by mutations in TPP1. There are no effective treatments, resulting in early childhood death. A couple with two affected children presented for reproductive genetic counselling and chose to undertake IVF and preimplantation genetic diagnosis (PGD) to avoid the possibility of another affected child. However, DNA testing revealed only one mutation in the proband inherited from mother. Linkage analysis identified five informative linked short tandem repeat markers to aid the genetic diagnosis. Following IVF, five cleavage-stage embryos were biopsied and blastomeres were first subjected to whole-genome amplification, then a series of down-stream molecular genetic analyses to diagnose TPP1 genotype and finally array comparative genomic hybridization (CGH) to assess the chromosomal ploidy of each embryo. Two unaffected euploid embryos were identified for transfer. One was transferred on day 5 resulting in an ongoing pregnancy. Confirmatory prenatal diagnosis by amniocentesis showed concordance of the embryo and fetal diagnosis. As far as is known, this is the first successful report of PGD for NCL-2 using double-factor PGD with simultaneous single-gene testing and array CGH to identify an unaffected and chromosomally normal embryo for transfer.

  14. Indications for acquisition of reductive dehalogenase genes through horizontal gene transfer by Dehalococcoides ethenogenes strain 195.

    PubMed

    Regeard, Christophe; Maillard, Julien; Dufraigne, Christine; Deschavanne, Patrick; Holliger, Christof

    2005-06-01

    The genome of Dehalococcoides ethenogenes strain 195, an anaerobic dehalorespiring bacterium, contains 18 copies of putative reductive dehalogenase genes, including the well-characterized tceA gene, whose gene product functions as the key enzyme in the environmentally important dehalorespiration process. The genome of D. ethenogenes was analyzed using a bioinformatic tool based on the frequency of oligonucleotides. The results in the form of a genomic signature revealed several local disruptions of the host signature along the genome sequence. These fractures represent DNA segments of potentially foreign origin, so-called atypical regions, which may have been acquired by an ancestor through horizontal gene transfer. Most interestingly, 15 of the 18 reductive dehalogenase genes, including the tceA gene, were found to be located in these regions, strongly indicating the foreign nature of the dehalorespiration activity. The GC content and the presence of recombinase genes within some of these regions corroborate this hypothesis. A hierarchical classification of the atypical regions containing the reductive dehalogenase genes indicated that these regions were probably acquired by several gene transfer events. PMID:15932990

  15. NGF has opposing effects on Na+ channel III and SNS gene expression in spinal sensory neurons.

    PubMed

    Black, J A; Langworthy, K; Hinson, A W; Dib-Hajj, S D; Waxman, S G

    1997-07-01

    Following sciatic nerve transection, the expression of sodium channel III (alpha-III) transcripts increases and SNS (alpha-SNS) transcripts decreases in small (< 25 microns diameter) dorsal root ganglion (DRG) neurons, which may reflect an interruption of retrograde transport of peripherally derived factor(s) involved in the regulation of these channels. To test the hypothesis that the neurotrophin nerve growth factor (NGF), which is abundant in peripheral targets, participates in the modulation of the expression of these sodium channel transcripts, we examined the hybridization signal of alpha-SNS and alpha-III mRNAs in small DRG neurons from adult rats that had been dissociated and maintained for 7 days in the absence or presence of exogenous NGF. Neurons maintained in control (no added NGF) cultures showed changes in alpha-III and alpha-SNS hybridization signal similar to those induced by axotomy, with increased alpha-III mRNA levels and decreased alpha-SNS mRNA levels, compared with those observed in small DRG neurons at 1 day in vitro. The addition of exogenous NGF to DRG cultures attenuated these alterations in transcript levels, decreasing alpha-III mRNA and increasing alpha-SNS mRNA expression. These results suggest that NGF participates in the regulation of membrane excitability in small DRG neurons by pathways that include opposing effects on different sodium channel genes. PMID:9243635

  16. Superior Cervical Ganglia Neurons Induce Foxp3+ Regulatory T Cells via Calcitonin Gene-Related Peptide

    PubMed Central

    Szklany, Kirsten; Ruiter, Evelyn; Mian, Firoz; Kunze, Wolfgang; Bienenstock, John; Forsythe, Paul; Karimi, Khalil

    2016-01-01

    The nervous and immune systems communicate bidirectionally, utilizing diverse molecular signals including cytokines and neurotransmitters to provide an integrated response to changes in the body’s internal and external environment. Although, neuro-immune interactions are becoming better understood under inflammatory circumstances and it has been evidenced that interaction between neurons and T cells results in the conversion of encephalitogenic T cells to T regulatory cells, relatively little is known about the communication between neurons and naïve T cells. Here, we demonstrate that following co-culture of naïve CD4+ T cells with superior cervical ganglion neurons, the percentage of Foxp3 expressing CD4+CD25+ cells significantly increased. This was mediated in part by immune-regulatory cytokines TGF-β and IL-10, as well as the neuropeptide calcitonin gene-related peptide while vasoactive intestinal peptide was shown to play no role in generation of T regulatory cells. Additionally, T cells co-cultured with neurons showed a decrease in the levels of pro-inflammatory cytokine IFN-γ released upon in vitro stimulation. These findings suggest that the generation of Tregs may be promoted by naïve CD4+ T cell: neuron interaction through the release of neuropeptide CGRP. PMID:27022966

  17. Shock wave permeabilization as a new gene transfer method.

    PubMed

    Lauer, U; Bürgelt, E; Squire, Z; Messmer, K; Hofschneider, P H; Gregor, M; Delius, M

    1997-07-01

    Uptake of naked functional DNA into mammalian cells can be achieved by a number of physical methods. However, for most of these techniques possibilities for therapeutic in vivo applications--especially to solid organs--are often limited. In this report, we describe shock wave permeabilization as a new physical gene transfer method, which can be easily applied, provides great flexibility in the size and sequence of the DNA molecules to be delivered, and which should exhibit an advantageous security profile in vivo. Upon exposure to lithotripter-generated shock waves eukaryotic cells display a temporary increase in membrane permeability. This effect was shown to be caused by cavitation resulting in the transient generation of cell pores which allows the direct transfer of naked plasmid DNA. Shockwave transfection of a variety of cell lines was demonstrated. Since shock waves can be well focused within particular body regions, future applications of extracorporally generated shock waves to tissues simultaneously perfused with DNA solutions might open up the possibility of achieving a regionally enhanced in vivo gene transfer.

  18. Phylogenomics of T4 cyanophages: lateral gene transfer in the 'core' and origins of host genes.

    PubMed

    Ignacio-Espinoza, J Cesar; Sullivan, Matthew B

    2012-08-01

    The last two decades have revealed that phages (viruses that infect bacteria) are abundant and play fundamental roles in the Earth System, with the T4-like myoviruses (herein T4-like phages) emerging as a dominant 'signal' in wild populations. Here we examine 27 T4-like phage genomes, with a focus on 17 that infect ocean picocyanobacteria (cyanophages), to evaluate lateral gene transfer (LGT) in this group. First, we establish a reference tree by evaluating concatenated core gene supertrees and whole genome gene content trees. Next, we evaluate what fraction of these 'core genes' shared by all 17 cyanophages appear prone to LGT. Most (47 out of 57 core genes) were vertically transferred as inferred from tree tests and genomic synteny. Of those 10 core genes that failed the tree tests, the bulk (8 of 10) remain syntenic in the genomes with only a few (3 of the 10) having identifiable signatures of mobile elements. Notably, only one of these 10 is shared not only by the 17 cyanophages, but also by all 27 T4-like phages (thymidylate synthase); its evolutionary history suggests cyanophages may be the origin of these genes to Prochlorococcus. Next, we examined intragenic recombination among the core genes and found that it did occur, even among these core genes, but that the rate was significantly higher between closely related phages, perhaps reducing any detectable LGT signal and leading to taxon cohesion. Finally, among 18 auxiliary metabolic genes (AMGs, a.k.a. 'host' genes), we found that half originated from their immediate hosts, in some cases multiple times (e.g. psbA, psbD, pstS), while the remaining have less clear evolutionary origins ranging from cyanobacteria (4 genes) or microbes (5 genes), with particular diversity among viral TalC and Hsp20 sequences. Together, these findings highlight the patterns and limits of vertical evolution, as well as the ecological and evolutionary roles of LGT in shaping T4-like phage genomes.

  19. Gene therapy: the role of cytoskeleton in gene transfer studies based on biology and mathematics.

    PubMed

    Notarangelo, Maria G; Natalini, Roberto; Signori, Emanuela

    2014-01-01

    Gene therapy is a promising approach for treating a wide range of human pathologies such as genetic disorders as well as diseases acquired over time. Viral and non-viral vectors are used to convey sequences of genes that can be expressed for therapeutic purposes. Plasmid DNA is receiving considerable attention for intramuscular gene transfer due to its safety, simplicity and low cost of production. Nevertheless, strategies to improve DNA uptake into the nucleus of cells for its expression are required. Cytoskeleton plays an important role in the intracellular trafficking. The mechanism regulating this process must be elucidated. Here, we propose a new methodological approach based on the coupling of biology assays and predictive mathematical models, in order to clarify the mechanism of the DNA uptake and its expression into the cells. Once these processes are better clarified, we will be able to propose more efficient therapeutic gene transfer protocols for the treatment of human patients.

  20. Interdomain lateral gene transfer of an essential ferrochelatase gene in human parasitic nematodes.

    PubMed

    Wu, Bo; Novelli, Jacopo; Jiang, Daojun; Dailey, Harry A; Landmann, Frédéric; Ford, Louise; Taylor, Mark J; Carlow, Clotilde K S; Kumar, Sanjay; Foster, Jeremy M; Slatko, Barton E

    2013-05-01

    Lateral gene transfer events between bacteria and animals highlight an avenue for evolutionary genomic loss/gain of function. Herein, we report functional lateral gene transfer in animal parasitic nematodes. Members of the Nematoda are heme auxotrophs, lacking the ability to synthesize heme; however, the human filarial parasite Brugia malayi has acquired a bacterial gene encoding ferrochelatase (BmFeCH), the terminal step in heme biosynthesis. BmFeCH, encoded by a 9-exon gene, is a mitochondrial-targeted, functional ferrochelatase based on enzyme assays, complementation, and inhibitor studies. Homologs have been identified in several filariae and a nonfilarial nematode. RNAi and ex vivo inhibitor experiments indicate that BmFeCH is essential for viability, validating it as a potential target for filariasis control.

  1. Interdomain lateral gene transfer of an essential ferrochelatase gene in human parasitic nematodes

    PubMed Central

    Wu, Bo; Novelli, Jacopo; Jiang, Daojun; Dailey, Harry A.; Landmann, Frédéric; Ford, Louise; Taylor, Mark J.; Carlow, Clotilde K. S.; Kumar, Sanjay; Foster, Jeremy M.; Slatko, Barton E.

    2013-01-01

    Lateral gene transfer events between bacteria and animals highlight an avenue for evolutionary genomic loss/gain of function. Herein, we report functional lateral gene transfer in animal parasitic nematodes. Members of the Nematoda are heme auxotrophs, lacking the ability to synthesize heme; however, the human filarial parasite Brugia malayi has acquired a bacterial gene encoding ferrochelatase (BmFeCH), the terminal step in heme biosynthesis. BmFeCH, encoded by a 9-exon gene, is a mitochondrial-targeted, functional ferrochelatase based on enzyme assays, complementation, and inhibitor studies. Homologs have been identified in several filariae and a nonfilarial nematode. RNAi and ex vivo inhibitor experiments indicate that BmFeCH is essential for viability, validating it as a potential target for filariasis control. PMID:23610429

  2. Adaptive Horizontal Gene Transfers between Multiple Cheese-Associated Fungi.

    PubMed

    Ropars, Jeanne; Rodríguez de la Vega, Ricardo C; López-Villavicencio, Manuela; Gouzy, Jérôme; Sallet, Erika; Dumas, Émilie; Lacoste, Sandrine; Debuchy, Robert; Dupont, Joëlle; Branca, Antoine; Giraud, Tatiana

    2015-10-01

    Domestication is an excellent model for studies of adaptation because it involves recent and strong selection on a few, identified traits [1-5]. Few studies have focused on the domestication of fungi, with notable exceptions [6-11], despite their importance to bioindustry [12] and to a general understanding of adaptation in eukaryotes [5]. Penicillium fungi are ubiquitous molds among which two distantly related species have been independently selected for cheese making-P. roqueforti for blue cheeses like Roquefort and P. camemberti for soft cheeses like Camembert. The selected traits include morphology, aromatic profile, lipolytic and proteolytic activities, and ability to grow at low temperatures, in a matrix containing bacterial and fungal competitors [13-15]. By comparing the genomes of ten Penicillium species, we show that adaptation to cheese was associated with multiple recent horizontal transfers of large genomic regions carrying crucial metabolic genes. We identified seven horizontally transferred regions (HTRs) spanning more than 10 kb each, flanked by specific transposable elements, and displaying nearly 100% identity between distant Penicillium species. Two HTRs carried genes with functions involved in the utilization of cheese nutrients or competition and were found nearly identical in multiple strains and species of cheese-associated Penicillium fungi, indicating recent selective sweeps; they were experimentally associated with faster growth and greater competitiveness on cheese and contained genes highly expressed in the early stage of cheese maturation. These findings have industrial and food safety implications and improve our understanding of the processes of adaptation to rapid environmental changes.

  3. Gene transfer by adenovirus in smooth muscle cells.

    PubMed

    Yu, M F; Ewaskiewicz, J I; Adda, S; Bailey, K; Harris, V; Sosnoski, D; Tomasic, M; Wilson, J; Kotlikoff, M I

    1996-08-01

    We report adenovirus-mediated gene transfer into airway smooth muscle cells in cultured cells and organ-cultured tracheal segments. Incubation of cultured rat tracheal myocytes with virus (5 x 10(8) pfu/ml) for 6 h resulted in beta-galactosidase expression in 94.8 +/- 2.5% of cells (n = 4). Following incubation of thin (less than 200 microns diameter) equine trachealis muscle segments with virus in organ culture (5 x 10(8)-5 x 10(10) pfu/ml) the average expression of the Lac Z gene was approximately 19 +/- 10% (n = 9). Expression was markedly improved, however, in segments from neonatal rats (13-21 days). In two experiments in which the mucosa and serosa were removed, nearly all cells expressed beta-galactosidase, whereas in a third experiment in which the tissue was not dissected, about 40% of cells were stained. Viral infection had no effect on tension development of strips following organ culture. In vitro gene transfer may provide a useful method to alter protein expression and examine the effect of this alteration on excitation/contraction coupling in smooth muscle.

  4. Gene transfer into mammalian cells by jet injection.

    PubMed

    Furth, P A; Shamay, A; Hennighausen, L

    1995-04-01

    Jet injection of DNA in solution is a technique that can be used to transfer DNA into tissues of living animals, where the introduced genes are expressed. The muscle, fat, skin, and mammary tissue of mice, and the fat, skin, and mammary tissue of sheep can be transfected with DNA. A jet injector, such as the Ped-o-jet (Stirn Industries, Dayton, NJ), is used to form a jet from 100 to 300 microliters of a DNA solution. This jet has sufficient force to travel into and through tissues of adult and juvenile animals. The introduced DNA is found in cells surrounding the path of the jet. When jet injection is performed through the surface of intact skin, underlying muscle, mammary, and fat, cells up to 2 cm distant from the point of injection are transfected with DNA. In this study, we demonstrate that the efficiency of DNA transfer is dependent upon the force of injection. Jet injection is an alternative to needle injection, lipofection, and particle bombardment for the introduction of "naked" DNA into the tissues of animals. This technique has potential for the introduction of genes into living organisms for genetic vaccination and gene therapy. PMID:7590772

  5. Roles of noncanonical Wnt/PCP pathway genes in neuronal migration and neurulation in zebrafish.

    PubMed

    Wada, Hironori; Okamoto, Hitoshi

    2009-03-01

    Noncanonical Wnt/planar cell polarity (PCP) pathways regulate oriented cell movement during development in both Drosophila and vertebrates. Recent studies have revealed similarities and differences between these pathways in the tissues on which they act. In zebrafish, PCP pathway genes regulate the directional migration of a specific population of motor neurons in the hindbrain, as well as morphogenesis of the neuroepithelium. In the present review, neuronal and neuroepithelial defects in zebrafish PCP pathway mutants are compared, and the possible cellular and molecular mechanisms underlying these phenotypes are discussed. Future analyses of zebrafish PCP mutants will reveal the general mechanisms underlying the development of the neuroepithelium and provide novel insights into both conserved and diverse functions of PCP pathway genes in vertebrate development.

  6. Distributive Conjugal Transfer: New Insights into Horizontal Gene Transfer and Genetic Exchange in Mycobacteria

    PubMed Central

    Derbyshire, Keith M.; Gray, Todd A.

    2014-01-01

    The last decade has seen an explosion in the application of genomic tools across all biological disciplines. This is also true for mycobacteria, where whole genome sequences are now available for pathogens and non-pathogens alike. Genomes within the Mycobacterium tuberculosis Complex (MTBC) bear the hallmarks of horizontal gene transfer (HGT). Conjugation is the form of HGT with the highest potential capacity and evolutionary influence. Donor and recipient strains of Mycobacterium smegmatis actively conjugate upon co-culturing in biofilms and on solid media. Whole genome sequencing of the transconjugant progeny demonstrated the incredible scale and range of genomic variation that conjugation generates. Transconjugant genomes are complex mosaics of the parental strains. Some transconjugant genomes are up to one-quarter donor-derived, distributed over 30 segments. Transferred segments range from ~50 bp to ~225,000 bp in length, and are exchanged with their recipient orthologs all around the genome. This unpredictable genome-wide infusion of DNA sequences is called Distributive Conjugal Transfer (DCT), to distinguish it from traditional oriT-based conjugation. The mosaicism generated in a single transfer event resembles that seen from meiotic recombination in sexually reproducing organisms, and contrasts with traditional models of HGT. This similarity allowed the application of a GWAS-like approach to map the donor genes that confer a donor mating identity phenotype. The mating identity genes map to the esx1 locus, expanding the central role of ESX-1 function in conjugation. The potential for DCT to instantaneously blend genomes will affect how we view mycobacterial evolution, and provide new tools for the facile manipulation of mycobacterial genomes. PMID:25505644

  7. Gene transfer with subsequent removal of the selection gene from the host genome.

    PubMed Central

    Dale, E C; Ow, D W

    1991-01-01

    A general method of gene transfer that does not leave behind a selectable marker in the host genome is described. A luciferase gene was introduced into the tobacco genome by using the hygromycin phosphotransferase gene (hpt) as a linked selectable marker. Flanked by recombination sites from the bacteriophage P1 Cre/lox recombination system, the hpt gene was subsequently excised from the plant genome by the Cre recombinase. The Cre-catalyzed excision event in the plant genome was precise and conservative--i.e., without loss or alteration of nucleotides in the recombinant site. After removal of the Cre-encoding locus by genetic segregation, plants were obtained that had incorporated only the desired transgene. Gene transfer without the incorporation of antibiotic-resistance markers in the host genome should ease public concerns over the field release of transgenic organisms expressing such traits. Moreover, it would obviate the need for different selectable markers in subsequent rounds of gene transfer into the same host. Images PMID:1660141

  8. In vivo Cytokine Gene Transfer by Gene Gun Reduces Tumor Growth in Mice

    NASA Astrophysics Data System (ADS)

    Sun, Wenn H.; Burkholder, Joseph K.; Sun, Jian; Culp, Jerilyn; Turner, Joel; Lu, Xing G.; Pugh, Thomas D.; Ershler, William B.; Yang, Ning-Sun

    1995-03-01

    Implantation of tumor cells modified by in vitro cytokine gene transfer has been shown by many investigators to result in potent in vivo antitumor activities in mice. Here we describe an approach to tumor immunotherapy utilizing direct transfection of cytokine genes into tumorbearing animals by particle-mediated gene transfer. In vivo transfection of the human interleukin 6 gene into the tumor site reduced methylcholanthrene-induced fibrosarcoma growth, and a combination of murine tumor necrosis factor α and interferon γ genes inhibited growth of a renal carcinoma tumor model (Renca). In addition, treatment with murine interleukin 2 and interferon γ genes prolonged the survival of Renca tumor-bearing mice and resulted in tumor eradication in 25% of the test animals. Transgene expression was demonstrated in treated tissues by ELISA and immunohistochemical analysis. Significant serum levels of interleukin 6 and interferon γ were detected, demonstrating effective secretion of transgenic proteins from treated skin into the bloodstream. This in vivo cytokine gene therapy approach provides a system for evaluating the antitumor properties of various cytokines in different tumor models and has potential utility for human cancer gene therapy.

  9. Variants of the elongator protein 3 (ELP3) gene are associated with motor neuron degeneration.

    PubMed

    Simpson, Claire L; Lemmens, Robin; Miskiewicz, Katarzyna; Broom, Wendy J; Hansen, Valerie K; van Vught, Paul W J; Landers, John E; Sapp, Peter; Van Den Bosch, Ludo; Knight, Joanne; Neale, Benjamin M; Turner, Martin R; Veldink, Jan H; Ophoff, Roel A; Tripathi, Vineeta B; Beleza, Ana; Shah, Meera N; Proitsi, Petroula; Van Hoecke, Annelies; Carmeliet, Peter; Horvitz, H Robert; Leigh, P Nigel; Shaw, Christopher E; van den Berg, Leonard H; Sham, Pak C; Powell, John F; Verstreken, Patrik; Brown, Robert H; Robberecht, Wim; Al-Chalabi, Ammar

    2009-02-01

    Amyotrophic lateral sclerosis (ALS) is a spontaneous, relentlessly progressive motor neuron disease, usually resulting in death from respiratory failure within 3 years. Variation in the genes SOD1 and TARDBP accounts for a small percentage of cases, and other genes have shown association in both candidate gene and genome-wide studies, but the genetic causes remain largely unknown. We have performed two independent parallel studies, both implicating the RNA polymerase II component, ELP3, in axonal biology and neuronal degeneration. In the first, an association study of 1884 microsatellite markers, allelic variants of ELP3 were associated with ALS in three human populations comprising 1483 people (P=1.96 x 10(-9)). In the second, an independent mutagenesis screen in Drosophila for genes important in neuronal communication and survival identified two different loss of function mutations, both in ELP3 (R475K and R456K). Furthermore, knock down of ELP3 protein levels using antisense morpholinos in zebrafish embryos resulted in dose-dependent motor axonal abnormalities [Pearson correlation: -0.49, P=1.83 x 10(-12) (start codon morpholino) and -0.46, P=4.05 x 10(-9) (splice-site morpholino), and in humans, risk-associated ELP3 genotypes correlated with reduced brain ELP3 expression (P=0.01). These findings add to the growing body of evidence implicating the RNA processing pathway in neurodegeneration and suggest a critical role for ELP3 in neuron biology and of ELP3 variants in ALS.

  10. Interaction between Conjugative and Retrotransposable Elements in Horizontal Gene Transfer

    PubMed Central

    Novikova, Olga; Smith, Dorie; Hahn, Ingrid; Beauregard, Arthur; Belfort, Marlene

    2014-01-01

    Mobile genetic elements either encode their own mobilization machineries or hijack them from other mobile elements. Multiple classes of mobile elements often coexist within genomes and it is unclear whether they have the capacity to functionally interact and even collaborate. We investigate the possibility that molecular machineries of disparate mobile elements may functionally interact, using the example of a retrotransposon, in the form of a mobile group II intron, found on a conjugative plasmid pRS01 in Lactococcus lactis. This intron resides within the pRS01 ltrB gene encoding relaxase, the enzyme required for nicking the transfer origin (oriT) for conjugal transmission of the plasmid into a recipient cell. Here, we show that relaxase stimulates both the frequency and diversity of retrotransposition events using a retromobility indicator gene (RIG), and by developing a high-throughput genomic retrotransposition detection system called RIG-Seq. We demonstrate that LtrB relaxase not only nicks ssDNA of its cognate oriT in a sequence- and strand-specific manner, but also possesses weak off-target activity. Together, the data support a model in which the two different mobile elements, one using an RNA-based mechanism, the other using DNA-based transfer, do functionally interact. Intron splicing facilitates relaxase expression required for conjugation, whereas relaxase introduces spurious nicks in recipient DNA that stimulate both the frequency of intron mobility and the density of events. We hypothesize that this functional interaction between the mobile elements would promote horizontal conjugal gene transfer while stimulating intron dissemination in the donor and recipient cells. PMID:25474706

  11. Precision Modulation of Neurodegenerative Disease-Related Gene Expression in Human iPSC-Derived Neurons

    PubMed Central

    Heman-Ackah, Sabrina Mahalia; Bassett, Andrew Roger; Wood, Matthew John Andrew

    2016-01-01

    The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and the subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Neurons differentiated from patient-specific iPSCs provide a means to recapitulate molecular phenotypes of neurodegenerative diseases in vitro. However, it remains challenging to conduct precise manipulations of gene expression in iPSC-derived neurons towards modeling complex human neurological diseases. The application of CRISPR/Cas9 to mammalian systems is revolutionizing the utilization of genome editing technologies in the study of molecular contributors to the pathogenesis of numerous diseases. Here, we demonstrate that CRISPRa and CRISPRi can be used to exert precise modulations of endogenous gene expression in fate-committed iPSC-derived neurons. This highlights CRISPRa/i as a major technical advancement in accessible tools for evaluating the specific contributions of critical neurodegenerative disease-related genes to neuropathogenesis. PMID:27341390

  12. Global control of motor neuron topography mediated by the repressive actions of a single hox gene.

    PubMed

    Jung, Heekyung; Lacombe, Julie; Mazzoni, Esteban O; Liem, Karel F; Grinstein, Jonathan; Mahony, Shaun; Mukhopadhyay, Debnath; Gifford, David K; Young, Richard A; Anderson, Kathryn V; Wichterle, Hynek; Dasen, Jeremy S

    2010-09-01

    In the developing spinal cord, regional and combinatorial activities of Hox transcription factors are critical in controlling motor neuron fates along the rostrocaudal axis, exemplified by the precise pattern of limb innervation by more than fifty Hox-dependent motor pools. The mechanisms by which motor neuron diversity is constrained to limb levels are, however, not well understood. We show that a single Hox gene, Hoxc9, has an essential role in organizing the motor system through global repressive activities. Hoxc9 is required for the generation of thoracic motor columns, and in its absence, neurons acquire the fates of limb-innervating populations. Unexpectedly, multiple Hox genes are derepressed in Hoxc9 mutants, leading to motor pool disorganization and alterations in the connections by thoracic and forelimb-level subtypes. Genome-wide analysis of Hoxc9 binding suggests that this mode of repression is mediated by direct interactions with Hox regulatory elements, independent of chromatin marks typically associated with repressed Hox genes.

  13. Highly efficient method for gene delivery into mouse dorsal root ganglia neurons.

    PubMed

    Yu, Lingli; Reynaud, Florie; Falk, Julien; Spencer, Ambre; Ding, Yin-Di; Baumlé, Véronique; Lu, Ruisheng; Castellani, Valérie; Yuan, Chonggang; Rudkin, Brian B

    2015-01-01

    The development of gene transfection technologies has greatly advanced our understanding of life sciences. While use of viral vectors has clear efficacy, it requires specific expertise and biological containment conditions. Electroporation has become an effective and commonly used method for introducing DNA into neurons and in intact brain tissue. The present study describes the use of the Neon® electroporation system to transfect genes into dorsal root ganglia neurons isolated from embryonic mouse Day 13.5-16. This cell type has been particularly recalcitrant and refractory to physical or chemical methods for introduction of DNA. By optimizing the culture condition and parameters including voltage and duration for this specific electroporation system, high efficiency (60-80%) and low toxicity (>60% survival) were achieved with robust differentiation in response to Nerve growth factor (NGF). Moreover, 3-50 times fewer cells are needed (6 × 10(4)) compared with other traditional electroporation methods. This approach underlines the efficacy of this type of electroporation, particularly when only limited amount of cells can be obtained, and is expected to greatly facilitate the study of gene function in dorsal root ganglia neuron cultures. PMID:25698920

  14. Gene expression for peptides in neurons of the petrosal and nodose ganglia in rat.

    PubMed

    Czyzyk-Krzeska, M F; Bayliss, D A; Seroogy, K B; Millhorn, D E

    1991-01-01

    In situ hybridization was used to determine whether genes for neuropeptides [substance P/neurokinin A (SP/NKA), calcitonin gene-related peptide (CGRP), somatostatin (SOM), neuropeptide tyrosine (NPY) and cholecystokinin (CCK)] are expressed in inferior ganglia of the vagus (nodose) and glossopharyngeal (petrosal) nerves. Synthetic oligodeoxyribonucleotides, complementary to the cognate, mRNAs were labeled with [32P] or [35S], and hybridized to 10 microns thick sections of unperfused tissue which were then processed for film and emulsion autoradiography. We found numerous, clustered neuronal perikarya throughout the nodose and petrosal ganglia that expressed preprotachykinin A (SP/NKA) and CGRP mRNAs to varying degrees. Neurons expressing preproSOM mRNA were less abundant and more scattered throughout both ganglia. Notably, we found mRNA for NPY in cells (usually 5-10 per section) in both ganglia. To our knowledge, this is first evidence for NPY in these sensory ganglia. In contrast to previous immunohistochemical findings, we found no evidence for expression of preproCCK in either the nodose or petrosal ganglia. The present findings demonstrate that cells of the nodose and petrosal ganglia express the genes for a number of neuropeptides that are presumably involved with transmission of visceral sensory afferent information to higher order neurons of the central nervous system. PMID:1708726

  15. Gene Transfer and Molecular Cloning of the Human NGF Receptor

    NASA Astrophysics Data System (ADS)

    Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

    1986-04-01

    Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

  16. Passive immunization against HIV/AIDS by antibody gene transfer.

    PubMed

    Yang, Lili; Wang, Pin

    2014-01-27

    Despite tremendous efforts over the course of many years, the quest for an effective HIV vaccine by the classical method of active immunization remains largely elusive. However, two recent studies in mice and macaques have now demonstrated a new strategy designated as Vectored ImmunoProphylaxis (VIP), which involves passive immunization by viral vector-mediated delivery of genes encoding broadly neutralizing antibodies (bnAbs) for in vivo expression. Robust protection against virus infection was observed in preclinical settings when animals were given VIP to express monoclonal neutralizing antibodies. This unorthodox approach raises new promise for combating the ongoing global HIV pandemic. In this article, we survey the status of antibody gene transfer, review the revolutionary progress on isolation of extremely bnAbs, detail VIP experiments against HIV and its related virus conduced in humanized mice and macaque monkeys, and discuss the pros and cons of VIP and its opportunities and challenges towards clinical applications to control HIV/AIDS endemics.

  17. Neuronal and glial expression of the multidrug resistance gene product in an experimental epilepsy model.

    PubMed

    Lazarowski, Alberto; Ramos, Alberto Javier; García-Rivello, Hernán; Brusco, Alicia; Girardi, Elena

    2004-02-01

    1. Failure of anticonvulsive drugs to prevent seizures is a common complication of epilepsy treatment known as drug-refractory epilepsy but their causes are not well understood. It is hypothesized that the multidrug resistance P-glycoprotein (Pgp-170), the product of the MDR-1 gene that is normally expressed in several excretory tissues including the blood brain barrier, may be participating in the refractory epilepsy. 2. Using two monoclonal antibodies against Pgp-170, we investigated the expression and cellular distribution of this protein in the rat brain during experimentally induced epilepsy. Repeated seizures were induced in male Wistar rats by daily administration of 3-mercaptopropionic acid (MP) 45 mg/kg i.p. for either 4 days (MP-4) or 7 days (MP-7). Control rats received an equivalent volume of vehicle. One day after the last injection, rats were sacrificed and brains were processed for immunohistochemistry for Pgp-170. As it was previously described, Pgp-170 immunostaining was observed in some brain capillary endothelial cells of animals from control group. 3. Increased Pgp-170 immunoreactivity was detected in MP-treated animals. Besides the Pgp-170 expressed in blood vessels, neuronal, and glial immunostaining was detected in hippocampus, striatum, and cerebral cortex of MP-treated rats. Pgp-170 immunolabeled neurons and glial cells were observed in a nonhomogeneous distribution. MP-4 animals presented a very prominent Pgp-170 immunostaining in the capillary endothelium, surrounding astrocytes and some neighboring neurons while MP-7 group showed increased neuronal labeling. 4. Our results demonstrate a selective increase in Pgp-170 immunoreactivity in the brain capillary endothelial cells, astrocytes, and neurons during repetitive MP-induced seizures. 5. The role for this Pgp-170 overexpression in endothelium and astrocytes as a clearance mechanism in the refractory epilepsy, and the consequences of neuronal Pgp-170 expression remain to be disclosed.

  18. Laterally Transferred Gene Recruited as a Venom in Parasitoid Wasps.

    PubMed

    Martinson, Ellen O; Martinson, Vincent G; Edwards, Rachel; Mrinalini; Werren, John H

    2016-04-01

    Parasitoid wasps use venom to manipulate the immunity and metabolism of their host insects in a variety of ways to provide resources for their offspring. Yet, how genes are recruited and evolve to perform venom functions remain open questions. A recently recognized source of eukaryotic genome innovation is lateral gene transfer (LGT). Glycoside hydrolase family 19 (GH19) chitinases are widespread in bacteria, microsporidia, and plants where they are used in nutrient acquisition or defense, but have previously not been known in metazoans. In this study, a GH19 chitinase LGT is described from the unicellular microsporidia/Rozella clade into parasitoid wasps of the superfamily Chalcidoidea, where it has become recruited as a venom protein. The GH19 chitinase is present in 15 species of chalcidoid wasps representing four families, and phylogenetic analysis indicates that it was laterally transferred near or before the origin of Chalcidoidea (∼95 Ma). The GH19 chitinase gene is highly expressed in the venom gland of at least seven species, indicating a role in the complex host manipulations performed by parasitoid wasp venom. RNAi knockdown in the model parasitoid Nasonia vitripennis reveals that-following envenomation-the GH19 chitinase induces fly hosts to upregulate genes involved in an immune response to fungi. A second, independent LGT of GH19 chitinase from microsporidia into mosquitoes was also found, also supported by phylogenetic reconstructions. Besides these two LGT events, GH19 chitinase is not found in any other sequenced animal genome, or in any fungi outside the microsporidia/Rozella clade.

  19. Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

    PubMed Central

    Woods, Georgia; Zito, Karen

    2008-01-01

    Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices. PMID:19066564

  20. Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.

    PubMed

    Woods, Georgia; Zito, Karen

    2008-02-13

    Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.

  1. CXCR4 gene transfer prevents pressure overload induced heart failure

    PubMed Central

    LaRocca, Thomas J.; Jeong, Dongtak; Kohlbrenner, Erik; Lee, Ahyoung; Chen, JiQiu; Hajjar, Roger J.; Tarzami, Sima T.

    2012-01-01

    Stem cell and gene therapies are being pursued as strategies for repairing damaged cardiac tissue following myocardial infarction in an attempt to prevent heart failure. The chemokine receptor-4 (CXCR4) and its ligand, CXCL12, play a critical role in stem cell recruitment post-acute myocardial infarction. Whereas progenitor cell migration via the CXCL12/CXCR4 axis is well characterized, little is known about the molecular mechanisms of CXCR4 mediated modulation of cardiac hypertrophy and failure. We used gene therapy to test the effects of CXCR4 gene delivery on adverse ventricular remodeling due to pressure overload. We assessed the effect of cardiac overexpression of CXCR4 during trans-aortic constriction (TAC) using a cardiotropic adeno-associated viral vector (AAV9) carrying the CXCR4 gene. Cardiac overexpression of CXCR4 in mice with pressure overload prevented ventricular remodeling, preserved capillary density and maintained function as determined by echocardiography and in vivo hemodynamics. In isolated adult rat cardiac myocytes, CXCL12 treatment prevented isoproterenol induced hypertrophy and interrupted the calcineurin/NFAT pathway. Finally, a complex involving the L-type calcium channel, β2-adenoreceptor, and CXCR4 (Cav1.2/β2AR/CXCR4) was identified in healthy cardiac myocytes and was shown to dissociate as a consequence of heart failure. CXCR4 administered to the heart via gene transfer prevents pressure overload induced heart failure. The identification of CXCR4 participation in a Cav1.2-β2AR regulatory complex provides further insight into the mechanism by which CXCR4 modulates calcium homeostasis and chronic pressure overload responses in the cardiac myocyte. Together these results suggest AAV9.CXCR4 gene therapy is a potential therapeutic approach for congestive heart failure. PMID:22668785

  2. Site-Specific Gene Expression in Vivo by Direct Gene Transfer into the Arterial Wall

    NASA Astrophysics Data System (ADS)

    Nabel, Elizabeth G.; Plautz, Gregory; Nabel, Gary J.

    1990-09-01

    A recombinant β-galactosidase gene has been expressed in a specific arterial segment in vivo by direct infection with a murine amphotropic retroviral vector or by DNA transfection with the use of liposomes. Several cell types in the vessel wall were transduced, including endothelial and vascular smooth muscle cells. After retroviral infection, a recombinant reporter gene was expressed for at least 5 months, and no helper virus was detected. Recombinant gene expression achieved by direct retroviral infection or liposome-mediated DNA transfection was limited to the site of infection and was absent from liver, lung, kidney, and spleen. These results demonstrate that site-specific gene expression can be achieved by direct gene transfer in vivo and could be applied to the treatment of such human diseases as atherosclerosis or cancer.

  3. Coe Genes Are Expressed in Differentiating Neurons in the Central Nervous System of Protostomes

    PubMed Central

    Demilly, Adrien; Simionato, Elena; Ohayon, David; Kerner, Pierre

    2011-01-01

    Genes of the coe (collier/olfactory/early B-cell factor) family encode Helix-Loop-Helix transcription factors that are widely conserved in metazoans and involved in many developmental processes, neurogenesis in particular. Whereas their functions during vertebrate neural tube formation have been well documented, very little is known about their expression and role during central nervous system (CNS) development in protostomes. Here we characterized the CNS expression of coe genes in the insect Drosophila melanogaster and the polychaete annelid Platynereis dumerilii, which belong to different subgroups of protostomes and show strikingly different modes of development. In the Drosophila ventral nerve cord, we found that the Collier-expressing cells form a subpopulation of interneurons with diverse molecular identities and neurotransmitter phenotypes. We also demonstrate that collier is required for the proper differentiation of some interneurons belonging to the Eve-Lateral cluster. In Platynereis dumerilii, we cloned a single coe gene, Pdu-coe, and found that it is exclusively expressed in post mitotic neural cells. Using an original technique of in silico 3D registration, we show that Pdu-coe is co-expressed with many different neuronal markers and therefore that, like in Drosophila, its expression defines a heterogeneous population of neurons with diverse molecular identities. Our detailed characterization and comparison of coe gene expression in the CNS of two distantly-related protostomes suggest conserved roles of coe genes in neuronal differentiation in this clade. As similar roles have also been observed in vertebrates, this function was probably already established in the last common ancestor of all bilaterians. PMID:21695052

  4. Eukaryotic origin of a metabolic pathway in virus by horizontal gene transfer.

    PubMed

    Wu, Dong-Dong; Zhang, Ya-Ping

    2011-11-01

    Horizontal gene transfer, the movement of genetic materials across the normal mating barriers between organisms occurs frequently and contributes significantly to the evolution of both eukaryotic and prokaryotic genomes. However, few concurrent transfers of functionally related genes implemented in a pathway from eukaryotes to prokaryotes are observed. Here, we did phylogenetic analyses to support that the genes, i.e. dihydrofolate reductase, glycine hydroxymethyltransferase, and thymidylate synthase involved in thymidylate metabolism, in Hz-1 virus were obtained from insect genome recently by independent horizontal gene transfers. In addition, five other related genes in nucleotide metabolism show evidences of horizontal gene transfers. These genes demonstrate similar expression pattern, and they may have formatted a functionally related pathway (e.g. thymidylate synthesis, and DNA replication) in Hz-1 virus. In conclusion, we provide an example of horizontal gene transfer of functionally related genes in a pathway to prokaryote from eukaryote.

  5. Synthetic Fatty Acids Prevent Plasmid-Mediated Horizontal Gene Transfer

    PubMed Central

    Getino, María; Sanabria-Ríos, David J.; Fernández-López, Raúl; Campos-Gómez, Javier; Sánchez-López, José M.; Fernández, Antonio; Carballeira, Néstor M.

    2015-01-01

    ABSTRACT Bacterial conjugation constitutes a major horizontal gene transfer mechanism for the dissemination of antibiotic resistance genes among human pathogens. Antibiotic resistance spread could be halted or diminished by molecules that interfere with the conjugation process. In this work, synthetic 2-alkynoic fatty acids were identified as a novel class of conjugation inhibitors. Their chemical properties were investigated by using the prototype 2-hexadecynoic acid and its derivatives. Essential features of effective inhibitors were the carboxylic group, an optimal long aliphatic chain of 16 carbon atoms, and one unsaturation. Chemical modification of these groups led to inactive or less-active derivatives. Conjugation inhibitors were found to act on the donor cell, affecting a wide number of pathogenic bacterial hosts, including Escherichia, Salmonella, Pseudomonas, and Acinetobacter spp. Conjugation inhibitors were active in inhibiting transfer of IncF, IncW, and IncH plasmids, moderately active against IncI, IncL/M, and IncX plasmids, and inactive against IncP and IncN plasmids. Importantly, the use of 2-hexadecynoic acid avoided the spread of a derepressed IncF plasmid into a recipient population, demonstrating the feasibility of abolishing the dissemination of antimicrobial resistances by blocking bacterial conjugation. PMID:26330514

  6. Gene transfer into mammalian cells by particle bombardment.

    PubMed

    Heiser, W C

    1994-03-01

    Using COS-7 and Chinese hamster ovary cells as model systems, I have examined the efficiency of gene transfer into mammalian cells by particle bombardment. The most important parameters affecting transformation efficiency are the size of the particles, the target distance, and the extent of chamber vacuum. The size of the cell culture plate also affects transformation efficiency. Factors which have little effect on transformation efficiency are the helium pressure, the gap distance, and the macrocarrier travel distance. Compared to several other gene transfer techniques, particle bombardment has the advantage of requiring a low amount of DNA and a low number of cells for successful expression, measured as either transient or stable. I also describe transformation of several murine cell lines which have not been successfully transformed, or have been transformed at only low levels using other methods. These cell lines include preadipocytes (BMS-2), macrophages (J774), and transformed pre-B cells (38B9 and 70Z/3). Compared to transformation by electroporation, lipofection, and diethylaminoethyl dextran, particle bombardment was found to give 50- to 240-fold higher levels of transient expression as measured by luciferase activity in cell extracts. PMID:8203746

  7. Horizontal gene transfer and mobile genetic elements in marine systems.

    PubMed

    Sobecky, Patricia A; Hazen, Tracy H

    2009-01-01

    The pool of mobile genetic elements (MGE) in microbial communities consists of viruses, plasmids, and associated elements (insertion sequences, transposons, and integrons) that are either self-transmissible or use mobile plasmids and viruses as vehicles for their dissemination. This mobilome facilitates the horizontal transfer of genes that promote the evolution and adaptation of microbial communities. Efforts to characterize MGEs from microbial populations resident in a variety of ecological habitats have revealed a surprisingly novel and seemingly untapped biodiversity. To better understand the impact of horizontal gene transfer (HGT), as well as the agents that promote HGT in marine ecosystems and to determine whether or not environmental parameters can effect the composition and structure of the mobilome in marine microbial communities, information on the distribution, diversity, and ecological traits of the marine mobilome is presented. In this chapter we discuss recent insights gained from different methodological approaches used to characterize the biodiversity and ecology of MGE in marine environments and their contributions to HGT. In addition, we present case studies that highlight specific HGT examples in coastal, open-ocean, and deep-sea marine ecosystems.

  8. Association of copy numbers of survival motor neuron gene 2 and neuronal apoptosis inhibitory protein gene with the natural history in a Chinese spinal muscular atrophy cohort.

    PubMed

    Qu, Yu-jin; Ge, Xiu-shan; Bai, Jin-li; Wang, Li-wen; Cao, Yan-yan; Lu, Yan-yu; Jin, Yu-wei; Wang, Hong; Song, Fang

    2015-03-01

    We evaluated survival motor neuron 2 (SMN2) and neuronal apoptosis inhibitory protein (NAIP) gene copy distribution and the association of copy number with survival in 232 Chinese spinal muscular atrophy (SMA) patients. The SMN2 and NAIP copy numbers correlated positively with the median onset age (r = 0.72 and 0.377). The risk of death for patients with fewer copies of SMN2 or NAIP was much higher than for those with more copies (P < .01). The survival probabilities at 5 years were 5.1%, 90.7%, and 100% for 2, 3, and 4 SMN2 copies and 27.9%, 66.7%, and 87.2% for 0, 1, and 2 NAIP copies, respectively. Our results indicated that combined SMN1-SMN2-NAIP genotypes with fewer copies were associated with earlier onset age and poorer survival probability. Better survival status for Chinese type I SMA might due to a higher proportion of 3 SMN2 and a lower rate of zero NAIP. PMID:25330799

  9. Association of copy numbers of survival motor neuron gene 2 and neuronal apoptosis inhibitory protein gene with the natural history in a Chinese spinal muscular atrophy cohort.

    PubMed

    Qu, Yu-jin; Ge, Xiu-shan; Bai, Jin-li; Wang, Li-wen; Cao, Yan-yan; Lu, Yan-yu; Jin, Yu-wei; Wang, Hong; Song, Fang

    2015-03-01

    We evaluated survival motor neuron 2 (SMN2) and neuronal apoptosis inhibitory protein (NAIP) gene copy distribution and the association of copy number with survival in 232 Chinese spinal muscular atrophy (SMA) patients. The SMN2 and NAIP copy numbers correlated positively with the median onset age (r = 0.72 and 0.377). The risk of death for patients with fewer copies of SMN2 or NAIP was much higher than for those with more copies (P < .01). The survival probabilities at 5 years were 5.1%, 90.7%, and 100% for 2, 3, and 4 SMN2 copies and 27.9%, 66.7%, and 87.2% for 0, 1, and 2 NAIP copies, respectively. Our results indicated that combined SMN1-SMN2-NAIP genotypes with fewer copies were associated with earlier onset age and poorer survival probability. Better survival status for Chinese type I SMA might due to a higher proportion of 3 SMN2 and a lower rate of zero NAIP.

  10. Real-Time qPCR Identifies Suitable Reference Genes for Borna Disease Virus-Infected Rat Cortical Neurons

    PubMed Central

    Zhang, Lujun; Liu, Siwen; Zhang, Liang; You, Hongmin; Huang, Rongzhong; Sun, Lin; He, Peng; Chen, Shigang; Zhang, Hong; Xie, Peng

    2014-01-01

    Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is the most commonly-used technique to identify gene expression profiles. The selection of stably expressed reference genes is a prerequisite to properly evaluating gene expression. Here, the suitability of commonly-used reference genes in normalizing RT-qPCR assays of mRNA expression in cultured rat cortical neurons infected with Borna disease virus (BDV) was assessed. The expressions of eight commonly-used reference genes were comparatively analyzed in BDV-infected rat cortical neurons and non-infected control neurons mainly across 9 and 12 days post-infection. These reference genes were validated by RT-qPCR and separately ranked by four statistical algorithms: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. Then, the RankAggreg package was used to construct consensus rankings. ARBP was found to be the most stable internal control gene at Day 9, and ACTB at Day 12. As the assessment of the validity of the selected reference genes confirms the suitability of applying a combination of the two most stable references genes, combining the two most stable genes for normalization of RT-qPCR studies in BDV-infected rat cortical neurons is recommended at each time point. This study can contribute to improving BDV research by providing the means by which to obtain more reliable and accurate gene expression measurements. PMID:25431926

  11. Tracing functional circuits using c-Fos regulated expression of marker genes targeted to neuronal projections.

    PubMed

    Murphy, Mark; Greferath, Ursula; Nag, Nupur; Nithianantharajah, Jess; Wilson, Yvette M

    2004-01-01

    We have developed novel techniques to trace functionally activated circuits and synaptic plasticity within the brain. We have generated transgenic mice, FTL, which contain a tau-lacZ fusion gene regulated by the promoter for c-fos. Following a particular nervous system stimulation in these mice, only neurons, which are functionally activated, will express LacZ, which is targeted to neuronal processes by the tau protein. In the FTL mice, we found highly inducible expression of lacZ by a range of different stimuli, and successful targeting of expression to neuronal cell bodies, axons and dendrites. To test if a functionally activated circuit could be visualized, the mice were deprived of water, which activates nuclei involved in body fluid homeostasis. LacZ was induced in these nuclei and their projections, allowing the mapping of a neuroendocrine circuit. Further studies have employed these mice in the analysis of neurons and circuits activated in vision, and learning and memory. We have also developed methods to measure markers of synaptic plasticity in the brain, and found significant experience dependent changes in the levels of these markers in different parts of the brain. We believe these techniques will aid in the identification of circuits for many different brain functions, and within those circuits, the locations of synaptic plasticity.

  12. Hox Genes Promote Neuronal Subtype Diversification through Posterior Induction in Caenorhabditis elegans.

    PubMed

    Zheng, Chaogu; Diaz-Cuadros, Margarete; Chalfie, Martin

    2015-11-01

    Although Hox genes specify the differentiation of neuronal subtypes along the anterior-posterior axis, their mode of action is not entirely understood. Using two subtypes of the touch receptor neurons (TRNs) in C. elegans, we found that a "posterior induction" mechanism underlies the Hox control of terminal neuronal differentiation. The anterior subtype maintains a default TRN state, whereas the posterior subtype undergoes further morphological and transcriptional specification induced by the posterior Hox proteins, mainly EGL-5/Abd-B. Misexpression of the posterior Hox proteins transformed the anterior TRN subtype toward a posterior identity both morphologically and genetically. The specification of the posterior subtype requires EGL-5-induced repression of TALE cofactors, which antagonize EGL-5 functions, and the activation of rfip-1, a component of recycling endosomes, which mediates Hox activities by promoting subtype-specific neurite outgrowth. Finally, EGL-5 is required for subtype-specific circuit formation by acting in both the sensory neuron and downstream interneuron to promote functional connectivity.

  13. Exploring the space of gene/species reconciliations with transfers.

    PubMed

    Chan, Yao-Ban; Ranwez, Vincent; Scornavacca, Céline

    2015-11-01

    Reconciliations between gene and species trees have important applications in the study of genome evolution (e.g. sequence orthology prediction or quantification of transfer events). While numerous methods have been proposed to infer them, little has been done to study the underlying reconciliation space. In this paper, we characterise the reconciliation space for two evolutionary models: the [Formula: see text] (duplication, loss and transfer) model and a variant of it-the no-[Formula: see text] model-which does not allow [Formula: see text] events (a transfer immediately followed by a loss). We provide formulae to compute the size of the corresponding spaces and define a set of transformation operators sufficient to explore the entire reconciliation space. We also define a distance between two reconciliations as the minimal number of operations needed to transform one into the other and prove that this distance is easily computable in the no-[Formula: see text] model. Computing this distance in the [Formula: see text] model is more difficult and it is an open question whether it is NP-hard or not. This work constitutes an important step toward reconciliation space characterisation and reconciliation comparison, needed to better assess the performance of reconciliation inference methods through simulations.

  14. Genome-wide experimental determination of barriers to horizontal gene transfer

    SciTech Connect

    Rubin, Edward; Sorek, Rotem; Zhu, Yiwen; Creevey, Christopher J.; Francino, M. Pilar; Bork, Peer; Rubin, Edward M.

    2007-09-24

    Horizontal gene transfer, in which genetic material is transferred from the genome of one organism to another, has been investigated in microbial species mainly through computational sequence analyses. To address the lack of experimental data, we studied the attempted movement of 246,045 genes from 79 prokaryotic genomes into E. coli and identified genes that consistently fail to transfer. We studied the mechanisms underlying transfer inhibition by placing coding regions from different species under the control of inducible promoters. Their toxicity to the host inhibited transfer regardless of the species of origin and our data suggest that increased gene dosage and associated increased expression is a predominant cause for transfer failure. While these experimental studies examined transfer solely into E. coli, a computational analysis of gene transfer rates across available bacterial and archaeal genomes indicates that the barriers observed in our study are general across the tree of life.

  15. FUS regulates genes coding for RNA-binding proteins in neurons by binding to their highly conserved introns

    PubMed Central

    Nakaya, Tadashi; Alexiou, Panagiotis; Maragkakis, Manolis; Chang, Alexandra; Mourelatos, Zissimos

    2013-01-01

    Dominant mutations and mislocalization or aggregation of Fused in Sarcoma (FUS), an RNA-binding protein (RBP), cause neuronal degeneration in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD), two incurable neurological diseases. However, the function of FUS in neurons is not well understood. To uncover the impact of FUS in the neuronal transcriptome, we used high-throughput sequencing of immunoprecipitated and cross-linked RNA (HITS–CLIP) of FUS in human brains and mouse neurons differentiated from embryonic stem cells, coupled with RNA-seq and FUS knockdowns. We report conserved neuronal RNA targets and networks that are regulated by FUS. We find that FUS regulates splicing of genes coding for RBPs by binding to their highly conserved introns. Our findings have important implications for understanding the impact of FUS in neurodegenerative diseases and suggest that perturbations of FUS can impact the neuronal transcriptome via perturbations of RBP transcripts. PMID:23389473

  16. The atRA-responsive gene neuron navigator 2 functions in neurite outgrowth and axonal elongation.

    PubMed

    Muley, P D; McNeill, E M; Marzinke, M A; Knobel, K M; Barr, M M; Clagett-Dame, M

    2008-11-01

    Neuron navigator 2 (Nav2) was first identified as an all-trans retinoic acid (atRA)-responsive gene in human neuroblastoma cells (retinoic acid-induced in neuroblastoma 1, RAINB1) that extend neurites after exposure to atRA. It is structurally related to the Caenorhabditis elegans unc-53 gene that is required for cell migration and axonal outgrowth. To gain insight into NAV2 function, the full-length human protein was expressed in C. elegans unc-53 mutants under the control of a mechanosensory neuron promoter. Transgene expression of NAV2 rescued the defects in unc-53 mutant mechanosensory neuron elongation, indicating that Nav2 is an ortholog of unc-53. Using a loss-of-function approach, we also show that Nav2 induction is essential for atRA to induce neurite outgrowth in SH-SY5Y cells. The NAV2 protein is located both in the cell body and along the length of the growing neurites of SH-SY5Y cells in a pattern that closely mimics that of neurofilament and microtubule proteins. Transfection of Nav2 deletion constructs in Cos-1 cells reveals a region of the protein (aa 837-1065) that directs localization with the microtubule cytoskeleton. Collectively, this work supports a role for NAV2 in neurite outgrowth and axonal elongation and suggests this protein may act by facilitating interactions between microtubules and other proteins such as neurofilaments that are key players in the formation and stability of growing neurites.

  17. Adaptive Horizontal Gene Transfers between Multiple Cheese-Associated Fungi

    PubMed Central

    Ropars, Jeanne; Rodríguez de la Vega, Ricardo C.; López-Villavicencio, Manuela; Gouzy, Jérôme; Sallet, Erika; Dumas, Émilie; Lacoste, Sandrine; Debuchy, Robert; Dupont, Joëlle; Branca, Antoine; Giraud, Tatiana

    2015-01-01

    Summary Domestication is an excellent model for studies of adaptation because it involves recent and strong selection on a few, identified traits [1–5]. Few studies have focused on the domestication of fungi, with notable exceptions [6–11], despite their importance to bioindustry [12] and to a general understanding of adaptation in eukaryotes [5]. Penicillium fungi are ubiquitous molds among which two distantly related species have been independently selected for cheese making—P. roqueforti for blue cheeses like Roquefort and P. camemberti for soft cheeses like Camembert. The selected traits include morphology, aromatic profile, lipolytic and proteolytic activities, and ability to grow at low temperatures, in a matrix containing bacterial and fungal competitors [13–15]. By comparing the genomes of ten Penicillium species, we show that adaptation to cheese was associated with multiple recent horizontal transfers of large genomic regions carrying crucial metabolic genes. We identified seven horizontally transferred regions (HTRs) spanning more than 10 kb each, flanked by specific transposable elements, and displaying nearly 100% identity between distant Penicillium species. Two HTRs carried genes with functions involved in the utilization of cheese nutrients or competition and were found nearly identical in multiple strains and species of cheese-associated Penicillium fungi, indicating recent selective sweeps; they were experimentally associated with faster growth and greater competitiveness on cheese and contained genes highly expressed in the early stage of cheese maturation. These findings have industrial and food safety implications and improve our understanding of the processes of adaptation to rapid environmental changes. PMID:26412136

  18. Amoebozoa possess lineage-specific globin gene repertoires gained by individual horizontal gene transfers.

    PubMed

    Dröge, Jasmin; Buczek, Dorota; Suzuki, Yutaka; Makałowski, Wojciech

    2014-01-01

    The Amoebozoa represent a clade of unicellular amoeboid organisms that display a wide variety of lifestyles, including free-living and parasitic species. For example, the social amoeba Dictyostelium discoideum has the ability to aggregate into a multicellular fruiting body upon starvation, while the pathogenic amoeba Entamoeba histolytica is a parasite of humans. Globins are small heme proteins that are present in almost all extant organisms. Although several genomes of amoebozoan species have been sequenced, little is known about the phyletic distribution of globin genes within this phylum. Only two flavohemoglobins (FHbs) of D. discoideum have been reported and characterized previously while the genomes of Entamoeba species are apparently devoid of globin genes. We investigated eleven amoebozoan species for the presence of globin genes by genomic and phylogenetic in silico analyses. Additional FHb genes were identified in the genomes of four social amoebas and the true slime mold Physarum polycephalum. Moreover, a single-domain globin (SDFgb) of Hartmannella vermiformis, as well as two truncated hemoglobins (trHbs) of Acanthamoeba castellanii were identified. Phylogenetic evidence suggests that these globin genes were independently acquired via horizontal gene transfer from some ancestral bacteria. Furthermore, the phylogenetic tree of amoebozoan FHbs indicates that they do not share a common ancestry and that a transfer of FHbs from bacteria to amoeba occurred multiple times. PMID:25013378

  19. Distinct epigenetic and gene expression changes in rat hippocampal neurons after Morris water maze training

    PubMed Central

    Carter, Sylvia D.; Mifsud, Karen R.; Reul, Johannes M. H. M.

    2015-01-01

    Gene transcription and translation in the hippocampus is of critical importance in hippocampus-dependent memory formation, including during Morris water maze (MWM) learning. Previous work using gene deletion models has shown that the immediate-early genes (IEGs) c-Fos, Egr-1, and Arc are crucial for such learning. Recently, we reported that induction of IEGs in sparse dentate gyrus neurons requires ERK MAPK signaling and downstream formation of a distinct epigenetic histone mark (i.e., phospho-acetylated histone H3). Until now, this signaling, epigenetic and gene transcriptional pathway has not been comprehensively studied in the MWM model. Therefore, we conducted a detailed study of the phosphorylation of ERK1/2 and serine10 in histone H3 (H3S10p) and induction of IEGs in the hippocampus of MWM trained rats and matched controls. MWM training evoked consecutive waves of ERK1/2 phosphorylation and H3S10 phosphorylation, as well as c-Fos, Egr-1, and Arc induction in sparse hippocampal neurons. The observed effects were most pronounced in the dentate gyrus. A positive correlation was found between the average latency to find the platform and the number of H3S10p-positive dentate gyrus neurons. Furthermore, chromatin immuno-precipitation (ChIP) revealed a significantly increased association of phospho-acetylated histone H3 (H3K9ac-S10p) with the gene promoters of c-Fos and Egr-1, but not Arc, after MWM exposure compared with controls. Surprisingly, however, we found very little difference between IEG responses (regarding both protein and mRNA) in MWM-trained rats compared with matched swim controls. We conclude that exposure to the water maze evokes ERK MAPK activation, distinct epigenetic changes and IEG induction predominantly in sparse dentate gyrus neurons. It appears, however, that a specific role for IEGs in the learning aspect of MWM training may become apparent in downstream AP-1- and Egr-1-regulated (second wave) genes and Arc-dependent effector mechanisms

  20. Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons

    PubMed Central

    Ji, Changyi; Kosman, Daniel J.

    2015-01-01

    The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and DMT1, and ferrireductases Steap2 and SDR2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co-localize, indicating these two proteins may function in Fe3+ reduction prior to Fe2+ permeation. Zip8, DMT1 and Steap2 co-localize with the transferrin receptor (TfR)/transferrin (Tf) complex, suggesting they may be involved in TfR/Tf-mediated iron assimilation. In brain interstitial fluid, transferring-bound iron (TBI) and non-transferrin-bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin-59Fe3+) and NTBI, whether presented as 59Fe2+-citrate or 59Fe3+-citrate; reductase-independent 59Fe2+ uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn2+ inhibition of Fe2+ uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of 59Fe from TBI relies at least in part on an endocytosis-independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons. PMID:25649872

  1. Ultrasound Gene Transfer into Fibroblast Cells using Microbubbles

    NASA Astrophysics Data System (ADS)

    Nakamura, Yoji; Hirayama, Kota; Yoshinaka, Kiyoshi; Tei, Yuichi; Takagi, Shu; Matsumoto, Yoichiro

    2009-04-01

    Ultrasound is widely applied in the medical field and offers the strong advantages of non-invasiveness and high-selectivity. Gene transfer using ultrasound, which is called sonoporation, is one application. Ultrasound has the potential to deliver therapeutic materials such as genes, drugs or proteins into cells. Microbubbles are known to be able to improve delivery efficiency. This is attributed to therapeutic materials passing through the cell membrane after permeability is increased by destruction or oscillation of microbubbles. The present study tried to deliver the GFP plasmids into fibroblast cells. Cells were cultured in 6-well culture plates and exposed to ultrasound (frequency, 2.1 MHz; wave pattern, duty cycle 10%; intensity, 0-26 W/cm2; time, 0-200 s) transmitted through medium containing microbubbles (Levovist® (void fraction, 8×10-5) or Sonazoid® (void fraction, 0-24×10-4)) and GFP plasmids at a concentration of 15 μg/mL. Density of microbubbles after ultrasound irradiation was measured. When ultrasound intensity was increased with Levovist® 8×10-4, transfection efficiency increased, cell viability decreased and microbubbles disappeared. With Sonazoid®, transfection efficiency and cell viability were basically unchanged and microbubbles decreased, but did not disappear. Transfection efficiency also improved with increased ultrasound irradiation time or microbubble density. Microbubble destruction appeared to have the main effect on gene transfection under Levovist® and microbubble oscillation had the main effect under Sonazoid®.

  2. Deletions of the survival motor neuron gene in unaffected siblings of patients with spinal muscular atrophy

    SciTech Connect

    Cobben, J.M.; Steege, G. van der; Grootscholten, P.

    1995-10-01

    DNA studies in 103 spinal muscular atrophy (SMA) patients from The Netherlands revealed homozygosity for a survival motor neuron (SMN) deletion in 96 (93%) of 103. Neuronal apoptosis inhibitory protein deletions were found in 38 (37%) of 103 and occurred most frequently in SMA type 1. SMN deletions have not yet been described to occur in healthy subjects. In this study, however, four unaffected sibs from two SMA families showed homozygosity for SMN deletions. Homozygosity for an SMN deletion in unaffected persons seems to be very rare. Therefore, demonstration of a homozygous SMN deletion in a clinically presumed SMA patient should be considered as a confirmation of the diagnosis, whether or not SMN is in fact the causal gene for SMA. 19 refs., 2 figs.

  3. Evolutionary analyses of the small subunit of glutamate synthase: gene order conservation, gene fusions, and prokaryote-to-eukaryote lateral gene transfers.

    PubMed

    Andersson, Jan O; Roger, Andrew J

    2002-04-01

    Lateral gene transfer has been identified as an important mode of genome evolution within prokaryotes. Except for the special case of gene transfer from organelle genomes to the eukaryotic nucleus, only a few cases of lateral gene transfer involving eukaryotes have been described. Here we present phylogenetic and gene order analyses on the small subunit of glutamate synthase (encoded by gltD) and its homologues, including the large subunit of sulfide dehydrogenase (encoded by sudA). The scattered distribution of the sudA and sudB gene pair and the phylogenetic analysis strongly suggest that lateral gene transfer was involved in the propagation of the genes in the three domains of life. One of these transfers most likely occurred between a prokaryote and an ancestor of diplomonad protists. Furthermore, phylogenetic analyses indicate that the gene for the small subunit of glutamate synthase was transferred from a low-GC gram-positive bacterium to a common ancestor of animals, fungi, and plants. Interestingly, in both examples, the eukaryotes encode a single gene that corresponds to a conserved operon structure in prokaryotes. Our analyses, together with several recent publications, show that lateral gene transfers from prokaryotes to unicellular eukaryotes occur with appreciable frequency. In the case of the genes for sulfide dehydrogenase, the transfer affected only a limited group of eukaryotes--the diplomonads--while the transfer of the glutamate synthase gene probably happened earlier in evolution and affected a wider range of eukaryotes.

  4. Cell-to-cell transfer of glial proteins to the squid giant axon. The glia-neuron protein trnasfer hypothesis.

    PubMed

    Lasek, R J; Gainer, H; Barker, J L

    1977-08-01

    The hypothesis that glial cells synthesize proteins which are transferred to adjacent neurons was evaluated in the giant fiber of the squid (Loligo pealei). When giant fibers are separated from their neuron cell bodies and incubated in the presence of radioactive amino acids, labeled proteins appear in the glial cells and axoplasm. Labeled axonal proteins were detected by three methods: extrusion of the axoplasm from the giant fiber, autoradiography, and perfusion of the giant fiber. This protein synthesis is completely inhibited by puromycin but is not affected by chloramphenicol. The following evidence indicates that the labeled axonal proteins are not synthesized within the axon itself. (a) The axon does not contain a significant amount of ribosomes or ribosomal RNA. (b) Isolated axoplasm did not incorporate [(3)H]leucine into proteins. (c) Injection of Rnase into the giant axon did not reduce the appearance of newly synthesized proteins in the axoplasm of the giant fiber. These findings, coupled with other evidence, have led us to conclude that the adaxonal glial cells synthesize a class of proteins which are transferred to the giant axon. Analysis of the kinetics of this phenomenon indicates that some proteins are transferred to the axon within minutes of their synthesis in the glial cells. One or more of the steps in the transfer process appear to involve Ca++, since replacement of extracellular Ca++ by either Mg++ or Co++ significantly reduces the appearance of labeled proteins in the axon. A substantial fraction of newly synthesized glial proteins, possibly as much as 40 percent, are transferred to the giant axon. These proteins are heterogeneous and range in size from 12,000 to greater than 200,000 daltons. Comparisons of the amount of amino acid incorporation in glia cells and neuron cell bodies raise the possibility that the adaxonal glial cells may provide an important source of axonal proteins which is supplemental to that provided by axonal transport

  5. Retroviral-mediated IL-2 gene transfer into murine neuroblastoma.

    PubMed

    Cho, H S; Song, J Y; Park, C Y; Lyu, C J; Kim, B S; Kim, K Y

    2000-02-01

    We used retroviral-mediated gene transfer of the human interleukin (IL)-2 gene into murine neuroblastoma cells to investigate whether locally-secreted IL-2 is able to influence the generation of anti-tumor immune responses. Supernatant obtained from cultures of approximately 1 x 10(6) IL-2 gene-transduced, G-418 selected neuro-2a cells was assayed for human IL-2 production by ELISA kit. First, to estimate whether the local secretion of IL-2 from the genetically-modified tumor cells would affect their tumorigenicity in vivo, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice and tumor growth was measured weekly. And to estimate whether IL-2 transfected neuroblastoma cells protect mice from tumor development after wild-type tumor cell challenge, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice. Seven days after IL-2 gene-transfected neuroblastoma cell injection, unmodified neuro-2a cells were s.c. injected into the contralateral site of A/J mice and tumor growth was measured weekly. Finally, to estimate IL-2 effect on pre-established large tumor burdens, IL-2-secreting neuro-2a cells were s.c. injected into A/J mice with established tumor and its growth was measured weekly. The IL-2 gene-transduced neuro-2a clones secreted 120.25-177.3 IU of IL-2 per ml per 10(6) cells during 24 hr. None of the mice injected with IL-2-secreting neuro-2a cells developed tumors within 6 weeks, while all of the mice injected with wild-type neuro-2a cells developed tumors. Immunization of mice with IL-2 gene-transfected, irradiated neuro-2a cells protected these animals against a subsequent challenge with wild-type tumor cells. Finally, the size of large neuroblastomas decreased after IL-2-secreting neuro-2a cell injection into mice. Local secretion of IL-2 gene-transduced tumor cells abrogates their tumorigenicity and induces protective immunity and may inhibit the growth of neuroblastoma.

  6. Structure and organization of the human survival motor neurone (SMN) gene

    SciTech Connect

    Buerglen, L.; Lefebvre, S.; Clermont, O.

    1996-03-05

    Spinal muscular atrophies (SMA) are characterized by degeneration of the anterior horn cells of the spinal cord and represent the second most common fatal autosomal-recessive disorder after cystic fibrosis. We have previously identified the survival motor neurone gene (SMN), a SMA-determining gene in the 5q13 region encoding a hitherto unknown protein. In this report, we describe the organization and structure of SMN. The gene is {congruent}20 kb in length and consists of nine exons. Sequence data of the 5{prime} end of the gene show that the dinucleotide repeat C272 is close to several putative binding sites for transcription factors, which will help to characterize the regulation of the SMN and {sup C}BCD541 gene expression. The availability of the human SMN and its highly homologous counterpart ({sup C}BCD541) gene structures and exon-intron boundaries will hopefully speed up the characterization of SMN gene mutations in SMA. 10 refs., 3 figs.

  7. Alternative splicing of the FMR1 gene in human fetal brain neurons

    SciTech Connect

    Tao Huang; Yan Shen; Xue-bin Qin; Guan-Yun Wu

    1996-08-09

    The alternative splicing expression of the FMR1 gene was reported in several human and mouse tissues. Five regions of FMR1 gene can be alternatively spliced, but the combination of them has not been investigated fully. We reported here the analysis of alternative splicing pattern of the FMR1 gene in cultured fetal human neurons, using a RT-PCR and cloning strategy. Eleven splicing types were cloned and different isoforms were not equally represented. The dominant isoform represents nearly 40%, and the other isoforms were relatively rare. One isoform has a different carboxyl-terminus. Most of the alternative spliced regions appear hydrophilic; thus, they may locate on the surface of the FMR1 protein. 16 refs., 2 figs.

  8. Extensive Intra-Kingdom Horizontal Gene Transfer Converging on a Fungal Fructose Transporter Gene

    PubMed Central

    Coelho, Marco A.; Gonçalves, Carla; Sampaio, José Paulo; Gonçalves, Paula

    2013-01-01

    Comparative genomics revealed in the last decade a scenario of rampant horizontal gene transfer (HGT) among prokaryotes, but for fungi a clearly dominant pattern of vertical inheritance still stands, punctuated however by an increasing number of exceptions. In the present work, we studied the phylogenetic distribution and pattern of inheritance of a fungal gene encoding a fructose transporter (FSY1) with unique substrate selectivity. 109 FSY1 homologues were identified in two sub-phyla of the Ascomycota, in a survey that included 241 available fungal genomes. At least 10 independent inter-species instances of horizontal gene transfer (HGT) involving FSY1 were identified, supported by strong phylogenetic evidence and synteny analyses. The acquisition of FSY1 through HGT was sometimes suggestive of xenolog gene displacement, but several cases of pseudoparalogy were also uncovered. Moreover, evidence was found for successive HGT events, possibly including those responsible for transmission of the gene among yeast lineages. These occurrences do not seem to be driven by functional diversification of the Fsy1 proteins because Fsy1 homologues from widely distant lineages, including at least one acquired by HGT, appear to have similar biochemical properties. In summary, retracing the evolutionary path of the FSY1 gene brought to light an unparalleled number of independent HGT events involving a single fungal gene. We propose that the turbulent evolutionary history of the gene may be linked to the unique biochemical properties of the encoded transporter, whose predictable effect on fitness may be highly variable. In general, our results support the most recent views suggesting that inter-species HGT may have contributed much more substantially to shape fungal genomes than heretofore assumed. PMID:23818872

  9. Evolutionary Origins of the Eukaryotic Shikimate Pathway: Gene Fusions, Horizontal Gene Transfer, and Endosymbiotic Replacements†

    PubMed Central

    Richards, Thomas A.; Dacks, Joel B.; Campbell, Samantha A.; Blanchard, Jeffrey L.; Foster, Peter G.; McLeod, Rima; Roberts, Craig W.

    2006-01-01

    Currently the shikimate pathway is reported as a metabolic feature of prokaryotes, ascomycete fungi, apicomplexans, and plants. The plant shikimate pathway enzymes have similarities to prokaryote homologues and are largely active in chloroplasts, suggesting ancestry from the plastid progenitor genome. Toxoplasma gondii, which also possesses an alga-derived plastid organelle, encodes a shikimate pathway with similarities to ascomycete genes, including a five-enzyme pentafunctional arom. These data suggests that the shikimate pathway and the pentafunctional arom either had an ancient origin in the eukaryotes or was conveyed by eukaryote-to-eukaryote horizontal gene transfer (HGT). We expand sampling and analyses of the shikimate pathway genes to include the oomycetes, ciliates, diatoms, basidiomycetes, zygomycetes, and the green and red algae. Sequencing of cDNA from Tetrahymena thermophila confirmed the presence of a pentafused arom, as in fungi and T. gondii. Phylogenies and taxon distribution suggest that the arom gene fusion event may be an ancient eukaryotic innovation. Conversely, the Plantae lineage (represented here by both Viridaeplantae and the red algae) acquired different prokaryotic genes for all seven steps of the shikimate pathway. Two of the phylogenies suggest a derivation of the Plantae genes from the cyanobacterial plastid progenitor genome, but if the full Plantae pathway was originally of cyanobacterial origin, then the five other shikimate pathway genes were obtained from a minimum of two other eubacterial genomes. Thus, the phylogenies demonstrate both separate HGTs and shared derived HGTs within the Plantae clade either by primary HGT transfer or secondarily via the plastid progenitor genome. The shared derived characters support the holophyly of the Plantae lineage and a single ancestral primary plastid endosymbiosis. Our analyses also pinpoints a minimum of 50 gene/domain loss events, demonstrating that loss and replacement events have been

  10. The medicinal leech genome encodes 21 innexin genes: different combinations are expressed by identified central neurons.

    PubMed

    Kandarian, Brandon; Sethi, Jasmine; Wu, Allan; Baker, Michael; Yazdani, Neema; Kym, Eunice; Sanchez, Alejandro; Edsall, Lee; Gaasterland, Terry; Macagno, Eduardo

    2012-03-01

    Gap junctional proteins are important components of signaling pathways required for the development and ongoing functions of all animal tissues, particularly the nervous system, where they function in the intracellular and extracellular exchange of small signaling factors and ions. In animals whose genomes have been sufficiently sequenced, large families of these proteins, connexins, pannexins, and innexins, have been found, with 25 innexins in the nematode Caenorhabditis elegans Starich et al. (Cell Commun Adhes 8: 311-314, 2001) and at least 37 connexins in the zebrafish Danio rerio Cruciani and Mikalsen (Biol Chem 388:253-264, 2009). Having recently sequenced the medicinal leech Hirudo verbana genome, we now report the presence of 21 innexin genes in this species, nine more than we had previously reported from the analysis of an EST-derived transcriptomic database Dykes and Macagno (Dev Genes Evol 216: 185-97, 2006); Macagno et al. (BMC Genomics 25:407, 2010). Gene structure analyses show that, depending on the leech innexin gene, they can contain from 0 to 6 introns, with closely related paralogs showing the same number of introns. Phylogenetic trees comparing Hirudo to another distantly related leech species, Helobdella robusta, shows a high degree of orthology, whereas comparison to other annelids shows a relatively low level. Comparisons with other Lophotrochozoans, Ecdyzozoans and with vertebrate pannexins suggest a low number (one to two) of ancestral innexin/pannexins at the protostome/deuterostome split. Whole-mount in situ hybridization for individual genes in early embryos shows that ∼50% of the expressed innexins are detectable in multiple tissues. Expression analyses using quantitative PCR show that ∼70% of the Hirudo innexins are expressed in the nervous system, with most of these detected in early development. Finally, quantitative PCR analysis of several identified adult neurons detects the presence of different combinations of innexin genes

  11. Applying horizontal gene transfer phenomena to enhance non-viral gene therapy.

    PubMed

    Elmer, Jacob J; Christensen, Matthew D; Rege, Kaushal

    2013-11-28

    Horizontal gene transfer (HGT) is widespread amongst prokaryotes, but eukaryotes tend to be far less promiscuous with their genetic information. However, several examples of HGT from pathogens into eukaryotic cells have been discovered and mimicked to improve non-viral gene delivery techniques. For example, several viral proteins and DNA sequences have been used to significantly increase cytoplasmic and nuclear gene delivery. Plant genetic engineering is routinely performed with the pathogenic bacterium Agrobacterium tumefaciens and similar pathogens (e.g. Bartonella henselae) may also be able to transform human cells. Intracellular parasites like Trypanosoma cruzi may also provide new insights into overcoming cellular barriers to gene delivery. Finally, intercellular nucleic acid transfer between host cells will also be briefly discussed. This article will review the unique characteristics of several different viruses and microbes and discuss how their traits have been successfully applied to improve non-viral gene delivery techniques. Consequently, pathogenic traits that originally caused diseases may eventually be used to treat many genetic diseases.

  12. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells.

    PubMed

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A F V

    2013-03-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.

  13. Applying horizontal gene transfer phenomena to enhance non-viral gene therapy

    PubMed Central

    Elmer, Jacob J.; Christensen, Matthew D.; Rege, Kaushal

    2014-01-01

    Horizontal gene transfer (HGT) is widespread amongst prokaryotes, but eukaryotes tend to be far less promiscuous with their genetic information. However, several examples of HGT from pathogens into eukaryotic cells have been discovered and mimicked to improve non-viral gene delivery techniques. For example, several viral proteins and DNA sequences have been used to significantly increase cytoplasmic and nuclear gene delivery. Plant genetic engineering is routinely performed with the pathogenic bacterium Agrobacterium tumefaciens and similar pathogens (e.g. Bartonella henselae) may also be able to transform human cells. Intracellular parasites like Trypanosoma cruzi may also provide new insights into overcoming cellular barriers to gene delivery. Finally, intercellular nucleic acid transfer between host cells will also be briefly discussed. This article will review the unique characteristics of several different viruses and microbes and discuss how their traits have been successfully applied to improve non-viral gene delivery techniques. Consequently, pathogenic traits that originally caused diseases may eventually be used to treat many genetic diseases. PMID:23994344

  14. Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells

    PubMed Central

    Holkers, Maarten; Maggio, Ignazio; Liu, Jin; Janssen, Josephine M.; Miselli, Francesca; Mussolino, Claudio; Recchia, Alessandra; Cathomen, Toni; Gonçalves, Manuel A. F. V.

    2013-01-01

    The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 ‘safe harbor’ locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform. PMID:23275534

  15. Studies of DEAE-dextran-mediated gene transfer.

    PubMed

    Yang, Y W; Yang, J C

    1997-02-01

    DEAE-dextran-mediated gene transfer was studied for the introduction of pSV2neo DNA into Fisher-rat 3T3 (FR3T3) cells. Zeta (zeta) potentials of the DEAE-dextran-DNA complexes and FR3T3 cells were found to be dependent on the concentration of DEAE-dextran in the medium. The maximum transfection efficiency occurred at a DEAE-dextran/DNA ratio of 50:1 or thereabouts. The interaction between DNA and cells is determined by the adsorption process. The results obtained, along with the correlation between the kinetic adsorption behaviour of 3H-labelled DNA and the transfection efficiency, indicated that adsorption of DEAE-dextran-DNA complexes to the negatively charged cell surfaces, due to electrostatic and dispersion attraction, plays the decisive role in determining the DNA transfection efficiencies.

  16. Measuring Immune Responses to recombinant AAV Gene Transfer

    PubMed Central

    Martino, Ashley T.; Herzog, Roland W.; Anegon, Ignacio; Adjali, Oumeya

    2013-01-01

    Following AAV-based gene transfer, the occurrence of adaptive immune responses specific to the vector or the transgene product is a major roadblock to successful clinical translation. These responses include antibodies against the AAV capsid, which can be neutralizing and therefore prevent the ability to repeatedly administer the vector, and CD8+ cytotoxic T lymphocytes, which can eliminate transduced cells. In addition, humans may have both humoral and cellular pre-existing immunity, as a result from natural infection with parent virus or related serotypes. The need for assays to detect and measure these anti-capsid immune responses in humans and in experimental animals is profound. Here, ELISPOT, immunocapture (ELISA), and neutralization assays are explained and provided in detail. Furthermore, such techniques can readily be adapted to monitor and quantify immune responses against therapeutic transgene products encoded by the vector genome. PMID:22034034

  17. Statistical Mechanics of Horizontal Gene Transfer in Evolutionary Ecology

    NASA Astrophysics Data System (ADS)

    Chia, Nicholas; Goldenfeld, Nigel

    2011-04-01

    The biological world, especially its majority microbial component, is strongly interacting and may be dominated by collective effects. In this review, we provide a brief introduction for statistical physicists of the way in which living cells communicate genetically through transferred genes, as well as the ways in which they can reorganize their genomes in response to environmental pressure. We discuss how genome evolution can be thought of as related to the physical phenomenon of annealing, and describe the sense in which genomes can be said to exhibit an analogue of information entropy. As a direct application of these ideas, we analyze the variation with ocean depth of transposons in marine microbial genomes, predicting trends that are consistent with recent observations using metagenomic surveys.

  18. Horizontal gene transfer: building the web of life.

    PubMed

    Soucy, Shannon M; Huang, Jinling; Gogarten, Johann Peter

    2015-08-01

    Horizontal gene transfer (HGT) is the sharing of genetic material between organisms that are not in a parent-offspring relationship. HGT is a widely recognized mechanism for adaptation in bacteria and archaea. Microbial antibiotic resistance and pathogenicity are often associated with HGT, but the scope of HGT extends far beyond disease-causing organisms. In this Review, we describe how HGT has shaped the web of life using examples of HGT among prokaryotes, between prokaryotes and eukaryotes, and even between multicellular eukaryotes. We discuss replacement and additive HGT, the proposed mechanisms of HGT, selective forces that influence HGT, and the evolutionary impact of HGT on ancestral populations and existing populations such as the human microbiome.

  19. Viral gene transfer of APPsα rescues synaptic failure in an Alzheimer's disease mouse model.

    PubMed

    Fol, Romain; Braudeau, Jerome; Ludewig, Susann; Abel, Tobias; Weyer, Sascha W; Roederer, Jan-Peter; Brod, Florian; Audrain, Mickael; Bemelmans, Alexis-Pierre; Buchholz, Christian J; Korte, Martin; Cartier, Nathalie; Müller, Ulrike C

    2016-02-01

    Alzheimer's disease (AD) is characterized by synaptic failure, dendritic and axonal atrophy, neuronal death and progressive loss of cognitive functions. It is commonly assumed that these deficits arise due to β-amyloid accumulation and plaque deposition. However, increasing evidence indicates that loss of physiological APP functions mediated predominantly by neurotrophic APPsα produced in the non-amyloidogenic α-secretase pathway may contribute to AD pathogenesis. Upregulation of APPsα production via induction of α-secretase might, however, be problematic as this may also affect substrates implicated in tumorigenesis. Here, we used a gene therapy approach to directly overexpress APPsα in the brain using AAV-mediated gene transfer and explored its potential to rescue structural, electrophysiological and behavioral deficits in APP/PS1∆E9 AD model mice. Sustained APPsα overexpression in aged mice with already preexisting pathology and amyloidosis restored synaptic plasticity and partially rescued spine density deficits. Importantly, AAV-APPsα treatment also resulted in a functional rescue of spatial reference memory in the Morris water maze. Moreover, we demonstrate a significant reduction of soluble Aβ species and plaque load. In addition, APPsα induced the recruitment of microglia with a ramified morphology into the vicinity of plaques and upregulated IDE and TREM2 expression suggesting enhanced plaque clearance. Collectively, these data indicate that APPsα can mitigate synaptic and cognitive deficits, despite established pathology. Increasing APPsα may therefore be of therapeutic relevance for AD.

  20. Ebi/AP-1 suppresses pro-apoptotic genes expression and permits long-term survival of Drosophila sensory neurons.

    PubMed

    Lim, Young-Mi; Hayashi, Shigeo; Tsuda, Leo

    2012-01-01

    Sensory organs are constantly exposed to physical and chemical stresses that collectively threaten the survival of sensory neurons. Failure to protect stressed neurons leads to age-related loss of neurons and sensory dysfunction in organs in which the supply of new sensory neurons is limited, such as the human auditory system. Transducin β-like protein 1 (TBL1) is a candidate gene for ocular albinism with late-onset sensorineural deafness, a form of X-linked age-related hearing loss. TBL1 encodes an evolutionarily conserved F-box-like and WD40 repeats-containing subunit of the nuclear receptor co-repressor/silencing mediator for retinoid and thyroid hormone receptor and other transcriptional co-repressor complexes. Here we report that a Drosophila homologue of TBL1, Ebi, is required for maintenance of photoreceptor neurons. Loss of ebi function caused late-onset neuronal apoptosis in the retina and increased sensitivity to oxidative stress. Ebi formed a complex with activator protein 1 (AP-1) and was required for repression of Drosophila pro-apoptotic and anti-apoptotic genes expression. These results suggest that Ebi/AP-1 suppresses basal transcription levels of apoptotic genes and thereby protects sensory neurons from degeneration. PMID:22666340

  1. Detecting rare gene transfer events in bacterial populations

    PubMed Central

    Nielsen, Kaare M.; Bøhn, Thomas; Townsend, Jeffrey P.

    2014-01-01

    Horizontal gene transfer (HGT) enables bacteria to access, share, and recombine genetic variation, resulting in genetic diversity that cannot be obtained through mutational processes alone. In most cases, the observation of evolutionary successful HGT events relies on the outcome of initially rare events that lead to novel functions in the new host, and that exhibit a positive effect on host fitness. Conversely, the large majority of HGT events occurring in bacterial populations will go undetected due to lack of replication success of transformants. Moreover, other HGT events that would be highly beneficial to new hosts can fail to ensue due to lack of physical proximity to the donor organism, lack of a suitable gene transfer mechanism, genetic compatibility, and stochasticity in tempo-spatial occurrence. Experimental attempts to detect HGT events in bacterial populations have typically focused on the transformed cells or their immediate offspring. However, rare HGT events occurring in large and structured populations are unlikely to reach relative population sizes that will allow their immediate identification; the exception being the unusually strong positive selection conferred by antibiotics. Most HGT events are not expected to alter the likelihood of host survival to such an extreme extent, and will confer only minor changes in host fitness. Due to the large population sizes of bacteria and the time scales involved, the process and outcome of HGT are often not amenable to experimental investigation. Population genetic modeling of the growth dynamics of bacteria with differing HGT rates and resulting fitness changes is therefore necessary to guide sampling design and predict realistic time frames for detection of HGT, as it occurs in laboratory or natural settings. Here we review the key population genetic parameters, consider their complexity and highlight knowledge gaps for further research. PMID:24432015

  2. Detecting rare gene transfer events in bacterial populations.

    PubMed

    Nielsen, Kaare M; Bøhn, Thomas; Townsend, Jeffrey P

    2014-01-01

    Horizontal gene transfer (HGT) enables bacteria to access, share, and recombine genetic variation, resulting in genetic diversity that cannot be obtained through mutational processes alone. In most cases, the observation of evolutionary successful HGT events relies on the outcome of initially rare events that lead to novel functions in the new host, and that exhibit a positive effect on host fitness. Conversely, the large majority of HGT events occurring in bacterial populations will go undetected due to lack of replication success of transformants. Moreover, other HGT events that would be highly beneficial to new hosts can fail to ensue due to lack of physical proximity to the donor organism, lack of a suitable gene transfer mechanism, genetic compatibility, and stochasticity in tempo-spatial occurrence. Experimental attempts to detect HGT events in bacterial populations have typically focused on the transformed cells or their immediate offspring. However, rare HGT events occurring in large and structured populations are unlikely to reach relative population sizes that will allow their immediate identification; the exception being the unusually strong positive selection conferred by antibiotics. Most HGT events are not expected to alter the likelihood of host survival to such an extreme extent, and will confer only minor changes in host fitness. Due to the large population sizes of bacteria and the time scales involved, the process and outcome of HGT are often not amenable to experimental investigation. Population genetic modeling of the growth dynamics of bacteria with differing HGT rates and resulting fitness changes is therefore necessary to guide sampling design and predict realistic time frames for detection of HGT, as it occurs in laboratory or natural settings. Here we review the key population genetic parameters, consider their complexity and highlight knowledge gaps for further research.

  3. Axon Growth and Guidance Genes Identify Nascent, Immature, and Mature Olfactory Sensory Neurons

    PubMed Central

    McIntyre, Jeremy C.; Titlow, William B.; McClintock, Timothy S.

    2016-01-01

    Neurogenesis of projection neurons requires that axons be initiated, extended, and connected. Differences in the expression of axon growth and guidance genes must drive these events, but comprehensively characterizing these differences in a single neuronal type has not been accomplished. Guided by a catalog of gene expression in olfactory sensory neurons (OSNs), in situ hybridization and immunohistochemistry revealed that Cxcr4 and Dbn1, two axon initiation genes, marked the developmental transition from basal progenitor cells to immature OSNs in the olfactory epithelium. The CXCR4 immunoreactivity of these nascent OSNs overlapped partially with markers of proliferation of basal progenitor cells and partially with immunoreactivity for GAP43, the canonical marker of immature OSNs. Intracellular guidance cue signaling transcripts Ablim1, Crmp1, Dypsl2, Dpysl3, Dpysl5, Gap43, Marcskl1, and Stmn1–4 were specific to, or much more abundant in, the immature OSN layer. Receptors that mediate axonal inhibition or repulsion tended to be expressed in both immature and mature OSNs (Plxna1, Plxna4, Nrp2, Efna5) or specifically in mature OSNs (Plxna3, Unc5b, Efna3, Epha5, Epha7), although some were specific to immature OSNs (Plxnb1, Plxnb2, Plxdc2, Nrp1). Cell adhesion molecules were expressed either by both immature and mature OSNs (Dscam, Ncam1, Ncam2, Nrxn1) or solely by immature OSNs (Chl1, Nfasc1, Dscaml1). Given the loss of intracellular signaling protein expression, the continued expression of guidance cue receptors in mature OSNs is consistent with a change in the role of these receptors, perhaps to sending signals back to the cell body and nucleus. PMID:20882566

  4. Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

    PubMed

    Peng, Yingmei; Cai, Jing; Wang, Wen; Su, Bing

    2012-01-01

    Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought. PMID:23251445

  5. Multiple inter-kingdom horizontal gene transfers in the evolution of the phosphoenolpyruvate carboxylase gene family.

    PubMed

    Peng, Yingmei; Cai, Jing; Wang, Wen; Su, Bing

    2012-01-01

    Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought.

  6. Multiple Inter-Kingdom Horizontal Gene Transfers in the Evolution of the Phosphoenolpyruvate Carboxylase Gene Family

    PubMed Central

    Wang, Wen; Su, Bing

    2012-01-01

    Pepcase is a gene encoding phosphoenolpyruvate carboxylase that exists in bacteria, archaea and plants,playing an important role in plant metabolism and development. Most plants have two or more pepcase genes belonging to two gene sub-families, while only one gene exists in other organisms. Previous research categorized one plant pepcase gene as plant-type pepcase (PTPC) while the other as bacteria-type pepcase (BTPC) because of its similarity with the pepcase gene found in bacteria. Phylogenetic reconstruction showed that PTPC is the ancestral lineage of plant pepcase, and that all bacteria, protistpepcase and BTPC in plants are derived from a lineage of pepcase closely related with PTPC in algae. However, their phylogeny contradicts the species tree and traditional chronology of organism evolution. Because the diversification of bacteria occurred much earlier than the origin of plants, presumably all bacterialpepcase derived from the ancestral PTPC of algal plants after divergingfrom the ancestor of vascular plant PTPC. To solve this contradiction, we reconstructed the phylogeny of pepcase gene family. Our result showed that both PTPC and BTPC are derived from an ancestral lineage of gamma-proteobacteriapepcases, possibly via an ancient inter-kingdom horizontal gene transfer (HGT) from bacteria to the eukaryotic common ancestor of plants, protists and cellular slime mold. Our phylogenetic analysis also found 48other pepcase genes originated from inter-kingdom HGTs. These results imply that inter-kingdom HGTs played important roles in the evolution of the pepcase gene family and furthermore that HGTsare a more frequent evolutionary event than previouslythought. PMID:23251445

  7. Gene rescue in plants by direct gene transfer of total genomic DNA into protoplasts.

    PubMed Central

    Gallois, P; Lindsey, K; Malone, R; Kreis, M; Jones, M G

    1992-01-01

    To study the possibility of gene rescue in plants by direct gene transfer we chose the Arabidopsis mutant GH50 as a source of donor DNA. GH50 is tolerant of chlorsulfuron, a herbicide of the sulfonylurea class. Tobacco protoplasts were cotransfected with genomic DNA and the plasmid pHP23 which confers kanamycin resistance. A high frequency of cointegration of the plasmid and the genomic DNA was expected, which would allow the tagging of the plant selectable trait with the plasmid DNA. After transfection by electroporation the protoplasts were cultivated on regeneration medium supplemented with either chlorsulfuron or kanamycin as a selective agent. Selection on kanamycin yielded resistant calluses at an absolute transformation frequency (ATF) of 0.8 x 10(-3). Selection on chlorsulfuron yielded resistant calluses at an ATF of 4.7 x 10(-6). When a selection on chlorsulfuron was subsequently applied to the kanamycin resistant calluses, 8% of them showed resistance to this herbicide. Southern analysis carried out on the herbicide resistant transformants detected the presence of the herbicide resistance gene of Arabidopsis into the genome of the transformed tobacco. Segregation analysis showed the presence of the resistance gene and the marker gene in the progeny of the five analysed transformants. 3 transformants showed evidence of genetic linkage between the two genes. In addition we show that using the same technique a kanamycin resistance gene from a transgenic tobacco could be transferred into sugar beet protoplasts at a frequency of 0.17% of the transformants. Images PMID:1508682

  8. Regulation of Neuronal Gene Expression and Survival by Basal NMDA Receptor Activity: A Role for Histone Deacetylase 4

    PubMed Central

    Chen, Yelin; Wang, Yuanyuan; Modrusan, Zora

    2014-01-01

    Neuronal gene expression is modulated by activity via calcium-permeable receptors such as NMDA receptors (NMDARs). While gene expression changes downstream of evoked NMDAR activity have been well studied, much less is known about gene expression changes that occur under conditions of basal neuronal activity. In mouse dissociated hippocampal neuronal cultures, we found that a broad NMDAR antagonist, AP5, induced robust gene expression changes under basal activity, but subtype-specific antagonists did not. While some of the gene expression changes are also known to be downstream of stimulated NMDAR activity, others appear specific to basal NMDAR activity. The genes altered by AP5 treatment of basal cultures were enriched for pathways related to class IIa histone deacetylases (HDACs), apoptosis, and synapse-related signaling. Specifically, AP5 altered the expression of all three class IIa HDACs that are highly expressed in the brain, HDAC4, HDAC5, and HDAC9, and also induced nuclear accumulation of HDAC4. HDAC4 knockdown abolished a subset of the gene expression changes induced by AP5, and led to neuronal death under long-term tetrodotoxin or AP5 treatment in rat hippocampal organotypic slice cultures. These data suggest that basal, but not evoked, NMDAR activity regulates gene expression in part through HDAC4, and, that HDAC4 has neuroprotective functions under conditions of low NMDAR activity. PMID:25392500

  9. Gene-transfer study approval awaits more data

    SciTech Connect

    Marwick, C.

    1988-11-18

    Approval of the gene-transfer study in cancer patients has been delayed. The proposal was recommended for approval by a National Institutes of Health (NIH) advisory committee, but has been put on hold by James B. Wyngaarden, MD, NIH director, pending submission in writing of further information. Some of this information, now forthcoming, had been withheld because data on preliminary studies had been submitted to peer-reviewed journals. The study involves placing the gene for neomycin-resistance to tumor-infiltrating lymphocytes as a marker. When these cells are injected into the patient, the presence of the marker should enable their fate to be studied over a prolonged period and an improved antitumor regimen could result. The use of tumor-infiltrating lymphocytes as immunotherapy has been studied for two years at the NIH's National Cancer Institute. The patients' tumors are removed and the tumor-infiltrating lymphocytes are cultivated to obtain several billion cells. These cells are then injected back into the patient. Early clinical experience has shown a substantial decease in tumor size in some patients, but not in all, an no one knows why.

  10. What tangled web: barriers to rampant horizontal gene transfer.

    PubMed

    Kurland, Charles G

    2005-07-01

    Dawkins in his The Selfish Gene(1) quite aptly applies the term "selfish" to parasitic repetitive DNA sequences endemic to eukaryotic genomes, especially vertebrates. Doolittle and Sapienza(2) as well as Orgel and Crick(3) enlivened this notion of selfish DNA with the identification of such repetitive sequences as remnants of mobile elements such as transposons. In addition, Orgel and Crick(3) associated parasitic DNA with a potential to outgrow their host genomes by propagating both vertically via conventional genome replication as well as infectiously by horizontal gene transfer (HGT) to other genomes. Still later, Doolittle(4) speculated that unchecked HGT between unrelated genomes so complicates phylogeny that the conventional representation of a tree of life would have to be replaced by a thicket or a web of life.(4) In contrast, considerable data now show that reconstructions based on whole genome sequences are consistent with the conventional "tree of life".(5-10) Here, we identify natural barriers that protect modern genome populations from the inroads of rampant HGT.

  11. Transgenic approach to express the channelrhodopsin 2 gene in arginine vasopressin neurons of rats.

    PubMed

    Ishii, Masahiro; Hashimoto, Hirofumi; Ohkubo, Jun-Ichi; Ohbuchi, Toyoaki; Saito, Takeshi; Maruyama, Takashi; Yoshimura, Mitsuhiro; Yamamoto, Yukiyo; Kusuhara, Koichi; Ueta, Yoichi

    2016-09-01

    Optogenetics provides a powerful tool to regulate neuronal activity by light-sensitive ion channels such as channelrhodopsin 2 (ChR2). Arginine vasopressin (AVP; also known as the anti-diuretic hormone) is a multifunctional hormone which is synthesized in the magnocellular neurosecretory cells (MNCs) of the hypothalamus. Here, we have generated a transgenic rat that expresses an AVP-ChR2-enhanced green fluorescent protein (eGFP) fusion gene in the MNCs of the hypothalamus. The eGFP fluorescence that indicates the expression of ChR2-eGFP was observed in the supraoptic nucleus (SON) and in the magnocellular division of the paraventricular nucleus (PVN) that is known to contain AVP-secreting neurons. The eGFP fluorescence intensities in those nuclei and posterior pituitary were markedly increased after chronic salt loading (2% NaCl in drinking water for 5days). ChR2-eGFP was localized mainly in the membrane of AVP-positive MNCs. Whole-cell patch-clamp recordings were performed from single MNCs isolated from the SON of the transgenic rats, and blue light evoked repetitive action potentials. Our work provides for the first time an optogenetic approach to selectively activate AVP neurons in the rat. PMID:27493075

  12. Center for fetal monkey gene transfer for heart, lung, and blood diseases: an NHLBI resource for the gene therapy community.

    PubMed

    Tarantal, Alice F; Skarlatos, Sonia I

    2012-11-01

    The goals of the National Heart, Lung, and Blood Institute (NHLBI) Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases are to conduct gene transfer studies in monkeys to evaluate safety and efficiency; and to provide NHLBI-supported investigators with expertise, resources, and services to actively pursue gene transfer approaches in monkeys in their research programs. NHLBI-supported projects span investigators throughout the United States and have addressed novel approaches to gene delivery; "proof-of-principle"; assessed whether findings in small-animal models could be demonstrated in a primate species; or were conducted to enable new grant or IND submissions. The Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood Diseases successfully aids the gene therapy community in addressing regulatory barriers, and serves as an effective vehicle for advancing the field.

  13. Genome-scale phylogenetic analysis finds extensive gene transfer among fungi

    PubMed Central

    Szöllősi, Gergely J.; Davín, Adrián Arellano; Tannier, Eric; Daubin, Vincent; Boussau, Bastien

    2015-01-01

    Although the role of lateral gene transfer is well recognized in the evolution of bacteria, it is generally assumed that it has had less influence among eukaryotes. To explore this hypothesis, we compare the dynamics of genome evolution in two groups of organisms: cyanobacteria and fungi. Ancestral genomes are inferred in both clades using two types of methods: first, Count, a gene tree unaware method that models gene duplications, gains and losses to explain the observed numbers of genes present in a genome; second, ALE, a more recent gene tree-aware method that reconciles gene trees with a species tree using a model of gene duplication, loss and transfer. We compare their merits and their ability to quantify the role of transfers, and assess the impact of taxonomic sampling on their inferences. We present what we believe is compelling evidence that gene transfer plays a significant role in the evolution of fungi. PMID:26323765

  14. Frequent, independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes.

    PubMed

    Bruto, Maxime; Prigent-Combaret, Claire; Luis, Patricia; Moënne-Loccoz, Yvan; Muller, Daniel

    2014-08-22

    Even genetically distant prokaryotes can exchange genes between them, and these horizontal gene transfer events play a central role in adaptation and evolution. While this was long thought to be restricted to prokaryotes, certain eukaryotes have acquired genes of bacterial origin. However, gene acquisitions in eukaryotes are thought to be much less important in magnitude than in prokaryotes. Here, we describe the complex evolutionary history of a bacterial catabolic gene that has been transferred repeatedly from different bacterial phyla to stramenopiles and fungi. Indeed, phylogenomic analysis pointed to multiple acquisitions of the gene in these filamentous eukaryotes-as many as 15 different events for 65 microeukaryotes. Furthermore, once transferred, this gene acquired introns and was found expressed in mRNA databases for most recipients. Our results show that effective inter-domain transfers and subsequent adaptation of a prokaryotic gene in eukaryotic cells can happen at an unprecedented magnitude.

  15. Immunoregulation by B7 and IL-12 gene transfer.

    PubMed

    Kato, K; Okumura, K; Yagita, H

    1997-04-01

    Our recent studies using various costimulatory molecules have demonstrated that antitumor effect could be induced by B7- or B70-transduced mouse tumors. To augment antitumor effect in vivo, the combination therapy with a costimulatory gene and a cytokine, interkeukin 12 (IL-12), gene to treat metastatic mouse lung tumor was investigated. We transfected with mouse B7 and/or IL-12 into mouse lung carcinoma 3LL, and three transfectants (IL-12/3LL, B7/3LL and IL-12/B7/3LL) were generated. CTL activity induced by the inoculation of IL-12/B7/3LL was increased about 10-fold compared with parental 3LL inoculation. We then examined the therapeutic efficacy of combination with B7 and IL-12-transduced tumors. Four weeks after 3LL inoculation, lung metastasis was significantly reduced by IL-12/B7/3LL post-inoculation, indicating that potent therapeutic antitumor immunity can be induced by combination with costimulators B7 and IL-12. Recently, it was reported that p40 subunit of IL-12 appeared to be a specific inhibitor for IL-12 heterodimer in vitro. To clarify the biological functions of p40 in vivo, we generated the myoblast transfectants which produced IL-12 p40 alone. Local production of IL-12 p40 from transfectant could suppress allogenic CTL induction and Th1-type antibodies (IgG2a/2b/3) production in vivo. Furthermore, IL-12 p40 producing myoblast are less susceptible to rejection compared with parental myoblast, indicating that IL-12 p40 gene transfer may be useful therapeutically in Th1-mediated transplantation and autoimmune disorders.

  16. Horizontal gene transfer of chlamydial-like tRNA genes into early vascular plant mitochondria.

    PubMed

    Knie, Nils; Polsakiewicz, Monika; Knoop, Volker

    2015-03-01

    Mitochondrial genomes of lycophytes are surprisingly diverse, including strikingly different transfer RNA (tRNA) gene complements: No mitochondrial tRNA genes are present in the spikemoss Selaginella moellendorffii, whereas 26 tRNAs are encoded in the chondrome of the clubmoss Huperzia squarrosa. Reinvestigating the latter we found that trnL(gag) and trnS(gga) had never before been identified in any other land plant mitochondrial DNA. Sensitive sequence comparisons showed these two tRNAs as well as trnN(guu) and trnS(gcu) to be very similar to their respective counterparts in chlamydial bacteria. We identified homologs of these chlamydial-type tRNAs also in other lycophyte, fern, and gymnosperm DNAs, suggesting horizontal gene transfer (HGT) into mitochondria in the early vascular plant stem lineages. These findings extend plant mitochondrial HGT to affect individual tRNA genes, to include bacterial donors, and suggest that Chlamydiae on top of their recently proposed key role in primary chloroplast establishment may also have participated in early tracheophyte genome evolution.

  17. Evaluation of biolistic gene transfer methods in vivo using non-invasive bioluminescent imaging techniques

    PubMed Central

    2011-01-01

    Background Gene therapy continues to hold great potential for treating many different types of disease and dysfunction. Safe and efficient techniques for gene transfer and expression in vivo are needed to enable gene therapeutic strategies to be effective in patients. Currently, the most commonly used methods employ replication-defective viral vectors for gene transfer, while physical gene transfer methods such as biolistic-mediated ("gene-gun") delivery to target tissues have not been as extensively explored. In the present study, we evaluated the efficacy of biolistic gene transfer techniques in vivo using non-invasive bioluminescent imaging (BLI) methods. Results Plasmid DNA carrying the firefly luciferase (LUC) reporter gene under the control of the human Cytomegalovirus (CMV) promoter/enhancer was transfected into mouse skin and liver using biolistic methods. The plasmids were coupled to gold microspheres (1 μm diameter) using different DNA Loading Ratios (DLRs), and "shot" into target tissues using a helium-driven gene gun. The optimal DLR was found to be in the range of 4-10. Bioluminescence was measured using an In Vivo Imaging System (IVIS-50) at various time-points following transfer. Biolistic gene transfer to mouse skin produced peak reporter gene expression one day after transfer. Expression remained detectable through four days, but declined to undetectable levels by six days following gene transfer. Maximum depth of tissue penetration following biolistic transfer to abdominal skin was 200-300 μm. Similarly, biolistic gene transfer to mouse liver in vivo also produced peak early expression followed by a decline over time. In contrast to skin, however, liver expression of the reporter gene was relatively stable 4-8 days post-biolistic gene transfer, and remained detectable for nearly two weeks. Conclusions The use of bioluminescence imaging techniques enabled efficient evaluation of reporter gene expression in vivo. Our results demonstrate that

  18. Differential calmodulin-modulatory and electron transfer properties of neuronal nitric oxide synthase mu compared to the alpha variant.

    PubMed

    Panda, Satya P; Li, Wenbing; Venkatakrishnan, Priya; Chen, Li; Astashkin, Andrei V; Masters, Bettie Sue S; Feng, Changjian; Roman, Linda J

    2013-12-11

    Neuronal nitric oxide synthase μ (nNOSμ) contains 34 additional residues in an autoregulatory element compared to nNOSα. Cytochrome c and flavin reductions in the absence of calmodulin (CaM) were faster in nNOSμ than nNOSα, while rates in the presence of CaM were smaller. The magnitude of stimulation by CaM is thus notably lower in nNOSμ. No difference in NO production was observed, while electron transfer between the FMN and heme moieties and formation of an inhibitory ferrous-nitrosyl complex were slower in nNOSμ. Thus, the insert affects electron transfer rates, modulation of electron flow by CaM, and heme-nitrosyl complex formation. PMID:24211446

  19. Differential Calmodulin-Modulatory and Electron Transfer Properties of Neuronal Nitric Oxide Synthase Mu Compared to the Alpha Variant

    PubMed Central

    Panda, Satya P.; Li, Wenbing; Venkatakrishnan, Priya; Chen, Li; Astashkin, Andrei V.; Masters, Bettie Sue S.; Feng, Changjian; Roman, Linda J.

    2014-01-01

    Neuronal nitric oxide synthase μ (nNOSμ) contains 34 additional residues in an autoregulatory element compared to nNOSα. Cytochrome c and flavin reductions in the absence of calmodulin (CaM) were faster in nNOSμ than nNOSα, while rates in the presence of CaM were smaller. The magnitude of stimulation by CaM is thus notably lower in nNOSμ. No difference in NO production was observed, while electron transfer between the FMN and heme moieties and formation of an inhibitory ferrous-nitrosyl complex were slower in nNOSμ. Thus, the insert affects electron transfer rates, modulation of electron flow by CaM, and heme-nitrosyl complex formation. PMID:24211446

  20. Gene Regulatory Mechanisms Underlying the Spatial and Temporal Regulation of Target-Dependent Gene Expression in Drosophila Neurons.

    PubMed

    Berndt, Anthony J E; Tang, Jonathan C Y; Ridyard, Marc S; Lian, Tianshun; Keatings, Kathleen; Allan, Douglas W

    2015-12-01

    Neuronal differentiation often requires target-derived signals from the cells they innervate. These signals typically activate neural subtype-specific genes, but the gene regulatory mechanisms remain largely unknown. Highly restricted expression of the FMRFa neuropeptide in Drosophila Tv4 neurons requires target-derived BMP signaling and a transcription factor code that includes Apterous. Using integrase transgenesis of enhancer reporters, we functionally dissected the Tv4-enhancer of FMRFa within its native cellular context. We identified two essential but discrete cis-elements, a BMP-response element (BMP-RE) that binds BMP-activated pMad, and a homeodomain-response element (HD-RE) that binds Apterous. These cis-elements have low activity and must be combined for Tv4-enhancer activity. Such combinatorial activity is often a mechanism for restricting expression to the intersection of cis-element spatiotemporal activities. However, concatemers of the HD-RE and BMP-RE cis-elements were found to independently generate the same spatiotemporal expression as the Tv4-enhancer. Thus, the Tv4-enhancer atypically combines two low-activity cis-elements that confer the same output from distinct inputs. The activation of target-dependent genes is assumed to 'wait' for target contact. We tested this directly, and unexpectedly found that premature BMP activity could not induce early FMRFa expression; also, we show that the BMP-insensitive HD-RE cis-element is activated at the time of target contact. This led us to uncover a role for the nuclear receptor, seven up (svp), as a repressor of FMRFa induction prior to target contact. Svp is normally downregulated immediately prior to target contact, and we found that maintaining Svp expression prevents cis-element activation, whereas reducing svp gene dosage prematurely activates cis-element activity. We conclude that the target-dependent FMRFa gene is repressed prior to target contact, and that target-derived BMP signaling directly

  1. Gene Regulatory Mechanisms Underlying the Spatial and Temporal Regulation of Target-Dependent Gene Expression in Drosophila Neurons

    PubMed Central

    Ridyard, Marc S.; Lian, Tianshun; Keatings, Kathleen; Allan, Douglas W.

    2015-01-01

    Neuronal differentiation often requires target-derived signals from the cells they innervate. These signals typically activate neural subtype-specific genes, but the gene regulatory mechanisms remain largely unknown. Highly restricted expression of the FMRFa neuropeptide in Drosophila Tv4 neurons requires target-derived BMP signaling and a transcription factor code that includes Apterous. Using integrase transgenesis of enhancer reporters, we functionally dissected the Tv4-enhancer of FMRFa within its native cellular context. We identified two essential but discrete cis-elements, a BMP-response element (BMP-RE) that binds BMP-activated pMad, and a homeodomain-response element (HD-RE) that binds Apterous. These cis-elements have low activity and must be combined for Tv4-enhancer activity. Such combinatorial activity is often a mechanism for restricting expression to the intersection of cis-element spatiotemporal activities. However, concatemers of the HD-RE and BMP-RE cis-elements were found to independently generate the same spatiotemporal expression as the Tv4-enhancer. Thus, the Tv4-enhancer atypically combines two low-activity cis-elements that confer the same output from distinct inputs. The activation of target-dependent genes is assumed to 'wait' for target contact. We tested this directly, and unexpectedly found that premature BMP activity could not induce early FMRFa expression; also, we show that the BMP-insensitive HD-RE cis-element is activated at the time of target contact. This led us to uncover a role for the nuclear receptor, seven up (svp), as a repressor of FMRFa induction prior to target contact. Svp is normally downregulated immediately prior to target contact, and we found that maintaining Svp expression prevents cis-element activation, whereas reducing svp gene dosage prematurely activates cis-element activity. We conclude that the target-dependent FMRFa gene is repressed prior to target contact, and that target-derived BMP signaling directly

  2. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen.

    PubMed

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L; Beauseigle, Stéphanie; Bernier, Louis; Copeland, Alex; Foster, Adam; Gill, Navdeep; Henrissat, Bernard; Herath, Padmini; LaButti, Kurt M; Levasseur, Anthony; Lindquist, Erika A; Majoor, Eline; Ohm, Robin A; Pangilinan, Jasmyn L; Pribowo, Amadeus; Saddler, John N; Sakalidis, Monique L; de Vries, Ronald P; Grigoriev, Igor V; Goodwin, Stephen B; Tanguay, Philippe; Hamelin, Richard C

    2015-03-17

    Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems.

  3. The Fusarium graminearum Genome Reveals More Secondary Metabolite Gene Clusters and Hints of Horizontal Gene Transfer

    PubMed Central

    Wong, Philip; Münsterkötter, Martin; Mewes, Hans-Werner; Schmeitzl, Clemens; Varga, Elisabeth; Berthiller, Franz; Adam, Gerhard; Güldener, Ulrich

    2014-01-01

    Fungal secondary metabolite biosynthesis genes are of major interest due to the pharmacological properties of their products (like mycotoxins and antibiotics). The genome of the plant pathogenic fungus Fusarium graminearum codes for a large number of candidate enzymes involved in secondary metabolite biosynthesis. However, the chemical nature of most enzymatic products of proteins encoded by putative secondary metabolism biosynthetic genes is largely unknown. Based on our analysis we present 67 gene clusters with significant enrichment of predicted secondary metabolism related enzymatic functions. 20 gene clusters with unknown metabolites exhibit strong gene expression correlation in planta and presumably play a role in virulence. Furthermore, the identification of conserved and over-represented putative transcription factor binding sites serves as additional evidence for cluster co-regulation. Orthologous cluster search provided insight into the evolution of secondary metabolism clusters. Some clusters are characteristic for the Fusarium phylum while others show evidence of horizontal gene transfer as orthologs can be found in representatives of the Botrytis or Cochliobolus lineage. The presented candidate clusters provide valuable targets for experimental examination. PMID:25333987

  4. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen

    PubMed Central

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L.; Beauseigle, Stéphanie; Bernier, Louis; Copeland, Alex; Foster, Adam; Gill, Navdeep; Henrissat, Bernard; Herath, Padmini; LaButti, Kurt M.; Levasseur, Anthony; Lindquist, Erika A.; Majoor, Eline; Ohm, Robin A.; Pangilinan, Jasmyn L.; Pribowo, Amadeus; Saddler, John N.; Sakalidis, Monique L.; de Vries, Ronald P.; Grigoriev, Igor V.; Goodwin, Stephen B.; Tanguay, Philippe; Hamelin, Richard C.

    2015-01-01

    Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems. PMID:25733908

  5. Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen.

    PubMed

    Dhillon, Braham; Feau, Nicolas; Aerts, Andrea L; Beauseigle, Stéphanie; Bernier, Louis; Copeland, Alex; Foster, Adam; Gill, Navdeep; Henrissat, Bernard; Herath, Padmini; LaButti, Kurt M; Levasseur, Anthony; Lindquist, Erika A; Majoor, Eline; Ohm, Robin A; Pangilinan, Jasmyn L; Pribowo, Amadeus; Saddler, John N; Sakalidis, Monique L; de Vries, Ronald P; Grigoriev, Igor V; Goodwin, Stephen B; Tanguay, Philippe; Hamelin, Richard C

    2015-03-17

    Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems. PMID:25733908

  6. Towards liver-directed gene therapy: retrovirus-mediated gene transfer into human hepatocytes.

    PubMed

    Grossman, M; Raper, S E; Wilson, J M

    1991-11-01

    Liver-directed gene therapy is being considered in the treatment of inherited metabolic diseases. One approach we are considering is the transplantation of autologous hepatocytes that have been genetically modified with recombinant retroviruses ex vivo. We describe, in this report, techniques for isolating human hepatocytes and efficiently transducing recombinant genes into primary cultures. Hepatocytes were isolated from tissue of four different donors, plated in primary culture, and exposed to recombinant retroviruses expressing either the LacZ reporter gene or the cDNA for rabbit LDL receptor. The efficiency of gene transfer under optimal conditions, as determined by Southern blot analysis, varied from a maximum of one proviral copy per cell to a minimum of 0.1 proviral copy per cell. Cytochemical assays were used to detect expression of the recombinant derived proteins, E. coli beta-galactosidase and rabbit LDL receptor. Hepatocytes transduced with the LDL receptor gene expressed levels of receptor protein that exceeded the normal endogenous levels. The ability to isolate and genetically modify human hepatocytes, as described in this report, is an important step towards the development of liver-directed gene therapies in humans. PMID:1767337

  7. The gene silencing transcription factor REST represses miR-132 expression in hippocampal neurons destined to die.

    PubMed

    Hwang, Jee-Yeon; Kaneko, Naoki; Noh, Kyung-Min; Pontarelli, Fabrizio; Zukin, R Suzanne

    2014-10-01

    The gene silencing transcription factor REST [repressor element 1 silencing transcription factor]/NRSF (neuron-restrictive silencer factor) actively represses a large array of coding and noncoding neuron-specific genes important to synaptic plasticity including miR-132. miR-132 is a neuron-specific microRNA and plays a pivotal role in synaptogenesis, synaptic plasticity and structural remodeling. However, a role for miR-132 in neuronal death is not, as yet, well-delineated. Here we show that ischemic insults promote REST binding and epigenetic remodeling at the miR-132 promoter and silencing of miR-132 expression in selectively vulnerable hippocampal CA1 neurons. REST occupancy was not altered at the miR-9 or miR-124a promoters despite the presence of repressor element 1 sites, indicating REST target specificity. Ischemia induced a substantial decrease in two marks of active gene transcription, dimethylation of lysine 4 on core histone 3 (H3K4me2) and acetylation of lysine 9 on H3 (H3K9ac) at the miR-132 promoter. RNAi-mediated depletion of REST in vivo blocked ischemia-induced loss of miR-132 in insulted hippocampal neurons, consistent with a causal relation between activation of REST and silencing of miR-132. Overexpression of miR-132 in primary cultures of hippocampal neurons or delivered directly into the CA1 of living rats by means of the lentiviral expression system prior to induction of ischemia afforded robust protection against ischemia-induced neuronal death. These findings document a previously unappreciated role for REST-dependent repression of miR-132 in the neuronal death associated with global ischemia and identify a novel therapeutic target for amelioration of the neurodegeneration and cognitive deficits associated with ischemic stroke. PMID:25108103

  8. snoU6 and 5S RNAs are not reliable miRNA reference genes in neuronal differentiation.

    PubMed

    Lim, Q E; Zhou, L; Ho, Y K; Wan, G; Too, H P

    2011-12-29

    Accurate profiling of microRNAs (miRNAs) is an essential step for understanding the functional significance of these small RNAs in both physiological and pathological processes. Quantitative real-time PCR (qPCR) has gained acceptance as a robust and reliable transcriptomic method to profile subtle changes in miRNA levels and requires reference genes for accurate normalization of gene expression. 5S and snoU6 RNAs are commonly used as reference genes in microRNA quantification. It is currently unknown if these small RNAs are stably expressed during neuronal differentiation. Panels of miRNAs have been suggested as alternative reference genes to 5S and snoU6 in various physiological contexts. To test the hypothesis that miRNAs may serve as stable references during neuronal differentiation, the expressions of eight miRNAs, 5S and snoU6 RNAs in five differentiating neuronal cell types were analyzed using qPCR. The stabilities of the expressions were evaluated using two complementary statistical approaches (geNorm and Normfinder). Expressions of 5S and snoU6 RNAs were stable under some but not all conditions of neuronal differentiation and thus are not suitable reference genes. In contrast, a combination of three miRNAs (miR-103, miR-106b and miR-26b) allowed accurate expression normalization across different models of neuronal differentiation.

  9. Microfluidic measurement of effects of ACF7/MACF1 gene on the mechanics of primary cortical neurons

    NASA Astrophysics Data System (ADS)

    Lee, Donghee; Ka, Minhan; Kim, Woo-Yang; Ryu, Sangjin

    2014-03-01

    Actin filaments and microtubules play important roles in determining the mechanics of cells, and ACF7/MACF1 (Actin Crosslinking Family 7/Microtubule And Actin Crosslinking Factor 1) gene seems to be closely related to connections between actin filaments and microtubules. To identify such roles of the ACF7/MACF1 gene of primary cortical neurons, we isolated neuronal cells from the cerebral cortex of the embryonic mouse brain, which is important in memory, language and perception. We exerted viscous shear flow to normal neuronal cells and ACF7/MACF1 gene knockout neuronal cells using rectangular microfluidic channels. While changing viscous shear stress on the cells, we recorded changes in the morphology of the two cell types using video microscopy. Having analyzed the deformation of the cells, we could quantitatively correlate differences in the morphological change between the both normal and ACF7/MACF1 gene knockout neuronal cells to the applied shear force, which will contribute toward identifying cell mechanical roles of the ACF7/MACF1 gene.

  10. Heterochromatin-mediated gene silencing facilitates the diversification of olfactory neurons

    PubMed Central

    Lyons, David B.; Magklara, Angeliki; Goh, Tracie; Sampath, Srihari; Schaefer, Anne; Schotta, Gunnar; Lomvardas, Stavros

    2014-01-01

    SUMMARY An astounding property of the nervous system is its cellular diversity. This diversity, which was initially realized by morphological and electrophysiological differences, is ultimately produced by variations in gene expression programs. In most cases these variations are determined by external cues. However, a growing number of neuronal types have been identified in which inductive signals cannot explain the few but decisive transcriptional differences that cause cell diversification. Here, we show that heterochromatic silencing, which we find is governed by histone methyltransferases G9a (KMT1C) and GLP (KMT1D), is essential for stochastic and singular OR expression. Deletion of G9a and GLP dramatically reduces the complexity of the OR transcriptome, resulting in transcriptional domination by a few ORs and loss of singularity in OR expression. Thus, in addition to its previously known functions, our data suggest that heterochromatin creates an epigenetic platform that affords stochastic, mutually exclusive gene choices and promotes cellular diversity. PMID:25437545

  11. The neuronal nicotinic acetylcholine receptor {alpha}7 subunit gene: Cloning, mapping, structure, and targeting in mouse

    SciTech Connect

    Orr-Urtreger, A.; Baldini, A.; Beaudet, A.L.

    1994-09-01

    The neuronal nicotinic acetylcholine receptor {alpha}7 subunit is a member of a family of ligand-gated ion channels, and is the only subunit know to bind {alpha}-bungarotoxin in mammalian brain. {alpha}-Bungarotoxin binding sites are known to be more abundant in the hippocampus of mouse strains that are particularly sensitive to nicotine-induced seizures. The {alpha}7 receptor is highly permeable to calcium, which could suggest a role in synaptic plasticity in the nervous system. Auditory gating deficiency, an abnormal response to a second auditory stimulus, is characteristic of schizophrenia. Mouse strains that exhibit a similar gating deficit have reduced hippocampal expression of the {alpha}7 subunit. We have cloned and sequenced the full length cDNA for the mouse {alpha}7 gene (Acra-7) and characterized its gene structure. The murine {alpha}7 shares amino acid identity of 99% and 93% with the rat and human {alpha}7 subunits, respectively. Using an interspecies backcross panel, the murine gene was mapped to chromosome 7 near the p locus, a region syntenic with human chromosome 15; the human gene (CHRNA7) was confirmed to map to 15q13-q14 by FISH. To generate a mouse {alpha}7 mutant by homologous recombination, we have constructed a replacement vector which will delete transmembrane domains II-IV and the cytoplasmic domain from the gene product. Recombinant embryonic stem (ES) cell clones were selected and used to develop mouse chimeras that are currently being bred to obtain germline transmission.

  12. Binding of TFIIIC to SINE Elements Controls the Relocation of Activity-Dependent Neuronal Genes to Transcription Factories

    PubMed Central

    Crepaldi, Luca; Policarpi, Cristina; Coatti, Alessandro; Sherlock, William T.; Jongbloets, Bart C.; Down, Thomas A.; Riccio, Antonella

    2013-01-01

    In neurons, the timely and accurate expression of genes in response to synaptic activity relies on the interplay between epigenetic modifications of histones, recruitment of regulatory proteins to chromatin and changes to nuclear structure. To identify genes and regulatory elements responsive to synaptic activation in vivo, we performed a genome-wide ChIPseq analysis of acetylated histone H3 using somatosensory cortex of mice exposed to novel enriched environmental (NEE) conditions. We discovered that Short Interspersed Elements (SINEs) located distal to promoters of activity-dependent genes became acetylated following exposure to NEE and were bound by the general transcription factor TFIIIC. Importantly, under depolarizing conditions, inducible genes relocated to transcription factories (TFs), and this event was controlled by TFIIIC. Silencing of the TFIIIC subunit Gtf3c5 in non-stimulated neurons induced uncontrolled relocation to TFs and transcription of activity-dependent genes. Remarkably, in cortical neurons, silencing of Gtf3c5 mimicked the effects of chronic depolarization, inducing a dramatic increase of both dendritic length and branching. These findings reveal a novel and essential regulatory function of both SINEs and TFIIIC in mediating gene relocation and transcription. They also suggest that TFIIIC may regulate the rearrangement of nuclear architecture, allowing the coordinated expression of activity-dependent neuronal genes. PMID:23966877

  13. ChIP-Seq Data Mining: Remarkable Differences in NRSF/REST Target Genes between Human ESC and ESC-Derived Neurons.

    PubMed

    Satoh, Jun-Ichi; Kawana, Natsuki; Yamamoto, Yoji

    2013-01-01

    The neuron-restrictive silencer factor (NRSF) is a zinc finger transcription factor that represses neuronal gene transcription in non-neuronal cells by binding to the consensus repressor element-1 (RE1) located in regulatory regions of target genes. NRSF silences the expression of a wide range of target genes involved in neuron-specific functions. Previous studies showed that aberrant regulation of NRSF plays a key role in the pathological process of human neurodegenerative diseases. However, a comprehensive set of NRSF target genes relevant to human neuronal functions has not yet been characterized. We performed genome-wide data mining from chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) datasets of NRSF binding sites in human embryonic stem cells (ESC) and the corresponding ESC-derived neurons, retrieved from the database of the ENCODE/HAIB project. Using bioinformatics tools such as Avadis NGS and MACS, we identified 2,172 NRSF target genes in ESC and 308 genes in ESC-derived neurons based on stringent criteria. Only 40 NRSF target genes overlapped between both data sets. According to motif analysis, binding regions showed an enrichment of the consensus RE1 sites in ESC, whereas they were mainly located in poorly defined non-RE1 sites in ESC-derived neurons. Molecular pathways of NRSF target genes were linked with various neuronal functions in ESC, such as neuroactive ligand-receptor interaction, CREB signaling, and axonal guidance signaling, while they were not directed to neuron-specific functions in ESC-derived neurons. Remarkable differences in ChIP-Seq-based NRSF target genes and pathways between ESC and ESC-derived neurons suggested that NRSF-mediated silencing of target genes is highly effective in human ESC but not in ESC-derived neurons. PMID:24324330

  14. Neuron type-specific miRNA represses two broadly expressed genes to modulate an avoidance behavior in C. elegans

    PubMed Central

    Drexel, Tanja; Mahofsky, Katharina; Latham, Richard; Zimmer, Manuel

    2016-01-01

    Two broad gene classes are distinguished within multicellular organisms: cell type-specific genes, which confer particular cellular properties, and ubiquitous genes that support general cellular functions. However, certain so-called ubiquitous genes show functionally relevant cell type-specific repression. How such repression is achieved is poorly understood. MicroRNAs (miRNAs) are repressors, many of which are expressed with high cell type specificity. Here we show that mir-791, expressed exclusively in the CO2-sensing neurons in Caenorhabditis elegans, represses two otherwise broadly expressed genes. This repression is necessary for normal neuronal function and behavior of the animals toward CO2. miRNA-mediated repression of broadly transcribed genes is a previously unappreciated strategy for cellular specialization. PMID:27688400

  15. Low-dose oral cadmium increases airway reactivity and lung neuronal gene expression in mice.

    PubMed

    Chandler, Joshua D; Wongtrakool, Cherry; Banton, Sophia A; Li, Shuzhao; Orr, Michael L; Barr, Dana Boyd; Neujahr, David C; Sutliff, Roy L; Go, Young-Mi; Jones, Dean P

    2016-07-01

    Inhalation of cadmium (Cd) is associated with lung diseases, but less is known concerning pulmonary effects of Cd found in the diet. Cd has a decades-long half-life in humans and significant bioaccumulation occurs with chronic dietary intake. We exposed mice to low-dose CdCl2 (10 mg/L in drinking water) for 20 weeks, which increased lung Cd to a level similar to that of nonoccupationally exposed adult humans. Cd-treated mice had increased airway hyperresponsiveness to methacholine challenge, and gene expression array showed that Cd altered the abundance of 443 mRNA transcripts in mouse lung. In contrast to higher doses, low-dose Cd did not elicit increased metallothionein transcripts in lung. To identify pathways most affected by Cd, gene set enrichment of transcripts was analyzed. Results showed that major inducible targets of low-dose Cd were neuronal receptors represented by enriched olfactory, glutamatergic, cholinergic, and serotonergic gene sets. Olfactory receptors regulate chemosensory function and airway hypersensitivity, and these gene sets were the most enriched. Targeted metabolomics analysis showed that Cd treatment also increased metabolites in pathways of glutamatergic (glutamate), serotonergic (tryptophan), cholinergic (choline), and catecholaminergic (tyrosine) receptors in the lung tissue. Protein abundance measurements showed that the glutamate receptor GRIN2A was increased in mouse lung tissue. Together, these results show that in mice, oral low-dose Cd increased lung Cd to levels comparable to humans, increased airway hyperresponsiveness and disrupted neuronal pathways regulating bronchial tone. Therefore, dietary Cd may promote or worsen airway hyperresponsiveness in multiple lung diseases including asthma.

  16. Low-dose oral cadmium increases airway reactivity and lung neuronal gene expression in mice.

    PubMed

    Chandler, Joshua D; Wongtrakool, Cherry; Banton, Sophia A; Li, Shuzhao; Orr, Michael L; Barr, Dana Boyd; Neujahr, David C; Sutliff, Roy L; Go, Young-Mi; Jones, Dean P

    2016-07-01

    Inhalation of cadmium (Cd) is associated with lung diseases, but less is known concerning pulmonary effects of Cd found in the diet. Cd has a decades-long half-life in humans and significant bioaccumulation occurs with chronic dietary intake. We exposed mice to low-dose CdCl2 (10 mg/L in drinking water) for 20 weeks, which increased lung Cd to a level similar to that of nonoccupationally exposed adult humans. Cd-treated mice had increased airway hyperresponsiveness to methacholine challenge, and gene expression array showed that Cd altered the abundance of 443 mRNA transcripts in mouse lung. In contrast to higher doses, low-dose Cd did not elicit increased metallothionein transcripts in lung. To identify pathways most affected by Cd, gene set enrichment of transcripts was analyzed. Results showed that major inducible targets of low-dose Cd were neuronal receptors represented by enriched olfactory, glutamatergic, cholinergic, and serotonergic gene sets. Olfactory receptors regulate chemosensory function and airway hypersensitivity, and these gene sets were the most enriched. Targeted metabolomics analysis showed that Cd treatment also increased metabolites in pathways of glutamatergic (glutamate), serotonergic (tryptophan), cholinergic (choline), and catecholaminergic (tyrosine) receptors in the lung tissue. Protein abundance measurements showed that the glutamate receptor GRIN2A was increased in mouse lung tissue. Together, these results show that in mice, oral low-dose Cd increased lung Cd to levels comparable to humans, increased airway hyperresponsiveness and disrupted neuronal pathways regulating bronchial tone. Therefore, dietary Cd may promote or worsen airway hyperresponsiveness in multiple lung diseases including asthma. PMID:27401458

  17. Ethanol-induced changes in poly (ADP ribose) polymerase and neuronal developmental gene expression.

    PubMed

    Gavin, David P; Kusumo, Handojo; Sharma, Rajiv P; Guizzetti, Marina

    2016-11-01

    Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that inhibition of PARP enzymatic activity increased PPARγ promoter binding, and this corresponded to increased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression. PMID:27497606

  18. SMN control of RNP assembly: from post-transcriptional gene regulation to motor neuron disease

    PubMed Central

    Li, Darrick K.; Tisdale, Sarah; Lotti, Francesco; Pellizzoni, Livio

    2014-01-01

    At the post-transcriptional level, expression of protein-coding genes is controlled by a series of RNA regulatory events including nuclear processing of primary transcripts, transport of mature mRNAs to specific cellular compartments, translation and ultimately, turnover. These processes are orchestrated through the dynamic association of mRNAs with RNA binding proteins and ribonucleoprotein (RNP) complexes. Accurate formation of RNPs in vivo is fundamentally important to cellular development and function, and its impairment often leads to human disease. The survival motor neuron (SMN) protein is key to this biological paradigm: SMN is essential for the biogenesis of various RNPs that function in mRNA processing, and genetic mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP assembly. We discuss advances in our understanding of SMN activity as a chaperone of RNPs and how disruption of SMN-dependent RNA pathways can cause motor neuron disease. PMID:24769255

  19. Neonicotinoid Insecticides Alter the Gene Expression Profile of Neuron-Enriched Cultures from Neonatal Rat Cerebellum

    PubMed Central

    Kimura-Kuroda, Junko; Nishito, Yasumasa; Yanagisawa, Hiroko; Kuroda, Yoichiro; Komuta, Yukari; Kawano, Hitoshi; Hayashi, Masaharu

    2016-01-01

    Neonicotinoids are considered safe because of their low affinities to mammalian nicotinic acetylcholine receptors (nAChRs) relative to insect nAChRs. However, because of importance of nAChRs in mammalian brain development, there remains a need to establish the safety of chronic neonicotinoid exposures with regards to children’s health. Here we examined the effects of long-term (14 days) and low dose (1 μM) exposure of neuron-enriched cultures from neonatal rat cerebellum to nicotine and two neonicotinoids: acetamiprid and imidacloprid. Immunocytochemistry revealed no differences in the number or morphology of immature neurons or glial cells in any group versus untreated control cultures. However, a slight disturbance in Purkinje cell dendritic arborization was observed in the exposed cultures. Next we performed transcriptome analysis on total RNAs using microarrays, and identified significant differential expression (p < 0.05, q < 0.05, ≥1.5 fold) between control cultures versus nicotine-, acetamiprid-, or imidacloprid-exposed cultures in 34, 48, and 67 genes, respectively. Common to all exposed groups were nine genes essential for neurodevelopment, suggesting that chronic neonicotinoid exposure alters the transcriptome of the developing mammalian brain in a similar way to nicotine exposure. Our results highlight the need for further careful investigations into the effects of neonicotinoids in the developing mammalian brain. PMID:27782041

  20. Ethanol-induced changes in poly (ADP ribose) polymerase and neuronal developmental gene expression.

    PubMed

    Gavin, David P; Kusumo, Handojo; Sharma, Rajiv P; Guizzetti, Marina

    2016-11-01

    Prenatal alcohol exposure has profound effects on neuronal growth and development. Poly-ADP Ribose Polymerase (PARP) enzymes are perhaps unique in the field of epigenetics in that they directly participate in histone modifications, transcription factor modifications, DNA methylation/demethylation and are highly inducible by ethanol. It was our hypothesis that ethanol would induce PARP enzymatic activity leading to alterations in neurodevelopmental gene expression. Mouse E18 cortical neurons were treated with ethanol, PARP inhibitors, and nuclear hormone receptor transcription factor PPARγ agonists and antagonists. Subsequently, we measured PARP activity and changes in Bdnf, OKSM (Oct4, Klf4, Sox2, c-Myc), DNA methylating/demethylating factors, and Pparγ mRNA expression, promoter 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC), and PPARγ promoter binding. We found that ethanol reduced Bdnf4, 9a, and Klf4 mRNA expression, and increased c-Myc expression. These changes were reversed with a PARP inhibitor. In agreement with its role in DNA demethylation PARP inhibition increased 5MC levels at the c-Myc promoter. In addition, we found that inhibition of PARP enzymatic activity increased PPARγ promoter binding, and this corresponded to increased Bdnf and Klf4 mRNA expression. Our results suggest that PARP participates in DNA demethylation and reduces PPARγ promoter binding. The current study underscores the importance of PARP in ethanol-induced changes to neurodevelopmental gene expression.

  1. Hipposeq: a comprehensive RNA-seq database of gene expression in hippocampal principal neurons

    PubMed Central

    Cembrowski, Mark S; Wang, Lihua; Sugino, Ken; Shields, Brenda C; Spruston, Nelson

    2016-01-01

    Clarifying gene expression in narrowly defined neuronal populations can provide insight into cellular identity, computation, and functionality. Here, we used next-generation RNA sequencing (RNA-seq) to produce a quantitative, whole genome characterization of gene expression for the major excitatory neuronal classes of the hippocampus; namely, granule cells and mossy cells of the dentate gyrus, and pyramidal cells of areas CA3, CA2, and CA1. Moreover, for the canonical cell classes of the trisynaptic loop, we profiled transcriptomes at both dorsal and ventral poles, producing a cell-class- and region-specific transcriptional description for these populations. This dataset clarifies the transcriptional properties and identities of lesser-known cell classes, and moreover reveals unexpected variation in the trisynaptic loop across the dorsal-ventral axis. We have created a public resource, Hipposeq (http://hipposeq.janelia.org), which provides analysis and visualization of these data and will act as a roadmap relating molecules to cells, circuits, and computation in the hippocampus. DOI: http://dx.doi.org/10.7554/eLife.14997.001 PMID:27113915

  2. Downregulation of immediate-early genes linking to suppression of neuronal plasticity in rats after 28-day exposure to glycidol.

    PubMed

    Akane, Hirotoshi; Saito, Fumiyo; Shiraki, Ayako; Takeyoshi, Masahiro; Imatanaka, Nobuya; Itahashi, Megu; Murakami, Tomoaki; Shibutani, Makoto

    2014-09-01

    We previously found that the 28-day oral toxicity study of glycidol at 200mg/kg/day in rats resulted in axonopathy in both the central and peripheral nervous systems and aberrations in the late-stage of hippocampal neurogenesis targeting the process of neurite extension. To capture the neuronal parameters in response to glycidol toxicity, these animals were subjected to region-specific global gene expression profiling in four regions of cerebral and cerebellar architectures, followed by immunohistochemical analysis of selected gene products. Expression changes of genes related to axonogenesis and synaptic transmission were observed in the hippocampal dentate gyrus, cingulate cortex and cerebellar vermis at 200mg/kg showing downregulation in most genes. In the corpus callosum, genes related to growth, survival and functions of glial cells fluctuated their expression. Immunohistochemically, neurons expressing gene products of immediate-early genes, i.e., Arc, Fos and Jun, decreased in their number in the dentate granule cell layer, cingulate cortex and cerebellar vermis. We also applied immunohistochemical analysis in rat offspring after developmental exposure to glycidol through maternal drinking water. The results revealed increases of Arc(+) neurons at 1000ppm and Fos(+) neurons at ≥300ppm in the dentate granule cell layer of offspring only at the adult stage. These results suggest that glycidol suppressed neuronal plasticity in the brain after 28-day exposure to young adult animals, in contrast to the operation of restoration mechanism to increase neuronal plasticity at the adult stage in response to aberrations in neurogenesis after developmental exposure.

  3. In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles.

    PubMed

    Lopes, Cátia Df; Oliveira, Hugo; Estevão, Inês; Pires, Liliana Raquel; Pêgo, Ana Paula

    2016-01-01

    A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies. PMID:27354797

  4. In vivo targeted gene delivery to peripheral neurons mediated by neurotropic poly(ethylene imine)-based nanoparticles

    PubMed Central

    Lopes, Cátia DF; Oliveira, Hugo; Estevão, Inês; Pires, Liliana Raquel; Pêgo, Ana Paula

    2016-01-01

    A major challenge in neuronal gene therapy is to achieve safe, efficient, and minimally invasive transgene delivery to neurons. In this study, we report the use of a nonviral neurotropic poly(ethylene imine)-based nanoparticle that is capable of mediating neuron-specific transfection upon a subcutaneous injection. Nanoparticles were targeted to peripheral neurons by using the nontoxic carboxylic fragment of tetanus toxin (HC), which, besides being neurotropic, is capable of being retrogradely transported from neuron terminals to the cell bodies. Nontargeted particles and naked plasmid DNA were used as control. Five days after treatment by subcutaneous injection in the footpad of Wistar rats, it was observed that 56% and 64% of L4 and L5 dorsal root ganglia neurons, respectively, were expressing the reporter protein. The delivery mediated by HC-functionalized nanoparticles spatially limited the transgene expression, in comparison with the controls. Histological examination revealed no significant adverse effects in the use of the proposed delivery system. These findings demonstrate the feasibility and safety of the developed neurotropic nanoparticles for the minimally invasive delivery of genes to the peripheral nervous system, opening new avenues for the application of gene therapy strategies in the treatment of peripheral neuropathies. PMID:27354797

  5. Molecular mechanisms of non-transferrin-bound and transferring-bound iron uptake in primary hippocampal neurons.

    PubMed

    Ji, Changyi; Kosman, Daniel J

    2015-06-01

    The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell-derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co-localize, indicating these two proteins may function in Fe(3+) reduction prior to Fe(2+) permeation. Zip8, DMT1, and Steap2 co-localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin-mediated iron assimilation. In brain interstitial fluid, transferring-bound iron (TBI) and non-transferrin-bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin-(59) Fe(3+)) and NTBI, whether presented as (59) Fe(2+) -citrate or (59) Fe(3+) -citrate; reductase-independent (59) Fe(2+) uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn(2+) inhibition of Fe(2+) uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of (59) Fe from TBI relies at least in part on an endocytosis-independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons. Analysis of the expression and localization of known iron uptake transporters demonstrated that Zip8 makes a major contribution to iron accumulation in primary cultures of rat embryonic hippocampal neurons. These cells exhibit uptake pathways for ferrous and ferric iron (non-transferrin-bound iron, NTBI in figure) and for transferrin-bound iron; the ferrireductases Steap2 and SDR2 support the uptake of ferric iron substrates. Zip8 and Steap2 are strongly expressed in the plasma membrane of both soma

  6. Transduction-like gene transfer in the methanogen Methanococcus voltae

    NASA Technical Reports Server (NTRS)

    Bertani, G.

    1999-01-01

    Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.

  7. Multimodality Imaging of Gene Transfer with a Receptor-Based Reporter Gene

    PubMed Central

    Chen, Ron; Parry, Jesse J.; Akers, Walter J.; Berezin, Mikhail Y.; El Naqa, Issam M.; Achilefu, Samuel; Edwards, W. Barry; Rogers, Buck E.

    2010-01-01

    Gene therapy trials have traditionally used tumor and tissue biopsies for assessing the efficacy of gene transfer. Non-invasive imaging techniques offer a distinct advantage over tissue biopsies in that the magnitude and duration of gene transfer can be monitored repeatedly. Human somatostatin receptor subtype 2 (SSTR2) has been used for the nuclear imaging of gene transfer. To extend this concept, we have developed a somatostatin receptor–enhanced green fluorescent protein fusion construct (SSTR2-EGFP) for nuclear and fluorescent multimodality imaging. Methods An adenovirus containing SSTR2-EGFP (AdSSTR2-EGFP) was constructed and evaluated in vitro and in vivo. SCC-9 human squamous cell carcinoma cells were infected with AdEGFP, AdSSTR2, or AdSSTR2-EGFP for in vitro evaluation by saturation binding, internalization, and fluorescence spectroscopy assays. In vivo biodistribution and nano-SPECT imaging studies were conducted with mice bearing SCC-9 tumor xenografts directly injected with AdSSTR2-EGFP or AdSSTR2 to determine the tumor localization of 111In-diethylenetriaminepentaacetic acid (DTPA)-Tyr3-octreotate. Fluorescence imaging was conducted in vivo with mice receiving intratumoral injections of AdSSTR2, AdSSTR2-EGFP, or AdEGFP as well as ex vivo with tissues extracted from mice. Results The similarity between AdSSTR2-EGFP and wild-type AdSSTR2 was demonstrated in vitro by the saturation binding and internalization assays, and the fluorescence emission spectra of cells infected with AdSSTR2-EGFP was almost identical to the spectra of cells infected with wild-type AdEGFP. Biodistribution studies demonstrated that the tumor uptake of 111In-DTPA-Tyr3-octreotate was not significantly different (P > 0.05) when tumors (n = 5) were injected with AdSSTR2 or AdSSTR2-EGFP but was significantly greater than the uptake in control tumors. Fluorescence was observed in tumors injected with AdSSTR2-EGFP and AdEGFP in vivo and ex vivo but not in tumors injected with AdSSTR2

  8. Dopaminergic neuron-specific deletion of p53 gene is neuroprotective in an experimental Parkinson's disease model.

    PubMed

    Qi, Xin; Davis, Brandon; Chiang, Yung-Hsiao; Filichia, Emily; Barnett, Austin; Greig, Nigel H; Hoffer, Barry; Luo, Yu

    2016-09-01

    p53, a stress response gene, is involved in diverse cell death pathways and its activation has been implicated in the pathogenesis of Parkinson's disease (PD). However, whether the neuronal p53 protein plays a direct role in regulating dopaminergic (DA) neuronal cell death is unknown. In this study, in contrast to the global inhibition of p53 function by pharmacological inhibitors and in traditional p53 knock-out (KO) mice, we examined the effect of DA specific p53 gene deletion in DAT-p53KO mice. These DAT-p53KO mice did not exhibit apparent changes in the general structure and neuronal density of DA neurons during late development and in aging. However, in DA-p53KO mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we found that the induction of Bax and p53 up-regulated modulator of apoptosis (PUMA) mRNA and protein levels by MPTP were diminished in both striatum and substantia nigra of these mice. Notably, deletion of the p53 gene in DA neurons significantly reduced dopaminergic neuronal loss in substantia nigra, dopaminergic neuronal terminal loss at striatum and, additionally, decreased motor deficits in mice challenged with MPTP. In contrast, there was no difference in astrogliosis between WT and DAT-p53KO mice in response to MPTP treatment. These findings demonstrate a specific contribution of p53 activation in DA neuronal cell death by MPTP challenge. Our results further support the role of programmed cell death mediated by p53 in this animal model of PD and identify Bax, BAD and PUMA genes as downstream targets of p53 in modulating DA neuronal death in the in vivo MPTP-induced PD model. We deleted p53 gene in dopaminergic neurons in late developmental stages and found that DA specific p53 deletion is protective in acute MPTP animal model possibly through blocking MPTP-induced BAX and PUMA up-regulation. Astrocyte activation measured by GFAP positive cells and GFAP gene up-regulation in the striatum shows no difference

  9. Dopaminergic neuron-specific deletion of p53 gene is neuroprotective in an experimental Parkinson's disease model.

    PubMed

    Qi, Xin; Davis, Brandon; Chiang, Yung-Hsiao; Filichia, Emily; Barnett, Austin; Greig, Nigel H; Hoffer, Barry; Luo, Yu

    2016-09-01

    p53, a stress response gene, is involved in diverse cell death pathways and its activation has been implicated in the pathogenesis of Parkinson's disease (PD). However, whether the neuronal p53 protein plays a direct role in regulating dopaminergic (DA) neuronal cell death is unknown. In this study, in contrast to the global inhibition of p53 function by pharmacological inhibitors and in traditional p53 knock-out (KO) mice, we examined the effect of DA specific p53 gene deletion in DAT-p53KO mice. These DAT-p53KO mice did not exhibit apparent changes in the general structure and neuronal density of DA neurons during late development and in aging. However, in DA-p53KO mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we found that the induction of Bax and p53 up-regulated modulator of apoptosis (PUMA) mRNA and protein levels by MPTP were diminished in both striatum and substantia nigra of these mice. Notably, deletion of the p53 gene in DA neurons significantly reduced dopaminergic neuronal loss in substantia nigra, dopaminergic neuronal terminal loss at striatum and, additionally, decreased motor deficits in mice challenged with MPTP. In contrast, there was no difference in astrogliosis between WT and DAT-p53KO mice in response to MPTP treatment. These findings demonstrate a specific contribution of p53 activation in DA neuronal cell death by MPTP challenge. Our results further support the role of programmed cell death mediated by p53 in this animal model of PD and identify Bax, BAD and PUMA genes as downstream targets of p53 in modulating DA neuronal death in the in vivo MPTP-induced PD model. We deleted p53 gene in dopaminergic neurons in late developmental stages and found that DA specific p53 deletion is protective in acute MPTP animal model possibly through blocking MPTP-induced BAX and PUMA up-regulation. Astrocyte activation measured by GFAP positive cells and GFAP gene up-regulation in the striatum shows no difference

  10. Selfish Operons: Horizontal Transfer May Drive the Evolution of Gene Clusters

    PubMed Central

    Lawrence, J. G.; Roth, J. R.

    1996-01-01

    A model is presented whereby the formation of gene clusters in bacteria is mediated by transfer of DNA within and among taxa. Bacterial operons are typically composed of genes whose products contribute to a single function. If this function is subject to weak selection or to long periods with no selection, the contributing genes may accumulate mutations and be lost by genetic drift. From a cell's perspective, once several genes are lost, the function can be restored only if all missing genes were acquired simultaneously by lateral transfer. The probability of transfer of multiple genes increases when genes are physically proximate. From a gene's perspective, horizontal transfer provides a way to escape evolutionary loss by allowing colonization of organisms lacking the encoded functions. Since organisms bearing clustered genes are more likely to act as successful donors, clustered genes would spread among bacterial genomes. The physical proximity of genes may be considered a selfish property of the operon since it affects the probability of successful horizontal transfer but may provide no physiological benefit to the host. This process predicts a mosaic structure of modern genomes in which ancestral chromosomal material is interspersed with novel, horizontally transferred operons providing peripheral metabolic functions. PMID:8844169

  11. Microbial Evolution Is in the Cards: Horizontal Gene Transfer in the Classroom

    ERIC Educational Resources Information Center

    Kagle, Jeanne; Hay, Anthony G.

    2007-01-01

    Horizontal gene transfer, the exchange of genetic material between bacteria, is a potentially important factor in the degradation of synthetic compounds introduced to the environment and in the acquisition of other characteristics including antibiotic resistance. This game-based activity illustrates the role of horizontal gene transfer in the…

  12. Cis- and Trans-Regulatory Mechanisms of Gene Expression in the ASJ Sensory Neuron of Caenorhabditis elegans

    PubMed Central

    González-Barrios, María; Fierro-González, Juan Carlos; Krpelanova, Eva; Mora-Lorca, José Antonio; Pedrajas, José Rafael; Peñate, Xenia; Chavez, Sebastián; Swoboda, Peter; Jansen, Gert; Miranda-Vizuete, Antonio

    2015-01-01

    The identity of a given cell type is determined by the expression of a set of genes sharing common cis-regulatory motifs and being regulated by shared transcription factors. Here, we identify cis and trans regulatory elements that drive gene expression in the bilateral sensory neuron ASJ, located in the head of the nematode Caenorhabditis elegans. For this purpose, we have dissected the promoters of the only two genes so far reported to be exclusively expressed in ASJ, trx-1 and ssu-1. We hereby identify the ASJ motif, a functional cis-regulatory bipartite promoter region composed of two individual 6 bp elements separated by a 3 bp linker. The first element is a 6 bp CG-rich sequence that presumably binds the Sp family member zinc-finger transcription factor SPTF-1. Interestingly, within the C. elegans nervous system SPTF-1 is also found to be expressed only in ASJ neurons where it regulates expression of other genes in these neurons and ASJ cell fate. The second element of the bipartite motif is a 6 bp AT-rich sequence that is predicted to potentially bind a transcription factor of the homeobox family. Together, our findings identify a specific promoter signature and SPTF-1 as a transcription factor that functions as a terminal selector gene to regulate gene expression in C. elegans ASJ sensory neurons. PMID:25769980

  13. Noise-assisted information transfer in crayfish mechanoreceptors: stochastic resonance in a neuronal receptor

    NASA Astrophysics Data System (ADS)

    Douglass, John K.; Wilkens, Lon A.; Moss, Frank

    1993-11-01

    Adding random noise to a weak periodic signal can enhance the flow of information through certain nonlinear physical systems, via a process known as stochastic resonance (SR). We have used crayfish mechanoreceptor cells to investigate the possibility that SR can be induced in neurophysiological systems. Various signal-to-noise ratio (SNR) measurements were derived from the action potentials (spikes) of single receptor cells stimulated with weak periodic signals. Spike noise was controlled by one of two methods: (1) adding external noise to the stimulus, or (2) altering internal noise sources by changing the temperature of the cell. In external noise experiments, an optimal noise level can be identified at which the SNR is maximized. In internal noise experiments, although the SNR increases with increasing noise, no SNR maximum has been observed. These results demonstrate that SR can be induced in single neurons, and suggest that neuronal systems may also be capable of exploiting SR.

  14. Inflammatory mediators release calcitonin gene-related peptide from dorsal root ganglion neurons of the rat.

    PubMed

    Averbeck, B; Izydorczyk, I; Kress, M

    2000-01-01

    The interactions between the inflammatory mediators bradykinin, serotonin, prostaglandin E(2) and acid pH were studied in rat dorsal root ganglion neurons in culture. For this purpose, the cultures were stimulated by inflammatory mediators (bradykinin, serotonin, prostaglandin E(2), 10(-5)M each) or acid solution (pH 6.1) for 5 min and the content of calcitonin gene-related peptide was determined in the supernatant before, during and after stimulation, using an enzyme immunoassay. Acid solution resulted in a threefold increase of the basal calcitonin gene-related peptide release which was entirely dependent on the presence of extracellular calcium. The release could not be blocked by the addition of the capsaicin antagonist capsazepine (10(-5)M). Bradykinin (10(-5)M) caused a 50% increase of the basal calcitonin gene-related peptide release which was again dependent on the presence of extracellular calcium, whereas serotonin and prostaglandin E(2) were each ineffective at 10(-5)M concentration. The combination of bradykinin, serotonin and prostaglandin E(2) led to a fivefold increase of the calcitonin gene-related peptide release which could not be further enhanced by acidification. The competitive capsaicin receptor antagonist capsazepine (10(-5)M) significantly reduced the release induced by the combination of bradykinin, serotonin and prostaglandin E(2). It is suggested that the inflammatory mediators co-operate and together may act as endogenous agonists at the capsaicin receptor to cause calcium influx and consecutive neuropeptide release. PMID:10858619

  15. A novel mutation in the MFSD8 gene in late infantile neuronal ceroid lipofuscinosis.

    PubMed

    Stogmann, E; El Tawil, S; Wagenstaller, J; Gaber, A; Edris, S; Abdelhady, A; Assem-Hilger, E; Leutmezer, F; Bonelli, S; Baumgartner, C; Zimprich, F; Strom, T M; Zimprich, A

    2009-02-01

    Neuronal ceroid lipofuscinoses (NCL) are lysosomal storage disorders and constitute the most common group of progressive neurodegenerative diseases in childhood. Most NCLs are inherited in a recessive manner and are clinically characterised by a variable age at onset, epileptic seizures, psychomotor decline, visual impairment and premature death. To date, eight causative genes have been identified to underlie various clinical forms of NCL. We performed a genome-wide linkage analysis followed by sequencing the recently described NCL gene MFSD8 in three affected and three unaffected members of a consanguineous Egyptian family with an autosomal recessively inherited progressive neurodegenerative disorder. The clinical picture of the patients was compatible with a late infantile NCL (LINCL); however, impairment of the visual system was not a cardinal symptom in the respective family. By linkage analysis, we identified two putative loci on chromosome 1p36.11-p35.1 and 4q28.1-q28.2. The latter locus (4q28.1-q28.2) contained the MFSD8 gene, comprising a novel homozygous missense mutation in exon 5 (c.362a>g /p.Tyr121Cys), which segregated with the disease in the three affected sibs. We describe a novel mutation in the previously identified MFSD8 gene in a family with a common phenotype of LINCL, but no clinical report of vision loss. Our results enlarge the mutational and perhaps the nosological spectrum of one of the recently identified subtypes of NCL, called CLN7. PMID:18850119

  16. The Neuronal Sortilin-Related Receptor Gene SORL1 and Late-Onset Alzheimer's Disease

    PubMed Central

    Lee, Joseph H.; Barral, Sandra; Reitz, Christiane

    2009-01-01

    Recent studies indicate that two clusters of single nucleotide polymorphisms in the neuronal sortilin-related receptor gene (SORL1) are causally associated with late-onset Alzheimer's disease (AD). At the cellular level, SORL1 is thought to be involved in intracellular trafficking of amyloid precursor protein. When this gene is suppressed, toxic amyloid β production is increased, and high levels of amyloid β are associated with a higher AD risk. Extending the cellular findings, gene expression studies show that SORL1 is differentially expressed in AD patients compared with controls. Furthermore, several genetic studies have identified allelic and haplotypic SORL1 variants associated with late-onset AD, and these variants confer small to modest risk of AD. Taken together, the evidence for SORL1 as a causative gene is compelling. However, putative variants have not yet been identified. Further research is necessary to determine its utility as a diagnostic marker of AD or as a target for new therapeutic approaches. PMID:18713574

  17. Neuron-specific specificity protein 4 bigenomically regulates the transcription of all mitochondria- and nucleus-encoded cytochrome c oxidase subunit genes in neurons.

    PubMed

    Johar, Kaid; Priya, Anusha; Dhar, Shilpa; Liu, Qiuli; Wong-Riley, Margaret T T

    2013-11-01

    Neurons are highly dependent on oxidative metabolism for their energy supply, and cytochrome c oxidase (COX) is a key energy-generating enzyme in the mitochondria. A unique feature of COX is that it is one of only four proteins in mammalian cells that are bigenomically regulated. Of its thirteen subunits, three are encoded in the mitochondrial genome and ten are nuclear-encoded on nine different chromosomes. The mechanism of regulating this multisubunit, bigenomic enzyme poses a distinct challenge. In recent years, we found that nuclear respiratory factors 1 and 2 (NRF-1 and NRF-2) mediate such bigenomic coordination. The latest candidate is the specificity factor (Sp) family of proteins. In N2a cells, we found that Sp1 regulates all 13 COX subunits. However, we discovered recently that in primary neurons, it is Sp4 and not Sp1 that regulates some of the key glutamatergic receptor subunit genes. The question naturally arises as to the role of Sp4 in regulating COX in primary neurons. The present study utilized multiple approaches, including chromatin immunoprecipitation, promoter mutational analysis, knockdown and over-expression of Sp4, as well as functional assays to document that Sp4 indeed functionally regulate all 13 subunits of COX as well as mitochondrial transcription factors A and B. The present study discovered that among the specificity family of transcription factors, it is the less known neuron-specific Sp4 that regulates the expression of all 13 subunits of mitochondrial cytochrome c oxidase (COX) enzyme in primary neurons. Sp4 also regulates the three mitochondrial transcription factors (TFAM, TFB1M, and TFB2M) and a COX assembly protein SURF-1 in primary neurons.

  18. Gene transfer in the evolution of parasite nucleotide biosynthesis.

    PubMed

    Striepen, Boris; Pruijssers, Andrea J P; Huang, Jinling; Li, Catherine; Gubbels, Marc-Jan; Umejiego, Nwakaso N; Hedstrom, Lizbeth; Kissinger, Jessica C

    2004-03-01

    Nucleotide metabolic pathways provide numerous successful targets for antiparasitic chemotherapy, but the human pathogen Cryptosporidium parvum thus far has proved extraordinarily refractory to classical treatments. Given the importance of this protist as an opportunistic pathogen afflicting immunosuppressed individuals, effective treatments are urgently needed. The genome sequence of C. parvum is approaching completion, and we have used this resource to critically assess nucleotide biosynthesis as a target in C. parvum. Genomic analysis indicates that this parasite is entirely dependent on salvage from the host for its purines and pyrimidines. Metabolic pathway reconstruction and experimental validation in the laboratory further suggest that the loss of pyrimidine de novo synthesis is compensated for by possession of three salvage enzymes. Two of these, uridine kinase-uracil phosphoribosyltransferase and thymidine kinase, are unique to C. parvum within the phylum Apicomplexa. Phylogenetic analysis suggests horizontal gene transfer of thymidine kinase from a proteobacterium. We further show that the purine metabolism in C. parvum follows a highly streamlined pathway. Salvage of adenosine provides C. parvum's sole source of purines. This renders the parasite susceptible to inhibition of inosine monophosphate dehydrogenase, the rate-limiting enzyme in the multistep conversion of AMP to GMP. The inosine 5' monophosphate dehydrogenase inhibitors ribavirin and mycophenolic acid, which are already in clinical use, show pronounced anticryptosporidial activity. Taken together, these data help to explain why widely used drugs fail in the treatment of cryptosporidiosis and suggest more promising targets. PMID:14973196

  19. Horizontal gene transfer in the acquisition of novel traits by metazoans

    PubMed Central

    Boto, Luis

    2014-01-01

    Horizontal gene transfer is accepted as an important evolutionary force modulating the evolution of prokaryote genomes. However, it is thought that horizontal gene transfer plays only a minor role in metazoan evolution. In this paper, I critically review the rising evidence on horizontally transferred genes and on the acquisition of novel traits in metazoans. In particular, I discuss suspected examples in sponges, cnidarians, rotifers, nematodes, molluscs and arthropods which suggest that horizontal gene transfer in metazoans is not simply a curiosity. In addition, I stress the scarcity of studies in vertebrates and other animal groups and the importance of forthcoming studies to understand the importance and extent of horizontal gene transfer in animals. PMID:24403327

  20. The C. elegans nuclear receptor gene fax-1 and homeobox gene unc-42 coordinate interneuron identity by regulating the expression of glutamate receptor subunits and other neuron-specific genes.

    PubMed

    Wightman, Bruce; Ebert, Bryan; Carmean, Nicole; Weber, Katherine; Clever, Sheila

    2005-11-01

    The fax-1 gene of the nematode C. elegans encodes a conserved nuclear receptor that is the ortholog of the human PNR gene and functions in the specification of neuron identities. Mutations in fax-1 result in locomotion defects. FAX-1 protein accumulates in the nuclei of 18 neurons, among them the AVA, AVB, and AVE interneuron pairs that coordinate body movements. The identities of AVA and AVE interneurons are defective in fax-1 mutants; neither neuron expresses the NMDA receptor subunits nmr-1 and nmr-2. Other ionotropic glutamate receptor subunits are expressed normally in the AVA and AVE neurons. The unc-42 homeobox gene also regulates AVA and AVE identity; however, unc-42 mutants display the complementary phenotype: NMDA receptor subunit expression is normal, but some non-NMDA glutamate receptor subunits are not expressed. These observations support a combinatorial role for fax-1 and unc-42 in specifying AVA and AVE identity. However, in four other neuron types, fax-1 is regulated by unc-42, and both transcriptional regulators function in the regulation of the opt-3 gene in the AVE neurons and the flp-1 and ncs-1 genes in the AVK neurons. Therefore, while fax-1 and unc-42 act in complementary parallel pathways in some cells, they function in overlapping or linear pathways in other cellular contexts, suggesting that combinatorial relationships among transcriptional regulators are complex and cannot be generalized from one neuron type to another.

  1. Ovarian steroids regulate gene expression related to DNA repair and neurodegenerative diseases in serotonin neurons of macaques

    PubMed Central

    Bethea, Cynthia L.; Reddy, Arubala P.

    2015-01-01

    Depression often accompanies the peri-menopausal transition and it often precedes overt symptomology in common neurodegenerative diseases (NDD; Alzheimer's, Parkinson's, Huntington, ALS). Serotonin dysfunction is frequently found in the different etiologies of depression. We have shown that ovariectomized (Ovx) monkeys treated with estradiol (E) for 28 days supplement with placebo or progesterone (P) on days 14-28 had reduced DNA fragmentation in serotonin neurons of the dorsal raphe nucleus; and long-term Ovx monkeys had fewer serotonin neurons than intact controls. We questioned the effect of E alone or E+P on gene expression related to DNA repair, protein folding (chaperones), the ubiquitin proteosome, axon transport, and NDD specific genes in serotonin neurons. Ovx macaques were treated with placebo, E or E+P (n=3/group) for 1 month. Serotonin neurons were laser captured and subjected to microarray analysis and qRT-PCR. Increases were confirmed with qRT-PCR in 5 genes that code for proteins involved in repair of strand breaks and nucleotide excision. NBN1, PCNA, GADD45A, RAD23A and GTF2H5 significantly increased with E or E+P treatment (all ANOVA p< 0.01). Chaperone genes HSP70, HSP60 and HSP27 significantly increased with E or E+P treatment (all ANOVA p<0.05). HSP90 showed a similar trend. Ubiquinase coding genes UBEA5, UBE2D3 and UBE3A (Parkin) increased with E or E+P (all ANOVA p<0.003). Transport related genes coding kinesin, dynein, and dynactin increased with E or E+P (all ANOVA p<0.03). SCNA (α synuclein) and ADAM10 (α secretase) increased (both ANOVA p<0.02), but PSEN1 (presenilin1) decreased (ANOVA p<0.02) with treatment. APP decreased 10-fold with E or E+P administration. Newman-Keuls posthoc comparisons indicated variation in the response to E alone versus E+P across the different genes. In summary, E or E+P increased gene expression for DNA repair mechanisms in serotonin neurons, thereby rendering them less vulnerable to stress-induced DNA

  2. Hematopoietic stem cell gene transfer for the treatment of hemoglobin disorders.

    PubMed

    Persons, Derek A

    2009-01-01

    Hematopoietic stem cell (HSC)-targeted gene transfer is an attractive approach for the treatment of a number of hematopoietic disorders caused by single gene defects. Indeed, in a series of gene transfer trials for two different primary immunodeficiencies beginning early in this decade, outstanding success has been achieved. Despite generally low levels of engrafted, genetically modified HSCs, these trials were successful because of the marked selective advantage of gene-corrected lymphoid precursors that allowed reconstitution of the immune system. Unlike the immunodeficiencies, this robust level of in vivo selection is not available to hematopoietic repopulating cells or early progenitor cells following gene transfer of a therapeutic globin gene in the setting of beta-thalassemia and sickle cell disease. Both preclinical and clinical transplant studies involving bone marrow chimeras suggest that 20% or higher levels of engraftment of genetically modified HSCs will be needed for clinical success in the most severe of these disorders. Encouragingly, gene transfer levels in this range have recently been reported in a lentiviral vector gene transfer clinical trial for children with adrenoleukodystrophy. A clinical gene transfer trial for beta-thalassemia has begun in France, and one patient with transfusion-dependent HbE/beta-thalassemia has demonstrated a therapeutic effect after transplantation with autologous CD34(+) cells genetically modified with a beta-globin lentiviral vector. Here, the development and recent progress of gene therapy for the hemoglobin disorders is reviewed.

  3. Gene transfer corrects acute GM2 gangliosidosis--potential therapeutic contribution of perivascular enzyme flow.

    PubMed

    Cachón-González, M Begoña; Wang, Susan Z; McNair, Rosamund; Bradley, Josephine; Lunn, David; Ziegler, Robin; Cheng, Seng H; Cox, Timothy M

    2012-08-01

    The GM2 gangliosidoses are fatal lysosomal storage diseases principally affecting the brain. Absence of β-hexosaminidase A and B activities in the Sandhoff mouse causes neurological dysfunction and recapitulates the acute Tay-Sachs (TSD) and Sandhoff diseases (SD) in infants. Intracranial coinjection of recombinant adeno-associated viral vectors (rAAV), serotype 2/1, expressing human β-hexosaminidase α (HEXA) and β (HEXB) subunits into 1-month-old Sandhoff mice gave unprecedented survival to 2 years and prevented disease throughout the brain and spinal cord. Classical manifestations of disease, including spasticity-as opposed to tremor-ataxia-were resolved by localized gene transfer to the striatum or cerebellum, respectively. Abundant biosynthesis of β-hexosaminidase isozymes and their global distribution via axonal, perivascular, and cerebrospinal fluid (CSF) spaces, as well as diffusion, account for the sustained phenotypic rescue-long-term protein expression by transduced brain parenchyma, choroid plexus epithelium, and dorsal root ganglia neurons supplies the corrective enzyme. Prolonged survival permitted expression of cryptic disease in organs not accessed by intracranial vector delivery. We contend that infusion of rAAV into CSF space and intraparenchymal administration by convection-enhanced delivery at a few strategic sites will optimally treat neurodegeneration in many diseases affecting the nervous system. PMID:22453766

  4. Gene Transfer Corrects Acute GM2 Gangliosidosis—Potential Therapeutic Contribution of Perivascular Enzyme Flow

    PubMed Central

    Cachón-González, M Begoña; Wang, Susan Z; McNair, Rosamund; Bradley, Josephine; Lunn, David; Ziegler, Robin; Cheng, Seng H; Cox, Timothy M

    2012-01-01

    The GM2 gangliosidoses are fatal lysosomal storage diseases principally affecting the brain. Absence of β-hexosaminidase A and B activities in the Sandhoff mouse causes neurological dysfunction and recapitulates the acute Tay–Sachs (TSD) and Sandhoff diseases (SD) in infants. Intracranial coinjection of recombinant adeno-associated viral vectors (rAAV), serotype 2/1, expressing human β-hexosaminidase α (HEXA) and β (HEXB) subunits into 1-month-old Sandhoff mice gave unprecedented survival to 2 years and prevented disease throughout the brain and spinal cord. Classical manifestations of disease, including spasticity—as opposed to tremor-ataxia—were resolved by localized gene transfer to the striatum or cerebellum, respectively. Abundant biosynthesis of β-hexosaminidase isozymes and their global distribution via axonal, perivascular, and cerebrospinal fluid (CSF) spaces, as well as diffusion, account for the sustained phenotypic rescue—long-term protein expression by transduced brain parenchyma, choroid plexus epithelium, and dorsal root ganglia neurons supplies the corrective enzyme. Prolonged survival permitted expression of cryptic disease in organs not accessed by intracranial vector delivery. We contend that infusion of rAAV into CSF space and intraparenchymal administration by convection-enhanced delivery at a few strategic sites will optimally treat neurodegeneration in many diseases affecting the nervous system. PMID:22453766

  5. Mechanisms by Which 17β-Estradiol (E2) Suppress Neuronal cox-2 Gene Expression

    PubMed Central

    Stacey, Winfred; Bhave, Shreyas; Uht, Rosalie M.

    2016-01-01

    E2 attenuates inflammatory responses by suppressing expression of pro-inflammatory genes. Given that inflammation is increasingly being associated with neurodegenerative and psychiatric processes, we sought to elucidate mechanisms by which E2 down-regulates a component of an inflammatory response, cyclooxygenase– 2 (COX-2) expression. Although inflammatory processes in the brain are usually associated with microglia and astrocytes, we found that the COX-2 gene (cox-2) was expressed in a neuronal context, specifically in an amygdalar cell line (AR-5). Given that COX-2 has been reported to be in neurons in the brain, and that the amygdala is a site involved in neurodegenerative and neuropsychiatric processes, we investigated mechanisms by which E2 could down-regulate cox-2 expression in the AR-5 line. These cells express estrogen receptors alpha (ERα) and beta (ERβ), and as shown here cox-2. At the level of RNA, E2 and the ERβ selective ligand diarylpropionitrile (DPN) both attenuated gene expression, whereas the ERα selective ligand propyl pyrazole triol (PPT) had no effect. Neither ligand increased ERβ at the cox-2 promoter. Rather, DPN decreased promoter occupancy of NF-κB p65 and histone 4 (H4) acetylation. Treatment with the non-specific HDAC inhibitor Trichostatin A (TSA) counteracted DPN’s repressive effects on cox-2 expression. In keeping with the TSA effect, E2 and DPN increased histone deacetylase one (HDAC1) and switch-independent 3A (Sin3A) promoter occupancy. Lastly, even though E2 increased CpG methylation, DPN did not. Taken together, the pharmacological data indicate that ERβ contributes to neuronal cox-2 expression, as measured by RNA levels. Furthermore, ER ligands lead to increased recruitment of HDAC1, Sin3A and a concomitant reduction of p65 occupancy and Ac-H4 levels. None of the events, however, are associated with a significant recruitment of ERβ at the promoter. Thus, ERβ directs recruitment to the cox-2 promoter, but does so in

  6. Mechanisms by Which 17β-Estradiol (E2) Suppress Neuronal cox-2 Gene Expression.

    PubMed

    Stacey, Winfred; Bhave, Shreyas; Uht, Rosalie M

    2016-01-01

    E2 attenuates inflammatory responses by suppressing expression of pro-inflammatory genes. Given that inflammation is increasingly being associated with neurodegenerative and psychiatric processes, we sought to elucidate mechanisms by which E2 down-regulates a component of an inflammatory response, cyclooxygenase- 2 (COX-2) expression. Although inflammatory processes in the brain are usually associated with microglia and astrocytes, we found that the COX-2 gene (cox-2) was expressed in a neuronal context, specifically in an amygdalar cell line (AR-5). Given that COX-2 has been reported to be in neurons in the brain, and that the amygdala is a site involved in neurodegenerative and neuropsychiatric processes, we investigated mechanisms by which E2 could down-regulate cox-2 expression in the AR-5 line. These cells express estrogen receptors alpha (ERα) and beta (ERβ), and as shown here cox-2. At the level of RNA, E2 and the ERβ selective ligand diarylpropionitrile (DPN) both attenuated gene expression, whereas the ERα selective ligand propyl pyrazole triol (PPT) had no effect. Neither ligand increased ERβ at the cox-2 promoter. Rather, DPN decreased promoter occupancy of NF-κB p65 and histone 4 (H4) acetylation. Treatment with the non-specific HDAC inhibitor Trichostatin A (TSA) counteracted DPN's repressive effects on cox-2 expression. In keeping with the TSA effect, E2 and DPN increased histone deacetylase one (HDAC1) and switch-independent 3A (Sin3A) promoter occupancy. Lastly, even though E2 increased CpG methylation, DPN did not. Taken together, the pharmacological data indicate that ERβ contributes to neuronal cox-2 expression, as measured by RNA levels. Furthermore, ER ligands lead to increased recruitment of HDAC1, Sin3A and a concomitant reduction of p65 occupancy and Ac-H4 levels. None of the events, however, are associated with a significant recruitment of ERβ at the promoter. Thus, ERβ directs recruitment to the cox-2 promoter, but does so in the

  7. Primary structure and neuronal effects of α-bag cell peptide, a second candidate neurotransmitter encoded by a single gene in bag cell neurons of Aplysia

    PubMed Central

    Rothman, Barry S.; Mayeri, Earl; Brown, Richard O.; Yuan, Pau-Miau; Shively, John E.

    1983-01-01

    A discharge of impulse activity in a group of neuroendocrine cells, the bag cells, produces several types of prolonged responses in various identified neurons of the abdominal ganglion of Aplysia. Two excitatory responses are almost certainly mediated by egg-laying hormone, but this peptide cannot account for other responses, such as inhibition of left upper quadrant neurons. We report here the isolation from bag cell clusters of three structurally similar peptides, seven, eight, and nine residues long, that are candidate transmitters for mediating bag cell-induced inhibition. They may also serve as autoexcitatory transmitters since the seven-residue peptide produces a slow depolarization of the bag cells similar to that which occurs during bag cell discharge. The amino acid sequence of the largest peptide, termed α-bag cell peptide[1-9], is H-Ala-Pro-Arg-Leu-Arg-Phe-Tyr-Ser-Leu-OH. The other two peptides are identical to α-BCP[1-9] except that they lack the COOH-terminal Ser-Leu or leucine residues. The three peptides inhibit left upper quadrant neurons at relative potencies of 10:30:1 (seven-, eight-, and nine-residue peptides, respectively). Recent molecular genetic analysis shows that both α-BCP[1-9] and egg-laying hormone are encoded by the same bag cell-specific gene. The multiple neuronal effects of bag cells are therefore likely to be mediated by at least two transmitters that are cleaved from a common precursor molecule. PMID:16593372

  8. Expression and transcriptional regulation of the GnRH receptor gene in human neuronal cells.

    PubMed

    Yeung, Chung-Man; An, Beum-Soo; Cheng, Chi Keung; Chow, Billy K C; Leung, Peter C K

    2005-11-01

    GnRH, acts via the GnRH receptor (GnRHR), plays a pivotal role in human reproduction by stimulating the synthesis and secretion of gonadotropins from pituitary gonadotropes. Studies have also suggested that it has other extra-pituitary functions. To date, the transcriptional regulation of human GnRHR gene in the brain remains largely unknown. Recently, the human cerebellar medulloblastoma cell line TE-671 is found to express GnRH. We report here for the first time that GnRHR is also expressed in this neuronal cell line. Treatment with GnRHR agonist stimulated the phosphorylation of both ERK1/2 and JNK in the cells. Moreover, transient transfection of various human GnRHR promoter-luciferase constructs into the cells identified an upstream promoter region located between -2197 and -1018. Important cis-acting regulatory elements were found at -1300/-1018 and -2197/- 1900, as deletion of either region caused a dramatic decrease in the promoter activity. An upstream GnRHR promoter element was identified to be important for basal transcription in the human neuronal TE-671 cells, in contrast to the previous finding that a downstream promoter is responsible for the gonadotrope-specific expression. Furthermore, we showed that antide (GnRHR antagonist) significantly stimulated the GnRHR promoter activity and inhibition of protein kinase C (PKC) pathway by staurosporine could also up-regulate the promoter activity in dose- and time-dependent manners. Taken together, these data suggest that activation of the GnRHR by interacting with GnRH may transcriptionally down-regulate itself via the PKC pathway in human neuronal cells.

  9. Glutamatergic neuron-targeted loss of LGI1 epilepsy gene results in seizures

    PubMed Central

    Boillot, Morgane; Huneau, Clément; Marsan, Elise; Lehongre, Katia; Navarro, Vincent; Ishida, Saeko; Dufresnois, Béatrice; Ozkaynak, Ekim; Garrigue, Jérôme; Miles, Richard; Martin, Benoit; Leguern, Eric; Anderson, Matthew P.

    2014-01-01

    Leucin-rich, glioma inactivated 1 (LGI1) is a secreted protein linked to human seizures of both genetic and autoimmune aetiology. Mutations in the LGI1 gene are responsible for autosomal dominant temporal lobe epilepsy with auditory features, whereas LGI1 autoantibodies are involved in limbic encephalitis, an acquired epileptic disorder associated with cognitive impairment. We and others previously reported that Lgi1-deficient mice have early-onset spontaneous seizures leading to premature death at 2–3 weeks of age. Yet, where and when Lgi1 deficiency causes epilepsy remains unknown. To address these questions, we generated Lgi1 conditional knockout (cKO) mice using a set of universal Cre-driver mouse lines. Selective deletion of Lgi1 was achieved in glutamatergic pyramidal neurons during embryonic (Emx1-Lgi1cKO) or late postnatal (CaMKIIα-Lgi1cKO) developmental stages, or in gamma amino butyric acidergic (GABAergic) parvalbumin interneurons (PV-Lgi1cKO). Emx1-Lgi1cKO mice displayed early-onset and lethal seizures, whereas CaMKIIα-Lgi1cKO mice presented late-onset occasional seizures associated with variable reduced lifespan. In contrast, neither spontaneous seizures nor increased seizure susceptibility to convulsant were observed when Lgi1 was deleted in parvalbumin interneurons. Together, these data showed that LGI1 depletion restricted to pyramidal cells is sufficient to generate seizures, whereas seizure thresholds were unchanged after depletion in gamma amino butyric acidergic parvalbumin interneurons. We suggest that LGI1 secreted from excitatory neurons, but not parvalbumin inhibitory neurons, makes a major contribution to the pathogenesis of LGI1-related epilepsies. Our data further indicate that LGI1 is required from embryogenesis to adulthood to achieve proper circuit functioning. PMID:25234641

  10. Glutamatergic neuron-targeted loss of LGI1 epilepsy gene results in seizures.

    PubMed

    Boillot, Morgane; Huneau, Clément; Marsan, Elise; Lehongre, Katia; Navarro, Vincent; Ishida, Saeko; Dufresnois, Béatrice; Ozkaynak, Ekim; Garrigue, Jérôme; Miles, Richard; Martin, Benoit; Leguern, Eric; Anderson, Matthew P; Baulac, Stéphanie

    2014-11-01

    Leucin-rich, glioma inactivated 1 (LGI1) is a secreted protein linked to human seizures of both genetic and autoimmune aetiology. Mutations in the LGI1 gene are responsible for autosomal dominant temporal lobe epilepsy with auditory features, whereas LGI1 autoantibodies are involved in limbic encephalitis, an acquired epileptic disorder associated with cognitive impairment. We and others previously reported that Lgi1-deficient mice have early-onset spontaneous seizures leading to premature death at 2-3 weeks of age. Yet, where and when Lgi1 deficiency causes epilepsy remains unknown. To address these questions, we generated Lgi1 conditional knockout (cKO) mice using a set of universal Cre-driver mouse lines. Selective deletion of Lgi1 was achieved in glutamatergic pyramidal neurons during embryonic (Emx1-Lgi1cKO) or late postnatal (CaMKIIα-Lgi1cKO) developmental stages, or in gamma amino butyric acidergic (GABAergic) parvalbumin interneurons (PV-Lgi1cKO). Emx1-Lgi1cKO mice displayed early-onset and lethal seizures, whereas CaMKIIα-Lgi1cKO mice presented late-onset occasional seizures associated with variable reduced lifespan. In contrast, neither spontaneous seizures nor increased seizure susceptibility to convulsant were observed when Lgi1 was deleted in parvalbumin interneurons. Together, these data showed that LGI1 depletion restricted to pyramidal cells is sufficient to generate seizures, whereas seizure thresholds were unchanged after depletion in gamma amino butyric acidergic parvalbumin interneurons. We suggest that LGI1 secreted from excitatory neurons, but not parvalbumin inhibitory neurons, makes a major contribution to the pathogenesis of LGI1-related epilepsies. Our data further indicate that LGI1 is required from embryogenesis to adulthood to achieve proper circuit functioning.

  11. A Functionally Conserved Gene Regulatory Network Module Governing Olfactory Neuron Diversity

    PubMed Central

    Okuwa, Sumie; Maciejewski, Abigail; Brandt, Alicia T.; Reinhold, Dominik; Jones, Corbin D.; Volkan, Pelin Cayirlioglu

    2016-01-01

    Sensory neuron diversity is required for organisms to decipher complex environmental cues. In Drosophila, the olfactory environment is detected by 50 different olfactory receptor neuron (ORN) classes that are clustered in combinations within distinct sensilla subtypes. Each sensilla subtype houses stereotypically clustered 1–4 ORN identities that arise through asymmetric divisions from a single multipotent sensory organ precursor (SOP). How each class of SOPs acquires a unique differentiation potential that accounts for ORN diversity is unknown. Previously, we reported a critical component of SOP diversification program, Rotund (Rn), increases ORN diversity by generating novel developmental trajectories from existing precursors within each independent sensilla type lineages. Here, we show that Rn, along with BarH1/H2 (Bar), Bric-à-brac (Bab), Apterous (Ap) and Dachshund (Dac), constitutes a transcription factor (TF) network that patterns the developing olfactory tissue. This network was previously shown to pattern the segmentation of the leg, which suggests that this network is functionally conserved. In antennal imaginal discs, precursors with diverse ORN differentiation potentials are selected from concentric rings defined by unique combinations of these TFs along the proximodistal axis of the developing antennal disc. The combinatorial code that demarcates each precursor field is set up by cross-regulatory interactions among different factors within the network. Modifications of this network lead to predictable changes in the diversity of sensilla subtypes and ORN pools. In light of our data, we propose a molecular map that defines each unique SOP fate. Our results highlight the importance of the early prepatterning gene regulatory network as a modulator of SOP and terminally differentiated ORN diversity. Finally, our model illustrates how conserved developmental strategies are used to generate neuronal diversity. PMID:26765103

  12. A Functionally Conserved Gene Regulatory Network Module Governing Olfactory Neuron Diversity.

    PubMed

    Li, Qingyun; Barish, Scott; Okuwa, Sumie; Maciejewski, Abigail; Brandt, Alicia T; Reinhold, Dominik; Jones, Corbin D; Volkan, Pelin Cayirlioglu

    2016-01-01

    Sensory neuron diversity is required for organisms to decipher complex environmental cues. In Drosophila, the olfactory environment is detected by 50 different olfactory receptor neuron (ORN) classes that are clustered in combinations within distinct sensilla subtypes. Each sensilla subtype houses stereotypically clustered 1-4 ORN identities that arise through asymmetric divisions from a single multipotent sensory organ precursor (SOP). How each class of SOPs acquires a unique differentiation potential that accounts for ORN diversity is unknown. Previously, we reported a critical component of SOP diversification program, Rotund (Rn), increases ORN diversity by generating novel developmental trajectories from existing precursors within each independent sensilla type lineages. Here, we show that Rn, along with BarH1/H2 (Bar), Bric-à-brac (Bab), Apterous (Ap) and Dachshund (Dac), constitutes a transcription factor (TF) network that patterns the developing olfactory tissue. This network was previously shown to pattern the segmentation of the leg, which suggests that this network is functionally conserved. In antennal imaginal discs, precursors with diverse ORN differentiation potentials are selected from concentric rings defined by unique combinations of these TFs along the proximodistal axis of the developing antennal disc. The combinatorial code that demarcates each precursor field is set up by cross-regulatory interactions among different factors within the network. Modifications of this network lead to predictable changes in the diversity of sensilla subtypes and ORN pools. In light of our data, we propose a molecular map that defines each unique SOP fate. Our results highlight the importance of the early prepatterning gene regulatory network as a modulator of SOP and terminally differentiated ORN diversity. Finally, our model illustrates how conserved developmental strategies are used to generate neuronal diversity. PMID:26765103

  13. A Functionally Conserved Gene Regulatory Network Module Governing Olfactory Neuron Diversity.

    PubMed

    Li, Qingyun; Barish, Scott; Okuwa, Sumie; Maciejewski, Abigail; Brandt, Alicia T; Reinhold, Dominik; Jones, Corbin D; Volkan, Pelin Cayirlioglu

    2016-01-01

    Sensory neuron diversity is required for organisms to decipher complex environmental cues. In Drosophila, the olfactory environment is detected by 50 different olfactory receptor neuron (ORN) classes that are clustered in combinations within distinct sensilla subtypes. Each sensilla subtype houses stereotypically clustered 1-4 ORN identities that arise through asymmetric divisions from a single multipotent sensory organ precursor (SOP). How each class of SOPs acquires a unique differentiation potential that accounts for ORN diversity is unknown. Previously, we reported a critical component of SOP diversification program, Rotund (Rn), increases ORN diversity by generating novel developmental trajectories from existing precursors within each independent sensilla type lineages. Here, we show that Rn, along with BarH1/H2 (Bar), Bric-à-brac (Bab), Apterous (Ap) and Dachshund (Dac), constitutes a transcription factor (TF) network that patterns the developing olfactory tissue. This network was previously shown to pattern the segmentation of the leg, which suggests that this network is functionally conserved. In antennal imaginal discs, precursors with diverse ORN differentiation potentials are selected from concentric rings defined by unique combinations of these TFs along the proximodistal axis of the developing antennal disc. The combinatorial code that demarcates each precursor field is set up by cross-regulatory interactions among different factors within the network. Modifications of this network lead to predictable changes in the diversity of sensilla subtypes and ORN pools. In light of our data, we propose a molecular map that defines each unique SOP fate. Our results highlight the importance of the early prepatterning gene regulatory network as a modulator of SOP and terminally differentiated ORN diversity. Finally, our model illustrates how conserved developmental strategies are used to generate neuronal diversity.

  14. Differential gene transfers and gene duplications in primary and secondary endosymbioses

    PubMed Central

    Zauner, Stefan; Lockhart, Peter; Stoebe-Maier, Bettina; Gilson, Paul; McFadden, Geoffrey I; Maier, Uwe G

    2006-01-01

    Background Most genes introduced into phototrophic eukaryotes during the process of endosymbiosis are either lost or relocated into the host nuclear genome. In contrast, groEL homologues are found in different genome compartments among phototrophic eukaryotes. Comparative sequence analyses of recently available genome data, have allowed us to reconstruct the evolutionary history of these genes and propose a hypothesis that explains the unusual genome distribution of groEL homologues. Results Our analyses indicate that while two distinct groEL genes were introduced into eukaryotes by a progenitor of plastids, these particular homologues have not been maintained in all evolutionary lineages. This is of significant interest, because two chaperone proteins always co-occur in oxygenic photosynthetic organisms. We infer strikingly different lineage specific processes of evolution involving deletion, duplication and targeting of groEL proteins. Conclusion The requirement of two groEL homologues for chaperon function in phototrophs has provided a constraint that has shaped convergent evolutionary scenarios in divergent evolutionary lineages. GroEL provides a general evolutionary model for studying gene transfers and convergent evolutionary processes among eukaryotic lineages. PMID:16640777

  15. Midbrain dopamine neurons in Parkinson's disease exhibit a dysregulated miRNA and target-gene network.

    PubMed

    Briggs, Christine E; Wang, Yulei; Kong, Benjamin; Woo, Tsung-Ung W; Iyer, Lakshmanan K; Sonntag, Kai C

    2015-08-27

    The degeneration of substantia nigra (SN) dopamine (DA) neurons in sporadic Parkinson׳s disease (PD) is characterized by disturbed gene expression networks. Micro(mi)RNAs are post-transcriptional regulators of gene expression and we recently provided evidence that these molecules may play a functional role in the pathogenesis of PD. Here, we document a comprehensive analysis of miRNAs in SN DA neurons and PD, including sex differences. Our data show that miRNAs are dysregulated in disease-affected neurons and differentially expressed between male and female samples with a trend of more up-regulated miRNAs in males and more down-regulated miRNAs in females. Unbiased Ingenuity Pathway Analysis (IPA) revealed a network of miRNA/target-gene associations that is consistent with dysfunctional gene and signaling pathways in PD pathology. Our study provides evidence for a general association of miRNAs with the cellular function and identity of SN DA neurons, and with deregulated gene expression networks and signaling pathways related to PD pathogenesis that may be sex-specific.

  16. C. elegans SoxB genes are dispensable for embryonic neurogenesis but required for terminal differentiation of specific neuron types

    PubMed Central

    Vidal, Berta; Santella, Anthony; Serrano-Saiz, Esther; Bao, Zhirong; Chuang, Chiou-Fen; Hobert, Oliver

    2015-01-01

    Neurogenesis involves deeply conserved patterning molecules, such as the proneural basic helix-loop-helix transcription factors. Sox proteins and specifically members of the SoxB and SoxC groups are another class of conserved transcription factors with an important role in neuronal fate commitment and differentiation in various species. In this study, we examine the expression of all five Sox genes of the nematode C. elegans and analyze the effect of null mutant alleles of all members of the SoxB and SoxC groups on nervous system development. Surprisingly, we find that, unlike in other systems, neither of the two C. elegans SoxB genes sox-2 (SoxB1) and sox-3 (SoxB2), nor the sole C. elegans SoxC gene sem-2, is broadly expressed throughout the embryonic or adult nervous system and that all three genes are mostly dispensable for embryonic neurogenesis. Instead, sox-2 is required to maintain the developmental potential of blast cells that are generated in the embryo but divide only postembryonically to give rise to differentiated neuronal cell types. Moreover, sox-2 and sox-3 have selective roles in the terminal differentiation of specific neuronal cell types. Our findings suggest that the common themes of SoxB gene function across phylogeny lie in specifying developmental potential and, later on, in selectively controlling terminal differentiation programs of specific neuron types, but not in broadly controlling neurogenesis. PMID:26153233

  17. Directed neuronal differentiation of mouse embryonic and induced pluripotent stem cells and their gene expression profiles.

    PubMed

    Chen, Xuesong; Gu, Qi; Wang, Xiang; Ma, Qingwen; Tang, Huixiang; Yan, Xiaoshuang; Guo, Xinbing; Yan, Hao; Hao, Jie; Zeng, Fanyi

    2013-07-01

    Embryonic stem cells (ESCs) may be useful as a therapeutic source of cells for the production of healthy tissue; however, they are associated with certain challenges including immunorejection as well as ethical issues. Induced pluripotent stem cells (iPSCs) are a promising substitute since a patient's own adult cells would serve as tissue precursors. Ethical concerns prevent a full evaluation of the developmental potency of human ESCs and iPSCs, therefore, mouse iPSC models are required for protocol development and safety assessments. We used a modified culturing protocol to differentiate pluripotent cells from a mouse iPS cell line and two mouse ES cell lines into neurons. Our results indicated that all three pluripotent stem cell lines underwent nearly the same differentiation process when induced to form neurons in vitro. Genomic expression microarray profiling and single-cell RT-qPCR were used to analyze the neural lineage differentiation process, and more than one thousand differentially expressed genes involved in multiple molecular processes relevant to neural development were identified.

  18. The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection.

    PubMed

    Wise, Andrew K; Tu, Tian; Atkinson, Patrick J; Flynn, Brianna O; Sgro, Beatrice E; Hume, Cliff; O'Leary, Stephen J; Shepherd, Robert K; Richardson, Rachael T

    2011-08-01

    A cochlear implant can restore hearing function by electrically exciting spiral ganglion neurons (SGNs) in the deaf cochlea. However, following deafness SGNs undergo progressive degeneration ultimately leading to their death. One significant cause of SGN degeneration is the loss of neurotrophic support that is normally provided by cells within the organ of Corti (OC). The administration of exogenous neurotrophins (NTs) can protect SGNs from degeneration but the effects are short-lived once the source of NTs has been exhausted. NT gene therapy, whereby cells within the cochlea are transfected with genes enabling them to produce NTs, is one strategy for providing a cellular source of NTs that may provide long-term support for SGNs. As the SGNs normally innervate sensory cells within the OC, targeting residual OC cells for gene therapy in the deaf cochlea may provide a source of NTs for SGN protection and targeted regrowth of their peripheral fibers. However, the continual degeneration of the OC over extended periods of deafness may deplete the cellular targets for NT gene therapy and hence limit the effectiveness of this method in preventing SGN loss. This study examined the effects of deafness duration on the efficacy of NT gene therapy in preventing SGN loss in guinea pigs that were systemically deafened with aminoglycosides. Adenoviral vectors containing green fluorescent protein (GFP) with or without genes for Brain Derived Neurotrophic Factor (BDNF) and Neurotrophin-3 (NT3) were injected into the scala media (SM) compartment of cochleae that had been deafened for one, four or eight weeks prior to the viral injection. The results showed that viral transfection of cells within the SM was still possible even after severe degeneration of the OC. Supporting cells (pillar and Deiters' cells), cells within the stria vascularis, the spiral ligament, endosteal cells lining the scala compartments and interdental cells in the spiral limbus were transfected. However, the

  19. Splitting hares and tortoises: a classification of neuronal immediate early gene transcription based on poised RNA polymerase II.

    PubMed

    Saha, R N; Dudek, S M

    2013-09-01

    Immediate early transcription is an integral part of the neuronal response to environmental stimulation and serves many brain processes including development, learning, triggers of programmed cell death, and reaction to injury and drugs. Following a stimulus, neurons express a select few genes within a short period of time without undergoing de novo protein translation. Referred to as the 'gateway to genetic response', these immediate early genes (IEGs) are either expressed within a few minutes of stimulation or later within the hour. In neuronal IEGs that are expressed rapidly, productive elongation in response to neuronal activity is jump-started by constitutive transcription initiation together with RNA polymerase II stalling in the vicinity of the promoter. IEGs expressed later in the hour do not depend on this mechanism. On the basis of this Polymerase II poising, we propose that the immediate early genes can be grouped in two distinct classes: the rapid and the delayed IEGs. The possible biological relevance of these classes in neurons is discussed.

  20. Identification of the class I genes of the mouse major histocompatibility complex by DNA-mediated gene transfer.

    PubMed

    Goodenow, R S; McMillan, M; Nicolson, M; Sher, B T; Eakle, K; Davidson, N; Hood, L

    1982-11-18

    DNA-mediated gene transfer was used to identify cloned class I genes from the major histocompatibility complex of the BALB/c mouse. Three genes encoding the transplantation antigens H-2 Kd, Dd and Ld were identified as well as genes encoding the Qa-2,3 and two TL differentiation antigens. As many as 10 putative novel class I genes were detected by the association of their gene products with beta 2-microglobulin. Alloantiserum prepared to one of the novel antigens was used to demonstrate the expression of the previously undetected antigen on spleen cells of various inbred, congeneic, and recombinant congeneic strains of mice. PMID:6815535

  1. Differential evolutionary rates of neuronal transcriptome in Aplysia kurodai and Aplysia californica as a tool for gene mining.

    PubMed

    Choi, Sun-Lim; Lee, Yong-Seok; Rim, Young-Soo; Kim, Tae-Hyung; Moroz, Leonid L; Kandel, Eric R; Bhak, Jong; Kaang, Bong-Kiun

    2010-07-01

    The marine mollusk Aplysia is a fascinating model organism for studying molecular mechanisms underlying learning and memory. However, evolutionary studies about Aplysia have been limited by the lack of its genomic information. Recently, large-scale expressed sequence tag (EST) databases have been acquired by sequencing cDNA libraries from A. californica and A. kurodai. The closeness between the two species allowed us to investigate rapidly evolving genes by comparing their orthologs. Using this method, we found that a subset of signal transduction genes in neurons showed rates of protein evolution higher than those of housekeeping genes. Moreover, we were also able to find several candidate genes that may be involved in learning and memory or synaptic plasticity among genes showing relatively higher K(a)/K(s) ratios. We also investigated the relationship between evolutionary rates and tissue distribution of Aplysia genes. They propose that the estimation of evolutionary rates cannot be a good marker to assess neuronal expression; however, it still can be an efficient way to narrow down the pool of candidate genes involved in neuronal functions for the further studies. PMID:20536287

  2. Gene transfer into Solanum tuberosum via Rhizobium spp.

    PubMed

    Wendt, Toni; Doohan, Fiona; Winckelmann, Dominik; Mullins, Ewen

    2011-04-01

    Agrobacterium tumefaciens-mediated transformation (ATMT) is the preferred technique for gene transfer into crops. A major disadvantage of the technology remains the complexity of the patent landscape that surrounds ATMT which restricts its use for commercial applications. An alternative system has been described (Broothaerts et al. in Nature 433:629-633, 2005) detailing the propensity of three rhizobia to transform the model crop Arabidopsis thaliana, the non-food crop Nicotiana tabacum and, at a very low frequency, the monocotyledonous crop Oryza sativa. In this report we describe for the first time the genetic transformation of Solanum tuberosum using the non-Agrobacterium species Sinorhizobium meliloti, Rhizobium sp. NGR234 and Mesorhizobium loti. This was achieved by combining an optimal bacterium and host co-cultivation period with a low antibiotic regime during the callus and shoot induction stages. Using this optimized protocol the transformation frequency (calculated as % of shoots equipped with root systems with the ability to grow in rooting media supplemented with 25 μg/ml hygromycin) of the rhizobia strains was calculated at 4.72, 5.85 and 1.86% for S. meliloti, R. sp. NGR234 and M. loti respectively, compared to 47.6% for the A. tumefaciens control. Stable transgene integration and expression was confirmed via southern hybridisation, quantitative PCR analysis and histochemical screening of both leaf and/or tuber tissue. In light of the rapid advances in potato genomics, combined with the sequencing of the potato genome, the ability of alternative bacteria species to genetically transform this major food crop will provide a novel resource to the Solanaceae community as it continues to develop potato as both a food and non-food crop.

  3. Migration and horizontal gene transfer divide microbial genomes into multiple niches.

    PubMed

    Niehus, Rene; Mitri, Sara; Fletcher, Alexander G; Foster, Kevin R

    2015-01-01

    Horizontal gene transfer is central to microbial evolution, because it enables genetic regions to spread horizontally through diverse communities. However, how gene transfer exerts such a strong effect is not understood. Here we develop an eco-evolutionary model and show how genetic transfer, even when rare, can transform the evolution and ecology of microbes. We recapitulate existing models, which suggest that asexual reproduction will overpower horizontal transfer and greatly limit its effects. We then show that allowing immigration completely changes these predictions. With migration, the rates and impacts of horizontal transfer are greatly increased, and transfer is most frequent for loci under positive natural selection. Our analysis explains how ecologically important loci can sweep through competing strains and species. In this way, microbial genomes can evolve to become ecologically diverse where different genomic regions encode for partially overlapping, but distinct, ecologies. Under these conditions ecological species do not exist, because genes, not species, inhabit niches. PMID:26592443

  4. Migration and horizontal gene transfer divide microbial genomes into multiple niches

    PubMed Central

    Niehus, Rene; Mitri, Sara; Fletcher, Alexander G.; Foster, Kevin R.

    2015-01-01

    Horizontal gene transfer is central to microbial evolution, because it enables genetic regions to spread horizontally through diverse communities. However, how gene transfer exerts such a strong effect is not understood. Here we develop an eco-evolutionary model and show how genetic transfer, even when rare, can transform the evolution and ecology of microbes. We recapitulate existing models, which suggest that asexual reproduction will overpower horizontal transfer and greatly limit its effects. We then show that allowing immigration completely changes these predictions. With migration, the rates and impacts of horizontal transfer are greatly increased, and transfer is most frequent for loci under positive natural selection. Our analysis explains how ecologically important loci can sweep through competing strains and species. In this way, microbial genomes can evolve to become ecologically diverse where different genomic regions encode for partially overlapping, but distinct, ecologies. Under these conditions ecological species do not exist, because genes, not species, inhabit niches. PMID:26592443

  5. Fast gene transfer into the adult zebrafish brain by herpes simplex virus 1 (HSV-1) and electroporation: methods and optogenetic applications

    PubMed Central

    Zou, Ming; De Koninck, Paul; Neve, Rachael L.; Friedrich, Rainer W.

    2014-01-01

    The zebrafish has various advantages as a model organism to analyze the structure and function of neural circuits but efficient viruses or other tools for fast gene transfer are lacking. We show that transgenes can be introduced directly into the adult zebrafish brain by herpes simplex type I viruses (HSV-1) or electroporation. We developed a new procedure to target electroporation to defined brain areas and identified promoters that produced strong long-term expression. The fast workflow of electroporation was exploited to express multiple channelrhodopsin-2 variants and genetically encoded calcium indicators in telencephalic neurons for measurements of neuronal activity and synaptic connectivity. The results demonstrate that HSV-1 and targeted electroporation are efficient tools for gene delivery into the zebrafish brain, similar to adeno-associated viruses and lentiviruses in other species. These methods fill an important gap in the spectrum of molecular tools for zebrafish and are likely to have a wide range of applications. PMID:24834028

  6. Apramycin resistance as a selective marker for gene transfer in mycobacteria.

    PubMed Central

    Paget, E; Davies, J

    1996-01-01

    We have explored the potential of using the apramycin resistance gene as a marker in mycobacterial gene transfer studies. Shuttle plasmids available for both electroporation and conjugation studies have been constructed, and we have successfully validated the use of the apramycin resistance gene as a component of cloning vectors for Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis. PMID:8892841

  7. uv excision-repair gene transfer in Chinese hamster ovary (CHO) cells

    SciTech Connect

    MacInnes, M.A.; Bingham, J.M.; Strniste, G.F.; Thompson, L.H.

    1983-01-01

    uvc-sensitive mutants of CHO cells provide a model system for molecular studies of DNA repair. We present our recent results which show that these mutants are competent recipients for plasmid marker gene transfer and incorporation of a putative CHO repair gene. The applicability and advantages of this system for interspecies human repair gene identification are discussed.

  8. Contribution of Horizontal Gene Transfer to the Evolution of Saccharomyces cerevisiae†

    PubMed Central

    Hall, Charles; Brachat, Sophie; Dietrich, Fred S.

    2005-01-01

    The genomes of the hemiascomycetes Saccharomyces cerevisiae and Ashbya gossypii have been completely sequenced, allowing a comparative analysis of these two genomes, which reveals that a small number of genes appear to have entered these genomes as a result of horizontal gene transfer from bacterial sources. One potential case of horizontal gene transfer in A. gossypii and 10 potential cases in S. cerevisiae were identified, of which two were investigated further. One gene, encoding the enzyme dihydroorotate dehydrogenase (DHOD), is potentially a case of horizontal gene transfer, as shown by sequencing of this gene from additional bacterial and fungal species to generate sufficient data to construct a well-supported phylogeny. The DHOD-encoding gene found in S. cerevisiae, URA1 (YKL216W), appears to have entered the Saccharomycetaceae after the divergence of the S. cerevisiae lineage from the Candida albicans lineage and possibly since the divergence from the A. gossypii lineage. This gene appears to have come from the Lactobacillales, and following its acquisition the endogenous eukaryotic DHOD gene was lost. It was also shown that the bacterially derived horizontally transferred DHOD is required for anaerobic synthesis of uracil in S. cerevisiae. The other gene discussed in detail is BDS1, an aryl- and alkyl-sulfatase gene of bacterial origin that we have shown allows utilization of sulfate from several organic sources. Among the eukaryotes, this gene is found in S. cerevisiae and Saccharomyces bayanus and appears to derive from the alpha-proteobacteria. PMID:15947202

  9. Proteomic profiling of salivary gland after nonviral gene transfer mediated by conventional plasmids and minicircles

    PubMed Central

    Geguchadze, Ramaz; Wang, Zhimin; Zourelias, Lee; Perez-Riveros, Paola; Edwards, Paul C; Machen, Laurie; Passineau, Michael J

    2014-01-01

    In this study, we compared gene transfer efficiency and host response to ultrasound-assisted, nonviral gene transfer with a conventional plasmid and a minicircle vector in the submandibular salivary glands of mice. Initially, we looked at gene transfer efficiency with equimolar amounts of the plasmid and minicircle vectors, corroborating an earlier report showing that minicircle is more efficient in the context of a physical method of gene transfer. We then sought to characterize the physiological response of the salivary gland to exogenous gene transfer using global proteomic profiling. Somewhat surprisingly, we found that sonoporation alone, without a gene transfer vector present, had virtually no effect on the salivary gland proteome. However, when a plasmid vector was used, we observed profound perturbations of the salivary gland proteome that compared in magnitude to that seen in a previous report after high doses of adeno-associated virus. Finally, we found that gene transfer with a minicircle induces only minor proteomic alterations that were similar to sonoporation alone. Using mass spectrometry, we assigned protein IDs to 218 gel spots that differed between plasmid and minicircle. Bioinformatic analysis of these proteins demonstrated convergence on 68 known protein interaction pathways, most notably those associated with innate immunity, cellular stress, and morphogenesis. PMID:25414909

  10. Horizontal Gene Transfer of Pectinases from Bacteria Preceded the Diversification of Stick and Leaf Insects

    PubMed Central

    Shelomi, Matan; Danchin, Etienne G. J.; Heckel, David; Wipfler, Benjamin; Bradler, Sven; Zhou, Xin; Pauchet, Yannick

    2016-01-01

    Genes acquired by horizontal transfer are increasingly being found in animal genomes. Understanding their origin and evolution requires knowledge about the phylogenetic relationships from both source and recipient organisms. We used RNASeq data and respective assembled transcript libraries to trace the evolutionary history of polygalacturonase (pectinase) genes in stick insects (Phasmatodea). By mapping the distribution of pectinase genes on a Polyneoptera phylogeny, we identified the transfer of pectinase genes from known phasmatodean gut microbes into the genome of an early euphasmatodean ancestor that took place between 60 and 100 million years ago. This transfer preceded the rapid diversification of the suborder, enabling symbiont-free pectinase production that would increase the insects’ digestive efficiency and reduce dependence on microbes. Bacteria-to-insect gene transfer was thought to be uncommon, however the increasing availability of large-scale genomic data may change this prevailing notion. PMID:27210832

  11. Horizontal Gene Transfer of Pectinases from Bacteria Preceded the Diversification of Stick and Leaf Insects.

    PubMed

    Shelomi, Matan; Danchin, Etienne G J; Heckel, David; Wipfler, Benjamin; Bradler, Sven; Zhou, Xin; Pauchet, Yannick

    2016-01-01

    Genes acquired by horizontal transfer are increasingly being found in animal genomes. Understanding their origin and evolution requires knowledge about the phylogenetic relationships from both source and recipient organisms. We used RNASeq data and respective assembled transcript libraries to trace the evolutionary history of polygalacturonase (pectinase) genes in stick insects (Phasmatodea). By mapping the distribution of pectinase genes on a Polyneoptera phylogeny, we identified the transfer of pectinase genes from known phasmatodean gut microbes into the genome of an early euphasmatodean ancestor that took place between 60 and 100 million years ago. This transfer preceded the rapid diversification of the suborder, enabling symbiont-free pectinase production that would increase the insects' digestive efficiency and reduce dependence on microbes. Bacteria-to-insect gene transfer was thought to be uncommon, however the increasing availability of large-scale genomic data may change this prevailing notion. PMID:27210832

  12. Horizontal gene transfer and the evolution of transcriptionalregulation in Escherichia coli

    SciTech Connect

    Price, Morgan N.; Dehal, Paramvir S.; Arkin, Adam P.

    2007-12-20

    Background: Most bacterial genes were acquired by horizontalgene transfer from other bacteria instead of being inherited bycontinuous vertical descent from an ancient ancestor}. To understand howthe regulation of these {acquired} genes evolved, we examined theevolutionary histories of transcription factors and of regulatoryinteractions from the model bacterium Escherichia coli K12. Results:Although most transcription factors have paralogs, these usually arose byhorizontal gene transfer rather than by duplication within the E. colilineage, as previously believed. In general, most neighbor regulators --regulators that are adjacent to genes that they regulate -- were acquiredby horizontal gene transfer, while most global regulators evolvedvertically within the gamma-Proteobacteria. Neighbor regulators wereoften acquired together with the adjacent operon that they regulate, sothe proximity might be maintained by repeated transfers (like "selfishoperons"). Many of the as-yet-uncharacterized (putative) regulators havealso been acquired together with adjacent genes, so we predict that theseare neighbor regulators as well. When we analyzed the histories ofregulatory interactions, we found that the evolution of regulation byduplication was rare, and surprisingly, many of the regulatoryinteractions that are shared between paralogs result from convergentevolution. Another surprise was that horizontally transferred genes aremore likely than other genes to be regulated by multiple regulators, andmost of this complex regulation probably evolved after the transfer.Conclusions: Our results highlight the rapid evolution of niche-specificgene regulation in bacteria.

  13. A new binding motif for the transcriptional repressor REST uncovers large gene networks devoted to neuronal functions.

    PubMed

    Otto, Stefanie J; McCorkle, Sean R; Hover, John; Conaco, Cecilia; Han, Jong-Jin; Impey, Soren; Yochum, Gregory S; Dunn, John J; Goodman, Richard H; Mandel, Gail

    2007-06-20

    The repressor element 1 (RE1) silencing transcription factor (REST) helps preserve the identity of nervous tissue by silencing neuronal genes in non-neural tissues. Moreover, in an epithelial model of tumorigenesis, loss of REST function is associated with loss of adhesion, suggesting the aberrant expression of REST-controlled genes encoding this property. To date, no adhesion molecules under REST control have been identified. Here, we used serial analysis of chromatin occupancy to perform genome-wide identification of REST-occupied target sequences (RE1 sites) in a kidney cell line. We discovered novel REST-binding motifs and found that the number of RE1 sites far exceeded previous estimates. A large family of targets encoding adhesion proteins was identified, as were genes encoding signature proteins of neuroendocrine tumors. Unexpectedly, genes considered exclusively non-neuronal also contained an RE1 motif and were expressed in neurons. This supports the model that REST binding is a critical determinant of neuronal phenotype.

  14. Transcriptomic signatures of neuronal differentiation and their association with risk genes for autism spectrum and related neuropsychiatric disorders.

    PubMed

    Chiocchetti, A G; Haslinger, D; Stein, J L; de la Torre-Ubieta, L; Cocchi, E; Rothämel, T; Lindlar, S; Waltes, R; Fulda, S; Geschwind, D H; Freitag, C M

    2016-01-01

    Genes for autism spectrum disorders (ASDs) are also implicated in fragile X syndrome (FXS), intellectual disabilities (ID) or schizophrenia (SCZ), and converge on neuronal function and differentiation. The SH-SY5Y neuroblastoma cell line, the most widely used system to study neurodevelopment, is currently discussed for its applicability to model cortical development. We implemented an optimal neuronal differentiation protocol of this system and evaluated neurodevelopment at the transcriptomic level using the CoNTeXT framework, a machine-learning algorithm based on human post-mortem brain data estimating developmental stage and regional identity of transcriptomic signatures. Our improved model in contrast to currently used SH-SY5Y models does capture early neurodevelopmental processes with high fidelity. We applied regression modelling, dynamic time warping analysis, parallel independent component analysis and weighted gene co-expression network analysis to identify activated gene sets and networks. Finally, we tested and compared these sets for enrichment of risk genes for neuropsychiatric disorders. We confirm a significant overlap of genes implicated in ASD with FXS, ID and SCZ. However, counterintuitive to this observation, we report that risk genes affect pathways specific for each disorder during early neurodevelopment. Genes implicated in ASD, ID, FXS and SCZ were enriched among the positive regulators, but only ID-implicated genes were also negative regulators of neuronal differentiation. ASD and ID genes were involved in dendritic branching modules, but only ASD risk genes were implicated in histone modification or axonal guidance. Only ID genes were over-represented among cell cycle modules. We conclude that the underlying signatures are disorder-specific and that the shared genetic architecture results in overlaps across disorders such as ID in ASD. Thus, adding developmental network context to genetic analyses will aid differentiating the pathophysiology

  15. Transcriptomic signatures of neuronal differentiation and their association with risk genes for autism spectrum and related neuropsychiatric disorders

    PubMed Central

    Chiocchetti, A G; Haslinger, D; Stein, J L; de la Torre-Ubieta, L; Cocchi, E; Rothämel, T; Lindlar, S; Waltes, R; Fulda, S; Geschwind, D H; Freitag, C M

    2016-01-01

    Genes for autism spectrum disorders (ASDs) are also implicated in fragile X syndrome (FXS), intellectual disabilities (ID) or schizophrenia (SCZ), and converge on neuronal function and differentiation. The SH-SY5Y neuroblastoma cell line, the most widely used system to study neurodevelopment, is currently discussed for its applicability to model cortical development. We implemented an optimal neuronal differentiation protocol of this system and evaluated neurodevelopment at the transcriptomic level using the CoNTeXT framework, a machine-learning algorithm based on human post-mortem brain data estimating developmental stage and regional identity of transcriptomic signatures. Our improved model in contrast to currently used SH-SY5Y models does capture early neurodevelopmental processes with high fidelity. We applied regression modelling, dynamic time warping analysis, parallel independent component analysis and weighted gene co-expression network analysis to identify activated gene sets and networks. Finally, we tested and compared these sets for enrichment of risk genes for neuropsychiatric disorders. We confirm a significant overlap of genes implicated in ASD with FXS, ID and SCZ. However, counterintuitive to this observation, we report that risk genes affect pathways specific for each disorder during early neurodevelopment. Genes implicated in ASD, ID, FXS and SCZ were enriched among the positive regulators, but only ID-implicated genes were also negative regulators of neuronal differentiation. ASD and ID genes were involved in dendritic branching modules, but only ASD risk genes were implicated in histone modification or axonal guidance. Only ID genes were over-represented among cell cycle modules. We conclude that the underlying signatures are disorder-specific and that the shared genetic architecture results in overlaps across disorders such as ID in ASD. Thus, adding developmental network context to genetic analyses will aid differentiating the pathophysiology

  16. Drosophila olfactory local interneurons and projection neurons derive from a common neuroblast lineage specified by the empty spiracles gene

    PubMed Central

    Das, Abhijit; Sen, Sonia; Lichtneckert, Robert; Okada, Ryuichi; Ito, Kei; Rodrigues, Veronica; Reichert, Heinrich

    2008-01-01

    Background Encoding of olfactory information in insects occurs in the antennal lobe where the olfactory receptor neurons interact with projection neurons and local interneurons in a complex sensory processing circuitry. While several studies have addressed the developmental mechanisms involved in specification and connectivity of olfactory receptor neurons and projection neurons in Drosophila, the local interneurons are far less well understood. Results In this study, we use genetic marking techniques combined with antibody labelling and neuroblast ablation to analyse lineage specific aspects of local interneuron development. We find that a large set of local interneurons labelled by the GAL4-LN1 (NP1227) and GAL4-LN2 (NP2426) lines arise from the lateral neuroblast, which has also been shown to generate uniglomerular projection neurons. Moreover, we find that a remarkable diversity of local interneuron cell types with different glomerular innervation patterns and neurotransmitter expression derives from this lineage. We analyse the birth order of these two distinct neuronal types by generating MARCM (mosaic analysis with a repressible cell marker) clones at different times during larval life. This analysis shows that local interneurons arise throughout the proliferative cycle of the lateral neuroblast beginning in the embryo, while uniglomerular projection neurons arise later during the second larval instar. The lateral neuroblast requires the function of the cephalic gap gene empty spiracles for the development of olfactory interneurons. In empty spiracles null mutant clones, most of the local interneurons and lateral projection neurons are lacking. These findings reveal similarities in the development of local interneurons and projection neurons in the olfactory system of Drosophila. Conclusion We find that the lateral neuroblast of the deutocerebrum gives rise to a large and remarkably diverse set of local interneurons as well as to projection neurons in the

  17. Role of horizontal gene transfer in the evolution of photosynthetic eukaryotes and their plastids.

    PubMed

    Keeling, Patrick J

    2009-01-01

    Plastids are the organelles derived from a cyanobacterium through endosymbiosis. Unlike mitochondria, plastids are not found in all eukaryotes, but their evolution has an added layer of complexity since plastids have moved between eukaryotic lineages by secondary and tertiary endosymbiotic events. This complex history, together with the genetic integration between plastids and their host, has led to many opportunities for gene flow between phylogenetically distinct lineages. Some intracellular transfers do not lead to a protein functioning in a new environment, but many others do and the protein makeup of many plastids appears to have been influenced by exogenous sources as well. Here, different evolutionary sources and cellular destinations of gene flow that has affected the plastid lineage are reviewed. Most horizontal gene transfer (HGT) affecting the modern plastid has taken place via the host nucleus, in the form of genes for plastid-targeted proteins. The impact of this varies greatly from lineage to lineage, but in some cases such transfers can be as high as one fifth of analyzed genes. More rarely, genes have also been transferred to the plastid genome itself, and plastid genes have also been transferred to other non-plant, non-algal lineages. Overall, the proteome of many plastids has emerged as a mosaic of proteins from many sources, some from within the same cell (e.g., cytosolic genes or genes left over from the replacement of an earlier plastid), some from the plastid of other algal lineages, and some from completely unrelated sources. PMID:19271204

  18. Horizontal gene transfer of an entire metabolic pathway between a eukaryotic alga and its DNA virus

    PubMed Central

    Monier, Adam; Pagarete, António; de Vargas, Colomban; Allen, Michael J.; Read, Betsy; Claverie, Jean-Michel; Ogata, Hiroyuki

    2009-01-01

    Interactions between viruses and phytoplankton, the main primary producers in the oceans, affect global biogeochemical cycles and climate. Recent studies are increasingly revealing possible cases of gene transfers between cyanobacteria and phages, which might have played significant roles in the evolution of cyanobacteria/phage systems. However, little has been documented about the occurrence of horizontal gene transfer in eukaryotic phytoplankton/virus systems. Here we report phylogenetic evidence for the transfer of seven genes involved in the sphingolipid biosynthesis pathway between the cosmopolitan eukaryotic microalga Emiliania huxleyi and its large DNA virus EhV. PCR assays indicate that these genes are prevalent in E. huxleyi and EhV strains isolated from different geographic locations. Patterns of protein and gene sequence conservation support that these genes are functional in both E. huxleyi and EhV. This is the first clear case of horizontal gene transfer of multiple functionally linked enzymes in a eukaryotic phytoplankton–virus system. We examine arguments for the possible direction of the gene transfer. The virus-to-host direction suggests the existence of ancient viruses that controlled the complex metabolic pathway in order to infect primitive eukaryotic cells. In contrast, the host-to-virus direction suggests that the serial acquisition of genes involved in the same metabolic pathway might have been a strategy for the ancestor of EhVs to stay ahead of their closest relatives in the great evolutionary race for survival. PMID:19451591

  19. Ultrasound -Assisted Gene Transfer to Adipose Tissue-Derived Stem/Progenitor Cells (ASCs)

    NASA Astrophysics Data System (ADS)

    Miyamoto, Yoshitaka; Ueno, Hitomi; Hokari, Rei; Yuan, Wenji; Kuno, Shuichi; Kakimoto, Takashi; Enosawa, Shin; Negishi, Yoichi; Yoshinaka, Kiyoshi; Matsumoto, Yoichiro; Chiba, Toshio; Hayashi, Shuji

    2011-09-01

    In recent years, multilineage adipose tissue-derived stem cells (ASCs) have become increasingly attractive as a promising source for cell transplantation and regenerative medicine. Particular interest has been expressed in the potential to make tissue stem cells, such as ASCs and marrow stromal cells (MSCs), differentiate by gene transfection. Gene transfection using highly efficient viral vectors such as adeno- and sendai viruses have been developed for this purpose. Sonoporation, or ultrasound (US)-assisted gene transfer, is an alternative gene manipulation technique which employs the creation of a jet stream by ultrasonic microbubble cavitation. Sonoporation using non-viral vectors is expected to be a much safer, although less efficient, tool for prospective clinical gene therapy. In this report, we assessed the efficacy of the sonoporation technique for gene transfer to ASCs. We isolated and cultured adipocyets from mouse adipose tissue. ASCs that have the potential to differentiate with transformation into adipocytes or osteoblasts were obtained. Using the US-assisted system, plasmid DNA containing beta-galactosidase (beta-Gal) and green fluorescent protein (GFP) genes were transferred to the ASCs. For this purpose, a Sonopore 4000 (NEPAGENE Co.) and a Sonazoid (Daiichi Sankyo Co.) instrument were used in combination. ASCs were subjected to US (3.1 MHz, 50% duty cycle, burst rate 2.0 Hz, intensity 1.2 W/cm2, exposure time 30 sec). We observed that the gene was more efficiently transferred with increased concentrations of plasmid DNA (5-150 μg/mL). However, further optimization of the US parameters is required, as the gene transfer efficiency was still relatively low. In conclusion, we herein demonstrate that a gene can be transferred to ASCs using our US-assisted system. In regenerative medicine, this system might resolve the current issues surrounding the use of viral vectors for gene transfer.

  20. Neuronal identity genes regulated by super-enhancers are preferentially down-regulated in the striatum of Huntington's disease mice.

    PubMed

    Achour, Mayada; Le Gras, Stéphanie; Keime, Céline; Parmentier, Frédéric; Lejeune, François-Xavier; Boutillier, Anne-Laurence; Néri, Christian; Davidson, Irwin; Merienne, Karine

    2015-06-15

    Huntington's disease (HD) is a neurodegenerative disease associated with extensive down-regulation of genes controlling neuronal function, particularly in the striatum. Whether altered epigenetic regulation underlies transcriptional defects in HD is unclear. Integrating RNA-sequencing (RNA-seq) and chromatin-immunoprecipitation followed by massively parallel sequencing (ChIP-seq), we show that down-regulated genes in HD mouse striatum associate with selective decrease in H3K27ac, a mark of active enhancers, and RNA Polymerase II (RNAPII). In addition, we reveal that decreased genes in HD mouse striatum display a specific epigenetic signature, characterized by high levels and broad patterns of H3K27ac and RNAPII. Our results indicate that this signature is that of super-enhancers, a category of broad enhancers regulating genes defining tissue identity and function. Specifically, we reveal that striatal super-enhancers display extensive H3K27 acetylation within gene bodies, drive transcription characterized by low levels of paused RNAPII, regulate neuronal function genes and are enriched in binding motifs for Gata transcription factors, such as Gata2 regulating striatal identity genes. Together, our results provide evidence for preferential down-regulation of genes controlled by super-enhancers in HD striatum and indicate that enhancer topography is a major parameter determining the propensity of a gene to be deregulated in a neurodegenerative disease.

  1. Sustained expression of a neuron-specific isoform of the Taf1 gene in development stages and aging in mice

    SciTech Connect

    Jambaldorj, Jamiyansuren; Makino, Satoshi; Munkhbat, Batmunkh; Tamiya, Gen

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer We identified the mouse homologue of neuron-specific TAF1 (N-Taf1). Black-Right-Pointing-Pointer Taf1 mRNA was expressed in most tissues and cell lines. Black-Right-Pointing-Pointer N-Taf1 mRNA was expressed in the brain and Neuroblastoma N2a cell lines. Black-Right-Pointing-Pointer Taf1 and N-Taf1 showed different expression profile in development stage and aging. -- Abstract: TATA-box binding protein associated factor 1 (TAF1) protein is the largest and the essential component of the TFIID complex in the pathway of RNA polymerase II-mediated gene transcription, and it regulates transcription of a large number of genes related to cell division. The neuron-specific isoform of the TAF1 gene (N-TAF1), which we reported previously, may have an essential role in neurons through transcriptional regulation of many neuron-specific genes. In the present study, we cloned the full-length cDNA that encodes the mouse homologue of N-TAF1 (N-Taf1) protein. By carrying out of real time RT-PCR, we investigated the expression analysis of the N-Taf1 mRNA in mouse tissues and cell lines. As well as the human N-TAF1, the N-Taf1 showed limited expression in the brain and neuroblastoma, whereas Taf1 expressed elsewhere. Furthermore, in mouse embryo head or mouse brain, mRNA expression of TAF1 changes dramatically during development but N-Taf1 showed sustained expression. Our result suggests that the N-Taf1 gene has an important role in non-dividing neuronal cell rather than in cell division and proliferation during neurogenesis.

  2. Gene Expression Profile of NF-κB, Nrf2, Glycolytic, and p53 Pathways During the SH-SY5Y Neuronal Differentiation Mediated by Retinoic Acid.

    PubMed

    de Bittencourt Pasquali, Matheus Augusto; de Ramos, Vitor Miranda; Albanus, Ricardo D Oliveira; Kunzler, Alice; de Souza, Luis Henrinque Trentin; Dalmolin, Rodrigo Juliani Siqueira; Gelain, Daniel Pens; Ribeiro, Leila; Carro, Luigi; Moreira, José Cláudio Fonseca

    2016-01-01

    SH-SY5Y cells, a neuroblastoma cell line that is a well-established model system to study the initial phases of neuronal differentiation, have been used in studies to elucidate the mechanisms of neuronal differentiation. In the present study, we investigated alterations of gene expression in SH-SY5Y cells during neuronal differentiation mediated by retinoic acid (RA) treatment. We evaluated important pathways involving nuclear factor kappa B (NF-κB), nuclear E2-related factor 2 (Nrf2), glycolytic, and p53 during neuronal differentiation. We also investigated the involvement of reactive oxygen species (ROS) in modulating the gene expression profile of those pathways by antioxidant co-treatment with Trolox®, a hydrophilic analogue of α-tocopherol. We found that RA treatment increases levels of gene expression of NF-κB, glycolytic, and antioxidant pathway genes during neuronal differentiation of SH-SY5Y cells. We also found that ROS production induced by RA treatment in SH-SY5Y cells is involved in gene expression profile alterations, chiefly in NF-κB, and glycolytic pathways. Antioxidant co-treatment with Trolox® reversed the effects mediated by RA NF-κB, and glycolytic pathways gene expression. Interestingly, co-treatment with Trolox® did not reverse the effects in antioxidant gene expression mediated by RA in SH-SY5Y. To confirm neuronal differentiation, we quantified endogenous levels of tyrosine hydroxylase, a recognized marker of neuronal differentiation. Our data suggest that during neuronal differentiation mediated by RA, changes in profile gene expression of important pathways occur. These alterations are in part mediated by ROS production. Therefore, our results reinforce the importance in understanding the mechanism by which RA induces neuronal differentiation in SH-SY5Y cells, principally due this model being commonly used as a neuronal cell model in studies of neuronal pathologies.

  3. Indirect Fitness Benefits Enable the Spread of Host Genes Promoting Costly Transfer of Beneficial Plasmids.

    PubMed

    Dimitriu, Tatiana; Misevic, Dusan; Lotton, Chantal; Brown, Sam P; Lindner, Ariel B; Taddei, François

    2016-06-01

    Bacterial genes that confer crucial phenotypes, such as antibiotic resistance, can spread horizontally by residing on mobile genetic elements (MGEs). Although many mobile genes provide strong benefits to their hosts, the fitness consequences of the process of transfer itself are less clear. In previous studies, transfer has been interpreted as a parasitic trait of the MGEs because of its costs to the host but also as a trait benefiting host populations through the sharing of a common gene pool. Here, we show that costly donation is an altruistic act when it spreads beneficial MGEs favoured when it increases the inclusive fitness of donor ability alleles. We show mathematically that donor ability can be selected when relatedness at the locus modulating transfer is sufficiently high between donor and recipients, ensuring high frequency of transfer between cells sharing donor alleles. We further experimentally demonstrate that either population structure or discrimination in transfer can increase relatedness to a level selecting for chromosomal transfer alleles. Both mechanisms are likely to occur in natural environments. The simple process of strong dilution can create sufficient population structure to select for donor ability. Another mechanism observed in natural isolates, discrimination in transfer, can emerge through coselection of transfer and discrimination alleles. Our work shows that horizontal gene transfer in bacteria can be promoted by bacterial hosts themselves and not only by MGEs. In the longer term, the success of cells bearing beneficial MGEs combined with biased transfer leads to an association between high donor ability, discrimination, and mobile beneficial genes. However, in conditions that do not select for altruism, host bacteria promoting transfer are outcompeted by hosts with lower transfer rate, an aspect that could be relevant in the fight against the spread of antibiotic resistance. PMID:27270455

  4. Indirect Fitness Benefits Enable the Spread of Host Genes Promoting Costly Transfer of Beneficial Plasmids

    PubMed Central

    Dimitriu, Tatiana; Misevic, Dusan; Lotton, Chantal; Brown, Sam P.; Lindner, Ariel B.; Taddei, François

    2016-01-01

    Bacterial genes that confer crucial phenotypes, such as antibiotic resistance, can spread horizontally by residing on mobile genetic elements (MGEs). Although many mobile genes provide strong benefits to their hosts, the fitness consequences of the process of transfer itself are less clear. In previous studies, transfer has been interpreted as a parasitic trait of the MGEs because of its costs to the host but also as a trait benefiting host populations through the sharing of a common gene pool. Here, we show that costly donation is an altruistic act when it spreads beneficial MGEs favoured when it increases the inclusive fitness of donor ability alleles. We show mathematically that donor ability can be selected when relatedness at the locus modulating transfer is sufficiently high between donor and recipients, ensuring high frequency of transfer between cells sharing donor alleles. We further experimentally demonstrate that either population structure or discrimination in transfer can increase relatedness to a level selecting for chromosomal transfer alleles. Both mechanisms are likely to occur in natural environments. The simple process of strong dilution can create sufficient population structure to select for donor ability. Another mechanism observed in natural isolates, discrimination in transfer, can emerge through coselection of transfer and discrimination alleles. Our work shows that horizontal gene transfer in bacteria can be promoted by bacterial hosts themselves and not only by MGEs. In the longer term, the success of cells bearing beneficial MGEs combined with biased transfer leads to an association between high donor ability, discrimination, and mobile beneficial genes. However, in conditions that do not select for altruism, host bacteria promoting transfer are outcompeted by hosts with lower transfer rate, an aspect that could be relevant in the fight against the spread of antibiotic resistance. PMID:27270455

  5. Reprogramming A375 cells to induced-resembled neuronal cells by structured overexpression of specific transcription genes

    PubMed Central

    Zhang, Hengzhu; Wei, Min; Jiang, Yangyang; Wang, Xiaodong; She, Lei; Yan, Zhengcun; Dong, Lun; Pang, Lujun; Wang, Xingdong

    2016-01-01

    Induced-resembled neuronal cells (irNCs) are generated by reprogramming human melanoma cells through the introduction of key transcription factors, providing novel concepts in the treatment of malignant tumor cells and making it possible to supply neural cells for laboratory use. In the present study, irNCs were derived from A375 cells by inducing the 'forced' overexpression of specific genes, including achaete-scute homolog 1 (Ascl1), neuronal differentiation factor 1 (Neurod1), myelin transcription factor 1 (Myt1), brain protein 2 (Brn2, also termed POU3F2) and human brain-derived neurotrophic factor (h-BDNF). irNCs induced from A375 cells express multiple neuronal markers and fire action potentials, exhibiting properties similar to those of motor neurons. The reprogramming procedure comprised reverse transcription-polymerase chain reaction and immunofluorescence staining; furthermore, electrophysiological profiling demonstrated the characteristics of the induced-resembled neurons. The present study obtained a novel type of human irNC from human melanoma, which secreted BDNF continuously, providing a model for neuron-like cells. Thus, irNCs offer promise in investigating various neural diseases by using neural-like cells derived directly from the patient of interest. PMID:27510459

  6. Mutations in the Drosophila pushover gene confer increased neuronal excitability and spontaneous synaptic vesicle fusion

    SciTech Connect

    Richards, S.; Hillman, T.; Stern, M.

    1996-04-01

    We describe the identification of a gene called pushover (push), which affects both behavior and synaptic transmission at the neuromuscular junction. Adults carrying either of two mutations in push exhibit sluggishness, uncoordination, a defective escape response, and male sterility. Larvae defective in push exhibit increased release of transmitter at the neuromuscular junction. In particular, the frequency of spontaneous transmitter release and the amount of transmitter release evoked by nerve stimulation are each increased two- to threefold in push mutants at the lowest external [(Ca{sup 2+})] tested (0.15 mM). Furthermore, these mutants are more sensitive than wild type to application of the potassium channel-blocking drug quinidine: following quinidine application, push mutants, but not wild-type, display repetitive firing of the motor axon, leading to repetitive muscle postsynaptic potentials. The push gene thus might affect both neuronal excitability and the transmitter release process. Complementation tests and recombinational mapping suggest that the push mutations are allelic to a previously identified P-element-induced mutation, which also causes behavorial abnormalities and male sterility. 43 refs., 5 figs., 1 tab.

  7. Bypassing hazard of housekeeping genes: their evaluation in rat granule neurons treated with cerebrospinal fluid of multiple sclerosis subjects

    PubMed Central

    Mathur, Deepali; Urena-Peralta, Juan R.; Lopez-Rodas, Gerardo; Casanova, Bonaventura; Coret-Ferrer, Francisco; Burgal-Marti, Maria

    2015-01-01

    Gene expression studies employing real-time PCR has become an intrinsic part of biomedical research. Appropriate normalization of target gene transcript(s) based on stably expressed housekeeping genes is crucial in individual experimental conditions to obtain accurate results. In multiple sclerosis (MS), several gene expression studies have been undertaken, however, the suitability of housekeeping genes to express stably in this disease is not yet explored. Recent research suggests that their expression level may vary under different experimental conditions. Hence it is indispensible to evaluate their expression stability to accurately normalize target gene transcripts. The present study aims to evaluate the expression stability of seven housekeeping genes in rat granule neurons treated with cerebrospinal fluid of MS patients. The selected reference genes were quantified by real time PCR and their expression stability was assessed using GeNorm and NormFinder algorithms. GeNorm identified transferrin receptor (Tfrc) and microglobulin beta-2 (B2m) the most stable genes followed by ribosomal protein L19 (Rpl19) whereas β-actin (ActB) and glyceraldehyde-3-phosphate-dehydrogenase (Gapdh) the most fluctuated ones in these neurons. NormFinder identified Tfrc as the best invariable gene followed by B2m and Rpl19. ActB and Gapdh were the least stable genes as analyzed by NormFinder algorithm. Both methods reported Tfrc and B2m the most stably expressed genes and Gapdh the least stable one. Altogether our data demonstrate the significance of pre-validation of housekeeping genes for accurate normalization and indicates Tfrc and B2m as best endogenous controls in MS. ActB and Gapdh are not recommended in gene expression studies related to current one. PMID:26441545

  8. Intrapleural 'outside-in' gene therapy: therapeutics for organs of the chest via gene transfer to the pleura.

    PubMed

    Heguy, Adriana; Crystal, Ronald G

    2005-10-01

    The pleural space is an attractive site for using viral vectors to deliver gene products to the lung parenchyma, other thoracic structures and the systemic circulation. The advantages of intrapleural gene transfer using viral vectors include: (i) easy accessibility; (ii) large surface area; (iii) ability to provide high concentrations of secreted gene products to chest structures; (iv) low risk of detrimental effects of possible vector-induced inflammation compared with intravascular delivery; and (v) because it is local, lower vector doses can be used to deliver therapeutic genes to thoracic structures than less efficient systemic routes. Examples of pleural gene transfer include the use of adenovirus vectors to treat mesothelioma by transiently expressing genes that encode toxic proteins, immunomodulatory molecules or anti-angiogenesis factors. Intrapleural delivery of adeno-associated viral vectors represents an efficient strategy to treat alpha1-antitrypsin (alpha1AT) deficiency, achieving high lung and systemic therapeutic levels of alpha1AT. Intrapleural delivery of gene transfer vectors holds promise for the treatment of diseases requiring transient, localized gene expression, as well as sustained expression of genes to correct hereditary disorders requiring localized or systemic expression of the therapeutic protein.

  9. Intrapleural 'outside-in' gene therapy: therapeutics for organs of the chest via gene transfer to the pleura.

    PubMed

    Heguy, Adriana; Crystal, Ronald G

    2005-10-01

    The pleural space is an attractive site for using viral vectors to deliver gene products to the lung parenchyma, other thoracic structures and the systemic circulation. The advantages of intrapleural gene transfer using viral vectors include: (i) easy accessibility; (ii) large surface area; (iii) ability to provide high concentrations of secreted gene products to chest structures; (iv) low risk of detrimental effects of possible vector-induced inflammation compared with intravascular delivery; and (v) because it is local, lower vector doses can be used to deliver therapeutic genes to thoracic structures than less efficient systemic routes. Examples of pleural gene transfer include the use of adenovirus vectors to treat mesothelioma by transiently expressing genes that encode toxic proteins, immunomodulatory molecules or anti-angiogenesis factors. Intrapleural delivery of adeno-associated viral vectors represents an efficient strategy to treat alpha1-antitrypsin (alpha1AT) deficiency, achieving high lung and systemic therapeutic levels of alpha1AT. Intrapleural delivery of gene transfer vectors holds promise for the treatment of diseases requiring transient, localized gene expression, as well as sustained expression of genes to correct hereditary disorders requiring localized or systemic expression of the therapeutic protein. PMID:16248279

  10. SAFETY AND EFFICIENCY OF MODULATING PARACELLULAR PERMEABILITY TO ENHANCE AIRWAY EPITHELIAL GENE TRANSFER IN VIVO

    EPA Science Inventory


    ABSTRACT

    We evaluated the safety of agents that enhance gene transfer by modulating paracellular permeability. Lactate dehydrogenase (LDH) and cytokine release were measured in polarized primary human airway epithelial (HAE) cells after luminal application of vehicle, ...

  11. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    NASA Astrophysics Data System (ADS)

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-09-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A -processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

  12. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    PubMed Central

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-01-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers. PMID:27608812

  13. Overexpression of glial cell line-derived neurotrophic factor induces genes regulating migration and differentiation of neuronal progenitor cells.

    PubMed

    Pahnke, Jens; Mix, Eilhard; Knoblich, Rupert; Müller, Jana; Zschiesche, Marlies; Schubert, Beke; Koczan, Dirk; Bauer, Peter; Böttcher, Tobias; Thiesen, Hans-Jürgen; Lazarov, Ludmil; Wree, Andreas; Rolfs, Arndt

    2004-07-15

    The glial cell line-derived neurotrophic factor (GDNF) is involved in the development and maintenance of neural tissues. Mutations in components of its signaling pathway lead to severe migration deficits of neuronal crest stem cells, tumor formation, or ablation of the urinary system. In animal models of Parkinson's disease, GDNF has been recognized to be neuroprotective and to improve motor function when delivered into the cerebral ventricles or into the substantia nigra. Here, we characterize the network of 43 genes induced by GDNF overproduction of neuronal progenitor cells (ST14A), which mainly regulate migration and differentiation of neuronal progenitor cells. GDNF down-regulates doublecortin, Paf-ah1b (Lis1), dynamin, and alpha-tubulin, which are involved in neocortical lamination and cytoskeletal reorganization. Axonal guidance depends on cell-surface molecules and extracellular matrix proteins. Laminin, Mpl3, Alcam, Bin1, Id1, Id2, Id3, neuregulin1, the ephrinB2-receptor, neuritin, focal adhesion kinase (FAK), Tc10, Pdpk1, clusterin, GTP-cyclooxygenase1, and follistatin are genes up-regulated by GDNF overexpression. Moreover, we found four key enzymes of the cholesterol-synthesis pathway to be down-regulated leading to decreased farnesyl-pyrophospate production. Many proteins are anchored by farnesyl-derivates at the cell membrane. The identification of these GDNF-regulated genes may open new opportunities for directly influencing differentiation and developmental processes of neurons. PMID:15212950

  14. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation

    NASA Astrophysics Data System (ADS)

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-09-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A –processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers.

  15. HMGB4 is expressed by neuronal cells and affects the expression of genes involved in neural differentiation.

    PubMed

    Rouhiainen, Ari; Zhao, Xiang; Vanttola, Päivi; Qian, Kui; Kulesskiy, Evgeny; Kuja-Panula, Juha; Gransalke, Kathleen; Grönholm, Mikaela; Unni, Emmanual; Meistrich, Marvin; Tian, Li; Auvinen, Petri; Rauvala, Heikki

    2016-01-01

    HMGB4 is a new member in the family of HMGB proteins that has been characterized in sperm cells, but little is known about its functions in somatic cells. Here we show that HMGB4 and the highly similar rat Transition Protein 4 (HMGB4L1) are expressed in neuronal cells. Both proteins had slow mobility in nucleus of living NIH-3T3 cells. They interacted with histones and their differential expression in transformed cells of the nervous system altered the post-translational modification statuses of histones in vitro. Overexpression of HMGB4 in HEK 293T cells made cells more susceptible to cell death induced by topoisomerase inhibitors in an oncology drug screening array and altered variant composition of histone H3. HMGB4 regulated over 800 genes in HEK 293T cells with a p-value ≤0.013 (n = 3) in a microarray analysis and displayed strongest association with adhesion and histone H2A -processes. In neuronal and transformed cells HMGB4 regulated the expression of an oligodendrocyte marker gene PPP1R14a and other neuronal differentiation marker genes. In conclusion, our data suggests that HMGB4 is a factor that regulates chromatin and expression of neuronal differentiation markers. PMID:27608812

  16. Age-related obesity and type 2 diabetes dysregulate neuronal associated genes and proteins in humans

    PubMed Central

    Daghighi, Mojtaba; Özcan, Behiye; Akbarkhanzadeh, Vishtaseb; Sheedfar, Fareeba; Amini, Marzyeh; Mazza, Tommaso; Pazienza, Valerio; Motazacker, Mahdi M.; Mahmoudi, Morteza; De Rooij, Felix W. M.; Sijbrands, Eric; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2015-01-01

    Despite numerous developed drugs based on glucose metabolism interventions for treatment of age-related diseases such as diabetes neuropathies (DNs), DNs are still increasing in patients with type 1 or type 2 diabetes (T1D, T2D). We aimed to identify novel candidates in adipose tissue (AT) and pancreas with T2D for targeting to develop new drugs for DNs therapy. AT-T2D displayed 15 (e.g. SYT4 up-regulated and VGF down-regulated) and pancreas-T2D showed 10 (e.g. BAG3 up-regulated, VAV3 and APOA1 down-regulated) highly differentially expressed genes with neuronal functions as compared to control tissues. ELISA was blindly performed to measure proteins of 5 most differentially expressed genes in 41 human subjects. SYT4 protein was upregulated, VAV3 and APOA1 were down-regulated, and BAG3 remained unchanged in 1- Obese and 2- Obese-T2D without insulin, VGF protein was higher in these two groups as well as in group 3- Obese-T2D receiving insulin than 4-lean subjects. Interaction networks analysis of these 5 genes showed several metabolic pathways (e.g. lipid metabolism and insulin signaling). Pancreas is a novel site for APOA1 synthesis. VGF is synthesized in AT and could be considered as good diagnostic, and even prognostic, marker for age-induced diseases obesity and T2D. This study provides new targets for rational drugs development for the therapy of age-related DNs. PMID:26337083

  17. Age-related obesity and type 2 diabetes dysregulate neuronal associated genes and proteins in humans.

    PubMed

    Rahimi, Mehran; Vinciguerra, Manlio; Daghighi, Mojtaba; Özcan, Behiye; Akbarkhanzadeh, Vishtaseb; Sheedfar, Fareeba; Amini, Marzyeh; Mazza, Tommaso; Pazienza, Valerio; Motazacker, Mahdi M; Mahmoudi, Morteza; De Rooij, Felix W M; Sijbrands, Eric; Peppelenbosch, Maikel P; Rezaee, Farhad

    2015-10-01

    Despite numerous developed drugs based on glucose metabolism interventions for treatment of age-related diseases such as diabetes neuropathies (DNs), DNs are still increasing in patients with type 1 or type 2 diabetes (T1D, T2D). We aimed to identify novel candidates in adipose tissue (AT) and pancreas with T2D for targeting to develop new drugs for DNs therapy. AT-T2D displayed 15 (e.g. SYT4 up-regulated and VGF down-regulated) and pancreas-T2D showed 10 (e.g. BAG3 up-regulated, VAV3 and APOA1 down-regulated) highly differentially expressed genes with neuronal functions as compared to control tissues. ELISA was blindly performed to measure proteins of 5 most differentially expressed genes in 41 human subjects. SYT4 protein was upregulated, VAV3 and APOA1 were down-regulated, and BAG3 remained unchanged in 1- Obese and 2- Obese-T2D without insulin, VGF protein was higher in these two groups as well as in group 3- Obese-T2D receiving insulin than 4-lean subjects. Interaction networks analysis of these 5 genes showed several metabolic pathways (e.g. lipid metabolism and insulin signaling). Pancreas is a novel site for APOA1 synthesis. VGF is synthesized in AT and could be considered as good diagnostic, and even prognostic, marker for age-induced diseases obesity and T2D. This study provides new targets for rational drugs development for the therapy of age-related DNs.

  18. The zebrafish trilobite gene is essential for tangential migration of branchiomotor neurons

    PubMed Central

    Bingham, Stephanie; Higashijima, Shin-ichi; Okamoto, Hitoshi; Chandrasekhar, Anand

    2009-01-01

    Newborn neurons migrate extensively in the radial and tangential directions to organize the developing vertebrate nervous system. We show here that mutations in zebrafish trilobite (tri) that affect gastrulation-associated cell movements also eliminate tangential migration of motor neurons in the hindbrain. In the wild-type hindbrain, facial (nVII) and glossopharyngeal (nIX) motor neurons are induced in rhombomeres 4 and 6, respectively, and migrate tangentially into r6 and r7 (nVII), and r7 (nIX). In all three tri alleles examined, although normal numbers of motor neurons are induced, nVII motor neurons are found exclusively in r4, and nIX-like motor neurons are found exclusively in r6. The migration of other neuronal and non-neuronal cell types is unaffected in tri mutants. Rhombomere formation and the development of other hindbrain neurons are also unaffected in tri mutants. Furthermore, tangential neuronal migration occurs normally in the gastrulation mutant knypek, indicating that the trilobite neuron phenotype does not arise non-specifically from aberrant gastrulation-associated movements. We conclude that trilobite function is specifically required for two types of cell migration that occur at different stages of zebrafish development. PMID:11820812

  19. Evolutionary Advantage Conferred by an Eukaryote-to-Eukaryote Gene Transfer Event in Wine Yeasts

    PubMed Central

    Marsit, Souhir; Mena, Adriana; Bigey, Frédéric; Sauvage, François-Xavier; Couloux, Arnaud; Guy, Julie; Legras, Jean-Luc; Barrio, Eladio; Dequin, Sylvie; Galeote, Virginie

    2015-01-01

    Although an increasing number of horizontal gene transfers have been reported in eukaryotes, experimental evidence for their adaptive value is lacking. Here, we report the recent transfer of a 158-kb genomic region between Torulaspora microellipsoides and Saccharomyces cerevisiae wine yeasts or closely related strains. This genomic region has undergone several rearrangements in S. cerevisiae strains, including gene loss and gene conversion between two tandemly duplicated FOT genes encoding oligopeptide transporters. We show that FOT genes confer a strong competitive advantage during grape must fermentation by increasing the number and diversity of oligopeptides that yeast can utilize as a source of nitrogen, thereby improving biomass formation, fermentation efficiency, and cell viability. Thus, the acquisition of FOT genes has favored yeast adaptation to the nitrogen-limited wine fermentation environment. This finding indicates that anthropic environments offer substantial ecological opportunity for evolutionary diversification through gene exchange between distant yeast species. PMID:25750179

  20. Direct Gene Transfer into Human Cultured Cells Facilitated by Laser Micropuncture of the Cell Membrane

    NASA Astrophysics Data System (ADS)

    Tao, Wen; Wilkinson, Joyce; Stanbridge, Eric J.; Berns, Michael W.

    1987-06-01

    The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in medium containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual human chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8 × 10-4-3 × 10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  1. Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane

    SciTech Connect

    Tao, W.; Wilkinson, J.; Stanbridge, E.J.; Berns, M.W.

    1987-06-01

    The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in media containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8x10-4-3x10-3. This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.

  2. Interleukin-6 gene transfer reverses body weight gain and fatty liver in obese mice.

    PubMed

    Ma, Yongjie; Gao, Mingming; Sun, Hao; Liu, Dexi

    2015-05-01

    Interleukin-6 (IL-6) is a multifunctional protein and has a major influence on energy metabolism. The current study was designed to assess the therapeutic effect of overexpression of Il-6 gene through gene transfer on high fat diet-induced obese mice. Hydrodynamic delivery of 1 μg pLIVE-IL6 plasmid per mouse into C57BL/6 obese mice resulted in peak level at 10 ng/ml of circulating IL-6 1 day after gene transfer and above 1n g/ml thereafter for a period of 6 weeks. Persistent Il-6 gene expression did not affect food intake but induced a significant reduction in body weight and improved obesity-associated hepatic steatosis. Il-6 gene delivery enhanced thermogenic gene expression and elevated protein levels of phosphorylated STAT3, PGC1α and UCP1 in brown adipose tissue. Il-6 overexpression elevated mRNA levels of lipolysis genes, triggered phosphorylation of STAT3, AMPK, and ACC, and increased expression of genes involved in fatty acid oxidation in skeletal muscle. IL-6 did not affect macrophage infiltration but maintained the M2 macrophage population in adipose tissue. Collectively, these results suggest that overexpression of the Il-6 gene by hydrodynamic gene delivery induces weight loss and alleviates obesity-induced fatty liver and insulin resistance, supporting the notion that gene transfer is a valid approach in managing obesity epidemics.

  3. Neuronal-type alpha-bungarotoxin receptors and the alpha 5-nicotinic receptor subunit gene are expressed in neuronal and nonneuronal human cell lines.

    PubMed Central

    Chini, B; Clementi, F; Hukovic, N; Sher, E

    1992-01-01

    alpha-Bungarotoxin (alpha Bgtx) is a toxin known to interact with muscle nicotinic receptors and with some neuronal nicotinic receptors. We show that alpha Bgtx binding sites are also expressed in nonmuscle and nonneuronal human cells, including small cell lung carcinoma and several epithelial cell lines. These receptors are immunologically related to the alpha Bgtx receptors of unknown function described in the nervous system and in the IMR32 neuroblastoma cell line and are distinct from muscle nicotinic receptors. We have also cloned from IMR32 cells the human alpha 5-nicotinic receptor subunit, which is supposed to participate in the formation of alpha Bgtx receptors. Transcripts corresponding to the alpha 5-subunit gene were found not only in neuroblastoma cells but also in all the cell lines expressing alpha Bgtx receptors, with the exception of the TE671 cell line, whose nicotinic receptor subunits are of the muscle type. We conclude that both alpha Bgtx receptors and the alpha 5-nicotinic subunit gene are not neuron-specific, as previously thought, but are expressed in a number of human cell lines of various origin. Images PMID:1542648

  4. Future directions in alcoholism research. Genomics and gene transfer.

    PubMed

    Brooks, P J; Lipsky, R H

    2000-01-01

    Alcohol affects the process by which genes direct the synthesis of proteins (i.e., expression). Therefore, patterns of gene expression in the presence of alcohol can help scientists identify the specific molecular sites of alcohol's actions within the brain. New technologies can detect and quantify changes in the expression of thousands of genes simultaneously by scanning microscopic gene arrays applied to glass or silicon chips an inch or so square. However, genes whose activity is altered in the presence of alcohol may either be contributing to alcoholism development or may be reacting to alcohol's presence. This question can be researched by observing the effects of manipulating the level of specific gene products. One way to accomplish this end is by means of viruses that have been engineered to express a specific gene in infected cells. This technique has been applied successfully in studying addictive behaviors. It is suggested that patterns of gene expression may become a diagnostic tool, with different disease states being characterized by distinct expression profiles.

  5. Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.

    PubMed

    Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

    2015-01-01

    Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002-0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer. PMID:26290434

  6. HDAC2 selectively regulates FOXO3a-mediated gene transcription during oxidative stress-induced neuronal cell death.

    PubMed

    Peng, Shengyi; Zhao, Siqi; Yan, Feng; Cheng, Jinbo; Huang, Li; Chen, Hong; Liu, Qingsong; Ji, Xunming; Yuan, Zengqiang

    2015-01-21

    All neurodegenerative diseases are associated with oxidative stress-induced neuronal death. Forkhead box O3a (FOXO3a) is a key transcription factor involved in neuronal apoptosis. However, how FOXO3a forms complexes and functions in oxidative stress processing remains largely unknown. In the present study, we show that histone deacetylase 2 (HDAC2) forms a physical complex with FOXO3a, which plays an important role in FOXO3a-dependent gene transcription and oxidative stress-induced mouse cerebellar granule neuron (CGN) apoptosis. Interestingly, we also found that HDAC2 became selectively enriched in the promoter region of the p21 gene, but not those of other target genes, and inhibited FOXO3a-mediated p21 transcription. Furthermore, we found that oxidative stress reduced the interaction between FOXO3a and HDAC2, leading to an increased histone H4K16 acetylation level in the p21 promoter region and upregulated p21 expression in a manner independent of p53 or E2F1. Phosphorylation of HDAC2 at Ser 394 is important for the HDAC2-FOXO3a interaction, and we found that cerebral ischemia/reperfusion reduced phosphorylation of HDAC2 at Ser 394 and mitigated the HDAC2-FOXO3a interaction in mouse brain tissue. Our study reveals the novel regulation of FOXO3a-mediated selective gene transcription via epigenetic modification in the process of oxidative stress-induced cell death, which could be exploited therapeutically.

  7. Lateral transfer of eukaryotic ribosomal RNA genes: an emerging concern for molecular ecology of microbial eukaryotes.

    PubMed

    Yabuki, Akinori; Toyofuku, Takashi; Takishita, Kiyotaka

    2014-07-01

    Ribosomal RNA (rRNA) genes are widely utilized in depicting organismal diversity and distribution in a wide range of environments. Although a few cases of lateral transfer of rRNA genes between closely related prokaryotes have been reported, it remains to be reported from eukaryotes. Here, we report the first case of lateral transfer of eukaryotic rRNA genes. Two distinct sequences of the 18S rRNA gene were detected from a clonal culture of the stramenopile, Ciliophrys infusionum. One was clearly derived from Ciliophrys, but the other gene originated from a perkinsid alveolate. Genome-walking analyses revealed that this alveolate-type rRNA gene is immediately adjacent to two protein-coding genes (ubc12 and usp39), and the origin of both genes was shown to be a stramenopile (that is, Ciliophrys) in our phylogenetic analyses. These findings indicate that the alveolate-type rRNA gene is encoded on the Ciliophrys genome and that eukaryotic rRNA genes can be transferred laterally.

  8. miR-128 regulates neuronal migration, outgrowth and intrinsic excitability via the intellectual disability gene Phf6.

    PubMed

    Franzoni, Eleonora; Booker, Sam A; Parthasarathy, Srinivas; Rehfeld, Frederick; Grosser, Sabine; Srivatsa, Swathi; Fuchs, Heiko R; Tarabykin, Victor; Vida, Imre; Wulczyn, F Gregory

    2015-01-01

    miR-128, a brain-enriched microRNA, has been implicated in the control of neurogenesis and synaptogenesis but its potential roles in intervening processes have not been addressed. We show that post-transcriptional mechanisms restrict miR-128 accumulation to post-mitotic neurons during mouse corticogenesis and in adult stem cell niches. Whereas premature miR-128 expression in progenitors for upper layer neurons leads to impaired neuronal migration and inappropriate branching, sponge-mediated inhibition results in overmigration. Within the upper layers, premature miR-128 expression reduces the complexity of dendritic arborization, associated with altered electrophysiological properties. We show that Phf6, a gene mutated in the cognitive disorder Börjeson-Forssman-Lehmann syndrome, is an important regulatory target for miR-128. Restoring PHF6 expression counteracts the deleterious effect of miR-128 on neuronal migration, outgrowth and intrinsic physiological properties. Our results place miR-128 upstream of PHF6 in a pathway vital for cortical lamination as well as for the development of neuronal morphology and intrinsic excitability.

  9. Reprogramming A375 cells to induced‑resembled neuronal cells by structured overexpression of specific transcription genes.

    PubMed

    Zhang, Hengzhu; Wei, Min; Jiang, Yangyang; Wang, Xiaodong; She, Lei; Yan, Zhengcun; Dong, Lun; Pang, Lujun; Wang, Xingdong

    2016-10-01

    Induced-resembled neuronal cells (irNCs) are generated by reprogramming human melanoma cells through the introduction of